Wmctc chromatography Workshop: 7th Feb 2017 Ian J. Shannon

Reporting TLC Data

• Rf – distance travelled by compound divided by distance travelled by eluent
• Reported to 2 d.p.
• Include eluent & visualisation

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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

Effect of Solvent on Rf

• In Hexane & Diethyl
• Ether

Solvent %Hexane in Ether	Rf Fluorene	Rf Fluorenone
100	0.54	0
95	0.72	0.22
90	0.81	0.40
80	0.83	0.54
60	0.84	0.70
40	0.86	0.77
20	0.97	0.91
0	1	1

TLC Video

• http://www.rsc.org/learn-chemistry/resource/res00001074/thin-layer-chromatography

• TLC Troubleshooting

• A

• B

• C

• D

• E

• F

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

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

A-Level Exemplar: Anadin Extra

• 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.

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

GC Video

• http://my.rsc.org/video/55

Schematic of Gas Chromatograph

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

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

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

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

HPLC

• High Pressure Liquid Chromatography
• High Performance Liquid Chromatography
• Allows Analysis and/or Purification

• 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

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

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

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