Sample Preparation for Chromatography of Amino Acids: Acid Hydrolysis of Proteins

Abstract

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.

Effect of static‐state fermentation on volatile composition in rapeseed meal

“…The samples of RSM and FRSM were ground, passed through a 60-mesh sieve, and then analyzed for crude protein, water-soluble protein, peptides, 9 water-soluble sugar, tannin, glucosinolates, phytic acid, 23 and crude fiber. The amino acid composition of RSM and FRSM was determined using an automatic amino acid analyzer (Agilent Technologies Inc., Santa Clara, CA, USA) after the samples had been hydrolyzed with 6 mol L −1 hydrochloric acid at 110°C for 24 h. 24 Electronic nose signal acquisition The electronic nose (FOX 3000; Alpha MOS, Toulouse, France) was equipped with 12 different gas sensors (LY2/LG, LY2/G, LY2/AA, LY2/GH, LY2/gCTL, LY2/gCT, T30/1, P10/1, P10/2, P40/1, T70/2, and PA/2) to analyze the volatile gas molecules using sensor array response and pattern recognition techniques. Samples of RSM (2.0 g) were placed in 20 mL sealed vials and then heated at 50°C for 10 min.…”

Development of value‐added nutritious crackers with high antidiabetic properties from blends of Acha (Digitaria exilis) and blanched Pigeon pea (Cajanus cajan)

“…The amino acid profiles were determined using the HPLC Pico-Tag system according to the method previously described by Bidlingmeyer, Cohen, and Tarvin (1984) after samples were digested with 6 M HCl for 24 h. The cysteine and methionine contents were determined after performic acid oxidation (Gehrke, Wall, Absheer, Kaiser, & Zumwalt, 1985), and the tryptophan content was determined after alkaline hydrolysis (Landry & Delhaye, 1992).…”

HPLC Method for Cyst(e)ine and Methionine in Infant Formulas

“…Although HPLC has been used in the study of amino acids, a review of published studies revealed that most of them have focused on the application of hydrolysis and previous treatment to protect CYS and MET in foods such as fish meal, maize corn meal, mixed feed, soybean meal, meat meal, whey powder, egg white and beef, etc.) (Sarwar et al, 1983;Elkin, 1984;Spindler et al, 1984;Gehrke et al, 1985;MacDonald et al, 1985;Elkin and Wasynczuk, 1987;Iwaki et al, 1987;Hagen et al, 1989;Cubedo Fernández-Trapiella, 1990;Van Der Meer, 1990) or pure proteins such as casein, ␤-lactoglobulin, lysozyme, etc.) (Sarwar et al, 1983;Bidlingmeyer et al, 1984;Cohen et al, 1984Cohen et al, , 1986Gehrke et al, 1985;MacDonald et al, 1985;Hagen et al, 1989).…”

“…(Sarwar et al, 1983;Elkin, 1984;Spindler et al, 1984;Gehrke et al, 1985;MacDonald et al, 1985;Elkin and Wasynczuk, 1987;Iwaki et al, 1987;Hagen et al, 1989;Cubedo Fernández-Trapiella, 1990;Van Der Meer, 1990) or pure proteins such as casein, ␤-lactoglobulin, lysozyme, etc.) (Sarwar et al, 1983;Bidlingmeyer et al, 1984;Cohen et al, 1984Cohen et al, , 1986Gehrke et al, 1985;MacDonald et al, 1985;Hagen et al, 1989). Very few studies have been reported on milk (Hanning et al, 1992) and infant formula (Sarwar et al, 1988(Sarwar et al, , 1989Astephen and Wheat, 1993).…”

“…The most frequently applied approach has been performic acid oxidation prior to acid hydrolysis. This treatment converts CYS and MET to cysteic acid (CYSCOOH) and methionine sulfone (ME-TONE), respectively, and since these compounds are stable during acid hydrolysis, losses of labile sulfur amino acids are overcome (Spindler et al, 1984;Gehrke et al, 1985;MacDonald et al, 1985;Csapó et al, 1986;Gehrke et al, 1987).…”