Departamento de Química Analítica, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain.
Anal Bioanal Chem. 2010 Apr;396(7):2647-56. doi: 10.1007/s00216-010-3493-2. Epub 2010 Feb 13.
Some of the optimization methods in reversed phase high-performance liquid chromatography (RP-HPLC) are based on resolution of the critical band pair. Mobile phase composition is changed systematically to establish those conditions giving an acceptable resolution for such a critical band pair, but sometimes the critical pair may change with the separation conditions, which obliges to identify it for each of those conditions. In the case of ionizable compounds, more than two bands may be involved in resolution, showing--in some cases--changes in the elution order when the mobile phase composition was modified. In this paper, an alternative way that does not identify the critical pair after changing experimental conditions is proposed. The relative separation of the three bands involved in two alternating critical band pairs is evaluated as a sort of conjugate or combined resolution, represented as contour maps vs. two variables (content of organic modifier and pH). These maps are obtained from data of chromatograms made under different separation conditions; these conditions were generated by experimental design and data was mathematically processed with a computer program. Analytes of three families that have acid-base properties, triazines, phenoxyacids, and phenols, were used for this purpose. The chromatographic behavior when elution order reversion of ionizable compounds exists is studied.
反相高效液相色谱（RP-HPLC）中的一些优化方法基于关键带对的分辨率。系统地改变流动相组成以建立为这种关键带对提供可接受分辨率的条件，但有时关键对可能随分离条件而变化，这就需要为每种条件识别它。对于可电离化合物，可能涉及两个以上的带参与分辨率，在改变流动相组成时，在某些情况下洗脱顺序会发生变化。在本文中，提出了一种在改变实验条件后无需识别关键对的替代方法。评估三个带之间的相对分离涉及两个交替关键带对作为一种共轭或组合分辨率，以两个变量（有机改性剂含量和 pH）的等高线图表示。这些图谱是从在不同分离条件下获得的色谱图数据中获得的；这些条件是通过实验设计生成的，数据通过计算机程序进行数学处理。为此目的使用了具有酸碱性质的三种类型的分析物：三嗪、苯氧酸和苯酚。研究了当存在可电离化合物的洗脱顺序反转时的色谱行为。
