Orthogonal chromatography techniques, coupled column

The second type of multidimensional chromatography is coupled column or continuous separation. As the name suggests, this technique relies on serially coupled chromatography columns for sample resolution. Partially resolved effluent fractions from the first column are sequentially directed to a second column, or series of columns, with different separation capabilities, for subsequent separation. It is important in this multidimensional method that the columns are carefully chosen to maximize their orthogonality to ensure optimum resolution is achieved. Furthermore, for the success of this method, it is important that the effluent cuts taken from the first column are sufficiently small so as to minimize the number of components in each cut, and therefore increase the probability of their ultimate separation. Typically, cuts are taken at about the duration of peak standard deviation timescale. This ensures the first dimension separation is not significantly degraded. 

Chromatographic and electrophoretic separations are truly orthogonal, which makes them excellent techniques to couple in a multidimensional system. Capillary electrophoresis separates analytes based on differences in the electrophoretic mobilities of analytes, while chromatographic separations discriminate based on differences in partition function, adsorption, or other properties unrelated to charge (with some clear exceptions). Typically in multidimensional techniques, the more orthogonal two methods are, then the more difficult it is to interface them. Microscale liquid chromatography (p.LC) has been comparatively easy to couple to capillary electrophoresis due to the fact that both techniques involve narrow-bore columns and liquid-phase eluents. 

Combination with other liquid chromatographic techniques is also reported by several workers. Orthogonal coupling of an SEC system to another high-performance liquid chromatography (HPLC) system K, Kj to achieve a desired cross-fractionation was proposed. It was an SEC-SEC mode, using the same polystyrene column, but the mobile phase in the first system was chosen to accomplish only a hydrodynamic volume separation, and the mobile phase in the second system was chosen so as to be a thermodynamically poorer solvent for one of the monomer types in the copolymer, in order to fractionate by composition under adsorption or partition modes as well as size exclusion. 

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