Kadlecová Zuzana, Kozlík Petr, Tesařová Eva, Gilar Martin, Kalíková Květa
Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, Prague 12843, Czech Republic.
Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 8, Prague 12843, Czech Republic.
J Chromatogr A. 2021 Jul 5;1648:462182. doi: 10.1016/j.chroma.2021.462182. Epub 2021 Apr 27.
In this work, two mixed-mode columns from a different manufacturers and one marketed as a reversed-phase column were characterized and compared in the terms of their interaction abilities, retentivity, peak symmetry, and applicability for peptide separation. All the tested columns contain octadecyl ligand and positively charged modifier, i.e. pyridyl group for the reversed-phase column XSelect CSH C18, quaternary alkylamine for mixed-mode column Atlantis PREMIER BEH C18 AX, and permanently charged moiety (details not available from the manufacturer) for mixed-mode column Luna Omega PS C18. For detailed characterization and comparison of their interaction potential, several approaches were used. First, a simple Walters test was performed to estimate hydrophobic and silanophilic interactions of the tested columns. The highest values of both parameters were observed for column Atlantis PREMIER BEH C18 AX. To investigate the effect of pH and buffer concentration on retention, mobile phases composed of acetonitrile and buffer (ammonium formate, pH 3.0; ammonium acetate pH 4.7 and pH 6.9) in various concentrations (5mM; 10mM; 15mM and 20mM) were used. The analysis of permanently charged compounds was used to describe the electrostatic interaction abilities of the stationary phases. The most significant contribution of electrostatic interactions to the retention was observed for Atlantis PREMIER BEH C18 AX column in the mobile phase with buffer of pH 3.0. A set of ten dipeptides, three pentapeptides and one octapeptide was used to investigate the effects of pH and buffer concentration on retention and peak symmetry. Each of the tested columns provides the optimal peak shape under different buffer pH and concentration. The gradient separation of the 14 tested peptides was used to verify the application potential of the tested columns for peptide separation. The best separation was achieved within 4 minutes on column Atlantis PREMIER BEH C18 AX.
在本研究中，对来自不同制造商的两根混合模式柱和一根市售反相柱进行了表征，并比较了它们的相互作用能力、保留性、峰对称性以及肽分离适用性。所有测试柱均含有十八烷基配体和带正电荷的改性剂，即反相柱XSelect CSH C18的吡啶基、混合模式柱Atlantis PREMIER BEH C18 AX的季铵烷基胺以及混合模式柱Luna Omega PS C18的永久带电部分（制造商未提供详细信息）。为详细表征和比较它们的相互作用潜力，采用了几种方法。首先，进行简单的沃尔特斯测试以估计测试柱的疏水和亲硅醇相互作用。柱Atlantis PREMIER BEH C18 AX的这两个参数值最高。为研究pH和缓冲液浓度对保留的影响，使用了由乙腈和不同浓度（5mM；10mM；15mM和20mM）的缓冲液（甲酸铵，pH 3.0；乙酸铵，pH 4.7和pH 6.9）组成的流动相。对永久带电化合物的分析用于描述固定相的静电相互作用能力。在pH 3.0缓冲液的流动相中，柱Atlantis PREMIER BEH C18 AX的静电相互作用对保留的贡献最为显著。使用一组十种二肽、三种五肽和一种八肽来研究pH和缓冲液浓度对保留和峰对称性的影响。每个测试柱在不同的缓冲液pH和浓度下均能提供最佳峰形。对14种测试肽进行梯度分离以验证测试柱用于肽分离的应用潜力。在柱Atlantis PREMIER BEH C18 AX上4分钟内实现了最佳分离。
