Sun L, Carr P W
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Anal Chem. 1995 Aug 1;67(15):2517-23. doi: 10.1021/ac00111a004.
Zirconia HPLC packing materials were found to be potentially advantageous for large-scale protein separations due to their excellent pH stability and mechanical stability. However, Lewis acid sites on zirconia's surface cause irreversible adsorption of proteins due to their interactions with hard Lewis bases such as the carboxyl groups in proteins. Although the Lewis acid sites can be effectively blocked by adsorbing phosphate ions onto zirconia's surface, proteins and peptides cannot be eluted using a typical reversed-phase mobile phase. In this work, we found that the separation of peptides on a phosphate-modified polybutadiene-coated zirconia (PBD-ZrO2) can be brought about by using a mobile phase containing both an organic modifier and a high concentration of sodium perchlorate. The salt is needed to cancel the Coulombic interactions between the negatively charged stationary phase and the positively charged proteins. To understand the retention mechanism of proteins and peptides on phosphate-modified PBD-ZrO2, this work was aimed at the study of the surface characteristics of the phosphate-modified PBD-ZrO2. We found that the phosphate-modified PBD-ZrO2 phase has both reversed-phase and cation-exchange characteristics under the acidic mobile-phase conditions used for protein and peptide separations. The PBD coating provides hydrophobic moieties, and the phosphate ions adsorbed on zirconia's surface provide cation-exchange sites. Reversed-phase separation of a peptide standard mixture and cation-exchange separation of a cationic peptide standard mixture on the same phosphate-modified PBD-ZrO2 column shows excellent column resolution in both modes. Although mixed-mode stationary phases provide unique selectivity, the secondary equilibrium on phosphate-modified PBD-ZrO2 can cause peak broadening. Applications of the phosphate-modified PBD-ZrO2 to peptide separations are demonstrated here.
由于具有出色的pH稳定性和机械稳定性，氧化锆高效液相色谱填充材料在大规模蛋白质分离方面具有潜在优势。然而，氧化锆表面的路易斯酸位点会因其与蛋白质中的羧基等硬路易斯碱相互作用，导致蛋白质发生不可逆吸附。尽管通过在氧化锆表面吸附磷酸根离子可有效阻断路易斯酸位点，但使用典型的反相流动相无法洗脱蛋白质和肽。在本研究中，我们发现，在含有有机改性剂和高浓度高氯酸钠的流动相作用下，磷酸改性聚丁二烯包覆氧化锆（PBD-ZrO₂）可实现肽的分离。需要这种盐来消除带负电的固定相和带正电的蛋白质之间的库仑相互作用。为了解蛋白质和肽在磷酸改性PBD-ZrO₂上的保留机制，本研究旨在研究磷酸改性PBD-ZrO₂的表面特性。我们发现，在用于蛋白质和肽分离的酸性流动相条件下，磷酸改性PBD-ZrO₂相同时具有反相和阳离子交换特性。PBD涂层提供疏水部分，吸附在氧化锆表面的磷酸根离子提供阳离子交换位点。在同一根磷酸改性PBD-ZrO₂柱上对肽标准混合物进行反相分离以及对阳离子肽标准混合物进行阳离子交换分离，在两种模式下均显示出出色的柱分辨率。尽管混合模式固定相具有独特的选择性，但磷酸改性PBD-ZrO₂上的二级平衡会导致峰展宽。本文展示了磷酸改性PBD-ZrO₂在肽分离中的应用。
