Departament de Química Analítica, Universitat de València, c/Dr. Moliner 50, 46100 Burjassot, Spain.
J Chromatogr A. 2013 Mar 15;1281:94-105. doi: 10.1016/j.chroma.2013.01.064. Epub 2013 Jan 22.
Although there is a great deal of stationary phases having different selectivities (even practically orthogonal selectivities), these very rarely are taken as a factor to be optimized during method development. The chromatographer selects the stationary phase usually in a trial-and-error fashion (or based on the solute nature and expected interactions), and then optimizes continuous factors as the mobile phase composition, pH, temperature and flow-rate. However, the optimization of the stationary phase nature and column length (which are discrete factors) may be interesting. In this regard, the optimization of the coupling of individual columns may yield separations that are not possible with a single column, based on the combined selectivities and the potential increase in plate count. The idea is highly attractive, but there are only few reports in the literature using this approach. The theory behind the use of serially coupled columns is indeed rather simple, but its implementation may be troublesome. The most problematic factor is the connection of the serial columns, which ideally should not modify the result of the sum of behaviors of the columns. A proper serial connection of columns needs a zero-dead volume (ZDV) union and a system to link tightly the columns to each other. In this work, two different approaches to solve this problem are compared using isocratic elution, one consisting in the use of holders, which are screwed to maintain attached the columns linked by ZDV junctions, and the other using ZDV fingertight column couplers, which are screwed directly to the columns maintaining them tightly attached without the need of column holders. The advantages and problems associated to these approaches are described, and information on the accuracy in the prediction of retention times, peak widths and asymmetries are given for the combined columns. Guidelines to prepare software to make reliable predictions are also presented. A set of 15 sulphonamides were used to probe the systems.
虽然有许多具有不同选择性的固定相（甚至是实际正交的选择性），但在方法开发过程中，这些固定相很少被视为需要优化的因素。色谱师通常通过反复试验（或根据溶质性质和预期相互作用）选择固定相，然后优化连续因素，如流动相组成、pH 值、温度和流速。然而，优化固定相性质和柱长（离散因素）可能会很有趣。在这方面，优化各个柱子的耦合可能会产生仅使用单个柱子无法实现的分离，这基于组合选择性和潜在的板数增加。这个想法非常有吸引力，但文献中只有很少的报道使用这种方法。串联柱使用背后的理论实际上相当简单，但它的实施可能会有麻烦。最成问题的因素是串联柱的连接，理想情况下，它不应改变柱子行为总和的结果。柱子的正确串联连接需要零死体积（ZDV）接头和一个将柱子紧密连接在一起的系统。在这项工作中，使用等度洗脱比较了两种不同的方法来解决这个问题，一种方法是使用接头，通过接头将 ZDV 连接的柱子拧在一起固定，另一种方法是使用 ZDV 手指紧的柱子接头，直接拧到柱子上，无需柱子接头。描述了这些方法的优缺点和问题，并提供了有关保留时间、峰宽和不对称性的综合柱子预测准确性的信息。还提出了编写可靠预测软件的指南。一组 15 种磺胺类药物被用于探测这些系统。
