Comprehensive Multidimensional Chromatographic Systems

In general, a comprehensive separation strategy implies the desire to resolve/analyze all components within a sample. In the specific context of a multidimensional chromatographic method, the term is more narrowly applied to indicate that all analytes introduced to the first-dimension separation are also subjected to a second-dimension separation. There are two basic configurations used by our laboratory to carry out comprehensive multidimensional (IEX/RP) protein separations—IEX— Dual Column RP system and IEX—Dual Trap RP system (Figs. 13.1 and 13.2), respectively. 

The IEX—Dual Column RP system configuration shown (Fig. 13.1) utilizes independent pumping systems for each separation dimension. One pumping system 

The IEX—Dual Trap RP system configuration (Fig. 13.2) directs first-dimension IEX effluent to two alternating reversed-phase trap columns (typically 2.1 x 10 mm 

FIGURE 13.1 Schematic of a comprehensive 2DLC (IEX/RP) configuration with alternating second-dimension analytical RP column sampling of first-dimension eluent. A salt diversion valve is present to divert salts from the IEX dimension to waste, and prevent contamination of downstream collected fractions or mass analyzer. 

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An example of the results obtained in the form of a chromatoelectropherogram can be seen in Figure 9.6. The contour type data display showed the three variables that were studied, namely chromatographic elution time, electrophoretic migration time, and relative absorbance intensity. Peptides were cleanly resolved by using this two-dimensional method. Neither method alone could have separated the analytes under the same conditions. The most notable feature of this early system was that (presumably) all of the sample components from the first dimension were analyzed by the second dimension, which made this a truly comprehensive multidimensional technique. 

As explained in Sections 16.4 and 16.5, the comprehensive characterization of complex polymer systems is hardly possible by the SEC alone. SEC employs only one retention mechanism which simnltaneonsly responds to all molecular characteristics of sample. Similarly, also the coupling of the different retention mechanisms within one single column only exceptionally allows fulfilling this task. Evidently several retention mechanisms should be applied in a tandem approach that is within at least two different on-line chromatographic systems. This is the basic idea of the two- and multidimensional polymer HPLC. In the present section, the principles of two-dimensional polymer HPLC, 2D polymer HPLC or (2D-LC) will be briefly elucidated. There are several reviews available [23-31,249,250] dealing with the 2D polymers. It is anticipated that also the three- and multidimensional polymer HPLC will be developed in future. 

Multidimensional methods thus involve a combination of single mechanisms and systems. In any multidimensional (usually 2D) approach, it is desirable that each dimension be as pure as possible in terms of selectivity of the separation mechanism. In comprehensive 2D separations, the precision (or chromatographic resolution) becomes a limiting factor and is ultimately determined by the quality of the separation in both dimensions. 

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