Size-exclusion chromatography (SEC) with wide application in peptide analysis presents challenges in determination of molecular weight distribution due to the relatively low resolution. In this study, a stop-flow reversed phase liquid chromatography (RPLC) × SEC system was constructed, aiming at improving the peptide separation in SEC. As the chromatographic dispersion during stop-flow operation might contribute to the band broadening in the first dimension (1st D) RPLC, the effects of different stop-flow operational parameters on the additional band broadening were quantitatively evaluated. Unlike analytes of large molecular size or long retention time with low effective diffusion coefficient (Deff), additional band broadening was evidently observed for analytes of small molecular size and short retention time (high Deff). Therefore, optimal flow rate, low column temperature and short stop-flow time were suggested for analyzing small molecules of short retention time. The established stop-flow two-dimensional liquid chromatography (2D-LC) was further tested on protein hydrolysates. The resolution was evidently improved for both heart-cutting and comprehensive 2D-LC analysis (despite additional band broadening in RPLC). Compared with heart-cutting analysis with higher 1st D resolution for selective fractions, comprehensive analysis could provide more complete information about the molecular weight distribution of the eluting solutes along RPLC.

中文翻译：

---

用于肽分离的停流反相液相色谱 × 尺寸排阻色谱

由于分辨率相对较低，在肽分析中广泛应用的尺寸排阻色谱 (SEC) 对分子量分布的测定提出了挑战。本研究构建了一个停流反相液相色谱 (RPLC) × SEC 系统，旨在改善 SEC 中的肽分离。由于停流操作期间的色谱分散可能有助于第一维 (1st D) RPLC 中的谱带展宽，因此定量评估了不同的停流操作参数对额外谱带展宽的影响。与具有低有效扩散系数 (Deff) 的大分子或长保留时间的分析物不同，对于小分子大小和短保留时间（高 Deff）的分析物，明显观察到了额外的谱带展宽。所以，建议使用最佳流速、低柱温和短停流时间来分析短保留时间的小分子。建立的停流二维液相色谱 (2D-LC) 进一步测试了蛋白质水解物。中心切割和综合二维液相色谱分析的分辨率明显提高（尽管 RPLC 中的谱带变宽了）。与选择性馏分具有更高一维分辨率的中心切割分析相比，综合分析可以提供关于洗脱溶质沿 RPLC 的分子量分布的更完整信息。建立的停流二维液相色谱 (2D-LC) 进一步测试了蛋白质水解物。中心切割和综合二维液相色谱分析的分辨率明显提高（尽管 RPLC 中的谱带变宽了）。与选择性馏分具有更高一维分辨率的中心切割分析相比，综合分析可以提供关于洗脱溶质沿 RPLC 的分子量分布的更完整信息。建立的停流二维液相色谱 (2D-LC) 进一步测试了蛋白质水解物。中心切割和综合二维液相色谱分析的分辨率明显提高（尽管 RPLC 中的谱带变宽了）。与选择性馏分具有更高一维分辨率的中心切割分析相比，综合分析可以提供关于洗脱溶质沿 RPLC 的分子量分布的更完整信息。