Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/optically gated capillary zone electrophoresis.

Multidimensional separation methods are attractive because of their potentially high peak capacities. Coupled-column systems in particular offer the advantages of on-line detection and automation. With the development of rapid two-dimensional (2D) analysis, it becomes possible to consider three-dimensional (3D) separation systems. In such a 3D system, effluent from a slow first dimension is repetitively sampled into a rapid 2D system. In the resultant data, each sample component has been subjected to three separative displacements, and the overall peak capacity is the product of that of each of the three dimensions. This paper demonstrates a comprehensive coupled-column 3D analysis of peptides. Size exclusion chromatography (SEC) is used as the first dimension to separate sample components by molecular weight, over an analysis time of several hours. The SEC effluent is repetitively sampled on-line into a rapid 2D reversed phase liquid chromatography/capillary zone electrophoresis (CZE) system with an analysis time of 7 min. Detection of sample zones is done only after the final CZE separation, by laser-induced fluorescence detection. Analysis data from this system consist of a series of 2D "slices" of the SEC effluent, which when stacked together give the 3D separation "volume".