Whole-Column-Imaging Detection for Capillary Isoelectric Focusing and Capillary Electrophoresis

Despite the significant advantages of capillary isoelectric focusing over gel isoelectric focusing, widespread acceptance of the technique as a replacement for gel isoelectric focusing has not occurred. Several factors contribute to capillary isoelectric focusing’s slower-than-expected adoption; perhaps the most important are the performance and procedural difficulties introduced when capillary isoelectric focusing is performed on conventional CE instruments. A conventional CE instrument is equipped with a single-point, on-column, UV–vis absorption detector that is located at one end of the capillary. Thus, most capillary isoelectric focusing is performed in a two-step operation (4–9). In the first step, samples such as proteins are focused (separated and concentrated) to stationary sharp zones according to their respective pI values in a coated capillary, in which electroosmotic flow is eliminated by the coating. Then as a second step, the focused protein zones are moved by electrophoretic or hydrodynamic mobilization so they can pass the detector point to be measured and recorded. The second mobilization step introduces several problems to capillary isoelectric focusing, such as uneven separation resolution, poor reproducibility, and increased analysis times. An alternative In this article, the authors review recent developments in the research of whole-column-imaging detection for capillary electrophoresis (CE). Wholecolumn-imaging detection was developed for capillary isoelectric focusing, for which it proved to be an ideal detector. Several whole-column-imaging detectors — including refractive index gradient imaging, UV-absorption imaging, and fluorescence imaging detectors — have been studied. The capillary isoelectric focusing UV-absorption imaging technique even has been commercialized. The development of whole-column-imaging detection itself facilitates CE studies in many directions such as in electrophoretic dynamics within narrow channels, new separation modes, and two-dimensional separations. Whole-column-imaging detection also finds application in capillary zone electrophoresis. Whole-Column-Imaging Detection for Capillary Isoelectric Focusing and Capillary Electrophoresis