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ο ο Chapter 4 Theoretical Background Gas Chromatography HPLC Quantitation, Calibration, Standardisation and Validation Review of Partitioning You need to be aware of the following concepts in order to have any idea about this chapter! ο ο Partitioning between two liquids (aqueous/organic) Why does the analyte partition? o Dynamic Process – Constant exchange at the interface o Partition Coefficients o Hydrophobic and Polar Functional Groups o Ions and Solvation o Influence through pH – changes ionisation state of molecule Chromatography physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. Stationary Phase one of the two phases forming a chromatographic system. It may be a solid, a gel or a liquid. If a liquid, it may be distributed on a solid. The liquid may also be chemically bonded to the solid (Bonded Phase) or immobilized onto it (Immobilized Phase). Mobile Phase fluid which percolates through or along the stationary bed, in a definite direction. It may be a liquid (Liquid Chromatography) or a gas (Gas Chromatography) or a supercritical fluid (Supercritical-Fluid Chromatography). In gas chromatography the expression Carrier Gas may be used for the mobile phase. In elution chromatography the expression Eluent is also used for the mobile phase. Solid – Liquid Interface Compound elution as a function of time Each component is characterised by its retention time at peak maximum tr In constant mobile phase tr can be converted into retention volume Vr Vr = Fvtr where Fv is flow rate tr t0 t’r w Retention time Hold-up time (void time) Adjusted retention time Peak width Plate Theory Useful chromatographic characteristics Neglects influence of diffusion and flow paths Rate Theory Accounts influence of diffusion and flow paths Predicts effects on column performance factors Partition Coefficient [π πππ’π‘π]π π‘ππ‘ππππππ¦ πβππ π πΎ= [π πππ’π‘π]ππππππ πβππ π Assumption: Independent on concentration, affected by temperature Large K means more time spent on the column Therefore: Increased elution time = Larger K Retention factor (k’) π‘π − π‘0 = π‘0 Measure of impact on stationary phase on analyte (how retained in column it is) π′ Related directly to K πΎππ ′ π = ππ Thermodynamic property Separation Factor α πΎπ π′π π‘ππ − π‘0 α= = = πΎπ π′π π‘ππ − π‘0 α≥1 Side effects of Longer Column How to increase α? Longer Column – Better Separation Peak broadens Increase in time for separation and quantity of mobile phase Plate Number N π‘π π = 16 π€ 2 π‘π = 5.54 π€0.5 2 Number of theoretical plates Separation power assessed by plate number Note tr and w MUST be measured in same units HETP Height equivalent to a theoretical plate πΏ π»= π L Length of column Resolution Rs π‘π2 − π‘π1 π π = 2 π€1 + π€2 π‘π2 − π‘π1 = 1.176 π€0.5,1 + π€0.5,2 Same separation, different resolution Band Broadening Affects peak width Governed through Kinetic Processes Diffusion Eddy Diffusion Molecular Diffusion Mass Transfer Time taken for partition between stationary and mobile phases Eddy Diffusion Effected by particle size and flow rate Therefore peak broadening Molecular Diffusion Effected by diffusion coefficient and flow rate Mass Transfer Rate of partitioning Faster Partitioning, decreased band broadening Van Deemter Equation π΅ π» = π΄ + + πΆπ + πΆπ π’ π’ A B C u H Eddy Diffusion Constant Molecular Diffusion Constant Mass Transfer Constant Flow Rate Height of theoretical plates effected by flow rate effected by flow rate Van Deemter Plot Determine optimum flow rate Maximum Resolution in Minimum Time They counter-act eachother – oh dear ο How does N, k’ and α impact these? ο ο ο ο ο What are k’, α, Rs, N (H)? How do you calculate them? Different factors contributing to band broadening and column efficiency The van Deemter equation – what do the different terms represent? The effect of altering different parameters on separation ability Theoretical Background Gas Chromatography HPLC Quantitation, Calibration, Standardisation and Validation ο Only works for volatile chemical species o Gas-Solid – ADSORPTION chromatography analysis of permanent gases (e.g O2 or N2O) o Gas-Liquid – PARTITION chromatography analysis of organic species Nitrogen, Hydrogen or Helium Must be of high purity Hydrogen preferred but generated in situ as needed ο ο Injected directly into heated port using micro-syringe Split Injection (left) Only 0.1-1% of sample enters column, remainder waste Splitless injection (right) All sample to column Good for trace analysis Packed (top) Liquid coated silica particles in glass tube Best for large scale Slow and Inefficient Capillary / Open Tubular (bottom) Wall coated thick liquid on inside of silica tube WCOT Support coated support particles coated with stationary phase SCOT Best for speed and efficiency Only small particles Immobilized ‘Liquid’ Stationary Phases ο Low volatility ο High decomposition temperature ο Chemically Inert ο Chemically attached to support ο Appropriate k’ and α for good resolution Stationary Phases ο Usually bonded or cross-linked Like attracts Like Non-Polar stationary phase for non-polar analytes Polar stationary phase for stationary analytes Minimum Temperature Required for analytes to get into vapour phase Higher Temperature Faster the analytes run Examples Thermal Conductivity Flame Ionisation Electron capture Flame photometric Nitrogen-Phosphorus Photoionization Hall Detector Mass Spectrometer Fourier Transform Infrared TC Detector (TCD) Simple Bulk property detector (responds to components and mobile phase) Universal (Sensitive to near all compounds) Non-Destructive Concentration based signal Not very sensitive Good for detecting permanent gasses (O2 or N2O etc) What is it? Measures change in thermal conductivity due to analyte gases eluting from column How? Pass elute over heated wire Temperature of wire changes as thermal conductivity of the effluent changes Signal is based on change in temperature Carrier Gas needs VERY LARGE thermal conductvity Hydrogen Highest of all – analyte will reduce thermal conductivity HeliumAnalyte detected as a negative (overall thermal conductivity increase) Simple Selective Destructive Signal dependent on MassFlow High temperature flame ionises the components Ions are collected and records a current Response Approximately proportional to number of carbon atoms in the compound Example ethane would be twice response of methane (per mole) Complex compounds, use table Contains Ionised Gas Creates conductive system Decrease in conductivity relates to compounds with high electron affinity E.G halogenated compounds, aromatics, alcohols Pre-concentrating Very dilute analytes to get a high enough concentration to measure If its non-volatile – use HPLC Derivatisation You can react analyte with compounds to make them volatile – HPLC simpler Analysis of ο Permanent Gases ο Volatile mixtures o Petrol, Perfumes ο Purity and content of volatile small molecules o Pesticides, Drug compounds ο Production processes o Alcohol in fermentation, conversions of petrochemicals ο Anything Volatile
