Chromatographie Methods
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1 Chromatographie Methods Fifth Edition A. BRAITHWAITE Department of Physical Sciences Nottingham Trent University and F. J. SMITH Department of Chemistry and Chemical Engineering University of Paisley BLACKIE ACADEMIC & PROFESSIONAL An tmprint of Chapman & Hall London Glasgow Weinheim New York Tokyo Melbourne Madras
2 Contents 1 Introduction Introduction to chromatography Definition of chromatography IUPAC definition of chromatography (1993) History of chromatography Paper chromatography Thin layer chromatography Ion exchange chromatography Gel permeation chromatography (size exclusion) Affinity chromatography Gas chromatography Supercritical fluid chromatography High Performance liquid chromatography Capillary (zone) electrophoresis Classification of Chromatographie methods According to Separation procedure According to development procedure 9 References 14 2 Theoretical considerations Introduction Distribution ratio and separations Factors influencing retention Coulomb's Law (like attracts like) Polar retention forces Non-polar retention forces Retention and equilibrium in chromatography Retention time, retention volume and retardation Retention factor Separation factor Separating efficiency of a column Peakwidth Column efficiency Peak asymmetry Band broadening processes Van Deemter model of band broadening A term ('eddy' diffusion and unequal pathways) B term (longitudinal diffusion) C terms (resistance to mass transfer) Optimum mobile phase velocity Alternative methods for band broadening Resolution % valley resolution Separation number Resolution and selectivity Quantification in chromatography Normalising peak areas 40
3 Vlll CONTENTS Internal Standard External Standards Standard addition 42 References 43 3 Planar chromatography Thin layer chromatography Theory and principles Outline of the method Surface adsorption processes and spot shape Comparison of thin layer with other forms of chromatography Adsorbents Preparation of plates Application of samples Documentation Development Overpressured thin layer chromatography Solvents Location of separated substances Preparative thin layer chromatography High Performance thin layer chromatography Paper chromatography Origin Overview of the technique Sample preparation Typesofpaper Solvents Equilibrium Development Sample application and detection Identification Quantitative methods Applications of paper chromatography Electrophoresis Procedure of zone electrophoresis Factors affecting migration rates Supporting media Techniques of electrophoresis Capillary electrophoresis Overview of instrument Operation Theory and principles Electroendosmotic flow Separation modes Instrumentation Applications 114 References Liquid phase chromatography on open columns Introduction Practical aspects and considerations Columns and packing procedures Sample application Elution procedures Modes of chromatography 119
4 CONTENTS IX 4.4 Adsorption chromatography Solvents Adsorbents Partition column chromatography Solid Supports Solvents Ion exchange chromatography Properties desirable in resins Ion exchange capacity Selectivity of resins Nature of the resin Separation methods Inorganic ion exchangers Gel ion exchangers Dextrans Agarose gels Cellulose Gel chromatography Column parameters and separations Nature of the gel Methodology Applications Affinity chromatography Principles Column materials Applications Covalent chromatography Chromatofocusing Ionic properties of amino acids and proteins Flash chromatography 162 References Gas chromatography Introduction Principles of gas chromatography Resolution % valley resolution Separation number Asymmetry Columns and stationary phase Gas chromatography columns and column oven Column oven and column temperature Packed columns Stationary phases for packed columns Specialised stationary phases Micropacked columns Choice of stationary phase Capillary column gas chromatography Band broadening and analysis time Optimum practical gas velocity Column efficiency Coating efficiency Wall coated open tubulär columns Stationary phases for wall coated open tubulär columns Phase ratio, film thickness and column internal diameter 193
5 X CONTENTS Performance of wall coated open tubulär columns Porous layer open tubulär columns Gas-solid chromatography Alumina adsorbents Silica gel Molecular sieves Carbon molecular sieves and graphitised carbon Microporous polymers 5.8 Gas chromatography Instrumentation Sampling techniques Carrier gas supply and control Column switching Sample inlet Systems Sample preparation Gaseous samples Liquid sample inlet Systems Capillary column gas chromatography injectors Derivatisation of samples Silylation Acylation Alkylation (esterification) Headspace sampling Sample introduction by pyrolysis Automated sample introduction Sampling by adsorption tubes 5.10 Detectors for gas chromatography Detector Performance Signal noise and detection limits Detector response factors Flame ionisation detector Electron capture detector Thermal conductivity detector Nitrogen-phosphorus detectors Flame Photometrie detector Photoionisation detector Miscellaneous detectors 5.11 Supercritical gas chromatography 5.12 Applications of gas chromatography References Headspace gas chromatography analysis Food analysis Drugs Pyrolysis gas chromatography Metal chelates and inorganic materials Dual detector applications Dual column applications Environmental analysis High Performance liquid chromatography Introduction Modes of chromatography Adsorption Bonded phase chromatography Ion exchange Ion pair partition Gel permeation-gel exclusion
6 CONTENTS XI Affinity Chiral chromatography Overview of high Performance liquid chromatography Instrumentation Packings Pumps Detectors Solvents Columns Injection Theory Retention Column efficiency Rate theory Extra column band broadening Temperature effects and difmsion Detailed discussion of high Performance liquid chromatography Instrumentation Solvent delivery system Equipment for gradient elution Sample introduction Detectors Specific property detectors Column packings and stationary phases for liquid chromatography Totally porous beads Pellicular packings Microporous particles Adsorption chromatography Solvent Systems Solvent optimisation Liquid-liquid partition chromatography Chemically bonded stationary phases for high Performance liquid chromatography Synthesis of bonded phase materials Chiral chromatography Chiral stationary phases Ion exchange chromatography Latex-based ion exchange materials Ion exchange with conductivity suppression Ion pairing Size exclusion chromatography Column packings Liquid chromatography method development Solvent optimisation techniques Quantitative analysis Preparative liquid chromatography Practical aspects Microcolumns in liquid chromatography Microbore packed columns Narrow bore packed columns Open tubulär columns Microcapillary packed columns Instrumentation Solvent delivery Injection Systems Microcolumn applications Applications 361 References 362
7 Xll CONTENTS 7 Chromatography and spectroscopic techniques Introduction Chromatographie requirements Chromatographie and mass spectrometry techniques Ion source and inlet System Mass analysers The quadrupole mass analyser Magnetic analysers Gas chromatography interfacing techniques Direct interfacing Indirect interfaces High Performance liquid chromatography interfacing Moving belt interface Thermospray interfaces Atmospheric pressure ionisation interfaces Electrospray interface Particle beam interface Chromatography and mass spectrometry data Systems Infrared spectrophotometry Data collection and processing Ultraviolet-visible spectrophotometry Atomic spectroscopy 393 References Processing Chromatographie data Introduction The chromatogram Chart recorders Integrators Data collection and processing Signal digitisation Data processing Computer hardware Chromatography Software Calculation of peak area Area percentage and area calculations Peak detection Baseline correction and overlapping peaks Method development and optimisation High Performance liquid chromatography optimisation Gas chromatography method development Expert Systems Laboratory Information management Systems 423 References Model or practical experiments in Chromatographie techniques Introduction List of experiments Section A. Paper chromatography Experiment 1. Separation of cobalt, manganese, nickel and zinc by ascending and horizontal development Experiment 2. Quantitative Separation of copper, cobalt and nickel by ascending development Experiment 3. Ascending chromatography on ion exchange paper 432
8 CONTENTS Xlll 9.4 Section B. Electrophoresis and related techniques Experiment 4. Horizontal low voltage electrophoresis of amino acids Experiment 5. Cellulose acetate and Polyacrylamide gel electrophoresis of proteins Experiment 6. Horizontal agarose gel electrophoresis of DNA Section C. Thin layer chromatography Experiment 7. Preparation of microplates and Separation of aromatic amines Experiment 8. Separation of simple organic Compounds on fluorescent silica plates Experiment 9. Separation of sugars on bisulphite and acetate modified silica Experiment 10. Analysis of analgesics using normal and reverse phase TLC Section D. Column chromatography Experiment 11. Separation of dichromate ion and permanganate ion using an alumina column Experiment 12. Determination of the exchange capacity and exchange efficiency of a cation exchange resin Experiment 13. Complex elution of iron and copper using a cation exchange resin Experiment 14. Purification ofproteins on DEAE-cellulose Experiment 15. Separation of a mixture of analgesics by flash column chromatography Section E. Gas chromatography Experiment 16. GC of alcohols Experiment 17. Determination of ethanol in an aqueous Solution by GC Experiment 18. Determination of barbiturates using an internal Standard Experiment 19. Determination of whisky congeners by capillary GC Experiment 20. Qualitative analysis by GC using retention data from two columns (polar and non-polar) Experiment 21. Study of some important parameters in GC Experiment 22. The determination of some chlorinated insecticides by capillary GC using an EC detector Experiment 23. Analysis ofpaint shop vapours Experiment 24. The determination of water content in solvents by capillary and packed GC using thermal conductivity detection Experiment 25. Analysis of mineral acids as their cyclohexyl derivatives by capillary GC and GC-MS Section F. High Performance liquid chromatography Experiment 26. Analysis of barbiturates by reverse phase isocratic chromatography Experiment 27. Ion pair chromatography of vitamins Experiment 28. Techniques in HPLC analysis of analgesics Experiment 29. Analysis of amino acids as their DNP derivatives Experiment 30. Analysis of paraben preservatives by HPLC with photodiode array detection Experiment 31. Determination of the amino acid composition of a peptide using pre-column derivatisation with o-phthalaldehyde and reverse phase HPLC and fluorescence detection Experiment 32. Analysis of inorganic anions in aqueous samples Section G. Capillary electrophoresis Experiment 33. Determination of inorganic cations by CE Experiment 34. Analysis of analgesics by CZE 507 References 509
9 XIV CONTENTS 10 Illustrative problems in chromatography Introduction Plate theory and resolution Van Deemter Volatility and temperature in gas chromatography Qualitative analysis Quantitative analysis Composite questions 523 Appendix 1 Glossary of Chromatographie terms 525 Appendix 2 Table of chromatography Symbols 546 Index 548
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