A nanogram and picogram method for amino acid analysis by gas-liquid chromatography

Abstract

A gas—liquid chromatographic method has been developed for the analysis of samples containing nanogram to picogram amounts of amino acids. The procedural details of the method are presented with the reaction conditions for conversion of 1 to 1000 ng of amino acids to their N-trifluoroacetyl n-butyl esters. The developed method was found to be essentially quantitative. Recoveries were greater than 80% when 5 ng of each amino acid were taken through the total method by comparison with diluted macro standard solutions. Also, an injection port solvent vent-chromatographic system was invented which allows injection of the total derivatized samples (up to 100 μl) on a standard packed analytical column. This device prevents the solvent and excess acylating reagent from traversing the column and entering the detector, while allowing quantitative transport of the amino acid derivatives through the column. Samples containing seventeen amino acids at the 50 and 5 ng levels were taken through the total derivatization and chromatographic procedure, and then analyzed by GLC incorporating the solvent vent device. To achieve higher sensitivities, the detection of the N-trifluoroacetyl and N-heptafluorobutyryl n-butyl esters of selected amino acids by 03Ni electron capture was studied. The minimum detectible amounts of various amino acid derivatives were determined by demonstrating that 1 to 50 pg can be clearly observed by this method. Studies were made on the N-TFA n-butyl ester derivatives of eight amino acids, and on the N-HFB n-butyl esters of methionine and cysteine. Analyses of the water extracts of the Returned Lunar Samples (Apollo flights 11 and 12) are presented with none of the common amino acids being detected above the background detection limit of ca. 4 to 5 ng/gram. However, several unidentified chromatographic peaks were observed, particularly in the Apollo 12 sample, which expected content of the compounds of interest is extremely small. The broad spectrum of application for this technique has already included the search for amino acids in the Returned Lunar Samples, and would range from studies at the cellular level, investigations on the molecular basis of disease, to the analysis for trace amounts of environmental pollutants.