自行加工的非对称流场流分离仪器对蛋白质分离的应用研究

Application of Self-made Asymmetric Flow Field Flow Fractionation Instrument in Protein Separation

目的设计并加工一套针对生物大分子分离的非对称流场流分离(asymmetric flow field-flow fractionation,As Fl FFF或AF4)仪器,对仪器性能进行初步研究,为下一步实际样品的分离提供依据和参考经验。方法为了解决样品聚焦(focus)问题,自行研究了一种新的聚焦方式。以标准牛血清白蛋白和乙醇脱氢酶作为混合样品,对仪器性能进行验证。并通过改变进样/聚焦时间、横向流动速度(cross-in flow)和横向流出速度(cross-out flow),考察各因素对分离效果(分离度R表示)的影响。结果混合蛋白的分离度分别随聚焦时间的减少和横向流动速度的增加而增加,而横向流出速度的影响并不显著。结论该非对称流场流分离仪器已基本实现对混合蛋白的分离,预计可用于更为复杂的样品分离。

OBJECTIVE To design and construct aset of a symmetric flow field flow fractionation instrument which can be used to separate biomacromoleeules, and preliminarily evaluate the instrument performance to provide practical experience for its application to real samples. METHODS The apparatus was made up of separator and tempered glass. Polycarbonate membrane was used as the accumulated wall of the channel. In order to solve the problem of focusing samples, a novel focus position was developed. The performance of the asymmetric flow field flow fractionation device was preliminarily studied by injecting samples consisting of standard bovine serum albumin and alcohol dehydrogenase. The impacts of three factors at six levels on separation （resolution R） were investigated, including injection/focus time, cross-in flow and cross-out flow. RESULTS Along with the decrease of focus time and increase of cross-in flow velocity, the resolution increased obviously. CONCLUSION The self-made asymmetric flow field flow fractionation in- strument can successfully separate protein mixtures and it is expected to separate more complex samples.