Principles of PC, TLC and HPLC and their uses.

Done by : Ain Nabilah Hj Awang. PU2E (2011)

PAPER CHROMATOGRAPHY (PC). Stationary phase : water molecules strongly adsorbed on the fibres of pure cellulose paper ; i.e the chromatography paper itself. Mobile phase : any suitable liquid solvent, may be a mixture of liquids chosen so as to give good separation of components. Procedure: The mixture must be dissolved in a solvent, such as water or ethanol. It is then spotted onto a piece of chromatography paper (stationary phase),which is supported vertically in an enclosed vessel with its lower end immersed in a solvent (mobile phase) the spot being above the initial level of the solvent. The solvent rises up the paper by capillary action. When it reaches the spots, equilibrium is set up as the components are repeatedly partitioned between the stationary phase and the mobile cluant. The components travel at different speeds with the less strongly adsorbed (more soluble) component moving further up the paper. The components of the mixture are thus separated and the solvent front is allowed to almost reach the top of the paper. The various components can be identified by their retention factor (Rf Values), which is the characteristic of each component in a given solvent.

Rf = distance moved by solute = x distance moved by solvent y

If the separated components are colourless , the paper is dried and sprayed with selective spray reagent (eg. Ninhydrin) which shows up the components as coloured spots.

Uses of PC: To analyse mixtures such as the dyestuffs in inks, the coloured additives in food and chlorophyll.

THIN LAYER CHROMATOGRAPHY (TLC). Stationary phase : a thin layer of powdered alumina (or silica) spread on to a piece of glass(or plastic) plate. Mobile phase : any suitable liquid solvent , may be a mixture of liquids chosen so as to give good separation of components on development. This technique is similar to paper chromatography. Good, accurate separation is obtained when carefully performed and the result is identical to that obtained by paper chromatography. In practice, TLC is more widely used than paper chromatography because 1. the solvent front moves more quickly and the chromatogram develops more rapidly. 2. both the solid absorbent and the solvent can be varied, thus facilitating optimum separation of the components in the mixture analysed. 3. the positions of colourless components may be detected using techniques which would damage or destroy paper. 4. TLC can be used with mixtures of corrosive nature. TLC has widespread applications in routine analysis: eg. - to check that only permitted colouring agents are present in foodstuffs. - to determine the amino acids present in blood samples of hospital patients.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC).

Stationary phase : finely divide material (eg. Powdered alumina or silica) packed into the a long column fitted with a tap. Mobile phase : any suitable liquid solvent, may be a mixture of liquids chosen as to give good separation of components.

This technique is similar to column chromatography except that 1. the column is much shorter and smaller (10cm to 30cm long, 4mm diameter). 2. a pump is used to flush the eluant through the column at a constant flow rate. This is because the column material is of a very small uniform particle size and high pressure is required to force the eluant through.

Procedure: The mixture is carefully applied to the top of the column which is kept saturated with the solvent ( to avoid air bubbles forming which would, otherwise, reduce the effective surface area of the powder in contact with the solution.) Thus, fresh solvent is added at the top of the column at the same rate as the solvent leaves the column at the bottom.

The components are washed down the column at different speeds. Due to different solubilities in the mobile phase and different adsorption onto the surface of the stationary phase. The least strongly adsorbed will travel most quickly and be collected separately as they pass out at the bottom of the column. The different components can then be identified from the time taken for each to reach the bottom of the column.

HPLC gives very rapid separation due to the very large surface area available for the small particles in the short column which is set up between the adsorbed and the dissolved components . HPLC is used in industry, hospital and scientific establishment. It is especially suited in detecting organic molecules whose boiling temperatures are too high for the use of gas/liquid chromatography (GC). eg - to identify suspected stimulants, doping and the drugs that may be present in people, athletes and racehorses. - to analyse pharmaceutical products before packaging n to check that undesirable impurities (even if in minute quantity) are not present.