An Automated Orthogonal Two-Dimensional Liquid Chromatograph

Abstract

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.

Comprehensive multi‐dimensional liquid chromatographic separation in biomedical and pharmaceutical analysis: a review

“…Flow was from one pump with splitting to feed all three columns. After the second-dimension the flow was coupled before the UV detector (Venkatramani and Zelechonok, 2003).…”

Properties of Monolithic Silica Columns for HPLC

“…66 The combination of normal-phase separation at 1st-D and reversed-phase separation on a monolithic-C18 column at 2nd-D was reported. 67 The use of a microbore column and a weak mobile phase at 1st-D and a monolithic column at 2nd-D was essential for successful operation.…”

Capillary columns in liquid chromatography: between conventional columns and microchips

“…Later Holland and Jorgenson separated amines with 0.10 mm anion exchange and reversed phase columns [120], and several others have followed up combining ion exchangers and reversed phase columns. Although conventional size columns can be used for 2D purposes [121,122], the large field of proteomics and LC-MS has directed the trend towards capillary columns. Figure 5 shows the connections of a comprehensive 2D system, consisting of a cation exchanger column in the first dimension, two reversed phase trapping columns working in sequence and one reversed phase analytical column.…”