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Appendix 1 Glossary of Chromatographic Terms

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Appendix 1 Glossary of Chromatographic Terms Appendix 1 Glossary of chromatographic terms Definition of chromatography, IUPAC (1993) "Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction." Adjusted retention time t~, also known as corrected retention time, takes into account the dead time tM of the column; see retention time and dead time. t~ = tR - tM Adsorption chromatography mode of separation in which a solute or sample components are attracted to a solid surface, the stationary phase, by adsorp­ tion retention forces, the mobile phase may be a gas or liquid. Adsorption retention forces attraction of a solute onto a solid stationary phase due to microporosity (pores 5~ 50 nm) and polar character (formation of van der Waal's forces and hydrogen bonding) of the surface, described by Langmuir isotherms (see isotherms). Affinity chromatography separation effected by affinity of solute molecules for a bio-specific stationary phase consisting of complex organic molecules bonded to an inert support material, e.g. separation of proteins on a bonded antibody stationary phase. The technique is really selective filtration rather than chromatography. Alumina A120 3, slightly basic adsorbent used in liquid chromatography, particularly TLC, as a less acidic alternative to silica gel. Anion exchange chromatography see ion exchange chromatography. Asymmetry, As term used to describe non-symmetrical peaks measured by obtaining the ratio at 10% peak height h of the forward part, a and the rear part, b of a peak measured from the perpendicular line drawn from the peak maxima to the baseline. b As = ~ at 10% h a Back-flushing used to remove components in a sample held strongly on the stationary phase at the beginning of a column by reversing the flow of mobile phase after completion of an analysis. 526 CHROMATOGRAPHIC METHODS Band (or zone) used to describe the distribution of a sample component (solute) as it travels through the chromatographic system. Band or peak broadening the process whereby a solute band or spot spreads out and is diluted as it moves through the chromatographic system due to: (i) variable pathways of solute through the stationary phase particles; (ii) movement of solute molecules in the mobile phase; (iii) mass transfer of solute molecules at the mobile phase-stationary phase boundary; band broadening is measured as peak width, Wb; see van Deemter equation. Band width see peak width. Bonded stationary phase a stationary phase consisting of organic molecules bonded to a stationary phase support material (micro particulate silica gel), usually through siloxane bonds. Bulk property detector an HPLC detector that responds to a bulk property of the mobile phase, such as refractive index, which is modified by the presence of solutes eluting from the column. CIS see octadecylsilane. Capacity factor or retention factor k, capacity of a stationary phase to attract a component, measured as the ratio of corrected retention time (t~) and column dead time (tM) k = (tR - tM) = t~ tM tM Capillary columns GC columns, e.g. WCOT, PLOT, having a bore ofO.l- 0.7 mm with a 0.1-5 f.lm film of stationary phase coated onto the inner wall of a pure silica glass column 1O-50m long, high column efficiency, Neff, and excellent resolution are achieved. Capacity of the stationary phase is very low so a sample injection splitter is used to introduce approximately 0.01 f.ll of sample onto the column. Capillary column gas chromatography gas chromatography using columns 10-100 m long with an internal diameter of 0.1-0.7 mm, usually with the stationary phase bonded to the internal wall (WCOT). Capillary columns have high efficiencies (Neff) and give rapid analysis times. The A term in the van Deemter equation is zero as the column does not contain stationary phase particles or packing. GLOSSARY 527 Capillary electrophoresis, CE a high efficiency separation technique which is a cross between HPLC and gel electrophoresis. The separations are achieved by applying a high voltage (>lOOOVcm- l ) across a narrow bore capillary column, 20-200 11m i.d. and 5-10cm long, the ends of which dip into a buffer solution. The resulting electroosmotic and electrophoretic flow of buffer solution transports ionic species to a ultraviolet detector, separation occurring according to the electrophoretic properties of the sample components and pH of the buffer. Column efficiency is high with over 500000 theoretical plates per column. Carrier gas inert mobile phase gas for gas-liquid or gas-solid chromatog­ raphy, usually nitrogen is used with packed columns, helium with capillary columns. Cation exchange chromatography see ion exchange chromatography. Chiral stationary phase used to separate optically active enantiomeric compounds, by bonding a stationary phase molecule that has enantiomeric properties to a solid support. The stationary phase therefore has specific optical retention characteristics; see bonded stationary phases. Chromatogram display of separated components: (i) column chromatography, chromatogram is obtained by plotting the detector signal against time to give a series of peaks each of which represents the distribution of an eluted component in the mobile phase; (ii) planar chromatography, chromatogram is a visualised display of the separated spots from a TLC or paper chromatographic separation. Coating efficiency CE, used as a measure of column quality for GC columns by comparing the theoretical minimum plate height (maximum column efficiency), H M1N , with the plate height achieved in practice, H eALe. CE = H eALe x 100 HMIN Column chromatography mobile phase flows through a column containing the stationary phase material, either as a particulate packing or as a film on the inner wall (WCOT) of the column tubing. Column efficiency N or Neff; defined by the number of theoretical plates or equilibrium steps in a column and reflects the column's ability to separate a mixture of components with good resolution. Column efficiency depends on the number of equilibrium steps in a column, length L, or planar system, smallest equilibrium steps or plate heights (H) give highest efficiencies and 528 CHROMATOGRAPHIC METHODS values of N (see H, HETP): N is the number of observed equilibrium steps calculated using t R ; Neff is the effective number of equilibrium steps calculated using t~ Column oven thermostatically controlled enclosure for the chromato­ graphic column, maintained to ±O.loC, may be isothermal or temperature programmed. Column packing stationary phase material in the form of small solid particles which may be coated with a liquid stationary phase. Particle size is 3-20Jlm in HPLC, 50-500Jlm (60-100 mesh) in Gc. Column switching use of two or more columns to effect a separation of a sample mixture. The mobile phase can be switched between the columns to direct specific fractions from the first column onto a second column, or switched to back-flush retained components off the first column. Corrected retention time see adjusted retention time. Counter-ion in ion pair chromatography, the ion of opposite charge to the component which forms a neutral ion pair that can be separated by reverse phase HPLC, also in ion chromatography the counter ion in solution that displaces the sample ion from the stationary phase. Dead-time tM, time taken for the mobile phase to pass through the column from injector to detector, also called the mobile phase hold-up time or void time. Dead volume volume of mobile phase between the injection port and detector which does not contain stationary phase, causes excessive band broadening. Degassing process to remove gases dissolved in an HPLC mobile phase by sparging with helium and/or using vacuum, to prevent micro bubbles forming in the detector causing signal noise. Detector response factors DRF, the response of a detector varies for different molecules and is related to the property being measured, e.g. ionisation in GLOSSARY 529 FIDs, UV absorption in HPLC ultraviolet detectors, and the molecular structure of the eluted components. DRFx is usually calculated with respect to a reference or internal standard: Ax CIS DRF =-x- x A IS Cx where A is the peak area, x is the amount/concentration, x is the component, IS is the internal standard, DRFx is the detector response factor for component x. Development separation procedure to obtain a chromatogram, also, visualisation of separated components in TLC, Pc. Diffusion coefficient DM or D s, movement of a molecule in solution, important in band broadening and is dependent on temperature, solvent viscosity and relative molar concentrations of solute and mobile phase (DM ) or stationary phase (Ds). Diode array detector HPLC multichannel ultraviolet-visible detector consisting of a linear array of photodiodes onto which the full spectrum falls, i.e. 200-400 nm ultraviolet, 400-800 nm visible regions. Thus a complete spectrum can be obtained in less than 0.1 s, also several wavelengths can be monitored simultaneously to achieve maximum sensitivity for each component. Displacement chromatography the separation process where a sample mixture is introduced at the start of the column and is then eluted with a mobile phase that is more strongly attracted to the stationary phase than the sample components. Individual components are therefore displaced by the mobile phase and by each other, in order of retention forces. Distribution ratio, equilibrium constant K, the term describing the distri­ bution of a solute between the mobile phase and stationary phase and reflects the retention forces. Also known as partition ratio Kp , for partition chromatography. K= Csp CMP Effective theoretical plates Neff, the effective or true number of separation steps in a column, takes into account the column dead time in the calculation of column efficiency (see column efficiency). Neff = (::r = 16( ~:r = 5.54( ~:r 530 CHROMATOGRAPHIC METHODS Efficiency see column efficiency.
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