Charge/polarity-dependent 2D separation: a new chromatography for monitoring biomolecules

Abstract

Thin-layer chromatography is a fast and easy tool for analyzing biomolecules, but opportunities for its use have up to now been limited due to its low resolution of separation. To overcome this problem, we have designed a new two-dimensional biomolecule analysis tool, in which analytes are first subjected to isoelectric focusing in a charge-specific-separation step, which is then followed by thin-layer chromatography in a polarity-specific-separation step. This new technique enables the separation of mixed large proteins as well as that of mixed synthetic oligopeptides that have similar physical properties. We propose here a newly developed thin-layer chromatographic technique that can be used to analyze various combinations of biomolecules, such as proteins, amino acids, and oligopeptides, within a single chip. The entire system is designed to be as rapid and easily applied as classic thin-layer chromatography to permit its use for high-throughput testing.