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 tr can be converted into retention volume Vr Vr = Fvtr where 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 𝐾= [𝑠𝑜𝑙𝑢𝑡𝑒] 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒 [𝑠𝑜𝑙𝑢𝑡𝑒] 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 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’) 𝑘 ′ = 𝑡 𝑟 − 𝑡 0 𝑡 0 Measure of impact on stationary phase on analyte (how retained in column it is) Related directly to K 𝑘 ′ = 𝐾 𝑉 𝑠 𝑉 𝑚 Thermodynamic property

11. Plate Theory
Separation Factor α α= 𝐾 𝑏 𝐾 𝑎 = 𝑘′ 𝑏 𝑘′ 𝑎 = 𝑡 𝑟𝑏 − 𝑡 0 𝑡 𝑟𝑎 − 𝑡 0 α≥1 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 𝑁=16 𝑡 𝑟 𝑤 2 =5.54 𝑡 𝑟 𝑤 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
𝐻= 𝐿 𝑁
L Length of column

13. Plate Theory
Resolution Rs 𝑅 𝑠 =2 𝑡 𝑟2 − 𝑡 𝑟1 𝑤 1 + 𝑤 2 =1.176 𝑡 𝑟2 − 𝑡 𝑟1 𝑤 0.5,1 + 𝑤 0.5,2
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 𝐻=𝐴+ 𝐵 𝑢 + 𝐶 𝑠 + 𝐶 𝑚 𝑢 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?
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
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)
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