Determination Of Protein Amino Acids Research Articles

Determination of protein amino acids as benzylthiocarbamyl derivatives compared with phenylthiocarbamyl derivatives by reversed-phase high-performance liquid chromatography, ultraviolet detection and precolumn derivatization

All 22 protein amino acids were successfully derivatized to benzylthiocarbamyl (BZTC) derivatives by precolumn reaction at 50°C for 30 min with benzylisothiocyanate (BZITC) and separated in 35 min on a reversed-phase Nova-Pak C 18 column (30 cm×3.9 mm, 4 μm). However, phenylthiocarbamyl-derivatives were not completely separated on this column; in particular cystine and cysteine were resolved into a single peak. The optimun wavelength for determination of BZTC derivatives was 246 nm although the strong absorption wavelengths were 220 nm and 238 nm. The relative standard deivations (R.S.D.) of the relative molar response to the internal standard (norleucine) were less than 5%, except for cysteine (7.22%); however for the PTC derivatives, glutamine, proline, threonine, alanine, leucine, cystine+cysteine and lysine the R.S.D. values exceeded 5%. Standard calibration curves showed good linearity in the measured range from 0.125 to 5 nmol. The correlation coefficient of cystine was the lowest, 0.952. The stability of BZTC derivatives of standard amino acids was about 120 h except for threonine, alanine, cystine and serine. The detection limit was about 3.9 pmol at 0.05 AUFS in both of BZTC and PTC derivatives. The compositions of amino acids of soybean and bovine serum albumin (BSA) analyzed with both derivatives were similar to the results found in the literature and food composition tables, except for cysteine. The BZTC derivatives of cystine and cysteine in soybean and BSA could be determined separately, but the PTC derivatives could not, nor could data on separate dertermination of cysteine and cystine be found in the literature studied.

Determination of protein amino acids as butylthiocarbamyl derivatives by reversed-phase high-performance liquid chromatography with precolumn derivatization and UV detection

Twenty-two protein amino acids were derivatized to butylthiocarbamyl derivatives by precolumn reaction at 40°C for 30 min with butyl isothiocyanate. All of the derivatives were quantitatively resolved in 27 min by RP-HPLC on a Nova-Pak C 18 column and detected at 250 nm with a UV detector. Asparagine and serine were resolved in a single peak. The relative standard deviations of the relative molar responses with respect to the methionine peak were less than 5% for all of the derivatives except cysteine. The calibration graphs showed good linearity in the range 0.5–2.5 nmol. Correlation coefficients for all of the calibration graphs were highly significant ( p < 0.001). The stability of the derivatives at room temperature was about 8 h.

Improved gas-liquid chromatographic method for the determination of protein amino acids

The gas-liquid chromatographic method previously reported by us was improved with regard to the gas chromatographic columns and their operating conditions. It became possible to perform more accurate quantification of the 22 protein amino acids, including asparagine and glutamine, in less than 25 min on a dual set of columns. At the same time, the method allowed determination of the amino acids in the range 0.5-10 μg of each by eliminating the contaminant peaks derived from solvents and reaction vials. A single sample could be analysed in an overall time of 1 h.

Gas-liquid chromatographic analysis of protein amino acids as N-isobutyloxycarbonylamino acid methyl esters

A practical method for the quantitative determination of protein amino acids by gas-liquid chromatography (GLC) is described. All of the common protein amino acids except arginine can be readily converted into their N-isobutyloxycarbonyl (N-isoBOC) methyl ester derivatives by a simple procedure involving isobutyloxycarbonylation with isobutyl chloroformate in aqueous medium, followed by methylation with diazomethane. Arginine was converted into N-isoBOC ornithine methyl ester by treatment with arginase, followed by the above derivatization procedure. The resulting N-isoBOC methyl esters of the amino acids have good GLC properties. Complete resolution of the derivatives of 20 protein amino acids was achieved by using a dual-column system consisting of a 0.65% Poly-A-101A column and a 0.70% FFAP-Poly-A-101A (1:1, w/w) column. The reproducibility of response was found to be good for derivatives carried through the entire chemical and chromatographic procedure. The calibration graphs were linear and showed no statistical bias. The results of recovery experiments with synthetic mixtures containing known amounts of the amino acids were satisfactory, the recoveries ranging from 94.3 to 106.2%.

Comparative determination of protein amino acids in plant materials by automated cation exchange and gas-liquid chromatography of the amino acid n-heptafluorobutyryl, n-propyl esters

Gas-liquid chromatographic (GLC) analysis of the amino acid N-heptafluoro-butyryl, n-propyl ester derivatives seems to offer the best potential for a single packed column technique of analyzing amino acids by GLC. A 25-min analysis on a 3.5 m × 2 mm I.D. glass column, packed with 3 ??? SE-30 on 80–100 mesh Gas-Chrom Q, gave good separation of all protein amino acids, and quantitative results for all except histidine. Procedures for amino acid analysis by GLC and automated cation exchange (CIE) were compared for accuracy, repeatability, and application to protein amino acids analysis in sorghum. In the latter instance, automated CIE and GLC were used to determine the dependence of protein quality in sorghum on nitrogen and sulphur fertilization. Identical conclusions were drawn from experimental data derived from both methods of analysis.

A source of error in amino acid analysis.

THE quantitative determination of amino-acids in proteins and tissues generally involves hydrolysis of the latter with acids, usually hydrochloric acid at a strength of about 6 N under a reflux condenser (about 108° C.) for 20—40 hr., or in a sealed tube at 120° C. for 10—12 hr.