Chromatography 2-dimensional

Becamel C, Galeotti N, Poncet J, et al. A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors. Biol Proc Online 2002 4 94-104. 

This recrystallised acid is pure in the norm y accepted sense of the word, namely it has a sharp m.p. and gives on analysis excellent values for carbon, hydrogen and nitrogen. If however it is subjected to one-dimensional paper chromatography (p. 53), the presence of traces of unchanged anthranilic acid can be detected, and repeated recrystallisation is necessary to remove these traces. 

The total phosphoms content of the sample is determined by method AOCS Ja 5-55. Analysis of phosphoUpid in lecithin concentrates (AOCS Ja 7-86) is performed by fractionation with two-dimensional thin-layer chromatography (tic) followed by acid digestion and reaction with molybdate to measure total phosphorous for each fraction at 310 nm. It is a semiquantitative method for PC, PE, PI, PA, LPC, and LPE. Method AOCS Ja 7b-91 is for the direct deterrnination of single phosphoHpids PE, PA, PI, PC in lecithin by high performance Hquid chromatography (hplc). The method is appHcable to oil-containing lecithins, deoiled lecithins, lecithin fractions, but not appHcable to lyso-PC and lyso-PE. 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

The limitations of one-dimensional (ID) chromatography in the analysis of complex mixtures are even more evident if a statistical method of overlap (SMO) is applied. The work of Davis and Giddings (26), and of Guiochon and co-workers (27), recently summarized by Jorgenson and co-workers (28) and Bertsch (29), showed how peak capacity is only the maximum number of mixture constituents which a chromatographic system may resolve. Because the peaks will be randomly rather than evenly distributed, it is inevitable that some will overlap. In fact, an SMO approach can be used to show how the number of resolved simple peaks (5) is related to n and the actual number of components in the mixture (m) by the following ... 

The values of n and the corresponding N which are necessary to resolve 50-90% of the constituents of a mixture of 100 compounds are listed in Table 1.5, thus making clear the limitations of one-dimensional chromatography. For example, to resolve over 80 % of the 100 compounds by GC would require a column generating 2.4 million plates, which would be approximately 500 m long for a conventional internal diameter of 250 p.m. For real mixtures, the situation is even less favourable to resolve, for example, 80 % the components of a mixture containing all possible 209 polychlorinated biphenyls (PCBS) would require over lO plates. 

Two-dimensional separations can be represented on a flat bed, by analogy with planar chromatography, with components represented by a series of dots . In fact, zone broadening processes in the two dimensions result in elliptically shaped spots centred on each dot . Overlap of the spots is then possible, but Bertsch (30) also showed how the contributors to the overall resolution, R, along the two axes, and Ry contribute to the final resolution according to the following ... 

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). 

W. Beitsch, Two-dimensional gas chromatography concept, instmmentation and appli-cations-Pait 1 fundamentals., conventional two-dimensional gas chromatography, selected applications , ]. High. Resolut. Chromatogr. 22 647 (1999). 

It is through observing examples of actual applications that the best understanding of GC-GC separation principles can be achieved. Over the past 30 years, there have been essentially three main areas where two-dimensional gas chromatography has been applied ... 

G. S. Frysinger, R. B. Gaines and E. B. Ledford-Jr, Quantitative determination of BTEX and total aromatic compounds in gasoline by comprehensive two-dimensional gas chromatography (GC X GC) , 7. High Resolut. Chromatogr. 22 195-200 (1999). 

Repeated chromatography in a third dimension after completion of two-dimensional development. Here, development in the first, second, and third dimensions can be envisaged as occurring on three plates arranged in the form of a cube the plate is again dried between developments. 

W. Markowski and K. L. Czapiriska, Computer simulation of the separation in one- and two-dimensional tliin-layer chromatography hy isocratic and stepwise gradient development ,/ Liq. Chromatogr. 18 1405-1427 (1995). 

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. 

Figure 9.5 The generic setup for two-dimensional liquid chromatography-capillary zone electrophoresis as used by Jorgenson s group. The LC separation was performed in hours, while the CZE runs were on a time scale of seconds.

Figure 9.6 Surfer-generated chromatoeletropherogram of fluorescamine-labeled tryptic digest of ovalbumin. Reprinted from Analytical Chemistry, 62, M. M. Bushey and J. W. Jorgenson, Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography/capillary zone electrophoresis, pp 978-984, copyright 1990, with permission from the American Chemical Society.

THREE-DIMENSIONAL SIZE EXCLUSION CHROMATOGRAPHY-REVERSE PHASE LIQUID CHROMATOGRAPHY-CAPILLARY ZONE ELECTROPHORESIS... 

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