Quantitative gas-liquid chromatography of sulfur containing amino acids

Abstract

This research describes the analytical methods for the preparation, yield, structural confirmation, RMR a.a./phen. values 2 2 RMR a.a/phen. = relative molar response = area amino acid/mole area phenanthrene/mole . , and substrate interaction for the volatile trimethylsilyl (TMS) derivatives of twelve sulfur amino acids: S-methyl- l-cysteine, taurine, cysteic acid, homocystine, djenkolic acid, ethionine, methionine sulfoxide and sulfone, and l-2-thiolhistidine. For reference and comparison purposes the TMS derivatives for methionine, cysteine, and cystine were also prepared. Bis(trimethylsilyl) acetamide (BSA) and bis(trimethylsilyl) trifluoracetamide (BSTFA) were employed as the silylating reagents. The TMS derivatives were also prepared on a macro scale, and the structural formulae were determined on the basis of elemental analysis and infrared spectra. The synthesized pure reference TMS derivatives were used in yield studies. The yields ranged from 95.5–99.2% with BSA as silylating reagent. Some difficulties due to oxidation of the sulfur atom were encountered during purification of the derivatives for the following sulfur amino acids: cysteic acid, l-2-thiolhistidine, cysteine, cystine, and homocystine. In this case working calibration curves were prepared. The TMS derivatives of the twelve sulfur amino acids can be quantitatively prepared by heating at 150° for five minutes in a closed tube. The silylation reagent-solvent ratio is 1 3 v/v. BSTFA is recommended as the silylation reagent for all of the named sulfur amino acids except methionine, methionine sulfoxide, and S-methyl- l-cysteine. The TMS derivatives were stable with respect to time in a closed tube. Separation of the derivatives was achieved on OV-1 and OV-17 columns. A reduced response was noted for methionine and l-2-thiolhistidine on an OV-22 column, and separation was not achieved for methionine sulfoxide and sulfone. The chromatographic and instrumental conditions were: Column 0.50 w/w% OV-1 coated on 80–100 mesh acid-washed dimethylchlorosilane (DMCS) treated Chromosorb G packed into a 1 m x 3.5 mm I.D. U-shaped column, and temperature programmed from 75°–200° with rise of 4.6°/min.