This paper discusses the development of an analytical method by an alternative separation approach, sequential elution liquid chromatography (SE-LC), to separate permanently charged ions (anions), weak acids, and neutral compounds using anion exchange and reversed-phase columns in tandem. SE-LC separates classes of compounds by group by employing two or more elution modes. Advantages to using SE-LC over conventional HPLC are a greater peak capacity and a reduced separation disorder. Importantly, the same HPLC as used for a conventional HPLC separation may be used to afford a successful SE-LC separation. Mobile phase selection and gradient optimization are integral for a successful SE-LC class separation of permanent anions, weak acids, and neutral compounds and will be discussed in detail in this paper. The most successful (best resolution and repeatability) SE-LC separation was achieved by applying isocratic elution at low pH to elute the weak acids, followed by an acetonitrile gradient to elute the neutral compounds, and last a sodium methanesulfonate gradient to elute the anionic compounds using a superficially porous C18 column coupled with a strong anion exchange (SAX) column. Repeatability (RSD) in the retention times and peak areas of the analytes was less than 0.25 % and 1.5 %, respectively.

中文翻译：

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使用串联柱连续洗脱液相色谱分离弱酸、中性化合物和永久阴离子


本文讨论了一种分析方法的开发，通过替代分离方法，即顺序洗脱液相色谱 (SE-LC)，使用阴离子交换柱和反相柱串联来分离永久带电离子（阴离子）、弱酸和中性化合物。 SE-LC 采用两种或多种洗脱模式按组分离化合物类别。与传统 HPLC 相比，使用 SE-LC 的优点是峰容量更大并减少分离紊乱。重要的是，可以使用与常规 HPLC 分离相同的 HPLC 来实现成功的 SE-LC 分离。流动相选择和梯度优化对于永久阴离子、弱酸和中性化合物的成功 SE-LC 类分离至关重要，本文将对此进行详细讨论。最成功（最佳分辨率和重复性）的SE-LC分离是通过在低pH值下应用等度洗脱来洗脱弱酸，然后使用乙腈梯度来洗脱中性化合物，最后使用甲磺酸钠梯度来洗脱阴离子化合物来实现的使用表面多孔 C18 柱和强阴离子交换 (SAX) 柱。分析物的保留时间和峰面积的重复性 (RSD) 分别小于 0.25% 和 1.5%。