Gradual Gradient Two-Dimensional Preparative Liquid Chromatography System for Preparative Separation of Complex Natural Products

  • Zheng Wang
  • Ting-Ting Xie
  • Xia Yan
  • Shi Xue
  • Jing-Wei Chen
  • Zhen Wu
  • Ying-Kun QiuEmail author


A low-cost and gradual gradient online two-dimensional preparative liquid chromatography system was developed for the preparative separation of compounds of interest from complex natural products. Medium-pressure liquid chromatography (MPLC) was applied as the first dimension and preparative high-performance liquid chromatography (HPLC) was employed as the second dimension. Two trapping columns and a makeup pump were also used. Each of the two dimension columns was packed with reversed-phase ostadecyl-silca (ODS), and eluted by an isocratic pump with a gradient mixing device, in which the solvent of the first and the second dimensions were gradiently changed to fit the demand of the sample elution. The instrument operation and the performance of this MPLC × preparative HPLC system were evaluated via gram-scale isolation of a crude methanol extract of toad venom from Bufo bufo gargarizans. As a result, in a single 2D separation run (345 min), 18 bufadienolides (118) were isolated from 0.5 g crude extract, and the purity of each compound was higher than 90%. We anticipate that this improved low-cost preparative 2D-LC system has broad applications for the simultaneous isolation and purification of multiple components from other complex sample matrices.


Two-dimensional preparative liquid chromatography Medium-pressure liquid chromatography × preparative high-performance liquid chromatography Bufo bufo gargarizans Toad venom Bufadienolides Gradual gradient 



This study was funded by the National Natural Science Foundation of China (Grant number 81773600).

Compliance with Ethical Standards

Conflict of Interest

The authors declare no conflicts of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10337_2018_3652_MOESM1_ESM.docx (135 kb)
Supplementary material 1 (DOCX 134 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of PharmacyZhongshan Hospital Affiliated to Xiamen UniversityXiamenChina
  2. 2.Fujian Provincial Key Laboratory of Innovative Drug Target Research, School of Pharmaceutical SciencesXiamen UniversityXiamenChina
  3. 3.Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical EngineeringXiamen UniversityXiamenChina
  4. 4.Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research CenterNingbo UniversityNingboChina

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