Appendix—Notes on the determination of amino acids by ion-exchange chromatography

Abstract

The method of Moore and Stein (J. biol. chem. 192, 663, 1951) was modified. A temperature of 60[degree] is employed throughout the elution of the 100 cm Dowex 50 column with sodium citrate buffer (pH 4.25), instead of a change from 50 to 75[degree] after the emergence of iso-leucine. The 0.1 [image] sodium phosphate buffer used to elute the 15 cm Dowex 50 column was reduced from pH 6.80 to 6.75 to separate histidine from both hydroxylsyine and lysine. The optical density of the colored solutions was measured in optical cells instead of in test tubes. This enabled a sensitive spectrophotometer having a linear scale up to at least 1.5 optical density units to be used, leading to increased accuracy and convenience in avoiding frequent dilution of samples. An empirical method for calculating tyrosine values is described which is applicable when there is considerable overlap by phenylalanine and only a small quantity of tyrosine present.