Two-Dimensional HPLC Systems

These columns offer the potential for creating a hybrid-silica monolith, which can be run on existing HPLC systems at high flow rates, that are temperature and pH resistant. By their very nature, these columns would be void free and the only column killers that they would suffer from would be particulates and bound organics. They probably could be reverse flushed for particulate wash out and bound materials could be washed off with strong solvents. 

Much of the pressure to develop automated sequential HPLC separations has come from the necessity to separate complex biological mixtures, especially protein mixtures. Traditionally, complex mixtures of proteins have been separated using two-dimensional gel electrophoresis (2D GEP). The first dimension gel separation is carried out with electrophoresis buffers, the gel plate is rotated 90° and the second SDS-PAGE separation is carried out under denaturing conditions, using sodium dilauryl sulfate. The separated spots are then visualized, scraped off the plate, and then extracted for further analysis. Protein analysis by MALDI time-of-flight mass spectrometry starts with this time- and labor-intensive 2D GEP separation mode. 

In theory, combining two HPLC modes sequentially would provide an online LC/LC/MS/MS and speed the analytical procedure. Bands from the first separations could be detected and collected with an automated loop-and-valve injector, and then individual bands could be passed to the second LC for 

The demand for such an LC/MS luggable would come from the field environmental chemist, from the arson investigator, and obviously from your local forensic CSI and drug enforcement teams. It would avoid the problem of 

I hasten to add that this system does not exist at the moment. You probably do not want to include it in your equipment proposal for this year. But, it is rapidly becoming a viable option for development. And, if successful, would the portable linear ion trap (LIT) based LC/MS/MS, for definitive compound identification by searching a MS database, be far behind  

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

D-Enantiomers of amino acids in living organisms are attributed to many important bioprocesses or can be markers of certain disorders. Therefore, accurate quantification of the low levels of the D-form in the presence of a large amount of L-form is of considerable interest. Branched D-amino acids in mammalian tissues and body fluids were quantified recently using a sophisticated two-dimensional HPLC system containing a narrow-bore reversed phase and t-BuCQN column for the enantioseparation of l- and D-enantiomers. Target analytes were determined as their fluorescent derivatives, precolumn labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole. D-Val, alio-lie, lie, and Leu were determined at nmol/ml level, making this a valuable method for quantification of D-enantiomers of amino acids [80]. 

Bandh C, Ishaq R, Broman D, et al. 1996. Separation for subsequent analysis of PCBs, PCDD/Fs and PAHs according to aromaticity and planarity using a two-dimensional HPLC system. Environ Sci Technol 30(1) 214-219. 

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 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... 

The determination of PCCD/Fs usually involves extensive clean-up to remove coextracted compounds, typically present in greater amounts. The PCDD/Fs and the most toxic non-ortho chlorine substituted CBs (Nos. 77, 126 and 169) are planar molecules. They can be separated from bulk PCBs and other interfering compounds by chromatography on activated charcoal (Jensen and Sundstrom, 1974 Huckins etal., 1980 Smith, 1981 Kannan et al., 1991). The advantages of HPLC techniques on activated carbon (PX21) dispersed with n-octadecane (Cig) was shown by Kannan etal. (1993) Feltz et al. (1995) described an automated HPLC fractionation of PCDDs and related compounds. Modern clean-up procedures comprise HPLC on 2-(l-pyrenyl)ethyl dimethyl silylated silica (PYE) columns (Haglund et al., 1990 Kannan et al., 1998), a two-dimensional HPLC system for the separation of PCDD/Fs, PCB and PAHs (Zebiihr et al., 1993) and the automated HPLC method by Bandh... 

Figure 3-6. Schematic illustration of two-dimensional HPLC system utilizing the monolithic HPLC columns as second-dimension separation media. Components seprarted on the first dimension are distributed alternately to the second-dimension columns by Switch bulb as indicated by solid and dotted lines.

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. 

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). 

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... 

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.

Mass spectrometry (MS) is increasingly being combined with reverse phase HPLC or CZE in order to add an additional dimension to the data that a traditional detection system would not provide. A two-dimensional EC-CZE system with mass... 

Gilar, M., Olivova, P., Daly, A.E., Gebler, J.C. (2005a). Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. J. Sep. Sci. 28, 1694. 

Phosphorus, fatty acids, carbohydrates, glycerol, and amino acids were analyzed by the method described in our previous paper [8] and references cited therein. SDS-PAGE [8], TLC [9], HPLC [9], determination of phos-phomonoester [8], reducing sugar analysis [13], methylation analysis [14], and hexose analysis [15] were performed as described in the respective literature. Two dimensional TLC was performed on silica-gel plate (Merck Silicagel 60 F254 No. 5715) using the solvent systems, chloroform-methanol-acetic acid (65/10/1, v/v/v) for the first development and chloroform-methanol-25% ammonia solution (65/10/1) for the second. 

With current commercially available equipment the ideal set-up for online analysis would be an HPLC-SPE system, a cryogenic flow probe (30 p,l active volume) that is in permanent use within an actively shielded magnet operating at 500 MHz or higher. The system would offer the optimum LC-NMR sensitivity (no dependency on LC peak volumes), and complex impurities as low as 0.1% could be identified by one- and two-dimensional NMR experiments, provided that the impurities are sufficiently stable to permit isolation on the SPE cartridges,... 

In conclusion, eluent gradient polymer HPLC represents a useful tool for separation of complex polymer system. It belongs to the important constituents of several two-dimensional polymer HPLC procedures. 

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. 

The contents of the test-tube are mixed and warmed at 55 °C for 1-5 h. The mixture is cooled and an aliquot portion is spotted on to a TLC plate for separation. Two-dimensional chromatography is carried out on silica gel layers with cyclohexane-ethyl acetate (1 1) and light petroleum-chloroform-diethyl ether-acetic acid (33 33 33 1). Chromatography on polyamide layers is accomplished with heptane-ethyl acetate-butanol (8 1 1). The Rp values of six NBD-amines in these systems [99] are given in Table 4.15. Amounts of less than 15 ng of NBD-amine per spot can be detected. HPLC of some NBD-amines has been carried out using Zipax coated with 0.5% 0,/3 -oxydipropionitrile and 1% tetra-hydrofuran in hexane as the mobile phase (see Section 4.2.4.2.2). 

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