Gas-liquid chromatography of protein amino acid trimethylsilyl derivatives

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Abstract

The purpose of the investigation was to make a thorough study of the chemistry of derivatization of the twenty protein amino acids as their N-trimethylsilyl trimethylsilyl (TMS) esters. Major emphasis was directed toward chromatographic separation of the derivatives, precision and accuracy of the method, silylation as a function of reaction temperature and time, molar excess of reactants, stability of the TMS derivatives, quantitative analysis of a synthetic amino acid mixture, and application to biological samples.

The gas-liquid chromatographic separation of the N-trimethylsilyl TMS esters of the twenty protein amino acids was achieved after evaluation of a number of combinations of siloxane liquid phases. The final chromatographic conditions used for the total separation on a single column for all twenty of the amino acids consisted of a mixed liquid phase of 3.0 w/w% OV-7 and 1.5 w/w% OV-22 coated on high-performance 100120 mesh Chromosorb G in a 1.75 m × 4 mm I.D. U-shaped borosilicate glass column. Phenanthrene was a suitable internal standard as it was completely resolved from the TMS amino acids. The instrumental settings were 75°, initial hold 7 min, program rate 2°/min, and carrier flow (N2) of 42 ml/min for fourteen of the amino acids, and 100° initial column temperature for the other six. Prior to chromatography, it is essential to analyze performance blanks under the same chromatographic and instrumental conditions to establish the purity of all chemical reagents.

The reaction conditions were investigated for the quantitative silylation of the twenty amino acids. Fourteen of the amino acids were reproducibly converted to the respective TMS derivatives in 15 min at 135° in a closed vial using a 30 molar excess of bis(trimethylsilyl)trifluoroacetamide (BSTFA)/total amino acids. A comparison of various silylation temperatures showed that silylation at 135° produced the most atmosphere in a closed tube. After evaporating the sample to dryness at room temperature with a rotary evaporator, the sample was transferred to a 50-ml volumetric flask and brought to volume with 0.1 N HCl. Five milliliter aliquots of this stock ribonuclease solution were then transferred to 16 mm × 75 mm glass reaction tubes, dried, and derivatized as described in the section experimental.

The chromatograms obtained on silylation at 135° for 15 min and 4 h, respectively, are presented in Figs. 11 and 12. The data obtained from three independent GLC analyses are given in Table X, and the results are in good agreement with those from classical ion-exchange analysis.

Preliminary investigations on the GLC analysis of cation and anion-exchange cleaned human urine by the TMS technique have shown that some problems still exist. The urine samples contained a large amount of glycine, and difficulty in obtaining a single peak for glycine was noted. Both the di-trimethylsilyl (GLY2) derivative and the tri-trimethylsilyl (GLY3, ca. 10%) derivative were obtained when the samples were derivatized at 135° for both 10 and 15 min. The GLY3 peak interfered with the resolution of TMS leucine and TMs proline due to the large quantity of glycine in the sample. Further investigations are needed to obviate this problem.

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Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 5759. Approved by the Director. Supported in part by grants from the National Acronautics and Space Administration (NGR 26004-011), the National Science Foundation (GB-7182), and the United States Department of Agriculture (12-14-100-8468(34)).

1

Professor, Experiment Station Chemical Laboratories.

2

Experimental data taken in part from Master's Thesis, University of Missouri, February 1969.

3

National Science Foundation Fellow.

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