By Joshi, Rashmi Atul
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According to the choice of solvent for the mobile phase, the separation may be based on adsorption, partition, gel filtration or ion exchange processes, or some combination of these. The advantages of thin layer chromatography is convenience, rapidity and high resolution has led to its routine use in the analysis of organic molecules. Q. What is Reverse Phase Chromatography (RPC)? Ans. Reverse phase chromatography is a form of liquid-liquid partition chromatography in which the polar character of the phases is reversed relatively to that of paper chromatography.
The stationary phase in this technique consists of beads of a hydrated sponge like material containing pores that span a relatively narrow size range of molecular dimensions. If an aqueous solution containing molecules of various sizes is passed through a column containing such molecular sieves, the molecules that are too large to pass through the pores are excluded from the solvent volume inside the gel beads. These larger molecules therefore traverse the column more rapidly, that is, in a smaller eluent volume, than the molecules that pass through the pores.
The basis for separation of the components of the volatile material is the difference in the partition coefficient of the components as they are carried through the column by an inert gas such as helium. The actual apparatus is quite simple and is shown in figure. The column is first flushed out with carrier gas to remove previously injected material and to form a stable baseline. The sample is introduced at A. pm5/Third proof/14-3-05 24 Question Bank of Biochemistry the liquid absorbent and separate, eventually a fraction passes through a suitable detecting device, which sends signals to a recorder, which in turn converts the signal into a useful sequence of peaks.