Retention behavior of proteins in size‐exclusion electrochromatography with a low‐voltage electric field perpendicular to the liquid phase streamline
Retention behavior of proteins in size‐exclusion electrochromatography with a low‐voltage electric field perpendicular to the liquid phase streamline
复制标题
DOI：
10.1002/elps.200500058
复制
发表时间：
2005-08
期刊：
影响因子：
2.9
通讯作者：
Guo-Min Tan;Qinghong Shi;Y. Sun
中科院分区：
文献类型：
--
作者：
Guo-Min Tan;Qinghong Shi;Y. Sun
提出了一种新型的制备尺寸排斥电色谱(PSEEC)，该电色谱具有垂直于液相流线的振荡低压电场(PSEEC)，柱设计为矩形截面。用Sephadex G-75填充柱，考察了不同条件下牛血清白蛋白(BSA)和肌红蛋白(Myo)的保留行为。结果表明，带电蛋白质的分配系数随电流强度的增大以及等电点与pH值之差的增大而显著增大。分配系数也随着流动相电导率的降低而增大。对于像BSA这样的凝胶排斥蛋白，蛋白质电迁移在凝胶表面引起的浓差极化(CP)是增加保留率的主要原因。对于像Myo这样的凝胶渗透性蛋白质，CP和固相中的电泳性迁移都有助于提高其保留率。进一步的结果表明，由于累积的蛋白质将回流到大块的液体相，因此极化将被扩散所抵消。因此，当电致质量通量等于扩散通量时，分配系数不随振荡电流周期的进一步增大而变化。最后，比较了pSEEC和SEC对BSA/Myo和BSA/Myo/溶菌酶混合蛋白的分离效果。结果表明，蛋白质混合物在pSEEC中有较好的分离度，柱短至12 cm。因此，pSEEC在制备蛋白质分离方面的潜力被证明。
A novel preparative size‐exclusion electrochromatography with an oscillatory low‐voltage electric field perpendicular to the liquid phase streamline (pSEEC) was proposed with a column design of rectangular cross‐section. The column of 12 cm length was packed with Sephadex G‐75, and the retention behavior of bovine serum albumin (BSA) and myoglobin (Myo) was extensively investigated under various conditions. The results indicated that the partition coefficient of a charged protein increased significantly on increasing the current strength as well as the difference between its pI and pH. The partition coefficient also increased on decreasing the mobile phase conductivity. For the gel‐excluded protein like BSA, the concentration polarization (CP) on the gel surface induced by the protein electromigration was the main reason for the increased retention. For a gel‐permeable protein like Myo, both the CP and electrophoretic migration in the solid phase contributed to its increased retention. Further results exhibited that the polarization would be offset by diffusion, because the accumulated protein would flux back to the bulk liquid phase. Therefore, when the electrically induced mass flux was equal to the diffusion flux, the partition coefficient did not change with a further increase of the oscillatory current cycle. Finally, pSEEC was compared with SEC in the separation of protein mixtures of BSA/Myo as well as BSA/Myo/lysozyme. The results showed much better resolutions of the protein mixtures in pSEEC with the column as short as 12 cm. The potential of pSEEC for preparative protein separation was therefore demonstrated.