摘要
【目的】建立稻米支链淀粉链长相对分子质量分布的高效液相体积排阻色谱法(high performance sizeexclusion chromatography,HPSEC)的测定方法。【方法】先采用丁醇沉降法分离提纯稻米支链淀粉,纯化后的支链淀粉经异淀粉酶酶解切开分支糖苷键,然后运用HPSEC测定其相对分子质量分布。建立了稻米支链淀粉链长相对分子质量分布的HPSEC分析方法:Shodex SB-G、Shodex SB-803(1 000—100 000)、Shodex SB-802.5(300—10 000)3根凝胶色谱柱串连,柱温40℃;流动相为0.1 mol.L-1Tris+0.1 mol.L-1NaCl,pH=7.40,流速0.5 mL.min-1;采用示差折光检测器(Differential refractive index detector,RI),检测器温度为37℃;待测样品浓度为5mg.mL-1,进样量20μL。【结果】本法提纯的稻米支链淀粉:淀粉含量为99.4%、蛋白残余为0.02%、支链淀粉碘复合物最大吸收峰为517 nm,为高纯度支链淀粉。供试大米样品脱分支支链淀粉的平均聚合度(DP)分布在2—120,无杂峰干扰。【结论】试验结果和方法验证表明本方法数据可靠、重复性好、简单易行。
【Objective】The aim of this study is to establish a sensitive high performance size exclusion chromatography with differential refractive index detector（HPSEC-RI） methods for measuring molecular weight distribution of rice amylopectin.【Method】The proposed method includes three parts： Firstly,separation of amylopectin by butanol precipitation method,Secondly,hydrolysis of rice amylopectin by isoamylase,and Thirdly,qualification of the molecular weight distribution by HPSEC-RI.The proposed parameters of HPSEC-RI are as follows： columns of Shodex SB-G,Shodex SB-803（1 000-100 000） and Shodex SB-802.5（300-10 000） being connected in tandem,column temperature being 40℃,the mobile phase being 0.1 M Tris＋0.1 M NaCl（pH=7.40） with a rate of 0.5 mL.min-1,detector temperature being 37℃,sample concentration being 5 mg.mL-1and the input volume being 20 μL.【Result】The rice amylopectin purified in this study is of high-purity,with the starch content being 99.4%,protein residue being 0.02%,and the peak of rice amylopectin-iodine complex absorption spectrum being 517 nm.The tested rice sample amylopectin chain-length distribution is in the range of DP 2-120.【Conclusion】The precision and repeatability verification test showed that the proposed method performed well in these aspects including good reproducibility,simple sample preparation,and high efficiency.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第16期3488-3495,共8页
Scientia Agricultura Sinica
基金
国家科技支撑计划(2012BAD04B08)
国家自然科学基金项目(31171485)
新世纪优秀人才支撑计划(NCET-10-0472)
