Biomedical subjects
Publications and source records attributed to J W Jorgenson.
Methods for sample introduction, capillary column fabrication, and on-column detection are discussed. A new cross-linked polymer stationary phase coating (based on similar coatings for capillary gas chromatography) is described. Results of separations of urinary components using 16-mum open-tubular columns and electrochemical detection and laser-induced fluorescence detection are shown.
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A simple theory of zone electrophoresis in open-tubular capillaries is developed. According to this theory, to achieve the highest resolution of zones, tubes with as small an inside diameter as possible should be used in combination with as high an applied voltage as feasible. To test this approach, we performed electrophoresis in glass capillaries with an internal diameter of 75 micron and a length of 100 cm. A special fluorescence detector was used to detect fluorescent zones while they migrated inside the capillary. With the application of 30 kV potentials to this system, rapid and efficient separations of amino acids, peptides, and urinary amines were demonstrated. In all cases fluorescent derivatives were necessary for detection. Preliminary results are encouraging, and with further development of sensitive detection devices, applications in clinical analyses may be feasible.
Analysis of biological samples is problematic because of their complex composition. Reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), and, more recently, capillary zone electrophoresis (CZE) are routinely used for the analysis of these samples, but are eventually limited because they are one-dimensional (1-D) methods. As sample complexity increases, the separation efficiency necessary to resolve a large number of sample components in one dimension becomes prohibitively high. A solution to this problem has been to use a two-dimensional (2-D) approach. Each dimension in a 2-D separation relies on a different separating mechanism. By expanding the separation into two dimensions, sample components unresolved in the first dimension can often be separated in the second. This circumvents the requirement for extremely high efficiencies in either dimension. Two-dimensional slab gel electrophoresis has been used successfully in this area, but a more instrumental approach is desired. In this paper we describe three coupled-column approaches to 2-D separations. First, microcolumn SEC-CZE is explored as a means of 2-D protein analysis. Next, RPLC-CZE is investigated for analysis of peptides in tryptic maps. Finally, RPLC is coupled with fast CZE (FCZE), a unique form of CZE analysis, for fast 2-D analysis of peptides. Details of the instrumentation used in these 2-D systems will be presented along with the results of some typical 2-D analyses.
A new sheath flow microelectrospray interface combines capillary electrophoretic separation of protein standards with condensation particle counting detection. Protein separations are performed in both coated and uncoated fused-silica columns. The electrospray needle utilizes a sheath flow rate between 0 and a few microL/min. Typically, the makeup flow is equal to or less than the electroosmotic flow from the separation capillary; thus there is minimal dilution while still providing pH adjustment and solid electrical contact with the separation capillary. While there are some inherent restrictions to a condensation particle counting detector, the microelectrospray needle performs well and has direct application to electrospray mass spectrometry.