Amino acid analysis:: Reduction of ninhydrin by sodium borohydride

Ninhydrin based monitoring systems remain the widely employed for the quantitative determination of amino acids subsequent to their separation by ion-exchange chromatography. In the reaction of ninhydrin with amino acids the presence of hydrindantin (reduced ninhydrin) is advantageous. The latter can be added to the reaction mixture directly or it can be formed by the addition of a reducing agent. In the present work, the stability of a ninhydrin reagent in which ninhydrin has been reduced with sodium borohydride is compared with that of commercial preparation using stannous chloride or titanium chloride as a reducing agent. Both agents are commonly used in automated amino acid analysis. The advantages of the sodium borohydride-containing preparation are as follows: (a) ninhydrin solution does not form precipitates in the flow lines of the analyser, (b) no waiting period for the maturation of the reagent is required, (c) very stable ninhydrin solution (at room temperature for a month, at 4-5 degrees C even longer), (d) no necessity to keep the solution in refrigerator, (e) amino acid colour yield is about 10% higher for 17 amino acids, (f) this modification based on methylcellosolve as a solvent buffered with 4 M sodium acetate (pH 5.5) overcome the increase of the base line at high pH (pH above 8) connected with ninhydrin reagent using dimethylsulfoxide-lithium acetate buffer.