Trajectory of isoelectric focusing from gels to capillaries to immobilized gradients in capillaries.

This review presents the need for replacing gels in 2D separations for proteomics, where speed, high-throughput, and the ability to characterize trace level proteins or small samples are the current desires. The theme of the review is isoelectric focusing, which is a valuable tool because it pre-concentrates proteins in addition to separating with high peak capacity. The review traces the technological progress from gel IEF to CIEF to packed capillaries with immobilized gradients for CIEF. Multiple capillary techniques are progressing toward meeting the current desires, providing extremely high sensitivity with regard to concentration and to small samples, integrated automation, and high peak capacity from multiple dimensions of separation. Capillaries with immobilized pH gradients for CIEF are emerging, which will alleviate interference from ampholytes and improve reproducibility in separation times when this valuable technique can be used as one of the dimensions.