Quantitative gas—liquid chromatography of histidine

Abstract

The quantitative gas—liquid chromatographic analysis of histidine was easily accomplished using two different methods and the mono and diacyl N-TFA n-butyl esters of histidine as the derivatives. To obtain quantitation, histidine must be present entirely as either the monoacyl or as the diacyl derivatives. There are certain advantages in using the diacyl derivative, but this derivative was not separated from the N-TFA n-butyl ester of aspartic acid on any of the siloxane columns evaluated (OV-1 to OV-25). A quantitative, reproducible method has been developed and is presented by which histidine can be analyzed as its diacyl N-TFA n-butyl ester. In this method the diacyl derivative formed during acylation was converted to the monoacyl derivative by evaporation of the excess acetylating reagent, TFAA. Thus, histidine is present as the monoacyl derivative when the sample is injected ∝on column’. Then, the monoacyl derivative was converted to the diacyl derivative by direct ∝on column’ injection of 4 μl of TFAA. When the TFAA was injected immediately after the methionine peak, the diacyl derivative synthesized on the column was precisely eluted between tyrosine and glutamic acid. No interference was observed with any of the other amino acid derivatives. The separation of the diacyl derivative of histidine from the aspartic acid deriviative was made possible because the retention temperature of the monoacyl derivative is different from that of the diacyl histidine derivative or the derivative for aspartic acid. The quantitative elution of all twenty of the protein amino acids as their N-TFA n-butyl esters has been achieved on a dual column system of 0.325 w/w % of stabilized EGA on heat-treated Chromosorb G and 1.5 w/w % OV-17 on high performance Chromosorb G as the stationary phases. Alternatively, for sixteen amino acids, a 1.5 m glass column with packing of 0.65 w/w % of stabilized EGA on 80 100 mesh acid-washed Chromosorb W dried at 140° for 12 h can be used. In another approach, sixteen of the protein amino acids can be determined as their N-TFA n-butyl esters on a 1.5 m × 4 mm I.D. glass column consisting of 0.325 w/w % of stabilized EGA on heat-treated Chromosorb G (or 0.65 w/w % of stabilized EGA on 80 100 mesh acid-washed Chromosorb W dried at 140° for 12 h) while arginine, histidine, tryptophan, and cystine are chromatographed as their trimethylsilyl (TMS) derivatives on a 1.75 m × 4 mm I.D. glass column containing a mixed phase of 3.0 w/w % OV-7 and 1.5 w/w % OV-22. The TMS derivative offers a unique advantage in that the derivatization reaction is completed at 135° for 4 h in a closed tube with no transfers or removal of reagents. Silylation of amino acids to their TMS derivatives holds considerable promise not only for the quantitative analysis of arginin, histidine, tryptophan, and known mixtures and ribonuclease as the TMS derivative is reported. An average recovery of 102.5% was obtained. The reaction conditions and chromatography of the TMS derivatives of the twenty protein amino acids and some non protein amino acids are the subject of a separate manuscript. A simplified determination of histidine can be made by a calculation of the AREA HIS on the OV-22 column from the AREA HIS+ASP on the same column.