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Sequential elution liquid chromatography using ion exchange stationary phases
Dissertation   Open access

Sequential elution liquid chromatography using ion exchange stationary phases

Lauren Kline Lovejoy
Doctor of Philosophy (Ph.D.), Drexel University
Feb 2026
DOI:
https://doi.org/10.17918/00011277
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Lovejoy_Lauren_20262.38 MBDownloadView
PDFOpen Access (License Unspecified),  Open Access

Abstract

High performance liquid chromatography Ion exchange chromatography Method development Method validation Sequential elution
Sequential Elution Liquid Chromatography (SE-LC) using ion exchange phases is a powerful technique for the separation of complex samples. SE-LC separates analytes by class with multiple elution modes, offering higher peak capacity and reduced separation disorder compared to conventional HPLC, while utilizing standard HPLC instrumentation. An SE-LC method was developed for the separation of permanently charged ions, weak acids, and neutral compounds by employing anion exchange and reversed-phase columns in tandem. Optimization of mobile phase composition, pH, and gradients was critical for effective class-based separation. The finalized method incorporated isocratic elution at low pH to elute the weak acids, followed by an acetonitrile gradient to elute the neutral compounds, and last a sodium methanesulfonate gradient to elute the anionic compounds using a superficially porous C18 column coupled with a strong anion exchange column. High repeatability (RSD < 0.25% for retention time and < 1.5% for peak area) was demonstrated. The above method was validated for quantitative analysis. The validation confirmed its specificity, linearity, accuracy, precision (repeatability), and the flow rate, starting mobile phase organic content, column temperature, mobile phase composition, and gradient start time. The method was demonstrated to be robust, specific, linear, accurate, and precise (repeatable) over the range of 6%-120% of the target analyte range. A second quantitative SE-LC method was developed to separate six pharmaceutical drug product excipients by their classes--amino acids (methionine, arginine, and histidine) and antimicrobial preservatives (sodium benzoate, methylparaben, and propylparaben). This method employed a tandem column setup with a cation exchange column and reversed-phase column (C18) in tandem and utilized a pH gradient to elute the amino acids followed by an acetonitrile gradient to elute the antimicrobial preservatives within 18 minutes. The method was also validated and demonstrated to be specific, linear, accurate, and precise (repeatable) over the range of 50%-150% of the target analyte concentrations. Method robustness was also assessed using a full factorial design of experiments for which the factors investigated were mobile phase concentration, acetonitrile gradient start time and starting percentage of acetonitrile. Based on the factorial results for the experiments, with respect to the relative retention time of the critical pair (sodium benzoate: arginine), the mobile phase concentration and acetonitrile were significant factors that affected the separation. This method could be applied in pharmaceutical laboratories to measure amounts of methionine, arginine, histidine, sodium benzoate, methylparaben, and propylparaben in drug products.

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