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ninhydrinaminoacidlab - Chemistry 365 Biochemistry...

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Chemistry 365 Biochemistry Laboratory Unit #2 Spectrophotometric Determination of Amino Acids with Ninhydrin References : Lee, Y.P.; Takahashi, T. Anal. Biochem. 1966 , 14 , pp 71-77. Robyt, J.F.; White, B.J. Biochemical Techniques Theory and Practice ; Waveland: Prospect Heights, IL, 1990. Solutions: 1) ninhydrin reagent - 63 % glycerol (volume), 37% 0.5 M citrate buffer, pH 6.5 containing 7.1 g ninhydrin per liter. 2) amino acid solutions - 0.002 M solutions of phenylalanine, glycine, and aspartic acid Laboratory: 1) Prepare a blank by mixing 3.8 mL ninhydrin reagent and 0.2 mL distilled water. 2) For each amino acid, prepare a range of four concentrations by mixing 3.8 mL ninhydrin reagent and varying amounts of amino acid stock solution and water that add to 0.2 mL. 3) Mix each solution well and heat in a boiling water bath for 45 minutes. 4) Cool the solutions and read the absorbances versus the blank at 570 nm. Report: 1) Write a brief summary of the experiment and spectrophotometric analysis in your notebook.
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