Liquid chromatography HPLC and LC-MS

Scalbert et al. (1989) described the chromatographic separation of cyanidin and delphinidin from methanolic extracts of oak heartwood using a C-18 Novapak column. The elution solvent was a mix of two solvents A and B that changed in composition from 0-100% B in a linear fashion over a period of 20 min. Solvent A was a mixture of 94 5 1 H20/methanol/H3P04, and solvent B was 99 1 methanol/H3P04, at a flow rate of 1 7mL/min. In this case a dual-channel spectrophotometer was used with two wavelengths selected for detection 280 and 530 nm. Under these conditions, and based on reference compounds, delphinidin eluted after 13.9 min and cyanidin after 14.8 min. 

These three examples illustrate technology developments over time (dual-channel detector, diode array detector, mass spectrometer). Note that while the overall methodology is very similar (methanolic extracts, methanol-based, acidified solvents used for HPLC, detection of eluted compounds), the exact conditions for successful separation need to be defined for each system. 

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