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Biochemistry - Lab Report Example

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The separation of amino acids depends on the rate at which it moves while in simultaneous contact the two phases. This migration…
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Biochemistry lab report
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Download file to see previous pages Paper chromatography is actually liquid-liquid chromatography, the paper should not be considered as solid phase, but the water molecules trapped in the cellulose of the paper form the ‘stationary’ phase. To saturate the cellulose, most paper chromatographic solvents have some amount of water in it. So the components with very high water solubility will move slower ( Paré and Bélanger, 1997).
Method: A 60:40 v/v acetonitrile: ammonium ethonate mobile phase is made, pH 7.2, and placed in a covered tall jar. Aqueous solutions of amino acids are spotted on the specified location (origin) on the stationary phase using a capillary and allowed to dry. The stationary phase is then put into the jar with mobile phase and allowed to run for 40 minutes. Mark the solvent front. Make sure the solvent stays well below the top edge. The stationary phase is then dried and sprayed with ninhydrin solution in the fume hood, and heated to allow the color to develop.
From the Rf values (Table 1) it seems neither molecular weight nor the polarity had any significant effect on the migration. Glycine being the smallest did not travel the farthest. Looking at both, Rf and the color developed the sample X is Lysine and Y is Proline. Finger prints are seen on both the lateral sides of the paper, probably at the places used to handle the paper. They appear due to reaction between ninhydrin and the terminal amines of the lysine de-bonded from the amino acid. Also the sweat-gland secretions in the ridges of the fingers has proteins too (Sens,Simmons and Spicer, 1985).
Conclusion: Paper chromatography can be used to separate amino acid from a mixture of amino acids. The migration of amino acids on the solid phase is a complex interplay between the molecular eight, shape, structure and polarity of the amino acids and their affinity towards the solid and the mobile phase. Use of ninhydrin to identify amino ...Download file to see next pagesRead More
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