柱前衍生高效液相色谱法分析制革皮屑水解物的氨基酸组成  

Analysis of Amino Acid Composition in Tannery Skin Bit Hydrolysates by HPLC with Pre-column Derivatization

作　　者：迟原龙[1] 张琦弦[1] 廖学品[1] 石碧[1] 

机构地区：[1]四川大学制革清洁技术国家工程实验室,四川成都610065

出　　处：《四川大学学报（工程科学版）》2012年第5期179-183,共5页Journal of Sichuan University (Engineering Science Edition)

基　　金：国家"863"计划资助项目(SQ2010AA0621629001);国家科技支撑计划资助项目(2011BAC06B11)

摘　　要：采用柱前衍生高效液相色谱法对制革皮屑及其4种水解物的氨基酸组成进行了检测分析。样品用6 mol/L HCl于110℃水解10 h,然后经异硫氰酸苯酯衍生后进行HPLC检测。色谱柱为Inertsil ODS-3 C18柱(250 mm×4.6 mm,5μm),流动相分别为乙腈和0.1 mol/L乙酸钠水溶液,进样量为20μL,柱温为40℃,流速为0.8 mL/min,检测波长为254 nm。实验结果显示,采用该分析方法可以准确地测定皮屑及其水解物的氨基酸组成。皮屑酶水解物的氨基酸组成与皮屑有一定的差异,经537酶、1398酶和2709酶制得的皮屑水解物的甘氨酸含量均低于皮屑,而它们的碱性氨基酸含量较皮屑高;经537酶和1398酶制得的皮屑水解物的酸性氨基酸含量低于皮屑,而采用2709酶制得的水解物的蛋氨酸含量约是皮屑的2倍。本研究为皮胶原样品中氨基酸组成的检测提供了一种有效的方法。The amino acid compositions of tannery skin bit and its hydrolysates were determined by HPLC with pre-column derivatization method.The samples were firstly hydrolyzed by 6 mol/L HCl at 110 ℃ or 10 h,and then pre-column derivatized with phenyl isothiocyanate and separated by HPLC under the condition of gradient elution using a Inertsil ODS-3 C18 column（250 mm ×4.6 mm,5 μm）.The mobile phase A and B were acetonitrile and 0.1 mol/L sodium acetate,respectively.The injection volume of sample solution was 20 μL.The column temperature was 40 ℃.The flow rate was 0.8 mg/mL and the wavelength for detection was 254 nm.It was found that the amino acid compositions of tannery skin bit and its hydrolysates were accurately determined by this method.Moreover,there were some differences in amino acid composition between tannery skin bit and its hydrolysates.The glycin content of skin bit hydrolysates prepared by protease 537,protease 1398 and protease 2709 was all lower than that of skin bit,but the content of basic amino acids in protease hydrolysates was higher while the acidic amino acid content in protease 537 and protease 1398 hydrolysates was lower compared with skin bit.Meanwhile,the methionine content of the skin bit hydrolysate prepared by protease 2709 was twice of that in skin bit.In general,this research provided an effective approach for the determination of amino acid composition of collagen and collagen hydrolysate.

关 键 词：制革废弃物 胶原 酶法水解 柱前衍生 高效液相色谱 氨基酸组成 

分 类 号：O657.7[理学—分析化学]