Multidimensional chromatography Subject

Most developments in the past two decades, however, have involved coupled column systems which are much more amenable to automation and more readily permit quantitative measurements, and such systems form the subject of this present book. A review on two-dimensional GC was published (43) in 1978 (and recently updated (29)), and the development by Liu and Phillips in 1991 of comprehensive 2D GC marked a particular advance (33). The fundamentals of HPLC-GC coupling have been set out (37) with great thoroughness by Grob. Other work on a number of other aspects of multidimensional chromatography have also been extensively reviewed (44,45). 

An inherent drawback of this methodology is that quantitative information is only obtained from those peptides which are subjected to MS/MS analysis. This fact stresses the necessity for efficient peptide separation via multidimensional chromatography (e.g., SCX/reversed-phase HPLC) prior to tandem MS analysis. Moreover, ion trap instruments cannot be employed in quantitative iTRAQ experiments since their peptide fragmentation spectra do not contain information... 

FIGURE 15.3 Outline of experimental protocol used for ICAT differential protein expression profiling. Protein mixtures from two cell populations are labeled with light or heavy isotopic versions of a cleavable ICAT reagent. Labeled proteins are combined, subject to multidimensional separation by SCX, RP, and avidin affinity chromatography, then analyzed by tandem MS for peptide and protein identification. Based on the relative ratio of the two isotopically labeled peptides, a relative abundance of protein expression can be determined. 

Practical implementation of two-dimensional separation is very easy in TLC. By definition, all the components of the sample are subjected to both separation dimensions, and components separated in the first dimension remain resolved in the second dimension. When a two-dimensional separation fulfills these requirements, it is considered comprehensive. From the practical point of view, multidimensional separations are much more difficult to implement in column chromatography. To perform the 2D separation in space, i.e., in a manner analogous to... 

Multidimensional gas chromatography may be defined as follows the process of selecting a (limited) region or zone of eluted compounds issuing from the end of one GC column, and subsequently subjecting the zone to a further GC displacement . 

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