Chromatography is used to analyze small quantities of a mixture of substances which are chemically similar to each other. It involves the partition of the components of the mixture between a stationary phase and a mobile phase. The mixture to be separated is introduced on the stationary phase which stays still. The mobile phase is then allowed to move over the stationary phase for separation. Partition depends on the different solubilities of the components in the mobile phase and the different adsorption forces of the components with the stationary phase. Adsorption is the temporary attraction of molecules of a gas or liquid to a solid surface. Components with greater solubilities will dissolve into the mobile phase and move along with it readily. Components with stronger adsorption forces will be held on the stationary phase and not move along readily with the mobile phase. The differences in solubilities and adsorption bring about separation.
In paper chromatography, a piece of filter paper or chromatography paper is used which consists of stationary water molecules embedded in a cellulose matrix. The water molecules act as the stationary phase. The mobile phase consists of a suitable solvent that travels up the stationary phase. The mixture to be separated is spotted a short distance from one end of the paper (the base line). The end below the spot is placed in the solvent. As the solvent moves along the paper it carries the mixture with it. The distance the solvent moves from the baseline is called the solvent front. Components of the mixture will separate readily according to how strongly they adsorb on the stationary phase and how readily they dissolve in the mobile phase. If the separated components are colorless, then a visualizing agent can be used to convert them into colored spots. The positions of certain substances can also be determined by fluorescing under a UV lamp. The ratio of the distance moved by a component of the mixture to the distance moved by the solvent is called retention factor. Rf = distance moved by a component distance moved by solvent Each component has a characteristic Rf value for a given solvent under controlled conditions. Thus Rf values of known substances can be used to identify components of a mixture. Paper chromatography is used to analyze mixtures such as dyes in ink, coloring in food additives and amino acids from protein hydrolysis. A visualizing agent such as ninhydrin is used to detect amino acids and amines.
Thin Layer Chromatography (TLC)
This method is similar to paper chromatography. The stationary phase is a thin layer of powered alumna or silica gel which s fixed on to a glass or plastic plate. Plates can be coated with a slurry of the powered adsorbent and then oven – dried. The mixture to be analyzed is spotted near the bottom of the plate. The end below the spot s placed in a suitable solvent. This solvent is the mobile phase and moves up the plate causing the components of the moving solvent. The separated components may be recovered for further analysis by scraping spots off the plate. Thin layer chromatography has the advantage that a variety of adsorbents can be used for separation. It is commonly used to separate amino acids in blood samples and for analysis of food dyes.
This method is similar to thin layer chromatography however the stationary phase is packed into a vertical glass column (diameter 1- 2cm) instead of being coated on a plate. A slurry of silica gel or alumina is commonly used for column chromatography. The mobile phase is a suitable solvent which is added to the top of the loaded column. The solvent flows down the column under gravity causing the components of the mixture to partition between the adsorbent and solvent. Each component emerges from the column at different times and can be collected separately. The time between addition of the sample at the top of the column and the emergence of a component at the bottom of the column is called the retention time of that component. Identical substances will have the same retention time under the same conditions thus retention times can be used to identify substances. Column chromatography has the advantage that larger quantities can be separated and therefore can be used to prepare compounds in addition to analyzing them. This method is used in biochemical research and in hospitals to identify amino acids, peptides and nucleotides.
High Performance Liquid Chromatography (HPLC)
This technique is similar to column chromatography however instead of gravity feed, high pressure is used to force the solvent through the column. Columns are smaller than those used in column chromatography, some being 10cm to 30cm long and 4mm in diameter. Retention times are shorter thus rapid analysis of substances can be made. HPLC s used n the industry and hospitals. It is also used to identify suspected stimulants, doping and drugs that may be present in athletes and racehorses.
Gas – Liquid Chromatography (GLC)
GLC uses a longer column than HPLC. It is usually packed with the stationary phase which is an inert powder coated with an non-volatile oil. The column is maintained at a constant, preset temperature in an oven. The mobile phase is an un-reactive gas. The sample to be analyzed has to be in the vapor state at the temperature at which the column is operated. The vaporized sample is carried through the column by the mobile phase. The sample is partitioned between the oil and the carrier gas A detector records each component as it leaves the column at different times. Emerging components can also be fed directly into a mass spectrometer for identification. GLC method of analysis is very sensitive and can be used in forensic testing, to monitor air and water pollution, to detect and identify traces of pesticides or agricultural chemicals in foodstuff and to check dosage of drugs in blood or urine samples.