An automated orthogonal two-dimensional liquid chromatograph.

A simple approach to two-dimensional liquid chromatography has been developed by coupling columns of different selectivity using a 12-port, dual-position valve and a standard HPLC system. The valve at the junction of the two columns enables continuous, periodic sampling (injection) of the primary column eluent onto the secondary column. The separation in the primary dimension is comparable to conventional HPLC, whereas the secondary column separation is fast, lasting several seconds. The high-speed separation in the secondary dimension enables the primary column eluent to be sampled with fidelity onto the secondary column throughout the chromatographic run. One might expect a coupled column liquid chromatography system operating in reverse-phase mode to be strongly correlated and, hence, inefficient. However, by applying a solvent gradient in the primary dimension and by progressively incrementing the solvent strength in the secondary dimension (tuning), the inefficiency or cross correlation between the two dimensions is minimized. In a tuned two-dimensional system, the influence of primary column retention (usually hydrophobicity) is minimal on secondary column retention. This enables subtle differences in component interaction with the two stationary phases to dominate the secondary column retention. The peaks are randomly dispersed over a retention plane rather than along a diagonal, resulting in an orthogonal separation. The peak capacity is multiplicative, and each component has a unique pair of retention times, enabling positive identification. In addition, the location of the component provides two independent measures of molecular properties. The 2D-LC system was evaluated by analyzing a test mixture made of some aromatic amines and non-amines on different secondary columns (ODS-AQ/ODS monolith, ODS/amino, ODS/cyano). The relative location of sample components in the two-dimensional plane varied significantly with change in secondary column. Among the secondary columns, the amino and cyano columns offered the most complementary separation, with the retention order of several components reversed in the secondary dimension. The theoretical peak capacity of the 2D-LC system was around 450 for a separation lasting 30 min. A 2D-LC system involving amino and cyano columns resulted in a high-speed separation of the test mixture, with most of the chemical components resolved within a few minutes.