First dimension separation

The prevailing aspect of the first dimension separation is miniaturisation, in order to keep the injection volume in the second dimension sufficiently low. The most important aspect of the second dimension is speed, in order to maintain the separation achieved in the first dimension as much as possible. 

Figure 2.1. Schematic illustration oftwo-dimensional gel electrophoresis. Proteins are extracted from the organism of interest and solubilized. The first dimension separates proteins based on isoelectric point. The pi strip is reduced and alkylated and applied to an SDS-PAGE gel for separation by molecular weight. Proteins canbe visualized using a number of staining techniques.

Ion exchange chromatography is another means to remove detergents and chaotropes from protein samples. This is one rarely mentioned, but well understood benefit of using ion exchange chromatography as a first dimension separation step in a two-dimensional LC experiment. 

FIGURE 5.1 Heart-cut 2DLC where a zone from the first-dimension separation is reinjected onto a second-dimension column for improved resolution of three analytes coeluting on the... 

FIGURE 5.3 2DLC configuration and sequence utilizing a protein A affinity column in the first dimension and an SEC column in the second dimension. In step 1, the sample is injected onto the affinity column and the first-dimension separation takes place while the SEC column is being equilibrated. In step 2, valve 1 moves to position 2 and a fraction of the affinity separation is collected into the loop. In step 3, valve 1 moves back to position 1 and the collected sample is injected onto the SEC column for MS analysis. In step 4, after the protein elutes from the SEC column valve 2 is switched to position 2 and the SEC column effluent is sent to waste to avoid salts from entering the MS. 

Another study (Bedani et al., 2006) starts from the multidimensional sampling theory (Murphy et al., 1998a), which is discussed in Chapter 2. This sampling theory states that one needs to sample the first dimension separation system at least three to four times per peak width for maximum resolution. Bedani et al. then equate the second-dimension total analysis time to the first-dimension narrowest peak standard deviation. This defines the second-dimension operational parameters. All other parameters can be derived from this balance and Bedani s study goes through this and discusses how the rest of these variables are obtained. 

Others have examined the necessary parameters that should be optimized to make the two-dimensional separation operate within the context of the columns that are chosen for the unique separation applications that are being developed. This is true for most of the applications shown in this book. However, one of the common themes here is that it is often necessary to slow down the first-dimension separation system in a 2DLC system. If one does not slow down the first dimension, another approach is to speed up the second dimension so that the whole analysis is not gated by the time of the second dimension. Recently, this has been the motivation behind the very fast second-dimension systems, such as Carr and coworker s fast gradient reversed-phase liquid chromatography (RPLC) second dimension systems, which operate at elevated temperatures (Stoll et al., 2006, 2007). Having a fast second dimension makes CE an attractive technique, especially with fast gating methods, which are discussed in Chapter 5. However, these are specialized for specific applications and may require method development techniques specific to CE. 

Because frequent sampling is necessary, the second dimension must be fast. Hence, the second-dimension technique must be developed first because it sets the stage for the type of performance that can be driven by the first dimension. This process has worked out well for a number of systems that we have studied. But the philosophy is simple Why develop a high efficiency first dimension separation if this efficiency is distorted by the sampling process Hence, the first dimension is matched in performance to the second-dimension system and related through the sampling criterion. 

The IEX chromatography possesses several attractive features as a first-dimension separation mode in an MDLC scheme. These features include the following ... 

In general, a comprehensive separation strategy implies the desire to resolve/analyze all components within a sample. In the specific context of a multidimensional chromatographic method, the term is more narrowly applied to indicate that all analytes introduced to the first-dimension separation are also subjected to a second-dimension separation. There are two basic configurations used by our laboratory to carry out comprehensive multidimensional (IEX/RP) protein separations—IEX— Dual Column RP system and IEX—Dual Trap RP system (Figs. 13.1 and 13.2), respectively. 

Two high-voltage power supplies are used to drive the separation. The first power supply applies high voltage through a platinum electrode to the injection buffer reservoir for the first dimension separation. The second power supply applies potential to the interface through its buffer reservoir. The sheath flow cuvette is held at ground potential. 

FIGURE 15.4 Computer record of a two-dimensional capillary electrophoresis analysis of a protein homogenate prepared from ahiopsy obtained from the fundus of a Barrett s esophagus patient. The data were generated hy performing 1 s transfers between capillaries and a 9 s second-dimension separation. The first-dimension separation employed the same buffer as the CSE separation in Fig. 15.1 and the second-dimension separation employed the same buffer as the MECC separation in Fig. 15.1. 

The most widespread among multidimensional HPFC techniqnes are two-dimensional (2D) methods, in which components migrate along two imaginary axes. Fignre 4.1 illustrates such a process, where five fractions of the first-dimension separation are snbjected to a second-dimension separation. 

Most of the traditional HPLC detectors can be applied to LCxLC analyses the choice of the detectors used in comprehensive HPLC setup depends above all on the nature of the analyzed compounds and the LC mode used. Usually, only one detector is installed after the second-dimension column, while monitoring of the first-dimension separation can be performed during the optimization of the method. Detectors for microHPLC can be necessary if microbore columns are used. Operating the second dimension in fast mode results in narrow peaks, which require fast detectors that permit a high data acquisition rate to ensure a proper reconstruction of the second-dimension chromatograms. 

Study 2 Pvridvl ketones. A narrower range of compounds, a group of pyridyl ketones, was examined by Southwick and Schiffman (. Two-dimensional cross-sections through the three-dimensional space achieved by the MDS are shown in Figures 3a and 3b. The first dimension separates the three 2-pyridyl ketones that have popcorn-nutty aromas from the six other compounds that are green-vegetable in character. 

FIGURE 6.41 Layout of the quartz chip for 2D separation. Inj. is the injection channel, Sep. 1 the first-dimension separation channel, Sep. 2 the second-dimension separation array of 500 parallel channels, Out 1 and Out 2 the waste collection channels. The channels cover an area of 16 X 16 mm, the overall chip size is 23 x 23 mm [150]. Reprinted with permission from the Institute of Physics Publishing. 

Fig. 5.5 GC X GC-TOF-MS chromatograms of a PP migration extract (a) view along the first dimension (separation on boiling point), (b) view on the second dimension (separation on polarity).

Resulting from our experience in the separation of a broad variety of samples with 2-DB, resolution strongly depends on the nature of the separated sample. Furthermore, upon long separation times, band broadening during first dimension separation can be observed. 

As an example, a bidimensional separation was performed for a segment in a separation of a cellulose pyrolysate. Figure 5.2.13(A) shows the chromatogram of a pyrolysate obtained at 600° C from microcrystalline cellulose. The results were obtained on a 30 m X 0.32 mm Carbowax column with 0.5 pm film thickness as a first dimension separation in a bidimensional system similar to the one shown in Figure 5.2.12. 

In this separation, a 10-mL sample (large volume) containing a solution in tert-butyl methyl ether (tBME) of the pyrolysate of 1 mg cellulose obtained at 600° C was injected (off-line pyrolysis). The PTV injector was programmed at 20° C initial temperature for 2 min. and ramped with 10° C/min at 250° C and kept at this temperature for 1 min. Then the injector was further heated at 300° C. The split vent purge time was 2.5 min. The oven temperature for the first dimension separation was kept at 35° C for 2.5 min. then heated with 30° C/min. at 55° C and further heated with 3° C/min. to 240° C. The detector used in the first dimension was an MS system, which allowed the identification of a series of compounds from this chromatogram. The peak identification is given in Table 5.2.2. 

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