Studies on soil humic acids II: Observations on the estimation of free amino groups. Reactions of humic acid and lignin preparations with nitrous acid

Abstract

1. Free amino groups in humic acid preparations isolated from 0·5M-sodium hydroxide and 0·1M-sodium pyrophosphate (pH 7·0) extracts of various soils have been estimated by the nitrous acid method of Van Slyke (1929) and the fluorodinitrobenzene technique of Sanger (1945). 2. The results obtained by the Van Slyke method using a reaction time of 15min. indicated that from 12 to 30% of the total nitrogen in the preparations examined was in the form of free amino groups. No free amino groups could be detected by the fluorodinitrobenzene technique. 3. It is shown that lignin interferes with the estimation of amino groups by the Van Slyke method, and it is suggested that lignin or ligninderived material may be largely responsible for the high apparent amino-nitrogen values obtained with humic acid preparations by this method. 4. The reaction of humic acid with nitrous acid resembles the reaction of lignin with nitrous acid in that it is accompanied by the fixation of nitrogen and the destruction of methoxyl groups. The reaction of lignin with nitrous acid is similar in many respects to its reaction with nitric acid. 5. Only about one-third of the nitrogen fixed by lignin in its reaction with nitrous acid is removed by prolonged hydrolysis with 6N-HC1, and most of the nitrogen so released is in the form of ammonia. A small amount of the nitrogen liberated by acid hydrolysis is in the form of hydroxylamine.