D. Roach, C. Gehrke
1969
Citations
1
Influential Citations
212
Citations
Quality indicators
Journal
Journal of Chromatography A
Abstract
Abstract The reaction conditions necessary for the “direct esterification” of the protein amino acids to their n-butyl esters are described. All of the amino acids were quantitatively esterified in n-butanol 3 N in hydrochloric acid at 100° with the exception of isoleucine. This “direct esterification” method with n-butanol permits a rapid derivatization and analysis by gas-liquid chromatography of the protein amino acids, thus, one of the major disadvantages of the earlier reported method has been removed. The amino acids were observed to dissolve very slowly in n-butanol 6 N in hydrochloric acid even when the samples were subjected to ultrasonic mixing. Fairly rapid dissolution occurred in 1.5 N hydrochloric acid but a longer esterification time was noted. The optimum concentration of hydrochloric acid was found to be 3 N because the amino acids dissolved quickly in this solution with ultrasonic mixing and short esterification times were obtained. The more insoluble amino acids were broken up by ultrasonic mixing, thus increased rates of solution and esterification to the n-butyl esters were achieved. The effect of temperature over the range of 90 to 120°, on the rate of esterification was investigated but little effect on the relative molar response values was observed. However, the time of esterification was quite significant for two amino acids, tryptophan and isoleucine. Nineteen of the amino acids were quantitatively esterified with n-butanol 3 N in hydrochloric acid in 15 min at 100°, but 35 min were required for the esterification of isoleucine. However, with the longer esterification time, tryptophan underwent some decomposition (ca. 15%). An esterification time of 35 min is recommended for samples which are to be analyzed for all of the amino acids including isoleucine and tryptophan; however, only 15 min are required if a slight error in the absolute value for isoleucine is permissible. In quantitative work, a reference calibration mixture is analyzed under exactly the same experimental conditions as are the samples thus correction is made for any tryptophan in the sample resulted from the low RMR for tryptophan in the calibration mixture. This resulted from heating tryptophan in an acidic n-butanolic solution for a total of 3 h with the corresponding losses. The RMR values for the amino acid derivatives which were obtained using the direct esterification procedure are presented in Table IV. The precision of the data is excellent as evidenced by the relative standard deviations, which in most cases are ca. 1% or less.