Isolation and characterization of chlorophylls and xanthophylls in grass by high-speed countercurrent chromatography

ABSTRACT

To isolate chlorophyll from grass extract by means of high-speed countercurrent chromatography, a solvent system composed of n-hexane–dichloromethane–ethanol–water 4:2:6:2 (v/v/v/v) was applied. The isolation of chlorophylls a, b, and pheophytins a, b was successfully performed from grass when dichloromethane was part of the solvent system. Comparatively, when chloroform is part of the stationary phase instead of dichloromethane, the xanthophyll separation showed better resolution compared to chlorophylls. Through the solvent system, we could demonstrate the meaningful difference of polarity between dichloromethane, a polar aprotic solvent, and chloroform, a non-polar solvent. Therefore, n-hexane–dichloromethane–ethanol–water 4:2:6:2 (v/v/v/v) is adequate for chlorophylls separation because it changes the system to yield more polarity. This hypothesis supports the fact that the elution mode length was 10 hr and the extrusion mode 2 hr, whereas with chloroform the elution mode length was 7 hr and the extrusion mode 4 hr. Online high-performance liquid chromatography in conjunction with atmospheric pressure chemical ionization mass spectrometry (HPLC/APCI-MS) was suitable to identify xanthophylls and chlorophylls fractions by means of mass spectra in positive (+) mode. Structure elucidation of chlorophylls and pheophytins was done by nuclear magnetic resonance (NMR) 1D/2D-NMR experiments.

GRAPHICAL ABSTRACT

KEYWORDS:

• Chlorophyll
• grass
• high-speed countercurrent chromatography
• LC-APCI spectrometric technique
• xanthophylls

Additional information

Funding

This study was supported by a grant from the German Academic Exchange Service (Deutscher Akademischer Austauschdienst, DAAD) fellowship A/06/32945 and COLCIENCIAS—Department of Science Technology and Innovation, Colombia—ALECOL program with Pontifical Xavierien University, Colombia.