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The separation and characterization of bioactive compounds from plant extract is the crucial step in the study of phytochemicals. Over the past few years, the applications of Liquid Chromatography coupled with Mass Spectrometry (LC/MS) in natural product analysis have been dramatically growing because of the increasingly improved separation and detection capabilities of LC/MS instruments. In particular, novel high-resolution hybrid instruments linked to ultra-High-Performance LC and the hyphenations of LC–MS with other separation or analytical techniques greatly aid unequivocal identification and highly sensitive quantification of natural products at trace concentrations in complex matrices. The resolution and efficiency of the chromatographic analysis can be even further increased by the usage of Two-Dimensional Liquid Chromatography, the technique in which two independent liquid phase separation systems are applied to the sample. From an analytical perspective it is very fruitful to think of the second column and its associated detector as comprising a very chemically selective detector for the separation on the first column. 2D-LC is mainly used to improve resolution and thereby decrease analysis time.
The characterization of certain UV inactive compounds, mostly the carbohydrates, are achieved by Agilent 1200 series Evaporative Light scattering Detector. Moreover the addition of Mass Spectrometry, HPLC, uHPLC and nanoLC along with an online Mascot server database enables in the identification and characterization of peptides and proteins.