电导法测定Sepharose 4FF凝胶层析柱柱效方法的验证  

Validation of conductivity method for determining column efficiency of Sepharose 4FF gel chromatography

作　　者：喻大鑫 丁文博 董继刚 马平 夏莲坤 杜送田 安建科 马国荣 YU Daxin;DING Wenbo;DONG Jigang;MA Ping;XIA Liankun;DU Songtian;AN Jianke;MA Guorong(The Third Bacterial Vaccine Department,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司菌苗三室,甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2023年第4期42-47,共6页Progress In Microbiology and Immunology

摘　　要：目的以氯化钠溶液为样品和洗脱液,对电导法测定Sepharose 4FF凝胶层析柱柱效方法进行验证,确定该方法测定柱效的最优条件。方法设计四因素三水平正交实验对电导法测定Sepharose 4FF凝胶层析柱柱效方法进行验证,考察不同样品体积、流速、样品浓度和洗脱液浓度对Sepharose 4FF凝胶层析柱柱效和对称性的影响。依据正交实验结果确定电导法测定柱效的最优条件并进行验证。结果9次实验测得柱效均>2800 N/m、对称性为1.020~1.160。通过单因素方差分析,样品体积对柱效的影响无统计学意义(P=0.123,P>0.05),对对称性的影响无统计学意义(P=0.735,P>0.05);流速对柱效的影响无统计学意义(P=0.600,P>0.05),对对称性的影响有统计学意义(P=0.032,P<0.05);样品浓度对柱效的影响无统计学意义(P=0.319,P>0.05),对对称性的影响无统计学意义(P=0.824,P>0.05);洗脱液浓度对柱效的影响无统计学意义(P=0.930,P>0.05),对对称性的影响无统计学意义(P=0.596,P>0.05)。通过极差分析,确定测定柱效的最优条件为:样品体积为600 mL、样品浓度为1.0 mol/L、洗脱液浓度为0.2 mol/L、流速为20 cm/h;重复性和耐用性考察中柱效RSD均<5%,对称性RSD均<5%。将丙酮对照实验结果和重复性实验结果进行t检验,柱效和对称性结果均无统计学意义(P均>0.05)。结论电导法可用于测定Sepharose 4FF凝胶层析柱柱效,确定的最优条件重复性、耐用性良好,可准确、真实地评估Sepharose 4FF凝胶层析柱的性能。Objective To verify the conductivity method for determining column efficiency of Sepharose 4FF gel chromatography with sodium chloride solution as sample and eluent,and to determine the optimal conditions for test the column efficiency of this method.Methods Four-factor and three-level orthogonal experiment was designed to verify the method of determining the column efficiency of Sepharose 4FF gel chromatography by conductivity method.The effects of different sample volume,flow rate,sample concentration and eluent concentration on the column efficiency and symmetry of Sepharose 4FF gel chromatography were investigated.According to the orthogonal test results,the optimal conditions for determination of column efficiency by conductivity method were determined and verified.Results The results of 9 experiments showed that the column efficiency was>2800 N/m and the symmetry was 1.020-1.160.Using one-way ANOVA,the effects of sample volume,sample concentration and elute concentration on column efficiency and symmetry were all not statistically significant(P=0.123,P>0.05 versus P=0.735,P>0.05),(P=0.319,P>0.05 versus P=0.824,P>0.05)and(P=0.930,P>0.05 versus P=0.596,P>0.05).The effect of flow rate on column efficiency was also not statistically significant(P=0.600,P>0.05),but it was statistically significant between the symmetry and different flow rate(P=0.032,P<0.05).Through range analysis,the optimal conditions were determined for test as follows:the sample volume,sample concentration,eluent concentration and flow rate were respectively 600 mL,1.0 mol/L,0.2 mol/L,and 20 cm/h.The relative standard deviation(RSD)of column efficiency and symmetry were both<5%for repeatability test.And,the RSD of column efficiency and symmetry were both<8%for durability test.The results of acetone control experiment and repeatability test were t-test,and the results of column effect(P=0.432,P>0.05)and symmetry(P=0.444,P>0.05)were not statistically significant.Conclusion The conductivity method can be used to determine the column efficiency o

关 键 词：Sepharose 4FF凝胶层析柱 柱效 电导法 单因素方差分析 极差分析 

分 类 号：R318[医药卫生—生物医学工程]