Department of Analytical Chemistry, University of Vienna, Währingerstrasse 38, 1090 Vienna, Austria.
J Chromatogr A. 2012 Jul 6;1245:175-82. doi: 10.1016/j.chroma.2012.05.023. Epub 2012 May 15.
Anion-exchange-type chiral stationary phases (CSPs) derived from quinine or quinidine were applied in subcritical fluid chromatography (SFC) for the direct separation of chiral acidic compounds. Employing subcritical (sc) mobile phase modes (CO₂ + methanol as co-solvent and acids and bases as additives) first the influence of type and amount of acidic and basic additives on separation performance was investigated. Secondly, water was tested as a neutral additive and the influence of temperature variation on enantioselectivity was studied. Thirdly, we could chromatographically confirm that the often verbalized "inherent acidity" of sc CO₂ + methanol is manifested by the in situ formation of methylcarbonic acids in the sc mobile phase and thus functioning as acidic additive. Accordingly the dissociated methylcarbonic acid, acting as a counterion, enables an anion exchange mechanism between the cationic CSP and the corresponding acidic analyte. In the absence of a dissociable acid in the mobile phase such an ion exchange mode would not work following a stoichiometric displacement model. This finding is further corroborated by the use of ammonia in methanol as co-solvent thus generating in situ the ammonium salt of methylcarbonic acid. In summary, we report on ion-exchange mediated chromatographic separations in SFC modes by merely using (i) sc CO₂ and MeOH, (ii) sc CO₂ and ammonia in MeOH, and (iii) sc CO₂ and MeOH plus acids and bases as additives. Comparisons to HPLC mode have been undertaken to evaluate merits and limitations. This mode exhibits high potential for preparative chromatography of chiral acids combining pronounced enantioselectivity with high column loadability and avoiding possibly troublesome mobile phase additives, as the in situ formed methylcarbonic acid disintegrates to CO₂ and methanol upon pressure release.
从奎宁或奎尼丁衍生而来的阴离子交换型手性固定相（CSP）被应用于亚临界流体色谱（SFC）中，用于直接分离手性酸性化合物。采用亚临界（sc）流动相模式（CO₂+甲醇作为共溶剂和酸、碱作为添加剂），首先考察了酸、碱添加剂的种类和用量对分离性能的影响。其次，考察了水作为中性添加剂对手性选择性的影响。然后，我们可以通过色谱法证实，sc CO₂+甲醇中经常被提及的“固有酸度”是通过 sc 流动相中甲基碳酸的原位形成来体现的，因此它作为酸性添加剂起作用。因此，作为抗衡离子的离解甲基碳酸使阳离子 CSP 和相应的酸性分析物之间发生阴离子交换机制。在流动相中不存在可离解酸的情况下，这种离子交换模式不会按照化学计量置换模型起作用。这一发现进一步得到了在甲醇中使用氨作为共溶剂的证实，从而原位生成甲基碳酸的铵盐。总之，我们报告了在 SFC 模式下通过仅使用（i）sc CO₂和 MeOH、（ii）sc CO₂和氨在 MeOH 中以及（iii）sc CO₂和 MeOH 加上酸和碱作为添加剂来实现的离子交换介导的色谱分离。与 HPLC 模式进行了比较，以评估其优缺点。该模式在手性酸的制备色谱中具有很大的潜力，它结合了高的手性选择性、高的柱载量和避免可能有问题的流动相添加剂，因为原位形成的甲基碳酸在压力释放时分解为 CO₂和甲醇。
