Systematic fractionation using Complementary Developing Solvent technique in flash chromatography: A key step in dereplication strategies for natural products

The Complementary Developing Solvent (CDS) technique, originally developed for High-Performance Thin Layer Chromatography (HPTLC), employs three solvent systems of varying polarities (low-polarity developing solvent, LPDS; medium-polarity developing solvent, MPDS; high-polarity developing solvent, HPDS) to achieve enhanced chromatographic resolution while covering a wide range of polarities and selectivities. This study demonstrates the successful adaptation of the CDS approach to normal-phase Flash chromatography, aiming to fractionate poorly studied plant extracts in a systematic manner and incorporate this approach into a broader dereplication strategy. Through extensive optimization, we validated the feasibility of this adaptation for two out of the three solvent systems: LPDS (toluene/ethyl-acetate 100:0-85:15) and HPDS (dichloromethane/methanol 100:0-40:60), both performed as step-gradients. The same System Suitability Test (SST) employed in HPTLC – namely the Universal HPTLC Mix (UHM), a mixture of eight compounds covering a wide range of polarity – was used to enable direct comparisons between the two techniques. The elution profiles observed in Flash chromatography were consistent with those obtained in HPTLC. Compound identities and elution order were verified by collecting each chromatographic peak and manually matching MS 2 spectra against reference standards. Additionally, we propose a systematic fractionation strategy of natural products by segmenting each elution gradient (LPDS, HPDS) into seven polarity “plateaus”, defined as distinct ranges of solvent polarity within the gradient where compounds with similar characteristics are likely to elute. This systematic approach of collecting a single fraction per plateau significantly reduces the number of samples for subsequent analysis, thereby accelerating the dereplication workflow. Publication History Article published online: 05 March 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany