A comprehensive two-dimensional (2-D) liquid chromatographic separation system is presented. The system uses a microbore cation exchange column, operated under gradient conditions, as the first dimension separation. Effluent from this first column alternately fills one of two loops on a computer-controlled eight-port valve. A second pump then forces loop material onto a second column, a size exclusion column. UV detection is used, and the system is applied to the separation of protein standards and serum proteins. The 2-D system has a higher resolving power and peak capacity than either of the two columns used alone. The entire first column effluent is analyzed on the second column in virtually the same time it takes to complete the first column separation, without the use of stopped flow methods. The entire system is automated and operated under computer control. Three-dimensional (3-D) data representation provides a means of viewing peak profiles in either separation dimension and contour mapping of the 3-D data provides a more reliable means of peak identification from run to run than that provided by single-column elution times.

A comprehensive two-dimensional (2-D) separation system utilizing reversed-phase (RP) chromatography as the first dimension separation and capillary zone electrophoresis (CZE) as the second dimension separation is presented. Effluent from the RP column, operated under gradient conditions, fills a loop on a computer-controlled six-port valve. A second pump flushes this loop material over the grounded (anode) end of a CZE capillary at specific intervals. Electromigration infections are performed on the CZE portion of the system from a flowing stream. Fluorescence detection is used on the CZE capillary. The system Is used for the analysis of peptide standards and fluorescently labeled peptide products from a tryptic digest of ovalbumin. The 2-D system has a much greater resolving power and peak capacity than either of the two systems used independently of each other. The entire system is automated and operated under computer control. Three-dimensional data representation provides a means of viewing peak profiles in either separation dimension, and contour mapping of the 3-D data provides a "fingerprint" of protein digests.

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