Comprehensive two-dimensional HPLC

The word says it comprehensive chromatography is an all-including technique, a method which allows to investigate the whole eluate in greater detail. In the case of 

Cohen and M.R. Schure, eds., Multidimensional Liquid Chromatography, John Wiley Sons, Ltd, 

Such chromatograms are often represented in two dimensions, namely as spots with contour lines or with coloured intensity gradation. We look at them as we look on maps, i.e. from above . 

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Stoll, D.R., Carr, P.W. (2005). Fast, comprehensive two-dimensional HPLC separation of tryptic peptides based on high temperature HPLC. J. Am. Chem. Soc. 127, 5034-5035. 

Hu, L., Chen, X., Kong, L., Su, X., Ye, M., Zou, H. (2005). Improved performance of comprehensive two-dimensional HPLC separation of traditional Chinese medicines by using a silica monolithic column and normalization of peak heights. J. Chromatogr. A 1092, 191 198. 

FIGURE 9.4 Schematic diagram of the on-line comprehensive two-dimensional HPLC system including an integrated sample preparation step. 

A new topic is now included Chapter 20 about quahty assurance. Part of it could be found before in chapter 19 but now the subject is presented much broadly and independent of Analytical HPLC . Two chapters in the appendix were updated and expanded by Bruno E. Lendi, namely the ones about the instrument test (now chapter 25) and troubleshooting (now chapter 26). Some new sections were created 1.7, comparison of HPLC with capillary electrophoresis 2.11, how to obtain peak capacity 8.7, van Deemter curves and other coherences 11.3, hydrophilic interaction chromatography 17.2, method transfer 18.4, comprehensive two-dimensional HPLC 23.3, fast separations at 1000 bar 23.5, HPLC with superheated water. In addition, many details were improved and numerous references added. 

Figure 6.5 Schematic representation of the on-line comprehensive two-dimensional HPLC system including an integrated sample preparation step [32]. Reprinted from Journal of Chromatography B, 803, Machtejevas, E., John, H., Wagner, K., Standker, L, Marko-Varga, C., Forssmann, W.C., Bischoff, R., Unger, K.K., Automated Multi-Dimensional Liquid Chromatography Sample Preparation and Identification of Peptides from Human Blood Filtrate, I2I 130. Copyright (2004), with permission from Elsevier...

A typical comprehensive two-dimensional HPLC separation is attained through the connection of two columns by means of an interface (usually a high-pressure switching valve), which entraps specific quantities of D eluate, and directs it onto a secondary colunm. This means that the first column effluent is divided into cuts that are transferred continuously to the D by the interface. The type of interface depends on the methods used, although multiport valve arrangements have been the most frequently employed. 

A comprehensive two-dimensional HPLC system, with an RP column as a primary column and an immobilized liposome chromatography (ELC) column as a secondary column, was developed for the screening and analysis of the membrane-permeable compounds in the traditional Chinese medicine... 

Hu L, Li X, Feng S, Kong L, Su X, Chen X, et aL Comprehensive two-dimensional HPLC to study the interaction of multiple components in Rheum pahmtum L. with HSA by coupling a silica-bonded HSA column to a silica monolithic OE6 column. J Sep Sci 2006 29 881-8. 

Figure 9.11 Schematic illustration of the transparent interface used to link the HPLC capillary to the CZE capillary. Reprinted from Analytical Chemistry, 69, T. E. Hooker and J. W. Jorgenson, A transparent flow gating interface for the coupling of microcolumn EC with CZE in a comprehensive two-dimensional system , pp 4134-4142, copyright 1997, with permission from the American Chemical Society.

Moore, A.W., Jorgenson, J.W. (1995a). Rapid comprehensive two-dimensional separations of peptides via RP-HPLC-optically gated capillary zone electrophoresis. Anal. Chem. 67, 3448-3455. 

Bushey, M.M., Jorgenson, J.W. (1990). Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins. Anal. Chem. 62,161-167. Cabrera, K. (2004). Applications of silica-based monolithic HPLC columns. J. Sep. Sci. 27, 843-852. 

Tanaka, N., Kimura, H., Tokuda, D., Hosoya, K., Ikegami, T., Ishizuka, N., Minakuchi, H., Nakanishi, K., Shintani, Y., Furuno, M., Cabrera, K. (2004). Simple and comprehensive two-dimensional reversed-phase HPLC using monolithic silica columns. Anal. Chem. 76, 1273-1281. 

For the analysis of complex mixtures, modern coupling techniques such as GC-MS, GC-FTIR, HPLC-MS, CE-MS and comprehensive two-dimensional GC [832, 836-838a] are valuable and sometimes essential tools. 

There is growing interest related to rapid screening and full characterization of the constituents of plants with medicinal properties. The high content in polyphenols accounts for in vitro and in vivo antioxidant activity of the extracts obtained from plants on the other hand, the high complexity of the samples extracted, depending on the method employed, may preclude complete resolution by conventional HPLC techniques. For this purpose, a comprehensive two-dimensional liquid chromatography (LCxLC) system, comprised of an RP-Amide first dimension and a partially porous octadecylsilica column in the second dimension, has been compared with a one-dimensional system (125). The chromatographic methods optimized in this research allowed the complete resolution and full characterization of polyphenols and xanthines in mate extracts. 

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). 

The only other group to have performed comprehensive multidimensional reverse-phase HPLC-CZE separations is at Hewlett-Packard. In 1996, a two-dimensional LC-CE instrument was described at the Erederick Conference on Capillary Electrophoresis by Vonda K. Smith (21). The possibility for a commercial multidimensional instrument may have been explored at that time. 

In 1995, Moore and Jorgenson used the optically gated CZE system to obtain extremely rapid separations with HPLC coupled to CZE. The rapid CZE analysis made possible more frequent sampling of the HPLC column, thus increasing the comprehensive resolving power. Complete two-dimensional analyses were performed in less than 10 min, with the CZE analyses requiring only 2.5s. A peak... 

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. 

New concepts presented in this edition include monolithic columns, bonded stationary phases, micro-HPLC, two-dimensional comprehensive liquid chromatography, gradient elution mode, and capillary electromigration techniques. The book also discusses LC-MS interfaces, nonlinear chromatography, displacement chromatography of peptides and proteins, field-flow fractionation, retention models for ions, and polymer HPLC. 

Many groups have used electrophoresis to enhance a primary chromatographic separation. These techniques can be considered to be two-dimensional, but they are not comprehensive, usually due to the loss of resolution in the interface between the two methods. For instance, capillary electrophoresis was used in 1989 by Grossman and co-workers to analyze fractions from an HPLC separation of peptide fragments. In this study, CE was employed for the separation of protein fragments that were not resolved by HPLC. These two techniques proved to be truly orthogonal, since there was no correlation between the retention time in HPLC and the elution order in CE. The analysis time for CE was found to be four times faster than for HPLC (12), which demonstrated that CE is a good candidate for the second dimension in a two-dimensional separation system, as will be discussed in more detail later. 

Moore and Jorgenson combined the rapid two-dimensional separation achieved by LC-CZE with SEC to make the first comprehensive three-dimensional separation involving an electrodriven component in 1995. Size exclusion chromatography separated the analytes over a period of several hours while the reverse phase HPLC-CZE combination separated components in only 7 min. A schematic diagram of the three-dimensional SEC-reverse phase HPLC-CZE instrument is shown in Figure 9.9 (18). A dilution tee was placed between the SEC column and the reverse phase HPLC injection loop in order to dilute the eluent from the SEC column, since it contained more methanol than was optimal for the reverse phase HPLC column. 

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