1. WMCTC Chromatography Workshop: 7th Feb 2017
Ian J. Shannon
School of Chemistry

2. Objectives General Concepts/ & Terminology Practical aspects of TLC
dips, artefacts, additives
columns
Advanced techniques GC
HPLC

3. Concepts & Terminology
Uses
Analysis and Purification
Separation of Components
Stationary Phase
Usually Silica/Alumina
Mobile Phase
Solvent, referred to as “Eluent”

4. TLC – Silica Silica supported on plate (Aluminium Foil)
Solvent Molecule
(Mobile Phase)
Hydroxyl-terminated Silica
(Stationary Phase)
Al Foil
(Backing Plate) 4

5. TLC – Paper Chromatography
Water molecules on Surface of Paper/Cellulose
Water molecules hydrogen-bond to surface to form Stationary Phase)
Solvent (Mobile Phase) moves across surface through capillary action
Paper/Cellulose
(“Backing Plate”) 5

6. TLC Uses Used for analysing mixtures Monitoring reactions
Analysing column chromatography fractions
e.g. separating menthol
and limonene

7. Preparation of a TLC Plate

8. Analysing a Reaction
A + B -> Products

9. Running a TLC Plate

10. What is Happening?

11. Completion of TLC
Don’t forget to mark the solvent front with a pencil at the end!

12. Visualisation UV – commonly 254 nm
Dips – not all spots are visible under UV
Iodine
KMnO4 solution
Vanillin solution
Ninhydrin
FeCl3 solution
Ceric Ammonium Molybdate (CAM)
Sulfuric Acid

13. Reporting TLC Data
Rf – distance travelled by compound divided by distance travelled by eluent
Reported to 2 d.p.
Include eluent & visualisation

14. Solvent Polarity Common Eluent Systems
Hexane & Diethyl Ether – Low polarity cpds
Hexane & Ethyl Acetate – Mid polarity cpds
Toluene & Acetone – Aromatic cpds
DCM & Methanol – High polarity cpds

15. Solvent %Hexane in Ether
Effect of Solvent on Rf
In Hexane & Diethyl
Ether
Solvent %Hexane in Ether
Rf Fluorene
Rf Fluorenone
100
0.54 95
0.72
0.22 90
0.81
0.40 80
0.83 60
0.84
0.70 40
0.86
0.77 20
0.97
0.91 1

16. TLC Video
chemistry/resource/res /thin-layer- chromatography

17. TLC Troubleshooting A B C D E F

18. TLC Troubleshooting Appearance Cause Fix B: Streaks, not spots
Too much sample
Dilute sample solution
F: Spot(s) smeared out
Acidic/Basic groups present in compound?
Use an additive in the eluent
E: Crescent-shaped ‘spot’
Silica disturbed during spotting
Be gentle when spotting sample
A: Curved solvent front with spots out of lane
Plate touching side of container
Plate not lowered level into eluent
Place plate in middle of container
Ensure plate level when lowering into eluent
C: Many blue spots/stripes
Origin marked in pen?
Only use pencil on TLC plates
D: No spots on plate
Sample too dilute
Wrong visualisation used
Remake sample/spot several times
Use alternative visualisation

19. Additives Carboxylic acids/amines interact strongly with silica
Use additive in the eluent to prevent streaking
Up to 2% v/v (20 mL additive in 1 L eluent)
Acetic acid for carboxylic acids
Triethylamine for amines

20. A-Level Exemplar: Anadin Extra
Caffeine, Aspirin, Ibuprofen, Paracetemol
Ethanol or Methanol as Solvent
Ethyl Acetate for TLC
Silica TLC plates & UV Visualisation
Variations/extensions could include different pain relief tablets, and identifying them based on the components present.
Anadin has Aspirin, Paracetemol and Caffeine but not Ibuprofen
UV Lamp (Fisher) ca 150pounds 20

21. Gas Chromatography (GC)
Mobile phase is a gas
Analysis only
Very good separation
Must be able to get components into gas phase
No good for bio- molecules

22. GC Video

23. Schematic of Gas Chromatograph
Temperature effects – higher temperature means more in gas phase 23

24. Choice of Mobile Phase
In GC, the choice of mobile phase is often limited
Often called the Carrier Gas
Inert to samples and instrumentation
Chosen for ease of purification/drying
Examples include N2, Ar, H2
Accurate flow control/measurement needed to obtain reproducibility

25. Types of Stationary Support
Packed columns have particles with surface coated or bonded stationary phase
Resistance flow restricts the length and hence resolution achievable
Capillary columns have surface coated or bonded stationary phase
As there is little flow resistance (hollow tube!) columns of >20 m are perfectly possible and therefore excellent resolution can be achieved
Not all stationary phases can be used in capillary columns
Packed – robust
Capillary – better but fragile 25

26. Choice of Stationary Phase
Requirements – Thermally stable, Inert, Low volatility
Polar stationary phase (capillary or packed column)
Non-polar Stationary Phase (capillary or packed column)
Intermediate Polarity Stationary Phases are available
Polarity of analytes should match polarity of stationary phase
If polarity match is good, elution reflects bpt of analytes
Talk about order of elution with example of alkane and alcohol 26

27. Chromatogram
A chromatogram is a graph showing the detector response as a function of elution time
The retention time, tr, is the time from the injection of the mixture onto the column until that component reaches the detector.
Eluent travels through the column in the minimum time possible Designated tm
The adjusted retention time, tr’, for a solute is the additional time for a solute to travel the length of the column
tr’ = tr - tm

29. HPLC High Pressure Liquid Chromatography
High Performance Liquid Chromatography
Allows Analysis and/or Purification

30. HPLC Small particle size – 2-10 μm (60 μm for ‘normal’ columns)
Greater surface area
High pressure
Needed to achieve sensible flow rate
Forces interactions with stationary phase

31. Normal and Reverse Phase
Normal Phase
Polar compounds move slower
Polar solvents have greater eluting power
Example Solvent: Hexane/iso-propanol
Reverse Phase
Silica is derivatised with a long chain hydrocarbon (C18)
Non-polar compounds move slower
Non-polar solvents have greater eluting power
Example Solvent: Water/acetonitrile

32. Gradient Solvent System
A gradual increase in the proportion of one solvent to increase/decrease the polarity
e.g. 10 % MeCN in water to 40 % MeCN in water over 15 min
Useful for multicomponent systems
Slower sample throughput
Can take longer to optimise

33. Isocratic Solvent System
A single solvent or constant solvent mixture is used for the chromatography
Improves sample throughput as the solvent in the system at the end is the same as at the beginning
Quicker to optimise
No issues relating to solvent mixing