An improved colorimetric determination of amino acids with the use of ninhydrin

✍ Scribed by Ya Pin Lee; Tunekazu Takahashi

• Publisher: Elsevier Science
• Year: 1966
• Tongue: English
• Weight: 335 KB
• Volume: 14
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(66)90057-1

✦ Synopsis

Attempts have been made to develop a simpler and more specific method for determining free a-amino acids in chemical and biological systems. The results obtained are presented in this paper.

METHODS

AND RESULTS

Reagents:

0.5 M citrate (Na+) buffer was prepared from analyticalgrade citric acid which was free of ammonia or amino acids. pH of the citrate buffer was adjusted to a desired value. A 1% ninhydrin solution was prepared by dissolving 1 gm of ninhydrin in 0.5M citrate buffer solution. Commercial ninhydrin preparations were found to be satisfactory without further purification. Some commercial glycerol preparations which were found to interfere with color development or to give a high blank reading were diluted with an equal amount of water to 50% (v/v) solution. The diluted solution was passed through a column of mixed resins of Amberlite ion exchanger to remove the contaminants. The effluent was concentrated to the anhydrous state (approximately 99.5%). Most of the amino acids, which were chromatographically pure, were obtained from Mann Research Laboratories, Inc., New York City. Amino acids, amines, or other compounds were dissolved in water, dilute NaOH, or dilute HCl solution.

Procedure: 1.0 ml of a reaction mixture which consisted of citrate buffer, ninhydrin, and glycerol was pipetted into a test tube. Less than or 1 ml of sample and additional compounds to be tested, if there was any, were introduced into the reaction mixture. The total volume was made up to 2.0 ml with water. After shaking, the test tube was placed in a boiling water bath for a certain time as described in each experiment. After cooling in a tap-water bath at room temperature, a reading was taken at 570 rnp within an hour. The reagent blank and standard amino 71