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Let us learn about Separation of Amino Acids by Paper Chromatography .
A mixture of unknown amino acids can be separated and identified by means of paper chromatography. The position of the amino acids in the chromatogram can be detected by spraying with ninhydrin, which reacts with amino acids to yield highly coloured products (purple).
Paper chromatography is a method of separating and analyzing a mixture For example, simple paper, chromatography can be used to separate a mixture of dyes. The filter paper, which contains a thin film of water trapped on it, forms the stationary phase. The solvent is called the mobile phase or eluant. The solvent moves up a piece of filter paper by capillary action.
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As the solvent moves past the simple spot of dye, there is a competition between:
1. The ability of dyes to attach to the adsorbed water, and
2. The ability of dyes to dissolve in the solvent
This is a type of partition between the stationary phase (the adsorbed water) and the mobile phase (the solvent). As different dyes have different partition between the mobile and the stationary phases they would be carried forward to different extents Dyes which are more soluble in the solvent than in water are carried further up by the mobile phase.
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Fresh solvent is continuously moving up and there is, in fact an enormous number of successive solvent extractions occur. At a suitable time, the experiment is stopped by removing the filter paper from the solvent. The retardation factor Rf, can be calculated as Fig. 22.8.
Rf = distance traveled by spot/distance traveled by the solvent (Note Rf value is always less than 1).
The Rf of component A and B can be determined as:
Rf of component A = d2/d1
Rf of component A = d3 /d 1,
The Rf value of any substance may be about the same whenever we use that particular solvent at a given temperature. However, the Rf value of a substance differs in different solvents and at different temperatures.
Chemicals:
2% ammonia, propan-2-ol, aluminium foil, ninhydrin spray (2% solution of ninhydrin in ethanol), for separate test tubes containing respectively 0.05M glycine, tyrosine, leucine and aspartic acid in 1.5% HCI.
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Appartus:
Capillary tube, chromatography paper, baker, oven,
Hazard Warning:
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Propan-1-ol is flammable. The ninhydrin solution should be kept off the body because it reacts with proteins in the skin to form a rather long-lasting purple discoloration.
Procedures:
1.10 cm3 of 2% ammonia solution is mixed with 20 cm3 of propan-2-ol in clean, 500 cm3 beaker, and covered tightly with a piece of aluminium foil. This is used as the solvent for the experiment.
2. On a clean sheet of chromatography paper with size about 12 cm by 22 cm, a light pencil line is marked to the bottom and about 1.5 cm away. Along this line ten light crosses (“x”) are marked at intervals of about 2 cm. Each cross is labeled. (“U” is represents the unknown amino acid mixture).
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3. Capillary tubes are used with appropriate solution and are placed on its two positions along the line on the chromatography paper. A spot larger than about 2 mm in diameter is avoided on the chromatography paper. The paper is let dry for a few minutes in air.
A second portion of the unknown is added to one of its two positions to make certain that sufficient quantities of each component of the unknown are presented for good visual observation when the paper is developed.
4. The paper is rolled into a cylindrical form. The ends are stapled together in such a fashion that they do not touch each other. Otherwise the solvent will flow more rapidly at that point and is formed an uneven solvent front.
5. When the spots on the cylindrical paper become dry, it might be necessary to place the paper in an oven at about 100°C for a short time. It is placed carefully in the beaker of solvent, and are covered carefully and tightly with the aluminium foil. The paper is carefully placed in order not to touch the wall of the beaker.
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6. The solvent is let to rise up the paper for at least 1.5 hours. If the time is shorter, the component might not be sufficiently separated for easy identification. The paper is removed and placed upside down on the desk top to dry. When most of the solvent had evaporated, the cylinder is opened by tearing it apart where it was stapled and hanged It in a fume cupboard.
The entire paper is lightly sprayed with a solution of nihydrin, and is left in the fume cupboard until the spray solution is dry.
7. The paper is placed in an oven at 100°-110°C for about 10 minutes, or until all the spots had been developed.
8. Each spot is circled with a pencil, and is measured the distance each spot traveled. The center of the spot is used for measurement. The distance is measured for the solvent traveled at each position, and the Rf values are calculated for each amino acid. The composition of the unknown is determined by visual comparison of spot colours and by comparing the Rf values.
