色谱与电色谱纯化头孢菌素C及蛋白质的研究
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摘要
色谱是分离纯化化学物质和生物物质的常用技术。电色谱,又称电层析,是电泳与色谱相结合的分离技术,即在色谱分离过程中引入电场以提高色谱的分离性能,是近年来国内外液相色谱技术的一个重要发展方向。
本文将色谱技术与轴向电场耦合,研究了此种电色谱在生物物质(包括小分子头孢菌素C和大分子蛋白)分离方面的作用,并与色谱技术的分离效果作了比较。
本文的实验内容,包括分离纯化头孢菌素C钠盐,从鸡卵清中分离溶菌酶,以及分离牛血清白蛋白、牛血红蛋白、牛胰核糖核酸酶和溶菌酶4种蛋白的混合物;用HPLC检测头孢菌素C,用Bradford法测定蛋白含量,用SDS-聚丙烯酰胺凝胶电泳检测蛋白的分离效果。
首先考察了在HPLC测定头孢菌素C的过程中,样品中有机溶剂含量的影响,发现乙醇等有机溶剂对测定有干扰,但可以通过用水稀释样品以减少这种干扰。
用HP-20大孔吸附树脂为固定相,探索了在通电和不通电情况下分离头孢菌素C的效果。结果发现,色谱分离能够使头孢菌素C钠盐水溶液的纯度从87.3%提高到93.5%,提取收率87.0%;电色谱可以将纯度从89.0%提高到93.3%,提取收率94.5%,收率比色谱有所提高。电色谱法在分离亲水性小分子头孢菌素C方面展示了良好的分离效果。
在蛋白分离方面,采用CM-52羧甲基纤维素为固定相,研究了从天然鸡卵清中分离溶菌酶,结果发现用聚焦电色谱法和普通离子交换色谱法从天然鸡卵清中分离溶菌酶都是可行的,且分离效果接近,然而聚焦电色谱法所需时间缩短了近一半,提高了分离效率。分离4种蛋白的混合物(牛血清白蛋白、牛血红蛋白、牛胰核糖核酸酶和溶菌酶),结果通电和不通电两种情况在各自的最适pH组合下的分离效果接近,但通电时牛血清白蛋白和溶菌酶的回收率明显提高。
综上所述,电色谱法在生物物质的分离方面有着良好的效果,特别是在产品回收率以及分离时间方面优势突出,表明了电色谱技术潜在的应用价值。同时也应看到,色谱与电泳技术的整合是一个复杂的过程,还需要更多的实验研究。
Chromatography is a common method to purify chemical and biological substances. Electrochromatography, a novel separation technique which has been attempted at home and abroad recently, couples liquid chromatography with electrophoresis by introducing an electric field into the column in order to improve the capacity of separation.
In this thesis, the role of electrochromatography in purification and separation of biological materials was investigated and compared with chromatography in many ways.
Experiments include purification of cephalosporin C, separation of lysozyme from egg white and separation of the mixture of bovine serum albumin, bovine hemoglobin, bovine pancreas ribonuclease and lysozyme. The amounts of cephalosporin C and protein were determined by HPLC and Bradford method respectively, and the separation effect of proteins was examined with SDS-polyacrylamide gel electrophoresis.
The influence of organic solvents on determination of cephalosporin C by HPLC was first investigated. Experimental results showed that ethanol had the greatest negative impact. However, by diluting the cephalosporin C solution containing ethanol to certain extent, this influence could be effectively eliminated.
Macroporous resin HP-20, which is commonly used to separate cephalosporin C, was attempted to purify CPC-Na in solution with/without electric field. Experimental results showed that the electrochromatography behaved better in separation of cephalosporin C than chromatography in terms of recovery. The purity of cephalosporin C was increased from 89.0% to 93.3% by electrochromatography, while from 87.3% to 93.5% by chromatography. Recovery rates by electrochromatography and chromatograph were 94.5% and 87.0% respectively. It was shown that electrochromatography had better performance on separation of hydrophilic small molecule cephalosporin C.
Carboxymethyl cellulose CM-52 was selected as the separation medium to separate lysozyme from egg white. Experimental results showed that although focusing electrochromatography had the same separation effect as ordinary ion-exchange chromatography, it only consumed nearly half of the time compared with the latter, thus greatly improving separation efficiency. The mixture of bovine serum albumin, bovine hemoglobin, bovine pancreas ribonuclease and lysozyme was separated with or without electric field respectively in most suitable series of pH. Experimental results showed that separation effects were close in both conditions. However, the recovery yields of bovine serum albumin and lysozyme were markedly increased with electric field.
In conclusion, electrochromatography has good separation performance of biological substances, especially in the product recovery and separation time, which illustrates its potential in industrial applications. However, it should also be seen that coupling liquid chromatography with electrophoresis is a complex process, and more experimental studies are needed in future.
本文将色谱技术与轴向电场耦合,研究了此种电色谱在生物物质(包括小分子头孢菌素C和大分子蛋白)分离方面的作用,并与色谱技术的分离效果作了比较。
本文的实验内容,包括分离纯化头孢菌素C钠盐,从鸡卵清中分离溶菌酶,以及分离牛血清白蛋白、牛血红蛋白、牛胰核糖核酸酶和溶菌酶4种蛋白的混合物;用HPLC检测头孢菌素C,用Bradford法测定蛋白含量,用SDS-聚丙烯酰胺凝胶电泳检测蛋白的分离效果。
首先考察了在HPLC测定头孢菌素C的过程中,样品中有机溶剂含量的影响,发现乙醇等有机溶剂对测定有干扰,但可以通过用水稀释样品以减少这种干扰。
用HP-20大孔吸附树脂为固定相,探索了在通电和不通电情况下分离头孢菌素C的效果。结果发现,色谱分离能够使头孢菌素C钠盐水溶液的纯度从87.3%提高到93.5%,提取收率87.0%;电色谱可以将纯度从89.0%提高到93.3%,提取收率94.5%,收率比色谱有所提高。电色谱法在分离亲水性小分子头孢菌素C方面展示了良好的分离效果。
在蛋白分离方面,采用CM-52羧甲基纤维素为固定相,研究了从天然鸡卵清中分离溶菌酶,结果发现用聚焦电色谱法和普通离子交换色谱法从天然鸡卵清中分离溶菌酶都是可行的,且分离效果接近,然而聚焦电色谱法所需时间缩短了近一半,提高了分离效率。分离4种蛋白的混合物(牛血清白蛋白、牛血红蛋白、牛胰核糖核酸酶和溶菌酶),结果通电和不通电两种情况在各自的最适pH组合下的分离效果接近,但通电时牛血清白蛋白和溶菌酶的回收率明显提高。
综上所述,电色谱法在生物物质的分离方面有着良好的效果,特别是在产品回收率以及分离时间方面优势突出,表明了电色谱技术潜在的应用价值。同时也应看到,色谱与电泳技术的整合是一个复杂的过程,还需要更多的实验研究。
Chromatography is a common method to purify chemical and biological substances. Electrochromatography, a novel separation technique which has been attempted at home and abroad recently, couples liquid chromatography with electrophoresis by introducing an electric field into the column in order to improve the capacity of separation.
In this thesis, the role of electrochromatography in purification and separation of biological materials was investigated and compared with chromatography in many ways.
Experiments include purification of cephalosporin C, separation of lysozyme from egg white and separation of the mixture of bovine serum albumin, bovine hemoglobin, bovine pancreas ribonuclease and lysozyme. The amounts of cephalosporin C and protein were determined by HPLC and Bradford method respectively, and the separation effect of proteins was examined with SDS-polyacrylamide gel electrophoresis.
The influence of organic solvents on determination of cephalosporin C by HPLC was first investigated. Experimental results showed that ethanol had the greatest negative impact. However, by diluting the cephalosporin C solution containing ethanol to certain extent, this influence could be effectively eliminated.
Macroporous resin HP-20, which is commonly used to separate cephalosporin C, was attempted to purify CPC-Na in solution with/without electric field. Experimental results showed that the electrochromatography behaved better in separation of cephalosporin C than chromatography in terms of recovery. The purity of cephalosporin C was increased from 89.0% to 93.3% by electrochromatography, while from 87.3% to 93.5% by chromatography. Recovery rates by electrochromatography and chromatograph were 94.5% and 87.0% respectively. It was shown that electrochromatography had better performance on separation of hydrophilic small molecule cephalosporin C.
Carboxymethyl cellulose CM-52 was selected as the separation medium to separate lysozyme from egg white. Experimental results showed that although focusing electrochromatography had the same separation effect as ordinary ion-exchange chromatography, it only consumed nearly half of the time compared with the latter, thus greatly improving separation efficiency. The mixture of bovine serum albumin, bovine hemoglobin, bovine pancreas ribonuclease and lysozyme was separated with or without electric field respectively in most suitable series of pH. Experimental results showed that separation effects were close in both conditions. However, the recovery yields of bovine serum albumin and lysozyme were markedly increased with electric field.
In conclusion, electrochromatography has good separation performance of biological substances, especially in the product recovery and separation time, which illustrates its potential in industrial applications. However, it should also be seen that coupling liquid chromatography with electrophoresis is a complex process, and more experimental studies are needed in future.
引文
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[3]陈红,张承红.超临界流体萃取及其在我国的研究应用进展[J].化学进展, 1999, 11(3): 227-238
[4]俞俊棠,唐孝宣.生物工艺学[M].上海:华东化工学院出版社, 1991年
[5]毛忠贵.生物工业下游技术[M].北京:中国轻工业出版社, 1999年
[6]严希康.生化分离工程[M].北京:化学工业出版社, 2001年
[7] Richardson P, Hoare M, Dunnill P. A new biochenmical engineering approach to the fractional precipitation of proteins [J]. Biotech Bioeng, 1990, 36(4): 354-366
[8] Cole KD. Preparative concentration and size fractionation of DNA by porous media using a combination of flow and low electric field strength [J]. Biotechnol Prog, 1997, 13: 289-295
[9] Nurok D. Planar electrochromatography [J]. J Chromatogr A, 2004, 1044: 83-96
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