Reversed phase liquid chromatography coupled with electrophoresis

Mao, Y., Zhang, X.M. (2003). Comprehensive two-dimensional separation system hy coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection. Electrophoresis 24, 3289-3295. 

As a rule, a separation method should be used for both purification and concentration of the sample. The classic method for peptides and proteins is a reverse-phase liquid chromatography preparation of the sample, followed by a concentration step (often lyophiliza-tion) of the fraction of interest. During those steps performed on very small quantities of sample, loss on the sample can occur if care is not taken to avoid it. Lyophilization, for instance, can lead to the loss of the sample absorbed on the walls of the vial. The use of separation methods on-line with the mass spectrometer often are preferred. Micro- or nano-HPLC [32,33] and capillary electrophoresis [34], both coupled mainly to electrospray ionization/mass spectrometry (ESI-MS), are used more and more. 

Clearly, the capabilities of modem chromatographic techniques have been vastly improved. Packed column GC has been replaced by capillary column GC. Similarly, the large columns of normal-phase liquid chromatography (LC) are replaced by microcolumn reverse phase LC columns. Capillary electrophoresis (CE) is an entirely new means of separating small amounts of more complex, and charged sample molecules, and has evolved into several distinct forms with unique capabilities. Mass spectrometry coupled with different forms of chromatography is now applied to the analysis of many mixtures, of higher complexity, and more disparate sample types. 

Identification and quantification of natural dyes need high performance analytical techniques, appropriate for the analysis of materials of complicated matrices containing a small amount of coloured substances. This requirement perfectly fits coupling of modern separation modules (usually high performance liquid chromatography in reversed phase mode, RPLC, but also capillary electrophoresis, CE) with selective detection units (mainly mass spectrometer). 

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