Multidimensional separations

Multidimensional separation involves techniques in which fractions from a primary separation system are transferred on-line to a secondary separation system. These techniques can utilize combinations of different chromatographic columns and has been practiced using LC, GC, SEC, CZE, and combinations of these methods (91). 

Multidimensional GC, for example, involves coupling of GC columns of different selectivities so that the primary column isolates the fraction of interest 

therefore, hardly surprising that multidimensional GC is a technique that will become increasingly important. 

Another exciting development in multidimensional separation involves coupling of LC to GC or even to CE. In these coupling systems, LC can play a role as a high-resolution procedure for preliminary separation and/or trace enrichment. LC-GC can thereby be carried out in the column-switching and in the precolumn-analytical column mode. Although on-line LC-GC has not yet advanced widely into routine analysis, this powerful technique holds promise for the future. 

Snyder and J.J. Kirkland, in Introduction to Modem Liquid Chromatography (L.R. Snyder and J.J. Kirkland, Eds.), 2nd ed., Wiley. New York (1979). 

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Multidimensional or hyphenated instmments employ two or more analytical instmmental techniques, either sequentially, or in parallel. Hence, one can have multidimensional separations, eg, hplc/gc, identifications, ms/ms, or separations/identifications, such as gc/ms (see CHROMATOGRAPHY Mass spectrometry). The purpose of interfacing two or more analytical instmments is to increase the analytical information while reducing data acquisition time. For example, in tandem-mass spectrometry (ms/ms) (17,18), the first mass spectrometer appHes soft ionization to separate the mixture of choice into molecular ions the second mass spectrometer obtains the mass spectmm of each ion. 

Pandey et al. [183] employed this idea of a sandwich configuration to transfer substances from one TLC plate to another for two- or multidimensional separations. 

In common with all multidimensional separations, two-dimensional GC has a requirement that target analytes are subjected to two or more mutually independent separation steps and that the components remain separated until completion of the overall procedure. Essentially, the effluent from a primary column is reanalysed by a second column of differing stationary phase selectivity. Since often enhancing the peak capacity of the analytical system is the main goal of the coupling, it is the relationship between the peak capacities of the individual dimensions that is crucial. Giddings (2) outlined the concepts of peak capacity product and it is this function that results in such powerful two-dimensional GC separations. 

G. Schomburg, H. Husmann and E. Hiibinger, Multidimensional separation of isomeric species of cWor inated hydrocarbons such as PCB, PCDD and PCDF , 7. High Resolut. Chromatogr. Chromatogr. Commun. 8 395-400 (1985). 

J. C. Giddings, Sample dimensionality a predictor of order- disorder in component peak distribution in multidimensional separation , J. Chromatogr. 703 3-15 (1995). 

J. C. Giddings, Concepts and comparison in multidimensional separation , ]. High Resolut. Chromatogr. Chromatogr. Comm. 10 319-323 (1987). 

Multidimensional chromatography brings together separations often based on different selectivity mechanisms. Although the forms of the mobile phase are not required to be different in the individual steps of a multidimensional separation, we usually strive to achieve orthogonal selectivity of these individual separation steps (1). 

In thinking about performing multidimensional separations within the framework of unified chromatography, we must think about using all available tuning opportunities to maximize the differences in the separation mechanisms in the successive parts of the process. The following is just one example. 

MD-PC is highly important in its own right, because this is the only real multidimensional separation method in which all compounds can be passed to a next dimension. It therefore serves as the reference system (7) against which all other multidimensional systems can be compared. 

Use of a bilayer plate (15) affords the special chromatographic possibility of being able to perform two different multidimensional separations on the same chromatographic plate (Figure 8.5), either with the same mobile phase or with mobile phases of different composition. 

Figure 8.15 Schematic diagrams of cross-sections of MD-TLC plates connected in series to ensure multidimensional separation on stationary phases of increasing polarity hatched lines, glass plate light shading, stationary phase A dark shading, stationary phase B.

Another means of realizing multidimensional separation is combination of two complementary separation techniques which use different methods of separation. In such multi-modal separation, different techniques can be coupled in which PC is used as the second dimension and another separation method, as the first. Some possible variations are as follows ... 

Multidimensional planar chromatographic separations, as we have seen, require not only a multiplicity of separation stages, but also that the integrity of separation achieved in one stage be transferred to the others. The process of separation on a two-dimensional plane is the clearest example of multidimensional separations. The greatest strength of MD-PC, when properly applied, is that compounds are distributed widely over two-dimensional space of high zone (peak) capacity. Another... 

Multidimensional separations allow for the analysis of complex mixtures, such as those from biological matrices with thousands of components that would be difficult or impossible to separate by utilizing only one method. Electrodriven separations have been employed to separate biological molecules for many years, due to the charged nature of amino acids and nucleic acids. The addition of an electrodriven component to a multidimensional separation is therefore desirable, especially for the separation of biological mixtures. 

This chapter will first cover the nature of electrophoretic separations, especially those concerning capillary electrophoresis. Comprehensive multidimensional separations will then be defined, specifically in terms of orthogonality and resolution. The history of planar and non-comprehensive electrodriven separations will then be discussed. True comprehensive multidimensional separations involving chromatography and capillary electrophoresis will be described next. Finally, the future directions of these multidimensional techniques will be outlined. 

Other groups have also used EC and CE to perform non-comprehensive multidimensional separations (15, 16). A three-dimensional separation was performed by Stromqvist in 1994, where size exclusion chromatography (SEC), reverse-phase HPLC, and CZE were used in an off-line manner to separate peptides (17). The most useful information gained from all of these non-comprehensive studies was knowledge of the orthogonality and compatibility of EC and CE. 

Electrodriven techniques are useful as components in multidimensional separation systems due to their unique mechanisms of separation, high efficiency and speed. The work carried out by Jorgenson and co-workers has demonstrated the high efficiencies and peak capacities that are possible with comprehensive multidimensional electrodriven separations. The speed and efficiency of CZE makes it possibly the best technique to use for the final dimension in a liquid phase multidimensional separation. It can be envisaged that multidimensional electrodriven techniques will eventually be applied to the analysis of complex mixtures of all types. The peak capacities that can result from these techniques make them extraordinarily powerful tools. When the limitations of one-dimensional separations are finally realized, and the simplicity of multidimensional methods is enhanced, the use of multidimensional electrodriven separations may become more widespread. 

Chromatography is the best technique for the separation of complex mixtures. Frequently, samples to be analysed are very complex, so the analyst has to choose more and more sophisticated techniques. Multidimensional separations, off-line and recently on-line, have been used for the analysis of such complex samples. 

There are two general types of multidimensional chromatography separation schemes those in which the effluent from one column flows directly on to a second column at some time during the experiment, and those in which some type of trap exists between the two columns to decouple them (off-line mode). The purpose of a trap is often to allow collection of a fixed eluate volume to reconcentrate the analyte zone prior to the second separation step, or to allow a changeover from one solvent system to another. The use of offline multidimensional techniques (conventional sample cleanup) with incompatible mobile phases, is common in the literature, and replacing these procedures with automated on-line multidimensional separations will require continuous development efforts. 

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