Appendix—A semi-micro method for the determination of amino acids by ion-exchange chromatography

Abstract

A semi-micro modification of the Moore and Stein ion-exchange method of amino acid determination is described, which enables a complete analysis to be carried out on 0.3-0.6 mg of protein. The increased sensitivity obtained results from a reduction of resin-column diameter from 0.9 to 0.4 cm with a corresponding reduction of the original volume of the fractions and reagents to one -fifth. A simple method of modifying a spectrophotometer for convenient measurements on small volumes of solution in standard optical cells is described. Preliminary separation of basic amino acids may be carried out on a column (2.2 cm x 0.4 cm) of Dowex 50 at pH 5.0. Separation of hydroxylysine from histidine is possible by combining the small column with a 15 cm column and eluting the two in series. Colorimetry on the reduced scale is closely similar to the original procedure, but the rate of fading is increased. The sharpness and resolution of amino acid peaks were not affected by reduction of column diameter. A comparison of amino acid values for a gelatin hydrolysate analysed by the original and semi-micro methods showe''d good agreement. The overall accuracy of the modified method is approximately 5%.