CNRS UMR 5623, UPS Laboratoire IMRCP, Université de Toulouse, 118 route de Narbonne, 31062 Toulouse, France.
Talanta. 2019 Jun 1;198:550-559. doi: 10.1016/j.talanta.2019.02.051. Epub 2019 Feb 11.
In this study, design of experiments was applied for the analysis of 6 reversed phase U-HPLC columns used for the separation of four tetracyclines (TCs): tetracycline, doxycycline, chlortetracycline and oxytetracycline in different elution conditions. In a first part, a fractional factorial design (2) was used to study the influence of four chromatographic parameters: column temperature, pH, flow rate and composition of the mobile phase (i.e. nature of the solvent used as the organic modifier), on the quality of the separation, which was evaluated in terms of peak width and resolution between two pairs of TCs. This experimental design revealed that the nature of the solvent: acetonitrile (ACN) or methanol (MeOH), and the mobile phase flow rate were the two main factors actually having the most influence on the quality of the separation. Moreover, these two factors presented an antagonistic influence according to the response considered: peak width or peak resolution. In order to understand this behavior, a Doehlert design was performed in the second part. It consisted in modeling the evolution of responses as a function of the two main factors: nature of the composition of the mobile phase (mix of ACN and MeOH, from 100% ACN to 100% MeOH) and mobile phase flow rate (from 0.3 to 0.8 mL min). For all the reversed phase columns studied, an inversion of the elution order of TCs and an increase of the retention factors was observed according to the composition of the organic mixture at the end of the gradient. To understand the modification of the interactions implied in the various retention modes related to the selectivity of the organic solvents used, a quantitative structure-property relationship (QSPR) study was achieved. In this final study, the molecular descriptors of each TCs were connected to its retention factor.
在这项研究中，设计了实验来分析 6 种反相 U-HPLC 柱，用于在不同洗脱条件下分离四种四环素（TCs）：四环素、强力霉素、金霉素和土霉素。在第一部分中，使用部分因子设计（2）研究了四个色谱参数对分离质量的影响，这四个参数分别是：柱温、pH 值、流速和流动相组成（即用作有机改性剂的溶剂种类）。分离质量通过峰宽和两对 TCs 之间的分辨率来评估。该实验设计表明，溶剂的性质（乙腈（ACN）或甲醇（MeOH））和流动相流速是对分离质量影响最大的两个主要因素。此外，根据考虑的响应，这两个因素表现出拮抗影响：峰宽或峰分辨率。为了理解这种行为，在第二部分进行了 Doehlert 设计。它包括将响应的演变建模为两个主要因素的函数：流动相组成的性质（ACN 和 MeOH 的混合物，从 100% ACN 到 100% MeOH）和流动相流速（从 0.3 到 0.8 mL/min）。对于所有研究的反相柱，根据梯度结束时有机混合物的组成，观察到 TCs 的洗脱顺序反转和保留因子增加。为了理解与所用有机溶剂的选择性相关的各种保留模式所涉及的相互作用的变化，进行了定量构效关系（QSPR）研究。在这项最终研究中，将每个 TCs 的分子描述符与其保留因子相关联。
