The acquisition of high-purity impurities is pivotal for structural identification and an origin analysis, thereby laying a critical foundation for subsequent toxicological evaluation, quality standard development, and process optimization. This study investigated the feasibility of using a solvent gradient twin-column recycling chromatography technique for the separation and purification of multiple trace impurities in iohexol. In this approach, a modifier with a weaker elution strength than the mobile phase is introduced between two chromatographic columns to form a step gradient solvent system. This gradient slows down the leading edge of the elution band relative to the rear edge, inducing a band compression effect that counteracts band broadening and enhances the chromatographic resolution. By optimizing parameters such as the mobile phase composition, elution mode, and modifier flow rate, three trace impurities were successfully separated and purified from iohexol. Their respective purities were improved from initial concentrations of 0.36%, 0.35%, and 0.15% to 97.82%, 91.56%, and 81.56%, respectively. Leveraging the band compression effect on the target components, the impurities were simultaneously purified and concentrated. These results demonstrate that the proposed method is highly effective for the rapid isolation and preparation of trace pharmaceutical impurities.
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