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<p>We have reported the development of a high-performance liquid chromatography (HPLC) system employing a phase-separation multiphase flow (PSMF) as the eluent—termed the phase-separation mode in HPLC. While previous studies demonstrated this system’s capability using a binary mixture of 1-naphthol and 2, 6-naphthalenedisulfonic acid, the present study extends its evaluation to a more complex sample comprising eight naphthalene derivatives. Separations were performed in the phase-separation mode at 20&#xa0;°C using a biphasic eluent of water/acetonitrile/NaCl, which was one of two-phase separation mixed solutions, and three column types: two core–shell silica columns (50&#xa0;mm and 150&#xa0;mm) and a 150&#xa0;mm triazole-modified porous silica column. Among these, the 150&#xa0;mm core–shell silica column exhibited the best performance, achieving baseline separation for nearly all components at 20&#xa0;°C in the phase-separation mode. The separation performance was evaluated and discussed based on the chromatographic data obtained. Furthermore, we investigated the separation mechanism in the phase-separation mode using the 150&#xa0;mm core–shell silica column, considering the distribution ratios of the analytes between the two phases at 20&#xa0;°C and the pH value of the eluent, both of which influence the chromatographic behavior. Collectively, these observations here supported the unique separation principle of the phase-separation mode in HPLC and its applicability to multicomponent analyte systems.</p>

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Development and Evaluation of a Phase-Separation Mode in HPLC Through the Analysis of Multicomponent Analytes

  • 期刊论文

  • 发表日期：

    2025年10月30日

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  • Ryuto Hirose,
  • Takeshi Iharada,
  • Kazuhiko Tsukagoshi

摘要

We have reported the development of a high-performance liquid chromatography (HPLC) system employing a phase-separation multiphase flow (PSMF) as the eluent—termed the phase-separation mode in HPLC. While previous studies demonstrated this system’s capability using a binary mixture of 1-naphthol and 2, 6-naphthalenedisulfonic acid, the present study extends its evaluation to a more complex sample comprising eight naphthalene derivatives. Separations were performed in the phase-separation mode at 20 °C using a biphasic eluent of water/acetonitrile/NaCl, which was one of two-phase separation mixed solutions, and three column types: two core–shell silica columns (50 mm and 150 mm) and a 150 mm triazole-modified porous silica column. Among these, the 150 mm core–shell silica column exhibited the best performance, achieving baseline separation for nearly all components at 20 °C in the phase-separation mode. The separation performance was evaluated and discussed based on the chromatographic data obtained. Furthermore, we investigated the separation mechanism in the phase-separation mode using the 150 mm core–shell silica column, considering the distribution ratios of the analytes between the two phases at 20 °C and the pH value of the eluent, both of which influence the chromatographic behavior. Collectively, these observations here supported the unique separation principle of the phase-separation mode in HPLC and its applicability to multicomponent analyte systems.