Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins

Abstract

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.