Department of Analytical Chemistry and Food Sciences, Institute of Research and Food Analyses, University of Santiago de Compostela, Spain.
J Chromatogr A. 2012 May 18;1238:91-104. doi: 10.1016/j.chroma.2012.03.055. Epub 2012 Mar 28.
Mixed-mode stationary phases are gaining adepts in liquid chromatography (LC) as more and more applications are published and new commercial columns appear in the market ought to their ability to retain and separate analytes with multiple functionalities. The increased number of adjustable variables gives these columns an enhanced value for the chromatographer, but, on the other hand, it complicates the process of developing satisfactory separations when complex samples must be analyzed. Thus, the availability of computer assisted methods development (CAMD) tools is highly desirable in this field. Therefore, the first specific tool for the CAMD of LC separations in mixed-mode columns is presented. The tool consists in two processes. The first one develops a retention model for peaks in a predefined experimental domain of pH and buffer concentration. In this domain, the retention as a function of the proportion of organic modifier is modeled using a two-stage re-calibration process departing from isocratic retention data and then, from gradient elutions. With this two-stage approach, reliability is gained. In the second process, the model is finally interpolated and used for the unattended optimization of the different possible elution modes available in these columns. This optimization process is driven by an evolutionary algorithm. The development and application of this new chemometrics tool is demonstrated by the optimization of a mixture of neutral and ionizable compounds. Hence, several different types of gradients were generated, showing a good agreement between simulated and experimental data, with retention time errors lower than 5% in most cases. On the other hand, classical CAMD tools, such as design of experiments, were unable to efficiently deal with mixed-mode optimizations, rendering errors above 30% for several compounds.
混合模式固定相在液相色谱（LC）中越来越受到重视，因为越来越多的应用被发表，并且新的商业柱在市场上出现，这要归功于它们能够保留和分离具有多种功能的分析物。可调变量的增加为色谱学家提供了更高的价值，但另一方面，当必须分析复杂样品时，它会使令人满意的分离开发过程变得复杂。因此，在这个领域中，计算机辅助方法开发（CAMD）工具的可用性是非常可取的。因此，提出了用于混合模式柱中 LC 分离的 CAMD 的第一个特定工具。该工具由两个过程组成。第一个过程为 pH 和缓冲浓度预定实验域中的峰开发保留模型。在此域中，使用两级重新校准过程来模拟保留作为有机改性剂比例的函数，该过程从等度保留数据开始，然后从梯度洗脱开始。通过这种两级方法，可以获得可靠性。在第二个过程中，最终对模型进行内插，并用于这些柱中可用的不同洗脱模式的无人值守优化。该优化过程由进化算法驱动。通过优化中性和可离子化化合物的混合物来演示这种新的化学计量学工具的开发和应用。因此，生成了几种不同类型的梯度，模拟数据与实验数据之间具有良好的一致性，在大多数情况下保留时间误差低于 5％。另一方面，经典的 CAMD 工具，如实验设计，无法有效地处理混合模式优化，导致几种化合物的误差超过 30％。
