Centrifugal extraction and determination of free amino acids in soil solutions by TLC using tritiated 1-fluoro-2, 4-dinitrobenzene

A method is described to separate and quantitate free amino acids from soil solutions extracted rapidly by centrifugation at 10,OOOg for 5 min. Centrifugation of soil samples with forces up to 20,000 g did not affect the content of ammonium and water soluble organic carbon in solution, therefore it was concluded that the integrity of microbial cells was unaffected. The method of amino acid analysis involved treatment of the soil solution with tritiated I-Fluoro-2,4_dinitrobenzene under alkaline conditions after addition of a known concentration of a standard mixture of amino acids. After extraction, the dinitrophenyl (DNP)-amino acids were separated by two-dimensional thin layer chromatography. Quantification was effected by counting the jH-activity of each spot and comparing it to that of spots of a standard amino acid mixture of known concentration. The method has been tested and found satisfactory on a variety of cultivated soils, except for an Andept soil with a high content of metallic ions in solution. The total free amino acid content in cultivated and virgin soil samples ranged between 0.32 and 4.72 pg g-' soil. Alanine, phenylalanine and threonine were the most abundant followed by valine and the aspartic-glutamic acid complex. Basic amino acids, those containing S (methionine and cystine), and the isoleucine-leucine pair were the least abundant and frequently not detected. With the Chernozemic order the total amino acid content decreased in the following order: Brown > Dark Brown > Black. Virgin soil samples contained more but not always a wider range of free amino acids than did cultivated soils. A Luvisolic soil cropped for 50 yr to a S-yr rotation contained fewer and a narrower range of free amino acids than did the same soil cropped to a wheat-fallow rotation.