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

Dowex 50. The resin, kindly supplied by Dow Chemical International Ltd., Midland, Michigan, U.S.A., was stated to be of nominal 12% cross-linking and 200-400 mesh. Sodium phosphate buffer pH 6-8, 0.1M. This buffer was adjusted to pH 6-75 in order to delay slightly the emergence of the histidine peak. At the lower pH, histidine emerges separated from and almost mid-way between hydroxylysine and lysine on the 15 cm. column. Thymol. The concentration of thymol incorporated in the buffer solutions was controlled at 0-25 g./l. Solutions kept for two years at room temperature showed no signs of mould growth. Thymol did not significantly alter the pH of the solutions but markedly affected the surface tension, even when detergent was present. BRIJ 35. A 33% (w/w) solution of BRIJ 35 (Honeywill and Stein Ltd., 21 St James's Square, London, S.W. 1) was freshly prepared each week, as some samples of detergent were contaminated with mould spores. Thiodiglycol. Technical-grade thiodiglycol (L. Light and Co. Ltd., Colnbrook, Bucks.) was freed from an impurity which gave a yellow colour with ninhydrin by the method described previously (Eastoe & Eastoe, 1954). Ethylene glycol monomethyl ether. 'Methyl oxitol' (Honeywill and Stein Ltd.) was redistilled to remove ninhydrin-positive impurities. To minimize risk of explosion due to decomposition of peroxides, 10 ml. of a solution of 30 g. of FeSO4,7H20 and 3 ml. of H2SO4 in 55 ml. of H20 were added per litre before distillation and the final 10% of the solvent was left undistilled and discarded. Ninhydrin. Ordinary-grade ninhydrin (British Drug Houses Ltd.) was purified in 87 % yield by the method of Moore & Stein (1948). The ninhydrin-stannous chloride reagent was prepared freshly each week, since the reagent, which was stored in the dark under nitrogen, showed signs of change after 2 weeks in hot weather. No significant change in the colour intensity produced on heating with amino acids could be observed during the first week after preparation. Diluent solution. Equal volumes of water and absolute ethanol were mixed (Moore & Stein, 1954). Fraction collector. The fraction collector used in this study was built in the laboratory and worked on the dropcounting principle, a fixed number of drops (20) being collected in each fraction after the operation of a capacity switch, thyratron relay and electromagnetic counter.