Rüttler Felix, Vetter Walter
University of Hohenheim, Institute of Food Chemistry, Department of Food Chemistry (170b), Garbenstraße 28, D-70599 Stuttgart, Germany.
University of Hohenheim, Institute of Food Chemistry, Department of Food Chemistry (170b), Garbenstraße 28, D-70599 Stuttgart, Germany.
J Chromatogr A. 2025 Aug 30;1757:466104. doi: 10.1016/j.chroma.2025.466104. Epub 2025 Jun 1.
Countercurrent chromatography (CCC) is an instrumental liquid-liquid chromatographic method that separates analytes according to their partitioning between the stationary and mobile phases (K value). CCC is valuable for the isolation of compounds in a biphasic solvent system, which provides K values between 0.4 and 2.5. However, limitations exist for very high K values, as this leads to long elution times and a high solvent consumption. Here, we show that these disadvantages can be overcome by a combination of conventional (CCC) and co-current (ccCCC) countercurrent chromatography. Specifically, separations were started in normal CCC mode followed by the fast elution in ccCCC mode (CCC+ccCCC). CCC+ccCCC experiments were performed with oleic acid methyl ester (18:1n-9-ME) and palmitic acid methyl ester (16:0-ME), which are difficult to separate by CCC and have K values of >10 in n-hexane/acetonitrile (1:1, v/v). Tests with different switching times between CCC to ccCCC were used to determine the linear range of CCC+ccCCC runs, which was crucial for the separation efficiency and the stability of the solvent system. By combining simulated data with the obtained experimental data, it was possible to reliably predict chromatographic parameters in CCC+ccCCC mode. As a result, 18:1n-9-ME and 16:0-ME were sufficiently separated with CCC+ccCCC. The yield of pure 18:1n-9-ME and 16:0-ME was further improved by performing CCC+ccCCC in heart-cut mode. Given their reproducibility and predictability, the combination of normal CCC mode, followed by the ccCCC mode (CCC+ccCCC), represents a promising complement to existing approaches to make compounds with unfavorable K values amenable to CCC.
逆流色谱法（CCC）是一种仪器液-液色谱方法，它根据分析物在固定相和流动相之间的分配情况（K值）来分离分析物。CCC对于在双相溶剂系统中分离化合物很有价值，该系统提供的K值在0.4至2.5之间。然而，对于非常高的K值存在局限性，因为这会导致洗脱时间长且溶剂消耗高。在这里，我们表明，通过常规（CCC）和并流（ccCCC）逆流色谱法的组合可以克服这些缺点。具体而言，分离首先在正常CCC模式下开始，然后在ccCCC模式下进行快速洗脱（CCC+ccCCC）。使用油酸甲酯（18:1n-9-ME）和棕榈酸甲酯（16:0-ME）进行了CCC+ccCCC实验，这两种物质难以通过CCC分离，并且在正己烷/乙腈（1:1，v/v）中的K值>10。通过在CCC至ccCCC之间使用不同的切换时间进行测试，以确定CCC+ccCCC运行的线性范围，这对于分离效率和溶剂系统的稳定性至关重要。通过将模拟数据与获得的实验数据相结合，可以可靠地预测CCC+ccCCC模式下的色谱参数。结果，使用CCC+ccCCC充分分离了18:1n-9-ME和16:0-ME。通过以中心切割模式进行CCC+ccCCC，进一步提高了纯18:1n-9-ME和16:0-ME 的产率。鉴于其重现性和可预测性，正常CCC模式后接ccCCC模式（CCC+ccCCC）的组合，是对现有方法的一种有前景的补充，可使具有不利K值的化合物适用于CCC。
