var textForPages = ["","","Selected Analytical Techniques of Solid State,                  Structure Identification, and Dissolution Testing                                          in Drug Life Cycle","","Selected  Analytical  Techniques  of  Solid  State,                Structure Identification, and Dissolution Testing in                 Drug Life Cycle                                                                                 Edited by:                                            Martin Čulen | Jiří Dohnal | Josef Jampílek                                                 Masaryk University Press                Brno 2023","Reviewed by:                  Witold Musial                Department of Physical Chemistry and Biophysics, Faculty of Pharmacy                Wroclaw Medical University, Wrocław, Poland                  Kin Tam                Faculty of Health Sciences                University of Macau, Macau, China                             The authors sincerely thank Ing. Kateřina Škrlová, Ph.D. (www.deart.cz) from VŠB-                Technical University Ostrava (Czech Republic) for the design and realization of the                book cover.                     CC BY 4.0                  © 2022 Masarykova univerzita, Josef Jampílek, Hana Brusová, Jaroslav Havlíček,                Izabela Jendrzejewska, Christelle Kadlec, Filip Kadlec, Bohumil Kratochvil, Lucie                Krumbholcová, Tomáš Pekárek, Anna Řezáčová, Dita Spálovská                 ISBN: 978-80-280-0193-3                 https://doi.org/10.5817/CZ.MUNI.O280-0193-2022","Table of contents                 PREFACE                                                                   1                 1 INTRODUCTION                                                            2                    1.1 BRIEF HISTORY                                                      2                    1.2 CLASSIFICATION OF ANALYTICAL METHODS AND TECHNIQUES  4                    1.3 PROCESS HARMONIZATION                                              6                 2 INFRARED SPECTROSCOPY                                                   9                    2.1 INTRODUCTION                                                       9                    2.2 IR SPECTROSCOPY IN MID-REGION                                    11                      2.2.1 Theoretical principles of MIR spectrometry                   11                      2.2.2 Experimental setup for MIR spectrometry                      13                      2.2.3 Techniques for measuring MIR spectra                         17                     2.2.4 Application of MIR spectrometry in pharmaceutical industry   19                    2.3 IR SPECTROSCOPY IN NEAR REGION                                   24                      2.3.1 Theoretical basics of NIR spectroscopy                       24                      2.3.2 Experimental arrangement of NIR spectroscopy                 26                      2.3.3 NIR spectra measurement techniques                           29                      2.3.4 NIR analysis strategies                                      35                      2.3.5 Application of NIR spectroscopy in pharmacy                  36                    2.4 RESOURCES AND RECOMMENDED LITERATURE                             50                 3 RAMAN SPECTROSCOPY                                                    54                    3.1 INTRODUCTION                                                     54                    3.2 EXPERIMENTAL SETUP                                               59                   3.3 APPLICATION OF RAMAN SPECTROSCOPY                                62                      3.3.1 Identification of unknown samples                            62","3.3.2 Identification of polymorphs, solvates and salts             64                      3.3.3 Raman mapping and imaging                                    66                    3.4 RESOURCES AND RECOMMENDED LITERATURE                             69                 4 X-RAY DIFFRACTION                                                     70                    4.1 INTRODUCTION                                                     70                    4.2 CRYSTAL STRUCTURE, ITS GEOMETRY AND SYMMETRY                     70                   4.3 X-RAYS                                                           74                      4.3.1 X-ray tube                                                   74                      4.3.2 Characterization of X-ray radiation                          77                    4.4 ANALYTICAL X-RAY DIFFRACTION METHODS                             78                      4.4.1 Interactions of X-rays with crystal                          82                      4.4.2 Powder methods                                               95                    4.5 APPLICATIONS OF X-RAY DIFFRACTION IN PHARMACY                   102                      4.5.1 Characterization and identification of unknown samples      102                      4.5.2 Crystalline phase control                                   105                      4.5.3 Negative proof of crystalline phase – amorphous material    107                      4.5.4 Observation of phase transformations                        108                     4.5.5 Study of substance behavior under defined conditions        110                      4.5.6 Determination of structures of unknown impurities or molecule chirality                                                                                       112                      4.5.7 Calculation of powder diffraction patterns                  112                      4.5.8 Determination of a 3D crystal structure from X-ray powder data   113                      4.5.9 Quantitative phase analysis                                 115                    4.6 SUMMARY                                                         116                    4.7 RESOURCES AND RECOMMENDED LITERATURE                            117","5 NMR SPECTROSCOPY                                                     119                    5.1 BASIC PRINCIPLES                                                119                    5.2 NMR SPECTROMETER                                                123                       1                   5.3 H NMR SPECTROSCOPY                                              126                     5.3.1 Signal position – chemical shift                            126                      5.3.2 Integral signal intensity                                   129                     5.3.3 Signal multiplicity                                         130                       13                   5.4 C NMR SPECTROSCOPY                                              134                   5.5 TWO-DIMENSIONAL NMR SPECTRA                                     138                    5.6  APPLICATION  OF  LIQUID  PHASE  NMR  SPECTROSCOPY  IN                   PHARMACY                                                            143                      5.6.1 Structural analysis of APIs                                 143                      5.6.2 Structural analysis of impurities                           146                    5.7 SOLID STATE NMR SPECTROSCOPY                                    148                     5.7.1 Theoretical introduction                                    148                       5.7.2 Application of solid state NMR spectroscopy in pharmacy     156                   5.8 CONCLUSION                                                      171                    5.9 RESOURCES AND RECOMMENDED LITERATURE                            172                 6 TERAHERTZ TIME-DOMAIN SPECTROSCOPY                                   174                    6.1 INTRODUCTION                                                    174                    6.2   OVERVIEW      OF    EXPERIMENTAL       TECHNIQUES       IN   THZ                   SPECTROSCOPY                                                        175                    6.3 PRINCIPLES OF THZ TIME-DOMAIN SPECTROSCOPY                      177                      6.3.1 Principle of THz pulse generation                           178                     6.3.2 Detection of THz pulses                                     179","6.4 THZ SPECTRA MEASURING TECHNIQUES AND DATA PROCESSING                                                                                       180                      6.4.1 Transmittance measurements                                  180                      6.4.2 Reflectance measurements                                    182                      6.4.3 Attenuated total reflection                                 183                     6.4.4 Time of flight measuring method                             184                      6.4.5 Imaging measurement                                         184                    6.5 APPLICATIONS OF THZ SPECTROSCOPY IN PHARMACY                    184                      6.5.1 Identification of substances using THz spectra              184                      6.5.2 Determination of hydration state of substances              186                      6.5.3 Determination of tablet coating thickness                   188                      6.5.4 Determination of grain size                                 188                    6.6 RESOURCES AND RECOMMENDED LITERATURE                            189                 7 MASS SPECTROMETRY                                                    191                   7.1 BASIC PRINCIPLES                                                191                     7.2 IONIZATION TECHNIQUES                                           193                     7.2.1 Electron ionization (EI)                                    195                      7.2.2 Chemical ionization (CI)                                    196                      7.2.3 Fast atom or ion bombardment ionization (FAB, FIB)          198                      7.2.4 Matrix assisted laser desorption/ionization (MALDI)         198                      7.2.5 Thermospray ionization (TSI)                                199                      7.2.6 Electrospray ionization (ESI)                               200                      7.2.7 Atmospheric pressure chemical ionization (APCI)             203                      7.2.8 Atmospheric pressure photoionization (APPI)                 204                      7.2.9 New ionization techniques                                   205","7.3 MASS ANALYZERS                                                  206                      7.3.1 Magnetic analyzer with simple magnetic focusing             207                      7.3.2 Sector analyzer with double ion focusing                    208                      7.3.3 Quadrupole (Q)                                              209                      7.3.4 Triple quadrupole analyzer (QqQ)                            209                      7.3.5 Ion trap (IT)                                               210                     7.3.6 Linear ion trap (LIT)                                       211                      7.3.7 Orbitrap                                                    212                      7.3.8 Time of flight analyzer (TOF)                               214                      7.3.9 Ion cyclotron resonance with Fourier transform (FT-ICR)     215                    7.4 ION DETECTION TECHNIQUES                                        216                    7.5 IDENTIFICATION AND DETERMINATION OF COMPOUNDS                   218                    7.6 STRUCTURAL MS ANALYSIS IN PHARMACY                              221                      7.6.1 Modern MS systems in pharmacy                               222                    7.7 RESOURCES AND RECOMMENDED LITERATURE                            225                 8 THERMAL ANALYSIS                                                     226                    8.1 DEFINITION OF THERMAL ANALYSIS METHODS                          226                   8.2 GENERAL PRINCIPLE OF THERMAL ANALYSIS METHODS                   227                    8.3 THERMAL METHODS                                                 229                      8.3.1 Basic measurement factors                                   229                      8.3.2 Measuring procedure                                         230                      8.3.3 Evaluation of thermoanalytical measurements                 231                      8.3.4 Differential scanning thermal analysis (DSC)                234                      8.3.5 Hyper DSC                                                   239                      8.3.6 Micro DSC                                                   240","8.3.7 Temperature modulated differential scanning calorimetry (TMDSC)  249                      8.3.8 Thermogravimetry (TGA)                                      254                      8.3.9 Thermally stimulated current (TSC)                          259                    8.4 RESOURCES AND RECOMMENDED LITERATURE                            262                 9 DISSOLUTION TESTING IN PHARMACEUTICAL INDUSTRY                       264                    9.1 BIOPHARMACEUTICAL CLASSIFICATION SYSTEM                         264                   9.2 DISSOLUTION TESTING OF SUBSTANCES                               266                      9.2.1 Intrinsic dissolution                                       266                      9.2.2 Apparent dissolution                                        271                    9.3 DISSOLUTION TESTING OF SOLID DOSAGE FORMS                       275                      9.3.1 Dissolution                                                 275                      9.3.2 Experimental dissolution methods                            277                      9.3.3 Dissolution apparatuses                                     279                      9.3.4 Advanced dissolution systems                                289                    9.4 RESOURCES AND RECOMMENDED LITERATURE                            293                 10 SUMMARY                                                             295                 11 ABBREVIATIONS                                                       298                12 AUTHORS                                                             303","Preface                 According  to  the  experience  of  the  authors  of  this  textbook,  the  activities  of  the                pharmaceutical  faculties  focus  primarily  on  preparing  students  for  the  work  in                community  pharmacy.  The  field  of  pharmaceutical  research,  development  and                industrial  applications,  on  the  other  and,  has  been  rather  neglected.                However,  the  modern  pharmaceutical  industry  primarily  needs  well-prepared                experts with solid knowledge across the entire pharmaceutical field. This book is                intended to achieve this precise goal.                 The  foundation  for  the  book  “Selected  Analytical  Techniques  of  Solid  State,                Structure  Identification,  and  Dissolution  Testing  in  Drug  Life  Cycle”  was  a                textbook  created almost  before  eleven  years  ago  as a  study  material  for  a  newly                approved  optional  class  “Analysis  in  pharmaceutical  industry”,  at  the  Faculty  of                Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno.                The textbook covered a very wide area – methods used in analytical development                and  quality  control  (QC)  of  pharmaceutical  products.  Examples  of  the  particular                methods  applied  in  solving  of  practical  problems  were  implemented  into  most                chapters.                The  current  version  is  focused  mainly  on  solid  state  and  structural  analysis.  The                topics  are  not  discussed  in  an  exhaustive  manner.  Only  the  most  important  and                prevalent methods and techniques are mentioned, and the text is simplified. On the                other  hand,  the  chapter  on  dissolution  was  extended  by  addition  of  special                dissolution methods. The textbook thus coveres three important areas: 1. structural                analysis  of  drugs,  2.  pharmaceutical  analysis  in  solid  state,  3.  dissolution  testing                methods.                 The structure and content of this textbook reflect the very experience of the authors                who  personally  contributed  or  introduced  the  described  approaches  and  methods                into  practice.  The  author  team  includes  senior  experts  who  were  involved  in  the                implementation of good practices in pharmacy and the according legislation, and                also  in  providing  the  related  technical  and  methodological  requirements.  At  the                same time, they were also at the very beginning of the harmonization process. The                younger  authors,  on  the  other  hand,  brought  innovative  solutions  to  many                problebms.                Most of the authors work in academy and have thus extensive teaching experience.                 It is up to the reader to assess whether we have accomplished to raise his the interest                in the given areas and to provide necessary information.                                                               1","1 Introduction                 1  Introduction                 Jiří Dohnal                   Motto:                 “The  laboratory  is  an  extension  of  our  senses,  enabling  us  to  obtain  data  on                substances beyond what we can see with naked eye and in amounts that our hands                could never grasp. These data are compiled into reports and are ultimately used for                making decisions, decisions that cannot be confirmed with our unaided senses. The                quality of any decision is absolutely dependent on the quality of the data; junk data                lead to junk decisions.“                J. M. Miller                   1.1  Brief history                 The pharmaceutical quality system was developed in steps that can be characterized                as follows:                        Period up to the 60’s of the past century, with a little alleviation, sometimes                also called “the dark age”.                The dominant methods in pharmaceutical analysis were volumetric and gravimetric                methods. Among the separation methods, thin layer chromatography showed more                intensive  use,  but  without  the  option  to  objectively  quantify  the  results,  in  the                beginnings.                Impurities in percentile quantities undetected by analysts were no exception! Little                attention  was  paid  to  chirality  and  polymorphism,  due  to  the  lack  of  proper                instruments for their differentiation. The analyses were in most cases carried out by                dissolving the analysed compounds, hence making some of their original properties                undetectable.                        70’s – cGMP                 This period was marked by the progress in separation methods, particularly the gas                chromatography (GC), and later also the high performance liquid chromatography                (HPLC) which constituted a revolution in pharmaceutical analysis. The importance                of this period also laid in the introduction of computational systems as operating                units for analytical instruments and data processing.                                                       2","1 Introduction                 The  introduction  of  good  laboratory  practice  (GMP)  and  other  good  practices  in                pharmaceutical development and production, together termed GxP, meant another                step forward in means  of safety  and  quality.  Stricter  limits for impurity contents                were established and the separation methods became more common.                 The  GC  analyses  however  had  their  disadvantages  and  often  required  a                derivatization of the quantified compounds, which was time consuming and with a                risk  of  artefact  creation.  The  gas  chromatography  is  of  course  still  in  use,  but                primarily  for  analysis  of  volatile  substances  and  for analysis  of  residual  solvents                when used in headspace technique.                        80’s – validation                 The  80’s  were marked  by  progress  achieved  in  instrumental  analysis, mainly  the                structural  analysis  methods,  such  as  cryogenic  nuclear  magnetic  resonance,  new                mass  spectrometry  methods  and  electrochemical  methods,  for  example  capillary                isotachophoresis, and later also other electromigratory methods.                 The term validation has a wider meaning, however due to the focus of this book, the                validation of analytical methods will be be only briefly communicated.                The first occurrence of the term validation is dated back to the turn of the 70’s and                80’s and is linked to common good manufacturing practice (cGMP) requirements in                drug production.                 Standard  requirements  on  registration  documentation  appointed  by  the  State                Institute for Drug Control (SUKL), defined on the basis of USP XXII and European                Community requirements on medicines, are valid since 1992 (former Republic of                Czechoslovakia).                 The validation policy was published in 1994 in the SUKL bulletin along with the                list of analytical parameters inspected during method validation, including a brief                procedure manual.                        2000 – continual quality verification                 Major part of quality control has been merged directly with production. Attributes                which  can  play  a  key  role  in  the  changes  of  product  quality  are  measured  and                analysed. The basis of Process Analytical Technology (PAT) is design, analysis and                management of production by in situ measuring of the above-mentioned attributes.                This was a revolutionary change in the approach to pharmaceutical analysis, as the                main interest was formerly the outcome analysis of the final product. These new                requirements  lead  to  selection  of  suitable  analytical  methods  which  are  often                                                         3","1 Introduction                 mistaken  with  the  PAT  itself.  The  PAT  depends  mainly  on  the  speed  and  the                capability of these methods for monitoring of selected attributes.                        Current approach – risk management-based approach                 Quality risk management ICH Q9 and Technical and regulatory considerations for                paharmaceutical  life  cycle  management  ICH  Q12.  Quality  risk  management  is  a                systematic process for the assessment, control, communication and review of risks                to the quality of the drug (medicinal) product across the product lifecycle.                                                     Figure 1.1. Overview of a typical quality risk management process (www.ich.org).                   1.2  Classification of analytical methods and techniques                1.   The selection of an appropriate analytical technique depends on the level of                    focus:                     •  Molecular  level:  infrared  spectrometry  techniques – attenuated  total                          reflectance  (ATR)  technique,  diffuse  reflectance  infrared  Fourier                          transform  (DRIFT)  technique,  Raman  spectrometry,  X-ray  structural                                                        4","1 Introduction                           analysis of single crystal or powder material, solid state nuclear magnetic                          resonance (ssNMR) etc.                     •  Intermolecular  level,  particles:  powder  X-ray  diffraction  (PXRD),  optical                          and electron microscopy – scanning electron microscopy (SEM) and its                          modification  –  electron  tomography,  transmission  electron  microscopy                          (TEM),  terahertz  spectroscopy  (applies  to  intermolecular  bond                          vibrations),  and  thermal  methods  –  differential  scanning  calorimetry                          (DSC),  micro DSC,  hyper DSC,  thermogravimetry  analysis  (TGA),                          thermally stimulated current (TSC).                     •  Bulk:  surface  region  –  inverse  gas  chromatography  (IGC),  high  energy                          regions,  thickness,  porousness,  flow  characteristics  (of  powders),                          dynamic  vapour  sorption  (DVS);  also  small  angle  X-ray  scattering                          (SAXS) for determination of particle size and shape etc.                2.   Based  on  the  physico-chemical  principles  and  due  to  historical  reasons,  the                    analytical methods in pharmaceutical analysis are divided as follows:                     •  Classic: volumetry, gravimetry etc.                     •  Electrochemical: potentiometry, amperometry, coulometry etc.                    •  Optical  methods:  refractometry,  polarimetry,  turbidimetry,  nephelometry                          etc.                     •  Spectral:  spectrometry  in  ultraviolet  and  visible  region,  spectrometry                          techniques  based  on  rotational  and  vibrational  transitions  [infrared                          spectrometry  in  mid  region  (MIR)  and  near  region  (NIR),  Raman                          spectrometry, terahertz spectrometry], circular dichroism, NMR, see Fig.                          1.1.                     •  Separation:  thin  layer  chromatography  (TLC),  HPLC,  GC,  supercritical                          fluid  chromatography  (SFC),  capillary  electrophoresis  (CE),  extraction                          methods, e.g., supercritical fluid extraction (SFE) etc.                     •  Techniques  based  on  light  scattering:  PXRD,  laser  diffraction,  dynamic                          diffraction, (Raman spectrometry) etc.                    •  Thermal methods: DSC, TGA, TSC etc.                                                                5","1 Introduction                                   Figure 1.2. Spectral analytical techniques, which use the electromagnetic radiation                                           of different wavelengths.                             (Source: http://www.infralytic.de/bilder/oel/spectrum_NG.gif, 1. 12. 2009.)                 3.   Less  common  classification  of  analytical  methods  (except  for  solid  state                    analysis) is based on the phase, in which the analysis is carried out:                    •  analysis in solutions (liquid state)                    •  analysis in solid state                    •  analysis in gas state                    •  analysis in plasma                   1.3 Process harmonization                A team of experts was appointed to perform international unification of the quality                system, and  the  most important  act  which  defined  tripartite  harmonization in the                quality field was the International Conference on Harmonization – Quality.                 It  is  a  list  of  guidelines  and  recommendations  intended  for  harmonization  of                requirements of regulatory authorities of USA, Europe and Japan.                  Another  authority  dealing  with  harmonization  is  the  Pharmocopeial  Discussion                Group  (PDG).  PDG  and  ICH  have  common  goals,  although  they  differ  in  the                manner of problem solving.                 To list the particular ICH Q guidelines would be inappropriately burdening for this                publication, taking into account its primary aims. The attention will be focused only                on three guidelines, which are directly related to the rest of the book content – ICH                Q2(R1), Q3 and Q6 and Q14                                                        6","1 Introduction                 The  first  is  the  ICH  Q2(R1)  „Analytical  Validations  –  Validation  of  Analytical                Procedures: Text and Methodology“.                 Formerly,  the  guideline  was  divided  to  Q2A  (the  primary  guideline  –  October                1994),  Q2B  (supplement  guideline  regarding  methodology  –  November  1996),                Q2(R1) – November 2005.                 The following items were subjects of validation:                   •   identity tests                   •   impurity quantification tests                  •   impurity control limit tests                   •   quantification tests for active pharmaceutical ingredient (API), dosage form                      (DF) or other selected product contents                 The  draft  of  the  guideline  ICH  Q14  –  “Analytical  Procedure  Development”  was                endorsed  on  24  March  2022  and  is  currently  under  public  consultation  and  a                consensus  guideline  was  published.  The  revised  Q2(R1)  will  include  validation                principles that cover analytical use of spectroscopic or spectrometry data (e.g., NIR,                Raman, NMR or MS), some of which often require multivariate statistical analyses.                These types of validations are especially useful for methods described in the new                edition of this book.                 The quality related harmonization process also comprises the impurity issues. The                Q series of the ICH guidelines belong to the most important ones and are divided as                follows:                    •  Q3A(R2) “Impurities in New Drug Substances“ (October 2006),                    •  Q3B(R2) “Impurities in New Drug Products“ (June 2006),                    •  Q3C(R8) “Impurities ‒ Guideline for Residual Solvents“ (1997, updates and                      appendices September 2002, October 2002 and November 2005).                    •  Q3D(R1) „Guideline for Elemental Impurities“ (March 2019)                 Guidelines Q3A(R2) and Q3B(R2) deal with classification of impurities, reasons for                impurity reporting and impurity control, which are divided into:                    •  organic impurities                    •  inorganic impurities                   •  solvents                                                       7","1 Introduction                 They also describe analytical procedures, impurity contents in batches, specification                list of impurities and classification of impurities. The guidelines include limits and                decision trees.                 The above mentioned guidelines do not concern polymorphism and optical purity.                These issues are partly discussed in guideline Q6A “Specifications: Test Procedures                and  Acceptance  Criteria  for  New  Drug  Substances  and  New  Drug  Products“                (October 1999).                 Guideline  Q6B  “Specifications:  Test  Procedures  and  Acceptance  Criteria  for                Biotechnological/Biological  Products”  (March  1999),  describes  the  testing                procedures and acceptation criteria for new biotechnological/biological products.                 The guideline Q6A can be divided into:                1.  General tests:                     •  description                     •  specific identification tests                     •  content quantification, selective tests for stability monitoring                     •  impurities                2.  Specific tests:                     •  physico-chemical characteristics (pH, Tt, refractive index etc.)                     •  particle size                     •  polymorphism (decision tree)                     •  set of tests for chiral API and DF                    •  water content                     •  inorganic impurities                     •  microbial limits                                                                  8","2 Infrared spectroscopy                 2  Infrared spectroscopy                 Tomáš Pekárek, Josef Jampílek, Dita Spálovská                   2.1  Introduction                 Spectroscopy in infrared (IR) region is an analytical optical method based on the                absorption of electromagnetic radiation from matter. Electron state of molecules is                not altered, but changes occur in vibrational-rotational energy states of molecules,                depending on changes in their dipole moment. The absorbed energy quanta increase                the  internal  molecular  energy,  which  is  related  to  atomic  bond  vibrations  and                rotations of the individual parts of a molecule.                The IR spectroscopy methods in pharmaceutical industry have wide application in                routine  quality  control  as  well  as  in  development.  The  main  advantages  are  the                measurement  speed  (seconds  to  minutes),  simple  or  no  sample  preparation,  and                relatively low instrument costs. For common measurements, an instrument may be                operated by a person without any deep knowledge of the principles of the method,                which  lowers  manpower  costs.  However,  when  dealing  with  more  complex                applications (such as measuring in complex dosage form matrices or quantification                of  components)  where  it  is  necessary  to  decide  on  the  most  appropriate                experimental arrangement, and also when evaluating spectra of unknown substances                or identifying structures, the knowledge of basic principles is necessary, as will be                explained  in  the  following  sections.  Since  dealing  with  molecular  (functional)                analysis,  the  IR  spectroscopy  can  provide  valuable  insight  into  the  structural                constitution  of  molecules,  functional  groups,  qualitative  and  quantitative                composition  of  mixtures  of  both  solid  and  liquid  samples  (including  suspensions                and emulsions). IR spectroscopy is also used for determination of gases. However,                the last application is not used in routine pharmaceutical practice, due to rarity of                gaseous drugs. Besides, in case of aerosols, it is possible to analyze the condensed                liquid or solid component.                IR techniques are recognized by state authorities as suitable methods for analysis of                pharmaceuticals.  Their  brief  description  with  instructions  for  calibration  and                validation of the instrument is listed in Pharmacopoeias (Ph. Eur.) and in guidelines.                 It can be said that IR and also Raman spectroscopies (see below) are techniques of                the first choice when a rapid, reliable, and validated analysis of drugs is required.                Their general versatility and only small limitations make them applicable in almost                each laboratory in both industry and development. Pharmaceutical development and                industry are no exceptions in this regard.                                                       9","2 Infrared spectroscopy                 IR spectroscopy uses interaction between infrared radiation and matter. IR radiation                is an electromagnetic radiation which can be considered both a wave and a stream                of  particles  (photons).  Radiation  can  be  characterized  by  speed  c,  wavelength  λ,                                                    −1                frequency  ν  [Hz],  wavenumber  υ  [cm ]  and  radiation  energy  E,  to  which  the                following relationships apply:                                                                h – Planck constant                            ℎ                            1                     = ℎ   =                      =            =                                                                                                                          Infrared   radiation   is   an                invisible thermal radiation which is generally emitted by all objects, especially at                higher temperatures. IR radiation is surrounded by visible light on one side of the                spectrum and by microwave radiation on the other side (Fig. 2.1). Its upper limit is                determined by spectral sensitivity of the human eye, the lower limit by ability to                create  and  detect  IR  radiation  with  the  longest  wavelengths.  IR  radiation  partly                overlaps  with  the  area  of  microwave  radiation  (electronically  produced                electromagnetic radiation) on its bottom boundary and is defined by wavelengths λ                in the range of 0.78‒1000 μm. However, wavenumber is used as a unit much more                                                                                         −1                frequently in infrared spectroscopy and is defined by the area of υ = 12800‒5 cm                (the boundaries are not sharp, different authors use different values).                                  Figure 2.1. Wavelengths of individual areas of electromagnetic spectrum.                   (Source: http://ipac.jpl.nasa.gov/media_images/screen_gif/background/spectrum_callout.gif, 10. 12. 2009.)                 Given the very broad energy range of photons of the IR spectrum they may cause                different  energy  transitions  when  they  collide  with  molecules.  Therefore,  the                infrared region is divided into three parts:                                                        −1                   •  Near-Infrared (NIR) 12800–4000 cm  (0.78–2.50 μm)                                                    −1                   •  Mid-Infrared (MIR) 4000–200 cm  (2.50–50 μm)                                                −1                   •  Far-Infrared (FIR) 200–5 cm  (50–1 000 μm)                IR  radiation  in  the  FIR  region  has  the  lowest  energy,  which  causes  changes  in                rotational energy of molecules. FIR is therefore primarily the region of rotational                                                     10","2 Infrared spectroscopy                 spectra  and  is  of  particular  importance  for  theoretical  chemistry  and  molecular                physics. It has practically no use in pharmaceutical industry.                 MIR region is the most important region in terms of pharmaceutics analyses. It is a                region  where  majority  of  the  fundamental  vibrations  appear  –  the  vibrational                transitions from the ground to the first excited vibrational state, as described below.                The focus of the analytical applications of these methods lies in the use in structural                diagnosis, characterization and identification of organic and inorganic substances.                 Photons  in  NIR  region  have  the  highest  energy  and  can  therefore  vibrationally                excite molecules into even higher excited vibrational states than the first level, i.e.,                the  second,  third,  and others.  These transitions are called  overtons.  Although the                overtons  are  generally  associated  with  lower  absorption  strength  than  the                fundamental  vibrations,  the  NIR  spectroscopy  has  great  analytical  importance                especially  in  many  areas  of  practical  and  applied  chemistry,  particularly  in                qualitative and quantitative analyses.                   2.2  IR spectroscopy in mid-region                2.2.1  Theoretical principles of MIR spectrometry                                                                                 −1                Mid-infrared region (MIR) uses radiation in the range of 4000‒200 cm . No bond                in molecules is rigid but still vibrates with a certain frequency. Non-linear molecule                with  N  atoms  has  3N  degrees  of  freedom,  three  of  which  represent  translational                motion  in  mutually  perpendicular  directions  (axes  x,  y,  z)  and  three  represent                rotational movement along the axes x, y, z. The remaining 3N-6 degrees of freedom                represent  the  number  of  options  how  a  non-linear  molecule  may  vibrate  –                vibrational modes. A linear molecule is deprived of rotary motion around an axis                intersecting the bonds because there is no regrouping of atoms, and the molecule                then has one extra vibrational mode (3N-5).                 As IR radiation passes through a sample, those parts of the radiation that match the                frequency of bond vibrations in the molecule are absorbed. A vibration must meet a                basic condition to be seen as a characteristic band in an IR spectrum and that is to                have non-zero dipole moment change matching normal coordinate Qi, that is:                                                                                                            ≠ 0                                                                        Furthermore, anharmonicity (more information can be found in textbooks of physics                and  physical  chemistry)  partially  suppresses  selection  rule,  forbidding  transitions                between  the  vibrational  levels  by  more  than  one  level.  Thus,  the  spectrum  may                                                      11","2 Infrared spectroscopy                 contain overtones, which is a case when a molecule transits to other than the next                higher  vibrational  energy  state  and  which  may  contain  combination  bands  when                combinations  of  various vibrational  modes  occur.  When interpreting fundamental                modes,  overtone  bands  are  usually  not  an  obstacle  as  they  have  low  intensity                compared to the fundamental transitions.                 A molecule can generally vibrate in several ways. The vibrations may be stretching                when a change in bond length occurs or deformation when a change in a bond angle                occurs.  These  basic  types  of  vibrations  have  several  subgroups.  Stretching                oscillations can be symmetrical or asymmetrical, deformation oscillations can be in-                plane or out-of-plane. In-plane vibrations can be scissoring or rocking, out-of-plane                can be wagging or twisting, as shown in Fig. 2.2. Stretching vibrations are denoted                by ν, deformation vibrations by δ. Added footnote can be used to describe the type                of the vibration, for example δω will represent deformation wagging vibration. The                symbol can be used alone to denote the vibration type, e.g., ω.                                     Stretching vibrational modes of a CH group                                                                    2                                              symmetrical   asymmetrical                                   Deformation vibrational modes of a CH group                                                                    2                               in-plane                              out-of-plane                       scissoring δ        rocking ρ            twisting γ        wagging ω                                          Figure 2.2. Vibrational modes.                         (Edited according to: Socrates G. Infrared and Raman Characteristic Group Frequencies.                                             John Wiley \& Sons, 2001.)                 In the region between 4000 and 1500 cm , vibrational transitions of atomic bonds                                                     −1                of particular functional groups occur (oscillating with a particular frequency), while                the rest of the molecule remains almost motionless, and positions of the particular                absorption bands correspond to the particular functional groups with influence from                the rest of the molecule. In other words, it is possible to deduce the presence of a                certain functional group based on positions of bands in spectrum. Correlation tables                are  used  for  this  purpose  to  simplify  orientation  in  spectra.  Examples  of  band                assigning will be discussed in the applications section.                                                        12","2 Infrared spectroscopy                 Skeletal vibrations are also found in the spectra, especially in region of 1500 to 200                   −1                cm , and they result from vibrations of many atoms in a molecule. Such vibrations                are not used to identify functional groups but can be used to distinguish structurally                similar molecules.                 To identify a molecule, it is not necessary to assign all the bands that appear in the                spectrum, but it is necessary to find all bands belonging to vibrational modes of a                particular  functional  group.  Above  mentioned  means  that  there  are  no  two                compounds with identical IR spectra. Nowadays, when instruments are controlled                by  computers,  it  is  possible  to  search  for  spectra  in  electronic  spectral  libraries,                which significantly speeds up the whole process.                  2.2.2  Experimental setup for MIR spectrometry                 All  optical  parts  of  an  instrument  must  be  made  from  IR  transparent  material,                mainly  alkali  metals  and  alkaline  earth  metals  salts  (KBr,  NaCl,  ZnSe).  Fig.  2.3                shows general configuration scheme of spectrometers with Fourier transform, which                are currently used almost exclusively.                 All spectrometers are composed of three basic parts:                    •  source of radiation                    •  detector                    •  interferometer                 MIR  spectroscopy  uses  silicon  carbide  or  NiCr  resistance  wire  heated  to  a  high                temperature  as  a  source  of  radiation.  Infrared  systems  use  two  basic  detection                systems  ‒  thermal  and  quantum.  In  MIR  spectroscopy,  an  example  of  a  thermal                detector  is  deuterated  triglycine  sulphate  (DTGS)  detector,  and  an  example  of  a                quantum detector is mercury cadmium telluride (Hgx-1CdxTe, MCT) detector. The                most important part of an instrument is an interferometer. Dispersion systems with a                dispersion element (prism, grating) used for separation of beam were used in the                past and are still being applied in some special applications. Common applications                use  almost  exclusively  interferometric  systems  with  Fourier  transform  (FT).  The                principle of interferometry can be easily explained for a case where there is only                one wave which is registered on the detector as a sine function. This simple function                is  used  for  monitoring  and  calibration  of  the  position  and  movement  of  mirrors                using  an  HeNe  laser.  High-intensity  radiation  proceeds  to  the  interferometer.  An                interferometer  consists  of  a  beamsplitter  that  reflects  half  of  the radiation  onto  a                fixed  mirror  and  another  half  passes  further  onto  a  moving  mirror.  The  moving                mirror reflects the radiation back, and the radiation thus travels a different optical                                                     13","2 Infrared spectroscopy                 path  than  the  radiation  reflected  from  the  fixed  mirror.  When  these  beams                recombine back on the beamsplitter and the distance of the fixed and the moving                mirror is such that the two waves are in phase, a constructive interference appears                and there is a high detector response. If the distance between the two mirrors is such                that the recombining waves are not in phase, there is a destructive interference at the                beamsplitter. Therefore, the response of the detector will be lower. This difference                in optical path is scanned and used for internal and absolute wavelength calibration.                                                fixed mirror                                         beam-splitter                                                                IR source                    moving                    mirror                                 x       0      -x                        He-Ne lasser                                                        lasser diode                                            sample                                                       detector                    Figure 2.3. Basic construction scheme of a spectrometer with Fourier transform.                          (Edited according to: Základy FT-NIR spektrometrie. Presentation. Nicolet CZ, 2008.)                 The model becomes more complicated for two waves but remains valid even  for                many waves (a source with a wide range of wavelengths is used).                 When  measuring  samples,  recombination  of  beams  occurs  on  the  beamsplitter                according to the above-mentioned rules, and the radiation is reflected further to the                sample. The interferometer modulates the IR radiation and creates an interferogram                containing the whole spectral information about the sample. The detector measures                intensity of the modulated radiation.                 Every moving part of the instrument is a subject to changes over time. To eliminate                this device instability, a \"Dynamic Alignment\" function may be used, which is a                continuous  correction  of  the  interferometer  optics  adjustment  and  is  based  on                measurements of three laser signals in three different places. Slightest change in the                movement leads to modulation of these signals, and the position of the fixed mirror                is continuously finely adjusted by three electromagnetic transducers. This correction                                                      14","2 Infrared spectroscopy                 is performed not only at the beginning of the measurement, but throughout its whole                course.                 To convert an interferogram into a spectrum, it is needed to change time domain                represented  by  the  interferogram  to  a  frequency  domain  (spectrum).  Fourier                transform uses two parameters in IR spectrometry application. One is addition of                zeros  into  the  interferogram  (zero  filling),  which  is  used  to  obtain  interpolated                spectrum  values.  And  the  second  parameter  is  apodization.  The  interferogram  is                truncated by the apodization function.                 Materials used in production of optical components of IR spectrometers are highly                hygroscopic. For this reason, it is required to keep the spectrometer under voltage so                the  whole  apparatus  is  permanently  heated  to  prevent  condensation  of  aqueous                vapors  on  the  optical elements.  Furthermore,  water  strongly absorbs  IR  radiation                and limits the use of IR spectroscopy for measurements of samples with high water                content.  This  also  results  in  the  need  to  measure  “background”  to  eliminate  the                effect  of  air  molecules  which  create  response  on  the  IR  detector  (air  humidity,                carbon dioxide etc.). An example of the atmospheric spectral background is shown                in  Fig.  2.4.  The  spectrum  contains  four  regions  of  evident  absorptions  (it  is  a                transmittance  measurement  and  the  absorption  region  in  the  background                measurement  leads  to  decrease  in  the  intensity  of  the  signal  incident  on  the                detector). The regions 1 and 2 can be therefore assigned to vibrations of molecules                of atmospheric humidity (stretching and deformation) and regions 3 and 4 belong to                stretching and deformation vibrations of carbon dioxide.                 In  total,  five  types  of  interactions  of  radiation  with  solid  sample  can  be                distinguished  (Fig.  2.5).  When  specific  radiation  passes  through  a  homogenous                environment, its initial intensity is attenuated by an extent which is absorbed by the                environment.  The  intensity  of  radiation  is  thus  reduced  by  absorption,  but  the                wavelength or the wave number remains unchanged.                                                                          15","2 Infrared spectroscopy                      36                    34                    32                    30                    28                    26                    24                    22                  Single Beam    20                    18                    16                    14                    12                    10                                                 2                     8                                 3                     6                                                           4                     4         1                     2                     4000      3500      3000      2500      2000      1500      1000      500                                                              -1                                                  Wavenumber (cm )                             Figure 2.4. MIR spectrum of an atmospheric background.                                                              Figure 2.5. Interactions of radiation with solid matter.                   (Edited according to: Giehl A. NIR reflectance spectroscopy in pharmaceutical drug product manufacture\"                                     Presentation. Bruker Optik GmbH Germany, 2006.)                                                       16","2 Infrared spectroscopy                 When  a  light  beam  with  intensity  Φ0  passes  through  a  layer  of  absorbing                environment  with  thickness  l,  a  part  of  the  beam  is  absorbed  and  a  part  with                intensity Φ passes through. The following relationships apply:                            Φ                 T – transmittance                       =                         Φ 0                                           A – absorbance                           1                      =                    100 T – transmittance [%]                                               For  reflectivity  measurements,  reflectance  R  [%]  is  often  used  and  is  defined  as                reflected radiation flow ΦR divided by incident radiation flow Φ0.                                                      Φ                                                    =                                                           Φ 0                                                                    ´                The reflectance is then formally converted to absorbance A  using the relationship:                                                         1                                                  ´                                                   =                                                                                  Beer-Lambert law implies that absorbance is a linear function of concentration. By                plotting dependence A = f(c) at a constant wavelength λ (and wave number υ), a                calibration line can be obtained. In presence of multiple components, the resulting                absorbance is given by the sum of the absorbances of the individual components.                 Beer-Lambert law:                                            ε – molar absorption coefficient [l/(mol.cm)]                                           c – molar concentration [mol/l]                       =                                                  l – layer thickness [cm]                   2.2.3  Techniques for measuring MIR spectra                 IR  spectra  in  mid  region  can  be  measured  as  attenuation  of  radiation  flow  after                passing  through  a  sample  (transmission  measurements)  or  after  reflection  of  the                radiation (reflection measurements).                                                         17","2 Infrared spectroscopy                 Transmission  measurements  are  used  for  analysis  of  gases,  liquids,  transparent                films, and solids. Liquid samples must be measured in special cells which are made                of,  for example, KBr,  NaCl,  CaF2,  BaF2,  ZnSe,  CsI, AgCl,  AgBr,  Ge,  silicon, or                mixed crystal of thallium iodide and bromide (KRS-5). Solvent selection must be                well considered as the above-mentioned cell materials have different solubility in                different  solvents.  A  cuvette  window  must  be  made  of  a  material  sufficiently                resistant to the applied solvent. The range of solvents is very wide and the most                used are halogenated solvents, such as chloroform and carbon disulfide, since they                provide a small number of characteristic bands in the spectrum. For measurements                of  samples  in  solid  state,  suspensions  in  paraffin  oil  or  tablets  created  by                compression of the sample with KBr are used.                 Reflectance  measurements  which  are  currently  the  most  widespread  in  practice                usually  use  ATR  (Attenuated  Total  Reflection)  technique.  Arrangement  of  this                experiment  involves  a  thin  layer  of  a  sample  pressed  on  crystal  mostly  made  of                ZnSe,  Ge,  or  Si,  or  diamond.  Each  material  has  different  spectral  and  physico-                chemical characteristics, and the crystal is chosen according to the requirements of                the analysis. Infrared radiation impacts a thin (1 μm) layer of the sample, and the                beam is reflected onto the crystal where it is refracted and continues through the                optical system to the detector. Refraction on a crystal may be multiple or only single                (Fig. 2.6). ATR technique enables measurement of solid samples, as well as liquids                or various semi-solid materials. It should be emphasized, however, that while the                transmission technique yields IR spectrum of an “averaged” sample, the ATR yields                spectrum  of  an  \"actual\"  sample  (sample  surface  facing  the  crystal).  Unlike  in                transmission spectra, the ATR technique leads to highlighted intensity of bands in                low  wavenumber  region,  contrary  to  the  bands  with  higher  wavenumbers.                Therefore,  the  spectra  obtained  by  ATR  technique  are  often  mathematically                corrected, to be closer to the transmission spectra.                                         Figure 2.6. Scheme of IR beam reflection on an ATR crystal.                 A very important and useful part of an IR spectrometer is a microscope connected                to the spectrometer. The mapping function which measures spectra of spatially very                close areas, point by point, and the imaging function which uses an area detector to                                                     18","2 Infrared spectroscopy                 display  an  image  of  the  studied  sample,  can  be  used  to  determine  the  size  and                distribution of individual components not only in powder mixtures but also directly                in dosage forms, which is with comparable spectral and spatial resolution currently                not possible by any other technique except Raman spectroscopy. This way, a spatial                resolution in orders of microns can be achieved. Besides, it is possible to measure                minimum sample amounts and detect foreign particles contained in mixtures and/or                dosage forms.                 Another advantage is the ability to lead the radiation outside of the spectrometer                using optical fibers, which is applied when measuring with probes. Radiation is led                from the spectrometer by optical fibers into a probe which measures in either the                absorption  or  ATR  arrangement.  Signal  attenuated  by  the  absorbed  part  of  the                radiation is led by the fibers to the detector. The probe measurement approach does                not  change  the  principle  of  the  method,  only  the  layout  of  the  experiment.  The                option to bring the spectrometer to the sample is mainly used for identification of                substances in packages or in cases where sample collection is not possible or very                difficult (reaction kinetics, samples packaged under controlled atmosphere, etc.).                 Further  details  of  experimental  configuration,  including  appropriate  sample                preparation, then depend on the measurement type and will not be discussed further                in this publication.                   2.2.4  Application of MIR spectrometry in pharmaceutical industry                 The  first  important  application  which  shall  be  mentioned  in  this  place  is  a                verification of molecule structures or also identification of unknown substances. In                production of substances or excipients, as well as when purchasing from a supplier,                it is necessary to verify not only the identity of final products but also identity of                intermediates and raw materials. The IR spectroscopy is a very suitable method for                characterization of unknown samples or confirmation of structures. An example of                assignment  of  characteristic  bands  of  vibrational  modes  of  functional  groups  is                shown for acetylsalicylic acid in Fig. 2.7.                 Besides  structure confirmation and identification  of unknown  compounds, the IR                spectroscopy is also used as a pharmacopeial method for identity confirmation using                standards. Spectrum of an examined substance is compared either with a record of a                standard,  provided  in  a  company  norm  with  positions  and  relative  intensities  of                bands, or it is compared using a software. In the latter case, computer compares the                records according to the assigned parameters and provides the matching result. Both                above  mentioned  applications  are  commonly  used  in  research,  development,  and                quality control laboratories.                                                      19","2 Infrared spectroscopy                 In  research  and  development  laboratories,  the  application  of  IR  is  even  more                extensive, because of the need of the most detailed information about the developed                formulation. In case of generic companies, a thorough comparison of the developed                formulation  with  original  pharmaceutical  is  also  required.  Here,  it  is  possible  to                apply the full potential of the IR spectroscopy. In analyses of active substances and                certain excipients, an issue of polymorphism also emerges.                      1,3                                                                               1188                     1,2                                                                     1692                     1,1                     1,0                                                                             1307                                                                    1754                     0,9                     0,8                                               1606   1221                  Absorbance   0,7                                        1458                      0,6                                                             917                     0,5                                                                           1419  1370                                            2871                                 1095   756                     0,4                        2699                               1013  840  667 705                     0,3                      0,2                     4000      3500      3000      2500      2000      1500       1000                                                    Wavenumbers (cm-1)                                 Figure 2.7. IR spectrum of acetylsalicylic acid.                                              −1                                                                     −1                 Bands represent: 3000–2600 cm  ν(O–H), 3100–3000 cm  ν(C–H) aromatic ring,                                                               −1                                                                                       −1                                −1                   3000–2800 cm  ν(C–H) aliphatic chain, 1754 cm  ν(C=O) esther, 1692 cm                                                                  −1                                                                                   −1                                    −1                 ν(C=O) acid, 1606 cm  ν(C=C) aromatic ring, 1458 cm  δ(CH3), 1221 cm  νas(C–                                                     −1                                          O), 1188 cm  νsym(C–O).                Crystalline substances can exist in different crystal modifications, as their atoms can                be arranged in different crystal structures. This is called polymorphism. Depending                on the type of the crystal modification, each polymorph of the same substance has                different properties,  such as melting point, dissolution rate, stability,  etc. Besides                other instrumental methods, such as X-ray powder diffraction and thermal analysis,                IR spectroscopy can also be used to detect the type of a crystal modification or to                distinguish  between  individual  crystal  modifications.  It  is  however  necessary  to                proceed  with  a  parallel  use  of  the  powder  X-ray  diffraction  which  is  the  only                technique  able  to  identify  polymorphic  arrangement  of  molecules  without  a                standard.  Samples  characterized  this  way  can  then  be  used  as  standards  for  IR                identification of polymorphs. An example of comparison of infrared spectra of two                polymorphic forms of one substance is shown in Fig. 2.8.                                                     20","2 Infrared spectroscopy                       95                     90                     85                  %R   80                     75                     70                     65                      95                     90                     85                     80                  %R   75                     70                     65                     60                     55                     4000      3500      3000      2500      2000      1500       1000                                                   Wavenumbers (cm-1)                     Figure 2.8. Example of comparison of two polymorphs of one compound.                 When analyzing samples containing more than one substance (for example dosage                forms),  it  is  necessary  to  confirm  the  identity  of  the  individual  components  and                especially of the active substance. One of the tests (besides separation techniques)                which can confirm the identity of an active substance, is IR spectroscopy. Ideally,                the content of the active substance is high enough so that the bands of the active                substance can be clearly identified in the spectra of the dosage form (Fig. 2.9). If the                bands of the active substance are overlapped by signals of other components, the                active substance must be extracted from the sample by an appropriate technique and                subsequently identified or determined in the given extract. For such identification, it                is  necessary  to  be  aware  of  the  possibility  of  polymorph  transformation  during                                                                                 −1                crystallization. Fig. 2.9 shows a spectrum in the range of 3600‒600 cm , where a                clear  similarity  between  red  spectrum  (working  standard)  and  magenta  spectrum                (dosage form) can be observed. These two spectra differ especially in the region                               −1                around 1000 cm  where an obvious contribution of blue spectrum (placebo) occurs.                In  other  words,  the  green  curve  is  the  sum  of  the  red  and  the  blue  curve,  in  an                appropriate  ratio.  This  implies  that  IR  spectrometry  can  be  also  used  for  semi-                quantitative  and  quantitative  analyses,  assuming  that  the  Beer-Lambert  law  is                applicable. For such measurements, it is however necessary to provide a calibration                curve or to use method of standard addition.                                                             21","2 Infrared spectroscopy                     90  Ibuprofenum                   80                  %R  70                    60                   50                   100                      Placebo of Ibalgin 600                   90                  %R  80                    70                       Ibalgin 600                   95                  %R                   90                    85                    4000      3500      3000      2500      2000      1500      1000                                                Wavenumbers (cm-1)                   Figure 2.9. Example of confirmation and identification of active substance in                  Ibalgin  600 dosage form. (Measurement in reflective setup – absorption regions                        ®                 oriented downwards). Working standard of the active substance (ibuprofen) – red,                                     dosage form – green, placebo – blue.                 IR spectroscopy can also distinguish different salts of one molecule. In production                of  dosage  forms,  the  active  substances  are  sometimes  injected  along  with  an                equimolar amount of, for example, sodium hydroxide, which leads to preparation of                a  sodium salt.  It is therefore  necessary to  monitor  whether the  salt  conversion is                fully completed and whether no unwanted ion exchange occurs. An example of the                difference between IR spectra of different salts of one active substance is shown in                Fig. 2.10                 In addition to the several above-mentioned examples, IR spectroscopy can also be                used  for  identification  of  excipients  and  packaging  materials  in  pharmaceutical                industry. Although arrangement and evaluation of the measurements then depend on                the objectives and the information to be obtained, the principle is still the same –                qualitative or quantitative determination as described above.                 Mapping  and  imaging  applications  using  connection  of  IR  spectroscopy  with  IR                microscope enable determination of very important information about particle size                of active substances in tablets, their distribution (Fig. 2.11), and for example coating                uniformity. The microscope also helps to easily identify even very small particles                that  stick  to  walls  of  ampoules,  without  the  need  for  difficult  separation  or                destructive analysis by other techniques.                                                      22","2 Infrared spectroscopy                    90                       API                   80                  %R  70                   60                   50                   40                          Sodium salt                   90                  %R                   80                    70                            Ammonium salt                   90                  %R  80                   70                   60                           1600        1400        1200         1000        800                                                               -1                                                Wavenumbers (cm )                      Figure 2.10. Comparison of different salts of one molecule; sodium salt,                                            ammonium salt, acid.                           Component 1                       Component 2                      Component 3                                    API                       Figure 2.11. Example of component distribution (FTIR map), of a pharmaceutical                                                   tablet.                                                             23","2 Infrared spectroscopy                 2.3  IR spectroscopy in near region                 2.3.1  Theoretical basics of NIR spectroscopy                 Spectroscopy in the near-infrared region (NIR) is a molecular spectroscopy method                                                                     −1                that uses radiation in the range between 12800 and 4000 cm .                The  physical  principle  for  creation  of  NIR  spectra  is  the  same  as  for  MIR                spectroscopy. Absorption of photons with frequencies identical to bond vibrations                leads to excitation of these bonds. Difference is in the higher energy of the NIR                radiation.  Absorption  of  radiation  in  the  NIR  region  is  usually  based  on  higher                energy  transitions  between  vibrational  levels  of  molecules,  namely  combination                transitions and overtones, and not on fundamental transitions which are dominant in                MIR region.                Combination transitions represent a simultaneous excitation of several vibrational                modes. Transition energy equals the sum of the energies of fundamental transitions                of  the  particular  vibrational  modes.  Overtone  then  represents  an  excitation  of  a                vibrational  mode  to  a  higher  excited  level.  The  first  overtone  corresponds                approximately to twofold of the fundamental transition energy, second overtone to                threefold,  third  overtone  to  fourfold  etc.  For  a  more  accurate  description,  it  is                necessary to consider anharmonicity of vibrational modes as mentioned in section                2.2.1.1.                         1                                         NIR region                          Absorbance  0.5                 combination         Fundamental                                                          transition                                                                             vibration                                                  1st overtone                                          2nd overtone                       (Mid IR)                                 3rd overtone                                   10000     8000     6000     4000  3000  2000  1000                                               Wavenumbers                  Figure 2.12. Intensity of absorption bands in NIR region compared to MIR region.                          (Source: Vzorkovací techniky v FT-NIR spektrometrii. Presentation. Nicolet CZ, 2008.)                                                      24","2 Infrared spectroscopy                 Assignment  of  absorption  bands  to  individual  combinational  transitions  and                overtones is difficult and often even impossible (Fig. 2.13), and the usual analysis is                therefore not used to identify a particular functional group as with MIR spectra. It is                possible  to  define  regions  where  specific  bands  are  dominant:  combinational                                        −1                transitions (4000–5300 cm ), first overtones (4600–7300 cm ), second overtones                                                                        −1                               −1                                                                   −1                (6000–10000 cm ) and third overtones (8800–12800 cm ), see Fig. 2.14.                                                             Figure 2.13. Absorption bands in NIR.                              (Source: Základy FT-NIR spektrometrie. Presentation. Nicolet CZ, 2008.)                              0.9                             0.8                                       Combination                            0.7                                                               1st Overtone                            0.6                                                      2nd Overtone                         Log(1/R)   0.5                            0.4                                          3rd Overtone                            0.3                            0.2                            0.1                                     10000    9000     8000     7000     6000     5000                                                    Wavenumbers (cm-1)                                   Figure 2.14. Example of a NIR spectrum.                              (Source: Základy FT-NIR spektrometrie. Presentation. Nicolet CZ, 2008.)                                                      25","2 Infrared spectroscopy                 2.3.2  Experimental arrangement of NIR spectroscopy                 NIR  spectrometers  are  less  demanding  on  build  materials  than  the  instruments                working in the MIR region, which is due to different radiation frequencies used in                the NIR spectrometry. NIR radiation passes through quartz glass, and therefore any                optics  or  cuvettes  can  be  made  of  quartz  that  is  commonly  used  in  UV-VIS                spectroscopy. Construction of NIR spectrometers with Fourier transform is the same                as for MIR spectrometers, as shown in  Fig. 2.3, described in section  2.2.1.2. All                NIR spectrometers consist of three basic components:                    •  radiation source                    •  detector                    •  interferometer                 Halogen  bulbs  are  used  as  radiation  sources.  Various  InGaAs,  PbSe,  PbS  or  Si                detectors are applied, and beamsplitters are made of quartz or CaF2. Similarly as in                MIR,  water  molecules  absorb  NIR  radiation,  and  it  is  therefore  necessary  to                measure  a  reference  spectrum  (spectrum  background)  before  the  analysis  to                eliminate the environmental effects, especially atmospheric humidity. Example of                atmospheric background is shown in Fig. 2.15.                                                         Figure 2.15. NIR spectrum of atmospheric background.                                                           26","2 Infrared spectroscopy                 Due to the nature of the near IR radiation, the intensity of NIR spectra is inferior to                MIR spectra. Fig. 2.16 shows MIR and NIR spectra of polystyrene. Fig. 2.17 then                shows comparison of the same spectra in the same scale.                 For qualitative purposes, it is necessary to compare the whole NIR spectrum or a                characteristic region with spectra of pure substances, standards, or spectral libraries.                Very often, the NIR spectra are sorted and classified using chemometric methods.                 Same  as  for  the  mid  IR  region,  a  radiation  passing  through  a  homogenous                environment is attenuated, and its initial intensity is lowered by an extent that is                absorbed by this material. The absorption thus reduces the radiation intensity. Due                to  the  nature  of  the  NIR  spectra  –  the  bands  are  usually  broad  even  for  pure                substances,  and  for  mixtures,  the  shape  of  absorption  bands  can  be  affected  by                overlapping  bands  of  different  components  or  mutual  effects  of  varying                concentrations of individual components. It is therefore not possible to apply the                simple Beer-Lambert law for this method.                       Pol yst yre ne measured as  a fil m                      Pol yst yre ne measured as  a fil m                      Pol yst yre ne measured as  a fil m                       Polystyren - spektrum v MIR oblasti                       Polystyren - spektrum v MIR oblasti                       Polystyren – MIR spectrum                     4, 5                     4, 5                     4, 5  Polystyren - spektrum v MIR oblasti                     4, 0                     4, 0                     4, 0                     3, 5                     3, 5                     3, 5                     3, 0                     3, 0                     3, 0                  Absorbance  Absorbance  Absorbance   2, 5                     2, 5                     2, 5                     2, 0                     2, 0                     2, 0                     1, 5                     1, 5                     1, 5                     1, 0                     1, 0                     1, 0                     0, 5                     0, 5                     0, 5                               350 0    300 0     250 0    200 0          150 0    100 0          500                               350 0                                                                     150 0                                                                              100 0                                                  250 0                                                           200 0                                                           200 0                                                                     150 0                                                  250 0                                        300 0                                                                                        500                               350 0                                                                              100 0                                                                                        500                                        300 0                                                   Wave numb ers  (cm-1)                                                   Wave numb ers  (cm-1)                                                   Wave numb ers  (cm-1)                      Pol yst yre n NI R                     0, 1 8                     0, 1 8  Pol yst yre n NI R                       Polystyren – spektrum v NIR oblasti                       Polystyren – spektrum v NIR oblasti                     0, 1 7 Polystyren – NIR spectrum                     0, 1 7                     0, 1 6                     0, 1 6                     0, 1 5                     0, 1 5                     0, 1 4                     0, 1 4                     0, 1 3                     0, 1 3                  Absorbance  Absorbance   0, 1 2                     0, 1 2                     0, 1 1                     0, 1 1                     0, 1 0                     0, 1 0                     0, 0 9                     0, 0 9                     0, 0 8                     0, 0 8                     0, 0 7                     0, 0 7                     0, 0 6                     0, 0 6                     0, 0 5                     0, 0 5                                                                                 500 0                                100 00                                          900 0                                                                       600 0                                100 00    900 0        800 0    700 0        600 0        500 0                                                             700 0                                                    800 0                                                   Wave numb ers  (cm-1)                                                   Wave numb ers  (cm-1)                         Figure 2.16. Spectrum of polystyrene in mid and near IR region.                              (Source: Základy FT-NIR spektrometrie. Presentation. Nicolet CZ, 2008.)                                                    27","2 Infrared spectroscopy                        Polystyren NIR Polystyrene - spectrum in MIR region                      Polystyrene measured as a film                     4,5                            Polystyrene – spectrum in NIR region                     4,0                     3,5                      3,0                  Absorbance   2,5                      2,0                      1,5                      1,0                      0,5                             10000        8000         6000         4000        2000                                                     Wavenumbers (cm-1)                 Figure 2.17. Comparison of a spectrum of polystyrene in near and mid IR regions.                                        (The same scale on the Y axis.)                              (Source: Základy FT-NIR spektrometrie. Presentation. Nicolet CZ, 2008.)                  Calibration for quantitative purposes in NIR spectrometry requires development of                calibration models with the use of advanced chemometric algorithms which require                an  extensive  set  of  standards  (at  least  30  calibration  samples).  The  set  must  be                sufficiently representative to cover the entire expected or predicted variability of the                sample  characteristics  which  are  to  be  quantified.  The  most  applied  methods  of                chemometric  algorithms  are  currently  the  methods  of  principal  component                regression (PCR), partial least squares (PLS), classical least squares (CLS), inverse                least squares (ILS) and multiple linear regression (MLR). Calibration model using                these  regression  methods  uses  an  entire  NIR  spectrum  or  a  spectrum  section,                whereas maximum absorbances of only selected bands are not sufficient. The aim is                to  find  a  relationship  between  the  multidimensional  spectral  information                (represented by a matrix of absorbance values in the selected spectral section of a                calibration  set)  and  the  composition  of  samples  (represented  by  a  matrix  of                concentration values of an observed group of analytes from the set of the calibration                samples).                 The  above  discussed  enables  a  wide  use  of  quantitative  NIR  analysis  in  many                industries,  such  as  food  processing,  petrochemical  and  paper  industry  and                pharmacy. It is also possible to quantify complex samples and to determine many                components or variables together, because the spectrum is evaluated as a whole and                practically  forms  a  \"fingerprint\" for  a  given mixture.  Acquisition  of  NIR  spectra                                                     28","2 Infrared spectroscopy                 requires  several  seconds  and  can  be  performed  during  manufacturing  process.                Therefore,  the  NIR  spectroscopy  is  used  for  process  analytical  technology  as  it                allows  fast  and  continuous  on-line  analysis  of  the  manufacturing  process,                monitoring  of  the  whole  production  state,  and  immediate  determination  of  any                deviation and its following corrections. This enables simultaneous determination of                fat, protein, lactose and urea contents in milk and dairy products (in various stages                of  their  processing),  or  carbohydrate  and  ethanol  content  in  alcoholic  beverages                (during ongoing fermentation), octane number in gasoline, etc.                   2.3.3  NIR spectra measurement techniques                 Main advantage of the NIR spectroscopy is the ability to measure samples through                glass  and  other  transparent  packaging  materials.  The  measurement  is  fast,  non-                destructive,  and  requires  no  special  sample  preparation.  This  minimizes  the                consumption of chemicals and disposable analytical material. Much more laborious                and  time-consuming  than  the  actual  spectra  measurement  is  the  subsequent                processing and evaluation of measured data.                 Radiation can interact with solid matter in several ways, as illustrated in Fig. 2.5.                The appropriate NIR measurement mode will be dictated by the optical properties                of the samples. Transparent materials are usually measured in transmittance mode                (A).  Turbid  liquids  or  semi-solids,  and  solids  may  be  measured  in  diffuse                transmittance (B), diffuse reflectance (C) or transflectance (D/E) modes, depending                on  their  absorption  and  scattering  characteristics.  NIR  radiation  is  specifically                modulated  by  interaction  of  a  diffuse  reflectance  with  particles  of  different  size                (Fig. 2.18).                 In  general,  NIR  spectra  can  be  measured  as  attenuation  of  radiation  flow  after                passage through a sample (transmittance measurements) or after reflection on the                sample (reflectance technique). The principle is illustrated in Fig. 2.19. Identically                as in the mid region, the spectra measured in transmittance or reflectance modes are                afterwards converted into absorbance.                                                                    29","2 Infrared spectroscopy                                                        Figure 2.18. NIR measuring modes: transmittance (A/B), diffuse reflectance (C)                                           and transflectance (D/E).                  (Edited according to: Reich G. Near-infrared spectroscopy and imaging: Basic principles and pharmaceutical                                   applications. Adv. Drug Deliv. Rev. 2005, 57, 1109–1143.)                                                        Transmittance                                                      Transmise                                                      (průchod záření vzorkem)                                                      measurement principle                                                               Reflectance                                                       Reflektance                                                       measurement prin                                                       (odraz záření od vzorku)ciple                          Figure 2.19. Principle of transmittance and reflectance measurements.                      (Edited according to Vzorkovací techniky v FT-NIR spektrometrii. Presentation. Nicolet CZ, 2008.)                                                      30","2 Infrared spectroscopy                 The  most  applied  principle  in  reflectance  techniques  is  the  diffuse  reflectance                infrared Fourier transform (DRIFT) where the incident radiation is reflected from                the surface of individual small powder particles (Fig. 2.20). This approach is most                often applied in pharmaceutical industry analysis.                                       Figure 2.20. Diffuse reflectance (DRIFT) principle and UpDrift adapter with                                       modification for sample rotation.                          (Source: Vzorkovací techniky v FT-NIR spektrometrii. Presentation. Nicolet CZ, 2008.)                 Transmittance measurements are used for measurement of liquids, mash samples,                and  polymeric  films.  Liquid  samples  must  be  measured  in  cells.  Optical  layer                thickness  of  cuvettes  ranges  from  1  to  10  mm,  and  their  selection  is  optimized                depending on the actual concentration of the analyte in solution and depending on                the optical properties of the solvent.                 Combination  of  the  two  above  mentioned  approaches  is  a  technique  called                “transflectance”. It can be used for measurements of thin suspensions, liquids, or                solutions. The technique uses special cells with bottom covered with by a totally                reflective (mirror) thin layer which reflects the radiation passed through the sample,                providing  repeated  passage  through  the  sample  and  continuation  to  the  detector                (Fig. 2.21).                                                                       31","2 Infrared spectroscopy                                                 Figure 2.21. Cuvettes for transfection NIR measurements and the measurement                                                 principle.                          (Source: Vzorkovací techniky v FT-NIR spektrometrii. Presentation. Nicolet CZ, 2008.)                 Besides the above-mentioned procedures where the sample is placed in a holder,                variously modified instruments (Fig. 2.22) for reflectance measurements often use                fiber  optics  with  different  probes  (Fig.  2.23)  that  can  be  placed  directly  in  a                production apparatus or in the measured sample (Fig. 2.24).                NIR techniques maintain a special position in PAT (process analytical technology).                Whole  NIR  spectrometers  can  be  attached  to  other  devices  so  that  an  entire                production process can be directly observed, and its course monitored on-line/in-                line. In pharmacy, an entire powder production process can be monitored this way.                This arrangement  used in PAT thus  represents  a  completely  non-contact analysis                and  offers  a  possibility  for  monitoring  of  standard  deviations  for  individual                components  and  the  final  mixture.  This  enables  an  on-line  control  of  powder                mixture uniformity or real-time analytical control of tablet blending and eliminates                losses caused by imperfect production control. The device is shown in Fig. 2.25.                                                                32","2 Infrared spectroscopy                                                             Figure 2.22. Various instruments for reflectance measurements.                          (Source: Vzorkovací techniky v FT-NIR spektrometrii. Presentation. Nicolet CZ, 2008.)                                                    Figure 2.23. NIR-Multiplexer system with different types of probes.                          (Source: Vzorkovací techniky v FT-NIR spektrometrii. Presentation. Nicolet CZ, 2008.)                                                     33","2 Infrared spectroscopy                                Figure 2.24. NIR analyzers for remote process monitoring with simultaneous fiber                  multiplexing technology and integrated communication for real-time feedback or                  direct control of the production process in real time by deployment to production                                                 equipment.                          (Source: Vzorkovací techniky v FT-NIR spektrometrii. Presentation. Nicolet CZ, 2008.)                                                     Figure 2.25. NIR analyzers connected to a homogenization apparatus. Integration                                   of in-line NIR analysis in a fluid bed dryer.                    (Source: Vzorkovací techniky v FT-NIR spektrometrii. Presentation. Nicolet CZ, 2008. Metrohm AG and                                               AZO Materials, 2022)                                                          34","2 Infrared spectroscopy                 2.3.4  NIR analysis strategies                 NIR instrumentation can be installed in a laboratory or directly implemented in a                manufacturing process or reactor. Overall, the most appropriate NIR measurement                mode and location of the NIR analyzer is dictated by the optical properties of the                sample, required analyte selectivity and sensitivity, duration of the process run, and                the  monitoring  and  control  requirements.  Analysis  strategies  can  thus  be                distinguished according to the general application:                     •  laboratory                     •  at-line                     •  on-line                     •  in-line                 Laboratory  analysis  is  represented  by  a  single  NIR  instrument  that  can  support                multiple  manufacturing  operations  from  raw  material  inspection  or  in-process                analysis  to  finished  product  release.  This  configuration  provides  the  greatest                flexibility as both the reflectance and transmittance measurements can be performed                on  processed  samples,  feedstock,  ingredients,  raw  materials,  effluents,  and  final                products. Laboratory strategies require manual sampling, and the turnaround time                can thus take up to 1 hour even though the NIR analysis requires less than a minute.                This  strategy  is  most  useful  for  supporting  pilot  plant/scale  up  facilities,  or                measurement  for  long  duration  runs  where  the  process  information  is  required                infrequently.                 For at-line analysis strategy, a NIR analyzer is located close to a process stream.                This  analyzer  configuration  is  dedicated  to  performing  a  particular  analysis  on  a                specific sample type, e.g., transmittance measurements of solid dosage forms. The                at-line  analysis  strategies  require  manual  sampling,  and  the  analyzer  must  meet                appropriate  classification.  This  strategy  is  well  fit  for  process  monitoring  and                control strategies, and for manufacturing operations.                When  a  NIR  analyzer  is  incorporated  into  a  process  using  a  sample-loop  and                performs  a  particular  analysis  on  a  particular  sample  type,  the  strategy  can  be                designated as on-line. Here, the NIR measurements are performed on a continuous                flow of a sample as it passes through a flow-cell. Side-streams provide a convenient                means  for  sample  conditioning  such  as  heating,  filtering,  or  degassing  or  for                maintenance, calibration, sample collection – all while the process is running. On-                line strategies provide automatic and near real-time (results in < 10 s) analyses of                specific media and are always optimized for one application. This strategy is well                                                        35","2 Infrared spectroscopy                 suited  for  closed-loop  monitoring  and  control  strategies  for  scale-up  and                manufacturing operations.                 With the in-line analysis strategy, a process NIR analyzer is implemented directly                into the process using a fiber optics and a stainless steel (or other material) probe                that is inserted directly into a port installed into the process stream or vessel. This                analyzer configuration is dedicated to performing a particular analysis on a specific                sample type, e.g., reflectance analysis in a dryer. A direct interface requires minimal                additional hardware, but its maintenance cannot be performed unless the process is                shut down. In addition, for transmittance analysis, the narrow optical path lengths                and  fluid  dynamics  of  the  process  streams  can  sometimes  make  this  analysis                strategy difficult to implement. An in-line analyses provides automatic, near real-                time (results in  <  10  s)  measurement  of  specific media and is  optimized  for  one                particular application. This strategy is well suited for closed-loop monitoring and                control strategies for scale-up and manufacturing operations.                   2.3.5  Application of NIR spectroscopy in pharmacy                                                                                   th                The application of NIR spectroscopy is expanding since the 90’s of the 20  century,                along with the development of new IR spectrometers with Fourier transformation                for  near  region.  FT-NIR  spectroscopy  is  a  fast  non-destructive,  non-invasive                analytical  method  which  currently  offers  a  wide  application  range  and  allows  to                replace more laborious and demanding methods in analytical chemistry. It can be                used for testing of final products, product uniformity monitoring, and verification of                raw materials. It allows on-line/in-line monitoring of production processes and is                widely used in PAT.                 The  measured  spectra  can  be  used  for  both  qualitative  and  quantitative  analysis.                Data  processing  depends  on  its  application.  The  spectra  obtained  for  qualitative                evaluation need to be compared with the spectra of a standard, and this approach is                suitable for small sample numbers. Large sets of spectra are better evaluated using                chemometric  statistical  data  processing.  Statistical  evaluation  of  qualitative  and                quantitative  data  is  possible  by  several  evaluation  methods.  The  most  general                categorization  of  statistical  methods  is  the  division  into  one-dimensional  and                multidimensional data analysis, and analysis of variance.                 In  pharmacy,  the  NIR  spectroscopy  is  used  for  both  qualitative  and  quantitative                application.  It  must  be  pointed  out  that  for  both  qualitative  and  quantitative                analyses,  it  is  required  to  measure  a  standard  or  series  of  standards.  The  data                processing  in  most  of  the  applications  requires  multivariate  calibration  models                which are often automated.                                                      36","2 Infrared spectroscopy                 The most common NIR applications are:                    •  identification of all raw materials                    •  identification of polymorphism of active substances and excipients                    •  API content monitoring                    •  content uniformity testing for batch units                    •  stability testing                    •  compatibility testing                    •  water content, residual solvents monitoring                   •  particle size determination                    •  hardness determination                    •  prediction of dissolution                    •  homogeneity determination, NIR-imaging (with microscope)                    •  on-line/in-line analyses, PAT                 NIR  spectroscopy  can  be  used  to  distinguish  structurally  related  molecules.  Fig.                2.26 shows examples of different alcoholic sugars, and  Fig. 2.27 shows common                hexoses.                                                                                 37","2 Infrared spectroscopy                                                                 Figure 2.26. NIR spectra of alcoholic sugars.                       glu c os e                     0.6  glucose                  Abs   0.4                     0.2                     0.4  ma nnos e                       mannose                  Abs   0.2                        galac tos e                     0.3  galactose                  Abs   0.2                     0.1                      allo s e                     0.3 allose                  Abs   0.2                     0.1                     10000       9000       8000       7000        6000       5000                                                    W avenumbers (cm-1)                                Figure 2.27. NIR spectra of hexoses (pyranoses).                 Discrimination between salts and free acids or bases, as well as identification of a                particular  salt  type  (sodium,  potassium  or  also  monosodium,  disodium)  is  very                important. Fig. 2.28 illustrates examples of tartaric acid and its various salts.                                                    38","2 Infrared spectroscopy                                                          Figure 2.28. NIR spectra of tartaric acid and its various salts.                 A very important application is the recognition of individual API polymorphs. Fig.                2.29 contains examples of NIR spectra of different polymorphs of one API.                         Risedronát_polymorf A                        form A                      0.8 form A                   Abs   0.6                      0.4                      0.8  Risedronát_polymorf H                        form B                        form B                      0.6                   Abs                      0.4                      0.2                       Risendronát_semikrystal                        form C                        form C                      0.2                   Abs                      0.1                     -0.0                       Risedronát_monohydrát                        form D                        form D                      0.5                   Abs   0.4                      0.3                           10000     9000      8000       7000      6000      5000                                                   Wavenumbers (cm-1)                          Figure 2.29. NIR spectra of different polymorphs of one API.                                                        39","2 Infrared spectroscopy                 Fig.  2.30  shows  examples  of  spectra  of  a  desired  API  polymorph  and                polymorphically impure API. All measured spectra were subsequently evaluated by                discriminant analysis and the record is shown in Fig. 2.31.                        0,24                      0,22                      0,20                       0,18                      0,16                   Absorbance   0,14                       0,12                       0,10                      0,08                      0,06                      0,04                       7500     7000      6500     6000     5500      5000     4500                                                    Wav enumbers (cm-1)                                                                   -1                  Figure 2.30. A clearly visible change in 5000–5300 cm  range for an API of the                        undesired polymorph in comparison with spectra of other samples.                                                                              different polymorph                           Figure 2.31. Resolution of polymorphically different API samples, 2D view.                  At  this  point,  it  should  be  noted  that  the  NIR  spectra  are  similar  to fingerprints.                Beer-Lambert law cannot be used for NIR spectrometry. Instead, a set of standards                is needed for accurate quantification, and the above-mentioned chemometrics must                be used. Both the solid phase form and the particle size, moisture (residual solvent                                                     40","2 Infrared spectroscopy                 content),  compression,  production  method,  impurities,  etc.,  are  projected  into  the                spectra. Figure 2.32 shows spectra processed by discriminant analysis for APIs that                meet  the  specification  criteria/limits  but  were  produced  by  two  different                 manufacturers.                                                                         manufacturer 2                                   manufacturer 1                          Figure 2.32. Resolution of the same API of two different manufacturers, 2D view.                 As  mentioned,  particle  size  has  great  influence  on  NIR  spectra  shape.  Fig.  2.33                shows spectra of sucrose compressed into tablets under high pressure, crystalline                sucrose, and a finely ground sucrose. Differences are apparent especially in height                of the particular bands.                       sucrose tablet                              crystalline sucrose                             powdered sucrose                                   Figure 2.33. NIR spectra of sucrose, differing in particle size.                                                       41","2 Infrared spectroscopy                 Fig. 2.34 shows the NIR spectra of different API batches with different particle size.                The  differences  are  evident  in  the  height  of  the  individual  bands.  All  measured                spectra were subsequently processed by chemometrics with the partial least square                method. The sample evaluation was based on the differences in the height of the                individual bands with a correlation for the particle size values measured by the laser                diffraction method. The result is shown in Fig. 3.35.                                                Figure 2.34. NIR spectra of different batches of an API with different particle size,                                   projected to the height of individual bands.                                                                              42","2 Infrared spectroscopy                                                   Figure 2.35. Sample evaluation based on differences in the height of individual                     bands vs. values from laser diffraction. Processed by partial least squares.                 Tablets of different hardness (i.e., tableted at different pressures) were measured by                NIR spectrometry. Calibration was performed based on the height of the selected                band  in  the  NIR  spectra  compared  to  the  measured  hardness.  Fig.  2.36  shows  3                groups  of  tablets  with  different  hardness,  as  evaluated  by  principal  component                analysis method.                                                                               43","2 Infrared spectroscopy                                                     Figure 2.36. Result of the evaluation of samples of tablets of different hardness                               processed by principal component analysis method.                 It is also possible to determine the content of residual solvents in APIs. Fig. 2.37                shows spectra of an API with various residual ethanol content. The exact ethanol                content was determined using gas chromatography. Subsequently, a calibration for                following  NIR-based  API  testing  was  created  through  assigning  NIR  spectra  to                specific ethanol content values (Fig. 2.38).                                                                             44","2 Infrared spectroscopy                                                    Figure 2.37. NIR spectra of an API with different residual ethanol content.                                                Figure 2.38. Evaluation of residual ethanol content in an API. The calibration and                  evaluation of samples is based on comparing the differences in individual spectra                           vs. gas chromatography. Processed by discriminant analysis.                                                      45","2 Infrared spectroscopy                 The evaluation of API content in tablets is widespread; an example is shown in Fig.                2.39.  For  this  purpose,  various  autosamplers  and  adapters  for  reflectance                measurements are preferably used (as shown, for example, in Fig. 2.22). This is an                excellent  quantification  method  because  it  can  be  fully  automated.  On  the  other                hand,  the  implementation  of  such  methodology  is  more  demanding  as  many                standards  must  be  prepared  so  the  methodology  is  suitable  for  frequent  routine                analyzes  of  large  product  volumes  within  the  output  quality  control.  A  crucial                condition for the application of the method is a sufficient API amount in the tablet                (at least 2%) and non-interfering excipients.                                                          Figure 2.39. Determination of API content in tablets.                        (Source: Základy NIR spektrometrie a její praktické využití. Presentation. Nicolet CZ, 2009.)                 Currently, dissolution represents one of the key methods for evaluation of solid oral                dosage  forms  and  their  release,  consistency,  similarity,  and  therapeutic  efficacy.                Dissolution  testing  however  involves  a  series  of  time-consuming  analytical  tasks                which  require  labor-intensive  protocols,  such  as  instrument  calibration,  media                preparation, sample collection, data collection, and drug assays. Some approaches                interconnecting dissolution testing and NIR spectroscopy were therefore developed:                    •  monitoring of the effect of humidity stress on dissolution quality                    •  monitoring of the effect of core coating layer on dissolution quality                    •  monitoring of the effect of particle size on dissolution quality                                                        46","2 Infrared spectroscopy                 All these methods correlate the NIR spectra and changes in monitored dissolution                parameter (% of released API).                 For  dissolution  quality  and  humidity  stress  monitoring,  the  NIR  spectra  are                measured  first  along  with  the  dissolution  of  an  unstressed  sample.  Then,  the                samples are treated in stability chamber at a defined humidity, and the NIR spectra                and dissolution of the stressed samples is measured in parallel at predefined time                intervals. Correlation of the changes in a selected dissolution parameter and the NIR                spectra  is  performed  after  dependence  determination.  Afterwards,  the  NIR                spectroscopy can be used for further analyses.                 Monitoring  of  the  effect  of  tablet  core  coating  layer  on  dissolution  requires  a                preparation  of  a  series  of  coated  tablets  with  a  defined  coating  layer  thickness.                Afterwards,  the  NIR  spectra  are  measured  along  with  dissolution  of  the  coated                cores, and the correlation between dissolution performance and the NIR spectra is                performed after dependence determination. Afterwards, the NIR spectroscopy can                be used for further analyses.                 For monitoring of particle size effect on the dissolution of a final drug formulation                manufactured with a standard process and a constant composition, the calibration                and sample evaluation is based on differences in height of individual bands.                 Chemical mapping is a technique combining spatial and chemical information. It                provides advantages for both powder mixture and drug formulation analyses, such                as  determination  of  component  size  and  distribution,  polymorphic  distribution,                hydration degree, detection of heterogeneous particles or determination of coating                type and thickness. Spatial resolution is in the order of micrometers. The acquired                data can be used for both understanding and optimization of the applied technology                and processes.                 NIR  enables  to  monitor  homogeneity  of  both  API  and  the  whole  sample  for                example  during  granule  production  or  tableting.  NIR  spectra  measured  using                traditional UpDrift (reflectance) are illustrated in Fig. 2.40. Several positions on a                tablet  are  measured  and  the  variance  in  individual  spectra  from  one  and  several                tablets is evaluated.                  Tablet  homogeneity  can  also  be  determined  by  NIR  imaging.  The  surface of the                sample is measured using a microscope coupled to a NIR spectrometer and then the                spectra  are  processed  by  chemometrics.  Examples  of  this  measurement  type  are                shown in Fig. 2.41.                                                           47","2 Infrared spectroscopy                                         homogeneous samples                non-homogenous samples                   Figure 2.40. Difference in spectra of homogeneous and non-homogenous tablets.                                                       homogeneous samples                                                             partially homogeneous samples                                                                non-homogenous samples                                   Figure 2.41. NIR imaging applied in tablet homogeneity evaluation.                                                          48","2 Infrared spectroscopy                 As indicated above, NIR is advantageously used in PAT. The non-contact analysis                is  used  primarily  for  manufacturing  process  control,  e.g.,  during  granulation  or                direct  compaction,  homogeneity  monitoring  (deviations  in  single  components,  as                well as of whole mixtures), see Fig. 2.42. The monitoring of these processes plays                an  important  role  in  quality  maintenance  for  various  “high-risk  formulations”.  A                disadvantage is the limit of detection, where the monitored component is required to                be present in at least 2%.                            start of process                          Process start                                 trouble-free homogenization of components                                problem-free component homogenization                                       start of process                         Process start                                           troubled homogenization                                  problematic component homogenization                                 Figure 2.42. The course of NIR analysis recording during homogenization.                                                                   49","2 Infrared spectroscopy                 2.4  Resources and recommended literature                   Basic principles of vibrational spectral methods                   1.    Smith B. Infrared Spectral Interpretation. CRC Press: Boca Raton, USA, 1999.                  2.    Socrates G. Infrared and Raman Characteristic Group Frequencies. John Wiley \&                        Sons: New York, USA, 2001.                                                                                          nd                  3.    Griffiths  P. R.,  de  Haseth  J. A.  Fourier  Transform  Infrared  Spectrometry,  2                        Edition.   John   Wiley   \&    Sons:   New    York,   USA,    2007.                        https://doi.org/10.1002/047010631X                   4.    Pivonka  D. E.,  Chalmers  J. M.  Application  of  Vibrational  Spectroscopy                        in  Pharmaceutical  Research  and  Development.  John  Wiley  \&  Sons:  New  York,                        USA, 2007.                  Infrared spectroscopy in mid region                   5.    Infračervená spektra a struktura molekul. Horák M., Papoušek D., Eds.; Akademia:                        Prague, Czech Republic, 1976.                  6.    Strauch B. Infračervená spektroskopie s Fourierovou transformací. SNTL: Prague,                        Czech Republic, 1988.                  7.    Roeges  N. P. G.  A  Guide  to  the  Complete  Interpretation  of  Infrared  Spectra  of                        Organic Structures. John Wiley \& Sons: New York, USA, 1994.                  8.    Silverstein  R. M.,  Webster  F.  X.,  Kiemle  D.  J.  Spectrometric  Identification  of                                          th                        Organic Compounds, 7  Edition. John Wiley \& Sons: New York, USA, 2005.                  9.    Rouessac F., Rouessac A. Infrared Spectroscopy. In Chemical Analysis – Modern                                                              nd                        Instrumentation  Methods  and  Techniques,  2   Edition;  John  Wiley  \&  Sons:                        Chichester, UK, 2007.                  Infrared spectroscopy in near region                   10.   Dreassi  E.,  Ceramelli  G.,  Corti  P.,  Lonardi  S.,  Perruccio  P.  L.  Near  Infrared                        Reflectance  Spectroscopy  in  the  Determination  of  the  Physical  State  of  Primary                        Materials  in  Pharmaceutical  Production.  Analyst.  1995,  120  (4),  1005–1008.                        https://doi.org/10.1039/an9952001005                   11.   Dreassi  E.,  Ceramelli  G.,  Corti  P.,  Massacesi  M.,  Perruccio  P. L.  Quantitative                        Fourier  Transform  Near-Infrared  Spectroscopy  in  the  Quality  Control  of  Solid                        Pharmaceutical   Formulations.   Analyst.   1995,   120   (9),   2361–2365.                        https://doi.org/10.1039/an9952002361                   12.   Kirsch J. D., Drennen J. K. Determination of Film-Coated Tablet Parameters by                        Near-Infrared Spectroscopy. J. Pharm. Biomed. Anal. 1995, 13 (10), 1273–1281.                        https://doi.org/10.1016/0731-7085(95)01562-Y                                                      50","2 Infrared spectroscopy                   13.   Pharmaceutical  Analytical  Sciences  Group.  Guidelines  for  the  development  and                        validation   of   near   infrared   (NIR)   spectroscopic   methods.                        http://www.pasg.org.uk/NIRmay01.pdf. May.2001.                   14.   Lyon R. C., Lester D. S., Lewis E. N., Lee E., Yu L. X., Jefferson E. H., Hussain                        A.  S.  Near-Infrared  Spectral  Imaging  for  Quality  Assurance  of  Pharmaceutical                        Products:  Analysis  of  tablets  to  Assess  Powder  Blend  Homogeneity.  AAPS                        PharmSciTech. 2002, 3 (3), 1-15. https://doi.org/10.1208/pt030317                  15.   Reich G.   Near-Infrared   Spectroscopy   and   Imaging:   Basic   Principles                        and  Pharmaceutical  Applications.  Adv.  Drug  Delivery  Rev.  2005,  57  (8),                        1109–1143.                   16.   Cogdill  R.  P.,  Anderson  C.  A.,  Delgado-Lopez  M.,  Molseed  D.,  Chisholm  R.,                        Bolton  R.,  Herkert  T.,  Ali  M.  Afnan,  and  Drennen  J.  K.  Process  Analytical                        Technology Case Study Part I: Feasibility Studies for Quantitative Near-Infrared                        Method  Development.  AAPS  PharmSciTech.  2005,  6  (2)  E263–273.                        https://doi.org/10.1208/pt060238                   17.   Cogdill  R.  P.,  Anderson  C.  A.,  Delgado-Lopez  M.,  Chisholm  R.,  Bolton  R.,                        Herkert T., Ali M. Afnan, and Drennen J. K. Process analytical Technology Case                        Study:  Part  II.  Development  and  validation  of  Quantitative  Near-Infrared                        Calibrations in support of a Process Analytical Technology Application for Real-                        Time   Release.   AAPS   PharmSciTech.   2005,   6   (2)   E273–283.                        https://doi.org/10.1208/pt060238                   18.   Blanco M., Valdes D., Llorente I., Bayod M. Application of NIR Spectroscopy in                        Polymorphic Analysis: Study of Pseudo-Polymorphs Stability. J. Pharm. Sci. 2005,                        94 (6), 1336–1342. https://doi.org/10.1002/jps.20362                   19.   Blanco  M.,  Alcala  M.  Content  uniformity  and  tablet  hardness  testing  of  intact                        pharmaceutical  tablets  by  near  infrared  spectroscopy:  A  contribution  to  process                        analytical  technologies.  Anal.  Chim.  Acta  2006,  557  (1–2),  353–359.                        https://doi.org/10.1016/j.aca.2005.09.070                   20.   Matějka P. Spektrometrie v blízké infračervené oblasti. Studijní materiály. Ústav                        analytické  chemie,  Fakulta  chemicko-inženýrská,  VŠCHT:  Prague,  Czech                        Republic, 2007.                   21.   Luypaert  J.,  Massart  D. L.,  van  der  Heyden  Y.  Near-Infrared  Spectroscopy                        Applications  in  Pharmaceutical  Analysis.  Talanta.  2007,  72  (3),  865–883.                        https://doi.org/10.1016/j.talanta.2006.12.023                   22.   Roggo  Y.,  Chalus  P.,  Maurer  L.,  Lema-Martinez  C.,  Jent  N.  A  Review  of  Near                        Infrared  Spectroscopy  and  Chemometrics  in  Pharmaceutical  Technologies.  J.                        Pharm.     Biomed.     Anal.     2007;     44      (3),    683–700.                        https://doi.org/10.1016/j.jpba.2007.03.023                                                       51","2 Infrared spectroscopy                   23.   Tabasi S. H., Fahmy R., D. Bensley, O’Brien C., Hoag S. W. Quality by Design,                        Part I: Application of NIR Spectroscopy to Monitor Tablet Manufacturing Process.                        J. Pharm. Sci. 2008, 97 (9), 4040–4051. https://doi.org/10.1002/jps.21303                   24.   Základy FT-NIR spektrometrie. Prezentace. Nicolet CZ, Thermo Scientific, 2008.                   25.   Vzorkovací  techniky  v  FT-NIR  spektrometrii.  Prezentace.  Nicolet  CZ,  Thermo                        Scientific, 2008.                   26.   Tabasia  S.  H.,  Moolchandania  V.,  Fahmy  R.,  Hoag  S.  W.  Sustained  Release                        Dosage Forms Dissolution Behavior Prediction: A study of Matrix Tablets Using                        NIR   Spectroscopy.   Int.   J.   Pharm.   2009,   382   (1–2),   1–6.                        https://doi.org/10.1016/j.ijpharm.2009.07.029                  27.   Základy  NIR  spektrometrie  a  její  praktické  využití.  Prezentace.  Nicolet  CZ,                        Thermo Scientific, 2009.                   28.   Hattori  Y.,  Otsuka  M.  NIR  spectroscopic  study  of  the  dissolution  process  in                        pharmaceutical   tablets.   Vib.   Spectrosc.   2011,   57   (2),   275–281.                        https://doi.org/10.1016/j.vibspec.2011.09.003                   29.   Metrohm.  NIR  Spectroscopy:  A  guide  to  Near-Infrared  Spectroscopic  Analysis  of                        Industrial   Manufacturing   Processes.   http://www.mep.net.au/wpmep/wp-                        content/uploads/2013/05/MEP_Monograph_NIRS_81085026EN.pdf. February 2013.                  30.   United States Pharmacopeia USP 35-NF 30, General chapter <1119> Near-Infrared                        Spectroscopy, 2013.                   31.   Pan D., Crull G., Yin S., Grosso J. Low level drug product API form analysis  –                        Avalide tablet NIR quantitative method development and robustness challenges. J.                        Pharm.      Biomed.       Anal.       2014;       89,      268–275.                        https://doi.org/10.1016/j.jpba.2013.11.011                   32.   European Medicines Agency. Guideline on the use of near infrared spectroscopy by                        the  pharmaceutical  industry  and  the  data  requirements  for  new  submissions  and                        variations.              https://www.ema.europa.eu/en/documents/scientific-                        guideline/guideline-use-near-infrared-spectroscopy-pharmaceutical-industry-data-                        requirements-new-submissions_en.pdf. January 2014.                   33.   McDowall  R.  D.,  Smith  P.  Quo  Vadis  Regulated  NIR  Analytical  Procedures?                        Spectroscopy 2015, 30 (12) 10–16.                  34.   Otto  M.  Chemometrics:  Statistics  and  Computer  Application  in  Analytical                        Chemistry,   3rd   ed.,   Wiley-VCH:   Weinheim,   Germany,   2017.                        https://doi.org/10.1002/9783527699377                  35.   FDA. Guidance for Industry: PAT - A Framework for Innovative Pharmaceutical                        Development,     Manufacturing,     and       Quality     Assurance,                        https://www.fda.gov/media/71012/download. August 2018.                                                     52","2 Infrared spectroscopy                   36.   Miller  J.,  Miller  J.  C.  Statistics  and  Chemometrics  for  Analytical  Chemistry.                        Pearson Education, 2018.                  37.   Sahoo N. K, Chandana G. M. H., Vaishnavi, G. Pharmaceutical Applications and                        Importance of Near Infrared Spectroscopy. Med. Analy. Chem. Int. J. 2020, 4 (1)                        000155.                   38.   Otsuka M. Near-Infrared Spectroscopy Application to the Pharmaceutical Industry.                        In:  Encyclopedia  of  Analytical  Chemistry:  Applications,  Theory  and                        Instrumentation, section Infrared Spectroscopy, Meyers R. A. (Ed.). John Wiley \&                        Sons: 2020. https://doi.org/10.1002/9780470027318.a9701                   39.   FDA:  Development  and  Submission  of  Near  Infrared  Analytical  Procedures                        Guidance for Industry, https://www.fda.gov/media/91343/download. August 2021.                   40.   Innopharma   Technology:   NIR   Spectroscopy   PAT   Sensors,   2022,                        https://www.innopharmatechnology.com/nir-spectroscopy-pat-sensors.                                                                                                   53","3 Raman spectroscopy                 3  Raman spectroscopy                 Tomáš Pekárek, Dita Spálovská                   3.1  Introduction                 Similarly  to  infrared  spectroscopy,  Raman  spectroscopy  is  one  of  the  vibrational                spectroscopy techniques. It is named after the Indian sir Chandrasekhara Venkata                Raman who, together with the sir Kariamanickam Srinivasa Krishnan, observed the                inelastic light scattering and was awarded the Nobel Prize in 1930 for this discovery                [1]. L. I. Mandelstam and G. S. Landsberg also observed this phenomenon in the                former USSR [2-5].                Raman spectroscopy offers the same options as infrared spectroscopy (see chapter                2.1.). Moreover, it enables easy measurement through the package, and even water                is not an obstacle here, whereas in IR spectroscopy water is a very unsuitable matrix                for measurements, as explained below. The higher cost of Raman spectrometers was                the main obstacle for their wider use. In recent years, the price continues to decline,                and although it is not at level with infrared spectrometers, the Raman spectrometers                are now more affordable.                 A molecule is not of a rigid shape, but it can be compared to weights connected to a                spring. All atoms of a functional group perform a permanent vibration. A non-linear                and a linear molecule can vibrate at 3N-6 and 3N-5 vibrational degrees of freedom                (normal vibrational modes), respectively, [6-9], where N is the number of atoms in                the molecule. The individual vibrational modes are characterized by the quantum                number n, which can acquire values 0, 1, 2, 3, etc. Transitions between individual                states are linked to energy changes and the energy of these vibrational transitions is                quantified according to the following equation:                                     1                            = hν (   + )       – vibrational transition frequency                                     2                                             h – Planck constant                 If the energy quantum is absorbed by a molecule, transitions that are shown in Fig.                3.1  take  place  (marked  as  IR)  and  can  be  observed  by  the  infrared  (IR)                spectroscopy. The second option is that the transitions occur during the scattering                effect,  which  can  be  described  as  absorption  of  a  photon  and  excitation  of  the                molecule  to  the  excited  energy  state  (virtual  levels)  followed  its  immediate                relaxation to a lower energy state linked with an emission of a scattered photon. The                whole process lasts no longer than 10 −15  second, which means that we cannot speak                about true absorption or emission, but the effect is considered as a non-elastic two-                                                     54","3 Raman spectroscopy                 photon scattering. If the molecule returns to the ground state after its excitation, the                scattered photon is of the same energy as the photon which was absorbed, and it                carries  no  information  about  the  vibrational  states  of  the  molecule  (Rayleigh                scattering) [6-9]. Stokes-Raman scattering is a process when the transition starts at                the  ground  vibrational level  and ends at  a higher  vibrational level,  whereas  anti-                Stokes-Raman scattering is observed when the molecule relaxes to a lower energy                level. The individual transitions are shown in Fig. 3.1.                                           Figure 3.1. Visualization of vibrational transitions of a molecule after its interaction                 with radiation. Arrows up indicate a molecule excitation by radiation; arrows down                  indicate molecule relaxation connected with “emission” of a photon; νvib – energy                                differences between individual vibrational levels.                 The Rayleigh radiation intensity is approximately six orders of magnitude higher                than the Raman bands intensity, resulting in high energy requirements for detection                of Raman spectra. The intensity of the Stokes and anti-Stokes lines is different as                well.  As  most  molecules  are  in  the  ground  vibrational  state  at  room  temperature                (Boltzmann distribution, see below), the anti-Stokes transitions are rare compared to                Stokes transitions and their bands have therefore lower intensity.                                                                 55","3 Raman spectroscopy                                         N0 – molecule abundance in the basic state                                       N1 – molecule abundance in the higher energy state                   N 1    −  hν vib    h – Planck constant                            kT                  N   = e                    0                                      υvib – vibrational frequency                                      k –Boltzmann constant                                      T – temperature                 Temperature of the sample during measurement can be calculated by the following                equation, which might be important especially in biological tissue analysis.                                                         Ianti-Stokes – band intensity in the Stokes                                                        region                                       4                  I anti-Stokes  = (  ν +ν vib ) e  −  hν vib                                0                                            kT                    I Stokes   ν -ν vib                 IStokes  –  band  intensity  in  the  anti-                                0                                                        Stokes region                                                        υ0 – incidental photon frequency                As depicted in Fig. 3.1, the differences in vibrational energy levels are important for                both IR and Raman spectroscopy. The IR spectroscopy observes a photon with the                energy of the particular vibrational energy transition, whereas Raman spectroscopy                deals with a two-photon process where the energy of the vibrational transition is                equal to the shift (difference) between the absorbed and the emitted photon, and the                techniques are complementary.                 For the IR vibration to be active, a non-zero dipole change must occur as it moves.                                       p – dipole moment                         ∂p            q – normal coordinate                         ∂q  ≠ 0                  In Raman spectroscopy, the functional group needs to change its polarizability to be                Raman active.                                        α  –  polarizability  –  the  ease  of  bond  (electron  cloud)                         ∂α            distortion by an external energy field                            ≠ 0                         ∂q                                                      56","3 Raman spectroscopy                   The  dipole  moment  is  induced  by  the  external  electric  field  E  given  by  the                following relationship:                                                   p = αE                 Intensity of the IR bands is proportional to the second power of the dipole change                                                  ∂p                                                    2                during particular vibrational motion ( ) , and Raman bands are proportional to the                                                  ∂q                                                        ∂α                                                           2                second power of the polarizability change ( ) . The modes with the high dipole                                                        ∂q                moment change that provide high intensity IR bands usually result in weak Raman                bands.  In  contrast,  vibrations  of  non-polar  functional  groups  appear  as  intensive                bands  in  the  Raman  spectra  and  as  weak  bands  in  the  IR  spectra.  For  instance,                carboxyl and carbonyl groups usually provide very strong bands in the IR spectrum                and  very  weak  bands  in  the  Raman  spectrum.  High  Raman  intensity  bands  are                usually found for double and triple bonds and for symmetric vibrations of functional                groups.  Example  is  shown  in  Fig.  3.2  with  the  IR  and  Raman  spectrum  of                acetylsalicylic  acid.  The  IR  spectrum  is  dominated  by  bands  1600‒1700  cm                                                                                         −1                assigned to the vibrations of carboxylic group. As two nonequivalent C=O groups                are  present  in  the  molecule  (ester  and  acid),  two  maxima  corresponding  to                stretching vibration are found in the spectrum. Another strong, broad band is found                in the region of 2500‒3500 cm  in the IR spectrum, assigned to the O‒H stretching                                            ‒1                vibration. Both mentioned spectral characteristics are weak in the Raman spectrum.                In  contrast,  Raman  spectrum  is  dominated  by  the  C=C  aromatic  ring  stretching                vibrations  near  1600  cm ,  and  there  are  also  intensive  bands  around  3000  cm                                       −1                                                                                         −1                corresponding to the C‒H stretching vibration.                                                                              57","3 Raman spectroscopy                                                  Figure 3.2. IR and Raman spectrum of the acetylsalicylic acid (IR spectrum red,                                               Raman violet).                 Raman spectroscopy is often used together with IR spectroscopy in the mid-infrared                region to obtain as much spectral information as possible. Raman spectroscopy can                be  a  supplementary  technique  for  the  structural  analysis  of  organic  molecules  in                combination  with  nuclear  magnetic  resonance  (NMR)  and/or  mass  spectrometry                (MS).                 Raman spectrometry provides the key information on the molecule structure. Band                positions  and  intensities  are  employed  in  functional  group  identification  of  the                sample or for identity confirmation [10]. Having the necessary knowledge, one can                identify chemical compounds or observe intermolecular interaction by evaluation of                Raman band positions and intensity changes. Spectral libraries serve as a tool which                makes sample identification easier by comparing the measured and library spectra.                 The  time  required  for  spectral  accumulation  depends  on  the  particular  Raman                system, and ranges from hours to nanoseconds with a special instrumentation.                 One of the biggest advantages over all other analytical techniques is that a sample                can be measured directly with very easy preparation or even without any sample                pretreatment. Issues with sample thickness appearing in transmission techniques do                not represent a problem. On the other hand, quantitative analysis is tricky for solid-                state samples as the optical pathlength is not exactly known.                                                      58","3 Raman spectroscopy                 In Raman spectroscopy, the ambient atmosphere (humidity and CO2) has a weak                influence  on  the  resulting  spectra,  so  there  is  no  need  to  measure  background                Samples can be measured directly in solution or suspension (as water has very weak                bands) or in packaging material (glass, polyethylene, etc.), thus, contamination of                both  sample  and  operator  can  be  avoided.  Next  advantage,  similar  to  IR                spectroscopy,  is  the  possibility  of  connecting  the  Raman  spectrometer  to  a                microscope with identical technical possibilities as mentioned for IR spectroscopy,                but  with  the  advantages  mentioned  above  (measurement  through  packaging                materials, water suspension measurements, and so on).                  3.2  Experimental setup                  The  basic  arrangement  of  Raman  spectrometer  is  depicted  in  Fig.  3.3.  Incident                radiation is emitted from the source and reaches the sample. Scattered photons are                passing through the collection optics to the diffraction element (usually grating) or                interferometer – based on spectrometer type – and are registered at the detector.                                                    Figure 3.3. Basic arrangement of a Raman spectrometer: 1 – Source, 2 – sample,                     3 – collection optic, 4 – interferometer or diffraction element, 5 – detector.                                                         59","3 Raman spectroscopy                 Lasers  are  usually  used  as  a  source  for  the  emission  of  high-energy  coherent                radiation [9]. Wavelength of the lasers used for Raman spectroscopy varies from IR                and  visible  to  UV  region.  Raman  signal  intensity  is  higher  with  lower  laser                wavelengths, but this is associated with a higher risk of continual sample emission –                fluorescence – which can overlap the whole Raman spectrum with its much stronger                signal (Fig. 3.4). Examples of lasers commonly used in Raman spectroscopy are as                follows:  Nd:YAG  (neodymium-doped  yttrium  aluminum  garnet)  with  the                wavelength of 1064 nm (NIR region) or doubled (532 nm), He-Ne gas laser (633                        +                nm),  Ar  (488  and 458  nm),  laser  semiconductor  diode  (780  and  785  nm),  color                lasers  (360–700  nm),  etc.,  and  lasers  with  adjustable  wavelength.  Most  of  the                routine applications employ continuous lasers. Pulsed lasers are used in the research                for time-resolved measurement.                 Depending  on  the  angle  between  the  excitation  and  the  scattered  light,  we  can                distinguish  90°  and  180°  arrangement  (geometry).  Some  special  applications  use                SORS (Spatially Offset Raman Spectroscopy) for measurement through packaging                material (glass, paper, or plastic bags, etc.). Sample can be placed into a vial, NMR                or UV cuvettes, on a plate or other holders. Handheld Raman systems can be used                for various in situ measurements. Optical fibers enable collection of spectra outside                of the spectrometer that can be used to monitor chemical reactions, crystallization,                and other processes. Special sample pretreatment is required for Surface Enhanced                (Resonance)  Raman  Scattering  (SER(R)S),  Tip-Enhanced  Raman  Spectroscopy                (TERS) and some other techniques employed mainly in primary research [6].                                                          Fig. 3.4. Example of fluorescence in the Raman spectrum.                                                     60","3 Raman spectroscopy                 In general, measured sample must be placed in a proper distance from the collection                optic (usually an objective) to collect the most scattered photons. Thus, sample is                focused  to  reach  the  maximum  signal.  This  setting  is  not  applicable  in  case  of                immersion probes or handheld systems. Furthermore, the laser spot is usually small,                and  several  accumulations  must  be  obtained  from  different  sample  locations  to                obtain  statistically  representative  spectrum  of  solid  state  and/or  inhomogeneous                samples.                 In contrary to IR spectroscopy which almost exclusively employs interferometers,                Raman spectroscopy uses both interferometers and dispersive elements (diffraction                gratings) [6]. The grating is usually a plate with hundreds to thousands of grids per                one  millimeter.  Fourier  transformation  is  used  for  signal  processing  in  case  of                systems with interferometers. Such systems are signed FT.                 Interferometric setup is equipped with a laser with an excitation wavelength of 1064                nm (NIR region). Dispersive (grating) systems are utilized with excitation source in                the UV, visible and NIR region. Dispersive systems provide better spatial resolution                and higher Raman signal intensity, while Fourier transform systems offer lower risk                of fluorescence.                 FT (interferometric) systems are usually connected to high-sensitivity germanium                detectors which need to be cooled by liquid nitrogen or InGaAs detectors working                in ambient laboratory temperature. CCD (Charge Coupled Device) are employed in                dispersive systems.                 Capabilities of Raman spectroscopy can be extended by connection to microscope                which enables measurement of small particles, approx. 1 µm, or mapping of surface                component  distribution  and  depth  profiling  [11].  Here,  the  basic  spectrometer                scheme as presented in Fig. 3.3 remains unchanged. Excitation radiation is brought                through microscope objective to the sample and scattered light is collected by the                same objective, i.e., 180° geometry is applied. Confocal microscopes can also be                employed  (basic  scheme  in  Fig.  3.5)  In  this  case,  the  sample  hit  by  excitation                radiation  emits  an  inelastically  scattered  light  and  this  light  is  focused  by  lens.                Combination  of  focusing  and  slit  position  enables  better  spatial  resolution  and                suppresses radiation from different sample layers. The Fig. 3.5 shows focusing on a                layer 2, whereas radiation from neighboring layers 1 and 3 is mostly blocked by the                slit shade. Only the radiation from level 2 can thus pass to the detector through the                slit, almost without any restriction. Depth profiling is suitable for sample analysis                where the upper level cannot by easily removed, or for mapping of components in a                matrix.                                                          61","3 Raman spectroscopy                                                            Figure 3.5. Scheme of a confocal microscope; P – slit, L – lens,                                       1,2,3 – individual sample levels.                 Spectrometer settings, such as sample placement, laser power, accumulation time                and other parameters, differ significantly between instruments and manufacturers.                The  sample  type  and requirements for spectral  quality  influence  the  instrumental                setting. Therefore, neither standard procedure nor general user guide is available for                every sample. One can only say higher laser energy, shorter laser wavelengths, and                longer  spectral  acquisition  can  increase  sample  temperature  and  cause  its                destruction  or  degradation  and  also  increase  fluorescence  risk.  Thus,  when                analyzing  sensitive  materials,  e.g.,  biological  tissues,  it  is  recommended  to  start                with short accumulations with low laser energy. The goal is to gain maximal signal                to noise ratio without sample degradation.                   3.3  Application of Raman spectroscopy                3.3.1  Identification of unknown samples                 Some general applications of Raman spectroscopy were stated above. This chapter                will provide more detail together with case studies.                                                       62","3 Raman spectroscopy                 Raman  spectroscopy  is  a  non-destructive  analytical  method.  Its  complementarity                with IR spectroscopy and characterization of the molecular and crystal structures                enables  its  utilization  for  identification  of  unknown  samples.  As  stated  in  the                chapter  on  IR  spectroscopy,  each  group  vibrates  at  a  specific  frequency.  These                vibrations are observed, and the location of peak (band) maxima corresponds to the                presence of particular functional groups. Moreover, functional group vibrations are                influenced  by  their  chemical  neighbors,  as  some  substituents  can  increase  or                decrease  vibration  frequency.  Therefore,  not  only  can  we  determine  which                functional groups are present in the sample, but also what substituents are present in                their  neighborhood,  e.g.,  if  there  is  only  one  isolated  aromatic  ring  or  if  it  is                substituted, etc. Fig. 3.6 shows spectra of two isomers differing only in the location                of one double bond.                                                                   Figure 3.6. Comparison of Raman spectra of two isomers.                  Similarly to IR spectroscopy, chemical structure of a sample can be determined by                the assignment of spectral bands to particular functional groups. Certain knowledge                and  experience  are  required  for  such  spectral  analysis.  Tables  and  charts  with                                                      63","3 Raman spectroscopy                 characteristic  vibration  energies  of  specific  functional  groups  are  a  useful  tool.                Chemical  structure  can  be  confirmed  by  NMR  spectroscopy,  comparison  with  a                standard, spectral library search, etc. As the spectrum of a mixture is a superposition                of  individual components’  spectra,  one  can  identify components contained in  the                mixture.  Individual  components  can  be  quantified  with  the  limitation  mentioned                above,  i.e.,  the  exact  depth  of  the  excitation  laser  penetration  is  not  known  and                therefore  calibration  curve  construction  can  be  complicated.  In  case  of  perfectly                homogenized liquid samples, calibration is easily applicable.                   3.3.2  Identification of polymorphs, solvates and salts                 Allotropy is a property of an element to exist in different structural modifications,                which can differ in their physical and chemical properties. In analogy, molecules                can also exist in different modifications and this property is called polymorphism.                Individual  polymorphs  can  have  different  specific  surface  area,  hardness,                dissolution  rate,  etc.  As  the  spatial  arrangement  of  functional  groups  in  different                crystal lattices varies, the neighborhood of functional groups varies also. This effect                influences  bond  vibration  frequency  and  lattice  vibrations  occur  at  lower                wavenumbers. That is why two different polymorphs of the same molecule do not                have the same Raman spectrum (Fig. 3.7), but the differences between polymorphs                can  have  various  intensity.  Therefore,  Raman  spectroscopy  is  not  suitable  for                determining  of  a  crystal  structure  without  standards.  In  such  case,  X-Ray                Diffraction (XRD) analysis will be the method of choice. Raman spectroscopy is                however  a  fast  and  non-destructive  technique  and  requires  smaller  amount  of                sample than thermal methods, e.g., Differential Scanning Calorimetry (DSC), which                is used for polymorph differentiation as well. Raman spectroscopy is also cheaper                compared to XRD. Thus, polymorph structure can be first identified by XRD and                the reference spectra recorded by Raman spectroscopy, and the further polymorph                screening  of  sample  sets  can  be  carried  out  only  by  Raman  spectroscopy  if  the                polymorph spectra are different enough. In that case, quantification of polymorphs                is also feasible by Raman spectroscopy.                 Besides the rapid identification of chemical structure and functional groups, Raman                spectroscopy serves as a tool for discrimination between different salts of a single                molecule. The salts can be distinguished and identified, and the ratio of dissociated                and  non-dissociated  carboxylic  groups  can  also  be  determined.  Fig.  3.8  shows                spectra of tartaric and succinic salts of one molecule. Even without deep knowledge                of Raman spectroscopy, the difference is easily recognizable.                                                         64","3 Raman spectroscopy                                                         Figure 3.7. Comparison of different polymorph Raman spectra.                                                 Figure 3.8. Comparison of Raman spectra of two different salts of one molecule –                                                                                      −1                   succinic and tartaric.  Major differences occur around 1700, 1450, 1050 cm .                                                      65","3 Raman spectroscopy                 Differentiation  between  salt,  solvate  or  cocrystal  is  also  feasible  by  Raman                spectroscopy. Solvate is formed by molecules of a sample and solvent arranged in                the crystal lattice. Hydrates are the special case of solvates when the solvent in the                lattice is water. Cocrystal contains two or more species when all of them are solids                under  normal  conditions,  and  the  arrangement  in  the  lattice  is  not  based  on  ion-                pairing but on weaker interactions such as hydrogen bonding, π-π stacking or van                der Waals interaction.                 Fiber optics and probes are used for in-situ reaction and crystallization monitoring.                Transition  of  polymorphs can also  be  directly  observed in suspension  or  through                glass by Raman spectroscopy. Furthermore, a heating stage can be placed under the                microscope objective to reveal the polymorphic changes during heating or cooling.                   3.3.3  Raman mapping and imaging                 Possibility  to  connect  microscope  with  a  spectrometer  was  already  mentioned                above.  The  arrangement  is  suitable  for  analysis  of  samples  with  minimal  size  of                approximately  one  micrometer.  With  special  instrumentation  like  TERS,  a                nanometer  resolution  can  be  reached,  which  is  more  often  used  in  the  primary                research. The same application as in case of the “macro” settings are applied, i.e.,                identification of unknown samples, including fibers, small impurities,  etc. One of                the biggest advantages of the microscope-spectrometer arrangement is the mapping                possibility. Spectra are accumulated on a sample surface in predefined points with                regular spacing. When the points form a line, the mapping is called line-mapping.                Line-mapping is suitable for evaluation of concentration gradient of one or more                components,  e.g.,  diffusion  of  one  component  into  another.  If  the  predefined                measurement points are spread over the part or over the whole surface, it is called                an “area mapping” or 2D mapping. Line- and area mapping require line and area                detectors, respectively. In combination with the depth profiling, a 3D mapping can                be obtained. Mappings are crucial in applications where component distribution and                surface homogeneity are to be observed and checked. Fig. 3.9 and 3.10 represent                Raman map of a pharmaceutical tablet.                 Spectra  accumulated  during  mapping  are  evaluated  by  multidimensional                mathematical and statistic methods, such as Principal Component Analysis (PCA),                and when the components are known and spectra available, maps can be evaluated                by comparison with spectral library records. It is possible to observe intensity, area,                bandwidth,  etc.,  as  well  as  distribution  of  only  one or  more  bands  which  do not                interfere  with  other  components.  The  obtained  map  or  image  is  consequently                visualized in different colors, 2D or 3D (Fig. 3.10), demonstrating the distribution                of one excipient in the tablet.                                                      66","3 Raman spectroscopy                                                         Figure 3.9. Cross-section Raman map of a pharmaceutical tablet. API particles are                highlighted on the left, one excipient in the middle, and blended picture on the right.                                                                                  67","3 Raman spectroscopy                                                                                    Figure 3.10. Distribution of an excipient in a tablet cross section (2D a 3D                                               visualization).                                                          68","3 Raman spectroscopy                 3.4  Resources and recommended literature                   1.     Sodomka L., Sodomková M., Nobelovy ceny za fyziku, Prague, Czech Republic,                         1997.                  2.     Raman C. V., A new radiation, Ind. J. Phys. 1928, 2, 387.                   3.     Landsberg  G.  S.,  Mandelstam  L.  I.,  New  phenomenon  in  scattering  of  light                         (preliminary report), Journal of the Russian Physico-Chemical Society, Physics                         Section 1928, 60, 335.                   4.     Landsberg  G.  S.,  Mandelstam  L.  I.,  Eine  neue  Erscheinung  bei  der                         Lichtzerstreuung  in  Krystallen,  Naturwissenschaften.  1928,  16,  557.                         https://doi.org/10.1007/BF01506807                   5.     Landsberg  G.  S,  Mandelstam  L.  I.,  Uber  die  Lichtzerstreuung  in  Kristallen,                         Zeitschrift fur Physik. 1928, 50, 769. https://doi.org/10.1007/BF01339412                   6.     Ferraro  J.  R.,  Nakamoto  K.,  Brown  C.W,  Introductory  Raman  Spectroscopy,                         Academic Press: London, UK 2003.                  7.     Long  D.  A.,  The  Raman  Effect,  Wiley:  Chichester,  UK  2002.                         https://doi.org/10.1002/0470845767                   8.     Pivonka  D.  E.,  Chalmers  J.  M.,  Griffiths  P.  R.  Applications  of  Vibrational                         Spectroscopy  in  Pharmaceutical  Research  and  Development,  Wiley:  Chichester                         UK, 2007.                   9.     Atkins P., de Paula J., Fyzikální chemie, VŠCHT, Praha 2013, 978-80-7080-830-                         6.                   10.    Socrates  G.,  Infrared  and  Raman  Characteristic  Group  Frequencies,  Wiley:                         Chichester, UK, 2001.                   11.    Salzer  R.,  Siesler  H.  W.,  Infrared  and  Raman  Spectroscopic  Imaging,  Wiley-                         VCH: Weinheim, Germany, 2009. https://doi.org/10.1002/9783527628230                                                                       69","4 X-ray diffraction                 4  X-ray diffraction                 Hana Brusová and Bohumil Kratochvíl                   4.1  Introduction                 X-ray diffraction is one of the main solid state analytical techniques, considered as                the  \"gold  standard\"  not  only  for  characterization  and  identification  of  crystalline                substances, but also for their discrimination.                 Originally, the X-ray diffraction was used mainly to study inorganic materials and                                                                                          th                minerals, where it was also observed for the first time, at the beginning of the 20                century. X-ray diffraction uses the ability of solids to scatter X-ray photons and thus                create a diffraction pattern which characterizes the internal structure of the studied                material. The main analytically usable quantities are the intensities and positions of                the diffracted X-ray beams in the diffraction pattern. The theory of X-ray diffraction                is based on the knowledge of crystallography, so this chapter therefore also includes                the basics of crystallography.                 Because  the  final  product  of  many  industrial  technologies  are  microcrystalline                powders,  the  X-ray  powder  diffraction  technique  is  the  most  discussed  in  this                chapter. The fact that the technique is well established in pharmaceutical industry is                also  demonstrated  by  its  inclusion  into  the  European  Pharmacopoeia  (Ph.  Eur.).                Besides,  other  control  authorities  also  require  this  test  as  a  standard  control  of                polymorphic  forms  of  active  substances  (APIs).  ICH  directive  (International                Conference  on  Harmonization  ‒  Quality)  Q6A  and  FDA  directives  list  X-ray                powder  diffraction  as  a  basic  analytical  method  for  resolving  different  crystal                structures  (polymorphs).  It  can  generally  be  assumed  that  different  internal                arrangements of molecules in a crystal are reflected by various physico-chemical                properties of the crystalline substances. Furthermore, the X-ray powder diffraction                enables non-destructive resolving of different forms of one API (polymorphs, salts,                hydrates, solvates and cocrystals).                  4.2  Crystal structure, its geometry and symmetry                 External  morphological  symmetry  of  crystals  has  long  been  speculated  as  a                manifestation  of  regularity  of  their  internal  arrangement.  This  was  for  example                observed in crystallization of arsenates into different crystal forms depending on the                crystallization conditions.                                                        70","4 X-ray diffraction                 Crystalline substances are indeed characterized by regular arrangement of particles                they consist of (atoms, ions, molecules or parts of molecules). The given structural                motif is three-dimensionally, translationally repeated throughout the whole structure                (model of the ideal crystal). If we approximate the structural motif by a point, then                we get an imaginary spatial lattice which represents a regular arrangement of points                in  space.  Nodal  points  lie  on  lines  and  planes  creating  a  spatial  lattice.  If  this                                                                            3                regularity is maintained throughout the whole volume (e.g., 1 mm ), the crystal is                called a single-crystal. Fig. 4.1 shows the crystal structure and the spatial lattice of                sodium chloride.                                Figure 4.1. Ionic crystal structure and spatial lattice of NaCl (Na  ions are green,                                                                           +                    -                  Cl  ions are red). The structural motif is formed by a pair of Na-Cl ions, with the                                                                        -                               lattice points always lying in the position of Cl  ions                                   (Source: https://en.wikipedia.org/wiki/Crystal_structure.)                The smallest parallelepiped of the spatial lattice is called a unit (elementary) cell,                which fills an ideal crystal without a residue when multiplied in three dimensions. It                represents a figure with parallel sides, having lattice points either only in its vertices                (primitive cell) or also in centers of its sides (face-centered, see e.g., the structure of                NaCl  in  Fig.  4.1)  or  in  vertices  and  the  middle  of  the  cell  (body-centered).  The                number of unit cell types is limited by the internal symmetry of the crystal structure.                The distribution of unit cells according to the arrangement of their nodes is shown                in Table 4.1.                A  unit  cell  is  characterized  by  lattice  parameters  a,  b,  c  and  ,  β,  γ  (lengths  of                edges, and the angles between them, respectively). The smallest fraction of the unit                cell is called the asymmetric unit, see Fig. 4.2. Thus, according to the symmetry of a                crystal structure, there can be distinguished 7 crystal systems, 14 Bravais lattices                (divided into crystal systems), 32 crystal classes, and 230 distinct space groups, see                Table 4.2.                                                            71","4 X-ray diffraction                     Table 4.1. Classification of unit cells according to the location of their lattice                                                   points.                        P – primitive (simple cell) – lattice points only in the vertices of                                            the cell                          I – spatially centered – lattice points in the vertices and the                                        center of the cell                         F – face centered – lattice points in the vertices and centers of                                       the sides of the cell                        C – base centered – lattice points in the vertices of the cell and                                   the centers of its opposite sides                                                                Figure 4.2. Scheme of an assessment of a crystal structure.                                                     72","4 X-ray diffraction                                       Table 20.2. List of Bravais lattices.                      7 crystal systems   14 Bravais lattices                                        P                      Triclinic                                           P          C                       Monoclinic                                           P          C           I           F                      Orthorhombic                                           P          I                      Tetragonal                                           P                      Rhombohedral                                          P                      Hexagonal                                           P (pcc)    I (bcc)     F (fcc)                      Cubic                                                             73","4 X-ray diffraction                  4.3  X-rays                 To  obtain  an  analyzable  diffraction  pattern,  the  probing  radiation  must  have  a                wavelength comparable to or lower than the distance between the scattering centers                (atoms – high electron density sites). X-rays (e.g., generated by a laboratory X-ray                tube, see Table 4.3) meet this requirement, and the result of a diffraction experiment                is the intensities and positions of diffraction spots, which encodes information about                the crystal structure and properties of the studied material.                 There are generally three options for X-ray generation:                     •  impact of an accelerated electron onto a solid surface – X-ray tube                    •  alternation of flight path of a relativistic electron moving in the synchrotron                      (so-called synchrotron radiation in the X-ray region)                    •  exited fluorescence radiation                   4.3.1  X-ray tube                 Radiation  in  an  X-ray  tube  is  created  by  a  sudden  collision  of  very  fast-moving                electrons with a high mass matter (high atomic number). The cathode in X-ray tube                is  heated.  Then,  if  voltage  is  applied  between  cathode  and  anode,  the  electrons,                emitted from the hot cathode, start to move towards the anode. Upon their impact                on  the  anode,  around  1%  of  their  kinetic  energy  converts  to  X-rays,  and  99%                converts to heat. Therefore, the anode must be cooled. Radiation is produced by 1%                of the accelerated electrons penetrating K and L electron levels of the anode (levels                near the core) where they knock other electrons out. Electrons from higher levels                move to fill these empty spaces, and the excess energy is radiated as X-rays. The                basic scheme of an X-ray tube is shown in Fig. 4.3.                                       Figure 4.3. Schematic description of an X-ray tube, with fixed anode.                                     (Source: https://en.wikipedia.org/wiki/X-ray_tube.)                                                     74","4 X-ray diffraction                 The radiation created by an impact of an electron onto a mass is divided into:                    •  braking  radiation  (Ger.  Bremsstrahlung,  see  German  nationality  of  W.C.                      Roentgen, discoverer of X-rays) – continuous, produced by electron velocity                      change                    •  X-ray  fluorescence  (defined  by  the  electron  energy  levels  of  the  chemical                      element)                Braking radiation is produced by an interaction of an electron and a nucleus anode                target atom. The deceleration is one-step and has the shortest wavelength (continual                braking creates longer wavelengths). By each interaction with the anode atoms, the                electrons  (denoted  A  and  B  in  Fig.  4.4)  lose  part  of  their  kinetic  energy  gained                between the two electrodes. The lost energy converts into X-rays. Since the energy                lost  in  the  collisions  has  various  intensities,  the  X-rays  will  be  represented  by  a                range of wavelengths from minimum value min to maximum value max. Therefore,                the radiation is termed continuous.                                                      Figure 4.4. Scheme of braking radiation formation.                       (Source: https://www.sciencedirect.com/topics/earth-and-planetary-sciences/bremsstrahlung.)                 X-ray fluorescence consists of discrete wavelengths. It is produced by an electron                with sufficient energy (E = eU), moving from cathode and knocking out an electron                from outer layers K and L of the anode. The vacant space is filled by an electron                with  higher energy  originating  from  level  farther  from  nucleus.  Excess  energy  is                released in the form of X-rays, see Fig. 4.5. The wavelength  of the X-ray radiation                emitted  during  the  transition  is  determined  by  the  difference  between  the  two                energy  levels.  Since  this  wavelength  is  characteristic  for  the  anode  material,  the                resulting radiation is called characteristic.                                                         75","4 X-ray diffraction                                                 Figure 4.5. Scheme of characteristic X-ray emission.                        (Source: https://www.sciencedirect.com/topics/medicine-and-dentistry/characteristic-x-ray.)                 Fig. 4.6 shows characteristic lines of monochromatic X-rays on a background of a                continuous  spectrum.  The  wavelength  of  a  characteristic  line  (denoted  K,  Kβ)                depends on the atomic number of the anode material.                             Energy levels                                  Intensity                                                                                    λ (ρm)                     Figure 4.6. Depiction of allowed electron transmissions between the individual                    energy levels in an electron cloud of an atom (anode) and a wave spectrum of                                    characteristic monochromatic radiation.                            (Source: Kratochvíl B. Chemie a fyzika pevných látek I. VŠCHT Praha, 1994.)                  4.3.1.1  Synchrotron radiation                 Synchrotron  radiation  (Fig.  4.7),  in  the  X-ray  region,  is  created  by  radial                acceleration of a relativistic electron. It movement on a circular path emits energy in                the form of a continuous spectrum with long wavelengths and with approximately                constant  intensity.  Undulating  and  bending  magnets  are  used  for  this  purpose.                                                     76","4 X-ray diffraction                 Synchrotron radiation is polarized in the orbit plane, and a deviation from this plane                enables  to  use  also  elliptically  polarized  radiation.  Other  characteristics  are:                coherence of the radiation, its time course (particles circulate in clusters), and about                two orders of magnitude higher energy than an X-ray tube.                                             Figure 4.7. Configuration scheme of an accumulation ring of a synchrotron with                             undulator and bending magnets. Work stations are called                                               „Beam Lines“.                  (Source: https://www.researchgate.net/figure/A-schematic-diagram-of-a-synchrotron-radiation-accelerator-hv-                                          photon-source-e_fig1_237806176.)                  4.3.2  Characterization of X-ray radiation                    •  It  is  an  electromagnetic  radiation  with  very  short  wavelengths                        -8                             -12                                                  -9                      10 –10  m. Wavelength of 10  m is used in radiodiagnostics.                   •  It is invisible and passes through matter where it is partially absorbed, and the                      absorbed amount depends on the composition of the matter (average atomic                      number, density, and thickness) and the quality of the radiation (wavelength).                      It  decreases  with  square  of  its  path  length.  It  is  radiated  linearly  from  its                      source.                    •  Its  wavelength  depends  on  the  material  of  anode  focal  point.  Radiation                      created by incidence of electrons on the focal point is called primary beam                      and has a conic shape.                                                         77","4 X-ray diffraction                 4.4  Analytical X-ray diffraction methods                 Two basic methods are discerned, based on the type of the analyzed sample:                    •  single-crystal X-ray diffraction (SCXRD)                    •  powder X-ray diffraction (PXRD)                 Their basic characteristics, advantages, and disadvantages are summarized in Table                4.4a and 3.4b.                 The X-ray structural analysis with single crystals allows a complete determination                of crystallographic characteristics. Diffraction on a single crystal yields diffraction                intensities  with  positions  which  enable  determination  of  the  complete  3D  crystal                                                                                4                                                                            2                structure. Number of collected diffraction spots varies between 10 –10 . In powder                diffraction,  max.  about  100  diffraction  lines  can  be  distinguished  at  most  in  a                debyegram  or  in  a  diffractogram.  Therefore,  X-ray  powder  diffraction  is  mainly                used to determine the phase composition of materials.                It  is  therefore  obvious  that  full  structural  analysis  of  crystals  with  complex                structures or low symmetries can be successfully performed only by diffraction on                single  crystals.  The  vast  majority  of  crystalline  substances  however  exist  in                polycrystalline  forms,  and  the  creation  of  single  crystals  is  often  difficult.                Moreover,  X-ray  powder  methods  are  irreplaceable  where  the  polycrystalline                structure  plays  a  critical  role  in  determination  of  material  properties  (phase                composition, grain size and orientation, internal stress, etc.).                                                                                   78","4 X-ray diffraction                              Table 20.4a. Comparison of basic diffraction methods.                      Name          Single crystal X-ray diffraction    X-ray powder diffraction                     Abbreviation   SCXRD                        PXRD                     Sample type   Single crystal                Powder                     Sample        1  single  crystal  with  size  of   100–500 mg                    amount        0.1–1 mm                                   Selection  of  a  suitable  single                    Sample        crystal  and  its  fixation  onto  the   Filling  the  holder  cavity  or                    preparation                                 possibly milling                                  goniometric head                     Sample        Non-destructive               Non-destructive                    consumption                      Measurement   Hours/days                    Minutes/hours                    time                                   Complete   information   about                                  molecule,   conformation,   bond                                  lengths,   chirality,   spatial                    Analysis   of   arrangement   of   molecules,   „Fingerprint“  of  the  crystal                    obtained data                               structure                                  interactions  between  molecules,                                  determination  and  location  of                                  solvents.                                   Monochromatic  X-rays  impact  a   Monochromatic   radiation                                  single   crystal   which   rotates                    Method                                      impacts  a  polycrystalline                    principle     according  to  three  axes  (the   material   which   rotates                                  principle   of      four-circle   around one axis.                                  diffractometer).                                   3D  crystal  structure  determination                    Practical     of   pharmaceutical   substances,   Phase   qualitative   and                    application                                 quantitative analyses.                                  proteins, and other materials.                                                                79","4 X-ray diffraction                 Selection  of  an  X-ray  tube,  i.e.,  wavelength,  depends  on  the  type  of  the  studied                material.  Excitation  of  an  X-ray  radiation  emission  in  the  sample,  called                fluorescence  radiation,  is  undesirable,  as  it  increases  the  background.  It  can  be                expected in cases where the absorption coefficients of atoms of the studied material                reach  high  values  with  the  radiation  wavelengths  of  the  particular  X-ray  tube.                Furthermore, a large number of diffraction lines are typical for crystals with low                symmetry  or  large  unit  cell.  Their  number  decreases  and  their  mutual  distance                increases when using an X-ray lamp with longer wavelength. The most commonly                used X-ray lamp is a lamp with a copper anode, whose Kα1 component wavelength                is approximately in the middle of the manufactured lamp wavelengths. The middle                wavelength Kα is used if the doublet is not discernible (Table 20.3).                       Table 20.3. Selected wavelengths of the most applied anode materials.                                                                     -10                              Components of characteristic radiation (10  m = 1 Å)                             anode       Kα1        Kα2        Kα         Kβ                                Cu      1.54050    1.54434    1.5418     1.39217                                Co      1.78889    1.792801   1.79019   1.620703                               Mo       0.70261    0.71354   0.706253   0.632253                 The  discovery  of  X-rays  in  1895  is  associated  with  the  name  of  the  German                physicist W.C. Roentgen. In 1912 Max von Laue expressed a belief that the regular                arrangement of atoms in crystals may act as a three-dimensional diffraction lattice.                The  prediction  was  confirmed  by  later  experiments.  These  showed  that  X-rays                provide a diffraction pattern with characteristic maxima and minima of diffraction                spot intensities, after passage. Less than a year after the Laue’s discovery, W. H.                Bragg and his son W. L. Bragg showed how to determine crystal symmetry, mutual                position of building blocks (atoms, ions), and their distances in crystal structures                based on the diffraction patterns. From the analysis of diffraction spot intensities on                films  they  interpreted  the  X-ray  diffraction  phenomenon  as  a  reflection  on  the                crystal planes placed one after another at certain distances, see Fig. 4.8.                                                                   80","4 X-ray diffraction                                          Figure 4.8. Depiction of three systems of imaginary planes with various interplanar                 lengths. The planes are denoted according to Miller indices hkl, depending on how                                          they intersect the unit cell.                          (Source: Kratochvíl B. Základy fyziky a chemie pevných látek I. VŠCHT Praha, 1994.)                 Each plane scatters only a small amount of radiation, but the resulting scatter from a                number  of  planes  provides  sufficient  intensity  for  observation.  If  parallel  X-ray                beams impact any two parallel planes with distance d, an interference maximum of                the diffracted beams can be detected if their path difference is equal to an integer                multiple of the incident radiation wavelength, as shown in Fig. 4.9                                 Figure 4.9. Schematic depiction of X-ray diffraction on a crystal structure showing                 the angle of incidence , interplanar distance d and phase shift of the individual rays                                                  2dsin .                       (Source: Kratochvíl B. Základy fyziky a chemie pevných látek I. VŠCHT Praha: Praha, 1994.)                 The path difference between beam 11' and beam 22' is equal to 2dsin , where the                angle   is the angle held between the incident beam and the given crystal plane. The                angle between the incident and diffracted beam is then  2. The condition for the                                                      81","4 X-ray diffraction                 occurrence  of  diffraction  maxima  for  beams  1'  and  2'  is  given  by  the  following                relationship – Bragg equation:                             2dsin  = nλ          n = 1, 2, 3… (order of reflection)                 If the wavelength λ and the angle   are known, the distances d between the crystal                planes can be determined, if all reflections are considered as of the first order. This                principle is the basis for the whole discipline of X-ray structural analysis.                 So  far,  only  one  set  of  planes  has  been  discussed.  By  turning  the  crystal  and                changing the angle of the incident radiation, the Bragg equation  – the diffraction                condition  –  will  be  met  by  various  sets  of  planes,  enabling  to  obtain  point                diffraction pattern of a single-crystal. In case of powdered samples, the diffraction                condition  is  simultaneously  met  by  multiple  sets  of  planes.  By  changing  the                diffraction  angle,  a resulting  diffraction  pattern  can be  obtained  in  the  form  of  a                system of conical areas with axes along the axis of the incident radiation and apex                angles 4  given by the interplanar distances between planes. It is therefore evident                that  measuring  of  the  diffraction  angle  enables  to  determine  the  interplanar                distances.                  4.4.1  Interactions of X-rays with crystal                 Many  physical  effects  occur  when  X-rays  impact  a  solid  matter.  The  radiation                incident  on  a  crystal  is  called  primary,  and  the  reflected  radiation  (irradiated  or                diffracted)  is  called  secondary.  Secondary  radiation  is  divided  into  fluorescent,                electron emission, and scattered.                 Scattering  of  X-ray  radiation  is  the  most  important  effect  for  X-ray  diffraction,                where  a  part  of  the  originally  parallel  beam  of  X-rays  is  scattered  sideways.                Electrons are the scattering centers in the material. If the electrons oscillate at the                same  frequency  as  the  primary  radiation,  the  scattering  of  radiation  is  elastic                (coherent).  The  oscillating  electrons  then  become  a  source  of  a  new  secondary                radiation with identical wavelength to that of the primary radiation. The diffraction                result is then a set of diffracted waves which are radiated from the crystal, but only                in  certain  directions.  The  spatial  distribution  of  the  diffracted  waves  forms  a                diffraction  pattern  of  the  crystal.  This  diffraction  pattern,  and  especially  the                intensity of the diffracted reflections, carry information about the internal structure                of the crystal.                 The effect of elastic scattering is critical for the formation of a diffraction pattern                which  enables  determination  of  the  distribution  of  atoms  or  ions  in  the  studied                crystalline material. In elastic scattering, the waves scattered (emitted) by individual                                                     82","4 X-ray diffraction                 atoms  have  an  invariant  phase  difference  –  these  atoms  form  a  set  of  coherent                sources (Fig. 4.10).                                                 Figure 4.10. Depiction of the interaction of X-ray photons with atoms in a crystal                                                 structure.                                      (Source: http://en.wikipedia.org/wiki/Bragg_law.)                 Interatomic distances in solids are comparable with wavelengths of X-rays, and an                observable interference effect may occur between the scattered waves that manifest                in certain directions, creating a diffraction pattern.                 If the primary radiation is assumed to be a flow of photons from the view of the                wave particle duality, then after collision of a photon and an electron, a part of the                energy is passed onto the released electron and a part is radiated as a photon with a                different, longer wavelength in another direction and participates on emergence of                the secondary radiation. This effect is called a Compton effect, and the scattering is                called inelastic (incoherent).                 Besides the diffraction effects, a luminescence effect can also be observed when the                impact of a radiation on a luminescent surface causes luminescence. There are two                types.                    •  Fluorescence  –  a  substance  luminesces  only  upon  incidence  of  a  short-                      wavelength radiation. Photon of the primary X-ray radiation with sufficient                      energy can release an electron from the inner atomic layer. Its place is then                                                      83","4 X-ray diffraction                       filled  up  by  another  electron  from  a  higher  layer,  and  an  emission  of  a                      fluorescence radiation quantum occurs.                    •  Phosphorescence – a substance luminesces for some time after the impact of                      the radiation.                 Luminescing materials called luminophores are, for example, cesium iodide, cesium                sulfide,  zinc,  etc.  During  luminescence,  an  X-ray  photon  knocks  out  an  electron                from an outer shell to a shell closer to the core. Lower energy is then emitted  in                form of a light which has a longer wavelength than the X-rays – termed as outer                layer  transitions.  Along  with  the  electron  transition  from  an  outer  surface  to  an                empty inner surface space, another outer electron may be expelled from the atom.                This  is  termed  as  a  non-luminescent  transition  –  Auger  effect  –  and  the  emitted                electrons are called Auger electrons.                 Photochemical  effect  can  be  another  occurring  effect.  It  affects  silver  halides                (AgBr),  and  the  radiation  causes  a  generation  of  individual  silver  and  bromine                atoms in this case, which is used in film development.                 An important factor which must be considered when working with X-ray radiation                is the biological effect as X-rays are harmful to living matter.                 In the case of single crystals, it is possible to meet the condition for Bragg equation                with a suitable crystal orientation towards the beam of incident radiation. In the case                of polycrystalline or powdered samples, with random orientation of crystal grains, it                is likely that some grains will be oriented so that the above-mentioned planes hkl                will  be  in  the  diffraction  position.  This  probability  will  be  greater  with  higher                number of grains in the irradiated space. The radiation diffracted by planes hkl of                one grain will further be radiated in a direction which holds an angle 2hkl with the                direction of the incident radiation. If the considered crystal grain is turned around an                axis identical with the direction of the incident radiation, the diffraction planes hkl                still remain in the diffraction position (since they still hold an angle hkl with the                incident beam), and the diffracted beams move over the circumference of a cone                with apex angle 4hkl and axis in the direction of the incident radiation. Diffracted                beams from all grains with planes hkl in the diffraction position will also be radiated                onto this conical area.                                                                    84","4 X-ray diffraction                              Table 20.4b. Comparison of X-ray diffraction methods.                       Information         Single crystal            Powder record                      Crystal size   0.1–0.8 mm              0.2–10 μm                      Number     of                     crystals      1                        From hundreds to thousands                      Sample        Selection of single crystal    Milling in agate mortar                     preparation                      Reflective    3 position rotation angles   1 angle (2θ)                     positions                      Individual    Clear  separation  of  each   Overlapping   of   intensities                     intensity   of                         caused by symmetry (absolute)                     reflexions    reflection               and close position of reflexions.                                                             Deformed by instrument flaws,                     Reflexion     Sharp, given by the shape of   tension  in  small  crystals,  and                     profiles      the crystal                                                            preferred orientation                                                            Presence  of  another  phase                     Phase purity   Always one phase        cannot  be  excluded  by  simple                                                            means.                       Preferred     Without problem          Distorted  information  about                     orientation                            intensity                      Number     of   Thousands,   suitable   for  Tens to hundreds. Low number                     observed                     reflections   evaluation   using   direct  for   direct   methods   using                                   methods                                                            statistics.                                3                     (V = 1000 Å )                The basic difference between single crystal and powder X-ray diffraction methods                lies  in  the  appearance  of  the  diffraction  patterns  which  are  obtained  during  the                measurements.  In  the  case  of  single-crystal  diffraction,  the  diffraction  pattern                consists of individual points corresponding to the reflections (Fig. 4.11a), while for                powder diffraction the pattern is formed by Debye-Scherrer rings (Fig. 4.11b).                                                             85","4 X-ray diffraction                        a                                         b                              Figure 4.11. Separate point diffractions for single crystal diffraction patterns (a)                             and diffraction rings for powder diffraction patterns (b).                                 (Source: https://www.britannica.com/science/X-ray-diffraction.)                   4.4.1.1  Structure determination methods                 In pharmaceutical applications, it is essential to determine the crystal and molecular                structure  of  active  substances  (polymorphs,  solvates,  salts,  cocrystals,  etc.)  with                high  precision.  Routine  single  crystal  X-ray  diffraction  analysis  is  now  available                with sophisticated experimental techniques (single crystal X-ray diffractometer) and                software packages for complete interpretation of measured data. The disadvantage                of  this  method  is  the  input  material:  a  single  crystal  of  sufficient  size,  which  is                difficult to obtain in some cases. Therefore, assessment of crystal structures from X-                ray  powder  data  is  being  developed  very  rapidly.  In  addition,  the  electron                diffraction  method  which  does  not  require  such  large  single  crystals  as  X-ray                diffraction is also being applied. Furthermore, in electron diffraction data collection,                it is not necessary to separate the single crystal from the mixture, and the structural                analysis can be performed directly in the matrix (compact or powder).                                                        86","4 X-ray diffraction                 4.4.1.2  Data collection on the single crystal X-ray diffractometer and the crystal                structure solution                                                                                   3                The input material for SCXRD is a single crystal, in the order of 0.01 mm  in size,                and monochromatic X-rays are used for analysis. The single crystal is placed on the                goniometric head of the X-ray diffractometer and a three-circle system allows the                single crystal to be rotated to all positions where the Bragg condition is satisfied                (see Bragg equation). A position-sensitive CCD detector rotates around the fourth                axis and collects diffraction beams from the measured crystal. The movement of the                crystal and the detector is controlled by a computer. (Fig. 4.14).                                       Figure 4.14. Schematic depiction and a photograph of a four-circle single-crystal                                     diffractometer with kappa geometry.                        (Source: http://serc.carleton.edu/research_education/geochemsheets/techniques/SXD.html.)                  The position of atoms in a unit cell affects the intensity of the the diffracted beams                from the individual crystal planes h,k,l. To determine the geometry and symmetry of                the  crystal  structure  –  the  lattice  parameters,  the  space  group,  and  the  atom                coordinates (x,y,z) – it is required to measure both the positions and intensities of                diffraction spots in the diffraction pattern (Fig. 4.15).                                                                     87","4 X-ray diffraction                                         Figure 4.15. Single-crystal ready for measuring, size 0.1×0.3 mm (a); acquired                                      single-crystal diffraction pattern (b).                             (Edited according to: http://en.wikipedia.org/wiki/X-ray_Crystallography.)                The principle of finding atomic coordinates in the asymmetric part of the unit cell is                described by the following equation:                   (x,y,z) = (1/Vcell)∑h∑k∑l Fhkl  exp [-2πi (hx + ky + lz)]                    Vcell – unit cell volume                 To  determine  the  unknown  graph  of  the  electron  density  function    (x,y,z),                assuming that knowledge of a sufficiently large number of Fhkl values, it is required                to perform a three-dimensional summation of the Fourier series over all values of                the  indices  h,k,l  in  the  elementary  cell  or  its  asymmetric  part,  respectively.  The                atoms  lie  in  the  found  maxima  of  the  function.  However,  there  is  one  important                consideration with this procedure – the structure factor Fhkl is a complex number, so                it is necessary to know not only the amplitude but also the phase hkl:                 Fhkl = |Fhkl | exp (ihkl);    Ihkl = |Fhkl | 2                 The phase values cannot be obtained directly from the diffraction experiment but                can  be  extracted  by  mathematical  procedures.  Experimentally,  only  the  Ihkl                intensities  are  available  from  which  the  Fhkl  values  needed  to  perform  Fourier                summation can be obtained. The whole procedure is described in Fig. 4.16.                                                              88","4 X-ray diffraction                                                                         Figure 4.16. Depiction of procedure for solving of crystal structure, based on                                         single-crystal measurements.                                  (Source: http://en.wikipedia.org/wiki/X-ray_crystallography.)                  4.4.1.3  Standard  outcome  and  presentation  of  results  of  a  single-crystal                diffraction                 Molecular structure is usually presented in form of images which also show thermal                movements  of  individual  atoms  in  the  form  of  thermal  ellipsoids.  Sets  of                interatomic  distances,  angles,  and  torsion  angles  are  also  exported  by  default.                                                     89","4 X-ray diffraction                 Information  on  molecular  packing  and  types  of  non-bonding  intermolecular                interactions  is  also  attached  to  the  solved  crystal  structure.  From  the  molecular                packing,  it  is  also  possible  to  identify  intramolecular  interactions  and  find  open                spaces  (channels  and  cavities)  in  the  structure.  Plenty  of  molecular  graphics                programs can be used for visualization (e.g., Mercury, WebLabViewer). The most                common  input  is  a  cif-file  (contains  lattice  parameters,  space  groups,  chemical                composition,  atomic  coordinates,  thermal-vibration  coefficients,  bond  distances,                etc.). A table of crystallographic parameters or a table of hydrogen bonds contained                in the studied structure are parts of the report for a solved structure, based on single-                crystal measurement, see Table 4.5, Table 4.6, Fig. 4.17, and 3.18.                 The resolved crystal structures are usually published together with synthetic data,                where the solved structure is the most illustrative confirmation. The published cif-                files  are  saved  in  Cambridge  Structural  Database  (CSD)  where  they  undergo  a                verification  process.  In  a  publication,  the  validated  structure  is  referred  to  by  a                CCDC  code.  In  the  CSD database,  the structures  are  saved  under  another  codes.                According to these references, it is possible to search for a structure, and the data                are publicly available per request (www.ccdc.cam.uk).                                                                             O4                                                      C34   C35    C29               C21                                               C33                   C20                                                         C30                                              C32   C31      O5  C19     N5            C22                            C12  C11                                 N4                                      C10           O1     O3                   C23                                                                         C24                  N1  C13                                             C25                             C14  C9          C7                                        C8                N3  C18    O2                               C4                    C16           C26                    C1  C2                         C6         C17           C27                                            C5                           C3           N2                                 C15                                                                         C282                                                               C281                         Figure 4.17. Depiction of a molecular structure of ergot alkaloid,                Obrázek 5.18. Ortep obrázek – znázornění molekulové struktury                         dihydroergocristine, using thermal ellipsoids of vibrating atoms.                  námelového alkaloidu prostřednictvím teplotních elipsoidů.                  (Source: Petříčková H. Structural study of morphine alkaloids and their complexes. Dissertation. UCT Prague,                                                    2007.)                                                         90","4 X-ray diffraction                  Table 20.5. Basic crystallography (left) and measurement parameters (right) for the                       structure of dihydroergocristine mesylate ethyl acetate monohydrate.                              Crystal parameters           Measurement parameters                                         C35H42N5O5                                          CH3O3S       Crystal           0.2  0.3                            Formula                                         2.C4H8O2      dimensions [mm]      0.4                                           H2O                                                       Range of h         -16 → 16                             a [Ǻ]       12.5090(3)            k           0 → 22                                                               l           0 → 25                                                       Total    number                             b [Ǻ]       17.7480(5)    of reflections      49251,                                                       measured, θ range   1.97–27.09                                                       Number       of                             c [Ǻ]       20.3050(5)    independent          8718                                                       reflections                                                       Criterion    for                             α [°]          90         observed          I > 1.96 (I o)                                                       reflections                                                       Minimized        Σw(| Fo | - | Fc                             β [°]          90                                                                              2                                                       function              |)                                                                         Chebychev                             γ [°]          90         Weighing scheme                                                                         polynomial                          Space group     P 212121     Parameters refined   595                                                       Values of R         0.0721                              Z             4          Rw                  0.0802                                                       S                   1.0480                                                        Ratio   of   the                                                       maximum    least-                                    3                        Unit cell V (Å )   4507.9(2)                       0.0002                                                       squares  shift  to                                                                st                                                       e.s.d in the l  cycle                                                       Max.  and  min.    -16 → 16                           μ [mm ]         0.142       heights in final ∆ρ   0 → 22                                 -1                                                       map                 0 → 25                                                        CSDC number         56324                                                         91","4 X-ray diffraction                               Table 20.6. Hydrogen bonds in the studied structure.                                   D–H..A       D–H    H..A   D..A  D–H..A    Symmetry                                 O5–H4..O1     0.9924  1.8143  2.702(3)  147.10                               N1–H1..Osolvent   0.9900  2.0142  2.944(5)  155.49                    Distances                    and angles   N2–H2..Omsf   0.9985  1.9366  2.907(4)  163.11                               N3–H3..O4     0.9951  2.4783  3.174(5)  126.61  -x,1/2 + y,-3/2-z                               OH2O–HH2O..Omsf  1.0048  1.9732  2.703(7)  127.31                               OH2O–HH2O..O4   1.0118  1.8011  2.576(7)  130.50  -x,1/2 + y,-3/2-z                                                       Figure 4.18. Overlap of two crystal structures of dihydroergocristine mesylate                 solvates, crystallized with various solvents built inside the structures. The positions                  of ergot alkaloid molecules are virtually identical and there is a large number of                 cavities in the structures where the solvent molecules (either two molecules of ethyl                               acetate or a single molecule of amyl acetate) can fit.                                                     92","4 X-ray diffraction                 4.4.1.4  Electron diffraction                Three-dimensional electron diffraction (3D ED) is a relatively new method that is                being introduced into structural analysis, and that requires only a small amount of                input material (nanocrystal). It has great potential for development and is certainly                interesting for application in pharmaceuticals.                 Due  to  the  wave  properties  of  elementary  particles  (Louis  de  Broglie,  1924),                electron  beam  incident  on  crystals  also  exhibits  diffraction  phenomena.  At                significantly higher incident electron energies, from approximately the first tens of                keV onwards, relativistic electrons can illuminate a thin layer of the substance under                investigation, e.g., of a nanocrystal, and diffract and provide analyzable diffraction                data  –  diffraction  intensities  and  positions  which  enable  the  crystal  structure                determination.                 The 3D ED experiment is performed on a transmission electron microscope (TEM)                in the diffraction mode; but specialized electron diffractometers are already being                                                                         -4                developed. Data are collected from a nanocrystal (volume 1–10  μm ). The size of                                                                              3                the nanocrystal is matched to the width of the probing electron beam which ranges                from units of nm to a few μm. Compared to X-ray single crystal analysis, the crystal                size  required  for  3D  ED  is  2–3  orders  of  magnitude  smaller,  which  is  very                significant for sample preparation.                Nanocrystals  measured  by  ED  are  of  course  very  sensitive  to  any  mechanical                instability of the TEM goniometer which can easily deflect the sample out of the                irradiated area. Most often, the electron beam follows the motion of the nanocrystal                according to a predetermined trajectory.                Protocols in  3D  ED  ensure  optimal  collection  of integrated  diffraction intensities                and  their  positions.  There  is  a  number  of  data  reduction  software  packages                specifically  designed  for  3D  ED  or  adapted  from  software  for  X-ray  diffraction                data.  Compared  to  X-ray  diffraction  however,  there  are  two  experimental                limitations with the 3D ED, so far: the positions of the diffractions on the detector                are  inaccurate,  and  multiple  electron  scattering  occurs  in  the  material  under                investigation,  which  distorts  the  diffraction  intensities.  In  contrast,  one  of  the                advantages of 3D ED is that the strong Coulomb interaction between the incident                electrons and the crystal mass allows a good signal-to-noise ratio even for very thin                samples,  making  it  easier  to  identify  light  atoms  such  as  lithium  and  hydrogen,                compared to X-ray diffraction. The 3D ED can thus determine the absolute structure                more reliably.                 Current pharmaceutical research is widely using the SCXRD method for structural                studies  of  molecular  crystals  (substances,  impurities,  intermediates,  etc.).  The                problem  however  is  that  some  substances  crystallize  poorly  –  in  submicroscopic                crystals of inappropriate shapes (filaments, leaflets, book-like crystals, etc.), or in                                                     93","4 X-ray diffraction                 imperfect crystals formed at phase interfaces. Polymorphism of substances and their                unexpected  polymorphic  transitions  are  also  a  persistent  problem  in  the                pharmaceutical industry.  It  is therefore  important to perform  structural  studies of                polymorphic  transitions  already  at  the  nucleation  stage,  and  then  in  complex                matrices, e.g., in a tablet or capsule. Recently, cocrystals have been the subject of                research in pharmaceutics. The boundary between the category of pharmaceutical                cocrystals  vs.  pharmaceutical  salts  is  loose  and  can  be  a  subject  of  litigation.  A                recognized criterion for clear classification of a substance into the correct category                is the determination of the position of the proton (hydrogen) between the donor and                acceptor. The occasional problems with determining the proton positions are known                in SCXRD. Knowledge of the absolute structure and the resulting chiral purity is                crucial  for  pharmaceuticals,  which  no  pharmaceutical  manufacturer  can  afford  to                ignore  today.  The  SCXRD  method  can  determine  the  absolute  structure  more                reliably when the structure contains a heavy atom (Fig. 4.19).                                                    Figure 4.19. Absolute crystal structure of cocrystal sofosbuvir–L-proline                                 determined by 3D electron diffraction method.                                       (Source: Palatinus et al. Chem. Listy., 2021.)                 In  all  of  these  pharmaceutical  applications,  3D  ED  can  be  successfully  applied.                Even  compared  to  SCXRD,  the  3D  ED  method,  in  combination  with  dynamic                refinement,  can  more  easily  and  reliably  determine  the  absolute  structure,  even                when consisting of only light atoms. Other examples of pharmaceutical applications                of  3D  ED  are  the  determination  of  the  active  substance  structure  directly  in  the                                                      94","4 X-ray diffraction                 dosage  form  (compact  matrix),  or  the  structural  study  of  rapid  polymorphic                transformations.                   4.4.2  Powder methods                 4.4.2.1  Debye-Scherrer method                 Polycrystalline  material  consists  of  many  small,  randomly  distributed,  and                independently  diffracting  single  crystals.  All  single  crystals  are  variously  rotated                towards each other. A polycrystalline sample can be a compact solid body (joint                crystal grains), a loose powder, or a thin surface layer. A basic and by far the most                applied method used for polycrystalline materials, is the Debye-Scherrer technique,                see Fig. 4.20.                                                            Figure 4.20. Principle of Debye-Scherrer method.                            (Source: https://encyclopedia2.thefreedictionary.com/Debye-Scherrer+Method.)                 When a monochromatic beam is incident on a polycrystalline sample, the crystal                planes that satisfy the Bragg equation diffract. The diffracted beams then form a                concentric cone with a vertex angle of 4. The intersection of the diffraction cone                with  a  planar  detection  film  placed  perpendicular  to  the  axis  of  the  cone  is  the                diffraction line which has the shape of a circle. The powder sample is spread on a                glass  fiber  with  a  diameter  of  about  0.3  mm  or  is  poured  into  a  glass  capillary                placed in the center of a cylindrical chamber, with a film strip placed along its inner                circumference. Here, the diffraction cones produced by diffraction of the incident                                                    95","4 X-ray diffraction                 beam  on  a  system  of  crystal  planes  with  varying  interplanar  lengths  dhkl  (and                                          )                therefore  different  angles   intersect  the  film  in  circles.  The  unrolled  film  then                shows a set of circular arcs with different radii. As an example, a Debye photograph                of powder silver obtained with radiation from a copper anode (CuKα  radiation) is                shown in Fig. 4.21.                                         Figure 4.21. Debye photograph of powdered silver.                                    (Source: http://www.xray.cz/krystalografie/str11a.htm.)                 Diffraction angles   are determined from measured positions of the diffraction lines,                and  their  determination  enables  to  calculate  the  interplanar  lengths  dhkl  using  the                Bragg equation. The chamber is designed so that the distance of diffraction lines                from the center of the right hole, expressed in millimeters, gives either the value of                  or 2  in degrees.                   4.4.2.2  Powder X-ray diffractometer with the Bragg-Brentano geometry                The powder diffractometer in Bragg-Brentano parafocusing geometry is currently                the conventional apparature for the study of powder diffraction on polycrystalline                materials.  It  enables  precise  measurement  of  diffraction  line  positions  and                intensities,  utilizing  an  extensive  control  and  application  software.  A  simple                diagram of the most common type of a powder diffractometer is shown in Fig. 4.22                A photo of the whole instrument and its goniometer is shown in Fig. 4.23.                 Sample  is  placed  in  the  middle  of  a  circular  plate,  see  Fig.  4.22,  which  rotates                around the main axis of the goniometer. A detector for diffracted radiation moves                on the perimeter of a circle with radius given by the geometric configuration, with                twice  the  angular  speed  of  the  sample.  Diffractogram  is  measured  either                continuously, where the angular dependence of registered pulse frequency is plotted                with  a  plotter,  or  in  steps,  with  data  output  maintained  using  a  printer  or  other                recording medium.                                                            96","4 X-ray diffraction                                                           sample                X-ray source                                                                     detector                  Figure 4.22. Scheme of a diffractometer in Bragg-Brentan parafocusing geometry.                       The primary incident beam forms 2  angle with the diffracted beam.                                    (Source: http://pd.chem.ucl.ac.uk/pdnn/inst1/focircle.htm.)                      a                                b                                          Figure 4.23. The whole X-ray powder diffractometer (a) and the separate                                          goniometer with arms (b).                                     (Source: https://www.malvernpanalytical.com/en/.)                                                        97","4 X-ray diffraction                 In  this  arrangement,  a  powder  sample  is  filled  into  a  flat  holder  located  in  the                goniometer  axis,  see  Fig.  4.24.  Scanning  is  carried  out  as  the  detector  moves  at                speed 2θ and the sample moves at speed . The diffraction is symmetrical as the                incident  angle  is  identical  with  the  diffraction  angle.  Thus,  diffraction  is  always                registered on planes hkl which meet the diffraction condition for the particular   and                are oriented parallel with the sample surface. This means that the information for                different hkl planes is obtained from different grains.                     a                                                b                          Figure 4.24. Samples prepared for measurement, placed in the most used holders in                   the X-ray powder diffraction with Bragg-Brentano geometry (a), raw powdered                                                 sample (b).                  A significant instrument error can occur with Bragg-Brentano diffraction, where the                most serious effect is the positioning of the sample away from axis. This leads to                both blurring of the diffraction line and also to its shifting. Diffracted beam is thus                focused in a place other than the position of the detector. A functional dependence                                                                          of  this  shift  on the  diffraction  angle  (        )  can  be  derived  from  a  geometrical                                                                            sketch. This can be used when evaluating lattice parameter, as shown in Fig. 4.25                 So  far,  only  a  monochromatic  incident  radiation  was  considered.  Acquisition  of                monochromatic  radiation  using  diffraction  on  a  crystal  –  a  monochromator,  is                associated with a decrease in intensity for the primary beam, and therefore the most                common measurements use the most intense parts of the characteristic spectrum of                an  employed  X-ray  tube:  doublet  K1,  K2.  The  wavelength  difference  of  this                doublet is so small that splitting of the diffractions occurs only at higher diffraction                angles. This implies directly from the Bragg equation. If this splitting is observed, it                is required to use corresponding wavelengths of the individual doublet components                for  the  interpretation.  It  should  be  emphasized  that  in  this  case,  there  are  two                diffractions  from  the  same  system  of  planes  but  for  two  different  wavelengths                contained in an incident beam of X-rays. Except the two most intense components,                there  are  also  other  components  in  the  X-ray  spectrum  of  an  X-ray  tube,  and                continuous  radiation  is  also  not  negligible.  These  components  are  inhibited  by                absorption filters or monochromators.                                                          98","4 X-ray diffraction                                                                       goniometer circle                                       ideal sample                                       position                                                          actual sample                                                            position                                                                                     opening                                      detector                                                               focused                                                               beam                                                  shift                  Figure 4.25. Scheme of an instrumental error of positioning the sample when using                                          Bragg-Brentano geometry.                                       (Source: http://www.xray.cz/kurs/debye.htm.)                                                                            99","4 X-ray diffraction                 4.4.2.3  Standard results of powder diffraction and their presentation                 A pattern of X-ray powder diffraction, shown in Fig. 4.26, is reported in a graphical                form  with  relative  intensities  (intensity  of  the  maximum  line  is  100  cps  and  the                other lines are calculated accordingly). Another addition to a graphical output is a                table of peak positions (2 and their relative intensities (Irel), value of d-spacing,                                         )                and the full width at half maximum (FWHM), see Table 4.7. In case of a control                pattern measurement, it is compared with a standard, and this comparison is only                visual (number of peaks and peak positions), with no available algorithm, such as                for FT-IR data comparison. Calibration curve and deviation are presented in case of                quantification. The measurement method is also reported as it has great influence on                the results, and the information is insufficient without the measurement parameters                 (wavelength, measurement geometry).             Counts                      Atomoxetin_1                 10000                             2500                                0                                10                 20                 30                                                 Position [°2Theta]                        Figure 4.26. X-ray powder pattern of a studied API. Diffractogram                       was measured using X´PERT PRO MPD PANalytical diffractometer                    under the following experimental conditions: radiation: CuKα (λ = 1.542 Å);                    monochromator: graphite; excitation voltage: 45 kV; anode current: 40 mA;                 measured range: 4–40˚ 2θ; step size: 0.008˚ 2θ; flat sample of area/thickness 10/0.5                                                    mm.                                                        100","4 X-ray diffraction                     Table 20.7. Values of peak positions (diffraction angles) 2θ, d-spacings, and                          relative intensities of characteristic peaks of a crystal API form.                                             2θ [°]     d [Å]     Irel                                           7.9229   11.14991   11.05                                          11.5267    7.67078    100                                          12.2892    7.19649   20.17                                          14.4072    6.14294   17.46                                          15.0082    5.8983    16.91                                           17.208    5.14891   49.72                                            19.841    4.47116   15.47                                          19.9785    4.4407    18.84                                          21.3222    4.1638    67.82                                          21.5503    4.12023   18.58                                          22.5418    3.9412    31.22                                           23.0286    3.85898   14.15                                           23.649    3.75912   46.14                                          24.6039    3.61534   12.33                                           24.726    3.59777   21.29                                          27.5295    3.23743   21.7                                          28.5065    3.12864   17.37                                          28.7475    3.10296   8.56                                           31.2385    2.86098   7.06                                                       101","4 X-ray diffraction                 4.5  Applications of X-ray diffraction in pharmacy                 Both single crystal and powder X-ray diffraction are widely used in pharmaceutical                research, development, manufacture, and control. For example, powder diffraction                is  used  to  monitor  the  reproducibility  of  production  batches  or  to  identify  and                determine  unknown  phases.  Single  crystal  diffraction  is  used,  for  example,  for                structural characterization of impurities or for absolute structure determination. In                addition,  data  obtained  from  both  methods  are  used  in  pharmaceutical                documentation, patent litigation, etc.                   4.5.1  Characterization and identification of unknown samples                 After  an  X-ray  pattern  measurement,  it  is  possible  to  immediately  determine                whether  the  substance  is  crystalline  or  amorphous.  In  the  case  of  a  crystalline                substance, a database (or specifications, literature, DMF [drug master file]) can be                used to compare results with reference data and identify the studied phase. With X-                ray powder pattern measurement and extraction of positions of individual peaks, a                set  of  2;  Irel  is  obtained,  and  this  set  of  data  is  then  compared  with  available                standard libraries (Fig. 4.27) and literature patents (Fig. 4.28). If a pattern library is                available, the peak positions can be assigned, and the crystal phase identified.                 When  searching  for  new  polymorphic  forms,  results  of  a  set  of  crystallization                experiments are considered, and similar patterns are looked up to create groups of                particular polymorphs. Cluster analysis proved useful for comparison of diffraction                patterns, see Fig. 4.29.                                                                                 102","4 X-ray diffraction             Counts                      Program1_181                 6400                                                                                                                              Podle PDF – 4 databáze:                  3600                                                                                                                            Finasterid                    1600                                                                                                                            –  ortorhombický  polymof I.                                                                                                                            –  (CSD reference code:                                                                                                                                 WOLXOK02)                  400                        0                                10                 20                 30                                                  Position [°2Theta]                  Figure 4.27. Identification of a crystalline phase using a library of X-ray patterns                   (database PDF-4): Finasterid, ortorhombic polymorph I, (CSD reference code:                                               WOLXOK02).                                     (Source: http://www.ccdc.cam.ac.uk/products/csd/.)                                                                                  103","4 X-ray diffraction                                           Intensity (counts)                       6400                       3600                       1600                       400                        0                           5       10        15       20       25       30       35                                                                                       2Theta (°)                         Figure 4.28. Identification of a polymorph according to a patent.                                         (Source: U. S. patent 2006/0148868A1.)                                                                104","4 X-ray diffraction                  Counts   Rimonabant_4 JC-RB59-sraz                            Forma NEW                                Counts   Rimonabant_8 JC-RB42-2K                             NEW form                  1000                                                                                  Forma I.                                                                                  form I.                                                                      2000                  500                                                                      1000                  0                       10   20   30                           Position [°2Theta] (Copper (Cu))                                                                      0                                                                           10   20   30                                                                               Position [°2Theta] (Copper (Cu))                                                                      Counts                 Counts                                                 Rimonabant_07_19 JC-RB103                    Rimonabant_10 JC-RB80-Me V                                                                      4000                                                                                  form II.                      amorphous form                     Amorfni forma                                                Forma II.                  400                                                 3000                                                                      2000                  200                                                                      1000                                                                      0                  0                                                       10   20   30                       10   20   30                                                                               Position [°2Theta] (Copper (Cu))                           Position [°2Theta] (Copper (Cu))                            Figure 4.29. Polymorph screening using cluster analysis.                 4.5.2  Crystalline phase control                X-ray powder diffraction is suitable also for routine control of the reproducibility of                production batches, with the possibility to control presence of unwanted crystalline                impurities  (unwanted  polymorphs),  see  Fig.  4.30  and  10.31.  For  this  type  of                experiments,  it  is  needed  to  find  a  suitable  window  in  the  diffractogram  where                peaks  of  one  of  the  studied  forms  always  occur.  The  particular  polymorph                modification can then be checked the same way, even after processing into a solid                dosage form, see Fig. 4.32.                                                                           105","4 X-ray diffraction                  Counts    Forma I bez ClopidoS_49                        6400                                                                      polymorf I                                                                       polymorph I                      3600                      1600                        400                         0   Forma II ClopidoS_48                                                                        polymorf II                                                                       polymorph II                      3600                      1600                        400                         0                                   10              20               30                                                  Position [°2Theta]                   Figure 4.30. X-ray powder patterns for two polymorphs of clopidogrel hydrogen                                                  sulphate.                                                      Peak 12.87  2θ                                                      Pík 12.87 2θ                 Counts                            Forma I ClopidoS_suma 68 69 70 71 72                            3% II ClopidoS_56_validace                            5% II ClopidoS_55_validace                            10% II ClopidoS_54_validace                            30% II ClopidoS_53_validace                            50% II ClopidoS_52_validace                            Forma II ClopidoS_48           12.89                     3600                                                                   13.6                                                       12.26   13.01                                  8.77    9.62                     1600                                               10.56                       400                           0                                           10                             15                                                   Position [°2Theta]                  Figure 4.31. Determination of the limit of detection for clopidogrel polymorph II                                          contained in polymorph I.                                                      106","4 X-ray diffraction                 Counts    ClopidoS_31                       22500                                                                             placebo                     10000                      2500                        0   ClopidoS_24                     10000                                                   tableta                        2500                        0                      6400   Forma I ClopidoS                      3600                                                   API                      1600                      400                       0                                  10               20               30                                                  Position [°2Theta]                   Figure 4.32. Control of crystalline clopidogrel after manufacturing into a solid                                                dosage form.                   4.5.3  Negative proof of crystalline phase – amorphous material                 To  obtain  a  diffraction  pattern,  the  studied  material  must  show  a  periodic                arrangement of molecules in its structure. If the periodic arrangement is missing or                present  only  in  short  distance,  the  X-ray  powder  pattern  will  show  only  wide                \"humps\", see Fig. 4.33. As shown in Fig. 4.34 and 10.35, crystalline or amorphous                phase can also be detected in a mixture.                  Counts   ATV_amorphous            Counts                    6400                                ATV_form I                                                      900                                                      400                    3600                                                      100                    1600                              0   ATV_form VIII                                                      900                    400                               400                                                      100                     0                                0                           10       20      30              10       20       30                                   Position [°2Theta] (Copper (Cu))  Position [°2Theta] (Copper (Cu))                Figure 4.33. Examples of X-ray patterns of an amorphous and a crystalline phase of                                                  an API.                                                     107","4 X-ray diffraction               Counts   ATV_validace53              Counts   ATV_validace_42                                                amorfní                   3600                        amorph     ATV_validace_43                                                          ATV_validace_43                 1600                                                          ATV_validace_46                 400                                 6400   ATV_validace_45                  0                                       ATV_validace_47                   ATV_validace57              1% krystal                                                 crystal                 3600                 1600                 400                  0   ATV_validace56                                                 crystal                 3600                          3% krystal                 1600                                4900                 400                  0                   ATV_validace55                                                 crystal                 3600                          5% krystal                 1600                 400                  0   ATV_validace54                                                 crystal                 3600                         10% krystal   3600                 1600                 400                  0                   ATV_validace58                                              30% krystal                                                 crystal                                                            0.5 – 5% crystal API in amorphous API                 2500                                     0.5 – 5% krystalické v amorfní API                  0                                                  crystal                10000   ATV_validace59         50% krystal   2500                 2500                  0                        10        20       30               10        15        20                                 Position [°2Theta]                   Position [°2Theta]                   Figure 4.34. Detection of a residual crystalline phase in an amorphous sample.                   Intensity (counts)                                            Counts                  22500                        4000   Solifen_08_112 krystal 0010108                                                    Solifen_08_107 95+5 1+2       100 % crystal                                                    Solifen_08_108 90+10 1+2                                                    Solifen_08_109 75+25 1+2       95 % crystal                                                    Solifen_08_110 60+40 1+2                                                    Solifen_08_111 50+50 1+2                                                    Solifen_08_113 amorf 23-114    90 % crystal                  10000                                                                                   75 % crystal                                               3000                                                                                   60 % crystal                                                                                   50 % crystal                  2500                                                                                   amorphous                  0                     5  10  15  20  25  30  35                                          2Theta (°)  2000                 Intensity (counts)                  900                                               1000                  400                  100                                                0                                                      10       15       20       25       30                                                                   Position [°2Theta] (Copper (Cu))                   0                    5   10  15  20  25  30  35                                          2Theta (°)                  Figure 4.35. Detection of an amorphous phase mixed with the crystalline phase.                 4.5.4  Observation of phase transformations                Changes  in  crystallization  parameters  of  an  API  can  produce  its  different                polymorph,  solvate,  or  an  amorph.  Similarly,  stability  testing  (Fig.  4.36)  or                processing of an API into a formulation (see Fig. 4.37 and 10.38) may lead to phase                transitions mentioned above,  i.e.,  changes  in  the molecular  packing.  These  phase                changes may occur due to mechanical processing (tableting, micronizing, milling),                or increased temperature, relative humidity, or combination of both.                                                    108","4 X-ray diffraction                 Counts    Losartan_1                      10000                                                                     anhydrous API                                                                      bezvodá API                     2500                         0    Losartan_25                     1600                      900                                                                       hydrát API                                                                      API hydrate                     400                      100                       0                                  10               20                30                                                  Position [°2Theta]                   Figure 4.36. Change in a crystal form of an API – formation of a hydrate during                   stability testing. After three-months, under 40 °C/75% RH in PE package, the                                  anhydrous API recrystallized into a hydrate.                Counts                     1500                            ClopidoS_07_17+18 0010307 2 min in ball mill                            ClopidoS_07_22 0010307 mlety 2min kulovymlyn                                       from ID disc                            ClopidoS_07_21 0010307 z ID disku                        1000                            500                             0                               10                20                30                                                  Position [°2Theta] (Copper (Cu))                       Figure 4.37. Amorphization of a crystalline sample caused by milling.                                                    109","4 X-ray diffraction               Counts                      10000                                            Suchá granulace                                                                       Dry granulation                                                                        Vlhká granulace                                                                       Wet granulation                                                                       Placebo                                                                       Placebo                         5000                                 0                                 10                20                30                                                   Position [°2Theta] (Copper (Cu))                    Figure 4.38. Phase conversion of a crystalline API into amorphate, caused by                                          wetting during granulation.                   4.5.5  Study of substance behavior under defined conditions                 Temperature-humidity chamber, shown in Fig. 4.39, enables monitoring of powder                sample behavior under different temperature and relative humidity. In this case, a                sample placed in this chamber is repeatedly irradiated by X-rays. The X-ray powder                monitoring of behavior of pharmaceutical substances, depending on the humidity, is                shown in Fig. 4.40.                                                                        110","4 X-ray diffraction                                                     Figure 4.39. Temperature-humidity chamber.                                        (Source: https://www.anton-paar.com/cz-cs/.)            Counts                         THC_40 forma I vstup                         THC_41 po 1h                         THC_42 po 2h                         THC_losartan po 3h 75%RH                         THC_losartan po 4h 75%RH                         THC_losartan po 5h 75%RH                 3600    THC_losartan po 6h 75%RH                         THC_losartan po 7h 75%RH                         THC_losartan po 8h 75%RH                         THC_losartan po 9h 75%RH                         THC_losartan po 10h 75%RH                         THC_losartan po 11h 75%RH                         THC_losartan po 12h 75%RH                         THC_losartan po 13h 75%RH                         THC_losartan po 14h 75%RH                         THC_losartan po 15h 75%RH                         THC_losartan po 16h 75%RH                         THC_losartan po 17h 75%RH                 1600    THC_losartan po 18h 75%RH                         THC_losartan po 19h 75%RH                         THC_losartan po 20h 75%RH                  400                          0                           5                              10                             15                                                 Position [°2Theta]           Counts                     THC_ClopidoHBr po 1h 75%RH                      Figure 4.40. Formation of hydrates in a temperature-humidity chamber.                 900                                                                                  1h                 400                 100                  0                     THC_ClopidoHBr po 4h 75%RH                 900                                                                                  4h                 400                 100                                111                   0   THC_ClopidoHBr po 8h 75%RH                 900                                                                                  8h                 400                 100                  0                     THC_ClopidoHBr po 12h 75%RH                 900                                                                                  12h                 400                 100                   0                               10                 20                  30                 40                                                 Position [°2Theta]","4 X-ray diffraction                 4.5.6  Determination of structures of unknown impurities or molecule chirality                 For  each  API,  it  is  necessary  to  characterize  the  impurity  profile  and  determine                impurity structures. Each impurity must be separately crystallized, and the resulting                single  crystal  subjected  to  a  structural  analysis.  In  most  cases,  the  single  crystal                diffraction analysis is able to determine the absolute structure, i.e., the configuration                of the chiral centers in the molecule (if present), see Fig. 4.41.                                              Figure 4.41. Molecular structure of an API with a determined configuration of the                    chiral center at the carbon atom, obtained by single-crystal X-ray diffraction.                   4.5.7  Calculation of powder diffraction patterns                 From  the  experimentally  determined  crystal  structure  (lattice  parameters,  space                group of symmetry, atomic coordinates), the so-called calculated theoretical powder                diffraction pattern of the substance can be calculated. Such a pattern is thus free                from  all  instrument  and  sample  influences  and  errors  (peak  asymmetry,  peak                extensions, preference orientation, etc.). The calculated theoretical powder pattern is                ideal  for  setting  of  a  substance  standard  (reference) X-ray  pattern,  see  Fig.  4.42.                Single-crystal  structure  can  therefore  serve  as  a  model  for  calculation  of  a                theoretical powder pattern – the optimal reference standard.                                                          112","4 X-ray diffraction                                         Figure 4.42. A calculated theoretical powder pattern of a known crystal structure,                                     determined from a single crystal data.                   4.5.8  Determination of a 3D crystal structure from X-ray powder data                 If a suitable single crystal for SCXRD cannot be crystallized, a powder sample with                a grain size of about 10 –10  m must be used. In this case, it is recommended to                                      -3                                          -5                solve the crystal structure from the powder diffraction data. The powder data are                measured on an advanced laboratory powder diffractometer or on a synchrotron.                However, this method has its limitations and is experimentally and computationally                more demanding than SCXRD. As already mentioned in Table 4.4b, a maximum of                about  100  diffraction  intensities  can  be  extracted  from  the  powder  diffraction                pattern. Unfortunately, these are biased because of their overlap and distorted by the                preferred  orientation.  To  get  the  undistorted  intensities,  they  must  be                mathematically corrected and refined (Le Bail refinement). However, the number of                intensities available may be lower than the number of refined structure parameters.                Therefore,  additional  structure  information  is  obtained  by  other  methods,  e.g.,                ssNMR,  theoretical  calculations,  etc.  For  example,  the  molecule  shape  can  be                optimized by quantum-chemical calculations and the position of the molecule (or                only part of it) is specified in the asymmetric part of the elementary cell as a single                object  (3  positional  parameters  and  3  angular  rotation  parameters),  without                specifying the positions of individual atoms, see Figure 4.43.                                                             113","4 X-ray diffraction                                           Figure 4.43. Refinement of the whole molecule position in the unit cell. DOF                                                parameters.                  In addition, only molecules that do not contain many internal rotation parameters                can be resolved (1…). These parameters are called DOF (degree of freedom), and                currently the limit to resolve structures from powder data is roughly DOF < 40.                  As  a  result,  refinement  of  the  structure  from  the  powder  provides  similar                verification parameters as for single crystal (R-factors). However, these are higher                for the powder than for the single crystal, and the powder structure model needs to                be  assessed  based  on  chemical  reality,  especially  intermolecular  bonding  (H-                bridges, etc.). An example of a solved structure from powder data is illustrated in                Fig. 4.44.                                     Figure 4.44. Structure of fluticasone solved from single crystal (left) and from                 powder (right). The differences are in the orientation of the terminal methyl groups.                                 (Source: The picture was kindly provided by Mr. Michal Hušák.)                                                    114","4 X-ray diffraction                   4.5.9  Quantitative phase analysis                 Quantitative phase analysis is currently performed using the Rietveld method. Its                principle is to calculate the theoretical powder patterns for all phases present in the                mixture.  Their  superposition  is  fitted  to  the  experimental  powder  profile  of  the                mixture  until  a  match  is  achieved.  The  least  squares  method  is  used  in  this                refinement.  The  theoretical  fitted  profile  then  gives  the  percentage  of  each                  component in the mixture (Fig. 4.45 and 10.46).                              QPA - crystalline/ amorphous solifenacin tartrate                250                                         1400                                              y = 126.87x - 3.1059  Counts  3000   Solifen_08_106 100408 exp 5mg 1+2                                                                  Solifen_08_125+126 5mg 100408 placebo jadra                                                2                                                                  Solifen_08_135+142 vz16 100408 75_25                       peak area               R  = 0.9962  1200   Solifen_08_134+141 vz15 100408 90_10                                                                  Solifen_08_136+143 vz17 100408 60_40                200                                               Solifen_08_130+144 vz18 100408 50_50                                                                  Solifen_08_78+79 5mg 100408                       peak intensity                       1000                       Lineární (peak intensity)                 intensity [cps]                   R  = 0.9944  2000                150    Lineární (peak area)       y = 23.2x - 7.8025  800                                                    2                100                                         600                                                            400                 50                                                            200                                                              1000                                                                17    17.50  18    18.50  19                                                                           Position [°2Theta] (Copper (Cu))                 0                                          0                  0.00  1.00  2.00  3.00  4.00  5.00  6.00  7.00  8.00  9.00  10.00                                   amount of crystal API [mg]                    Figure 4.45. Quantitative determination of components in a mixture using a                                              calibration curve.                                                                               115","4 X-ray diffraction                                                    Figure 4.46. Quantitative determination of components in a mixture, using Rietveld                                      method in HighScore Plus software.                                      (Source: https://www.malvernpanalytical.com.)                   4.6  Summary                X-ray diffraction is a unique analytical method for studying of inner structure of                crystalline solids. X-ray powder diffraction is used mainly for quick resolution of                various  solid  forms  (polymorphs,  salts,  bases,  acids,  hydrates,  solvates)  and  is                rightly  recognized  as  the  gold  standard.  In  its  applications,  it  is  a  very  powerful                method,  but  its  use  is  not  simple  and  requires  a  certain  detailed  knowledge  of                crystallography  and  at  least  a  basic  understanding  of  elementary  principles  and                limitations for divergent X-ray beams. For advanced users, special optics come into                consideration, such as mirror optics for generating of parallel X-ray beams, as the                most important one. The X-ray diffraction technique cannot be in any case used as a                \"black box\". An obtained diffractogram reflects the molecular packing in a crystal,                and  therefore  the  individual  peaks  cannot  be  assigned  to  specific  parts  of  the                molecule, as in spectroscopy. Modern trends in analytical diffraction techniques are                represented by electron diffraction and the further development of structure solving                from powder data.                                                     116","4 X-ray diffraction                 This chapter presents the principles of X-ray diffraction and its application mainly                for  pharmaceutical  analysis.  Mathematical  basics,  hidden  under  the  applications                themselves, are almost not mentioned in the text. There are, however, many high-                quality literature sources listed, from worldwide renowned authors and \"fathers\" of                crystallography, powder diffraction, and single-crystal diffraction.                   4.7  Resources and recommended literature                   1.   Palatinus L., Kratochvíl B.: Electron diffraction  – a new toll for crystal structure                        solutions. Chem. Listy. 2021, 115, 368-374.                   2.   Siegrist T. X-Ray Structure Analysis. Gruyter, Walter de GmbH: Berlin, Germany,                       2021. https://doi.org/10.1515/9783110610833                   3.   Kraus I., Fiala J. Krystalografie. Nakladatelství ČVUT: Prague, Czech Republic,                       2021.                   4.   Giaccovazzo C., Monaco H. L., Viterbo D., Scordari F., Gilli G., Zanotti G., Catti                       M. Fundamentals of Crystallography. The second edition. Oxford University Press:                       New York, USA, 2002.                  5.   Kratochvíl  B.  Základy  fyziky  a  chemie  pevných  látek  I. VŠCHT  Praha:  Prague,                       Czech Republic, 1994.                   6.   Polymorphism  in  the  pharmaceutical  industry,  Hilfiker  R.  (Ed.).  Wiley-VCH:                       Weinheim, Germany, 2006.                   7.   Byrn S.R., Pfeiffer R.R., Stowell J.G. Solid state chemistry of drugs. The second                       edition. SSCI, Inc: West Lafayette, USA 1999.                  8.   The  Rietveld  Metod,  Young  R.A.  (Ed.).  Oxford  University  Press:  Oxford,  UK,                       1993.                   9.   Jenkins R., Snyder R. L. Introduction to X-Ray Powder Diffractometry. Chemical                       Analysis.    Wiley    Interscience:   New    York,    USA,     1996.                       https://doi.org/10.1002/9781118520994                  10.   Bruno J., Cole J. C., Edgington P. R., Kessler M. K., Macrae C. F., McCabe P.,                       Pearson  J.,  Taylor  R.  New  Software  for  Searching  the  Cambridge  Structural                       Database and Visualising Crystal Structures. I. Acta Crystallogr. 2002, B58, 389–                       397. https://doi.org/10.1107/S0108768102003324                   11.   Hovmöller S., Abrahams J. P Gemmi M., Mugnaioli E., Gorelik T. E., Kolb U.,                       Palatinus L., Boullay P. 3D Electron Diffraction: Nanocrystallography Revolution.                       ACS Cent. Sci. 2019, 5, 1315-1329. https://doi.org/10.1021/acscentsci.9b00394                                                       117","4 X-ray diffraction                   12.   Kratochvíl  B.,  Hušák  M.,  Korotkova  E.  I.,  Jegorov  A.  The  importance  of  X-ray                       crystal structure determination for pharmacy. Chem. Listy. 2016, 110, 40-47.                  13.   Solid  State  Characterization  of  Pharmaceuticals.  Zakrzewski  A.,  Zakrzewski  M.                       (Eds.); Assa: Danbury, USA, 2006.                   14.   Newman  A.  W.,  Byrn  S.  R.  Solid  State  Analysis  of  the  Active  Pharmaceutical                       Ingredient  in  Drug  Products.  Drug  Discov.  Today.  2003,  8  (19),  898–905.                       https://doi.org/10.1016/S1359-6446(03)02832-0                   15.   Macrae C. F., Edgington P. R., McCabe P., Pidcock E., Shields G. P., Taylor R.,                       Towler  M.,  van  de  Streek  J.  Merkury:  Visualization  and  Analysis  of  Crystal                       Structures.   J.   Appl.    Cryst.    2006,   39     (3),   453–457.                       https://doi.org/10.1107/S002188980600731X                   16.   Petříčková  H.  Structural  study  of  morphine  alkaloids  and  their  complexes.                       Dissertation. UCT Prague: Prague, Czech Republic, 2007.                   17.   Bruker  company.  https://www.bruker.com/en/landingpages/baxs/x-ray-structural-                       and-elemental-analysis.html.                   18.   The     Cambridge     Crystallographic   Data    Centre    (CCDC).                       https://www.ccdc.cam.ac.uk/solutions/csd-core/components/csd/.                   19.   U.S. Food and Drug Administration. https://www.fda.gov/.                   20.   The  International  Council  for  Harmonisation  of  Technical  Requirements  for                       Pharmaceuticals for Human Use, https://www.ich.org/.                   21.   European Medicines Agency. http://www.ema.europa.eu.                   22.   Státní ústav pro kontrolu léčiv. www.sukl.cz.                                                                             118","5 NMR spectroscopy                 5  NMR spectroscopy                 Jaroslav Havlíček                   5.1  Basic principles                 NMR spectroscopy (nuclear magnetic resonance) is a method, where energy state of                nuclear  spins  is  changed  in  a  strong  magnetic  field,  after  absorption  of                radiofrequency radiation.                 The basic building blocks of atomic nuclei – protons and neutrons, rotate around                their axes and thus have a momentum p, called spin. Protons and neutrons have a                spin  quantum  number  I  =  1/2.  Nuclei  of  isotopes  with  an  even  number  of  both                protons and neutrons have paired particle spins, so the resulting nuclear spin I = 0.                                12                                    16                Such nuclei, e.g.,  C,  O, have zero spin, and do not provide NMR signals. Nuclei                with  an  odd  number  of  protons, neutrons,  or both  types  of particles  do  not  have                paired spins and their I > 0. Because the movement of electrically charged particles                on a closed path, is associated with emergence of magnetic field, the nuclei with I >                       1                0 (e.g.,  H,  C,  O) have their own magnetic moments:                          13                              17                          =  p       – gyromagnetic ratio, characterising each isotope                If a sample containing an isotope with a non-zero magnetic moment is placed into a                strong magnetic field with induction B0, the energy levels of its nuclear spins are                split (Fig. 5.1).                 The potential energy of a nucleus E is given by:                                                   E = –B0                 and for a component of magnetic moment in the direction of field B0 applies:                                   mh               m – magnetic quantum number                               =                                  2               h – Planck constant                 Magnetic  quantum  number  acquires  values  m  = I,  I–1,  –I+1,  –I.  The number of                created energy levels is defined by the value of spin quantum number I (number of                                                                      1                                                                             13                states = 2I+1), therefore in isotopes with I = 1/2, such as  H and  C, the nuclear                spins acquire two energy states with magnetic quantum number m = + ½ and m = +                ½. Their energy can then be calculated by the relationship:                                  1 ℎ   0                                    1 ℎ   0                              1 = − (     )                               1 = + (    )                          +      2  2                                −      2  2                             2                                          2                                                     119","5 NMR spectroscopy                                                          Figure 5.1. Nuclear magnetization M0 in equilibrium. Magnetic moments that are                 directed according to the outer magnetic field B0 belong to spins in +1/2 state, and                             oppositely directed moments belong to spins in 1/2 state.                 According to the laws of quantum mechanics, each spin can permanently  occupy                only  one  energy  level,  and  so  it  can  only  have  one  of  two  possible  orientations.                Transitions between these levels can be caused by radiation whose frequency meets                the condition:                      E =  E − E  = h         E – energy difference between levels                          − 1  + 1                           2    2                Using the previously mentioned relationships, the following can be stated:                                                      E  B                                                =     =   0                                                0                                                    h      2                                                           120","5 NMR spectroscopy                 This relationship is known as the resonance condition, and the frequency 0 is a so-                called Larmor frequency, often stated in the form:                               = B0                             – angular speed                 Since  the  difference  E  between  the  energy  levels  is  small,  energy  of  radio-                frequency radiation (frequency in orders of MHz) is enough to excite these nuclei.                 Lower energy level is occupied by nuclei whose projection of a nuclear magnetic                moment    matches the orientation of an external magnetic field B0. This orientation                corresponds to the magnetic quantum number m = +1/2. Nuclei at a higher energy                level have the resulting magnetic moment oriented oppositely to the direction of the                magnetic field m = –1/2 (Fig. 5.1).                 Comment.  At  an  equilibrium  state,  temperature  T  =  300  K,  and  magnetic  field                induction B = 1T, the ratio of the number of proton spins at an energy level matches                the Boltzmann distribution law:                                                                   1                                                                          ∆                                1                                                   1                                                                 +                     1 (   = + )    and       1 (   = − ),    hence:      2  = exp ( ) = 1.000006                    +         2          −         2              1                                2                    2                      −                                                                 2                                           k – Boltzmann constant                Population of nuclei at a lower energy level is therefore only slightly larger than at                the  higher  energy  level.  The  result  of  this  difference  is  a  vector  sum  of  all  the                nuclear  spin  magnetic  moments  ‒  nuclear  magnetization  M0.  Its  value  is                proportional to the excess of nuclei at the lower energy level.                When measuring NMR spectra using pulse methods, the population of both energy                levels is equalized by a suitable radiofrequency pulse, and the magnetization M0 is                deflected from its equilibrium position (from the direction of the axis of the external                magnetic  field  B0).  Since  the  magnetization  is  associated  with  the  momentum  of                nuclei, it does not return to the direction of the magnetic field immediately after the                termination of the pulse but behaves as a flywheel mounted outside its centre of                gravity whose rotation axis is tilted towards the gravitational field. The result is a                precession in which the magnetization retains its intensity and an angle towards the                magnetic field and rotates around the direction of the magnetic field B0 with Larmor                frequency (Fig. 5.2).                 The relaxation processes also gradually restore the disrupted equilibrium. After a                certain time characterized by the period of the so-called spin-lattice relaxation T1,                the equilibrium distribution of spins on both energy levels is once again stabilized.                This  restores  the  magnetization  component  in  the  direction  of  the  field.  A                magnetization component perpendicular to the direction of the magnetic field on the                                                      121","5 NMR spectroscopy                 other hand decreases down to zero during the so-called spin-spin relaxation interval                T2.                 Similarly,  as  a  rotating  magnet  induces  an  alternating  current  in  a  stator  coil,                rotating magnetization vector induces AC voltage in a receiver coil that is wound                around a sample in an NMR spectrometer. The time course of the voltage (signal)                induced by the nuclear magnetization in the coil is commonly referred to as FID                (free induction decay) and is shown in Fig. 5.3. It can be thought of as a fading tone                after pressing a piano key.                                                       Figure 5.2. Return of magnetization M into an equilibrium position, after being                                       tilted by a radiofrequency pulse.                                                                      122","5 NMR spectroscopy                       a                                  b                                       Figure 5.3. FID – dependence of signal s and time t for: one (a)   and two (b) types                        of nuclei with one or two signals in the spectrum, correspondingly.                   5.2  NMR spectrometer                 A  modern  NMR  spectrometer  with  Fourier  transform  (FT-NMR)  is  a  very                complicated device that has three basic parts:                    •  magnet                    •  electronic part of the spectrometer                    •  computer system with peripherals                 The main part of each NMR spectrometer is a magnet with a probe. Electromagnets                with iron core were used earlier. Currently, there are used superconducting magnets                allowing to reach higher magnetic induction (4.7 to 28.2 T, which corresponds to a                                    1                working frequency of  H 200–1200 MHz). Superconducting magnet is formed by a                solenoid coil made of superconducting material (e.g., NbTi or NbSn alloy) which is                placed  in  a  cryostat  with  liquid  helium  that  provides  cooling  of  the  solenoid  to                temperature of 2–4 K. Sample of a measured substance in a glass tube is placed into                the  so-called  probe  (Fig.  5.4b).  The  sample  tube  rotates  in  the  probe  to  increase                homogeneity. The main parts of the probe are coils and electrical circuits used to                supply energy to the sample using a radio-frequency pulse from a transmitter and to                detect  absorption  of  this  energy  in  receiver  circuits  (FID  scan).  Furthermore, the                probe  also  contains  circuits  used  for  irradiation  of  a  sample  by  another                radiofrequency radiation used for decoupling (see below).                                                     123","5 NMR spectroscopy                 The  electronics  of  the  spectrometer  provide  excitation  of  the  spin  system  and  a                subsequent detection of the signal from the sample. An NMR spectrometer usually                contains three independent high-frequency channels:                     •  observation (measuring) – for nuclei excitation and response detection                   •  locking (stabilization) – for resonance condition stabilization, usually using a                      resonance signal of deuterium from deuterated solvents                    •  decoupling  –  for  sample  irradiation  with  another  high-frequency  radiation                      (the so-called decoupling method)                 Computational  system  provides  control  of  all  spectrometer  functions  and  the                processing  of  acquired  data.  The  experiment  itself  is  performed  by  a  pulse                programmer  or  a  separate  acquisition  processor.  The  computer  controls  the                spectrometer,  operation  of  the  pulse  programmer  or  the  acquisition  processor,                mathematical processing of measured data (FIDs) and their Fourier transform and                provides  output  of  the  acquired  spectra  (along  with  necessary  parameters)  onto                screen and printer (Fig. 5.4).                                                                  Figure 5.4a. Scheme of a superconducting magnet.                                                         124","5 NMR spectroscopy                                           Figure 5.4b. Photo of measuring probes and NMR sample tubes for liquid state                          analysis (from the left: 5 mm, 1 mm and 10 mm sample tube).                If  a sample contains  only one type  of  atoms  of  one isotope,  for example  CHCl3                (Fig.  5.3a), the  decrease  of  amplitude  of  muted  oscillations  is exponential  and is                characterized  by  the  duration  of  the  spin-spin  relaxation  T2.  The  oscillation                frequency is the Larmor frequency. For more complex samples, the resulting FID is                an interferogram or a superposition of FIDs originating from different nuclei of the                same  isotope  (Fig.  5.3b).  To  obtain  the  resonant  frequencies  and  an  intensity  of                signal response for individual nucleus types, it is necessary to first convert the FID                from signal time dependence to a frequency dependence, i.e., the NMR spectrum.                This conversion is done by a computer using Fourier transform.                 The  NMR  spectrum  then  contains  information  about  the  structure  of  substances,                stored in three parameters:                    •  position of signals (chemical shifts)                    •  integral intensity of signals                    •  multiplicity (fine structure) of signals                                                                  125","5 NMR spectroscopy                      1                5.3   H NMR spectroscopy                5.3.1  Signal position – chemical shift                 Relationship between frequency of absorbed radiation and magnetic induction B0 is                expressed in the resonance condition:                                                      E  B                                                =     =   0                                                0                                                    h      2                According to this condition, resonant frequency should be constant for all nuclei of                the same isotope. However, the nuclei in the molecule are bound into groups and                are  therefore  influenced  by  different  electron  environments.  The  electron                surrounding alters (\"shields\") the magnetic field at the nucleus, and the following                relationship applies for magnetic induction Blok:                           Blok = B0(1 – )                     – shielding constant                 Determination of the shielding constant is experimentally demanding and therefore                relative values are used in practice for resonant frequency, related to a frequency                standard. As an internal standard, tetramethylsilane (TMS) is usually directly added                to  the  sample.  Distance  length  of  resonance  signals  from  the  TMS  signal  is                measured in Hz. Because this value is dependent on B0, a chemical shift  [ppm]                was defined as a quantity independent on magnetic field induction:                                      – frequency of measured nucleus                         −                      =      st 10 6                           0       st – frequency of measured standard                                     o – working frequency of spectrometer for the given isotope                 Chemical shift is expressed in ppm (parts per million). The value of a chemical shift                of TMS, according to the following relationship, is equal to 0 ppm.                                     1 ℎ   0                             1 ℎ   0                                  1 = − (    )                        1 = + (    )                             +      2  2                         −      2  2                                2                                   2                The range of chemical shifts in hydrogen  H NMR spectra is usually 0–20 ppm.                                                     1                Table  7.1  lists  chemical  shifts  of  protons,  according  to  which  signals  can  be                assigned to specific structural groups. If protons are bound identically, and therefore                have the same \"surrounding\", they show identical resonant frequencies. Such nuclei                are then called magnetically equivalent and are always chemically equivalent (but                not vice versa). For example, in the benzene ring of cyclohexane, all protons are                                                                    1                chemically  equivalent  due  to  the  symmetry,  and  in  a  H  NMR  spectrum  they                                                     126","5 NMR spectroscopy                 provide  only  one  signal.  Similarly,  in  the  –CH3  group,  all  three  protons  are                equivalent due to the free rotation around the single bond, and they will match only                a single signal in the spectrum. Also, the compounds tetramethylsilane Si(CH3)4 and                symmetrically substituted ethane X–CH2–CH2–X (with free rotation around single                                                                          1                bond and also molecule symmetry) provide only one signal in a  H NMR spectrum,                corresponding to twelve and four equivalent protons in the case of tetramethylsilane                and ethane, respectively.                 Chemical  shift  is  influenced  by  many  factors  (electronegativity  of  substituents,                magnetic  anisotropy,  steric  effects,  temperature,  concentration,  solvent,  etc.).  The                most important of these is the electronegativity of substituents. A rule applies that                with  increasing  electronegativity  of  neighbouring  atoms  or  groups,  shielding                decreases and the value of chemical shift  increases. Temperature, concentration,                and solvent have the highest effect on the value  of the chemical shift of protons                bound to heteroatoms (–OH, –NH–, –SH).                                 1                Fig. 5.5 shows a  H NMR spectrum of ethyl formate. The spectrum contains three                signals  (1.3  ppm,  4.2  ppm,  and  8.0  ppm)  which  correspond  to  three  groups  of                protons contained in the ethyl formate, i.e., CH3–, –CH2– and H–CO–. They differ                in their chemical shifts which are consistent with the values given in Table. 4.1. A                signal with chemical shift of 7.26 ppm, which is also contained in the spectrum,                corresponds to a residual signal of CHCl3 in CDCl3, which was used as a solvent in                this                                                                  case.                Comment. Solvents used for NMR are not 100% deuterated and thus signals of non-                                                    1                deuterated solvents always appear in the  H NMR spectra.                                                                                  127","5 NMR spectroscopy                                             1                 Table 5.1. Chemical shifts of  H protons (values are related to TMS, concentration                             or temperature dependent shifts are marked with cross).                                                                                                                        128","5 NMR spectroscopy                 5.3.2  Integral signal intensity                    1                In  H NMR spectra, the  areas of resonance signals are proportional to the number                of protons in the molecule. For this purpose, an integral record is acquired, which                has  a  shape  of  a  wave  placed  over  the  individual  signals.  Height  of  the  integral                wave corresponds to the areas of individual signals, and the values of these areas are                usually  found  in  the  spectrum,  below  the  scale  of  chemical  shifts.  To  know  the                number of protons, contained in the individual signals, the ratio of the area sizes                must be adjusted to the ratio of small integers.                 For example, in the case of ethylformate (Fig. 5.5), the signal area (wave height) at                1.3 ppm must be considered equivalent to three protons. In this area, there almost                always appear only the signals of –CH3 groups. Thus, it can be easily determined,                what area size corresponds to one proton. The remaining areas can then be divided                by this value, and the resulting numbers rounded. In the spectrum of ethylformate in                Fig. 5.5, this ratio is 3:2:1 after rounding, from right to left. This implies that the                signal with the relative intensity 3 corresponds to the group –CH3, the signal with                relative  intensity  2  to  group  –CH2–,  and  the  last  signal  with  relative  intensity  1                corresponds to H–CO–.                                                                                           1                                  Figure 5.5. Ethylformate H NMR spectrum.                                                      129","5 NMR spectroscopy                 5.3.3  Signal multiplicity                    1                In  H  NMR  spectrum  of  ethylformate  (Fig.  5.5),  the  signals  differ  not  only  in                different  position  caused  by  different  chemical  shift  of  individual  groups  in  the                molecule  and  in  the  varying  intensity  given  by  the  number  of  protons  in  the                individual groups, but also in fine structure (splitting, multiplicity) of the signals.                The cause of this splitting is a mutual interaction of spins of individual protons –                spin-spin  interaction  –  that  is  transmitted  by  bond  electrons  (not  space).  It  is                therefore  most  important  usually  for  the  closest  neighbouring  groups.  Spin-spin                interaction is mutual, i.e., if a signal from A protons is split by protons matching the                signal B, then the signal of the B protons must also be split by protons matching the                signal A.                 Spin-spin interaction is characterized by the value of a spin-spin coupling constant                J,  whose  value  is  expressed  in  Hz  and  is  independent  on  induction  of  external                magnetic field. Its value is not directly shown in the spectrum but can be calculated.                The  positions  of  lines  of  multiplets  are  stated  in  Hz  above  these  multiplets.                Assuming  that  the  interpreted  spectrum  is  a  first  order  spectrum,  i.e.,  it  meets                condition   /J  >  6,  where   is  the  difference  between  resonant  frequencies  of                 interacting nuclei, the coupling constants can be calculated as the differences in the                frequencies of lines of individual multiplets. Values of some spin-spin interaction                constants are listed in Table 5.2.                 In general, simple rules apply in the first order spectra for the shape of multiplets of                protons and nuclei with spin quantum number I = 1/2:                     •  If  there  are  groups  containing  different  nuclei  with  different  coupling                      constants than the observed nucleus in their vicinity, then the signal of the                      observed nucleus is split to a maximum of (n1  + 1) × (n2  + 1) components                      (lines), where n1 and n2 are numbers of the nuclei of the neighbouring groups                      with coupling constants J1 and. J2. However, if J1 = J2, then the signal is split                      to (n +1) components, where n is the total number of nuclei of the adjacent                      group or groups with the same coupling constant, see Table 5.3.                    •  For a multiplet resulting from an interaction with a group of n magnetically                      equivalent  nuclei,  the  relative  line  intensities  have  ratios  of  binomial                                                     n                      development  coefficients  (a  +  b) ,  which  can  be  obtained  from  Pascal's                      triangle, i.e., doublet 1:1, triplet 1:2:1, quartet 1:3:3:1, etc.                 In the spectrum of ethylformate (Fig. 5.5), there can be observed two multiplets, a                triplet with a chemical shift of 1.3 ppm, and a quartet with chemical shift of 4.2                ppm.  The  triplet  corresponds  to  –CH3,  as  this  group  is  directly  adjacent  to  two                equivalent  protons  of  the  –CH2–  group.  The  quartet  corresponds  to  the  –CH2–                                                      130","5 NMR spectroscopy                 group, because the methylene group is directly adjacent to three equivalent protons                of –CH3 groups. The proton in H–CO– group has no proton on the adjacent carbon,                and hence the signal with a chemical shift of 8.0 ppm for this group is a singlet in                the spectrum of the ethylformate.                                                           1                The  value  of  spin-spin  coupling  constant  in  H  NMR  spectra  decreases  with  the                number of bonds between the interacting nuclei. Therefore, there are distinguished                                                              2                                                                       3                geminal H–C–H coupling constants, denoted with  J, vicinal  J constants (H–C–C–                                                           9                                                   4                H), and long-range coupling constants  J up to  J. It is appropriate to point out the                oddity of aromatic protons. In a substituted aromatic ring, the hydrogen atoms are                not equivalent. Depending on the nature of the substituent present, the condition for                the first order spectrum is often not met (differences in chemical shifts are often                small) and spectrometers with lower working frequency cannot distinguish between                these  hydrogen  atoms.  Therefore,  a  series  of  signals  with  fine  structures  can  be                found in the spectrum, in 6–9 ppm range. Since these signals can be mistaken for                the signals of double bonds (the ranges of chemical shifts can overlap), the relative                intensities of multiplets and the values of spin-spin interaction constants (Table 7.2)                which  are  lower  than  for  a  double  bond  should  be  considered  when  analysing  a                spectrum.                                                                                            131","5 NMR spectroscopy                    Table 7.2. The values of spin-spin interaction constant proton-proton [Hz] for                                       selected molecular arrangements.                                             Interaction constant J 1,2                                                 Maximum range        Typical range                                                                                                               132","5 NMR spectroscopy                                                                  1                           Table 7.3. A scheme of signal splitting in  H NMR spectra.                                J1          J1 = J2      J1 > J2       J1 = J2 = J3                                       doublet        triplet     doublet of      quartet                              (d)           (t)       doublet (dd)       (q)                               J1 > J2 = J3        J1 = J2 > J3       J1 > J2 > J3                                                                             doublet of dublet of                          doublet of triplet (dt)  triplet of doublet (td)                                                                    dublet (ddd)                 Comment. In case of more multiplets, the weakest side signals of a multiplet can be                overlapped by noise (e.g., for nonet, the theoretical ratio between the weakest and                the strongest signal in the multiplet is 1:70 according to Pascal's triangle).                                                                              133","5 NMR spectroscopy                 Multiplicity                                     Intensity of bands                 singlet                                                           1                 doublet                                                       1    1                 triplet                                                       1    2    1                 quartet                                                  1    3    3    1                 quintet                                               1    4    6    4    1                sextet                                              1    5   10  10   5    1                 septet                                           1    6   15  20  15   6    1                 octet                                         1    7   21  35  35  21   7    1                 nonet                                     1    8   28  56  70  56  28   8    1                        13                5.4   C NMR spectroscopy                              12                                                                                 13                Since  isotope  C  is  not  magnetically  active,  only  the  carbon  isotope  C  can  be                measured  as  its  spin  quantum  number  is  the  same  as  for  protons,  i.e.,                                    13                                                                                         1                I  =  1/2.  The  carbon  C  NMR  spectra  have  several  differences  compared  to  H                NMR.                   •  The range of chemical shifts is approximately 300 ppm. Usually, the signals                      of  carbon  atoms  from  organic  compounds  appear  in  the  interval  of  0–220                      ppm. This enhances the transparency and simplifies the interpretation of these                      spectra since overlapping of signals is not as frequent as in the case of proton                      spectra. Chemical shift of carbons as well as for protons is related to TMS                      and  is  also  affected  by  many  factors.  Its  value  depends  primarily  on  the                                                                                     3                                                                            2                      hybridization of carbon atoms and decreases in this order: sp  > sp > sp .                   •  The  greatest  chemical  shifts  are  however  those  of  carbonyl  and  often  of                      quaternary carbon atoms. The chemical shift is further affected by inductive                      effect of substituents. Substituents attracting electrons cause shift of C and                      C carbons to a lower field, i.e., to a higher chemical shift, while the effect is                      opposite in C and usually negligible in C. Chemical shift is also affected by                                                                               13                      steric,  conjugation,  and  other  effects.  Basic  overview  of  the  C  chemical                      shifts is summarized in Table 5.4.                                                       134","5 NMR spectroscopy                                          13                   •  Signal intensity A of  C nuclei depends on gyromagnetic ratio, spin quantum                      number  I  and  natural  occurrence  of  nucleus  n  (%)  according  to  the                      relationship:                                                     3                                               A =  nI(I + 1)                                                    13                As a result of the natural occurrence of  C nuclei (1.11%) and a four times smaller                                             1                gyromagnetic  ratio  than  of  a  H  nucleus,  the  relative  sensitivity  for  carbons  is                approximately  5700  times  lower  than  for  protons.  Therefore,  the   13 C  NMR                spectroscopy has become a common method only after the introduction of pulse FT-                NMR spectrometers, which enable rapid spectra accumulation. Because the signal                to noise ratio increases with square root of the number of accumulations (scans), it                is  necessary  to  perform  hundreds  or  even  several  tens  of  thousands  of                accumulations to obtain quality carbon spectra, depending on the structure of the                molecule and concentration of the sample.                 Unlike  in  proton  spectra,  the  intensities  in  C  NMR  spectra  are  not  always                                                           13                proportional  to  the  number  of  corresponding  carbon  nuclei.  This  complicates the                          13                use of the  C NMR spectroscopy for quantitative purposes and sometimes also the                interpretation of the spectra. The reason is the unequal relaxation times of various                carbon nuclei which depend on the surroundings of the given atoms. It is useful to                know that signals of quaternary carbons provide weaker signals than carbons with                                                            13                hydrogen  atoms.  The  intensity  of  signals  in  C  NMR  spectra  is  furthermore                dependent on the manner (according to the pulse sequence) of spectrum acquisition.                                                                                    135","5 NMR spectroscopy                                                  13                       Table 7.4. Chemical shifts of  C carbons (values are related to TMS).                                                                                                                         136","5 NMR spectroscopy                                                13                                           13                Unlike in proton spectra, the  C– C interaction in carbon spectra does practically                                                                           13                not appear due to the small probability of the occurrence of two  C isotope nuclei                in one molecule in proximity. The carbon spectra however show significant spin-                                                                        1                                1                                   13                spin interactions  H– C. The most important are the direct  J coupling constants                which depend on hybridization of the carbon atom and its value which changes in                                            2                           3                the order: sp  (120–150 Hz), sp (150–250 Hz) and sp (250–320 Hz). This fact eases                the assignment of signals to carbons in a molecule, but on the other hand worsens                the  transparency  of  spectra  when  there  is  little  difference  in  chemical  shifts  of                                                                                  13                specific carbons. Therefore, a method known as decoupling is used. The  C NMR                spectra  are  measured  under  a  constant  irradiation  of  all  protons  in  the  molecule.                This  eliminates  all  splitting  induced  by  spin-spin  carbon-proton  interactions.                Moreover, it increases signal intensity of carbon atoms in a spectrum because the                whole intensity is then  concentrated  into one  signal peak.  This  is  documented in                Fig.  5.6  which  shows  normal  (non-decoupled)  and  decoupled  spectrum  of  p-(2-                butyl)phenol.  Only  unsplit  signals  appear  in  the  decoupled  spectrum.  They  are                characterized by different chemical shifts and correspond to the carbon atoms found                in the p-(2-butyl)phenol molecule. In the non-decoupled spectrum, it is possible to                observe  two  quartets  matching  two  –CH3  groups,  and  a  triplet  and  a  doublet                matching –CH2– and –CH–, respectively. The signals of quaternary carbon atoms                are  not  split  by  direct  H–C  binding  interactions.  This  spectrum,  unlike  the                decoupled one, also contains an information about the type of the carbon (primary,                secondary,  tertiary,  or  quaternary),  but  the  sensitivity  of  this  experiment  is  very                small  and  there  is  a  possibility  of  signal  overlap.  APT  (attached  proton  test)  is                therefore  a  compromise  between  both  techniques,  where  one  line  corresponds  to                each carbon in the spectrum, and carbon signals are discriminated according to the                number of attached protons, odd or even by up or down oriented signals.                                                                                137","5 NMR spectroscopy                                                            Figure 5.6. Comparison of  C NMR spectra for p-(2-butyl)phenol.                                                13                  5.5  Two-dimensional NMR spectra                 1D NMR experiment is schematically shown in Fig. 5.7. After the relaxation period                which can be denoted as d1, a 90° radio-frequency pulse follows, and its response is                detected  as  FID  over  detection  time  t2.  An  NMR  spectrum  as  a  function  of                frequency 2, is obtained using Fourier transform.                                              Figure 5.7. Scheme of a 1D NMR experiment.                       (Source: Friebolin H. Basic One- and Two-Dimensional NMR Spectroscopy. Wiley-VCH, 1998.)                                                      138","5 NMR spectroscopy                 Addition of another time variable – evolution time t1, between two radiofrequency                pulses, is typical for 2D NMR experiments (Fig. 5.8).                                               Figure 5.8. Scheme of a 2D NMR experiment.                       (Source: Friebolin H. Basic One- and Two-Dimensional NMR Spectroscopy. Wiley-VCH, 1998.)                 If  the  time  delay  t1  between  the  pulses  systematically  changes,  a  dependence  on                time  variables  t1  and  t2  is  obtained  and  is  then  converted  by  double  Fourier                transform into a spectrum which is a dependency of intensity on two frequencies 1                and 2 (Fig. 5.9). The most common type is a contour plot. This view corresponds to                a section of a three-dimensional view of the peaks of a 2D NMR spectrum (stacked                plot) which has intensity as its third dimension (similarly as the 1D NMR spectrum                has intensity as the second dimension). The advantage of the contour plots is their                transparency, which simplifies interpretation.                 Methods using 2D NMR are generally divided into the so-called correlated (peaks                correlate  with  frequencies 1  and 2) and resolved  (frequencies  2 are \"resolved\"                according  to  coupling  constant  J  (J-resolved)  and  the  y-axis  shows  the  coupling                constant instead of frequency 1). Both types can then have either homonuclear or                heteronuclear form.                                                                              139","5 NMR spectroscopy                                                             Figure. 6.9. Calculation of 2D NMR spectra (contour).                       (Source: Friebolin H. Basic One- and Two-Dimensional NMR Spectroscopy. Wiley-VCH, 1998.)                 Fig. 5.8 shows a scheme of the simplest homonuclear correlated 2D method called                   1                1 H- H COSY (correlated spectroscopy). Fig. 5.10 shows a 2D COSY spectrum of p-                (2-butyl)phenol,  with  chemical  shifts  of  protons  in  both  dimensions.  It  can  be                considered as a series of experiments with selective decoupling, during which all                protons are gradually decoupled. The advantage is that all can be obtained in one                step,  and  the  protons  can  be  selectively  decoupled  without  a  limitation,  as  their                signals often tend to be overlapped.                Two  types  of signals  appear  in  a  COSY  spectrum. These are the diagonal  peaks                representing the 1D spectrum and the so-called cross peaks indicating interactions                between  nuclei.  The  cross  peaks  which  are  usually  symmetrical  according  to  a                diagonal  are  therefore  very  important  for  finding  interactions  between  individual                groups.  A  cross  peak  in  the  spectrum  of  p-(2-butyl)phenol  denoted  as  H7-H6                represents  interaction  of  CH3(7)  and  CH2(6)  groups.  The  cross  peak  H8-H5                represents interaction  of  CH3(8)  group  with CH(5); H6-H5  denotes interaction  of                CH2(6) and CH(5); H2-H3 denotes interaction of aromatic protons (2) and (3).                                                              140","5 NMR spectroscopy                                 7      8                             6  5                                4                            3                                                 H7-H6                            2                                1                               OH                                     H6-H5   H8-H5                                9                                          H2-H3                        Figure 5.10. 2D COSY NMR spectrum of p-(2-butyl)phenol in CDCl3.                 Diagram  in  Fig.  5.11  shows  a  pulse  sequence  of  a  heteronuclear  form  of  a  2D                COSY  spectrum,  known  as  H,C-COSY  or  heteronuclear  H,C-correlated  spectra                                                                    1                (heteronuclear shift correlated spectroscopy, HETCOR).  H signals are correlated                on one axis and  C signals on the other axis. Similarly as in homonuclear scenario,                               13                                                 13                it  replaces  a  series  of  complicate  C  experiments  with  selective  decoupling  of                hydrogens. Because on each axis there are different chemical shifts, these spectra do                not have diagonal peaks nor any symmetry.                                             Figure 5.11. Scheme of a pulse sequence of 2D HETCOR.                       (Source: Friebolin H. Basic One- and Two-Dimensional NMR Spectroscopy. Wiley-VCH, 1998.)                                                         141","5 NMR spectroscopy                 Fig. 5.12 shows a 2D HETCOR spectrum of p-(2-butyl)phenol. It is apparent which                                       1                13 C signal corresponds to  H signal. It is thus easy to recognize, which signal in the                13 C NMR spectrum corresponds to the CH3 group 7 or 8 and which correspond to                CH2  and  CH.  Assignment  can  also  be  performed  in  a  similar  manner  for  the                aromatic  region.  This  experiment,  however,  cannot  be  used  to  assign  signals  of                quaternary carbon atoms.                             7      8                                         H7-C7                          6  5                                  H8-C8                             4                         3                                           H6-C6                          2                             1                        H5-C5                            OH                             9                                H2-C2                              H3-C3                       Figure 5.12. 2D HETCOR NMR spectrum of p-(2-butyl)phenol in CDCl3.                  Currently, the heterocorrelated experiments are carried out in an “inverse” manner,                where the protons are detected as the more sensitive of the isotopes. In this form,                and  especially  with  the  use  of  magnetic  field  gradients,  inversion  probes,  or                cryoprobes, it is possible to obtain heterocorelated experiments over several bonds                within few minutes, from even few milligrams of a substance.                                                                     142","5 NMR spectroscopy                 5.6  Application of liquid phase NMR spectroscopy in pharmacy                 NMR spectroscopy is not used for routine analyses in pharmacy, but still, its role in                development is indispensable and has a very wide application. The most important                applications are:                    •  structural analysis of APIs                    •  structural analysis of impurities                   5.6.1  Structural analysis of APIs                 The use of NMR spectroscopy in liquid phase analysis of APIs will be demonstrated                                                         1                on acetylsalicylic acid. Fig. 5.13 comprises a  H NMR spectrum of this substance in                DMSO-d6. Except signals belonging to the solvent and water, there are five other                signals in the spectrum. Assignment of all protons and carbons in the molecule was                performed with the use of 2D NMR spectra.                            7             1         3       2      4                          COOH                                  8                           6    OCOCH 3                       1            9                               5                       2       4                           3                                                                                   9                                                          1                             Figure 5.13.  H NMR spectrum of acetylsalicylic acid.                 1 H NMR spectrum contains one signal in the aliphatic area at 2.25 ppm, besides the                signals  of  solvent  and  water,  which  corresponds  with  a  moiety  from  the  acetyl                group, in the means of position, intensity and multiplicity of –CH3. In the aromatic                                                     143","5 NMR spectroscopy                 area, there are 4 signals that can be expected from the structure of the molecule. All                signals  have  the  same  intensity,  and  the  multiplicity  of  signals  does  not  allow                unambiguous assignment. It is therefore necessary to use further experiments.                             13                Spectrum of C APT NMR (Fig. 5.14), also allows a clear assignment of the –CH3                group  from  the  aliphatic  region.  Furthermore,  there  can  be  seen  four  signals                oriented  downwards,  i.e.,  CH  signals  of  the  aromatic  carbons,  and  four  signals                oriented upwards, i.e., two quaternary aromatic signals and two signals of carbonyl                groups. This spectrum once again does not allow further definite assignment of all                the signals.                                                          7                                                        COOH                                                               8                                                         6   OCOCH 3                                                                 9                                                    1                            7                               5                                  5        6                          8                         2       4                                                        3                                                                                           9                                      3                                             4                                       1   2                                        13                           Figure 5.14.  C APT NMR spectrum of acetylsalicylic acid.                 2D COSY spectrum (Fig. 5.15) contains the diagonal peaks and three cross peaks,                making it apparent which signal pairs are next to each other. Again, the signals of                protons 1 and 4 cannot be assigned without NMR spectra simulation or the use of                13 C heterocorrelated spectra.                 A  heterocorrelated  spectrum,  called  heteronuclear  single  quantum  coherence  (2D                HSQC), allows to form CH pairs in the aromatic region (Fig. 5.16).                                                          144","5 NMR spectroscopy                                       1               3             2        4                           4                      2                                         7                                         COOH                                                8                                          6    OCOCH 3                    3                1            9                                             5                                                                    3-2      3-4                                     2       4                                         3                    1                                                                   1-2                                         1                          Figure 5.15.  H COSY NMR spectrum of acetylsalicylic acid.                                        1             3           2       4                                      7                         4       COOH                                       4                                        8                                 6    OCOCH 3                         2   1       5    9                                                                    2                             2       4                                 3                          1                 1                          3                              3                                          1                        Figure 5.16.  H– C HSQC NMR spectrum of acetylsalicylic acid.                                       13                                                    145","5 NMR spectroscopy                                     2                                          9                               1       4                                  3                             4                                             7                   6                                                   COOH                         2                                                     8                                                                        6    OCOCH 3                     1                                                           9                                                                   1                                                                            5                         3                                                                   2        4                                                                        3                    5                                                                            C5-H9                      7                    8         C7-H1                                                                             C8-H9                           Figure 5.17.  H– C HMBC NMR spectrum of acetylsalicylic acid.                                    1                                       13                Another  heterocorrelated  spectrum  –  heteronuclear  multiple  bond  correlation                (HMBC) – allows to correlate carbons and hydrogens over multiple bonds, usually                over  three  or  four  bonds  (Fig.  5.17).  Therefore, the carbons  C8  and  C7,  and  the                proton H1 can be definitely assigned. This then allows unambiguous assignment of                the  COSY  spectrum, all  protons  in the molecule, and  also the assignment  of the                remaining  CH  carbons  from  the  HSQC  experiment.  The  remaining  quaternary                carbon atoms can then also be assigned from the HMBC experiment.                  5.6.2  Structural analysis of impurities                 The next example in Fig. 5.18 will demonstrate the possibility to clearly distinguish                between  two  positional  isomers.  These  cannot  be  distinguished  by  common  MS                spectrometry – as they have identical molecular weight.                As can be seen from a heterocorrelated NMR spectrum (HSQC) and the attached                1D  experiments,  isotibolone  has,  contrary  to  tibolone,  a  signal  of  the  proton  4                located  in  the  proton  spectrum,  and  the  carbon  4  located  in  the  aromatic  region,                which could not be provided by tibolone. The heterocorrelated spectrum contains                the  corresponding  crosspeak.  The  number  of  cross  peaks  and  the  size  of  the                                                     146","5 NMR spectroscopy                 molecule  show  that  a  complete  assignment  of  the  molecule  is  not  a  matter  of                minutes.                                                                    13                            Figure 5.18.  H– C HSQC NMR spectrum of isotibolone.                                       1                                                                   6           3,1                                    6    10                                 1          9                                       5                                       4   8                                  2                                   3                             12  HO  3   N H  O 11  10   6                                         7                                      9                                                                        1                            6    10                         1           9                                5                                4   8                          2                      HO         N                     12     3    H     O 11                                  7                                                                                 10   9                                                           3,1                               7       12           6                      7        12              10  6   3   1   9                                 1                 Figure 5.19.  H NMR spectrum of 7-hydroxy-3,4-dihydroquinolin-2(1H)-on and its                                                 impurities.                                                          147","5 NMR spectroscopy                 The  last  example  shows  and  impurity  analysis.  Molecule  of  7-hydroxy-3,4-                dihydroquinolin-2(1H)-on,  as  the  main  component,  does  not  have  both  rings                unsaturated and thus provides two signals in the aliphatic region, corresponding to                CH2 groups 9 and 10. Aromatic region indicates a presence of an impurity, with a                content  around  5%.  From  the  expanded  proton  spectrum  and  multipliticity  of                signals, a clear assignment of the molecule of 7-hydroxyquinolin-2(1H)-on can be                provided.                   5.7  Solid state NMR spectroscopy                 5.7.1  Theoretical introduction                 NMR  spectroscopy  in  liquid  phase  and  its  indisputable  advantages  have  been                explained in the previous chapter. This extensive chapter will focus exclusively on                the NMR spectroscopy in solid phase on nuclei with spin quantum number I = 1/2.                Unlike in liquid phase, where signals are very narrow due to isotropic movement of                molecules, in solid state the signals are significantly wider due to interactions that                cannot  be  observed  in  the  liquid  phase.  While  in  the  liquid  phase  half-width  of                signals  is  often  less  than  1  Hz,  in  solid  state  the  half-width  can  reach  several                                            13                                                                   13                thousand Hz. Fig. 5.20 shows  C NMR spectrum of a 1- C (10%) marked glycine                measured in aqueous solution. Signal of CO group is split by C(α) protons into a                triplet, CH2 carbon is split to a td-triplet of a doublet (α protons cause splitting into a                triplet and the carbon from carbonyl group to a doublet).                 13 C NMR spectrum of the 1- C (10%) marked glycine in solid state is shown in                                           13                Fig. 5.21. While the CO group signal in solution is very sharp, in solid state it is                approx. 200 ppm wide and the spectrum is virtually not interpretable.                 Two major factors that cause the broadening of bands in solid state are:                    •  dipolar interaction                    •  anisotropy of chemical shift                                                                     148","5 NMR spectroscopy                                                                              H                                                                   H 2 N                                                                           C                                                                     H                                                                             C     O                                                                         HO                                                                    13                   Figure 5.20.  C NMR spectrum of 1- C (10%) marked glycine in H2O/D2O.                               13                                    (Source: Laws D.D. et al. Angew. Chem. Int. Ed., 2002.)                                                                13                   Figure 5.21.  C NMR spectrum of 1- C (10%) marked glycine in solid state.                                                     13                                    (Source: Laws D.D. et al. Angew. Chem. Int. Ed., 2002.)                                                     149","5 NMR spectroscopy                 Dipolar interactions                 In solution, free movement of molecules eliminates interactions of nuclei. In solid                state, this interaction is very important and is characterized by dipolar interaction                constant D given by relationship:                                                      h                                                  =                                           D     0       A  X                                                4    2   r 3 AX                 Dipolar interaction constant  is  proportional to  gyromagnetic ratios  (gyromagnetic                ratio  of  hydrogen  is  4  times  higher  than  of  carbon)  and  decreases  with  the third                power of the distance between nuclei. The quantity µ0 is permeability of vacuum. In                contrast to liquid phase, dipolar interaction constant in solid state depends also on                orientation of the nuclei connecting line to the magnetic field and is proportional to                             2                a factor (3 cos  θ – 1), as shown in Fig. 5.22.                                                  Figure 5.22. Angle θ is held between internuclear vector C–H and direction of                                              magnetic field B0.                                    (Source: Laws D.D. et al. Angew. Chem. Int. Ed., 2002.)                 Unlike  bond  scalar  interaction  which  requires  a  chemical  bond,  the  dipolar                interaction is a spatial interaction and often occurs also between nuclei of different                                                        150","5 NMR spectroscopy                 molecules.  It  exists  either  between  nuclei  of  the  same  type  –  homonuclear,  or                between various types of nuclei ‒ heteronuclear.                                                       2                And when the following applies  – (3 cos  θ – 1) = 0  θ = 54.74° – then, at this                precise  angle  (the  magic  angle  which  corresponds  to  the  angle  between  a  body                diagonal and the edge in a cube), the dipolar interaction is eliminated. The second                option, how to eliminate the dipolar interaction, especially the heteronuclear one, is                by using a decoupling technique.                 Chemical shift anisotropy                 Orientation dependence, or chemical shift anisotropy, on a magnetic field (CSA) is                shown  in  Fig.  5.23.  It  is  based  on  the  fact  that  distribution  of  electron  density                around nuclei or molecules is not spherical (ball) but ellipsoidal. The value of CSA                                                     2                is  also  proportional  to  the  factor  (3 cos  θ-1)  and  is  thus  also  eliminated  by  the                magic angle. Otherwise, it may reach values of up to several hundred ppm.                                                        Figure 5.23. Schematic depiction of the CSA principle. Position of each resonance                 depends on the orientation of an ellipsoid of chemical bond electron density to the                                             magnetic field axis.                                    (Source: Laws D.D. et al. Angew. Chem. Int. Ed., 2002.)                                                     151","5 NMR spectroscopy                 The value of the isotropic chemical shift, observed in a solution, is then defined by                the relationship:                                               =  1 ( +  +   )                                            iso                                                 3  11   22   33                In  a  powder  sample,  with  many  random  orientations,  it  is  possible  to  obtain  the                “powder pattern” which has basically no practical importance, and in real-life, the                chemical shift anisotropy must be eliminated.                 Magic angle spinning                 If  a  sample  is  placed  at  an  angle  of  54.74°  towards  the  magnetic  field                (Fig. 5.24), the elimination of the element (3 cos  θ – 1) occurs, and if the rotation is                                                           2                sufficiently high, it leads to elimination of the heteronuclear dipolar interactions and                 of the chemical shift anisotropy.                      Z                                                      54.7°                                  θ          Y                                         X                              Obrázek 6.24. Rotace pod magickým úhlem (MAS).                                      Figure 5.24. Magic angle spinning.                Magic angle spinning (MAS) is then combined with the heteronuclear decoupling                technique  and  the  result  is  an  NMR  spectrum  with  a  very  good  resolution  (Fig.                5.25).                                                                   152","5 NMR spectroscopy                                                                                           *        *                   Figure 5.25. Comparison of  C NMR spectra of alanine under static conditions (no                                          13                   rotation), and magic angle spinning without and with heteronuclear decoupling.                                   Symbol * denotes the spinning sidebands.                 A necessary addition is a technique called cross-polarization (CP/MAS), which is                                                                                       13                used to transfer polarization from protons to isotopically poor nuclei, such as  C,                        29                15 N,  or  Si.  This  required  the  fulfilment  of  the  Hartmann-Hahn  condition.  The                result  is  a  significant  increase  in  sensitivity  of  the  NMR  experiment.  Spinning                sidebands  appear in the  spectrum  if the  rotation is  not  high  enough,  which  often                complicates the interpretation of NMR spectra (Fig. 5.26). Only when the speed is                sufficient,  the  spectrum  has  a  signal  count  corresponding  to  isotropic  chemical                shifts.  If  it  is  not  possible  to  achieve  adequate  rotation  speed  due  to  technical                reasons, the rotational sidebands can be suppressed by pulse techniques, such as a                TOSS sequence (total sidebands suppression).                                                                        153","5 NMR spectroscopy                                    7kHz                                        5kHz                                      4kHz                                      2,5kHz                                     13                  Figure 5.26.  C CP/MAS NMR spectrum of hexamethylbenzene under various                                              spinning speeds.                 Spinning speed is determined by rotor size (Fig. 5.27). The smaller the diameter of                an NMR rotor, the higher the possible spinning speed but the lower the sensitivity                of measurement. Maximum spinning speed of a 4 mm rotor is 15 kHz, for 2.5 mm                rotor the speed reaches up to 35 kHz.                                                                  154","5 NMR spectroscopy                                                     Figure 5.27. NMR rotors designed for solid state measurements.                  Currently,  there  are  already  available  probes  with  cell  size  of  1.3  mm,  enabling                rotation of 70 kHz. Yet, the rotation speed is still not sufficient for suppression of                strong  homonuclear  dipolar  interactions  and  acquisition  of  well-differentiated                proton  NMR  spectra.  The  technical  realization  is  shown  in  Fig.  5.28,  where  the                rotor is spun by air flow and the velocity is detected by optical fibre.                                             Figure 5.28. Technical realization of rotor spinning, under magic angle.                                     (Source: Short CP/MAS. Prezentace. Varian, 2001.)                                                        155","5 NMR spectroscopy                 5.7.2  Application of solid state NMR spectroscopy in pharmacy                 Solid state NMR spectroscopy is not used for routine analyses in pharmacy, but its                role  in  development  is  indispensable.  It  has  a  very  wide  application.  The  most                important applications can be divided into the following groups:                    •  API structural analysis                    •  polymorphism                    •  dosage form analysis                    •  solvate analysis                    •  salt analysis                   5.7.2.1  API structural analysis                 Similarly as in  liquid  phase, many  types  of  solid  state  NMR  experiments can be                performed to obtain information about the molecule structure. The most common is                the measurement of  C CP/MAS NMR spectra, which provides basic information                                   13                about  a  molecule.  As  in  liquid  phase,  this  can  then  be  complemented  by  other                experiments performed to obtain information on the types of carbons included in the                molecule.  One  of  the  experiments  is  a  NQS  experiment  (non-quaternary                suppression). Signals of carbon atoms with directly bound protons, especially CH                and CH2 are supressed in the spectra. The CH3 signals remain unsuppressed due to                free rotation, which also applies for acetylsalicylic acid (Fig. 5.29).                                                                                156","5 NMR spectroscopy                                                        7                                                       COOH                                                               8                                                        6    OCOCH 3             a)                                                                 9                                                   1                                                            5                                                   2        4                                                       3                                                                                         b)                            8    7    5          7  6                                                                           9                                                                                       13                                            13                 Figure 5.29. Comparison of a  C CP/MAS NMR spectrum (a) and a  C NQS (b)                                       spectrum of acetylsalicylic acid.                As  already  mentioned,  measurements  of  proton  spectra  in  solid  state  are  not                common.  Therefore,  a  suitable  complement  to  a  C  CP/MAS  experiment  is  a                                                               13                heterocorrelated  NMR  spectrum,  from  which  it  is  indirectly  possible  to  acquire                information about chemical shifts of protons (Fig. 5.30).                                                                               157","5 NMR spectroscopy                                     1   2                                       9                                      3    4                                 7                           COOH                                    8                             6    OCOCH 3                       1               9                                 5                        2        4                            3                                      Figure 5.30. 2D heterocorrelated NMR spectrum of acetylsalicylic acid.                    5.7.2.2  Polymorphism                 Polymorphism can be defined as an ability of compounds to exist in more than one                crystalline state, each differing in arrangement or conformation of molecules in the                crystal  structure.  Polymorphs  are  chemically  identical  but  differ  in  their  physical                (spectral)  properties.  The  NMR  spectroscopy  in  solid  state  is  one  of  the  few                methods  that  allows  clear  identification  of  polymorphs  and  detection  of                polymorphic  purity.  In  addition,  one  of  the  biggest  advantages  of  NMR                spectroscopy  is  the  possibility  to  measure  spectra  of  different  isotopes,  which                                                                         13                provides  complementary  information.  Usually,  measuring  of  C  CP/MAS  NMR                spectra is the first and the most common choice.                 Fig.  5.31  shows  comparison  of  two  crystalline  polymorphs  and  one  amorphous                form. The spectra, particularly of the crystalline forms, can be clearly interpreted,                and  the  duplication  of  lines  indicates  the  presence  of  two  symmetrically                inequivalent molecules.                                                     158","5 NMR spectroscopy                                                                          Figure 5.31. Comparison of  C CP/MAS NMR spectra of three polymorphic forms                                          13                                           of calcium atorvastatine.                Molecule of atorvastatin contains two nitrogen atoms. Fig. 5.32 shows a comparison                   15                of  N CP/MAS spectra of the same molecule in different polymorphs. Sensitivity                       15                of  the  N  nucleus  is  however  50-fold  lower,  and  acquisition  of  such  spectra                requires  appropriate  equipment  and  sufficient  experimental  time.  Duplication  of                signals again indicates the presence of two symmetrically inequivalent molecules in                one unit cell.                                                     159","5 NMR spectroscopy                                                         Figure 5.32. Comparison of  N CP/MAS NMR spectra of three polymorphic forms                                          15                                           of calcium atorvastatine.                 Atorvastatin molecule also contains a fluorine atom. This allows measurement of                    19                                                                                   19                the  F isotope, which has a natural occurrence of 100%, enabling quality  F NMR                spectra measurement in a relatively short time. Fig. 5.33 compares the same three                atorvastatin  forms,  as  previously.  The  duplication  of  lines  is  similar  as  in  the                previous cases.                 The  advantage  of  the  presence  of  a  fluorine  atom  can  be  applied  in  quantitative                                                                                        19                analysis  or  in  detection  of  polymorphic  purity  of  APIs.  Fig.  5.34  shows  F                CP/MAS NMR spectra of atorvastatin form I, its amorphous form, and their model                mixture.  Similar  spectra  can  be  obtained  when  measuring  dosage  forms.  In  this                case, the fluorine atom represents an advantage not only for sensitivity but also for                selectivity,  since  no  excipient  contains  fluorine  atoms  and  thus  the  polymorphic                purity of the API can be studied also in formulations, without signals of API being                                                                         13                overlapped by signals of excipients, which can be the case in  C CP/MAS NMR                spectra.                                                     160","5 NMR spectroscopy                                                                         19                 Figure 5.33. Comparison of  F CP/MAS NMR spectra of three polymorphic forms                                           of calcium atorvastatine.                                             Figure 5.34.  F CP/MAS NMR spectra of a model mixture of calcium atorvastatine                            19                   (5 % of form I and 95 % of amorphous form), and of pure crystalline and pure                                              amorphous forms.                                                      161","5 NMR spectroscopy                 For comparison, the Fig. 5.35 contains an example of a measurement of a similar                model mixture in a dosage form but using the carbon spectra. The time required for                this experiment is much longer than for the fluorine NMR spectra.                                                      Figure 5.35. Comparison of  C CP/MAS NMR spectra of model mixtures of                                             13                 calcium atorvastatine (5% of form I and 10% amorphous form) with spectra of pure                                       crystalline and amorphous forms.                   5.7.2.3  Dosage form analysis                    13                The  C CP/MAS NMR spectra are very often used for identification of polymorphs                in formulations. The comparison can be carried out only in those parts of the NMR                spectra which do not contain signals of excipients. If the API content in a dosage                form is low, the acquisition of this information can be very time consuming and                sometimes  lasting  several  tens  of  hours.  Fig.  5.36  shows  comparison  of  an                atorvastatine  formulation  containing  crystalline  form I  with two  pure  atorvastatin                forms.                                                          162","5 NMR spectroscopy                                                                            13                 Figure 5.36. Comparison of a  C CP/MAS NMR spectrum of atorvastatin as a pure                   amorphous form, pure crystalline form I, and a dosage form containing calcium                                       atorvastatine as crystalline form I.                 If  a  dosage  form  contains  an  API  with  a  hetero  atom,  such  as  fluorine  or                phosphorus, the identification of the API form is significantly easier. An example                with fluorine was already shown in Fig. 5.34. Fig. 5.37 shows a situation where it is                possible  to  find  out  whether  dosage  form  contains  an  amorphous  form  of                risedronate sodium polymorph A, H, or a mixture of the polymorphs A and H, in a                few  minutes.  To  determine  whether  a  sample  represents  a  pure  API  form  or  a                                31                dosage form, the  P spectra are not sufficient, and the  C CP/MAS NMR spectra                                                                  13                must be measured, or the results must be complemented by other methods, such as                X-ray diffraction, IR or Raman spectroscopy. In both cases, the  P CP/MAS NMR                                                                          31                spectra  will  not  differ,  and  the  difference  will  only  lie  in  the  sensitivity  of  the                experiment, especially when the API content in the formulation is low.                                                                  163","5 NMR spectroscopy                                                              Figure 5.37. Comparison of  P CP/MAS NMR spectrum of an amorphous form of                                          31                        sodium risendronate and its polymorphs A and H, and a mixture of                                            polymorphs A and H.                 Analysis of dosage forms does not include only identification of API forms, but also                qualitative  and  quantitative  analysis  of  excipients.  In  this  case,  it  is  necessary  to                                13                analyse  also  the  C  CP/MAS  NMR  spectra.  The  analysis  of  dosage  forms  can                sometimes be very difficult because the signals of excipients and APIs very often                overlap. Usually, however, it is possible to identify the excipients at least according                to some of the signals (Fig. 5.38 and 6.39). Fig. 5.40 shows, how well it is possible                to distinguish some types of cellulose.                                                             164","5 NMR spectroscopy                                                                   13                      Figure 5.38.  C CP/MAS NMR spectrum of a dosage form containing                           excipients – microcrystalline cellulose Avicel and Povidone.                                                                          13                  Figure 5.39. Comparison of  C CP/MAS NMR spectra of a placebo containing                 excipients ‒ microcrystalline cellulose Avicel, Povidone, and Lactose monohydrate.                                                     165","5 NMR spectroscopy                          HPMC                               HPC                             Avicel                                                      13                      Figure 5.40. Comparison of  C CP/MAS NMR spectra of excipients ‒                      microcrystalline cellulose (Avicel), hydroxypropylcellulose (HPC), and                                    hydroxypropylmethylcellulose (HPMC).                   5.7.2.4  Solvate analysis                Analysis  of  solvates  is  nowadays  a  common  requirement.  The  reason  is  that                solvents are very often incorporated in crystal structures. Such compounds are then                called solvates, pseudopolymorphs, or solvatomorphs. Hydrates are another special                group, and the hydration process can be very well monitored. Desolvation, in some                cases,  goes  easily  and  does  not  change  the  crystalline  form  of  the  API,  but                sometimes it leads to a collapse of the crystal structure. Since desolvation can also                occur during production, formulation, and storage of drugs, its monitoring is very                important.                 Most solvents are of organic nature, and therefore it is suitable to use  C CP/MAS                                                                               13                NMR spectra. Fig. 5.41 shows a comparison of spectra of three different solvates of                one API.                 The  H NMR spectroscopy in a solution may be a good addition, since it can both                     1                identify and quantify the solvent by comparing the signal intensities for the API and                the given solvent (Fig. 5.42).                                                     166","5 NMR spectroscopy                                                                         MTBE                                                                                        C 2 H 4 Cl 2                                                                                   CH 2 Cl 2                                                  13                  Figure 5.41. Comparison of  C CP/MAS NMR spectra of three solvates formed                   with dichlormethane, 1,1-dichlorethane, and methyl-terc-butyl ether (MTBE).                                                                                  167","5 NMR spectroscopy                                                                           IPA                                            Figure 5.42.  H NMR spectrum of a solvate of a studied API with propane-2-ol                              1                                                   (IPA).                   5.7.2.5  Salt analysis                 NMR spectroscopy is of great importance in salt analysis. Salts can basically appear                in two types, either as salts of organic acids (API) with metal cations (Na , K  etc.)                                                                                  +                                                                                      +                or  with  organic  cations  (e.g.,  meglumine),  or  salts  of  organic  bases  (API)  with                inorganic anions (e.g., hydrochloride) or organic anions (e.g., maleate). Fig. 5.43                shows  differences  between  C  CP/MAS  spectra  of  sodium  and  ammonium  salts                                          13                and a pure API.                                                                         168","5 NMR spectroscopy                                                                               13                   Figure 5.43. Comparison of  C CP/MAS NMR spectra of meloxicam and its                                         sodium and ammonium salts.                 15 N CP/MAS spectroscopy can be a good addition in case of ammonium salts, as it                allows  identification  of  ammonium  salts  in  pure  API  samples  and  also  in                formulations. This is documented in Fig. 5.44 where three nitrogen atoms can be                found in an NMR spectrum of meloxicam, while in the case of its ammonium salt, a                signal of the NH4 group also appears.                                                                             169","5 NMR spectroscopy                                                       Figure 5.44.  N CP/MAS NMR spectra of meloxicam and its amorphous and                               15                                         crystalline ammonium salts.                 13 C CP/MAS NMR spectroscopy is very useful for measuring of salts of bases with                organic acids. This is documented in Fig. 5.45, which contains spectra of one base                and three organic salts.                 A good complement for determination of the base/acid ratio can be the  H NMR                                                                                   1                spectroscopy in solution.                                                                          170","5 NMR spectroscopy                                                                             13                 Figure 5.45. Comparison of  C CP/MAS NMR spectra of a base and salts of three                                of organic acids – oxalic, lactic, and hexanedioic.                   5.8  Conclusion                The  text  provides  a  brief  demonstration  that  the  use  of  ssNMR  spectroscopy  in                pharmacy is very broad and can be conveniently combined with other spectroscopic                techniques, including NMR spectroscopy in liquid phase.                                                                         171","5 NMR spectroscopy                 5.9  Resources and recommended literature                 Principles and basics of NMR                   1.    Holzgrabe U., Wawer I., Diehl D. NMR Spectroscopy in Drug Development and                        Analysis.     Wiley-VCH:       Weinheim,       Germany,       1999.                        https://doi.org/10.1002/9783527613649                  2.    Buděšínský M., Pelnař J. Fyzikálně chemické metody  – NMR, 25. svazek cyklu                        Organická chemie. ÚOCHB: Prague, Czech Republic, 2000.                  3.    Keller J. Understanding NMR Spectroscopy. John Wiley \& Sons: Chichester, UK,                        2005.                  4.    Rouessac  F.,  Rouessac  A.  Nuclear  Magnetic  Resonance  Spectroscopy.                        In  Chemical  Analysis  –  Modern  Instrumentation  Methods  and  Techniques,  2                                                                                          nd                        Edition; John Wiley \& Sons: Chichester, UK, 2007, pp. 327–365.                  5.    Holzgrabe U., Wawer I., Diehl D. NMR Spectroscopy in Pharmaceutical Analysis.                        Elsevier: Amsterdam, The Netherlands, 2008. https://doi.org/10.1016/B978-0-444-                        53173-5.00005-6                 Application of NMR in liquid phase                   6.    Pretch  C.,  Seidle  S.  Spectral  Data  for  Structure  Determination  of  Organic                                   nd                        Compounds, 2  Edition. Springer-Verlag: Berlin, Germany, 1989.                  7.    Friebolin  H.  Basic  One-  and  Two-Dimensional  NMR  Spectroscopy,                        3  Revised Edition. Wiley-VCH: Weinheim, Germany, 1998.                         rd                  8.    Silverstein  R. M.,  Webster  X. F.,  Kiemle  D. J.  Spectrometric  Identification  of                                          th                        Organic Compounds, 7  Edition. John Wiley \& Sons: New York, USA, 2005.                Application of NMR in solid state                   9.    West  A. R.  Basic  Solid  State  Chemistry.  John  Wiley  \&  Sons:  Chichester,  UK,                        1999.                10.     Duer M. J. Solid-State NMR Spectroscopy, Principles and Applications. Blackwell                        Science: Oxford, UK, 2002. https://doi.org/10.1002/9780470999394                11.     Laws D. D., Bitter H. M. L., Jerschow A. Solid-State NMR Spectroscopic Methods                        in Chemistry.  Angew.  Chem.  Int.  Ed.  2002,  41  (17),  3096–3129.                        https://doi.org/10.1002/1521-3773(20020902)41:17%3C3096::AID-                        ANIE3096%3E3.0.CO;2-X                12.     Klinowski J. New Techniques in Solid-State NMR. Springer-Verlag: Heidelberg,                        Germany, 2005. https://doi.org/10.1007/b94544                 13.     Zakrzewski  A.,  Zakrzewski  M.  Solid  State  Characterization  of  Pharmaceuticals.                        Eds.; Assa: Danbury, CT, USA, 2006.                                                     172","5 NMR spectroscopy                 14.     Hartus R. K. NMR Studies of Organic Polymorphs \& Solvates. Analyst 2006, 131                        (3), 351–373. https://doi.org/10.1039/b516057j                                                                                                                          173","6 Terahertz time-domain spectroscopy                 6  Terahertz time-domain spectroscopy                  Filip Kadlec, Christelle Kadlec                   6.1  Introduction                  The terahertz (THz) spectral region is one of the still relatively little known parts of                the electromagnetic spectrum, situated in the zone adjacent to the infrared (optical)                region,  on  one  side,  and  to  the  microwave  (electronic)  region  on  the  other.  Like                other spectral regions, the range of the THz region is not defined precisely exactly,                but  its  use  associated  with  several  specific  experimental  techniques.  These                techniques cover a wide interval, which can be roughly delimited by frequencies of                100 GHz and 3 THz, corresponding to wavelengths between 3 and 0.1 mm,  i.e.,                                             -1                wave numbers from 3 to 100 cm . The THz region hence partially overlaps with the                far-infrared  region.  The  characteristic  frequency  of  1  THz  can  be  equivalently                expressed in other spectroscopic units, as shown below.                                               -1                               1 THz   33 cm   0.3 mm   48 K   4.1 meV                           frequency wavenumber wavelength temperature photon energy                                                                                    th                The boom of the THz research occurred gradually in the nineties of the 20  century,                after the invention of THz time domain spectroscopy. The decisive factor was the                development of optical femtosecond lasers which present the basis for most current                laboratory  and  commercial  THz  spectrometers.  Until  then,  there  had  been  only                limited  options  for  generation  and  detection  of  THz  radiation  since  there  is  no                access  to  the  THz  field  measurements  using  either  classical  optical  spectroscopy                (intensity of optical radiation sources declines, similarly as blackbody radiation) or                traditional electronic sources (electronic components are not capable of operation at                such high frequencies).                 The attractiveness of the THz field is linked to several specific possibilities of using                the  electromagnetic  radiation  with  these  frequencies.  The  applications  can  be                divided  into  three  main  categories:  spectroscopy,  imaging,  and  data  transfer                (telecommunications).  The  applications  from  the  first  two  groups  are  gradually                finding their way into the pharmaceutical industry, mostly in analysis of solid active                ingredients.  In  most  cases,  however,  these  are  only  pilot  studies,  designed  to                demonstrate  the  suitability  of  a  certain  method  for  a  specific  purpose.  It  can  be                                                      174","6 Terahertz time-domain spectroscopy                 therefore expected that some other applications of the THz radiation in pharmacy                will only be discovered in upcoming years.                 Solid substances often exhibit specific interactions in the THz region. While some                crystals are transparent in this range, many others show low-frequency oscillations                of  the  crystal  lattice  (called  phonon  bands),  with  characteristic  frequencies                determined  by  short-range  inter-atomic  interactions  and  regular  long-range                arrangement  of  atoms.  Since  amorphous  materials  have  significantly  different                atomic  arrangements,  the  THz  spectra  of  amorphous  and  crystalline  phases  are                significantly different even with identical chemical composition.                  In  the  cases  of  molecular  crystals,  liquids,  and  gases,  the  THz  region  includes                molecular  movements  ‒  particularly  the  intermolecular  motions,  when  entire                molecules  or  their  large  parts  vibrate,  librate,  or  rotate  together.  The  vibrational                frequencies  of  molecules  are  only  slightly  affected  by  their  incorporation  into  a                crystal  lattice  and  thus  the  shifts  of  the  absorption  bands  are  relatively  small                compared to shifts of free molecules.                  The  THz  properties  of  weakly  doped  semiconductors  are  related  mainly  to  the                movement of free electrons. The THz radiation is continuously and very strongly                reflected  by  metals  and  strongly  absorbed  by  liquid  water.  On  the  other  hand,                isolated water molecules, such as water vapor or crystal water, show characteristic                narrow absorption bands.                   6.2  Overview of experimental techniques in THz spectroscopy                  Due to the interest in the THz spectral region and its specific features and expected                applications, there was a rapid development of the appropriate sources, detectors,                and  measurement  approaches,  in  the  recent  decades.  By  now,  there  are  so  many                experimental techniques that it would be pointless to describe them all in detail in                this text. Therefore, only the main options will be briefly discussed.                 One of the most intensive THz radiation sources are synchrotrons. These are large                and very expensive instruments working on the principle of acceleration of charged                particles (mainly electrons) on an approximately circular trajectory with a diameter                of tens to hundreds of meters. The irradiation of electromagnetic waves occurs due                to  bending  of  particle  trajectories  in  a  magnetic  field,  thus  generating  a  broad                continuous spectrum of which the THz radiation constitutes only a very small part.                Since  the  synchrotron  measurement  time  is  limited  and  highly  demanded,  the                                                       175","6 Terahertz time-domain spectroscopy                 studies of substances using synchrotron radiation are limited to well justified cases                with the aim of making breakthrough discoveries.                 Gas  lasers  working  in  the  THz  region  have  been  used  for  many  decades.  Their                active media consist of some selected gases (e.g., methanol, formic acid, ammonia)                with  rotational  levels  that  enable  to  choose  the  appropriate  lines.  Their  output                spectra are very narrow, and their performance can typically reach a few mW. A                disadvantage  of  the  THz  lasers  is  their  size  and  complex  operation.  Since  their                output  wavelengths  are  practically  not  tunable,  these  sources  are  not  suitable  for                spectroscopic purposes.                  Backward  wave  oscillators  were mainly  developed  in  the  Soviet  Union  since the                               th                fifties of the 20  century. They are composed of electron-tubes placed in a strong                magnetic field where an accelerated electron beam passes through a so-called slow-                wave structure, creating a monochromatic THz radiation. It is possible to tune the                frequency  by  changing  the  accelerating  high  voltage.  To  cover  the  whole  THz                spectral range, it is necessary to use several different oscillators and thus a wide-                spectrum measurement can prove quite lengthy. The advantage is high resolution                (the width of a frequency line is typically several units of MHz).                  Quantum cascade lasers belong to the so far less applied sources, but also the most                promising  ones.  They  constitute  optoelectronic  components  based  on  a  series  of                alternating thin layers of semiconductors with larger and smaller band gap energies                and  with  well-chosen  thicknesses  (on  the  nanoscale).  Applying  a  suitable  low                voltage  to  such  structures  creates  a  cascade  of  radiation  transitions.  They  cause                moving  electrons  to  lose  their  energy,  and  to  emit  photons  in  the  THz  region.                Although  the  quantum  cascade  lasers  operating  in  the  infrared  region  are  very                reliable,  they  have  a  fundamental  limitation  in  the  THz  region,  which  is  the                operating  temperature  of  their  components.  If  the  thermal  energy  of  particle                oscillations  is  greater  than  the  target  photon  energy  (the  room  temperature                corresponds  to  the  energy  of  26  meV,  about  6.3  THz),  the  energy  losses  are                increased  to  such  an  extent  that  no  laser  amplification  is  possible.  So  far  it  is                therefore necessary to cool the THz quantum cascade lasers to low temperatures. If                this  limitation  can  be  overcome  in  the  future,  these  lasers  will  likely  be  used                commercially, as they are very compact and their operation is simple.                  One of the most versatile and most widely applied techniques in the THz region is                the  THz  time-domain  spectroscopy.  It  is  a  set  of  methods  based  on  nonlinear                conversion of energy from ultrashort laser pulses into the THz region. Their rapid                development  began  around  the  early  nineties,  when  the  first  THz  semiconductor                switch was developed. These methods enable relatively poor frequency resolution                (maximum of units of GHz) and operate with low output (typically units of μW).                                                     176","6 Terahertz time-domain spectroscopy                 However,  their  advantages  include  namely  the  possibility  of  a  simultaneous                measurement  over a  wide spectral range,  phase sensitivity  of  detection,  and  time                domain measurement (possibility to distinguish reflections of pulses from individual                interfaces).  The  time-domain  THz  spectroscopy  methods  are  discussed  in  the                following  section.  More  detailed  information  can  be  found  for  example  in  the                monograph [1].                  6.3  Principles of THz time-domain spectroscopy                  Most often, the THz time-domain spectroscopy uses transmittance measurements;                the usual experimental arrangement is shown in Fig. 6.1. A sequence of ultrashort                pulses  is  generated  using  a  femtosecond  laser  (oscillator),  usually  a  titanium-                sapphire oscillator, which provides pulses with a wavelength of approximately 800                nm and duration of approx. 20–100 fs at a repetition rate of around 80 MHz. The                beam  is  then  split  into  two  parts  using  a  semitransparent  beamsplitter,  with  the                largest part of the output impacting the THz emitter (see below). The THz radiation                is emitted in a pulsed mode, in forward direction, and the pulses have duration of                approximately 1 ps.                 Radiation pulses with a waveform E0(t) are focused using a metallic mirror into a                focal  point  where  the  sample  can  be  placed,  and  the  pulses  passed  through  the                sample  have  a  waveform  E(t).  The  radiation  spreading  farther  is  focused  by  a                second mirror onto a sensor. The detection of THz electric field intensity requires                that two pulses fall simultaneously onto a detection sensor – the terahertz pulse and                the femtosecond laser pulse. The waveform of the THz field E(t) is measured point                by point; the length of one of the laser beam branches is changed for this purpose                using  an  exactly  positioned  delay  line  (with  a  sliding  mirror  retroreflector).  An                electronic  synchronous  detection  is  typically  used  to  increase  the  signal  to  noise                ratio. It is also advantageous if the entire optical path of the THz beam is placed in a                vacuum  chamber,  since,  when  the  measurement  is  performed  in  ambient  air,  an                absorption  on  the  rotational  lines  of  water  vapor  molecules  occurs,  which                complicates the subsequent interpretation of the spectra.                                                                  177","6 Terahertz time-domain spectroscopy                                                     Figure 6.1. Typical experimental configuration for THz transmittance                                               measurements.                   6.3.1  Principle of THz pulse generation                 The  principle  of  THz  pulse  generation  is  schematically  shown  in  Fig.  6.2a.  The                emitter consists of a thin plate made of a semiconductor with transversely applied                DC voltage. In unlit state, the emitter has high resistance, therefore no current can                pass through it.                 If an ultrashort laser pulse comprising photons with energy higher than the band gap                energy  of  the  semiconductor  impacts  the  emitter,  free  electron-hole  pairs  are                generated near its surface. These are then immediately accelerated by the  applied                electric  field  into  mutually  opposite  directions.  The  semiconductor  material  is                fabricated in a way that the lifetime of these electric charge carriers is very short ‒                usually less than 1 picosecond.                 Thus, after the arrival of the femtosecond pulse, a very short current impulse occurs                in the direction of the applied voltage, in the semiconductor. The THz pulse emitted                by the accelerated charges has a shape corresponding approximately to one cycle of                the electric field (Fig. 6.2b). The spatial emitting characteristic is closely related to                the size of the laser beam, and in some cases a silicon lens is attached to the emitter                for a better focusing of the THz beam.                                                     178","6 Terahertz time-domain spectroscopy                                          Figure 6.2. Principle of THz pulse generation. (a) function of a semi-conducting                    emitter, (b) typical time course of a THz pulse electric field (spectrum of an                         amplitude obtained by Fourier transformation of the curve E0(t)).                  6.3.2  Detection of THz pulses                 As already mentioned, a great advantage of the THz time-domain spectroscopy is                the  possibility  to  use  a  phase-sensitive  detection.  In  other  words,  while  optical                methods in the infrared, visible, and ultraviolet regions mainly use power detectors,                in the THz region, it is possible to directly detect the intensity of the electric field E.                Two different techniques can be used for this purpose. The common feature of both                is  that  the  THz  pulse  (with  a  duration  of  usually  a  few  ps)  and  the  optical                (femtosecond) pulse simultaneously impact the detection element.                The first method is based on the use of a semiconductor receiver antenna, similar to                the  emitter  shown  in  Fig.  6.2a.  In  this  case,  however,  there  is  no  bias  voltage                applied on the antenna. If an optical pulse impacts the antenna, free charge carriers                are generated. If the THz pulse impacts the antenna during the short time period                when the charge carriers move in the conduction band of the semiconductor, then                the electric  component  E(t)  of  the  pulse,  oriented in  the  direction connecting  the                electrodes, accelerates the charge carriers. A small but easily detectable current with                magnitude directly proportional to the output of the optical pulse and to the intensity                of the THz field E(t) then passes through the external circuit containing the antenna.                                                           179","6 Terahertz time-domain spectroscopy                                Figure 6.3. Principle of electrooptic detection of THz radiation. THz – propagating                   field E(t); fs – femtosecond pulse with circular polarization; EO – electrooptic                 crystal (sensor); W – Wollaston prism; PD1,2 – detection photodiodes with a slow                                           response (typically μs).                 The second option (Fig. 6.3) is to use the electro-optic effect, also called the Pockels                effect, during which a change in the polarization state of optical radiation occurs in                a  so-called  non-linear  environment  (in  a  crystal,  for  example  ZnTe),  due  to  an                applied electric field. The applied femtosecond pulses are always linearly polarized                but can be easily converted to circularly polarized pulses. If these pulses impact an                electro-optic crystal simultaneously with the THz pulse, their polarization changes                to a slightly elliptical one, due to instantaneous value of the intensity of the electric                (THz) field.                  The size and the sign of the ellipticity are directly proportional to the size of the                field E. Using a Wollaston prism, the optical beam can be split into a vertical and a                horizontal component which have the same intensities in the absence of the THz                field. The value of the THz field E at a given time point (i.e., for a given mutual                position of the optical and THz beam) then corresponds to the voltage difference on                the detection photodiodes. The E(t) function is measured point by point in a wide                range for different values of the delay t, corresponding to the shift of the delay line.                    6.4  THz spectra measuring techniques and data processing                 6.4.1  Transmittance measurements                Transmittance measurements belong to the most frequently used techniques in THz                spectroscopy.  A  diaphragm  is  placed  into  the  beam  focal  point,  and  two                measurements  are  performed:  E0(t)  corresponding to  the reference  waveform  and                E(t) carried out with the sample which has a shape of a homogeneous plane-parallel                plate. Compared to measurement without a sample, the interaction of the THz pulse                with  the  sample  generally  leads  to  a  signal  delay,  its  broadening  in  time  and  a                                                     180","6 Terahertz time-domain spectroscopy                 reduction  of  its  amplitude.  While  a  part  of  the  pulse  passes  directly  through  the                sample,  reflections  will  occur  on  both  its  surfaces.  The  reflected  pulses  are                propagating  backwards,  which  leads  to  further  multiple  reflections.  At  every                reflection,  a  part  of  the  THz  wave  passes  through  the  outer  surface  toward  the                detector (see Fig. 6.4).                                             Figure 6.4. Interaction of THz radiation with a sample in a transmittance                   measurement. While the reference waveform contains a single pulse, multiple                 reflections on the sample surfaces appear as a sequence of pulses at regular intervals                                            in the waveform E(t).                 Using  Fourier  transform,  the  two  complex  spectra  are  calculated,  and  their  ratio                yields the spectrum of a complex transmittance:                     At the same time, the transmittance of a homogeneous plane-parallel plate is given                by the relationship:                     where N = n + i κ denotes the complex refractive index of the sample, d its                thickness, c the speed of light, and m the number of measured reflections in the time                domain. The task of finding the spectrum of a sample N(ω) from the experimentally                measured transmittance t^(ω) must be solved numerically.                                                        181","6 Terahertz time-domain spectroscopy                 The permittivity (dielectric function) of the material can then be determined from                the complex refractive index spectrum as:                   The  above-described  procedure  for  evaluation  of  experimental  data  is                straightforward and easily applicable to homogeneous solid samples.                 The  situation  is  somewhat  different  in  the  case  of  powder  samples.  Their                measurement requires the use of cuvettes with parallel windows transparent in the                THz region and a reference measurement performed with an empty cuvette. If the                grain  size  is  much  smaller  than  the  wavelength  of  the  THz  radiation,  i.e.,  if  the                grains do not exceed several tens of microns, then the sample can be considered                homogeneous.  Obtaining  of  quantitatively  correct  THz  spectra  of  a  powder                substance itself is however complicated by the fact that the sample behaves as an                effective medium ‒ a mixture of the  substance with air. The measured refractive                index is then determined not only by the properties of the solid phase, but also by                the porosity of the sample, and also by the shape of the grains. Hence, if it is needed                to  determine  the  refractive  index  of  the  sample  itself,  it  is  necessary  to  use                expressions describing the response of an effective medium. These expressions are                always  based  on  certain  assumptions,  corresponding  to  the  specific  microscopic                arrangement  of  a  mixture.  These  assumptions,  however,  are  not  always  entirely                valid in practice, and the choice of an appropriate mixture model can be difficult.                When  measuring  powdered  substances  with  grain  sizes  comparable  with  the                wavelength  of  the  THz  radiation  (greater  than  approx.  50  μm),  the  quantitative                evaluation of the spectra is practically impossible to perform, as scattering of the                radiation passing through the sample plays a significant role, especially at higher                frequencies.  Even  then,  however,  it  is  possible  to  identify  in  the  spectra  the                absorption maxima, characteristic for the particular substance.                   6.4.2  Reflectance measurements                 In general, THz reflectance measurements of solid samples in the THz region are                performed  less  often  than  the  transmittance  measurements,  as  the  possibility  to                exactly  determine  the  phase  of  a  reflected  radiation  is  critically  dependent  on  a                perfect reference measurement. This is very difficult compared to the transmittance                geometry.  In  fact,  the  mirror  replacing  the  sample  in  the  reference  measurement                must not only show a perfect reflectance; at the same time, its surface must be in the                same position as the surface of the measured substance, with an accuracy better than                1 μm [2]. Otherwise, the phase of the reflected radiation is increased by a phase                                                      182","6 Terahertz time-domain spectroscopy                 corresponding to free-space wave propagation at a small but unspecified distance,                which impedes the determination of complex optical constants from the measured                data.                  Nevertheless,  in  certain  cases,  the  phase  sensitivity  can  be  achieved  also  in  the                reflectance  measurements.  The  simplest  way  is  to  place  the  sample  behind  a                transparent window, where the reflection from its front side can serve as a reference                signal, while the second interface includes also the measured sample, the spectrum                of which can then be easily determined. This procedure is particularly suitable for                measuring of liquids, which will perfectly adapt their shape to the surface of the                window. By contrast, for both bulk and powdered solids, it is not easy to ensure                their full contact with the window, and even a small air gap can cause significant                errors in the measurement.                 In  practice,  the  reflectance  measurements  are  mostly  used  in  cases  when  it  is                sufficient  to  measure  the  reflected  amplitude.  An  indisputable  advantage  of  the                method  is  its  suitability  for  measuring  of  thick  samples,  where  transmittance                measurements would not be feasible.                    6.4.3  Attenuated total reflection                 Basics of the attenuated total reflection (ATR) method are described in the section                2.2.1.3, within the chapter on infrared spectroscopy. Also in the THz region, the                advantage of the method is an easy sample preparation, as it suffices to place the                sample on the reflective surface of the ATR prism. If the sample exhibits vibrational                modes in the THz region, it is possible to identify them as absorption maxima in the                ATR  spectrum,  which  can  then  be  unambiguously  assigned  to  a  particular                substance.                  Unlike  the  geometry  for  transmittance  measurements,  the  common  ATR  method                variant does not allow determination of complex spectra of optical constants. This                can  be  achieved  when  the  measured  substance  (a  bulk  sample  or  a  tablet)  is                separated from the ATR prism, with a thin layer of air, the thickness of which is                defined with micron accuracy and is gradually changed [3].                 ATR spectra of powder samples can be obtained from amounts of less than 1 mg                [4].  At  the  same  time,  however,  it  is  necessary  to  ensure  that  the  amount  of  the                studied substance is not too small, as the penetration of the THz radiation 1/α rises                towards low frequencies and, in the THz region with the longest wavelengths, it can                reach hundreds of μm. To obtain spectra with visible absorption maxima and with                                                     183","6 Terahertz time-domain spectroscopy                 optimal contrast, it is necessary to use a sufficient compression force (of around 5                MPa [4]). At the same time, it is necessary to verify that the pressure does not result                in changes of the structure of the studied substance.                  Modules  for  the  ATR  measurements  are  offered  among  products  supplied  by                commercial suppliers of THz spectrometers (e.g., TeraView company).                   6.4.4  Time of flight measuring method                  Thanks  to  high  time  resolution,  the  THz  spectroscopy  in  reflectance  mode  can                employ time of flight measuring method. The method utilizes the fact that every                planar interface of two materials, with different refractive indexes, reflects a part of                the radiation. The time of flight, for the reflected radiation, is longer for those parts                of  the  pulse  that  penetrate  deeper  into  the  sample.  The  reflections  from  various                interfaces can then be distinguished in the curves of electric field E(t) waveforms,                and the thickness of the respective layers (i.e., presence of interfaces) can then be                determined from their positions in time.                    6.4.5  Imaging measurement                 All the aforementioned THz spectroscopy methods can be, in principle, performed                in  imaging  arrangement.  In  this  case,  the  radiation  is  spatially  restricted  by  a                relatively small diaphragm (minimum approx. 0.5 mm), the sample is mounted on a                sliding table, and the spectra are recorded depending on its position. When a spatial                resolution significantly better than 0.5 mm is needed, the use of smaller diaphragms                would  lead  to  a  substantial  signal  decrease  and  to  the  prolongation  of  the                measurement  time.  Therefore,  in  these  cases,  a  special  class  of  methods,  called                near-field imaging, is chosen.                    6.5  Applications of THz spectroscopy in pharmacy                6.5.1  Identification of substances using THz spectra                  Similarly as in the mid-infrared region, many crystalline substances show structured                spectra,  characteristic  for  the  given  chemical  composition.  In  cases  when  the                absorption of a substance is too high, and the sample transmits only very little of the                radiation,  it  is  suitable  to  use  a  powder  mixture  of  the  substance  with  grains  of                                                     184","6 Terahertz time-domain spectroscopy                 polyethylene.  After  compaction  of  the  mixture  into  tablets,  it  is  appropriate  to                check, by using X-ray diffraction, that the compression did not adversely affect the                crystal structure of the sample. Since the THz spectrum of polyethylene is flat, only                absorption maxima of the studied substance will appear.                  In  principle,  for  samples  prepared  this  way,  it  is  possible  to  directly  measure                complex  optical  constants  of  the  mixtures.  However,  the  porosity  of  the  sample,                which  leads  to  radiation  scattering  on  the  grains,  may  adversely  influence  the                measured  spectra,  but  this  is  usually  neglected.  The  impact  of  porosity  on  the                obtained data can be estimated for example by using Kramers-Kronig relations that                allow to transform the real part of a complex refractive index to the imaginary part,                and vice versa [5] and thus allow to verify the consistency of the spectra.                 By  contrast,  in  porous  tablets  with  a  known  composition,  the  dependence  of  the                refractive index on the air volume content can be used to determine their porosity,                which may be directly linked to tensile strength of the sample. Measuring the tablet                reflectance appears as the most appropriate method, allowing to map their surface                refractive index which may be influenced for example by the compressive force in                tablet production [6]. This may be important e.g., in tablets with a convex surface,                as their tensile strength is not based on a well-known empirical dependence on the                manufacturing compression force.                  In many cases, the THz spectra also reflect crystal polymorphism of APIs (active                pharmaceutical ingredients) [7] besides their chemical composition. A pilot study                [8] has shown that THz spectroscopy can also be used for real-time monitoring of                changes  occurring  in  crystal  structures.  Transmission  THz  spectra  of  an  active                substance (Carbamazepine) were measured at 165 °C, for approximately 2 hours.                After this period, a complete conversion of polymorphism has been detected. Due to                good sensitivity of THz spectroscopy, and short acquisition time for each spectrum,                it is possible to observe even significantly faster changes, occurring at the scale of                minutes, or even tens of seconds.                  It is known that vibrational modes of amorphous and crystalline substances are very                different. This was experimentally proven in THz spectra, for example, of samples                of  crystalline  and  amorphous  saccharides  [9].  The  absence  of  sharp  vibrational                modes  that  can  be  observed  in  crystalline  form  was  later  also  confirmed in  THz                spectra of indomethacin [7]. A comparison of spectra of a complex refractive index                of atorvastatin is shown in Fig. 6.5. Although the spectra of the crystalline phase do                not contain any distinct vibrational bands, there is an obvious contrast between the                spectra of the crystalline and the amorphous phases, respectively. It is evident that                the  differences  in  the  real  and  the  imaginary  parts  are  much  higher  than  the                                                      185","6 Terahertz time-domain spectroscopy                 experimental  inaccuracies  associated  with  grain  size  and  deviations  caused  by                imperfect compaction of powdered samples.                                                     Figure 6.5. THz spectra of the complex refractive index of atorvastatin powder                     samples. Full symbols: crystalline phase, open symbols: amorphous phase.                     Measurements were performed in cuvettes, and with different grain sizes.                   6.5.2  Determination of hydration state of substances                  Interaction of the THz radiation with molecules of water is generally very strong.                Furthermore,  it  is  usually  possible  to  clearly  determine  the  phase  of  the  water                present,  as  the  spectra  are  different  in  the  case  of  water  vapor  molecules  (very                narrow  bands),  liquid  water  (broad  absorption),  or  crystal  water  (which  usually                causes shifts of existing lines and changes in their intensity). Therefore, during the                measurements, as mentioned above, the THz beam is usually enclosed in a space                with controlled atmosphere, to eliminate the effects of ambient humidity.                 In  the  liquid  phase,  water  absorbs  the  THz  radiation  so  strongly  that  for                transmittance  measurements  of  aqueous  solutions,  it  is  necessary  to  use  special                cells,  where  the  optical  path  of  the  radiation  passes  through  the  solution  with  a                                                      186","6 Terahertz time-domain spectroscopy                 thickness  not  exceeding  100  µm.  If  a sufficient  sample volume  is  available,  it  is                possible to use for such measurements a special nozzle with freely flowing water                [10].                  Likewise, the  presence  of small quantities  of  water in  solid  samples  is  also  very                strongly apparent in THz spectra. Therefore, the THz spectroscopy is a very suitable                method  for  determination  of  hydration  state  of  samples  [11].  It  is  suitable  for                analysis of raw active substances, as well as for monitoring of production processes,                where it is possible to determine gradual changes in the drug water content.                                               Figure 6.6. THz spectra of relative amplitude transmittance of powder lactose.                 Unless stated otherwise, the samples were placed in a cuvette with 2 mm thickness.                  Transmittance can exceed value of 1, since it is related to a spectrum of an empty                    cell, and inserting the studied substance reduces reflection losses at internal                       interfaces. The d variable denotes the grain size of the monohydrate.                 An  example  of  the  difference  between  the  spectra  of  anhydrous  and  hydrated                substances is shown in Fig. 6.6. The spectra belong to powder lactose samples with                different grain sizes. Two anhydrous samples and three monohydrate samples were                measured. The spectra are visibly separated into two groups according to their water                content. Within these groups, there are also differences based on some other factors                ‒ optical path length of the cuvette, and grain size (see section 6.5.4), but these are                clearly smaller than the effect of hydration.                                                        187","6 Terahertz time-domain spectroscopy                 6.5.3  Determination of tablet coating thickness                 An important parameter, ensuring a correct dissolution profile, is the thickness of                coating,  which  is  particularly  important  in  tablets  with  sustained  release.  THz                spectroscopy  provides  a  non-destructive  way  of  measuring  of  coating  thickness,                with the use of time-resolved detection of THz waves (time of flight of reflected                radiation, see section 6.4.4). It is possible to detect the presence of hidden defects in                the  coating  of  a  tablet  that  would  be  otherwise  difficult  to  detect.  A  study  was                performed where coating thickness of separate tablets was measured [12], and the                results could be partly linked to their dissolution performance. It was also proven                that due to a relatively large penetration depth of the THz radiation, it is possible to                analyze multilayer coatings [13] with this method.                 Because of considerable time-demands of imaging measurements, the analyzes of                this  type  that  involve  surface  mapping  are  not  suitable  for  production  process                monitoring. However, analysis of a small group of tablets with different parameters                may help to optimize the production technology. By contrast, measuring of coating                thickness in one point or small area can be very fast (< 100 ms), which, in principle,                provides the potential for a technology of simplified control of coating thickness in                the production process.                   6.5.4  Determination of grain size                 As already mentioned, for powder crystalline substances with grain sizes exceeding                tens of µm, the scattering of THz radiation plays an important role, as it prevents                accurate  quantitative  evaluation  of  the  measured  spectra.  On  the  contrary,  the                average particle size can be determined based on the dependence of the intensity of                scattering  on  particle  size.  Fig.  6.6  shows  an  example  of  spectra  of  the  same                substance, but with different grain sizes. The sample is lactose monohydrate, the                transmittance  of  which  is  significantly  dependent  on  grain  size,  especially  in  the                region  of  higher  THz  frequencies  where  the  radiation  wavelength  is  comparable                with the size of the grains. The scattering mechanism can be generally described                using the Mie theory. In practice, however, it can be expected that this description                will be usually difficult to use, as the shape of grains and their size distribution can                be very varying. Therefore, the most appropriate approach seems to be a calibration                of  spectra  for  several  known  samples  of  a  substance,  enabling  subsequent                determination  of  the  grain  size  of  unknown  samples,  based  on  the  calibration                curves.                                                        188","6 Terahertz time-domain spectroscopy                 6.6  Resources and recommended literature                   1.   Terahertz Spectroscopy, Dexheimer S. L., (Ed.). Principles and Applications CRC                       Press: Boca Raton, USA, 2008.                  2.   Pashkin  A.,  Kempa  M.,  Němec  H.,  Kadlec  F.,  Kužel  P.  Phase-sensitive  time-                       domain  terahertz  reflection  spectroscopy,  Rev.  Sci.  Instr.  2003,  74  (11),  4711–                       4717. https://doi.org/10.1063/1.1614878                   3.   Hirori  H.,  Yamashita  K.,  Nagai  M.,  Tanaka  K.  Attenuated  Total  Reflection                       Spectroscopy in Time Domain Using Terahertz Coherent Pulses, Jap. J. of Appl.                       Phys. 2004, 43 (10A), L1287–L1289. https://doi.org/10.1143/JJAP.43.L1287                   4.   Newnham  D.  A.,  Taday  P.  F.  Pulsed  Terahertz  Attenuated  Total  Reflection                       Spectroscopy,   Appl.   Spectroscopy.   2008,   62    (4),   394–398.                       https://doi.org/10.1366/000370208784046731                   5.   Tuononen H., Gornov E., Zeitler J. A., Aaltonen J., Peiponen K.-E. Using modified                       Kramers–Kronig  relations  to  test  transmission  spectra  of  porous  media  in  THz-                       TDS, Opt. Lett. 2010, 35 (5), 631–633. https://doi.org/10.1364/OL.35.000631                   6.   May R. K., Su K., Han L. H., Zhong S. C., Elliott J. A., Gladden L. F., Evans M.,                       Shen  Y.  C.,  Zeitler  J.  A.  Hardness  and  density  distributions  of  pharmaceutical                       tablets measured by terahertz pulsed imaging, J. Pharm. Sci. 2013, 102 (7), 2179-                       2186. https://doi.org/10.1002/jps.23560                   7.   Strachan C. J., Rades T., Newnham D. A., Gordon K. C., Pepper M., Taday P. F.                       Using  terahertz  pulsed  spectroscopy  to  study  crystallinity  of  pharmaceutical                       materials,    Chem.     Phys.     Lett.    2004,     390,     20–24.                       https://doi.org/10.1016/j.cplett.2004.03.117                  8.   Zeitler J. A., Newnham D. A., Taday P. F., Strachan C. J., Pepper M., Gordon K.                       C.,  Rades  T.  Temperature  dependent  terahertz  pulsed  spectroscopy  of                       carbamazepine,    Thermochim.     Acta.     2005,     436,    71–77.                       https://doi.org/10.1016/j.tca.2005.07.006                   9.   Walther  M.,  Fischer  B.  M.,  Jepsen  P.  U.  Noncovalent  intermolecular  forces  in                       polycrystalline and amorphous saccharides in the far infrared, Chem. Phys. 2003,                       288, 261–288. https://doi.org/10.1016/S0301-0104(03)00031-4                   10.   Tauber M. J., Mathies R. A., Chen X., Bradforth S. E. Flowing liquid sample jet for                       resonance Raman and ultrafast optical spectroscopy, Rev. Sci. Instr. 2003, 74 (11),                       4958–4960. https://doi.org/10.1063/1.1614874                   11.   Zeitler  J.  A.,  Kogermann  K.,  Rantanen  J.,  Rades  T.,  Taday  P.  F.,  Pepper  M.,                       Aaltonen J., Strachan C. J. Drug hydrate systems and dehydration processes studied                       by  terahertz  pulsed  spectroscopy,  Int.  J.  of  Pharm.  2007,  334,  78–84.                       https://doi.org/10.1016/j.ijpharm.2006.10.027                                                      189","6 Terahertz time-domain spectroscopy                   12.   Spencer J. A., Gao Z., Moore T., Buhse L. F., Taday P. F., Newnham D. A., Shen                       Y., Portieri A., Husain A. Delayed Release Tablet Dissolution Related to Coating                       Thickness by Terahertz Pulsed Image Mapping, Pharm. Technol. 2007, 97, 1543–                       1550. https://doi.org/10.1002/jps.21051                  13.   Zeitler  J.  A.,  Shen  Y.,  Baker  C.,  Taday  P.  F.,  Pepper  M.,  Rades  T.  Analysis  of                       Coating  Structures  and  Interfaces  in  Solid  Oral  Dosage  Forms  by  Three                       Dimensional Terahertz Pulsed Imaging, J. of Pharm. Sci. 2006, 96 (2), 330–340.                       https://doi.org/10.1002/jps.20789                                                                                                              190","7 Mass spectrometry                 7  Mass spectrometry                 Lukáš Plaček                   7.1  Basic principles                 Mass  spectrometry  (MS)  is  a  physico-chemical  method  that  uses  separation  of                accelerated ionized particles (ions) based on their mass during their passage through                a magnetic and an electric field in vacuum. The main processes occurring in the                mass spectrometer are ionization of the analyte, separation of the resulting ions, and                subsequent detection. It is a destructive but highly sensitive method with very low                sample  consumption.  The  method  can  be  used  for  many  purposes  such  as                identification  of  unknown  compounds,  quantification  of  known  compounds,                structure determination of ions in a gas phase, and determination of physical and                chemical properties of ions and molecules. The method is formally categorized as a                spectral  technique,  although  it  does  not  belong  to  this  category  by  definition.                Spectral  techniques  observe  interaction  of  radiation  and  matter  and  describe  the                energy  differences  between  quantum  states.  MS  was  categorized  as  a  spectral                method only for the similarity of obtained records ‒ mass spectra ‒ and for similar                application in structural analysis. The mass spectrometry is traditionally not a solid-                state  analysis  method  as  most  of  the  ionization  techniques  require  a  solubilized                sample  (sample  preparation  requirements  are  discussed  separately  for  each                ionization technique, see below). The solid-state analysis is now much easier with                the newer, so-called ambient ionization techniques.                 Mass spectra are usually displayed in a normalized form, which means that the most                intense peak in the spectrum is assigned a 100% relative intensity and the intensities                of other peaks are then normalized accordingly. Normalized form is used for both                diagram  and  the  tabular  form  which  is  mainly  found  in  literature  dealing  with                spectra of electron ionization (Fig. 7.1). Mass spectrum is a relationship between                intensity and mass/charge ratio (m/z). The charge is equal to one, except for some                cases such as electrospray ionization (ESI ‒ explained below), which implies that                the value on the x axis corresponds to molar mass.                                                                  191","7 Mass spectrometry                                  Figure 7.1. Record of a mass spectrum of p-methylbenzoic acid, obtained by                              electron ionization, with included table of intensities.                 Mass  spectrometer  is  an  ion-optical  device  that  consists  of  three  main  parts                (Fig. 7.2), which are:                    •  ion source                    •  mass analyzer                    •  detector                                               Figure 7.2. Scheme of a mass spectrometer.                 Besides the main components, the operation of a spectrometer requires other parts.                In  case  of  direct  measurements  without  a  preceding  separation  technique,  it  is                mainly the input system or a probe that serves as a device for injection of a sample                into the ion source. Heated cartridges are used for both liquid and solid samples. In                combination  with  separation  techniques  (GC-MS,  HPLC-MS,  CE-MS,  etc.,  ‒                explained  below)  special  interfaces  are  used,  according  to  the  applied  ionization                technique. Another important part is a vacuum system since the separation of ions                based on the m/z ratio in a gas phase is carried out under high vacuum (10 –10 -                                                                                      -3                6   Pa, depending on the type of the analyzer). Ion cyclotron resonance – as the most                                                                 -7                                                                     -9                demanding – needs even higher vacuum of about 10 –10  Pa. Some ion sources                also  operate  under  vacuum  (EI,  CI,  MALDI,  etc.,  see  below).  The  employed                vacuum  prevents  collisions  with  neutral  atoms  and  thus  maintains  ions  with                sufficiently  long  mean  free  path.  Development  of  the  combination  of  LC-MS                                                      192","7 Mass spectrometry                 resulted in ionization techniques operating at atmospheric pressure (e.g., ESI, APCI,                APPI ‒ explained below).                 The  vacuum is  created by  a  multistep  pumping,  using  different  types  of  vacuum                pumps. The pumps can be generally divided into positive displacement, momentum                transfer, and entrapment pumps. Positive displacement pumps include rotary vane                pumps, diaphragm, and scroll pumps. Momentum transfer type includes diffusion                pumps. Entrapment pumps include sorption pumps, cryopumps, etc. An important                part  of  the  vacuum  system  is  a  vacuummeter,  which  must  have  sufficient  range,                sensitivity, and minimal effect on the measured environment (temperature increase,                formation of chemical compounds in discharges, etc.). Ionization gauge and thermal                conductivity vacuummeters may be stated as an example. Ion optics for focusing                and accelerating of ions are also indispensable, as is high-performance electronics                and a control unit.                  7.2  Ionization techniques                  Ion  source  is  used  in  a  mass  spectrometer  to  convert  neutral  analyte  to  charged                particles (ions), which are subsequently separated by the mass analyzer. Ionization                techniques  can  be  divided  into  soft  and  hard  techniques,  according  to  the  input                energy,  as  shown  in  Fig.  7.3.  Hard  techniques  form  primarily  molecular  radical                          +                cations  M •  which  are  subjects  to  further  fragmentation,  due  to  large  excess  of                internal  molecular  energy  obtained  within  the  ionization  process.  This  may  also                result in an absent molecular ion in some cases.                 Soft ionization techniques form only ions with even number of electrons. Because                                                                 +                                                                           +                                                                                     +                of ion-molecular reactions, these ions are, e.g., [M+H] , [M+Na] , [M+NH4] , [M-                H]  or others, depending on the particular ionization technique. Lack of structural                   -                information is compensated by collision-induced dissociation in the ion source, or                            n                by  using  MS   techniques  (tandem  mass  spectrometry).  The  ionization  techniques                can  be  approximately  arranged  according  to  their  \"hardness\",  =  according  to  an                excess of internal energy leading to fragmentation of ionized molecules, as follows:                ESI < MALDI < TSI < FAB/FIB < APCI < CI < EI; individual abbreviations are                explained further in the text.                                                                  193","7 Mass spectrometry                                                                Figure 7.3. Soft and hard ionization.                  Selection of an ionization technique (Fig. 7.4) depends on sample properties such as                volatility, polarity, thermal stability, and molecular weight which is partly related to                volatility.  An  important  consideration  for  selection  of  an  ionization  technique  is                whether the sample is a single compound or a mixture. Polarity of the ionization                techniques  can  be  set  by  software.  While  negative  ions  are  mainly  provided  by                sulfonic and carboxylic acids, or also polyhydroxylated analytes, positive ions can                be recorded for most substances.                                                                                 194","7 Mass spectrometry                                                      Figure 7.4. Application options for ionization techniques ESI, APCI and APPI                                           depending on analytes.                                  (Source: http://holcapek.upce.cz/teaching.htm, 10. 12. 2009.)                   7.2.1  Electron ionization (EI)                 Electron  ionization  is  the  hardest  and  the  oldest  ionization  technique.  The                requirement  for  measuring  mass  spectra  with  electron  ionization  is  a  sufficient                sample  volatility,  and  because the  ionization occurs  in  gas phase  at temperatures                typically around 150–400 °C, also a sufficient thermostability. Heated cathode (W                or  Re  filament)  emits  electrons  which  are  detected  at  anode  upon  their  passage                through the ion source (Fig. 7.5). As the emitting electron approaches the valence                electrons of a sample molecule, their magnetic fields are affected, which can lead to                release of a valence electron and thus creation of a radical cation M •. The created                                                                              +                ions are expelled from the ion source by a repeller. The ion beam is then further                focused  and  accelerated  by  other  electrodes  towards  the  analyzer.  Accelerating                potential between cathode and anode determines the energy of the electrons and is                expressed in electronvolts. To be able to create libraries and compare MS spectra, it                was necessary to choose an established and generally accepted value, and thus the                accelerating  energy  of  70  eV  was  chosen.  This  provides  highest  sensitivity,  but                since the energy needed to ionize most organic compounds is between 7–16 eV, the                                                    195","7 Mass spectrometry                 excessive  internal  energy  leads  to  a  massive  fragmentation  or  eventually                transformation and subsequent fragmentation of the molecule. Such spectra can be                too complicated, but they carry valuable information about substance structure.                                                              Figure 7.5. Scheme of an electron ionizer.                                  (Source: http://holcapek.upce.cz/teaching.htm, 10. 12. 2009.)                   7.2.2  Chemical ionization (CI)                 Chemical  ionization  was  developed  as  an  alternative  to  EI  which  is  a  very  hard                technique that causes strong fragmentation, making some substances impossible to                identify.  Design  of  a  CI  ion  source  is  analogous  to  EI,  and  the  analyte  is  also                converted  to  gas  phase.  The  source,  however,  contains  a  reaction  gas  which  is                present  in  excess  amounts  compared  to  the  analyte,  approximately  10000:1.                Pressure,  around  100  Pa  must  guarantee  sufficient  number  of  collisions.  The                ionizing electrons first ionize molecules of the reaction gas which then ionize the                                                                                     -                                                                           +                analyte  molecules  via  ion-molecular  reactions  and  form  [M+H]   or  [M-H]   ions.                The  reaction  gas  is  often  methane,  isobutene,  or  ammonia.  Examples  of  the                reactions  may  be  protonation,  hydride  abstraction,  charge  exchange,  or                condensation.                 The  examples in  Fig.  7.6 and  7.7  show  differences between the  electron  and  the                chemical ionization.                                                           196","7 Mass spectrometry                                               Figure 7.6. EI spectrum of 2-bromo-2-phenylacetonitrile; molecular                                        +                                      M  ion is missing in the spectrum.                                              Figure 7.7. CI (methane) spectrum of 2-bromo-2-phenylacetonitrile;                                                        81                       +                                  79                [M+H]  ions 196 of  Br isotope and 198 of  Br isotope are present in the spectrum.                                                                 197","7 Mass spectrometry                 7.2.3  Fast atom or ion bombardment ionization (FAB, FIB)                 These two techniques are very similar to each other in their principle, application,                sample preparation, and acquired results, and differ only in the presence or absence                of a charge of the bombarding particles. FAB (fast atom bombardment) uses high-                energy, neutral molecules of rare gases, usually argon or xenon, in a viscous matrix                which extends the life of the resulting ions. The matrix must meet some specific                requirements. The sample must be soluble in the chosen matrix, the matrix should                have low volatility, and should be chemically inert to the analyzed samples.  It is                also important that the ions coming from the matrix do not have the same m/z values                as  the  analyte  ions.  The  most  used  matrices  are  glycerol,  thioglycerol,  and                triethylamine. Nevertheless, other special matrices, such as liquid metals (Ga, In),                can  be  employed.  Accelerated  atoms  are  obtained  using  a  so-called  ion  cannon.                First, the atoms are ionized by electron ionization and then accelerated by voltage of                around 8–10 kV.                 The accelerated ions are then introduced into a chamber with high pressure, where                they collide with neutral atoms. The charge exchange does not cause loss in kinetic                energy, which results in a stream of high-energy accelerated atoms. These are led                onto a target with the sample placed in the matrix. Charged particles cannot get into                the  ion  source  as  they  are  diverted  by  an  electrode.  In  the  case  of  FIB  (fast  ion                                            +                bombardment), accelerated Cs  ions are used most often. Their formation can be                described as follows. Alkali aluminosilicates are heated to about 1000 °C, triggering                a thermal desorption of ions from surface. The resulting ions are then accelerated by                an electric field and focused onto a target with the sample placed in a matrix.                   7.2.4  Matrix assisted laser desorption/ionization (MALDI)                 MALDI (Fig. 7.8), is one of the softest ionization techniques, which enabled mass                spectrometry  analysis  of  such  molecules  as  peptides,  proteins,  oligonucleotides,                                                           6                nucleic acids, and biopolymers, up to MR ≈ 10 , triggering a rapid development in                the  field  of  biochemistry.  The  sample  is  first  dissolved,  mixed  with  a  suitable                matrix,  and  finally  deprived  of  the  solvent.  Its  surface  is  then  bombarded  under                vacuum by short laser pulses lasting in orders of nanoseconds or microseconds at                maximum. Energy absorbed by the matrix is then transferred on analyte and causes                ionization,  desorption  of  sample  ions,  and  ion-molecular  reactions.  It  is  a  pulsed                ionization  technique  which  is  most  often  associated  with  time  of  flight  analyzer                (TOF).                 UV lasers such as 337 nm nitrogen laser, 355 nm Nd:YAG, less frequently IR 2.94                µm  Er:YAG,  are  the  most  common  sources  of  laser  pulses.  Matrix  choice                considerably  affects  the  quality  of  ionization  as  the  matrix  must  absorb  the                                                     198","7 Mass spectrometry                 wavelength  of  the  laser  and  must  form  the  desired  crystal  with  the  analyte.                Furthermore, the matrix must be sufficiently stable, non-reactive with the analyte,                and  not  very  volatile.  The  most  commonly  used  matrices  are  organic  aromatic                carboxylic  acids,  e.g.,  dihydroxybenzoic  acid,  cinnamic  acid,  etc.  Sample                preparation also plays a key role, with several methods that have been developed.                Samples are prepared on targets which can be stored for a long time.                                                      Figure 7.8. Matrix assisted laser desorption/ionization – MALDI.                     (Source: http://www.magnet.fsu.edu/education/tutorials/tools/images/ionization-maldi.jpg, 7. 1. 2008.)                   7.2.5  Thermospray ionization (TSI)                 TSI (thermospray ionization) was the first ionization technique developed for the                HPLC-MS connection but is now receding. It can be used with flow rates reaching 1                ml/min and with mobile phases having high water content. Since it is a very soft                technique,  fragmentation  occurs  only  very  rarely,  and  most  ions  have  an  even                electron  number.  Analyte  dissolved  in  a  suitable  solvent  is  brought  via  metal                capillary into the ion source. The capillary end is heated in a thermostat to 150–300                °C, triggering a partial evaporation of the solvent in the capillary and thus creating a                supersonic flow of the solvent vapors and small droplets containing the analyzed                substance.  Evaporation  of  the  solvent  from  the  surface  of  the  droplets  decreases                their surface area and volume and increases their surface charge density. When the                charge density reaches a critical limit, it causes a so-called Coulomb explosion, i.e.,                shattering  of  the  charged  droplet  into  a  number  of  smaller  droplets  also  with  a                                                      199","7 Mass spectrometry                 charge. This is repeated until the droplets are small enough to enable desorption of                protonated or deprotonated molecules from their surface. This effect is called ion                evaporation.  Temperature of  the  heated capillary  depends on the  volatility  of the                analyzed  compound,  mobile  phase  flow,  and  the  solvents  applied.  Therefore,  for                increased  water  content,  it  is  necessary  to  use  a  higher  temperature.  An  overall                optimization is necessary, but in theory TSI can be used for any molecule that is                present  in  the  form  of  ions  when  already  in  the  solution.  TSI  is  generally  not                suitable for low-polar substances and large molecules.                   7.2.6  Electrospray ionization (ESI)                 ESI (electrospray ionization) is currently the most applied ionization technique for                the HPLC-MS connection, which is due to its compatibility with a wide range of                chromatographical  conditions.  It  is  suitable  for  moderately  polar  to  ionic                compounds with low- to high-molecular mass. It is considered the softest technique                and  is  therefore  preferably  used  also  in  analysis  of  thermolabile  substances  and                biopolymers. An analyte, dissolved in a suitable solvent, is brought to the ion source                via a metal capillary with high voltage of 3–5 kV. As a result, the created droplets                possess a high number of charges after they are sprayed at the end of the capillary                (Fig. 7.9). Dispersion of the eluate is maintained by coaxial inflow of nitrogen gas,                serving as a nebulization gas. Afterwards, the solvent evaporation causes Coulomb                explosion as in TSI. Evaporation of droplets and ion formation are facilitated by                formation  of  small  starting  droplets.  Therefore,  mobile  phases  with  high  surface                tension and/or high viscosity are not suitable. The droplets created by this method                carry high number of charges on their surface, which enables formation of ions with                multiple charges, currently used in analysis of biopolymers (large value of z allows                display in a smaller m/z range). Fig. 7.10 and 7.11 show examples of ESI spectra.                 ESI  in  HPLC-MS  connection  use  flow  rates  reaching  1  ml/min.  With  such  high                flow  rates,  a  flow  divider  is  used  to  decrease  the  amount  of  eluent  entering  the                ionizer.  Inflowing  liquid  is  split  into  two  flows  according  to  pressure  resistance                ratios  of  both  flows.  Therefore,  microcolumn  or  capillary  HPLC  are  ideal  for                connection with ESI, as their flow rates are consistent with the optimal flow rates                for ESI. Moreover, in this case there is no sensitivity reduction as with the flow                divider. There is also a special ESI modification, without the nebulizing gas and                with  lower  temperature  of  the  drying  gas,  called  nanoelectrospray.  Mobile  phase                flow  rates  range  from  units  to  hundreds  of  nl/min,  in  this  method.  The                nanoelectrospray uses a specially adjusted end of the spraying capillary. Extremely                low  sample  consumption  allows  in  vivo  experiments.  This  method  has very high                sensitivity  (concentrations  in  orders  of  attomoles  and  zeptomoles,  i.e.,  only                hundreds  of  molecules).  Nanoelectrospray  is  more  tolerant  to  salt  content  in  the                                                     200","7 Mass spectrometry                 sample,  reducing the  requirements for  sample  preparation.  The  disadvantages  are                high experimental demands, and low robustness.                                                                                                  Figure 7.9. Scheme of an electrospray.                  (Edited according to: Schalley C. A. Analytical Methods in Supramolecular Chemistry. Wiley-VCH, 2006., and                                     http://holcapek.upce.cz/teaching.htm, 10. 12. 2009.)                                                    201","7 Mass spectrometry                                                    Figure 7.10. ESI spectrum of polypropylene glycol in positive mode.                                              Figure 7.11. ESI spectrum of a highly sulfated α-cyclodextrin in negative mode.                                                              202","7 Mass spectrometry                 7.2.7  Atmospheric pressure chemical ionization (APCI)                 APCI (atmospheric pressure chemical ionization) is currently one of the standard                ionization techniques for the HPLC-MS. It is a gentle technique, except for certain                very unstable molecules. Electrode is placed near a heated column end and has a                high voltage pulse input of 3–4 kV, resulting in corona discharge which first ionizes                molecules of the mobile phase, because of their great abundance. The subsequent                ion-molecular reactions cause ionization of the analyte molecules. Counter-current                of drying gas, usually nitrogen, is used in a heated chamber to evaporate the carrier                liquid and to break any non-covalent clusters and associates. This technique is not                suitable for ionic and low volatility substances. Due to its suitability for ionization                of medium- and low-polar compounds, it is de facto a complementary technique to                ESI. It may be used with flow rates of up to 2 ml/min that is directly compatible                with conventional HPLC columns. It is tolerant to the presence of buffers in mobile                phases  and  to  changes  in  experimental  conditions  including  gradient  elution.  Its                limitation  is  the  impossibility  of  use  at  very  low  flow  rates.  A  scheme  of  the                interconnection  between  HPLC  and  MS  detector  is  shown  in  Fig.  7.12,  and  an                example of a spectrum is given in Fig. 7.13.                                               Figure 7.12. Scheme of the interface of atmospheric pressure chemical ionization.                                  (Source: http://holcapek.upce.cz/teaching.htm, 10. 12. 2009.)                                                            203","7 Mass spectrometry                                                           Figure 7.13. APCI spectrum of coffee with sugar.                  7.2.8  Atmospheric pressure photoionization (APPI)                 APPI (atmospheric pressure photoionization) is analogous to APCI, with the only                difference of ultraviolet radiation used instead of corona discharge (Fig. 7.14). The                source of UV radiation is usually a krypton lamp with photon energies of 10 or 10.6                eV. This energy is higher than ionization energies of most organic molecules, but                lower  than  the  energy  needed  to  ionize  atmospheric  oxygen  or  molecules  of  a                mobile  phase  such  as  acetonitrile,  methanol,  or  water.  This  enables  selective                ionization of the analyte. Unlike in APCI and ESI, there may emerge ions with an                odd  number  of  electrons,  which  can  complicate  the  interpretation.  Thus,  it  is                preferable to use dopant, which is usually toluene or benzene, or methoxybenzene                for RP-HPLC for nonpolar substances. Dopant is a substance with ionization energy                of  less  than  10  eV.  Dopant  molecules  react  by  ion-molecular  reactions  with  the                analyte  but  not  with  the  mobile  phase,  which  increases  sensitivity.  Combined                alternative APCI/APPI ion sources are now commonly used.                                                                   204","7 Mass spectrometry                                                     Figure 7.14. Scheme for interface of atmospheric pressure photoionization.                                  (Source: http://holcapek.upce.cz/teaching.htm, 10. 12. 2009.)                   7.2.9  New ionization techniques                 With the expansion of mass spectrometry in biological disciplines during the last                years, there are growing demands for minimum preparation and treatment of native                samples.  This  led  to  a  development  of  ambient  ionization  techniques  where  the                surface of often untreated sample is exposed to desorption medium under laboratory                conditions. The ambient techniques are undergoing a turbulent evolution, and the                unnecessity for sample preparation means that MS can now be more widely used for                a solid-state analysis. Some of the commercial ambient techniques are listed below,                while many others are currently in development:                    •  ionized spray in the case of electrospray desorption ionization (DESI)                    •  current of charged gas in the case of direct analysis in real time (DART)                    •  charged  agent  produced  by  nano-electrospray  in  Super  Secondary  electro-                      spray ionization (SESI)                    •  nano ESI                                                       205","7 Mass spectrometry                 Ions desorbed by these methods are then led to the analyzer (Fig. 7.15).                                                 Figure 7.15. Principle of DESI (upper part) and DART ionization (lower part).                                        (Source: Cooks R. G. et al. Science, 2006.)                   7.3  Mass analyzers                 A mass analyzer separates ions according to m/z ratio. It is located behind the ion                source so the incoming molecules are already converted into ions. The ions can be                separated based on their m/z according to several physical principles:                    •  flight trajectory bending in magnetic or electric field                    •  different  oscillation  stabilities  of  ions  in  two-  or  three-dimensional                      combination of DC and high-frequency AC voltage                    •  different time of flight in an area without a field                    •  different energy absorption during cycloidal movement of ions in combined                      magnetic and electric field                 An  important  parameter  for  evaluation  of  analyzer  properties  is  mass  resolving                power (RP) which is expressed as a ratio of mass of ion m1 and the difference in                mass of ions m1 and m2 which have a unit charge, while the peaks of both ions are                equally high and the gap between the peaks is 10%.                                                     206","7 Mass spectrometry                                                                                                            =     1                                                   (   −    )                                                           2                                                      1                An alternative definition of RP applies especially for quadrupoles and ion traps – a                ratio of ion mass m and the width of the peak of this ion Δm at its mid height.                                                                                                                =                                                                          Analyzer type significantly affects both the quality of acquired mass spectra and the                price of the mass spectrometer. High-resolution spectra obtained by sector analyzer                with double ion focusation or by a spectrometer with ion-cyclotron resonance allow                to determine elemental composition. On the other hand, low-resolution spectra only                allow resolution of ions differing at most by a unit of m/z. Several basic types of                analyzers  can  be  distinguished,  but  there  are  also  hybrid  devices  that  use                combinations  of  different  types  of  analyzers.  Examples  of  spectra  of  a  single                compound obtained by various types of analyzers are provided in Fig. 7.16.                   7.3.1  Magnetic analyzer with simple magnetic focusing                 This type of analyzers operates on the principle of bending the flight path of ions                which  pass  through  magnetic  field.  Positive  ions  with  a  certain  value  of  m/z  are                accelerated by a  negative potential  V and  enter  the magnetic  field  with  magnetic                induction B, which results in bending the ion trajectory to a trajectory with a certain                radius r. Stronger trajectory bending occurs for ions with lower m/z value as heavier                ions have greater inertia and their trajectory is bent less. A continuous change, i.e.,                scanning of B (magnetic scan) or V (potential scan) at a constant radius given for the                particular device forces all the ions to pass successively through the exit slot and                continue  to  the  detector  where  the  intensities  of  the  ions  with  specific  m/z  are                recorded, resulting in a mass spectrum.                                                                          207","7 Mass spectrometry                          ORBITRAP                                                               C 9 H 35 O 2 N 3 B 18 Co                                      465        467        469        471        473        475        477                                                     m/z                         ION TRAP                                       465        467        469        471        473        475        477                                                     m/z                     Figure 7.16. Comparison of resolution capabilities of Orbitrap and Ion trap                            demonstrated on mass spectrum of a cobalt-boron cluster.                   7.3.2  Sector analyzer with double ion focusing                 Sector  analyzer  with  double  ion  focusing  works  on  a  similar  principle  as  the                previous analyzer. In addition to the magnetic focusing, it also features electrostatic                focusing which significantly increases RP. If ions with different Ek and m/z enter an                electric field, their paths bend according to their Ek, regardless of m/z. Since ions                generated in the ion source have a certain energy distribution, explaining the width                                                      208","7 Mass spectrometry                 of the  obtained  peaks, it is  necessary to energetically  unite  the ions to achieve  a                greater  resolution.  This  can  be  achieved  by  an  electric  analyzer  which  yields  a                mono-energetic beam. The combination of magnetic and electric ion focusing can                                   5                achieve RP above 10 . There are four basic types of geometric arrangement of the                magnetic and the electric focusing in sector analyzers: according to Mattauch and                Herzog, Nier and Johnson, Matsuda, and inverse geometry. The advantage of this                type  of  analyzers  is  the  possibility  of  high-energy  activation  and  especially  the                possibility  of  measuring  also  the  so-called  metastable  ions.  These  are  ions  with                                  -4                                       -6                lifetime  around  10 –10  s,  which  means  that  they  break  down  on  their  way                between the ion source and the detector. Their presence causes formation of broad                diffuse  peaks  at  non-whole  m/z  values.  Their  detection  is  important  because  it                enables to study the fragmentation pathways.                   7.3.3  Quadrupole (Q)                Quadrupole  analyzer  consists  of  four  identical  metal  rods  of  circular  or  better                hyperbolic cross-section shape, 20–30 cm long. Two opposite bars have a positive                DC voltage U applied and the two other bars have a negative DC voltage U applied.                All four bars are then superimposed with high-frequency AC voltage V (Fig. 7.17).                An ion introduced into the center of the axis of the quadrupole begins to oscillate.                At a certain time and a certain U/V ratio, the oscillations are stable only for ion with                a particular value of m/z. Only this ion passes through the quadrupole and reaches                the detector. All other ions are captured on the quadrupole bars. Continuous change                – scanning – of the ratio of DC voltage and AC voltage amplitude U/V allows for                successive release of all ions into the detector, being a mass filter by its nature. For                maximum RP, the ideal cross-section of bars is hyperbolic. At the same time, higher                sensitivity  can  be  achieved  by  reducing  the  range.  Quadrupole  is  a  simple  and                relatively inexpensive mass analyzer which has become widespread also for GC-                MS and HPLC-MS combinations.                  7.3.4  Triple quadrupole analyzer (QqQ)                 The  triple  quadrupole  analyzer  is  probably  the  most  commonly  used  MS/MS                instrument. It consists of three quadrupoles arranged in a line where the middle one                (q) serves as a collision cell. The first quadrupole serves for isolation, the second for                dissociation, and the third for analysis of the resulting fragments. Introduction of a                collision  gas  into  the  second  quadrupole  causes  a  collision  activation  of  ions                selected by the first quadrupole, and their subsequent fragmentation. Unlike in ion                trap, this method can lead to repeated activated collisions, which means that it is                possible to observe more fragmented ions than in MS/MS measurements performed                                                     209","7 Mass spectrometry                 with ion traps. Triple quadrupole also allows measurements of precursor scans and                                    3                neutral loss scans. MS  measurements would require connecting of five quadrupoles                (QqQqQ), which is used very rarely. For high resolution MS measurements, it is                usually better to connect ion trap or cyclotron resonance with Fourier transform.                                                  Figure 7.17. Scheme of a quadrupole analyzer.                 (Source: Hillenkamp F. et al. MALDI MS: A practical Guide to Instrumentation, Methods and Applications. Wiley-                                                  VCH, 2007.)                  7.3.5  Ion trap (IT)                 Spherical  3D  ion  trap  is  composed  of  two  end  cap  cylindrical  electrodes  and  a                central ring electrode, as schematically depicted in Fig. 7.18. Ions are delivered into                the trap by a short voltage pulse through an inlet opening of the end cap electrode.                Here, they are captured and successively expelled towards the detector according to                their m/z. It is possible to use external ionization (ESI, APCI), as well as internal                ionization  in  the  ion  trap.  Accumulation  times  for  ion  capturing  range                approximately from 10 µm to 200 ms, depending on the amount of the introduced                ions. Time of excitation and collisions in the trap is around 20–60 ms. The ions are                retained  inside  the  ion  trap  by  using  appropriate  ratios  of  DC  and  AC  voltage                applied on the ring and the end cap electrodes. The ions are forced to move inside                the ion trap in closed orbits. With gradually increasing voltage amplitude, the ions                with increasing m/z get to unstable trajectories and leave the ion trap through an                outlet towards the detector. Helium is introduced into the trap as a dumping gas at a                                      -2                pressure of around 5.10  Pa to moderate unwanted oscillations and resulting in a                significant increase in RP and improved ion capture. The number of ions let into the                trap must be regulated because trap overfilling may lead to a discharge known as a                “space  charge”,  which  results  in  worse  resolution  and  sensitivity.  Ion  traps  can                perform tandem mass spectrometry without any other connected analyzer. Selected                precursor ion is retained in the trap and all other ions are expelled. This leads to                collision  activation  with  helium  atoms  present  in  the  trap  and  thus  also  to  ion                fragmentation. Subsequently, an MS/MS spectrum is recorded. The same procedure                                                     210","7 Mass spectrometry                 can be repeated many times, but in reality, it is limited by lifetime of the ions and                                                     5                sensitivity. It is practically used up to MS , which is entirely sufficient for structural                analysis. The process is of course fully automated. Ion trap often yields only one                step  fragmentations,  i.e.,  the  spectrum  contains  less  fragments,  which  simplifies                interpretation.  On  the  other  hand,  fragment  ions  may  be  missing  in  the  low                molecular region, called \"cut-off\", in one-third of the m/z of the precursor ion.                                                                       Figure 7.18. Scheme of an ion trap.                                  (Source: http://holcapek.upce.cz/teaching.htm, 10. 12. 2009.)                   7.3.6  Linear ion trap (LIT)                LIT (linear ion trap) consists of four parabolic bars, similarly as quadrupole, which                are  however  divided  into  three  parts  ‒  front,  center, and  back  (Fig.  7.19).  These                parts with appropriately selected DC and AC voltages are analogous to cylindrical                end cap and ring electrodes in the 3D trap. During scanning, the ions in unstable                states are expelled from the trap through slots which are parts of two opposite bars                of the central part of the trap. The advantages over 3D trap are obvious: because of                its length, the linear trap excels in better ion entrapment (about 70% of the injected                ions  are  stabilized  in  the  trap  compared  to  30%  in  3D)  and  higher  sensitivity,                because of the presence of two exit slots and two ion detectors.                                                      211","7 Mass spectrometry                                                            Figure 7.19. Scheme of a linear ion trap.                                  (Source: http://holcapek.upce.cz/teaching.htm, 10. 12. 2009.)                  7.3.7  Orbitrap                 Orbitrap  is  a  type  of  analyzer  somewhere  between  ion  trap  and  ion  cyclotron                resonance.  Ions  with  different  m/z  values  rotate  around  a  central  asymmetric                electrode that is placed in an inhomogeneous electric field which forces the ions not                only to circle, but also to oscillate. The frequency of oscillation is proportional to                the ratio of mass and charge of the given ion. Oscillating ions induce alternating                current  in  the  external  electrodes,  and  this  current  is  then  further  amplified  and                digitized. Dependence of the passage of the ions through the left and the right part                of the orbitrap on time is recorded as an electric current. After Fourier transform                (FT), the time  domain is converted  to frequency  (and  to mass,  after  calibration),                which  makes  the  spectrum  much  more  transparent.  Since  the  electric  current                measurement in time can be done very accurately and quickly, as well as the FT                processing, the resolution reaches values of up to 10 . Orbitrap does not reach the                                                                6                accuracy and resolution of a cyclotron, but its cost is significantly lower and it also                does  not  require  a  sophisticated  magnet/magnetic  field  accommodation  and                maintenance. An orbitrap is shown in Fig. 7.20.                                                                 212","7 Mass spectrometry                                                            0 0    5 5   10 10  15 15  20 20  25 25                                                                             čas [ms]                                                                             čas [ms]                                                                          time [ms]                                                                          FFT                                                                         FFT                                                                                       m/z                                                                              m/z                                                                             350                                                                                      500                                                                                  450                                                                               400                                                    0 0  50 50  100  150  200  250  300  350  400  450  500                                                                     250                                                                        300                                                                 200                                                           100                                                              150                                                                  Figure 7.20. Orbitrap.                     (Edited according to: http://aston.chem.purdue.edu/files/images/research/orbitrap1.png, 7. 1. 2008.)                                                           213","7 Mass spectrometry                 7.3.8  Time of flight analyzer (TOF)                 TOF analyzer is the simplest mass analyzer. It works on a principle that ions with                smaller m/z, but the same kinetic energy, move faster and reach the detector sooner.                It  is  a  typical  pulse  analyzer.  Ions  are  first  accelerated  by  a  very  short  pulse  at                entrance  into  the  analyzer  tube,  and  afterwards  the  time  required  for  the  ions  to                reach the detector is measured, determining their m/z. The scanning is fast, and the                mass range is in theory not limited and depends only on the time during which the                                                          6                impact of the ion occurs, which can be up to 10  m/z. Several TOF modifications are                used to increase resolution.                 Reflection time of flight  (r-TOF)  uses an ion mirror  (reflectron)  which  equalizes                different  kinetic  energies  of  ions  with  the  same  m/z  (Fig.  7.21).  As  already                mentioned  above,  during  ionization,  the  ions  gain  kinetic  energy  with  a  certain                distribution, which leads to a widening of their peaks and thus to decrease in RP.                The  principle  of  the  ion  mirror,  which  is  a  system  of  circular  electrodes  with                increasing voltage, is based on proportionality of Ek of ions (but not m/z) and the                depth of their penetration into an electric field of the reflectron. Ions with greater                kinetic  energy  penetrate  deeper  into  the  reflecting  electric  field  before  they  are                bounced back, and thus they are delayed in comparison with the ions with lower Ek,                leading to levelling of total paths for ions with different Ek.                 Another method is the delayed extraction which is based on delayed activation of                extraction voltage. A laser pulse at a time t = 0 causes an expansion of ions in a                deactivated  extraction  field,  followed  by  a  delayed  rapid  field  activation.  The                MS/MS  analysis  can  be  performed  also  with  the  TOF  analyzer.  The  repelling                electrode deflects all the unwanted ions, and the chosen precursor ion is selected for                the following collision-induced fragmentation. The ion can be fragmented during its                flight – thus forming ions with the same speed, but lower kinetic energy. In devices                without  reflectron,  all  these  ions  reach  the  detector  at  the  same  time  and  are                therefore not separated. However, with a reflectron, ions with different energies are                distinguished and can be separated according to their m/z. This is termed a \"post-                source decay\", as the fragments are formed only after extraction of the ions from the                ion source.                 Connection    of   quadrupole   with   the   TOF   analyzer,   i.e.,   QqTOF                (quadrupole/quadrupole/TOF) is also advantageous. The device is partially based on                a triple quadrupole, but the last quadrupole is replaced by a TOF analyzer.                 TOF analyzer can analyze all the ions that enter the analyzer at a given moment,                unlike the other analyzers which successively scan the given m/z region. Therefore,                the instrument allows continual full MS/MS spectrum measurement of products of                                                        214","7 Mass spectrometry                 each  ion  coming  from  the  source.  The  only  disadvantage  can  be  its  inability  to                provide precursor scans and neutral loss scans as the QqQ.                                                                       Figure 7.21. TOF analyzer scheme.                                  (Source: http://holcapek.upce.cz/teaching.htm, 10. 12. 2009.)                   7.3.9  Ion cyclotron resonance with Fourier transform (FT-ICR)                 Ion cyclotron resonance with Fourier transform (Fig. 7.22), completely differs from                the other analyzers in its parameters, price, resolution, and applied vacuum. A bulk                of ions is injected by a short pulse from the ion source into the cyclotron trap which                is placed in a strong magnetic field with induction up to 7 T. The ions rotate in a                plane  perpendicular  to  the  magnetic  induction  vector,  at  a  frequency  which  is  a                function of their m/z. If a potential radio-frequency signal, with a frequency of any                of the present ions, is inserted on a transmitter plate which forms the cyclotron trap,                the given ion starts to resonate. The radius of its trajectory increases, and its rotation                can be detected on a receiver plate as an induced current. If the frequency of the                inserted  voltage  is  scanned  (frequency  value  of  the  inserted  voltage  increases                linearly),  all  the  present  ions  will  successively  resonate.  After  mathematical                processing with Fourier transform a highly resolved mass spectrum with RP > 10  is                                                                                        6                obtained.                                                       215","7 Mass spectrometry                                                                 Figure 7.22. Scheme of ion cyclotron resonance with Fourier transform.                                  (Source: http://holcapek.upce.cz/teaching.htm, 10. 12. 2009.)                  7.4  Ion detection techniques                 Ion  detector  detects  ions  after  their  separation,  based  on  their  m/z,  and  it  also                determines  the  relative  intensities  of  the  individual  ions.  Detectors  for  mass                spectrometry can be categorized into two groups:                 1.  Detectors for direct measurements: detect electric current generated by direct                      incidence  of  ions.  These  detectors  are  necessary  for  determination  of  the                      exact isotopic representation of elements and are used in specialized systems.                       •  photographic plate – used in the past. Intensity was assessed according to                         the intensity of blackening.                                                       216","7 Mass spectrometry                       •  Faraday cups – ions are captured and discharged, and the current decrease                         is  detected  and  amplified.  It  is  a  very  accurate  measuring  used  for  the                         above-mentioned isotopic measurements.                 2.  Amplifying  detectors:  use  amplification  of  electrons  released  from  the  first                      conversion dynode after an ion impact. This is the most often applied type of                      detectors in mass spectrometry, as they can yield a sufficiently measurable                      signal for individual ions.                       •  discrete  dynode  electron  multipliers  –  consist  of  a  series  of  metal                         electrically connected dynodes. After the voltage is applied between the                         first  and  the  last  dynode,  the  electrons  are  successively  accelerated                         towards the following dynodes and are finally captured by collector. The                         incidence  of  ions  on  the  first  conversion  dynode  causes  secondary                         emission  of  electrons  from  its  material,  and  the  number  of  electrons  is                         multiplied after incidence on the following dynodes. The multipliers with                         discrete  dynodes  can  even  have  twenty  steps  and  can  achieve                                                    8                                                7                         amplification values of 10 –10 .                      •  continual dynode electron multipliers – consist of a curved tube from lead                         glass with a high electrical resistance. The tube is covered on the inside                         with a layer of beryllium or aluminum oxide. Contacts at the entrance and                         at the end of the tube are connected to a high voltage source. After an ion                         impacts, there are electrons emitted from the material of the tube which                         are  further  accelerated  by  the  electric  field  in  the  direction  towards  the                         collector.  Repeated  collisions  of  the  electrons  on  the  tube  wall  cause                         further  emissions  and  their  number  grows  exponentially.  Dimensions  of                         the multipliers with continuous dynodes can be advantageously reduced.                         The effectiveness of these systems is comparable to the discrete systems,                         but their life and sensitivity to ions with higher mass is lower.                       •  electron multiplier  –  is  probably  the most  used  currently.  Its lifetime  is                         one or two years, and it also exists in an arrangement with a field, which                         offers a higher amplification.                       •  scintillation  photomultiplier  detector  –  impacting  ions  are  converted  to                         photons  on  a  scintillation  material  and  then  detected  by  a  common                         photomultiplier. A thin metal film on the scintillation surface prevents the                         growth  of  a  surface  charge,  which  prevents  free  ion  incidence.                         Photomultiplier is also a very frequently used detector, with a longer life.                         Its great advantage is the possibility of measuring outside of high vacuum.                                                        217","7 Mass spectrometry                 7.5  Identification and determination of compounds                 Mass spectrometry is used for identification and quantification of substances. In the                former case, the spectrometer becomes a tool of structural analysis, and in the latter                case, the spectrometer serves as a detector, the signal of which is proportional to the                amount of the monitored substance. Mass spectrometer can be used for both tasks,                but  it  is  worth  mentioning  that  in  structural  analysis,  it  is  important  to  scan  a                spectrum  with  a  suitable  range,  while  for  the  selective  and  sensitive  quantitative                analysis with low detection limit, it is useful to monitor only the selected ion of a                known  structure,  or  its  fragment  (especially  in  chromatographic  separation;  Fig.                7.23).                                                             Figure 7.23. Schematic depiction of an MS/MS technique.                 Various scan types are distinguished in mass spectrometry (Fig. 7.24 and Fig. 7.25):                    •  Full  scan  is  a  measurement  of  a  mass  spectrum  in  the  whole  studied  m/z                      range; in the case of pre-connected separation technique, the scanning is done                      periodically during the whole separation and the mass spectra are recorded in                      time; after the measurement, it is possible to display the chromatogram as a                      dependence of a software selected ion on time, or as total ion current in time                      (TIC).                                                        218","7 Mass spectrometry                    •  Selected ion monitoring (SIM) measures only dependence of a selected ion                      signal  on  time;  only  one  or  few  ions  are  selected,  which  significantly                      increases the signal/noise (S/N) ratio.                    •  Product  ion  scan  results  from  measuring  MS/MS  spectrum  of  a  chosen                      precursor.                    •  Precursor  scan  monitors  original  precursor  ions  for  a  selected  fragment                      (typical for triple quadrupoles).                    •  Neutral  loss  scan  monitors  characteristic  ion  pairs  of  precursors  and                      fragments,  for  which  a  cleavage  of  a  selected  structural  unit  occurred;  for                      example Δ m/z = 18 is characteristic for water molecule cleavage.                    •  Selected reaction monitoring (SRM) uses a selection of a precursor ion by                      first analyzer and its following fragmentation in a collision cell. It does not                      record  a  full  spectrum  as  in  the  case  of  production  ion  scan,  but  only  the                      selected  fragmented  ion  or  several  ions  (transitions  ‒  several  successive                      fragments).  Along  with  SIM,  the  SRM  is  particularly  suitable  for  trace                      analysis of HPLC-MS or GC-MS. It is not a measurement of mass spectra,                      but  only  a  recording  of  a  signal  corresponding  to  a  selected  ion,  or  a                      fragmented  ion  in  SRM.  The  selected  transition,  or  more  transitions,                      markedly increase the S/N ratio and selectivity of the detection.                                                Figure 7.24. Comparison of a LC-MS technique depending on the scan type.                                                        219","7 Mass spectrometry                                                Figure 7.25. Chromatographical record of determination of methylester of                    methanesulfonic acid, at 15 ppm in an API, by GC-MS method (SIM mode).                 A  separation  preceding  a  quantitative  MS,  despite  high  selectivity  of  the                determination, is used to overcome a so-called “matrix effect” – an effect where the                analyte signal is not only a function of its concentration but can be influenced by                other substances in the sample. If the analyte signal is not affected by the matrix, a                flow injection analysis (FIA) technique can be applied, where the sample is injected                into a flow-through system without a separation column (Fig. 7.26).                                                                                   220","7 Mass spectrometry                                                  Figure 7.26. FIA-MS/MS; determination of meglumine in a dosage form, ten                                              sample injections.                   7.6  Structural MS analysis in pharmacy                 The  field  of  pharmaceutical  structural  analysis  includes  tasks  as  identification  of                main  substances  and  impurities,  from  small  organic  molecules  such  as  residual                solvents,  through  synthetic  active  pharmaceutical  ingredients  (API)  and  their                impurities  with  relative  molecular  mass  of  approximately  200–600,  to                macromolecular  substances,  e.g.,  determination  of  amino  acid  sequence  of  an                isolated peptide or other biopharmaceuticals. Pure and isolated substances can be                injected directly into the MS system, while the spectra of minor impurities should                be  measured  after  a  chromatographical  separation.  Selection  of  an  appropriate                technique for acquisition of mass spectra for subsequent identification of substances                plays  a  key  role.  Recommendations  for  selection  of  techniques  are  provided  in                Table 7.1.                 Interpretation  of  the  obtained  spectra  depends  on  the  ionization  and  the                measurement  technique  ‒  each  has  its  own  specifics  and  possible  methods  of                detection  or  clarification  of  the  given  structure.  In  case  of  any  doubts,  it  is                                                                                       n                appropriate  to  operatively  choose  another  ionization  technique,  perform  MS ,  or                obtain elementary composition of the molecular ion or its fragments.                                                     221","7 Mass spectrometry                    Table 7.1. Options for MS technique selection and connection with separation.                                         Separation       Ionization                    Substance type                                       MS analyzer                                       technique        technique                      Solvents           GC, headspace    EI, CI           IT, LIT, Q                                                                          IT,   LIT,   Q,                    Volatile substances   GC, split/splitless   EI, CI                                                                         Orbitrap                                                                           IT, LIT, Q, TOF,                    API, excipients    HPLC             ESI, APCI, APPI                                                                         Orbitrap, ICR                     Macromolecules                                       Orbitrap,  TOF,                                       HPLC             ESI, MALDI                    /Biopharmaceuticals                                  ICR                  Despite  great  variability  of  techniques,  it  is  possible  to  outline  basic  rules  for                spectra interpretation:                    •  finding the molecular ion and molecular adducts                    •  estimation of elementary composition based on the m/z of the molecular peak                      and the intensities of its isotopic satellites                    •  assessment of the most probable elementary composition if a value of highly                      resolved molecular ion mass is available (also from estimation in the previous                      step)                                                                                         n                   •  fragment mapping and neutral loss calculation (possible performing of MS )                Based on the information from the assignee, and the knowledge gained from the                interpretation,  a  possible  structure  is  suggested,  and  it  should  be  confirmed  or                specified by another structural analysis technique.                   7.6.1  Modern MS systems in pharmacy                 As  mentioned  above,  MS  systems  in  laboratories  of  pharmaceutical  structural                analysis should enable measurements of spectra of a wide range of sample types.                GC-MS  system  can  be  appropriately  set  with  a  headspace  gas  injector  or  with  a                split/splitless  injector  for  liquid  samples.  Capillary  columns  should  not  have  an                                                     222","7 Mass spectrometry                 overly  thick  layer  of  a  stationary  phase,  so  that  it  does  not  contaminate  the  ion                source ‒ commercial manufacturers differentiate between the use of GC columns                for MS and other detector types, and the information is provided in their catalogues.                Ion source of choice for GC-MS is EI in combination with CI, and as analyzer, it is                useful to apply ion trap (3D or linear), triple quadrupole for easy tandem MS or                orbital trap for structure elucidation. The inlet interface into an MS system may not                only be from a separation technique, but it is also practical to expect direct heated                probe use.                 HPLC systems for MS identification of substances contain a stable gradient pump                with a wide flow rate range, quality injector, sample magazine and a column space                with thermostat. For practical reasons, it is recommended to include a UV detector                for substance elution and impurity monitoring, as shown in Fig. 7.27. The mobile                phase  should  be  easily  evaporated  and  contain  water  or  aqueous  solution  of                ammonium  formate  or  acetate,  in  a  gradient  with  methanol  or  acetonitrile.  Any                parameter reversed-phase columns can be used, when providing the desired quality                of separation and ionization. For direct introduction of solutions into the ion source,                it is suitable to connect a syringe pump to the MS system. ESI and APCI ionization                techniques  should  not  be  missing  in  a  standard  LC/MS  apparatus  arrangement,                either as separate interchangeable units or combined robust sources. The analyzer                should be chosen with regards to the range of the m/z region (traps, quadrupoles,                and orbitrap only up to approx. 2000 m/z), simplicity of tandem MS, and the use of                high resolution. Although DESI and DART ionization techniques are not common,                they may be desired in structural analysis and MS imaging techniques.                 The  biopharmaceutical  industry  is  increasing  the  development  of  protein-based                biotherapeutics (such as monoclonal antibodies or vaccines). Complexity of these                drugs and their biological production by living cells requires their thorough analysis                to guarantee safety and efficacy. Intact protein analysis, peptide mapping, charge                variants,  oligonucleotides,  glycans  or  host  cell  protein  analysis  are  important                characterization steps for biotherapeutic proteins and they confirm that product with                the  correct  molecular  weight  has  been  expressed.  High-resolution,  accurate-mass                (HRAM)  Orbitrap  mass  spectrometry  has  been  shown  to  be  essential  for  this                technique.                 However, the suitability of techniques applied for a specific purpose should always                be  considered and  the  equipment selected  according to  the  analysis requirements                assigned by the client.                                                             223","7 Mass spectrometry                                                        Figure 7.27. In identification of impurities by LC-MS, it is useful to connect an UV                                     detector before the mass spectrometer.                                                                                  224","7 Mass spectrometry                 7.7  Resources and recommended literature                   1.   McLafferty  F. W.,  Tureček  F.  Interpretation  of  Mass  Spectra,  4   Edition.                                                                                  th                       University Science Books: Sausalito, USA, 1993.                  2.   Cole  R. B.  Electrospray  Ionization  Mass  Spectrometry,  Fundamentals,                       Instrumentation and Applications, John Wiley \& Sons: New York, USA, 1997.                   3.   Willoughby R., Sheehan E., Mitrovich, S. A Global View of LC/MS. Global View                       Publishing: Pittsburg, USA, 1998.                   4.   Ardrey R. E.  Liquid  Chromatography – Mass  Spectrometry:  An  introduction,                       Analytical Techniques in the Science. John Wiley \& Sons: Chichester, UK, 2003.                       https://doi.org/10.1002/0470867299                   5.   Kolektiv autorů. Sborník přednášek 3. ročníku školy HPLC-MS. Doubice, 2005.                   6.   Analytical  Methods  in  Supramolecular  Chemistry.  Schalley  C.  A.  (Ed.).  Wiley-                       VCH: Weinheim, Germany, 2006.                   7.   Cooks R. G., Ouyang Z., Takats Z., Wiseman J. M. Ambient Mass Spectrometry.                       Science 2006, 311 (5767), 1566–1570. https://doi.org/10.1126/science.1119426                   8.   Kogej  M.,  Schalley  C. A.  Mass  Spectroscopy  and  Gas  Phase  Chemistry                       of Supramolecules. In: Analytical Methods in Supramolecular Chemistry, Schalley                       C. A., (Ed.). Wiley-VCH: Weinheim, Germany, 2007, pp. 104–162.                   9.   MALDI     MS:    A   Practical   Guide   to   Instrumentation,   Methods                       and  Applications.  Hillenkamp  F.,  Peter-Katalinic  J.,  (Eds.).  Wiley-VCH:                       Weinheim, Germany, 2007.                   10.   Rouessac  F.,  Rouessac  A.  Mass  Spectrometry.  In  Chemical  Analysis  –  Modern                       Instrumentation  Methods  and  Techniques,  2   Edition;  John  Wiley  \&  Sons:                                                              nd                       Chichester, UK, 2007, pp. 369–418.                   11.   Aplikační listy. Thermo Fisher Scientific.                  12.   Hmotnostní   spektrometrie   (MS),   http://tomcat.bf.jcu.cz/sima/analyticka_                       chemie/vybranemet.htm. December 2009.                   13.   Hillenkamp F., Katalinic J. P. MALDI MS: A practical Guide to Instrumentation,                       Methods   and   Applications.   Wiley-VCH:   Weinheim,   Germany,   2007.                       https://doi.org/10.1002/9783527610464                   14.   Holčapek M. – Mass   Spectroscopy    Group,     http://holcapek.upce.cz/                       teaching.htm. December 2009.                                                         225","8 Thermal analysis                 8  Thermal analysis                 Lucie Krumbholcová, Jiří Dohnal, Izabela Jendrzejewska                    8.1  Definition of thermal analysis methods                 The  principle  of  thermal  analysis  is  heating  a  solid  sample  and  recording  the  physical                changes and chemical reactions that occur under the influence of the supplied heat. During                the  heating,  several  physicochemical  phenomena  characteristic  for  the  tested  substance                occur, such as:                    •  change of the physical state (melting, crystallization, sublimation)                   •  phase  transformations  (formation  of  a  crystal  lattice,  polymorphic                      transformations)                    •  chemical reactions (thermal dissociation, reactions between substrates in the                      solid phase)                  These  phenomena  are  accompanied  by  a  change  in  enthalpy  (  H).  If  we  are                dealing with an endothermic reaction (H > 0), heat is absorbed, e.g., dehydration,                reduction.  An  exothermic  reaction  (H  <  0)  is  related  to  heat  release,  e.g.,                crystallization or oxidation.                 Each substance can be characterized by free enthalpy content (G), which is given                by the relationship:                                            H – change of enthalpy                   ∆   = ∆    −    ∆       T – temperature                                            S – change of entropy                 Each  system  attempts  to  achieve  a  state  corresponding  to  a  lower  free  enthalpy                content  at  a  given  temperature.  The  heating  or  cooling  of  a  substance  leads  to                reversible or irreversible changes in its size, depending on its initial dimensions and                temperature.  Also,  analysis  of  gaseous  products  of  chemical  reactions  and                monitoring  other  physico-chemical  parameters,  such  as  electrical  and  thermal                conductivity,  optical  properties,  dielectric  constant,  thermoelectric  voltage,                magnetic  properties,  etc., are the  principles  of  thermal  analysis  methods.  Since  a                chemical  reaction  or  a  phase  change is accompanied  by simultaneous  changes in                several  physico-chemical  parameters,  various  complementary  thermal  analysis                                                     226","8 Thermal analysis                 methods can be used to obtain more results to ensure a better match of experimental                conditions.                 One of the oldest thermal analysis methods is thermogravimetry analysis (TGA) and                differential thermal  analysis  (DTA).  First  works  with  thermogravimetry character                were already carried out in 1893 and 1914, even if the scales used at that time were                not  termed  thermobalances.  Thermobalance  is  a  device  allowing  to  express  or                graphically  record  weight  changes  of  a  sample  subjected  to  heating  or  cooling,                depending on temperature or time. The beginnings of DTA date back to the same                period  as  the  thermogravimetric  method  itself.  In  1886,  it  was  first  used  by  Le                Chatelier for studying calcite and later for studying clay materials. The technique                was advanced in 1891 by Roberts-Austen by introducing differential thermoelectric                cells which measured thermoelectric voltage difference between a cell located in the                studied sample and a cell located in the standard sample which was not subject to                changes.                   8.2  General principle of thermal analysis methods                  Thermal  analysis  belongs  to  experimental  material  analysis.  New  methods  for                monitoring  material  changes  depending  on  temperature  are  still  developed.                Generally, there are three quantities of interest:                    •  Time – changes in time are observed during isothermal heating. In practice,                      the isothermal heating of a studied material is combined with non-isothermal                      heating. In non-isothermal heating, a heating rate is determined, and the time                      is then given by the heating rate and the temperature interval.                     •  Temperature  –  besides  isothermal  conditions,  the  variable  temperature  is                      often used in thermal methods. Primarily, a constant heating rate is used, but                      the current level of regulatory and information technology also allows other                      types,  such  as  periodic  temperature  increase  or  decrease  in  a  chosen                      temperature range where the temperature rise can have a constant rate. The                      decrease may be spontaneous or controlled along with the heating. Another                      option is a periodic heating rate change with a sinusoidal or another period.                     •  Material properties – thermal analysis techniques are classified according to                      the observed property type. An overview of the essential techniques is listed                      in Table 8.1.                                                           227","8 Thermal analysis                          Table 8.1. A list of the most common thermal analysis techniques.                       Method name                      Studied quantity                        Differential   thermal   analysis  thermal  difference  between  the                     (DTA)                            studied and a standard sample                       Differential  scanning  calorimetry  thermal   energy   provided   for                     (DSC)                            compensation    of   temperature                                                      between the studied and a standard                                                      sample                       Thermogravimetry       analysis  weight change                     (TGA)                        Differential   thermogravimetric  the first derivation of weight change                     analysis (DTGA)                        Thermomechanical       analysis  mechanical    property    change                     (TMA)                            (module, hardness)                        Dilatometry                      volume change                       Effluent gas analysis            studied gas volume                        Pyrolysis                        pyrolysis products                       Thermal luminescence analysis    light emission                        Electric conductivity analysis   electric conductivity change                                                            228","8 Thermal analysis                 8.3  Thermal methods                  Thermal analysis is a broad term referring to methods that measure the physical and                chemical properties of a substance, a mixture, or a reaction mixture as a function of                temperature or time during a controlled temperature program. The sample may also                be exposed to other effects (e.g., reactive atmosphere, static or dynamic mechanic                load). The application field of thermal analysis in pharmacy is wide.                   8.3.1  Basic measurement factors                  The  variables  affecting  measurements  can  be  divided  into  the  three  following                groups.                    •  Instrumental factors:                       o  thermal source and the thermoregulatory system                       o  temperature measuring system                       o  thermoanalytical curve recording system                      o  detection system                    •  Methodical factors:                       o  temperature program                       o  the geometrical arrangement of the measuring system                       o  atmospheric effect                       o  sample placement and preparation                    •  Properties of measured substances:                      o  physical and chemical properties of the measured sample                       o  particle size and shape                       o  sample thermal conductivity                       o  density and thermal capacity                       o  the effect of humidity and (residual) solvents                      o  crystallinity and sample purity degree                                                      229","8 Thermal analysis                 8.3.2  Measuring procedure                 8.3.2.1  Instrument calibration                 Accurate  temperature  determination  is  crucial,  and  because  of  the  different                instrument  construction  and  temperature  measuring  systems,  there  must  be                internationally  accepted  temperature  calibration  standards.  The  international                confederation  for  thermal  analysis  and  calorimetry  (ICTAC)  includes  a  scientific                committee  coordinating  standardization  and  nomenclature  activities.  There  exist                some standards that have been comparatively measured in laboratories worldwide.                These standards are high purity, low molecular substances with precisely defined                thermal characteristics: boiling point, melting point, solidification temperature.                  The calibration is performed by inserting a standard sample into the measuring cell,                and an empty capsule is used to prepare the standard into the reference cell. The                phase transition is sharp and can be accurately read. The standards are supplied with                the  instruments.  Calibration  is  performed  in  the  same  range  and  intervals  as  the                following measurements, as the instrument properties may change over time. For                some  methods  (DSC),  enthalpy  is  also  calibrated  as  one  of  the  outputs.  In                thermogravimetry,  calibration  of  scales  is  essential,  making  it  necessary  to                determine  whether the measured  weight  of  the  standard  at  different  temperatures                corresponds to the actual weight.                    8.3.2.2  Sample preparation before analysis                 Due to the different nature of the thermoanalytical measurements, it is clear that the                sample  weight and  dimensions are  important for measurement outcomes, as they                affect the heat flux between the environment and the measured sample. Generally, it                is good to use identically large samples spread evenly over a pan or a measuring cell                to achieve reproducibility.                   8.3.2.3  Measurement atmosphere                  The  type  of  gas  used  for  creating  the  measuring  environment  depends  on  the                measurement  type.  Inert  nitrogen  is  used  for  melting  temperature  measurements,                polymorph screening, glass transition temperature measurement, etc. The nitrogen                flow also removes gaseous products formed during the heating. Oxygen is used to                measure degradation products during oxidation or other processes. The oxygen must                then have access to the sample, i.e., the lid of the closed pan is pierced.                                                     230","8 Thermal analysis                 8.3.2.4  Heating rate                 The  heating  rate  depends  on  the  information  that  is  to  be  obtained.  Typical                measurements are performed at a rate of 10 °C/min. However, to determine glass                transition temperature, it is often preferable to choose a higher rate of up to 500                °C/min. High sensitivity measurement changes, at speeds of up to 1.2° C/min, are                enabled by microcalorimetry. In practice, an unknown sample is recommended to                be  measured  at  several  heating  rates  to  better  interpret  the  ongoing  processes.                Instrument  calibration  must  be  performed  after  every  heating  rate  adjustment.                Different behavior of one substance at different heating rates is documented in Fig.                8.1.                                                       Figure 8.1. DSC for one sample measured at different heating rates.                                   (Source: Nicoara, et al., J. Therm. Anal. Calorim., 2016.)                   8.3.3  Evaluation of thermoanalytical measurements                 Two  curve  characteristics  are  typically  evaluated  in  thermoanalytical                measurements:                    •  Thermal process position, i.e., the temperature at which the process occurs.                      Peak  value  and  onset  (tangent  line  to  the  curve)  is  obtained.  Next,  the                                                      231","8 Thermal analysis                       inflexion point is determined – typically for glass transitions of polymers or                      amorphous materials (Fig. 8.2 and 8.3.).                    •  Peak  area  proportional  to  weight  change  in  TGA  and  enthalpy  process  in                      DSC; generally determined by integration, see Fig. 8.4a and 8.4b.                                            Figure 8.2. A typical record of a DSC curve for crystalline substance melting.                                       Evaluation – peak, onset and H.                                      (Source: Araujo et al. Braz. J. Pharm. Sci., 2010.)                                                Figure 8.3. Glass transition curve record for an amorphous substance.                                              Evaluation – Tg.                                       (Source: Joseph et al. Energy Procedia, 2011.)                                                    232","8 Thermal analysis                                                Figure 8.4a. TGA curve record for a pharmaceutical substance hydrate                 (CaC2O4·H2O). The curve with a distinct minimum represents the first derivation of                                   a TGA curve. Evaluation – water content.                   (Source: https://www.sciencedirect.com/topics/materials-science/differential-thermal-analysis, 6.02.2022.)                                                Figure 8.4b. DSC record of the substance from Fig.8.4a, showing decomposition of                 hydrated calcium oxalate (CaC2O4·H2O) in three stages. The ignition temperature of                                           838–1025°C is evident.                    (Source: https://www.sathyabama.ac.in/sites/default/files/course-material/2020-10/SCY2.pdf, 6.02.2022.)                                                    233","8 Thermal analysis                 8.3.4  Differential scanning thermal analysis (DSC)                 8.3.4.1  Theoretical basics of DSC                 Differential  scanning  thermal  analysis  (DSC)  is  one  of  the  most  applied  thermal                analysis methods in pharmaceutical development. In DSC, the sample is subjected                to linear heating, and the heat flux rate in the sample is proportional to the actual                specific heat and is continuously measured. Two symmetrical containers are placed                inside the measuring jacket, typically maintained at room temperature. The primary                temperature control system consists of a resistance thermometer and a heating unit                embedded  in  the  sample  holder.  The  secondary  temperature  control  system                measures  the  temperature difference  between  both  carriers,  and  this  difference  is                adjusted to zero value by controlling the measured  heat flux. In other words, the                sample  temperature  is  maintained  isothermally  with  the  comparative  sample  (or                block) supplied with heat. The amount needed to maintain isothermal conditions is                recorded  as  a  measure  of  time  or  temperature.  The  electric  input  required  to                maintain the isothermal conditions is measured instead of differential temperature                as  in  classical  DTA.  The  use  of  small  samples  (milligram  quantities)  placed  on                metal foils reduces the thermal gradient to a minimum. The small heat capacity of                the whole system allows the use of high heating rates (tens to hundreds of °C/min)                and provides a high-resolution capability. The amount of released (seized) heat is                thus  proportional  to  the  amount  of  electric  energy  used  to  heat  the  sample                (standard). It is, therefore, a calorimetric method. Devices and their principles are                shown in Fig. 8.5‒8.7.                                                   Figure 8.5. DSC Pyris 1 and TGA6 by Perkin Elmer company.                                                        234","8 Thermal analysis                                                                  Figure 8.6. Measuring cell in DSC Pyris 1.                                     (Source: Aplikace DSC Pyris1. Perkin Elmer, 1997.)                                                Figure 8.7. Closing of measuring room for its isolation from extraneous influence.                                     (Source: Aplikace DSC Pyris1. Perkin Elmer, 1997.)                                                      235","8 Thermal analysis                 Besides  the  measured  quantity,  methods  can  be  characterized  based  on  thermal                balance also by the basic thermal flow between:                    •  sample and surroundings                    •  reference and surroundings                    •  sample and reference                 The following applies for sample enthalpy change:                                               ∆HS – measured quantity                                              qS – enthalpy term                   ∆HS = qS +  ∆q  + QS        ∆q – thermal conductivity term                                               QS – inertion term + (supplement term)                    8.3.4.2  Sample preparation                 It  should  always  be  kept  in  mind  that  a  sample  is  heated  or  cooled  during  its                preparation through  direct contact  with the  platform on  which it  is  placed.  Good                thermal contact with the platform is therefore necessary. The sample must be placed                evenly  on  the  bottom  of  the  crucible  or  tamped  onto  it,  and  it  should  have  a                minimum  thickness.  Weight  depends  on  the  expected  thermal  effect.  In  general,                greater  sample  weight  reduces  the  instrument  sensitivity.  When  studying  subtle                effects  (glass  transition,  heat  capacity  measurements,  etc.),  the  sample  amount                should  be  greater  than  10  mg.  The  sample  mass  should  be  about  2‒6  mg  when                determining  purity  or  phase  transition  enthalpy.  The  scales  should  be  selected                accordingly when weighing samples for the DSC measurements.                 The selection of an appropriate pan, see Fig. 8.8, is another crucial step in sample                preparation before measurement. The crucibles are made of different materials, and                according to the sample characteristics, they may be non-hermetic and hermetic or                high-pressure.  The  most  common  crucibles  are  aluminum  that  can  be  used  for                measurements up to 550 °C. Less common crucibles for special applications can be                made of, e.g., graphite, copper, gold, alumina or platinum crucibles. Generally, non-                hermetic aluminum crucibles can be used for non-volatile samples, and hermetic or                high-pressure crucibles must be used for liquid samples and organic materials.                                                         236","8 Thermal analysis                                                            Figure 8.8. DSC measuring crucibles.                    8.3.4.3  Application of DSC                 The  most  common  applications  of  DSC  in  pharmaceutical  analysis  include  the                determination  of  API  purity  and  the  study  of  API  polymorphism  and  excipients.                Some examples are shown in Fig. 8.9‒8.11.                                                                 Figure 8.9. Purity of phenacetine.                                                                         TM                         (Source: TA Instruments, Cassel RB, Purity determination and DSC Tzero  Technology)                                                      237","8 Thermal analysis                                             Figure 8.10. DSC sample curves for polymorphic forms of carbamazepine.                                            (Source: https://www.hitachi-                    hightech.com/file/global/pdf/products/science/appli/ana/thermal/application_TA_079e.pdf, 20. 02. 2022.)                                               Figure 8.11. Evaluation of crystallinity using DSC: pure fenofibrate (blue curve),                  physical mixture of 20% fenofibrate with Klucel LF (green curve), recrystallized                      solid dispersion (purple curve), amorphous solid dispersion (red curve).                                     (Source: Feng et al. Drug. Dev. Ind. Pharm., 2014.)                                                            238","8 Thermal analysis                 8.3.5  Hyper DSC                 Hyper  DSC  offers  a  much  more  sensitive  evaluation  of  changes  during  rapid                temperature  increase  to  up  to  500  °C/min  (Fig.  8.12).  This  technique  provides                additional and often crucial information about the tested material.                Advantages of Hyper DSC:                     •  speed, simplicity                   •  lower occurrence of effects typical for lower temperature increase rates that                      may  lead  to  incorrect  result  interpretation  (water  evaporation,                      recrystallization, or chemical degradation)                    •  detection of effects with lower kinetic energy (glass transition or melting)                 Disadvantages of Hyper DSC:                    •  calibration for every individual heat rate                 Application of Hyper DSC:                    •  more accessible study of polymorphism                    •  improved identification of glass transition                    •  realistic simulation of polymorphism behavior                    •  protein analysis                                                                                239","8 Thermal analysis                                              Figure 8.12. Diamond Hyper DSC by Perkin Elmer company (with autosampler).                   8.3.6  Micro DSC                 Another thermal analysis technique used in pharmaceutical development is a purely                calorimetric  method.  It  monitors  the  enthalpy  and  thermal  capacity  of  processes                during the measurement, and it also enables the measurement of mixtures of two                substances  and  observation  of  their  reactions.  The  sample  is  heated  or  cooled,                resulting in structural and chemical composition changes. These changes are usually                associated with heat exchange. Micro DSC is a highly sensitive technique with a                special place among other DSC techniques.                   8.3.6.1  Micro DSC III (SETARAM)                 Micro DSC III device by SETARAM (Fig. 8.13) has a temperature range of 20 ~                120  °C.  Its  detection  limit  is  significantly  lower than  in  commonly  applied  DSC                techniques,  and  it  can  detect  calorimetric  changes  of  less  than  1  W,  allowing                analysis of small sample amounts, with a very low temperature increase rate from                0.001 to 1.2 °C/min.                                                            240","8 Thermal analysis                                               Figure 8.13. Micro DSC by SETARAM (from left: external cooling, unit with a                      calorimetry block, and CS 32 unit for collection and data transfer to PC).                  Technical arrangement                 The  calorimetric  block  consists  of  gold-coated  cylinders  with  high  thermal                conductivity.  This  arrangement  provides  excellent  temperature  homogeneity  and                precise control. There are two cavities in a block that serves as a measuring space                for experimental cells. A three-dimensional thermal flow sensor (3D sensor) enables                the  Micro  DSC  III  to  achieve  high  sensitivity  (detection  limit  of  0.2  mW)  with                perfect precision.                  The calorimetric block (Fig. 8.14) is very well protected against thermal influences                from the external environment, which ensures the ability to detect minimal thermal                effects in the sample.                                                                       241","8 Thermal analysis                                                                              Figure 8.14. Calorimetry block.                                    (Source: Aplikační listy. SETARAM Instrumentation.)                 Types of measurement cells                 Several  experimental  cells  ensure  the  flexibility  of  Micro  DSC  III.  There  are                insertion \"Batch\" types or flow-through types of measuring cells. All cells can be                used for isothermal as well as for DSC measurements. They are made of unique                                                                         3                \"Hastelloy  C\"  (brand  name)  steel,  have  a  volume  of  1  cm ,  and  are  quickly                changeable and easy to clean.                 The  most  common  cells  in  pharmaceutical  development  are  closed  \"Batch\"  cells                and mixing \"Batch\" cells.                 A closed \"Batch\" cell (Fig. 8.15a) for analysis of raw solid and liquid samples. A                screw-on  ensures  good  sealing  with  a  gasket  that  can  withstand  the  maximum                internal  pressure  of  20  bar.  The  cell  is  used  for  studies  of  protein  denaturation,                phase transitions, gelling processes, and compatibility studies for active substances                with excipients.                                                      242","8 Thermal analysis                 The  mixing  \"Batch\"  cell  (Fig.  8.15b)  has  two  separate  compartments  for  the                insertion of substances for which the mixing processes are to be observed. The cell                also  allows  manual  mixing  and  is  used  for  studying  reactions  between  solid  and                liquid substances.                           a                                  b                                               Figure 8.15. Closed \"Batch \"cell (a); mixing \"Batch \"cell (b).                                    (Source: Aplikační listy. SETARAM Instrumentation.)                  8.3.6.2  Application of Micro DSC III                 DSC mode                     •  Denaturation  ‒  aggregation  of  collagen  solutions.  Collagen  is  a  fibrous                      protein  with  three  chains  bound  in  a  triple  helix.  Like  other  proteins,  it                      denatures when heated. At the same time, a subtle endothermal effect occurs,                      which can be measured only by sensitive methods, such as micro DSC (Fig.                      8.16).                                                                    243","8 Thermal analysis                                                      Figure 8.16. Denaturation – aggregation of collagen solutions.                                    (Source: Aplikační listy. SETARAM Instrumentation.)                     •  Starch gelatinization. Starch is a hydrocompatible polymer, naturally forming                      water-insoluble crystalline granules. If such suspension is heated, a transition                      of crystalline state occurs, and a gel is formed. It is once again accompanied                      by a small endothermal effect that is detectable by micro DSC (Fig. 8.17).                    •  Phase  transformations  of  phospholipids.  Phospholipids  are  one  of  the  cell                      membrane  building  blocks.  Upon  their  dispersion  in  water,  two  thermal                      effects can be observed: i) gel/gel transition appearing at a lower temperature,                      accompanied by a molecular rearrangement, ii) and the following transition                      between  the  gel  and  the  crystalline  phase,  accompanied  by  melting                      hydrocarbon chains (Fig. 8.18).                                                                          244","8 Thermal analysis                                                                     Figure 8.17. Starch gelatinization.                                    (Source: Aplikační listy. SETARAM Instrumentation.)                                                              Figure 8.18. Phase transitions of phospholipids.                                    (Source: Aplikační listy. SETARAM Instrumentation.)                                                         245","8 Thermal analysis                     •  Denaturation and aggregation of immunoglobulin solution. Immunoglobulin                      is a protective protein in blood plasma, the lymphatic system, and some other                      body fluids. Like other protein structures, immunoglobulin solution begins to                      denature  during  heating.  An  endothermal  effect  accompanies  denaturation,                      and  the  temperature  characterizes  the  protein's  thermal  stability.  A  higher                      denaturation  temperature  means  a  more  stable  protein.  Fig.  8.19  shows  an                      aggregation accompanied by an exothermal effect.                                               Figure 8.19. Denaturation and aggregation of an immunoglobulin solution.                                    (Source: Aplikační listy. SETARAM Instrumentation.)                 The examples mentioned above show that micro DSC can measure minimal thermal                changes which cannot be measured by classical thermal analysis methods, such as                DSC.                 Isothermal mode                     •  Compatibilities  between  active  substances  and  excipients.  Whether  in                      original or generic products, selection of proper excipients in pharmaceutical                      development  is  necessary.  Micro  DSC  can  speed  up  the  selection  by                      revealing  possible  reactions  between  an  excipient  and  an  active  substance                      (Fig. 8.20). The information could otherwise be obtained later from long-term                      stability tests where product degradation will likely occur by, e.g., formation                      of  similar  undesirable  substances  after  a  long  time  (at  least  three  months).                      The  micro  DSC  measurement  is  relatively  simple,  and  it  is  based  on                      measurements of two-component mixtures at predefined ratios and gradually                                                      246","8 Thermal analysis                       increasing  temperature.  The  mixture  is  heated  for  at  least  4  hours  at  each                      selected temperature point.                                               Figure 8.20. Compatibilities of an active substance and excipients. The curve of                       excipient C remains at zero heat flux value, showing no interaction with the                      active substance, demonstrating compatibility of the substances. Another two                            excipients A and B deviate from zero heat flux value and are thus                           incompatible with the active substance and not suitable for product                                                   formulation.                                       (Source: Aplikační listy. SETARAM Instrumentation.)                     •  Stability of pharmaceutical products. Stability tests are one of the main tests                      for pharmaceutical, and micro DSC can accurately and rapidly predict their                      outcome. Fig. 8.21 shows an analysis of two different formulations with the                      same active ingredient at 80 °C. PREP 1 exhibits a thermal reaction during                      the test, as a degradation process occurs. This formulation is not acceptable                      and would be unsatisfactory in long-term stability tests.                    •  Enzyme  reactions.  Micro  DSC  can  also  be  used  to  determine  optimum                      temperatures for enzymes and to model enzyme reactions. Such a reaction is                      continually  monitored  using  a  mixing  cell.  Fig.  8.22  shows  a  study  of  the                      transformation of maltose by glucoamylase in water.                                                             247","8 Thermal analysis                                                               Figure 8.21. Stability of pharmaceutical products.                                    (Source: Aplikační listy. SETARAM Instrumentation.)                                                Figure 8.22. Study of glucoamylase induced maltose transformation in water.                                    (Source: Aplikační listy. SETARAM Instrumentation.)                                                       248","8 Thermal analysis                 8.3.7  Temperature modulated differential scanning calorimetry (TMDSC)                 8.3.7.1  Nomenclature and method origin                                                                        th                The  method  came  into  use  in  the  early  nineties  of  the  20   century  and  has  no                established name. Besides temperature modulated differential scanning calorimetry                (TMDSC),  also  other  terms  are  used,  e.g.,  modulated  differential  scanning                calorimetry  (MDSC),  alternating  differential  scanning  calorimetry  (ADSC),  or                dynamic differential scanning calorimetry (DDSC). The same heating process can                be  similarly  used  for  thermal  gravimetry,  thus  getting  modulated  differential                thermogravimetry  analysis  (MDTA).  Because  the  principle  is  the  same,  only                TMDSC will be discussed.                   8.3.7.2  Method principle                  As  mentioned  before,  the  temperature  and  the  heat  flux  associated  with  phase                changes are measured in DSC as a function of time and temperature in a defined                atmosphere.  The  heating  of  the  sample  is  performed  at  a  constant  rate.  The                measuring  principle  is  the  same  as  in  classical  DSC,  but  the  sample  undergoes                modulated heating by a periodically changing heat flux. It has a sinusoid shape and                superimposes the heat flux from classical DSC, and it linearly varies with time. The                result  of  a  classical  DSC  is  a  total  heat  flux.  In  TMDSC,  the  total  heat  flux  is                interpreted by two terms that characterize reversible (e.g., melting) and irreversible                (e.g., crystallization) processes that occur during the measurements. TMDSC allows                a more specific study of substance properties or analysis of mixtures that usually                cannot be interpreted using DSC.                 This measurement provides qualitative and quantitative information about physical                and chemical changes with either exothermal or endothermal character or only the                thermal capacity of the material changes.                  The measurements start by heating at least 2 minutes before a phase transformation,                e.g., with 10 °C/min heat rate, the start should be at least 20 °C below the expected                transformation. The heating is performed for at least two minutes to reach the phase                transition. Table 8.2 shows the effect of heating rate on analysis.                                                                 249","8 Thermal analysis                                  Table 8.2. Effect of heat rate on the course of Tg.                              Heating rate [°C/min]   Sensitivity   Reproducibility                                        5                low          very good                                        20               good           good                                       40            very good          low                    8.3.7.3  Division of TMDSC curve into reversible and irreversible part                 A comparison of heat flux in DSC and TMDSC is shown in Fig. 8.23 and 8.24.                 Heat flux  consists  of sample  heating  and the  following  phase  transition.  DSC  is,                however,  only  able  to  measure  their  sum.  TMDSC  can  separate  these  two                components.                 For all TMDSC experiments, the signal is derived from three measured parameters:                    •  time                    •  modulated temperature (as a stimulator)                    •  modulated heat flux (as a response)                  The  average  value  of  the  modulated  heat  flux  is  qualitatively  and  quantitatively                consistent with the average flow value in a conventional DSC method. A calculation                using Fourier transform yields an average value.                 Reversible heat flux is a capacitive component of the total heat flux. The conversion                of specific capacity gives a component corresponding to a classical heat flux.                                    Reversible heat flux = -Cp . average heat rate                 Irreversible heat flux is a kinetic part of the total heat flux.                                        Irreversible part = Total – Reversible                                                      250","8 Thermal analysis                 Thus:                                                     dQ/dt – total heat flux                                                      Cp – measured capacity                                                    =        +   (  ,   )         dT/dt – average rate                                                                                   Cp (dT/dt) – reversible part                                                     f(T,t) – irreversible part                                                                          Figure 8.23. Continued.                                                                        251","8 Thermal analysis                                               Figure 8.23. Input signal in DSC and TMDSC, (a) temperature as a function of time                 for a typical DSC and TMDSC experiment; (b) temperature increase (heating rate)                         as a function of time for a typical DSC and TMDSC experiment.                                             Figure 8.24. The middle curve represents a curve obtained by the classical DSC                   method. The other two curves are reversible and irreversible parts in TMDSC.                                                         252","8 Thermal analysis                 8.3.7.4  Advantages of TMDSC                    •  increased detection sensitivity for glass transition                    •  increased  resolution  capability  –  the  process  happens  at  two  rates                      simultaneously                    •  ability to divide capacity change processes and kinetic processes                    •  the  reversible  component  is  suitable  for  glass  transition  and  melting  point                      measurements                    •  the  irreversible  component  is  suitable  for  measuring  enthalpic  relaxation,                      evaporation, crystallization, degradation, some types of melting                    •  ability  to  measure  thermal  capacity  changes  even  during  reactions  (phase                      transitions)                    •  a  better  interpretation  of  crystallization  processes  and  crystal  structures  of                      semicrystalline polymers                     •  possibility to identify recrystallization even during melting                   •  measuring of very low thermal conductivities                     •  elimination  of  the  overlap  of  different  thermal  effects  (separation  of                      reversible and irreversible processes)                    •  mixture analysis                    •  high sensitivity                    8.3.7.5  Application of TMDSC in combination with DSC                   •  measuring mixtures of various substances                     •  measuring  mixtures  of  crystalline  forms,  including  combinations  with  a                      content of one amorphous substance (Fig. 8.25)                    •  TMDSC  does  not  replace  classical  DSC  but  complements  the  information                      obtained by DSC                    •  it is always advised to start with analyses using the classical DSC and use the                      TMDSC method in case of ambiguous results                                                     253","8 Thermal analysis                    •  TMDSC  complements  the  information  from  classical  DSC  for  mixtures  or                      compounds with low Tg                                                 Figure 8.25. Dependence of thermal capacity on the content of an amorphous phase                                    in a mixture with a crystalline substance.                   8.3.8  Thermogravimetry (TGA)                 Thermogravimetric  analysis,  or  thermogravimetry  in  short  (TG  or  TGA),  is  a                thermal  analysis  that  quantitatively  monitors  sample  weight  changes  (increase,                decrease) during temperature changes. The immediate weight  w is evaluated in a                static  configuration  depending  on  time  t  at  a  constant  temperature  T  (isothermal                technique). See the below-listed relation:                                        w = f(t)                      T – constant                 A  problem  that  can  affect  the  analysis  in  an  undetermined  manner  is  the  initial                samle heating to a working temperature. Another problem is the extent to which the                heating to isothermal temperature changes the properties of the studied sample.                                                          254","8 Thermal analysis                 Thermogravimetry usually refers to a dynamic process when the sample weight w is                recorded depending on a programmed temperature increase:                                                   w = f(t)                  Temperature T is a linear function of time in most commercially available devices:                                                                          = Ø                       Ø – heat flux                                                      Hence:                     T = T0 + Ø t           T0 – initial sample, i.e., measurement, temperature                 Some  devices  currently  enable  periodic  heating  rate  change,  i.e.,  an  analogy  to                TMDSC, which can be regarded as MDTA.                 Apparatuses for TGA, i.e., thermobalances, are very accurate scales currently based                on  a  compensation  principle  ‒  sample  weight  change  is  compensated                electromagnetically and thus quickly recorded. There are two configuration types of                thermobalances:  horizontal  and  vertical (more frequent).  Each  has its  advantages                and technical limitations. The construction of the instrument must allow operation                under a defined atmosphere. An example of a TG built is shown in Fig. 8.26.                 The measurement result is a thermogravimetric curve that states the actual sample                weight  dependent  on  temperature  and  time.  The  curve  shape  is  affected  by  the                heating rate. The higher the heating rate, the narrower the temperature interval for                the  weight  change  to  occur.  High  heating  rates  may,  however,  cause  that  minor                changes  are  not  displayed  in  the  curve.  An  evaluation  may  include  the  first                derivation of the obtained curve, allowing better resolution of individual processes,                see Fig. 8.27.                                                                         255","8 Thermal analysis                                                                           Balance purge                                                                   Furnace housing                                                                    Sample pan                                                                  Furnace                                                                   Furnace purge                                                                   Thermocouple                                            Figure 8.26. Design of TG.                                    (Source: Aplikační listy. SETARAM Instrumentation.)                 Thermogravimetry measurement is affected by many factors based on:                    •  instrument construction and materials                    •  experimental  design (heating  rate,  heat transfer,  atmosphere in  the reaction                      space, gas flow, a temperature measuring method, etc.)                    •  physical  and  chemical  properties  of  the  sample  (weight,  particle  size,                      geometry, preparation method, and material history)                                                                        256","8 Thermal analysis                                      Acetaminophen (Sigma Aldrich)                             0                                                                            100                             -1                                             80                           Heat Flow (mW/mg)  -2                            60  TG (%)                                                                                    40                            -3                               -4                          ----- DTG           20                                   100        200        300         400                                                           o                                          Temperature ( C)                          Figure 8.27. DSC and TGA curve with the first derivation DTG.                                      (Source: Jendrzejewska et al. Molecules, 2020.)                The following figures, Fig. 8.28–8.31, contain TGA and DSC curves dealing with                the study of hydrates of a sample in development.                                          Figure 8.28. TG and DTG curves of a sample with water content around 8% (about                  1–2 moles of H2O after calculation). Sample decomposition occurs at around 270                                                    °C.                                                       257","8 Thermal analysis                                         Figure 8.29. TG and DTG curves of a sample with a water content of around 18%                    (about 4–5 moles of H2O after calculation). Sample decomposition occurs at                                            approximately 282 °C.                                           Figure 8.30. TG and DTG curves of a sample with a water content of around 26 %                    (about 7–8 moles of H2O after calculation). Sample decomposition occurs at                                               about 273 °C.                                                              258","8 Thermal analysis                                            Figure 8.31. TG and DTG curves of a sample containing no water. Sample                                    decomposition occurs at around 254 °C.                   8.3.9  Thermally stimulated current (TSC)                 This unique thermal method (thermally stimulated current, TSC) uses the so-called                molecular  mobility  which  provides  information  about  the  substance  structure,                dynamic parameters H and S, and relaxation (release) time .                 In TSC, the substance is heated to a temperature TP in an electric field with intensity                EP  for  a  sufficiently  long  time  tP,  allowing  differently  moving  particles  of  the                studied substance to reach identical orientation in the electric field. In this state, the                sample is  rapidly  cooled  to  a  temperature  T0,  ensuring  zero  particle  motion.  The                effect  of  the  electric  field  is  then  deactivated,  and  the  substance  is  kept  at                temperature  T0  for  time  t0.  The  temperature  then  linearly  increases,  the  substance                returns to the previous state, and current depolarization ID is recorded as a function                of  temperature.  Temperature  Tmax  characterizes  each  depolarization  peak  with                intensity  Imax  (Fig.  8.32  and  8.33).  This  technique  is  particularly  suitable  for                substances with polar character.                 Application:                    •  interactions between the active substance and an excipient                     •  determination of the ratio of crystalline and amorphous phases in a sample                    •  detection of a low concentration of amorphous phase (< 1%; see Fig. 8.34)                                                     259","8 Thermal analysis                    •  determination of crystallinity degree, polymorph resolution (Fig. 8.35).                                                        Figure 8.32. Schematic record of a TSC measurement.                                    (Source: Aplikační listy. SETARAM Instrumentation.)                                                                      Figure 8.33. Scheme of a TSC instrument.                                    (Source: Aplikační listy. SETARAM Instrumentation.)                                                     260","8 Thermal analysis                                                Figure 8.34. Purity of crystalline substances. Detection of amorphous phase                                               content < 1%.                                  (Source: Aplikační listy. SETARAM Instrumentation.)                                             Figure 8.35. Resolution of polymorphic forms A (continuous line) and B (dashed                    line) of irbesartan using the TSC method. Classical DSC does not allow this                   differentiation. The peak between 100 and 150 °C is visible also in DSC. Peaks                      between -150 and -50 °C are only detectable by the TSC method and are                      characteristic of polymorph A. The α, β, γ denote molecular mobilities.                       (Source: Sample measurement record from SETARAM Instrumentation application laboratory.)                                                      261","8 Thermal analysis                 8.4  Resources and recommended literature                   1.   Nicoara M., Locovei C., Opris C., Ursu D., Vasiu  R., Stoica M. Optimizing the                       Parameters for In-situ Fabrication of Hybrid Al-Al2O3 Composites. J. Therm. Anal.                       Calorim. 2016. https://doi.org/10.1007/s10973-016-5595-3                   2.   Araújo  A.  A.  S.,  Bezerra  M.  S.,  Storpirtis  S.,  Matos  J.  R.  Determination  of  the                       melting temperature, heat of fusion, and purity analysis of different samples of zidovudine                       (AZT) using DSC. Braz. J. Pharm. Sci. 2010, 46 (1). https://doi.org/10.1590/S1984-                       82502010000100005                   3.   Joseph K., Asuvathraman R., Madhavan R. R., Jena H. Studies on Novel Matrices                       for  High  Level  Waste  from  Fast  Reactor  Fuel  Reprocessing.  Energy  Procedia.                       2011, 7, 518-524. https://doi.org/10.1016/j.egypro.2011.06.071                   4.   Brittain H. G. Polymorphism in Pharmaceutical Solids. Marcel Dekker: New York,                       USA, 1999.                  5.   Comprehensive  Handbook  of  Calorimetry  and  Thermal  Analysis.  Sorai  M.  (Ed.)                       John Wiley \& Sons: New York, USA, 2004.                   6.   Vaníček  J.  Metody  termické  analýzy.  Studijní  materiály.  Katedra  textilních                       materiálů, Fakulta textilní, Technická univerzita v Liberci: Liberec, Czech republic,                       2007.                   7.   Řehula,  M.  Příručka  termoanalytických  charakteristik  pomocných  látek.  Studijní                       materiály.  Katedra  farmaceutické  technologie,  Farmaceutická  fakulta  v  Hradci                       Králové, Univerzita Karlova v Praze: Prague, Czech Republic, 2007.                  8.   Gabbott P. Principles and Applications of Thermal Analysis. Blackwell Publishing:                       Singapore, 2008. https://doi.org/10.1002/9780470697702                   9.   Feng X., Ye X., Park J. B., Lu W. Evaluation of the recrystallization kinetics of                       hot-melt extruded polymeric solid dispersions using an improved Avrami equation.                       Drug       Dev.      Ind.     Pharm. 2014,     41       (9),     1-9.                       https://doi.org/10.3109/03639045.2014.958755                   10.   Jendrzejewska I., Goryczka T., Pietrasik E., Klimontko J., Jampilek J. X-Ray and                       Thermal Analysis of Selected Drugs Containing Acetaminophen. Molecules, 2020,                       25 (2020), 5909. https://doi.org/10.3390/molecules25245909                   11.   Qi  S.  Analytical  Techniques  in  the  Pharmaceutical  Sciences.  Eds.;  Müllertz  A.,                       Perrie Y., Rades T. Springer: New York, USA, 2016.                   12.   Chiu M. H., Prenner E. J. Differential scanning calorimetry: An invaluable tool for                       a  detailed  thermodynamic  characterization  of  macromolecules  and  their                       interactions.   J.   Pharm.   Bioallied.   Sci.   2011,   3   (1),   39-59.                       https://doi.org/10.4103/0975-7406.76463                                                      262","8 Thermal analysis                   13.   Craig  D.  Q.  M.  Reading  M.  Thermal  Analysis  of  Pharmaceuticals.  CRC  Press:                       Boca Raton, USA, 2006. https://doi.org/10.1201/9781420014891                  14.   Ebeid E. Z. M., Zakaria M. B. Thermal Analysis: From Introductory Fundamentals                       to   Advanced   Applications.   Elsevier:   Amsterdam,   Netherlands,   2021.                       https://doi.org/10.1016/B978-0-323-90191-8.00004-X                   15.   Zainal  N.  F.  A.,  Saiter  J.  M.,  Halim  S.  I.  A.,  Lucas  R.,  Chan  C.  H.  Thermal                       analysis: basic concept of differential scanning calorimetry and thermogravimetry                       for  beginners.  Chemistry  Teacher  International.  2021,  3  (2),  59-75.                       https://doi.org/10.1515/cti-2020-0010                                                                                                           263","9 Dissolution testing in pharmaceutical industry                 9  Dissolution testing in pharmaceutical industry                 Martin Čulen, Anna Řezáčová                   9.1  Biopharmaceutical classification system                 Biopharmaceutical classification system (BCS) proposed by Amidon et al. in 1995                divides drugs into four different classes depending on their solubility and permeability                (Table 9.1). The aim of this system was to create a theoretical basis for correlation of                in vitro dissolution profiles with in vivo drug bioavailability. A drug is considered                highly soluble when its highest dose is soluble in 250 ml of dissolution medium (the                usual volume of juices in stomach during bioavailability testing) at a physiological pH                range (1.0–7.5). A drug is classified as highly permeable if its absorbed amount (from                a solution) is greater than 90%.                                Table 9.1. Biopharmaceutical classification system.                                     Class       Solubility     Permeability                                    Class I     high           high                                     Class II    low            high                                    Class III   high           low                                    Class IV    low            low                  BCS is based on a simple absorption model in which the intestine is represented by a                cylindrical  pipe  where  the  absorption  takes  place.  In  this  model,  there  are  round                identically  sized,  inert  (does  not  allow  metabolism)  particles.  Solubility  is                independent on the size of these particles and on the intestinal pH gradient, and there                is no possibility of aggregation. Amidon et al. demonstrated that key parameters that                control  the  absorption  of  drugs  are  three  variables:  An  –  absorption  number,  Dn  –                dissolution number, Do – dose number.                                                          264","9 Dissolution testing in pharmaceutical industry                 These  parameters  represent  the  basic  processes,  such  as:  permeability  of                membranes, dissolution rate and drug dose:                                              M0 – administered drug dose                              /                                                           =                     V0 – standardized stomach volume ( 250 ml)                                                                                                        Cs  –  saturation  (balanced)  solubility  of  a                                              substance, in mg/ml                                                              =                    tres  –  average  drug  residence  time  drug  in  the                                                                        gastrointestinal tract (GIT) ( 180 min)                                               tdiss – time needed to dissolve the dosage form,                                                          =                      Peff – effective permeability                                                                                                          R – radius of the intestinal segment                 Pharmaceuticals  of  the  BCS  class  I  exhibit  good  solubility  and  permeability  and                have very good absorption in general. Immediate release (IR) tablets containing an                API from this class are expected to have 100% intestinal absorption, provided that                at  least  85%  of  the  total  amount  is  dissolved  in  30  minutes  within  the  whole                physiological  pH  range.  For  this  reason,  these  products  do  not  have  to  undergo                bioequivalence studies.                 Class II substances are poorly soluble and highly permeable compounds. They are                characterized  by  high  An  and  Do.  Here,  dissolution  significantly  affects  the                absorption  rate  and  extent,  which  may  lead  to  incomplete  absorption  and                bioavailability  despite  good  membrane  permeability  (An).  These  drugs  may  be                influenced by the length of their GIT transit time. The longer the transit time at the                absorption site, the greater the expected absorption. Absorption can be limited by                equilibrium  or  kinetics  type.  For  drugs  with  high  Do,  the  API  dose  is  too  high,                considering the solubility of the substance in the GIT fluids. For drugs with Dn  1,                the  dissolution  process  is  too  slow  to  be  completed  before  the  drug  leaves  the                intestinal  absorption  site.  For  these  drugs,  the  in  vitro  –  in  vivo  correlation  is                expected  to  be  good  when  in  vitro  dissolution  rate  is  similar  to  the  in  vivo                dissolution rate. As a general rule, any factor that increases the speed and extent of                in  vivo  dissolution  of  class  II  drugs  also  increases  their  bioavailability.  It  is                therefore assumed that administration of high-fat meals increases solubility and thus                also bioavailability of class II drugs.                                                           265","9 Dissolution testing in pharmaceutical industry                 Class III substances have high solubility but poor permeability. Intestinal absorption                of  these  substances  is  limited  by  the  speed  of  their  permeation  through  the                membrane, while their dissolution is fast.                 Class IV substances are characterized by low solubility and low permeability and                exhibit large inter- and intrapersonal variability.                   9.2  Dissolution testing of substances                 9.2.1  Intrinsic dissolution                 These tests determine the rate of true (intrinsic) dissolution of pure solid substances                (APIs) or their mixtures with excipients. The rate of true dissolution is a theoretical                quantity related to a chemically pure solid substance with no porosity. In reality, the                tests are conducted with samples of pure APIs or APIs in mixtures which do not                have a non-zero but still a minimal porosity achieved by compaction (into tablets).                The  rate  of  true  dissolution  (dm/dt)  is  defined  as  a  mass  of  tested  substance                                                                2                dissolved per time from a unit of surface (mg/min·cm ):                                                  S – API solubility                                                    A – release surface for the API                                                      =     = −    (   −   )     m – sample mass                                                                                k – rate of true dissolution                                                    C – concentration in time t                 Physical properties of solid materials such as polymorphism, crystal shape, particle                size, specific surface, etc., all affect dissolution rate. The rate is also influenced by                environmental conditions, i.e., experiment parameters. Thus, the dissolution rate is                affected by compacting pressure, temperature, pH, medium composition, medium                ionic strength, and buffer capacity. A critical parameter of true dissolution is tablet                preparation – compaction – which must meet the dissulution requirements. A basic                requirement is that no phase changes occur (crystal form, solvate formation, etc.)                during  the  whole  sample  preparation  and  dissolution  process.  This  must  be                confirmed  by  X-ray  diffraction  or  another  suitable  analysis  at  all  stages  of  the                dissolution  test  (sample  at  input,  after  compaction,  remaining  material  after                dissolution). The experiment ends after the comprimate is released or falls out of its                matrix (see below), because the process no longer meets the condition of a constant                contact surface.                                                      266","9 Dissolution testing in pharmaceutical industry                 9.2.1.1  Experimental design                 Weighed  amount  of  a  powdered  material  is  compressed  into  a  special  metallic                                             2                matrix with a hole sized 0.5 cm  (Fig. 9.1). The matrix with the sample is fixed to                the  dissolution  instrument axis and immersed into  a selected  dissolution medium                (defined  composition,  pH,  ionic  strength,  temperature,  surfactants,  etc.)  which  is                stirred  with  a  predefined  speed.  Samples  of  the  medium  are  collected  at                predetermined time intervals during a selected part of the test. The total testing time                varies depending on the nature of the tested material. As a rule, the sampling should                be maintained until at least 10% of the sample is transferred into solution.                                                                               Figure 9.1. Continued.                                                                     267","9 Dissolution testing in pharmaceutical industry                                              Figure 9.1. Apparatus for intrinsic dissolution, dimensions in millimetres.                      (Edited according to: European Pharmacopea 10.0 and commercial materials of Varian company.)                  Calculations  are  based  on  integrated  form  of  modified  Noyes-Whitney  equation.                Providing  that  the  immersion  condition  is met,  the  concentration  gradient  can be                considered  as  constant.  Data  on  the  cumulative  dissolved  amount  from  each                sampling  must  be  corrected  by  the  loss  of  the  medium  during  sampling.  To                calculate the true dissolution rate, cumulative amount of the solved substance per                area unit of the comprimate is plotted against time needed to dissolve 10% of the                sample. The cumulative amount of the dissolved sample per area unit is given by                the ratio of the cumulative amount of substance solved during the specified time and                the area of the comprimate exposed to the solvent solution (usually a matrix of 0.5                   2                                                     2                cm ). The dissolution rate in mg/min·cm , at the given rotation speed, is then given                by the direction of the calculated regression line (Fig. 9.2).                Results must always be interpreted according to the experimental conditions. The                method  is  not  absolute,  but  comparative,  and  therefore  suitable  for  comparing                quality  of  substances  or  intermediates  from  specific  stages  of  pharmaceutical                production. The experiment parameters are set to achieve discrimination between                different sample types (Fig. 9.3–9.6).                                                               268","9 Dissolution testing in pharmaceutical industry                                                    Figure 9.2. Evaluation of intrinsic dissolution – calculated regression line.                                                   Figure 9.3. Comparison of dissolution profiles of one substance at different pH.                                                        269","9 Dissolution testing in pharmaceutical industry                                            Figure 9.4. Comparison of dissolution profiles of different salts of one substance, in                                     phosphate buffer of pH 6.8, at 37 °C.                                            Figure 9.5. Comparison of dissolution profiles of different salts of one substance, in                                            0.01M HCl, at 37 °C.                                                             270","9 Dissolution testing in pharmaceutical industry                                                   Figure 9.6. Effect of different SDS (surfactant) concentrations and different                                           compaction parameters.                    9.2.2  Apparent dissolution                This method is used for testing of substances, powder mixtures, or grained powders.                It uses flow-through dissolution method (USP apparatus IV). The system with the                flow-through  cell  is  also  used  for  sustained-release  formulations,  or  formulations                containing poorly soluble APIs. The flow-through cell system is used for dosage                forms  such  as  tablets  and  capsules.  Dissolution  rate  of  pure  substances  can  be                characterized using specific cells designed for powder samples.                 Method advantages:                    •  elimination of problematic behavior of powder samples in USP I and USP II                      apparatuses, i.e., floatation in the vessel or sticking to the spindle                    •  immediate wetting of the studied powders                    •  simple changing of the medium pH                    •  prevention of settling of heavier granulates in USP II apparatuses                                                         271","9 Dissolution testing in pharmaceutical industry                    •  improved  alternative  for  true  (intrinsic)  API  dissolution  without  possible                      unwanted modifications occurring during API compaction                    •  comparison  of  different  dissolution  rates  as  pure  substance  kinetics;  these                      differences  in  dissolution  rates  can  be  caused  by  various  crystal                      configurations, particle morphologies, etc.                   9.2.2.1  Experimental procedure                 All  parts  of  the  instrument  that  come  into  contact  with  the  formulation  or  the                dissolution  medium  must  be  chemically  inert,  i.e.,  not  absorbing  the  tested                substances, non-reactive, and not interfering with the tested sample. All metal parts                that come into contact with the product or dissolution medium are made of stainless                steel or coated in a suitable inert material that prevents reaction or interference with                the product or the dissolution medium.                 Eluate is filtered at cell exit and can be analyzed directly (on-line) or collected by a                fraction collector (off-line). Dissolved gases can cause formation of bubbles in the                medium,  which  may  bias  the  results.  In  such  case,  the  dissolved  gases  must  be                removed prior the test. The problem of API agglomeration can be prevented by using                surfactants.                                                       Figure 9.7. Scheme of a flow-through cell apparatus.                                     (Edited according to: European Pharmacopea 10.0.)                                                           272","9 Dissolution testing in pharmaceutical industry                                                             Figure 9.8. Flow-through cell for powders.                                 (Edited according to commercial materials by Sotax company.)                Open circuit: fresh dissolution medium is continuously pumped through the cells                while a known amount of the eluate is analyzed and the rest is pumped out as  a                waste. Open circuit enables simple pH changes, analysis of low soluble substances,                working with small API amounts, maintaining sink conditions (i.e., preventing drug                saturation), in vivo – in vitro correlations even with multiple pH changes (as there is                no method means for automatic medium change in the USP).                 Closed circuit: the medium circulates in the system after flowing through the cells.                In this case it is possible to work with small volumes – less than 400 ml (or even 5                ml), large volumes – higher than 1000 ml (depending on the cell type), to detect                very low API concentrations, and to perform dissolution of special dosage forms                (powders,  pellets,  suspensions,  soft  gelatin  capsules,  implants,  gels  and  creams,                etc.).                 Samples are collected at the cell exit, regardless of whether the circuit is open or                closed.  The  amount  of  the  released  active  substance  during  a  defined  time  is                expressed as a percentage of the declared amount.                This is the only dissolution method to express dissolution rate as kinetics, not as an                equilibrium state. In the case of a closed circuit, the data have a shape of a classic                dissolution  curve  –  cumulative  (like  for  USP  apparatus  I  and  II).  With  an  open                system, the curve has a shape of a differential shape (see Fig. 9.9).                                                          273","9 Dissolution testing in pharmaceutical industry                      A.                                  B.                            Figure 9.9. A. Dissolution profile from a closed circuit. B. Dissolution profile from                                               an open circuit.                                    (Source: commercial presentation by Sotax company)                                                 Figure 9.10. Comparison of dissolution profiles of one substance originating from                 different production sites, tested in a closed circuit in phosphate buffer with pH 6.8                                                  at 37 °C.                                                                 274","9 Dissolution testing in pharmaceutical industry                 9.3  Dissolution testing of solid dosage forms                 9.3.1  Dissolution                 Before  an  API  is  absorbed,  it  must  be  first  released  from  the  dosage  form.                Depending on the dosage form, the following processes occur:                     •  disintegration  (disintegration  of  a  solid  dosage  form  to  smaller  particle                      clusters – aggregates)                    •  disaggregation (disintegration of aggregates into undissolved API particles)                    •  dissolution (dissolution of solid API particles in the digestion fluid)                 Solid  dosage  forms  consisting  of  coarse  grains  are  subjected  to  all  three  steps                whereas an API that is already dissolved in a dosage form is only mixed with the                juice in the digestive tract.                Dissolution of a solid compound can be described as a two-step process. First, the                API molecules are released from the solid dosage form surface into the surrounding                environment of the dissolution medium. At this stage, a saturated layer is formed,                i.e., a diffusion layer adjacent to the API surface. Second, the API diffuses into the                whole  solution  volume  according  to  the  concentration  gradient  –  from  area  with                high API concentration to areas with low concentrations. Dissolution rate in time is                described by Noyes-Whitney equation:                                            Dx/dt – dissolution rate                                           A – surface area of the dissolving dosage form                                            D – diffusion coefficient                                              – thickness of the diffusion layer surrounding the                      =       − (   )      dosage form                                                                               Cs  –  saturation  (equilibrium)  dissolution  of  active                                           substance in mg/ml                                            Xd  –  amount  of  dissolved  substance  in  time  t  and                                           volume of the dissolution medium V                                                              275","9 Dissolution testing in pharmaceutical industry                 Dissolution rate is influenced by the physico-chemical properties of the tested API                and the prevailing physiological conditions in the gastrointestinal tract (GIT) that                are different for individual patients and depend on fed or fasted state. The aim of                formulation strategies is to change these properties so that the dissolution rate of                low soluble active substances is increased. These strategies include micronization,                preparation  of  nanosuspensions,  microemulsions,  oil  formulations,  and  use  of                complexing substances, such as cyclodextrins.                       Table 2.2. List of physico-chemical and physiological characteristics which                                    influence dissolution of an API in GIT.                   Parameter                    Physico-chemical       Physiological                                             characteristic         characteristic                   Surface area (A)             Particle         size,  Surfactants  contained  in                                             wettability            gastric fluid and bile                  Diffusion coefficient (D)    Molecular  size  and  Viscosity  of  luminal                                             shape                  content                   Diffusion layer thickness ()                       Motility type, flow rate                  API solubility (Cs)          Hydrophilicity,  crystal  pH, buffer capacity, bile,                                             arrangement,           food composition                                             solubilization, pKa                   Amount of dissolved API (Xd)                        API      removal     by                                                                    absorption    –    sink                                                                    condition                  Solvent volume (V)                                  Secretion,  administered                                                                    fluids                  According  to  the  equation  above,  any  changes  in  the  dissolution  medium  which                effectively  increase  the  solubility  of  the  studied  formulation  should  theoretically                lead to higher dissolution rate. If the substance dissolution rate is < 1, it is required                                                      276","9 Dissolution testing in pharmaceutical industry                 to  use  a  dissolution  medium  similar  to  the  GIT  juices  in  order  to  achieve  an                adequate  in  vitro  –  in  vivo  correlation  (correlation  between  test  results  and                bioavailability in vivo). However, the use of surfactants in dissolution media, aimed                to increase solubility and simulate environment similar to GIT, conversely leads to                slowing of the dissolution rate. This effect indicates that dissolution of substances                with  poor  water solubility  is more complex in  such  media  than  described  by the                equation.                   9.3.2  Experimental dissolution methods                 Dissolution  test  determines  the  quantity  of  an  API  released  from  a  solid  dosage                form  (e.g.,  tablet,  capsule,  or  suppository).  The  following  dosage  form  types  are                discerned according to API release rates:                    •  immediate release (also conventional release)                    •  extended release (also prolonged release)                    •  delayed release (also sustained release, enteric coated, gastro-resistant)                 Such test is used to determine compliance with the requirements for dissolution of                orally administered solid dosage forms. The dissolution test is required for all solid                dosage forms as an output quality control. Dissolution test must be discriminatory,                precise, accurate, robust, and transferrable.                 Selection  of  an  instrument  depends  on  the  physico-chemical  properties  of  the                dosage  form.  The  following  four  types  of  dissolution  apparatuses  are  commonly                used for dissolution testing:                    •  basket                    •  paddle                    •  reciprocating cylinder                    •  flow-through cell                 The following parameters are specified when determining dissolution rate of an API                from a solid dosage form:                    •  apparatus; in case of a flow-through cell, it is required to state the cell type                    •  composition, volume, and temperature of the dissolution medium                                                       277","9 Dissolution testing in pharmaceutical industry                    •  rotation or dissolution medium flow rate                    •  duration,  method,  and  amount  of  the  testing  solution  used  for  sample                      collection, or conditions of continuous monitoring                    •  analysis method                    •  acceptance criteria                   9.3.2.1  Experimental procedure                 The procedure for a basket, paddle, and reciprocating cylinder apparatus is usually                designed  to  maintain  sink  conditions.  This  represents  an  approach  where  the                material that is already in solution does not significantly affect the release rate of                the  rest  of  the  API.  \"Sink\"  condition  normally  occurs  when  the  volume  of  the                dissolution medium is 3–10 times higher than the volume of a saturated solution.                  The  dissolution  medium  is  usually  aqueous.  The  composition  of  the  medium  is                selected  according  to  physico-chemical  properties  of  the  API  and  the  excipients,                and  falls  in  the  range  of  biorelevant  conditions  that  the  dosage  form  would  be                subjected to after administration. This applies especially to pH and ionic strength of                the dissolution medium. The pH of a dissolution medium is usually set in the range                from 1 to 8. In certain cases, a higher pH value may be required. Hydrochloric acid                with concentration of 0.1 mol/l is used to maintain lower pH in the acid region. Pure                water  is  recommended  as  a  dissolution  medium,  only  if  pH  does  not  affect                dissolution.  In  specific  cases,  upon  approval  by  a  legitimate  authority,  the                dissolution  medium  may  contain  enzymes,  surfactants,  and  other  inorganic  and                organic substances. Testing of products with poorly water-soluble active substances                may  require  a  necessary  adjustment  of  the  medium.  In  these  cases,  it  is                recommended to use low concentrations of a surfactant and to avoid use of organic                solvents.  Gases  dissolved  in  the  dissolution  medium  may  also  affect  dissolution                results.  This  applies  especially  to  the  flow-through  cell,  where  it  is  necessary  to                degas the dissolution medium to prevent bubble formation in the cell. In paddle and                basket apparatuses, the volume of a dissolution medium is usually 500–1000 ml.                Stirring  speed  is  usually  between  50  and  100  rev/min  and  must  not  exceed  150                rev/min. The flow rate of the medium in a flow-through cell is usually between 4                and 50 ml/min.                                                           278","9 Dissolution testing in pharmaceutical industry                 9.3.3  Dissolution apparatuses                 The selection is based on the physico-chemical characteristics of the tested dosage                form.  All  parts  of  the  device  that  come  into  contact  with  the  product  or  the                dissolution medium must be chemically inert, i.e., non-absorbing, non-reactive, and                not interfering with the tested sample. All metal parts that meet the product or the                dissolution  medium  must  be  made  of  stainless  steel  or  coated  in  a  suitable  inert                material that prevents reactions or interference with the product or the dissolution                medium.  No  part  of  the  apparatus  or  its  accessories  may  exhibit  vibrations  or                fluctuations, and the rotation of the spindle and the flow through the cell must be                continuous.                The  dissolution  apparatuses  are  typically  termed  according  to  US  pharmacopeia                (USP).                   9.3.3.1  USP 1 apparatus – basket                 The  instrument  consists  of:  a  vessel  made  of  glass  or  other  inert,  transparent                material and may be covered with a lid; engine; drive shaft and a cylindrical basket                (mixing unit). The vessel is partially immersed in a water bath of an adequate size                or equipped with a suitable heating equipment, such as a heating jacket. Water bath                or  heating  equipment  enables  to  maintain  temperature  of  37 ± 0.5 °C  inside  the                vessel.  A  see-through  instrument  that  allows  free  observation  of  the  processes  is                more appropriate. The engine allows speed adjustment and its maintenance within ±                4%. The dosage form is put into a dry basket at the beginning of each experiment.                The distance between the inner bottom of the vessel and the bottom of the basket is                kept at 25 ± 2 mm during the test.                                                                              279","9 Dissolution testing in pharmaceutical industry                                                                                                Figure 9.11. Continued.                                                                      280","9 Dissolution testing in pharmaceutical industry                                                    Figure 9.11. A sectional view and a photo of a USP 1 dissolution apparatus                                         (dimensions in millimeters).                      (Edited according to: European Pharmacopea 10.0, commercial materials by Varian company, and                                         www.cecilinstruments.com, 18.6.2010.)                  9.3.3.2  USP 2 apparatus – paddle                 The same apparatus is used as with USP 1, with the exception that the mixing unit                consists  of  a  paddle  attached  to  the  shaft.  The  shaft  must  be  centered  with  the                vertical axis of the vessel, and its rotation must be smooth and without noticeable                vibrations that could affect the results. The vertical central axis of the paddle stirrer                passes through the axis of the shaft so that the bottom of the paddle is in level with                the bottom of the shaft. The paddle must meet the specifications given in Fig. 9.12.                The distance between the bottom of the paddle and the inside bottom of the vessel is                maintained at 25 ± 2 mm. The paddle is made of metal or a suitable inert, inflexible                material,  and  forms  a  single  unit  with  the  shaft.  To  prevent  the  floating  of  the                dosage form,  a  small,  free  piece  of  a  non-reactive  material  such  as  a  small wire                spiral can be used. The dosage form is then kept in this spiral or a similar housing,                as shown in Fig. 9.13. Other proven attachment tools can also be applied.                 The limitations of USP methods 1 and 2 are that they represent traditional closed                models where the dissolved API remains and accumulates in the system, opposed to                                                     281","9 Dissolution testing in pharmaceutical industry                 in vivo conditions where the API is rapidly absorbed, and the concentration gradient                is renewed. Floating of the dosage form on the surface or its sticking to the bottom                of the basket may also occur. Another disadvantage is the unequal hydrodynamics                in different parts of the system.                                                                                                   Figure 9.12. Continued.                                                           282","9 Dissolution testing in pharmaceutical industry                                           Figure 9.12. A sectional view and a photo of a USP 2 dissolution apparatus                                         (dimensions in millimeters).                      (Edited according to: European Pharmacopea 10.0, and commercial materials by Varian company)                                                             Figure 9.13. A sectional view and a photo of an alternative holding cage                                         (dimensions in millimeters).                     (Edited according to: European Pharmacopea 10.0, and commercial materials by LabHut company.)                                                    283","9 Dissolution testing in pharmaceutical industry                 9.3.3.3  Dissolution testing procedure for USP 1 and 2 apparatuses                 Solid dosage forms with normal and sustained release                An apparatus is assembled, and the vessels are filled with a specified volume of a                dissolution medium which is then heated to 37 ± 0.5 °C. Dosage form is placed into                the vessel. It is necessary to carefully avoid formation of air bubbles on the surface                of  the  tested  product.  The  rotation  is  switched  on  at  a  specified  rotation  speed.                Collection of samples is performed at each specified time or time interval. In the                case of repeated samplings, the volume removed for analysis is replaced with the                same volume of a fresh dissolution medium with temperature of 37 °C. In cases,                where  the  medium  replacement  is  proven  unnecessary,  the  volume  change  is                corrected in the calculations. During the test, the vessel is covered with a lid and the                medium  temperature  of  the  medium  is  controlled  at  appropriate  intervals.  The                analysis  is  carried  out  using  appropriate  analytical  methods  for  quantitative                determination of the given active substance. The test is repeated using other dosage                forms.                 Solid dosage forms with sustained release                The  test  consists  of  a  phase  in  an  acidic  environment  and  then  a  phase  with  a                buffered solution. It may differ in the dosage form  transfer between the buffered                solutions – addition to the acid environment (method A) or total replacement of the                medium (method B).                   9.3.3.4  USP 3 apparatus – reciprocating cylinder                The apparatus consists of a set of cylindrical glass vessels with a flat bottom, a set                of reciprocating glass cylinders, inert clips and meshes designed to cover the upper                and the lower parts of the reciprocating cylinders, engine and a drive component for                the reciprocating vertical movement of the cylinders inside the vessels or also for                their  horizontal  movement  into  another  vessel  series.  The  vessels  are  partially                submerged in a water bath which allows to maintain a temperature of 37 ± 0.5 °C.                An apparatus allowing observation of the product and the reciprocating cylinders is                preferred. The vessels are equipped with evaporation lids, which remain in place                during  the  test.  Unless  specified  otherwise,  the  dimensions  of  components                correspond to the values shown in Fig. 9.14.                 The apparatus enables easier simulation of the GIT conditions (pH changes and the                corresponding  transit  times).  The  release  of  an  API  from  a  product  occurs  in  6                media with different pH. The method is particularly suitable for sustained release                (SR) products and verification of the resistance of a dosage form against a strong                mechanical stress.                                                      284","9 Dissolution testing in pharmaceutical industry                                                                               Figure 9.14. A sectional view and a photo of a USP 3 dissolution apparatus                                         (dimensions in millimeters).                      (Edited according to: European Pharmacopea 10.0, and commercial materials by Varian company)                                                            285","9 Dissolution testing in pharmaceutical industry                 9.3.3.5  USP 4 apparatus – flow-through cell                 The apparatus consists of a storage tank, a pump driving the dissolution medium, a                flow-through cell, and a water bath which keeps the dissolution medium at 37 ± 0.5                °C. The cells have specified dimensions. The pump which propels the dissolution                medium  through  the  cell  must  maintain  a  constant  flow  and  a  sinusoidal  flow                profile. The flow-through cell (Fig. 9.15‒1.17) is made of a transparent and inert                material  and  is  vertically connected  with  a  filter  system  that  prevents  leakage of                undissolved particles from the top of the cell. Standard cell diameters are 12 mm                and 22.6 mm. The bottom conical part of the cell is usually filled with small glass                beads, and one bead that is placed into the tip of the cone to prevent entry of the                small glass beads (if used) into the cell. There are special holders designed for some                special types of dosage forms (Fig. 9.15 and 1.16).                 If the dissolution medium is formed by a buffer solution, its pH is adjusted to ± 0.05                pH  units  of  the  specified  value.  Dissolved  gases  must  be  removed  from  the                dissolution medium as they may result in formation of bubbles released during the                test and may affect the progress and results.                                                                                               286","9 Dissolution testing in pharmaceutical industry                                                                                                         Figure 9.15. Continued.                                                             287","9 Dissolution testing in pharmaceutical industry                                        Figure 9.15. A sectional view and a photo of a USP 4 dissolution apparatus: large                    cell for tablets and capsules (upper picture part), tablet holder (dimensions in                                                millimetres).                      (Edited according to: European Pharmacopea 10.0, and commercial materials by Sotax company)                                                    Figure 9.16. A sectional view of a USP 4 dissolution apparatus: small cell for                 tablets and capsules (upper picture part), tablet holder for small cell (lower picture                                       part), dimensions in millimeters.                                     (Edited according to: European Pharmacopea 10.0)                                                      288","9 Dissolution testing in pharmaceutical industry                 Samples are collected at the cell outlet regardless of closed or open circuit type. If                the  collection  is  repeated,  the  removed  volume  is  always  replaced  with  an  equal                volume of the dissolution medium or its loss is considered in calculations, except                for automatic evaluation where the dissolution fluid is returned to the vessel. The                amount of API released during a prescribed time is expressed as a percentage of the                declared amount.                                                      Figure 9.17. Different types of cells for flow-through systems.                                     (Source: commercial materials by Sotax company).                   9.3.4  Advanced dissolution systems                Special  dissolution  instruments  were  developed  for  research  and  development                purposes to answer the need for more accurate simulation of the in vivo dissolution.                In body, the drug passes through the different parts of the GIT and faces various                environments and conditions – from acidic pH in stomach to slightly basic distal                parts of SI; gastric secretions with pepsin and later pancreatic enzymes and bile in                duodenum;  high  liquid  content  in  stomach  followed  by  air-pockets  in  small                intestine;  intense  mixing  in  stomach  and  progressively  decreasing  peristaltics                strength in SI. Moreover, the GIT processes are not always regular. For example,                the frequency and amplitude of peristaltics is not regular but consists of occasional                short housekeeping waves to expel food from stomach, followed by longer periods                of more regular low-intensity motility. There is also a large intra- and inter-subject                variability,  for  example  in  the  frequency  of  gastric  emptying.  Simulation  of  fed-                                                     289","9 Dissolution testing in pharmaceutical industry                 state  is  even  more  complex  as  food  administration  increases  dynamics  and                variability of the GI processes. All of these examples underline the complexity of                the GIT and show that a faithful in vitro simulation of GI conditions is tricky and                requires in-depth knowledge and complex instrumentation.                 One of the most renowned artificial GIT systems is the TIM-1, introduced by Dutch                company  TNO  in  the  early  ‘90s,  which  still  remains  one  of  the  most  advanced                instruments to date. It includes one gastric and three small intestinal compartments,                with simulation of pH, gastric, biliary, and pancreatic secretions, and a controlled                emptying of all the individual compartments. It works with a more realistic motility                provided  by  flexible  tubes  and  more  physiological  medium  volumes  and                composition than the commonly used dissolution apparatuses (USP 2–4). The TIM-                1 measures drug absorption, which also maintains sink condition. The device can be                used to study pure APIs, binary mixtures of APIs with various excipients, as well as                finalized formulations. The acquired values give information about the solubility at                various  pH,  about  stability  of  coating  layers,  about  release  profile  of  the  active                substance, and consequently also about the availability of the drug for absorption.                Moreover, there is the possibility to monitor all these parameters in interaction with                food. Because of these characteristics, the device is also recognized by the FDA. Its                complexity  however  requires  laborious  cleaning  and  maintenance,  which  led  to                development  of  a  simplified  version  with  only  one  SI  compartment,  named                TinyTIM.  Other  similarly  complex  multicompartmental  dissolution  apparatuses                include  Engineered  Stomach  and  small  INtestine  (ESIN)  model  and  Golem                apparatus.                 A number of other advanced apparatuses have been developed to simulate only one                part  of  the  GIT.  For  precise  simulation  of  the  complex  mechanical  forces  in  the                stomach,  systems  such  as  Gastric  Digestion  Simulator  (GDS),  Advanced  Gastric                Simulator  (AGS),  Gastric  Simulation  Model  (GSM),  and  GastroDuo  have  been                designed.  Most  originate  from  nutrition  research  and  vary  in  build,  stress-force                simulation,  and  presence  or  absence  of  enzymatic  secretion.  Accurate  gastric                simulation  finds  application  for  formulations  where  liberation  is  dictated  by                mechanical forces (hydroxypropyl methylcellulose, HPMC, matrices) or in case of a                gastro-resistant controlled release. Novel approach to simulation of small intestine                has brought several apparatuses that mimic the tube geometry and peristaltics of SI,                such as the Intestine Model for Simulating the Peristaltic Action (IMSPA). Another                apparatus, the Human Duodenal Model (HDM) then focuses only on the simulation                of sigmoidal duodenum but with addition of enzymatic secretion and absorption.                                                             290","9 Dissolution testing in pharmaceutical industry                                                                                           Figure 1.18. A scheme and a photo of TNO TIM-1 instrument.                                     (Source: commercial materials by TNO company.)                                                      291","9 Dissolution testing in pharmaceutical industry                                                                                 Heated case                                                                      Easily removable ports                                                                             pH electrode                                                                       V-shaped paddle                                                                          Heated platform                                                                       Connecting tubes                                                                           Peristaltic pumps                  Figure 1.19. A photo of Golem v2 apparatus. Numbers 1-4 designate compartments                              – stomach, duaodenum, jejunum, ileum, respectively.                  (Source: Stupak I. Optimalizace disolučních kompartmentů k nové verzi přístroje pro biorelevantní disoluce –                                         Golem v2. Masaryk University, 2021.)                                                                                              292","9 Dissolution testing in pharmaceutical industry                 9.4  Resources and recommended literature                   1.     Amidon  G.  L.,  Lennemas  H.,  Shah  V.  P.,  Crison  J.  R.  A  theoretical  basis                         for a biopharmaceutic drug classification: the correlation of in vitro drug product                         dissolution and in vivo bioavailability. Pharm. Res. 1995, 12.                   2.     Martinez M. N., Amidon G. L. A mechanistic approach to understand the factors                         affecting  drug  absorption:  a  review  of  fundamentals,  J.  Pharmacokinet.                         Pharmacodyn. 2002, 42. https://doi.org/10.1177/00970002042006005                   3.     Dressman  J.  B.,  Amidon  G.  L.,  Reppas  C.,  Shah  V.  P.  Dissolution  testing                         as a prognostic  tool  for  oral  drug  absorption:  immediate  release  dosage  forms.                         Pharm. Res. 1998, 15.                   4.     Český lékopis 2009. Grada Publishing a.s., Prague, Czech Republic, 2009.                  5.     Pharmeuropa Vol. 16, No. 1, 2004.                   6.     European pharmacopeia 5.7, EDQM, 2006.                   7.     Viegas  T.  X.,  Curatella  R.  U.,  Van  Winkle  L.  L.,  Brinker  G.  Measurement  of                         Intrinsic Drug Dissolution Rates Using Two Types of Apparatus, Pharm. Tech.                         2001.                   8.     Bhattachar S. N., Wesley J. A., Fioritto A., Martin P. J., Babu S. R. Dissolution                         testing  of  a  poorly  soluble  compound  using  the  flow-through  cell  dissolution                         apparatus,   Int.   J.   Pharm.   236,   2002.   https://doi.org/10.1016/S0378-                         5173(02)00027-3                   9.     E.  Beyssac.  Examining  recent  developments  in  powder  dissolution  testing:                         prezentace  na konferenci  Dissolution  testing  symposium,  Prague,  Czech                         Republic, 2008.                   10.    Brown C. K., Chokshi H. P., Nickerson B., Reed R. A., Rohrs B. R., Shah P. A.                         Acceptable  Analytical  Practices  for  Dissolution  Testing  of  Poorly  Soluble                         Compounds, Pharm. Tech. 2004. https://doi.org/10.14227/DT120405P6                   11.    Cardot  J.  M.,  Beyssac  E.,  Alric  M.  In  Vitro–In  Vivo  Correlation:  Importance                         of Dissolution   in   IVIVC,    Dissolution   Technologies,   2007.                         https://doi.org/10.14227/DT140107P15                   12.    E.  Beyssac  and  J.  Lavigne.  Dissolution  Study  of  Active  Pharmaceutical                         Ingredients  Using  the  Flow  Through  Apparatus  Usp  4,  Dissolution  Technol.                         2005. https://doi.org/10.14227/DT120205P23                  13.    Haddouchi S., SPS Pharma. Spotlight session – flow through dissolution testing                         for low dose products, prezentace na konferenci Dissolution testing symposium,                         Prague, Czech Republic, 2008.                                                      293","9 Dissolution testing in pharmaceutical industry                   14.    Glantzmann J. M., Raton J. L. Dissolution tests with the flow through method.                         www.pharmaquality.com, 2005.                  15.    www.dissolutiontech.com. June 2010.                   16.    Culen M., Rezacova, A. Jampilek J., Dohnal J. Designing a Dynamic Dissolution                         Method:  A  Review  of  Instrumental  Options  and  Corresponding  Physiology  of                         Stomach  and  Small  Intestine.  J.  Pharm.  Sci.  2013,  102  (9),  2995-3017.                         https://doi.org/10.1002/jps.23494                   17.    Minekus M.. Chapter 5 The TNO Gastro-Intestinal Model (TIM). In: The Impact                         of Food Bioactives on Health: in vitro and ex vivo models. Verhoeckx K., Cotter                         P., López-Expósito I., et al. (Ed.). Springer: 2015. https://doi.org/10.1007/978-3-                         319-16104-4_5                   18.    Guerra A., Denis S., le Goff O., Sicardi V., François O., Yao A.F., Garrait G.,                         Manzi A.P., Beyssac E., Alric M., Blanquet-Diot S. Development and validation                         of a new dynamic computer-controlled model of the human stomach and small                         intestine.   Biotechnol.   Bioeng.   2016,   113    (6),   1325-35.                         https://doi.org/10.1002/bit.25890                   19.    Culen M., Dohnal J. Advances in dissolution instrumentation and their practical                         applications. Drug. Dev. Ind. Pharm. 2014, 40 (10), 1277-82.                         https://doi.org/10.3109/03639045.2013.841184                   20.    Stupak  I.,  Bilik  T.,  Vyslouzil  J.,  Culen  M.,  Dohnal  J.  Prehľad  a  vývoj                         biorelevantných  dynamických  disolučných  prístrojov.  Chem.  Listy.  2021,  115                         (11), 602-608.                                                                                  294","10 Summary                 10  Summary                 Jiří Dohnal                   Pharmacy is one of the most regulated fields and the choice of appropriate quality                and safety assurance of pharmaceuticals depends, to a large extent, on the approval                of the regulators. This is especially true if adhering to GMP, which may lead to a                certain  conservatism.  The  application  of  modern  analytical  methods  ranges  from                routine quality control to highly specialized research and development analyses, and                they also find place in works of a normative nature, such as pharmacopoeias. In the                field of research and development a higher degree of freedom can be applied in the                selection and use of methods for process monitoring and optimization. Regarding                pharmaceutical control, it is important to emphasize that the focus is increasingly                shifting  from  final  control  to  in-process  control  and  continual  monitoring  of                production processes to obtain a quality, effective, and safe final product.                 The following text provides a brief summary of the real-life use of the individual                methods  in pharmaceutical  areas.  Since  the  selection  of  an  appropriate analytical                technique  depends  mainly  on  the  desired  level  of  focus,  the  following  text  is                structured accordingly, into three parts:                 Molecular level                 Here, probaby the most important are infrared spectrometry techniques – attenuated                total  reflectance  (ATR)  technique,  diffuse  reflectance  infrared  Fourier  transform                (DRIFT) technique, Raman spectrometry, X-ray structural analysis of single crystal                or powder material, solid state nuclear magnetic resonance (ssNMR).                 Application of MIR lies mostly in the development and partially also in QC.                 NIR  spectroscopy  is  used  in  pharmacy  in  both  qualitative  and  quantitative                applications.  Main  advantage  of  the  NIR  spectroscopy  is  the  ability  to  measure                samples through glass and other transparent packaging materials. The measurement                is fast, non-destructive, and requires no special sample preparation. This minimizes                the consumption of chemicals and disposable analytical material. NIR techniques                maintain  a  special  position  in  PAT  (process  analytical  technology).  Whole  NIR                spectrometers can be attached to other devices so that an entire production process                can be directly observed, and its course monitored on-line/in-line.                 The  expansion  of  Raman  spectroscopy  is  closely  related  to  the  development  of                lasers  and  their  higher  availability.  Raman  spectroscopy  is  a  non-destructive                analytical method. Its complementarity with IR spectroscopy and characterization                                                      295","10 Summary                 of  the  molecular and  crystal  structures  enables  its  utilization  for  identification of                unknown samples. Raman spectroscopy is often used together with IR spectroscopy                in  the  mid-infrared  region  to  obtain  as  much  spectral  information  as  possible.                Raman spectroscopy can be a supplementary technique for the structural analysis of                organic molecules in combination with nuclear magnetic resonance (NMR) and/or                mass  spectrometry  (MS).  Within  the  pharmaceutical  field,  very  important                applications are those such as Raman mapping and imaging. These techniques are                crucial where component distribution and surface homogeneity are to be observed                and assessed.                 For  certain  reasons,  Raman  spectroscopy  is  not  suitable  for  determining  crystal                structures without standards. In such a case, X-Ray Diffraction (XRD) analysis will                be the method of choice. Raman spectroscopy is however a fast and non-destructive                technique  and  requires  smaller  amount  of  sample  than  thermal  methods,  e.g.,                Differential  Scanning  Calorimetry  (DSC)  which  is  used  for  polymorph                differentiation as well. Raman spectroscopy is also cheaper compared to XRD.                 Solid  state  NMR  and terahertz spectroscopy  are  typically  used in pharmaceutical                development.  They  share  some  general  characteristics,  such  as  expensive                instrumentation and requirement for highly specialized personnel. Solid state NMR                offers  broad  application  options,  namely  polymorph  characterization,  excipient                analysis, and selective and highly attractive analysis of APIs containing heteroatoms                directly in mixtures or finished dosage forms.                The terahertz spectroscopy originates from military setting and is gradually being                adopted in pharma industry, where it may find a widespread application potential.                 Intermolecular level, particles                 At this level, the techniques of interest are, for example powder X-ray diffraction                (PXRD), optical and electron microscopy  – scanning electron microscopy (SEM)                and  its  modification  –  electron  tomography,  transmission  electron  microscopy                (TEM),  terahertz  spectroscopy  (applies  to  intermolecular  bond  vibrations),  and                thermal  methods  –  differential  scanning  calorimetry  (DSC),  micro  DSC,  hyper                DSC, thermogravimetry analysis (TGA), thermally stimulated current (TSC).                  Of  the  X-ray  techniques,  powder  diffraction  is  well  established  and  is  used  for                determination  of  polymorphic  purity  of  active  substances  in  development                laboratories and routinely in QCs, besides other applications.                 Thermal analysis represents standard techniques that are typically used for detection                of solvents in APIs, determination of API purity (DSC techniques). Its disadvantage                is the limited possibility of analysis of mixtures.                                                       296","10 Summary                 Bulk, surface region                 The  suitable  methods  include  inverse  gas  chromatography  (IGC),  techniques  for                finding  high  energy  regions,  thickness,  porousness,  flow  characteristics  (of                powders), dynamic vapor sorption (DVS); also small angle X-ray scattering (SAXS)                for determination of particle size and shape etc.                 Mass spectrometry is amply used as detection in LC-MS setting. In QC, it is often                used  for  QC  impurity  detection.  In  development,  it  is  used  for  example  for                characterization of large biomolecules, such as monoclonal antibodies.                 Dissolution testing is routinely used for quality testing of complete dosage forms,                but  at  the same  time  it  is irreplaceable  in formulation  development  to  assess  the                dissolution of APIs and dosage forms and to predict bioavailability of the product.                 Based  on  the  above  mentioned,  it  is  clear  that  without  these  modern  analytical                methods, it would not be possible to ensure control during the entire life cycle of the                drug.                                                                                               297","11 Abbreviations                 11  Abbreviations                 ADSC Alternating Differential Scanning Calorimetry                 APCI Atmospheric Pressure Chemical Ionization                 API Active Pharmaceutical Ingredient                 APPI Atmospheric Pressure Photoionization                 APT Attached Proton Test                 ATR Attenuated Total Reflectance                 CCD Charge Coupled Device                 CCDC Cambridge Crystallographic Data Centre                 CDCl3 Deuterochloroform                CE-MS Capillary Electrophoresis – Mass Spectrometry                 cGMP common Good Manufacturing Practice                 CI Chemical Ionization                 COSY Correlated Spectroscopy                 CP/MAS Cross Polarization Magic Angle Spinning                 CSA Chemical Shift Anisotropy                 CSD Cambridge Structural Database                 D2O Deuterium Oxide (Heavy Water)                 DART Direct Analysis in Real Time                 DDSC Dynamic Differential Scanning Calorimetry                 DESI Electrospray Desorption Ionization                DF Dosage Form                 DMSO-d6 Hexadeuterodimethyl Sulfoxide                 DRIFT Diffuse Reflectance Infrared Fourier Transform                 DRIFTS Diffuse Reflectance Infrared Fourier Transform Spectroscopy                                                     298","11 Abbreviations                 DSC Differential Scanning Calorimetry                 DTA Differential Thermal Analysis                 DTGA Differential Thermogravimetric Analysis                 EI Electron Ionization                 EMEA European Medicines Agency                 ESI Electrospray Ionization                FAB Fast Atom Bombardment                 FDA Food and Drug Administration                 FIA Flow Injection Analysis                 FIB Fast Ion Bombardment                 FID Free Induction Decay                 FIR Far-Infrared                 FT Fourier Transform                 FT-ICR Fourier Transform – Ion Cyclotron Resonance                 FT-IR Fourier Transform – Infrared                 FT-NMR Fourier Transform – Nuclear Magnetic Resonance                 FWHM Full Width at Half Maximum                GC Gas Chromatography                 GC-MS Gas Chromatography – Mass Spectrometry                 GIT Gastrointestinal Tract                 GMP Good Manufacturing Practice                 HETCOR Heteronuclear Shift Correlated Spectroscopy                 HMBC Heteronuclear Multiple Bond Correlation                 HPLC High Performance Liquid Chromatography                 HPLC-MS High Performance Liquid Chromatography – Mass Spectrometry                                                     299","11 Abbreviations                 HPMC Hydroxypropyl Methylcellulose                 HSQC Heteronuclear Single Quantum Coherence                 ICH Q International Conference on Harmonization – Quality                 IR Infrared                 ISO International Organization for Standardization                 IT Ion Trap                LC-MS Liquid Chromatography – Mass Spectrometry                 LIT Linear Ion Trap                 MALDI Matrix-Assisted Laser Desorption/Ionization                 MAS Magic Angle Spinning                 MDSC Modulated Differential Scanning Calorimetry                 MDTA Modulated Differential Thermogravimetry Analysis                 MIR Mid-Infrared                 MS Mass Spectrometry                 MS/MS (MSn) Tandem Mass Spectrometry                 NIR Near-Infrared                 NMR Nuclear Magnetic Resonance                NQS Non-Quaternary Suppression                 PAT Process Analytical Technology                 PCR Principial Component Regression                 PDG Pharmocopeial Discussion Group                 PE Polyethylene                 Ph. Eur. European Pharmacopoeia                 PLS Partial Least Squares                 PXRD Powder X-Ray Diffraction                                                     300","11 Abbreviations                 Q Quadrupole                 QqTOF Quadrupole/Quadrupole/Time of Flight                 QqQ Triple Quadrupole                 RP Reversed Phase                 RP-HPLC Reversed Phase – High Performance Liquid Chromatography                 r-TOF Reflection Time of Flight                SDS Sodium Dodecyl Sulphate                 SIM Selected Ion Monitoring                 SRM Selected Reaction Monitoring                 SSI Sonic Spray Ionization                 ssNMR Solid State Nuclear Magnetic Resonance                 TEM Transmission Electron Microscopy                 TG(A) Thermogravimetry Analysis                 TIC Total Ion Current                 TMA Thermomechanical Analysis                 TMDSC Temperature-Modulated Differential Scanning Calorimetry                 TMS Tetramethyl silane                TOF Time of Flight                 TOSS Total Sidebands Suppression                 TSC Thermally Stimulated Current                 TSI Thermospray Ionization                 USP United States Pharmacopeia                 UV Ultraviolet                 UV-VIS Ultraviolet – Visible                 VIS Visible                                                     301","11 Abbreviations                 XRD X-Ray Diffraction                 YAG Yttrium Aluminum Garnet Y3Al5O12                                                                                                                       302","12 Authors                 12  Authors                   Editors                Martin Čulen                Department of Internal Medicine, Hematology and Oncology, Faculty of Medicine                Department of Chemical Drugs, Faculty of Pharmacy Masaryk University                Kamenice 5, 625 00 Brno, Czech Republic                233554@mail.muni.cz                 Jiří Dohnal                Department of Applied Pharmacy                Masaryk University, Brno, Czech Republic                 Josef Jampílek                Department of Analytical Chemistry, Faculty of Natural Sciences                Comenius University, Bratislava, Slovakia                  Authors                Hana Brusová                Zentiva, Prague, Czech Republic                 Martin Čulen                Department of Internal Medicine, Hematology and Oncology, Faculty of Medicine                Department of Chemical Drugs, Faculty of Pharmacy                Masaryk University, Brno, Czech Republic                 Jiří Dohnal                Department of Applied Pharmacy, Faculty of Pharmacy                Masaryk University, Brno, Czech Republic                 Jaroslav Havlíček                Zentiva, Prague, Czech Republic                 Josef Jampílek                Department of Analytical Chemistry, Faculty of Natural Sciences                Comenius University, Bratislava, Slovakia                                                      303","12 Authors                 Izabela Jendrzejewska                Institute of Chemistry                University of Silesia in Katowice, Poland                 Christelle Kadlec                Department of Dielectrics, Institute of Physics                Czech Academy of Sciences, Prague, Czech Republic                 Filip Kadlec                Department of Dielectrics, Institute of Physics                Czech Academy of Sciences, Prague, Czech Republic                 Bohumil Kratochvíl                Department of Solid State Chemistry                University of Chemistry and Technology Prague, Czech Republic                 Lucie Krumbholcová                Zentiva, Prague, Czech Republic                 Tomáš Pekárek                Zentiva, Prague, Czech Republic                 Lukáš Plaček                Pragolab, Prague, Czech Republic                 Anna Řezáčová                Icon Clinical Research, Prague, Czech Republic                 Dita Spálovská                Zentiva, Prague, Czech Republic                                                                   304","Translation: Martin Čulen                 Design and typesetting: Martin Čulen                Cover design: Kateřina Škrlová (www.deart.cz)                   Published  by  Masaryk  University  Press,  Žerotínovo  nám.  617/9,  601  77  Brno,                Czech Republic                  First electronic edition, 2023                 ISBN: 978-80-280-0193-3                https://doi.org/10.5817/CZ.MUNI.O280-0193-2022",""];