Planta Med 2013; 79 - PI87
DOI: 10.1055/s-0033-1352176

Innovative strategies for the efficient isolation of natural at the preparative scale

EF Queiroz 1, S Challal 1, W Kloeti 1, D Guillarme 1, JL Wolfender 1
  • 1University of Geneva, University of Lausanne, School of Pharmaceutical Sciences, Geneva, Switzerland

In natural product research the isolation of compounds at the milligram scale is key to assess their bioactivity in vivo. Conventional isolation approaches are often time-consuming and employ relatively complex schemes that involve different preparative chromatographic methods. One efficient strategy consists in the transfer of extract profiling gradient conditions from HPLC to semi-prep HPLC [1]. In order to further increase the sample loading, Medium Pressure Liquid Chromatography (MPLC) represents an interesting alternative since grams of crude extract can be separated in one step and we have developed models for accurate gradient HPLC-MPLC transfer. MPLC separations are however limited by the maximal flow rate and the pressure allowed by the glass columns used and implies that the separations are carried out over several days. To overcome this problem the pressure drop was minimised by designing a dedicated column oven for preparative separation and perform separations under relatively high temperatures. The speed of separation was enhanced by maximising the flow and using a new way of controlling pressure at the column inlet. For a comprehensive survey of the isolation, MPLC fractions were also monitored post-chromatographically by ultra-fast UHPLC/TOF/MS and provided 2D LC x LC matrices that contain all information enabling an optimal fraction combination. The improvements of separation obtained are illustrated with the separation of different crude plant extracts each containing different classes of molecules. In these different cases a majority of pure NPs could be obtained in mg amounts in about one to two days.

Reference:

[1] Glauser G, Guillarme D, Grata E, Boccard J, Thiocone A, Carrupt P-A, Veuthey J-L, Rudaz S, Wolfender J-L, J Chrom A 2008, 1180, 90.