Planar Two-Dimensional Separations

Two-dimensional planar eleetrophoresis was first used in 1951 (8), while eleetrophoresis was eoupled with thin-layer ehromatography (TLC) in 1964 to separate mixtures of nueleosides and nueleotides (9). These teehniques were novel and led to other great diseoveries, but did not survive the test of time, and they are no longer eommonly used. TLC-eleetrophoresis in partieular was an awkward teehnique to 

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. 

In 1989, Yamamoto et al. developed the first technique that directly coupled chromatography to capillary electrophoresis, although again in a non-comprehensive fashion. Low-pressure gel permeation chromatography, which separates analytes based on differences in molecular size, was combined with capillary isotachophore-sis, which separates according to electrophoretic mobility. Capillary isotachophoresis 

Yamamoto et al. also coupled gel permeation HPLC and CE in an on-line fashion in 1990, where capillary isotachophoresis was again used in the second dimension. This technique was also not comprehensive due to the loss of resolution between the techniques. It was also not particularly fast, with a 23 min CE cycle, which was repeated 90 times throughout the HPLC run (14). Volume incompatibility between HPLC and CE was one problem not addressed in this study, in which a large HPLC column was coupled to an electrophoresis capillary. 

Other groups have also used LC 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 LC and CE. 

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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 ... 

Two-dimensional separations in planar chromatography are rather trivial to perform. All unidimensional multiple development techniques employ successive repeated development of the layer in the same direction, with removal of the mobile phase between developments. The main variants are multiple chromatography and incremental multiple development. The basis for automated multiple development (AMD) is the automation of unidimensional, incremental, multiple development with a reverse solvent strength gradient [998]. 2D TLC finds limited use, and is mainly a qualitative technique. 

Sajewicz, M. Pietra, R. Drabik, G. Namyolo, E. Kowalska, T. On the stereochemistry peculiar two-dimensional separation of 2-arylpropionic acids by chiral TLC. J. Planar Chromatogr. Mod. TLC 2006, 19, 273-277... 

A representation of the peak capacity of a planar two-dimensional system is presented in Fig. 1. A multidimensional ITPLC system in which the entire first dimension column effluent (not merely an interesting region) is reanalyzed as discrete fractions at regular intervals in the second dimension is referred to as using the comprehensive concept If only the components of interest from the first column effluent are subjected to separation on the second column, this is referred to as the heart-cutting technique. Heart-cutting techniques require that the retention properties of the analytes in the first dimension are known in advance. This technique is appropriate when only one or a few components need to be isolated. 

Unquestionably, most practical planar chromatographic (PC) analytical problems can be solved by the use of a single thin-layer chromatographic (TLC) plate and for most analytical applications it would be impractical to apply two-dimensional (2-D) TLC. One-dimensional chromatographic systems, however, often have an inadequate capability for the clean resolution of the compounds present in complex biological samples, and because this failure becomes increasingly pronounced as the number of compounds increases (1), multidimensional (MD) separation procedures become especially important for such samples. 

In planar ehromatography, the fraetions are not always transferred to another separation system, but rather a seeondary separation is developed, orthogonally on the same ehromatographie plate. Therefore, for all substanees not eompletely separated it is possible that baseline separation ean be aehieved by means of a seeond separation proeess with an appropriate mobile (stationary) phase. Figure 8.2 shows that in the seeond dimension a theoretieally unlimited number of seeondary eolumns ean be applied. Beeause of this, the terminology two-dimensional PC is not suffieiently... 

Multidimensional planar chromatographic separations, as we have seen, require not only a multiplicity of separation stages, but also that the integrity of separation achieved in one stage be transferred to the others. The process of separation on a two-dimensional plane is the clearest example of multidimensional separations. The greatest strength of MD-PC, when properly applied, is that compounds are distributed widely over two-dimensional space of high zone (peak) capacity. Another... 

P. Flarmala, L. Botz, O. Sticher and R. Fliltunen, Two-dimensional planar chromatographic separation of a complex mixture of closely related coumarins from the genus Angelica, J. Planar Chromatogr. 3 515-520 (1990). 

Figure 7. Two-dimensional cuts through the potential energy surface for planar HF-HF collisions including vibration. The quantity plotted in the figure is the total potential (in hartrees), which is defined as the sum of the interaction potential and the two diatomic potentials, with the zero of energy corresponding to two infinitely separated HF molecules, each at its classical equilibrium separation. This figure shows cuts through the r. plane (in bohrs) for 0 = 0 = = 0 and...

Unlike the continuous zone development mechanism utilized in a planar separation experiment, comprehensive MDLC is a sequential operation in which finite volumes of eluant are injected into the next dimension column. Because of this finite volume aspect, the mechanism and consequences of sampling eluant from one column with subsequent injection into the next column must be understood. Undersampling would lead to a loss in two-dimensional resolution and oversampling would lead to excessively long run times as the second dimension column would be used in a very inefficient way. 

This effect is illustrated in Fig. 17.1. Multidimensional chromatography separations can be done in planar systems or coupled-column systems. Examples of planar systems include two-dimensional thin-layer chromatography (TLC) (Consden et al., 1944 Grinberg et al., 1990), where successive one-dimensional TLC experiments are performed at 90° angles with different solvents, and 2D electrophoresis, where gel electrophoresis is run in the first dimension followed by isoelectric focusing in the second dimension (O Farrell, 1975 Anderson et al., 1981 Celis and... 

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