Multidimensional HPLC

Multidimensional HPLC offers very high separation power when compared to monodimensional LC analysis. Thus, it can be applied to the analysis of very complex mixtures. Applications of on-line MD-HPLC have been developed, using various techniques such as heart-cut, on-column concentration or trace enrichment applications in which liquid phases on both columns are miscible and compatible are frequently reported, but the on-line coupling of columns with incompatible mobile phases have also been studied. 

In on-line multidimensional HPLC (MDHPLC) two relatively high-efficiency columns are coupled in an instrument, via the use of valves, traps and other means. In LC-LC the precolumn is used for sample cleanup and prefractionation, before introduction of the fraction of interest to the analytical column. Much of the instrumentation for MDHPLC is the same as that in conventional one-dimensional experiments. However, the additional complexity of MDHPLC experiments leads to greater difficulties than those found in conventional HPLC ... 

Coupled LC-LC can separate high-boiling petroleum residues into groups of saturates, olefins, aromatics and polar compounds. However, the lack of a suitable mass-sensitive, universal detector in LC makes quantitation difficult SFC-SFC is more suitable for this purpose. Applications of multidimensional HPLC in food analysis are dominated by off-line techniques. MDHPLC has been exploited in trace component analysis (e.g. vitamin assays), in which an adequate separation for quantitation cannot be achieved on a single column [972]. LC-LC-GC-FID was used for the selective isolation of some key components among the irradiation-induced olefinic degradation products in food, e.g. dienes and trienes [946],... 

Wagner, K., Miliotis, T., Marko-Varga, G., Bischoff, R., Unger, K.K. (2002). An automated online multidimensional HPLC system for protein and peptide mapping with integrated sample preparation. Anal. Chem. 74, 809-820. 

FIGURE 16.11 Multidimensional HPLC-CE separation of cytochrome c/myoglobin enzymatic digest presented in 2D format. For experimental details see text (reprinted with permission from Electrophoresis). 

In parallel with recent developments in GC, multidimensional HPLC (LC x LC) is now also finding application in environmental analysis.33 The combination of two sufficiently different separation dimensions (e.g., NP-HPLC x RP-HPLC or IC x RP-HPLC), however, remains difficult because of the solvent compatibility issues discussed above. Here, too, HILIC may bring about a significant improvement, since its mobile phase requirements are much closer to RP-HPLC than those of other liquid chromatographic techniques.34 In contrast to GC x GC, LC x LC cannot be implemented with a (thermal) modulator that collects the analytes after the first separation dimension and reinjects them into the second column it is most practically realized with a double-loop interface that alternately collects and transfers the analytes from the first to the second dimension (Figure 13.7). Even though the second dimension chromatogram is also very fast, detection is not normally a problem since the peak widths in the second dimension are usually still of the order of 1-2 s. 

S. McSheehy, P. Pohl, R. Fobinski, J. Szpunar, Investigation of arsenic speciation in oyster test reference material by multidimensional HPLC-ICP MS and electrospray tandem mass spectrometry (ES MS/MS), Analyst, 126 (2001), 1055D1062. 

B. Gammelgaard, K. E. Madsen, J. Bjerrum, L. Bendahl, O. Jons, J. Olsen, U. Sidenius, Separation, purification and identification of the major selenium metabolite from human urine by multidimensional HPLC-ICP-MS and APCI-MS, J. Anal. Atom. Spectrom., 18 (2003), 65-70. 

McSheehy, S., Pohl, P., Lobmski, R., Szpunar, J. Complementarity of multidimensional HPLC-ICP-MS and electrospray MS-MS for speciation analysis of arsenic in algae. Anal. Chim. Acta 440, 3-16 (2001)... 

The prefractionation technique provides improvements in separation resolution, increased sensitivity, and enhanced ability of loading a much higher amount of sample in any narrow pH interval in gel electrophoresis. Once the complexity of proteins is reduced by prefractionation, individual fractions can be subjected to either 2D gel electrophoresis or multidimensional HPLC for further separation, followed by MS analysis. 

FIGURE 2.7 Isoform analysis of phosphorylated HI histone by multidimensional HPLC-CZE. Panel (a) shows the separation of HI histones by RP-HPLC on a NucleosU 300-5 C4 column. Panel (b) shows the separation of the HI.5 RP-HPLC fraction by CZE. The CE analytical system included a Beckman-Coulter P/ACE 2100 instrument operated in normal polarity at 12 kV with UV detection at 200 nm and capillary cooling at 25°C. Nonphosphorylated (pO) and mono-, di-, and tri-phosphorylated (pi, p2, p3) H1.5 isoforms were separated on the basis of miz using a 57 cm long (50 cm to detector) by 75 ixm ID untreated fused-silica capillary. Separation media contained 0.1 M sodium phosphate, 0.02% hydroxypropyhnethylceUulose, pH 2.0. Samples were injected for 2 s. (From Sarg B, et al. J Biol Chem 2006 281 6573-80. With permission.)... 

J.V. Posluszny and R. Weinberger, Optimization of Multidimensional HPLC for the Determination of Drugs in Plasma by Direct Injection, Micellar Cleanup and Photodiode Array Detection, J. Chromatogr., 507 267 (1990). 

Reliable hardware and software tools for coupling techniques and system control have enabled the establishment of more complex two- or multidimensional HPLC systems, which allow consecutive chromatographic steps performed online on different stationary phases also including steps of analyte trapping or sample preparation. 

With the hnman genome now sequenced (primarily by capillary electrophoresis technology), scientists are turning their attention to proteomics. The term pro-teome has been introdnced to define the full complement of proteins in a cell. It also reqnires a description of the localization, concentration, and multisubunit association of each of these proteins (25). The multidimensional hplc with automated column switching technology can successfully complement the more conventional approaches to protein separation, such as two-dimensional gel electrophoresis, which nsnally involve many manual steps and are not conducive to high throughput applications. 

As a general rule, the approach to separation must be tailored to the separation goals i.e., purification of a single peptide from a complex mixture will require a different approach from that necessary for separating all components of a mixture. Whereas the former approach may require only the application of a single HPLC mode, the latter will require a combination of separation modes (multistep or multidimensional HPLC) for efficient resolution of all desired peptides. 

In multidimensional HPLC, several columns with different selectivities are switched parallel to each other with the help of two high-pressure switching valves (see Fig. 39 C). This technique is... 

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