The retention of a compound often differs considerably between experiments and laboratories due to variations of the eluent, the stationary phase, temperature, and the setup. It is therefore important to compare the retention of the test compound to that of one or more standard compounds under absolutely identical conditions.
The plate theory describes the chromotography system, the mobile and stationary phases, as being in equilibrium. The partition coefficient K is based on this equilibrium, and is defined by the following equation:. K is assumed to be independent of concentration, and can change if experimental conditions are changed, for example temperature is increased or decreased.
As K increases, it takes longer for solutes to separate. See the article paper chromatography. A small spot of solution containing the sample is applied to a strip of chromatography paper about one centimetre from the base. This sample is adsorbed onto the paper. This means that the sample will contact the paper and may form interactions with it. Any substance that will react with and thus bond to the paper cannot be measured using this technique. The paper is then dipped in to a suitable solvent such as ethanol or water and placed in a sealed container.
As the solvent rises through the paper it meets the sample mixture which starts to travel up the paper with the solvent.
Different compounds in the sample mixture travel different distances according to how strongly they interact with the paper. Paper chromatography takes some time and the experiment is usually left to complete for some hours. The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes.
Two-way paper chromatography involves using two solvents and rotating the paper 90 o inbetween. This is useful for separating complex mixtures of similar compounds. In thin layer chromatography or TLC the stationary phase consists of a thin layer of adsorbent like silica gel , alumina , or cellulose on a flat carrier like a glass plate, a thick aluminum foil, or a plastic sheet. The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different adsorbents.
TLC is a standard laboratory method in organic chemistry. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products. TLC plates are made by mixing the adsorbent with a small amount of inert binder like calcium sulfate gypsum and water, spreading the a thick slurry on the carrier, drying the plate, and activation of the adsorbent by heating in an oven.
The thickness of the adsorbent layer is typically around 0. Once visible, the spots can be quantified by way of calculating their R f values. These values should be the same regardless of the extent of travel of the solvent, and in theory are independant of a single experimental run. A pencil line is drawn, and spots of ink or plant dye are placed on it. There is a container of solvent, such as water or ethanol. The paper is lowered into the solvent. The solvent travels up through the paper, taking some of the coloured substances with it.
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