Second-dimension elution process

Mass spectrometers that use electrospray ionization (ESI) do not function well if the eluent contains low volatility salts. This is a major concern when an ion-exchange column is used as a first-dimension column and the salt concentration is used to modulate the retention in this column. In this case, another valve can be connected between the second-dimension column and the detector so that any salt from the second-dimension elution process that is either unretained or weakly retained can be diverted prior to feeding zones to the mass spectrometer. 

In complex samples, when the range of elution times may not be known beforehand, there is the possibility of wraparound where components from the previous run are still eluting on the next second-dimension elution (Micyus et al., 2005). This situation is of concern and should be eliminated in the method development process for all but the most exploratory of work. This may require collecting fractions and injecting these fractions into the second-dimension column to determine the most retained compound retention time as part of the method development process. 

For isocratic LC, the solute does not need to fully elute from the second-dimension column prior to the next sampling period. This is illustrated in Fig. 6.4, where it is shown that more than one sample can be resident in the column at one time. Using the chromatograms shown in Fig. 6.5, which show the effect of various injection volumes that will be discussed later, it is not necessary to wait for the full 2 min of sampling time. This significantly helps to speed up the sampling process and is most useful for SEC, where short elution time ranges are typically found and short times between the injection and nonretained peaks are typical of column operation. 

In the second RP dimension, gradient elution is required to elute proteins with a wide range in hydrophobicity. As gradient elution is typically a slow process, several approaches have been proposed to speed up the second-dimension separation, such as the use of short columns, high flow rates, and a parallel column setup [68]. 

Figure 1 Schematic diagram of a multidimensional chromatography system. The two systems correspond to the methods employed, e.g., the GC or LC system. The separation channel may be an elution column or separation plane. Between the two systems is the process for transferring solute from the first to second dimensions. It may be a heart-cut process (GC-GC), a fraction collection step (LC-GC), a modulation process (GC X GC), or a plate positional change for planar TLC. Some systems will allow or require a detection step between the two systems, such as a monitor detector in MDGC. In the figure, step 1 refers to the sample application/injeclion step 2 to the interface or intermediate sample-processing device and step 3 to the elution of the separated sample into a detection system.

The SDS gel slab, when stained at the end of the second dimension run, is the final 2D map displaying all the polypeptide chains spread randomly in the pI/Mf plane. For high sensitivity, this gel slab is in general stained with silver stains, deemed to be the most sensitive today available. However, as the 2D map has to be further processed, with elution of the various spot and their analysis by a variety of (MS methods, the silver stains employed cannot use glutaraldehyde or other aldehydes that could irreversibly modify the proteins, impeding proper... 

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