Chromatography

1. Chromatography Chapter 4

2. Best Broken into four categories Theoretical Background Gas Chromatography HPLC Quantitation, Calibration, Standardisation and Validation

3. Theory Review of Partitioning You need to be aware of the following concepts in order to have any idea about this chapter! • Partitioning between two liquids (aqueous/organic) • Why does the analyte partition? • Dynamic Process – Constant exchange at the interface • Partition Coefficients • Hydrophobic and Polar Functional Groups • Ions and Solvation • Influence through pH – changes ionisation state of molecule

4. Definitions 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.

5. Chromatographic Process Solid – Liquid Interface

6. Chromatograms Compound elution as a function of time Each component is characterised by its retention time at peak maximum tr In constant mobile phase trcan be converted into retention volume Vr Vr = Fvtrwhere Fv is flow rate

7. Chromatogram tr Retention time t0 Hold-up time (void time) t’r Adjusted retention time w Peak width

8. Types of Theory 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

9. Plate Theory Partition Coefficient Assumption: Independent on concentration, affected by temperature Large K means more time spent on the column Therefore: Increased elution time = Larger K

10. Plate Theory Retention factor (k’) Measure of impact on stationary phase on analyte(how retained in column it is) Related directly to K Thermodynamic property

11. Plate Theory Separation Factor α How to increase α? Longer Column – Better Separation Side effects of Longer Column Peak broadens Increase in time for separation and quantity of mobile phase

12. Plate Theory Plate Number N 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 L Length of column

13. Plate Theory Resolution Rs Same separation, different resolution

14. Rate Theory 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

15. Rate Theory Eddy Diffusion Effected by particle size and flow rate Therefore peak broadening

16. Rate Theory Molecular Diffusion Effected by diffusion coefficient and flow rate

17. Rate Theory Mass Transfer Rate of partitioning Faster Partitioning, decreased band broadening

18. Rate Theory Van Deemter Equation A Eddy Diffusion Constant B Molecular Diffusion Constant effected by flow rate C Mass Transfer Constant effected by flow rate u Flow Rate H Height of theoretical plates

19. Rate Theory Van Deemter Plot Determine optimum flow rate

20. What Do We Want Maximum Resolution in Minimum Time They counter-act eachother – oh dear  How does N, k’ and α impact these? • Optimising k’ does not ensure separation of two compounds • Separation factor needs to be considered • How can we increase ? • In GC – vary temperature • In HPLC – vary mobile phase composition - vary stationary phase material (selectivity mechanism) • Resolution increases with increasing k’ • Retention time also increases with increasing k’ • What is an optimum k’? • largest increases in resolution occur with k’<5 • largest increases in retention time occur with k’>5 • Suggestion – Keep k’>1, but also k’<10 k’ is changed by altering the stationary phase material to increase/decrease analyte interaction or altering running conditions such as GC column temperature or HPLC mobile phase composition • Highest possible plate number = better resolution • How can we increase N? • Optimisation of flow rate (u) • Increase the column length (both methods also increase the retention time) • reduce particle size of stationary phase (increases cost and back pressure in HPLC) • Reduce stationary phase thickness in GC (reduces column capacity and hence detection limit) Higher separation efficiency is ALWAYS a trade-off against other factors

21. Key Points • 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

22. Best Broken into four categories Theoretical Background Gas Chromatography HPLC Quantitation, Calibration, Standardisation and Validation

23. Gas Chromatography • Only works for volatile chemical species • Gas-Solid – ADSORPTION chromatography analysis of permanent gases (e.g O2 or N2O) • Gas-Liquid – PARTITION chromatography analysis of organic species

24. Carrier Gas • Nitrogen, Hydrogen or Helium Must be of high purity Hydrogen preferred but generated in situ as needed

25. Injectors • 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

26. Columns 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

27. Stationary Phases Immobilized ‘Liquid’ Stationary Phases • Low volatility • High decomposition temperature • Chemically Inert • Chemically attached to support • Appropriate k’ and α for good resolution Stationary Phases • Usually bonded or cross-linked Like attracts Like Non-Polar stationary phase for non-polar analytes Polar stationary phase for stationary analytes

28. Elution Control using Temp Minimum Temperature Required for analytes to get into vapour phase Higher Temperature Faster the analytes run

29. Detectors Examples Thermal Conductivity Flame Ionisation Electron capture Flame photometric Nitrogen-Phosphorus Photoionization Hall Detector Mass Spectrometer Fourier Transform Infrared

30. Thermal Conductivity 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)

31. Thermal Conductivity 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 Helium Analyte detected as a negative (overall thermal conductivity increase)

32. Flame Ionisation Simple Selective Destructive Signal dependent on Mass-Flow High temperature flame ionises the components Ions are collected and records a current

33. Flame Ionisation 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

34. Electron Capture Detector Contains Ionised Gas Creates conductive system Decrease in conductivity relates to compounds with high electron affinity E.G halogenated compounds, aromatics, alcohols

35. Sample Prep - GC 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

36. GC Uses Analysis of • Permanent Gases • Volatile mixtures • Petrol, Perfumes • Purity and content of volatile small molecules • Pesticides, Drug compounds • Production processes • Alcohol in fermentation, conversions of petrochemicals • Anything Volatile