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Separation of Amino Acids by Paper Chromatography

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Separation of Amino Acids by Paper Chromatography ( separation-amino-acids-by-paper-chromatography )

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to their separation on the paper. The affinities of these amino acids for the mobile phase can be correlated to the solubility of the different amino acids in the solvent (i.e., an amino acid that is highly soluble in the eluting solvent will have a higher affinity for the mobile phase than an amino acid that is less soluble in the solvent.). When the solvent front comes near the top of the filter paper, the paper is removed from the beaker and allowed to dry. At this point, the various amino acids are invisible. The acids can be visualized by spraying the paper with a compound called ninhydrin. Ninhydrin reacts with amino acids to form a blue-violet compound. Therefore, the sprayed filter paper should show a number of spots, each one corresponding to an amino acid. The further the spot from the starting line, the higher the affinity of the amino acid for the mobile phase and the faster its migration. The relative extent to which solute molecules move in a chromatography experiment is indicated by Rf values. The Rf value for a component is defined as the ratio of the distance moved by that particular component divided by the distance moved by the solvent. Figure 1 represents the migration of two components. Measurements are made from the line on which the original samples were applied to the center of the migrated spot. In the figure, dA is the distance traveled by component A, dB is the distance traveled by component B, and dsolv is the distance traveled by the eluting solution. In all three cases, the travel time is the same. Figure 1: Paper chromatography - migration of two components. pencil line with spot of mixture solvent level in beaker solvent front solvent front A B dA dsolv dB Thus the Rf values for components A and B are Rf(A) = dA/dsolv Rf(B) = dB/dsolv Note that Rf values can range from 0 to 1. In this example, Rf(A) is obviously larger than Rf(B). Although Rf values are not exactly reproducible, they are reasonably good guides for identifying the various amino acids. Paper chromatography is most effective for the identification of unknown substances when known samples are run on the same paper chromatograph with unknowns.

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