A number of variations were evaluated in the techniques and procedures of the classical 6N hydrochloric acid, 110°C, 24 h hydrolysis of protein. Variations included the use of glass tubes with Teflon-lined screw caps as the hydrolysis vessel, high-temperature short-time hydrolysis, performic acid oxidation of cystine and methionine, multiple hydrolysis times at 145°C, and interlaboratory preparation of hydrolysates. A diverse sample set used in the study included a range of protein-containing matrices, and automated ionexchange chromatography was used for the amino acid analysis. Results show that for hydrolysis in glass tubes with Teflon-lined screw caps at 110°C for 24 h, recoveries of amino acids were in good agreement with recoveries by classical hydrolysis in sealed glass ampoules at reduced pressure. Recoveries from a higher temperature hydrolysis, i.e., 145°C for 4 h and using sealed ampoules, were also in agreement with 110°C, 24 h, sealed ampoule results; the former procedure yielded increased isoleucine and valine and decreased serine and threonine values. Glass tubes with Teflon-lined screw caps for hydrolysis were found to be a practical and convenient alternative to sealed glass ampoules; the improved precision with the former was probably due to the simplicity of the method. The average recovery of cystine from a wide range of matrices without the use of performic acid was 55.5% compared with results obtained with performic acid oxidation. Similarly, methionine is preferably analyzed as methionine sulfone. Interlaboratory evaluation of 145°C, 4 h hydrolysis, in which one laboratory used sealed ampoules and the other laboratory used Teflon-lined screw-cap tubes, demonstrated excellent agreement of amino acid values.
The conditions used to hydrolyze proteins are vital in determining amino acid compositions because they necessarily represent a compromise aimed at yielding the best estimate of amino acid composition. Variations in ease of peptide bond cleavage, differences in amino acid stabilities, and matrix effects from nonproteinaceous components all militate against a single set of hydrolysis conditions that quantitatively hydrolyze every peptide bond and concurrently cause no destruction of any amino acid. This presentation summarizes and reviews an extensive study which evaluated a number of variations in the techniques and procedures of the classical 6N HC1, 110°C, 24 h hydrolysis of protein. The objectives of the recent investigation were: (/) to compare hydrolysis at 145°C, 4 h with 110°C, 24 h for proteins in a wide range of different sample matrixes; (2) to compare protein hydrolysis at 110°C, 24 h conducted in sealed glass ampoules after vacuum removal of air with hydrolysis in glass tubes with Teflon-lined screw caps after removal of air by vacuum, nitrogen purge, and sonication; (3) to evaluate a performic acid oxidation procedure before hydrolysis for the analysis of cystine and methionine in the different sample matrixes; (4) to evaluate multiple hydrolysis times at 145°C; (5) to evaluate the variation of interlaboratory hydrolysates prepared at 145°C, 4 h in 2 different laboratories on the amino acid analysis of an array of protein-containing matrixes. The major sources of inaccuracy and lack of precision arising from the application of ion-exchange or gas chromatography, both of which provide excellent accuracy and precision, are prechromatographic sample handling and the method used for hydrolysis of the protein sample itself. Hydrolysate preparation is the area that requires the most attention to solve problems of variability of amino acid analyses.
This presentation describes amino acid analysis with the gas chromatographic method and experimental conditions using the N-trifluoroacetyl n-butyl ester derivatives; the study we describe here was undertaken to compare gas chromatographic (GC) and ion-exchange chromatographic (IEC) analyses of amino acids in hydrolysates of 9 diverse sample types to gain insight into effects of these 2 chromatographic methods of analysis on variation in amino acid results. Our study showed that values for samples prepared by 2 separate laboratories using the same procedure were generally in good agreement when all of the hydrolysates were analyzed by a single laboratory using a single method of analysis. To compare results from gas chromatography with those from ion-exchange chromatography analyses were performed by 2 different laboratories on the same hydrolysates and on different hydrolysates prepared by the same method by both laboratories. The data demonstrate that GC and IEC can be expected to yield essentially identical results when applied to the same hydrolysate. Agreement is so close that interlaboratory differences in hydrolysate preparation of the same sample contribute as much to variation in amino acid results as does the method of analysis, a fact which should be noted in planning collaborative studies.
Applications of an improved gel-solvent system for cleanup of pesticide residues by gel permeation chromatography (GPC) were investigated. Elution characteristics using Bio-Beads SX-3 gel and a toluene-ethyl acetate (1+3) elution solvent were determined for 16 nonicnic chlorinated pesticides, 3 polychlorinated biphenyls (PCBs), 14 chlorophenoxy herbicide esters, and 7 organophosphate insecticides. Elution patterns for vegetable and animal lipids were also studied. Quantitative recoveries of the pesticides were achieved. No liquid-liquid partitioning cleanup steps were required with any type of nonionic chlorinated pesticide or sample matrix. Only GPC cleanup was required for the nonionic chlorinated pesticides, PCBs, and organophosphate pesticide residues in chicken and turkey fat samples. Electron capture and flame photometric detectors were used in the gas chromatographic method for the respective pesticides. Samples containing up to 0.5 g lipid each were processed at the rate of one every 30–40 min with the automated system. Results were in excellent agreement with those from accepted manual partitioning methods and were achieved with significant savings of both labor and chemicals.
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