Chromatography

Chromatography

• Separation technique that relies on the affinity of components of a mixture to:-– A Mobile Phase– A Stationary Phase

• All types of chromatography rely on this– Paper chromatography– thin layer chromatography, – high performance liquid chromatography, – gas chromatography

Chromatography

• Mobile Phase– This is the phase that is a carrier for the

components as they travel through the stationary phase

• Stationary Phase– This is the phase that is stationary!!

Types of Chromatography

• Paper Chromatography• Thin layer chromatography (TLC)• High Performance Liquid Chromatography

(HPLC)• Gas Chromatography (GC)

there are more......................

Paper Chromatography

• Basic technique• Stationary Phase – Paper• Mobile Phase – Usually water• Sample preparation• Good for separating water soluble inks• Limited types of mobile phases can be used

therefore limit to what can be separated• Can be slower • Detection – naked eye or UV light

Paper chromatography.rv

Thin Layer Chromatography

• Improvement on paper technique• Stationary Phase – silica or alumina based

– Acidic, basic, neutral• Mobile Phase – solvents (varied polarities)• Good for:-

– Monitoring progress of a reaction– Identification of components in a mixture– Purity determination– Conditions for column separation techniques

• Semi-quantative • Can be quicker than paper – phases• Detection – UV light, chemical spray, iodine tank

Rf Values (retention factor)

• Distance travelled by component

• Distance travelled by mobile phase

• Specific to each plate• Comparable• Each component on

plate has unique Rf value

High Performance Liquid Chromatography

• Mobile phase – solvents/water/mixes• Stationary phases – silica based/many types for certain

applications• Sample preparation• Precise injection volumes• Composition/flow of mobile phase controlled• Oven temperature• Widely used in analytical laboratories• Quantify components – non volatile mixtures• Retention times

Gas Chromatography

• Mobile phase – unreactive gas (helium, nitrogen)• Stationary phases – polymer or liquid on an inert

solid support• Sample preparation• Precise injection volumes• Temperature/gas flow control• Widely used in analytical laboratories• Quantify components – volatile mixtures• Retention times

Columns

Injector

Autosampler

Detector

Data collection software

Column oven

AutosamplerData collection software

Column oven Keypad

Injector

Detector

Applications in Pharma Industry

Chromatogram

The Future• Predictive software• Screening• Saving resources