cIEF Revolution

cIEF REVOLUTION

The Promise Realized
In 1994, Pawliszyn and Wu demonstrated a breakthrough technology - capillary isoelectric focusing (cIEF), performed using whole column detection, without a mobilization phase. This approach combined the benefits of gel like separation with the automation and quantitation of column based separation, but at a significantly faster speed. Convergent Bioscience commercialized this patented imaged cIEF technology with its introduction of the iCE280 IEF Analyzer.

The Original Promise of Isoelectric Focusing
There was significant interest when IEF, as performed in polyacrylamide slab gels, was first commercially available in the early 1970’s. IEF was seen as a special electrophoresis technology performed in a pH gradient that could be a powerful tool for profiling protein charge heterogeneity. In the pH gradient, ampholytes such as proteins are separated and focused into narrow bands at their isoelectric points (pIs) under an electrical field. IEF had superior resolution compared to other charged based separation technologies for proteins.

The Promise of cIEF
Gel IEF became a routine technique; however, there were limitations regarding its lack of quantitation, automation and speed. It appeared that these would be overcome by Capillary Isoelectric Focusing (cIEF), as described in 1985 by Hjerten and Zhu, who reported high resolution separations performed in a capillary format. Capillary electrophoresis (CE) manufacturers introduced kits and accessories offering cIEF analysis capabilities on conventional CE instruments.

The Promise Lost
Despite cIEF’s inherent appeal, its widespread acceptance to replace gel IEF has not occurred, due to performance and procedural difficulties when cIEF is performed on conventional CE instruments. These instruments use a single point detection system located at one end of the capillary. After the IEF process is complete, a mobilization phase is required to move the focused protein zones past the single point detector. This can result in uneven separation resolution, poor reproducibility and significantly longer time to develop methods and analyze samples.