Ix

iiiii

1st dimension retention time

1st dimension retention time

2nd dimension chromatograms

1st dimension retention time

(c) 2nd dimension chromatograms

FIGURE 3.25 The concept of multidimensional GC.26 (a) Single heart-cut GC analysis, in which a portion of the effluent from the primary column containing analytes of interest is diverted to the second dimension column and subjected to additional separation over an extended period of time. (b) Dual heart-cut GC analysis, in which two regions with coelutions are diverted to the second dimension column, with less time to perform each separation. (c) Comprehensive two dimensional GC analysis, in which the sizes of the sequential heart-cut fractions are very small, and the time to develop each sequential second dimension chromatogram is very short.

in one-dimensional GC. However, rather than reach a detector, the effluent from the primary column enters a special interface (modulator) placed between the first and second column. This modulator collects the material from the first column for a short period of time, and then injects the entire fraction which it has collected into the second dimension column as a short chromatographic pulse. It then collects another fraction of the effluent from the first column while the previous fraction is being separated on the second dimension column. This process of effluent collection and injection is repeated frequently throughout the entire analysis.26 The second dimension column is short, so that the separation in this column can be completed before first components of subsequent fraction reach the detector (a few seconds). The stationary phase in the second column must have different selectivity than the first column to fulfill the condition of orthogonality. The material exiting the second dimension column is passed to the detector, so that a series of sequential short second dimension chromatograms is obtained. In order to preserve the separation achieved in the first dimension, each peak eluting from the first dimension should be sampled at least three times.27 For example, if the peaks eluting from the first dimension have a width of 18s, the modulation period must be no longer than 6s.

The multiple second dimension chromatograms are recorded by the system as a single linear chromatogram. In this form, it is exceedingly difficult to interpret. For this reason, the data are usually converted into a three-dimensional plot with primary retention plotted along the X axis, secondary retention plotted along the Y axis and peak intensity plotted along the Z axis. This 3D plot is usually displayed as a top-down view in the form of a contour plot. The construction of such a plot is outlined in Figure 3.26. An appropriate software package uses the modulation period of the interface and the times at which the pulses to the second dimension column occur (11, t2 and i3 in Figure 3.26a) to slice the original chromatographic signal into its component second dimension chromatograms (Figure 3.26b). These chromatograms are then aligned side-by-side to form GC X GC retention plane (Figure 3.26c), which is then plotted top-down as in Figure 3.26d. The time at which a modulation pulse occurs provides the primary retention time for all of the peaks which elute between that pulse and the following pulse. The second dimension retention time of a peak is then its original (1D) retention time minus the primary dimension retention time.

The heart of any GC X GC system is the modulator. There are two basic types of modulators currently in use: thermal modulators and valve-based modulators. Thermal modulators are more popular; in fact, the commercial GC X GC systems are all based on thermal modulation. Early thermal modulators required moving parts, which made them not always reliable. Today, most thermal modulators are based on cryocooling, with no moving parts inside the oven. These are reliable enough to be used in routine applications. An example of a modern cryogenic modulator utilizing liquid CO2 as the cryocoolant28 is presented in Figure 3.27. When the downstream CO2 jet (D) is on and the upstream jet (U) is off, material is focused into a narrow band within a cooled segment of the second dimension column. The upstream jet is then turned on so that it can trap the material eluting from the primary column while the downstream jet turns off to launch the focused band into the second dimension column. The downstream jet turns back on, before the upstream jet turns off, so that the material released from the upstream cold spot is retrapped in the downstream cold spot prior to injection into the second column. This two-stage mode of operation prevents breakthrough of the analytes through the trap while any of the jets is off.

GC X GC offers unparalleled resolving power. It can separate components of very complex mixtures, for example all 209 PCB congeners,29 which is impossible using 1D GC. It can also potentially simplify sample preparation before chromatographic analysis by eliminating the need for extensive sample clean-up when the analytes of interest can be chromatographically separated from the matrix components. Consequently, GC X GC has tremendous potential in environmental analysis, especially in combination with TOF mass spectrometry.

FIGURE 3.26 The interpretation of GC X GC data and generation of contour plots.26 (a) Raw GC X GC chromatogram consisting of a series of short second dimension chromatograms; 1i, t2, and t3 indicate the times when injections to the second dimension column occurred. (b) The computer uses these injection times to slice the original signal into the individual second dimension chromatograms. (c) The second dimension chromatograms are aligned on a three dimensional plane with primary retention time and secondary retention time as the X and Y axes, respectively, and signal intensity as the Z axis. (d) When viewed from above, the peaks appear as rings of contour lines or color-coded spots.

FIGURE 3.26 The interpretation of GC X GC data and generation of contour plots.26 (a) Raw GC X GC chromatogram consisting of a series of short second dimension chromatograms; 1i, t2, and t3 indicate the times when injections to the second dimension column occurred. (b) The computer uses these injection times to slice the original signal into the individual second dimension chromatograms. (c) The second dimension chromatograms are aligned on a three dimensional plane with primary retention time and secondary retention time as the X and Y axes, respectively, and signal intensity as the Z axis. (d) When viewed from above, the peaks appear as rings of contour lines or color-coded spots.

B. Coupled Column Liquid Chromatography

In liquid chromatography, two dimensional separations in the vast majority of cases are not comprehensive. While comprehensive 2D-LC separations (LC X LC) can be accomplished and have been demonstrated (e.g., Refs. 30 and 31), the technique is not very popular. Probably one of the main reasons for this is the inability to perform very fast separations in liquid chromatography. In GC X GC, a typical second dimension separation can be completed in a few seconds. In LC, the separation time required is much longer. The problem can be overcome by stopping the flow in the first dimension column while the second dimension separation proceeds, but this causes the overall analysis times to be very long.

FIGURE 3.27 Schematic diagram of the dual cryojet interface.26 When the downstream jet (D) is on and the upstream jet (U) is off, material from the primary column is trapped as a narrow band within the second dimension column. It is then released by turning the downstream jet off, and retrapped by the upstream jet. The downstream jet is turned back on before desorption from the second stage is effected to prevent breakthrough.

FIGURE 3.27 Schematic diagram of the dual cryojet interface.26 When the downstream jet (D) is on and the upstream jet (U) is off, material from the primary column is trapped as a narrow band within the second dimension column. It is then released by turning the downstream jet off, and retrapped by the upstream jet. The downstream jet is turned back on before desorption from the second stage is effected to prevent breakthrough.

In LC-LC, two columns are linked via a switching valve so that any component flowing through the first column can be directed to the detector or to the second column. Two types of arrangements are used: the two columns may have the same stationary phase, but a different length, or they may have similar length, but different stationary phase. For reasons explained at the beginning of this section, only the second approach can be classified as two-dimensional separation. The most important applications of LC-LC include trace enrichment and sample clean-up. Both of them are important from the point of view of environmental analysis. In many cases, both sample clean-up and trace enrichment are employed in the same LC-LC scheme. Trace enrichment is based on the fact that the analytes may be retained as a narrow zone at the head of the first (preconcentrating) column while a large sample volume is pumped through this column. For example, nonpolar or weakly-polar analytes can be preconcentrated from aqueous solutions on a reversed phase column because water is a weak eluent in this scenario. This will also result in partial sample clean-up, as the polar sample components will not be retained. The preconcentrated sample can then be eluted with a stronger eluent into the second (analytical) column, where the proper separation takes place.

Apart from analyte preconcentration and sample clean-up, LC-LC can also be used to improve the separation of critical sample components. This is done by using heart-cut techniques similar in principle to those used in GC-GC. A high-resolving LC-LC system can be implemented by using columns packed with stationary phases offering different separation mechanisms. Examples of the possible combinations include size exclusion-ion-exchange; size exclusion-reversed phase; ion-exchange-reversed phase; reversed phase (alkyl ligand)-reversed phase (ion-pairing eluent); reversed phase-affinity, etc.32 The resolving power of the system can be enhanced even further by coupling the LC-LC system with mass spectrometry.33'34 It is also possible to couple the LC-LC system to other separation techniques like capillary zone electrophoresis, which creates a three-dimensional separation system.35 However, such couplings are outside the scope of this chapter.

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