Pat Sandra
Research Institute for Chromtaography, Kortrijk, Belgium, This email address is being protected from spambots. You need JavaScript enabled to view it.
Optimization of the separation performance of fluid-based separation techniques, and especially of liquid chromatography (LC), is essential for analysis of the complex samples encountered in today’s research (omics, food, environmental, natural product, etc. samples).
Around a decade ago, tremendous improvements were made in LC column technology (sub-2 µm porous particles, core-shell particles) and in instrumentation (pressures up to 1200 bar) opening new possibilities in terms of speed and resolution to LC practitioners for tackling complex samples. A now well-accepted metric of separation power in gradient elution, mandatory for samples of high complexity, is the peak capacity (nc). nc is the maximum number of peaks that can fit side-by-side between the first and last peak of interest with resolution 1. With state-of-the-art LC, peak capacities of ca. 600 (in ca. 1 h) in conventional one-dimensional LC (1D-LC) can routinely be obtained.
It is, however, wishful thinking that such peak capacities are sufficient to separate very complex mixtures. Peak capacity should significantly outstrip the number of components in a sample; the statistical theory of peak overlap (developed by Giddings) indicates to resolve 98% of randomly distributed sample components, nc should exceed the number of components by a factor 100 ! This means that a nc value of 10.000 corresponding to ca. 1x108 theoretical is needed to “chromatographically” resolve a sample containing 100 components.
One straightforward approach to increasing nc is multidimensional LC, especially two-dimensional LC or 2D-LC. In 2D-LC the sample is subjected to two different separation mechanisms and this in an off-line or on-line way. On-line 2D-LC is divided in heart-cutting LC (LC-LC), multiple heart-cutting or parking LC (mLC-LC) and comprehensive LC (LC×LC). LC-LC and mLC-LC are applied to better resolve components in a selected retention time window or windows, while in LC×LC, the entire sample is subjected to two separation mechanisms.
The recent developments in 2D-LC hardware design, software approaches and column configurations will be presented. The figures of merit of LCxLC will be discussed and illustrated with analysis of several complex matrices including some of QA/QC.
