Recent Developments in Two-Dimensional Liquid Chromatography: Fundamental Improvements for Practical Applications

Liquid chromatography (LC) is an incredibly successful analytical separation tool. Its versatility is unprecedented, because of the many different separation modes (reversed-phase LC, ion-exchange chromatography, size-exclusion chromatography, etc.) and because almost all samples can be dissolved in some kind of solvent, ranging from water to organic solvents to strong acids or bases. Conditions (mobile and stationary phases, additives, pH, temperatures, etc.) can be found to separate almost all pairs of analytes. For example, LC is immensely successful in the separation of enantiomers. Good selectivities can be accompanied by high efficiencies in a very short time, using contemporary ultra-high-performance liquid chromatography (UHPLC) instrumentation and (short) columns packed with sub-2-μm particles. However, LC cannot deliver very high efficiencies in a short time. Unlike other techniques, such as gas chromatography (GC) or capillary electrophoresis (CE), plate counts exceeding 100,000 are not routinely obtained in LC. As a result, LC cannot easily deal with complex mixtures that contain more than a few dozen analytes. While the selectivity between any pair of analytes can be maximized, these peaks may then start to overlap with other relevant analytes or with matrix compounds. There simply is not enough room in LC chromatograms to separate very many compounds that behave "statistically"1 and the attainable peak capacity does not suffice to separate complex samples. As a rule of thumb, LC offers a high probability of success for separating samples containing 10 or 20 components in 1 or 2 hours, or up to 50 components in about 10 hours2,3.