Instrumental Analysis in the Biological Sciences

1 Introduction to instrumental methods of analysis.- 1.1 Precision and accuracy.- 1.2 Speed of analysis.- 1.3 Cost.- 1.4 Safety.- 1.5 Automation.- Recommended general texts.- 2 Liquid chromatography.- 2.1 Introduction.- 2.2 Theory of liquid chromatography.- 2.2.1 Chromatography.- 2.2.2 Band broadening.- 2.3 Modes of chromatography.- 2.3.1 Adsorption chromatography.- 2.3.2 Liquid—liquid partition chromatography.- 2.3.3 Ion-exchange chromatography.- 2.3.4 Size-exclusion chromatography.- 2.3.5 Affinity chromatography.- 2.4 Chromatographic techniques.- 2.4.1 Paper chromatography.- 2.4.2 Thin-layer chromatography.- 2.4.3 Column chromatography.- 2.4.4 High-performance liquid chromatography.- 2.5 Sample preparation.- 2.5.1 Extraction.- 2.5.2 Sample clean-up.- References.- 3 Gas chromatography.- 3.1 Introduction.- 3.2 Principles.- 3.3 The chromatographic system.- 3.4 GLC columns.- 3.5 Principles of separation.- 3.5.1 Chromatographic retention.- 3.5.2 Band broadening.- 3.5.3 Separation of poorly resolved peaks.- 3.6 Stationary phases.- 3.7 Gas—solid chromatography.- 3.8 Detectors.- 3.8.1 Flame ionization detector (FID).- 3.8.2 Thermionic detector (TD).- 3.8.3 Flame photometric detector (FPD).- 3.8.4 Electron capture detector (FCD).- 3.8.5 Thermal conductivity detector (TCD).- 3.8.6 Mass spectrometer.- 3.8.7 Infrared detector.- 3.8.8 Other detectors.- 3.9 Sample preparation.- 3.9.1 Headspace analysis.- 3.9.2 Derivatization.- 3.10 Quantification.- References.- 4 Electrophoresis.- 4.1 Introduction.- 4.2 Effect of pH on charge.- 4.3 Techniques of electrophoresis.- 4.3.1 Moving-boundary electrophoresis.- 4.3.2 Paper electrophoresis.- 4.3.3 Cellulose-acetate electrophoresis.- 4.3.4 Rod-gel electrophoresis.- 4.3.5 Slab-gel electrophoresis.- 4.3.6 Immunoelectrophoresis.- 4.4 Isotachophoresis.- References.- 5 Introduction to spectroscopy.- 5.1 Spectroscopy.- 5.2 The electromagnetic spectrum.- 5.3 Molecular energy states.- 5.4 Molecular transitions.- 5.5 Quantitative analysis.- 5.6 Determination of a spectrum.- Further reading.- 6 UV—visible spectrophotometry.- 6.1 Introduction.- 6.2 Electronic energy levels.- 6.3 Electronic transitions.- 6.3.1 Solvent effects.- 6.3.2 Effect of conjugation.- 6.4 Qualitative analysis.- 6.5 Quantitative analysis.- 6.6 Calibration of spectrophotometers.- 6.7 Sample presentation.- 6.8 Difference spectrophotometry.- 6.9 Spectrophotometric titrations.- 6.10 Derivative spectrophotometry.- 6.11 Dual-wavelength spectrophotometry.- 6.12 Spectrophotometers and colorimeters.- 6.12.1 Radiation source.- 6.12.2 Monochromators.- 6.12.3 Filters.- 6.12.4 Detectors.- 6.13 Turbidimetry and nephelometry.- 6.14 Colour and gloss of solid samples.- References.- 7 Fluorescence and phosphorescence spectrophotometry.- 7.1 Introduction.- 7.2 Fluorophores.- 7.3 Excitation and emission spectra.- 7.4 Quantitative measurements.- 7.5 Factors affecting fluorescence spectra.- 7.5.1 Quenching of fluorescence.- 7.5.2 Solvent effects.- 7.5.3 The effect of pH.- 7.5.4 Polarization effects.- 7.5.5 Fluorescence lifetimes.- 7.6 Instruments for fluorescence studies.- 7.6.1 Light sources.- 7.6.2 Monochromators.- 7.6.3 Sample details.- 7.6.4 Filter fluorometers.- 7.7 Applications of fluorescence spectrophotometry.- References.- 8 Infrared spectroscopy.- 8.1 Introduction.- 8.2 Molecular vibrations.- 8.3 Qualitative analysis.- 8.4 Quantitative analysis.- 8.5 Instrumentation.- 8.5.1 Sources.- 8.5.2 Monochromators.- 8.5.3 Detectors.- 8.5.4 Double-beam spectrometers.- 8.5.5 Fourier transform infrared spectroscopy (FT-IR).- 8.6 Sample presentation.- 8.7 Attenuated total reflectance.- 8.8 Near-infrared reflectance analysis.- References.- 9 Nuclear magnetic resonance spectroscopy.- 9.1 Introduction.- 9.2 Principles.- 9.2.1 Nuclear energy levels.- 9.2.2 Magnetic resonance.- 9.2.3 Relaxation processes.- 9.2.4 Principles of NMR measurement.- 9.3 Pulse NMR spectrometer.- 9.3.1 The magnet.- 9.3.2 Radiofrequency generator.- 9.3.3 Probe unit and sample.- 9.3.4 Field/frequency lock.- 9.3.5 Computer.- 9.4 Chemical shifts.- 9.5 Spin—spin coupling.- 9.6 Integration.- 9.7 Further techniques for elucidation of NMR spectra.- 9.7.1 Recording the spectrum at higher field strength.- 9.7.2 Addition of D2O.- 9.7.3 Double-resonance experiments.- 9.7.4 Shift reagents.- 9.7.5 Two-dimensional NMR.- 9.8 Wide-line NMR.- 9.9 In-vivo NMR.- 9.9.1 Topical NMR.- 9.9.2 Surface coils.- 9.9.3 NMR imaging.- References.- 10 Electron spin resonance.- 10.1 Principles.- 10.2 ESR spectra.- 10.2.1 The g-factor.- 10.2.2 Hyperfine splitting.- 10.3 ESR spectrometer.- 10.4 Sample preparation.- 10.5 Spin labelling.- 10.6 Quantitative analysis.- References.- 11 Flame techniques.- 11.1 Introduction.- 11.2 Flame emission spectrometry (FES).- 11.2.1 Interference effects.- 11.2.2 Quantitative measurements.- 11.3 Atomic absorption spectrometry (AAS).- 11.3.1 Interference effects.- 11.3.2 Quantitative measurements.- 11.3.3 Alternative sampling techniques.- 11.4 Applications.- References.- 12 Mass spectrometry.- 12.1 Introduction.- 12.2 Mass spectrometer.- 12.2.1 Inlet systems.- 12.2.2 Ion sources.- 12.2.3 Mass analysis.- 12.2.4 Detectors.- 12.2.5 Data handling and display.- 12.3 Analysis of mixtures.- 12.3.1 GC—MS interfaces.- 12.3.2 LC—MS interfaces.- 12.3.3 Tandem mass spectrometry.- 12.4 Determination of molecular structures.- 12.4.1 Bond cleavage.- 12.4.2 Rearrangements.- 12.4.3 Metastable peaks.- References.- 13 Electrochemical techniques.- 13.1 Introduction.- 13.1.1 Nature of solutions.- 13.1.2 Electrode reactions.- 13.2 Conductivity of solutions.- 13.2.1 Measurement of conductivity.- 13.2.2 Analytical applications.- 13.3 Voltammetry.- 13.3.1 Polarography.- 13.3.2 Amperometric titrations.- 13.4 Potentiometric measurements.- 13.4.1 pH measurement.- 13.4.2 Ion-selective electrodes.- 13.4.3 Potentiometric titrations.- 13.4.4 Oxygen electrodes.- References.