P Campns Falc and R Herrez Hernndez

Universidad de Valencia, Valencia, Spain

Copyright © 2000 Academic Press

Trace organic separations are often made difficult by the large number of substances present in various kind of samples, by the similarities among the analytes, and by the need to remove major components in the sample. The advantages of using hyphenated systems to tackle such problems have been demonstrated and sequential separation techniques are also well suited. A characteristic feature of these methods is the use of two or more columns for the separation.

Separation in two dimensions has a substantial history. While methods such as two-dimensional (2D) electrophoresis and thin-layer chromatography are 2-D in space, coupled column techniques are 2-D in time. In offline techniques, the fractions of the first column are collected in vials and reinjected onto the second column later. Interest in these techniques has been revived by increased automation. In 1973 Huber et al. proposed a two-channel multicolumn system which allowed an imitation of 2-D chromatography with columns as they existed at that time. In online techniques, manual or automatic switching by a valve directs fractions between columns. Electrically controlled valves greatly facilitate the full automation of the chromatographic process, thus increasing the speed and the work capacity of the high performance liquid chromatography (HPLC) system. The terms column switching, coupled column chromatography and multidimensional chromatography are used interchangeably.

Column switching includes, in the widest sense, all techniques in which the flow path of the mobile phase is changed by valves, so the effluent from a primary column is passed to a secondary column for a defined period of time. An unlimited number of columns can theoretically be incorporated in a chromatographic network. However, in each successive step, the transferred fraction must be reconcentrated to reduce the dispersion of the analyte in the chromatographic system, and the dead volumes in the conections between columns and in any switching valves must be minimized to achieve maximum separation efficiency. The band spread problem can be approached by making a judicious choice of the order in which the dimensions are sequenced.

Multidimensional separations have been defined by Giddings as having only two criteria. The first is that sample components must be displaced by two or more separation techniques based on different separating mechanisms. The second is that components separated by any single separation dimension must not be recombined in any further separation dimension. Most coupled-column approaches proposed only subject a portion of the sample to full 2-D or n-D analysis and are useful for the resolution of a single fused peak from the first dimension. They do not permit a comprehensive 2-D or n-D separation of the entire sample. The term linear heart-cut approach, used by Deans, describes the use of on-off valves in order to isolate an effluent segment which is then injected into a subsequent column. Comprehensive automated systems are useful for the greater resolution of multiple fused peaks from the first dimension column and resolved in the second-dimension orthogonal separation system.

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