Welinder B S, Sørensen H H
Hagedorn Research Laboratory, Gentofte, Denmark.
J Chromatogr. 1991 Jan 11;537(1-2):181-99. doi: 10.1016/s0021-9673(01)88894-4.
The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.
对于基于二乙烯基苯（DVB）的固定相，评估了将高含量乙酸用作流动相添加剂用于几种蛋白质和生物组织提取物的反相高效液相色谱（RP-HPLC），并将在乙腈、异丙醇或水中使用乙酸梯度获得的分离结果与在硅胶C4上使用三氟乙酸（TFA）-乙腈进行的经典多肽RP-HPLC进行了比较。用TFA-乙腈洗脱的C4柱上和用乙酸-乙腈洗脱的DVB柱上，重组衍生的白细胞介素-1β（IL-1β）的分离模式相似，但只有聚合物柱能够分离碘化IL-1β制剂中存在的组分。两种洗脱液对RP-HPLC后分离出的IL-1β的生物活性均无有害影响。当聚合物柱用乙腈、异丙醇或水中的乙酸梯度洗脱时，几种标准蛋白质可以被分离，尽管水中乙酸的分离效率低于与经典有机改性剂组合时的效率，但胰岛素、胰高血糖素和人生长激素（hGH）以尖锐、对称的峰洗脱。所有三种流动相的胰岛素和hGH回收率相当（80-90%）。使用有或没有有机改性剂的乙酸梯度分析正常和糖尿病患者人胰腺的粗乙酸提取物获得的分离模式，与在硅胶C4柱上用TFA中的乙腈梯度洗脱获得的分离模式相似且可比。主要差异源于使用了不同的紫外波长（TFA-乙腈为215nm，乙酸为280nm）。用水中的乙酸梯度洗脱的DVB柱上分离了重组衍生的产hGH大肠杆菌的乙酸提取物。尽管起始材料是高度复杂的混合物，但经过这一步纯化后分离出的hGH出人意料地纯（通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳判断）。因此，在不使用有机改性剂的情况下，在用水中的乙酸梯度洗脱的聚合物固定相上分离了几种（纯）多肽和复杂生物样品。
