Laboratoire Sciences Analytiques, Bioanalytiques et Miniaturisation UMR-CNRS-UPMC-PECSA, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, ParisTech, Paris, France.
J Chromatogr A. 2011 May 20;1218(20):2963-75. doi: 10.1016/j.chroma.2011.03.031. Epub 2011 Apr 13.
Selectivity of phase system is of primary concern when designing a bidimensional chromatographic system and looking for the highest degree of orthogonality between the two separations. Several statistical or geometrical criteria can potentially be used to measure the degree of orthogonality. A comparison of eight candidate criteria has been carried out in this study. Analysis of variance (ANOVA) was used to evaluate the relevance of each criterion and its ability to reveal the significance of the influence of factors like pH, stationary phase, and organic modifier. Experimentally, a set of 32 chromatographic systems was evaluated by the same generic gradient with 63 probe solutes, likely to be present in biological and/or environmental samples and covering a wide range of physico-chemical properties: acidic, basic and neutral compounds with different pKa, molecular mass and hydrophobicity (logP). Each chromatographic system was defined by the nature of the stationary phase (8 different silica or grafting chemistries), the pH of the aqueous fraction of the mobile phase (2.5 or 7.0) and the nature of the organic modifier (acetonitrile or methanol). The orthogonality of the 496 couples of chromatographic systems was evaluated and ranked using the eight different approaches: the three correlation coefficients (Pearson, Spearman and Kendall), two geometric criteria characterizing the coverage of the 2D separation space, Slonecker's information similarity and two chi-square statistics of independence between normalized retention times. In fact, there were only seven distinct criteria, since we established the analytical equivalence between the rankings with the likelihood ratio statistics and Slonecker's information similarity. Kendall's correlation coefficient appeared to be the best measure of orthogonality since, according to ANOVA, it exhibited the highest sensitivity to all experimental factors. The chi-square measures, and hence Slonecker's information similarity, performed equally well provided the discretization of the separation space was carried out appropriately. Finally, from the compared study of the factors acting upon orthogonality carried out by ANOVA, it is possible to draw the conclusion that the pH of the mobile phases has the highest impact on the selectivity followed by the type of stationary phase and finally by the organic modifier.
当设计二维色谱系统并寻找两个分离之间最大程度的正交性时，首要关注的是相系统的选择性。可以使用几种统计或几何标准来衡量正交性的程度。本研究比较了八种候选标准。方差分析 (ANOVA) 用于评估每个标准的相关性及其揭示 pH、固定相和有机改性剂等因素影响的重要性的能力。在实验中，使用相同的通用梯度评估了一组 32 个色谱系统，这些系统可能存在于生物和/或环境样品中，并涵盖了广泛的物理化学性质：具有不同 pKa、分子量和疏水性（logP）的酸性、碱性和中性化合物。每个色谱系统由固定相的性质（8 种不同的硅胶或接枝化学）、流动相水相的 pH（2.5 或 7.0）和有机改性剂的性质（乙腈或甲醇）定义。使用八种不同的方法评估和排名 496 对色谱系统的正交性：三个相关系数（皮尔逊、斯皮尔曼和肯德尔）、两个几何标准，用于表征二维分离空间的覆盖范围、斯隆克的信息相似度和两个标准化保留时间之间独立性的卡方统计。实际上，只有七个不同的标准，因为我们根据似然比统计和斯隆克的信息相似度建立了排名的分析等效性。肯德尔相关系数似乎是正交性的最佳衡量标准，因为根据 ANOVA，它对所有实验因素表现出最高的敏感性。卡方测量值，因此斯隆克的信息相似度，只要分离空间的离散化得到适当执行，效果就一样好。最后，根据 ANOVA 对正交性作用因素的比较研究，可以得出结论，流动相的 pH 对选择性的影响最大，其次是固定相的类型，最后是有机改性剂。
