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

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

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

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

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

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

Preparation of a TLC Plate

Analysing a Reaction A + B -> Products

Running a TLC Plate

What is Happening?

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

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

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

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

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

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 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

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 Temperature effects – higher temperature means more in gas phase 23

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 Packed – robust Capillary – better but fragile 25

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

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