Influence of pressure on the retention and separation of insulin variants under linear conditions
Influence of pressure on the retention and separation of insulin variants under linear conditions
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DOI：
10.1021/ac0205964
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发表时间：
2003-08-15
影响因子：
7.4
通讯作者：
Guiochon, G
中科院分区：
文献类型：
--
作者：
Liu, XD;Zhou, DM;Guiochon, G
在YMC-ODS C-18反相色谱柱上，在线性条件下，研究了压力对胰岛素异构体保留行为的影响。当平均柱压从55 bar增加到250 bar时，这些变体的保留因子增加近2倍，而它们的分离因子几乎保持不变。该效应通过与其吸附相关的胰岛素变体的偏摩尔体积的变化来解释，当乙腈的流动相浓度从29%增加到33%（v/v）时，其从-99 mL/mol降低到-80 mL/mol。该体积变化比低分子量化合物观察到的体积变化大得多。对于相同的压力变化，从蛋白质和吸附后的固定相取代的乙腈分子的平均数Z从22增加到23.3。赖脯胰岛素的压力诱导项B[S]/[D-0]（Z）（对应于吸附等温线的初始斜率）的相对增加约为猪胰岛素的两倍。由于胰岛素的结合常数随着压力的增加而降低，这表明固定相上结合位点的数量增加得更快。最后，观察到在高于0.6 mL/min的流速下，柱效率随着压力的增加而略微增加。这表明，这些观察结果也是有效的其他蛋白质在RPLC分析。
The effect of pressure on the retention behavior of insulin variants in RPLC on a YMC-ODS C-18 column was investigated under linear conditions. The retention factors of these variants increase nearly 2-fold when the average column pressure is increased from 55 to 250 bar while their separation factors remain nearly unchanged. This effect is explained by a change of the partial molar volume of the insulin variants associated with their adsorption that decreases from -99 to -80 mL/mol for mobile-phase concentrations of acetonitrile increasing from 29 to 33% (v/v). This volume change is much larger than the one observed with low molecular weight compounds. For the same pressure variation, the average number Z of acetonitrile molecules displaced from the protein and the stationary phase upon adsorption increases from 22 to 23.3. The pressure-induced relative increase of the term b[S]/[D-0](Z) (which corresponds to the initial slope of the adsorption isotherm) is approximately twice as large for Lispro than for porcine insulin. Because the binding constant of insulin decreases with increasing pressure, this suggests that the number of binding sites on the stationary phase increases even faster. Finally, it was observed that the column efficiency at flow rates higher than 0.6 mL/min increases slightly with increasing pressure. It is suggested that these observations are also valid for other proteins analyzed in RPLC.