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纸层析法分离氨基酸 Isolation and identification of Amino acid by paper chromatography
4 (b) Isolation and identification of Amino acid by paper chromatography Next Biochemistry Practical * Materials required Filter paper Beaker, Petri dish, Scotch tape, Capillary tube, Pencil, Measuring ruler. White plastic board Oven Hydrolyzed protein solution (Containing amino acids:- Leucine, Cysteine, Valine, Serine) Known amino acid solutions:- a) Leucine solution, b) Cysteine solution, c) Valine solution, d) Serine solution, e) Proline solution. Sample of unknown amino acid solution. [a)Serine solution, b) Proline solution] Eluting solution [Butyl alcohol + Formic acid + Water (15:3:2) (v/v)] Ninhydrin reagent with sprayer. * Procedure Take three sheets of filter papers, and draw a faint pencil line about 1cm from one of the long edges and parallel to that edge of each paper. This will be the bottom of the chromatogram. Mark off five equally spaced points along this line. (They should be separated by about 1 cm). Your samples will be applied to these spots. The samples can be applied to the paper by using a narrow capillary tube. Place one spot of each of the five known amino acids on the separate points that you previously marked on a filter paper. Apply samples of your unknown to two of the points of previously marked another filter paper. Apply sample of hydrolyzed protein solution to one of the points of previously marked another filter paper. Be careful not to contaminate either the solutions or the spots. Label each spot (with pencil and below the starting line) to indicate its identity. Finally, it’s a good idea to avoid getting fingerprints on the chromatographic paper. * When you have finished spotting your paper, allow it to dry by waving those in the air or using a oven at 400 C. (Don’t get it too hot.) Meanwhile, take 20 mL of the eluting solution [Butyl alcohol + Formic acid + Water (15:3:2) (v/v)] into each of three clean, dry 600 mL beakers and cover the beakers with Petri dishes. When the sample spots have dried, roll each paper i
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