Separation Methods

This section begins with a brief overview of the aspects of separation methods that are of general concern in the interface to MS. Then each subsection covers traits that are specific to particular methods. As a sample introduction system, the purpose of the chromatography column is to transport single, separated components of the mixture efficiently and completely into the source of the mass spectrometer. The relevant questions are therefore simple. How much material is transported? How fast is it coming through? In what form are the sample components? How well is each component separated in time from other components of the mixture? To summarize, these issues are scale, flux, phase, and purity. Therefore, the descriptions that follow will not be comprehensive overviews of how the various chromatographic interfaces to the mass spectrometer were developed or how they are operated, but will concentrate on these four central issues.

Gas chromatography The characteristic of GC that makes the interface to MS (electron ionization (EI) and chemical ionization (CI)) especially straightforward is the fact that the sample molecules are already in the gas phase, and that they are transported into the source of the mass spectrometer by a carrier gas with substantially different physical characteristics from those of the sample molecules themselves. In packed column GC, the flow of helium carrier gas was so high under the conditions normally used for separation that an 'enrichment' device had to be used to remove most of the helium, and therefore increase the concentration of sample molecules in the gas flow entering the source. The higher diffus-ivity of helium gas formed the basis for most of these separators. As higher pumping speeds became available with improved vacuum technology, and as the use of capillary column GC cut the flux of helium into the source of the mass spectrometer by a factor of ten, it was found that the flow of helium gas (and entrained sample molecules) could be handled directly by the improved pumping in the source of the mass spectrometer, maintaining the pressure at 10~5-10~6 torr.

The helium was present in the source in excess, but the low mass of helium was an advantage in that most mass spectra were recorded only to a lower mass limit of about m/z 45. So the ions from helium were not recorded, and neither were ions from nitrogen, oxygen, argon, water, and carbon dioxide, all of which constituted residual molecules in the vacuum system. As there was no separate enrichment device, the efficiency of sample transport into the source was 100%, and the sample molecules were in the gas phase. As packed columns were replaced by capillary columns in GC, the influx of sample molecules changed from nanogram-microgram levels of sample per peak to picogram-nanogram levels of sample per peak. Higher amounts of sample overloaded the capillary column, and compromised separation, but these picogram-nanogram amounts of sample material were still within the detection range of the mass spectrometer. As the widths of the peaks in capillary columns were decreased relative to the widths generated by packed column chromatography, the flux in terms of amount of material(s) was still similar, even though the total amount of material was reduced. Of course, with reduced peak widths and higher separation resolution, the chances of any given peak being completely resolved were also increased. Issues that remain relevant are the need to scan the mass analyzer fast enough that representative mass spectra of a narrow peak can be recorded, and the increased demands upon a data system that is called upon to record thousands of mass spectra for hundreds of resolved sample mixture components.

Liquid chromatography Interfaces for liquid chromatography-mass spectrometry (LC-MS) must deal with transport issues that are additionally complicated by the fact that the sample is a solute in the liquid phase, the transfer into the gas phase produces large volumes of solvent vapour, and the samples are likely to be those that are relatively nonvolatile in the first place (otherwise GC would be used). Although the separation resolution may not be as high as in the best capillary GC, peaks are usually still only a few seconds wide and the amount of sample to be transported is also in the picogram-nanogram range, so the flux of material into the source is similar to that in GC-MS. The purity of the sample assessed relative to other mixture components is also similar, with separations designed to produce clean, well-resolved peaks. However, the solvent is often a mixture (as in reversed-phase gradient LC) and buffers and additives may be added to the solvent system. There is a background signal contribution from these components, and this contribution may change during the course of a chromatographic separation. As detailed in the appropriate sections, EI and CI MS act upon sample molecules in the gas phase. This is not the form in which the sample molecules are found in LC, and it is difficult to transfer nonvolatile sample molecules into the gas phase without thermal degradation. Several ionization processes have been developed that do not rely on the sample being in the gas phase. Thermospray ionization, continuous flow fast atom bombardment, and discharge ionization sources have been developed and optimized. However, the most widely used ionization method is electrospray ionization (ESI). In this technique, a combination of progressive desolvation and field-assisted ion extraction creates a series of multiply charged ions from the sample molecules, even if those sample molecules are 'nonvolatile' and thermally fragile. The flow and flux ranges accommodated by the ESI sources overlay the range of flow and flux in modern LC, endorsing the combination.

Capillary electrophoresis In capillary electrophoresis (CE) the movement of sample molecules (often charged, but neutral molecules move through the column as well) is induced by a combination of elec-trophoretic and electroosmotic flow. The small differences in mobility exhibited by molecules result in different retention times within the 0.5-1-m-long columns usually used. These columns have a small diameter (50 |im) capillary to efficiently dissipate the heat produced by the high potential difference (30 kV, for example) between the front and the back of the column. The flow profile in the column is not parabolic (as in pressure-driven systems) but is essentially flat, leading to very high resolution separation. There are few instances of overlapped peaks. The small column diameter limits the amount of material that can be loaded onto the column, with loadings 10-100 times lower than in LC. Peak widths are still a few seconds wide, so the instantaneous concentration of sample is lower than in GC or LC. A small volume of sample solution (picogram-nanogram levels of sample in 10 nL of solvent) is injected at the positive end of the capillary and the separated components are detected near the negative end of the capillary. Detection is accomplished with all of the same detectors as in LC, including mass spectrometers. However, the dynamic ranges of CE and MS are not as extensively overlapped as in GC or LC coupled with MS. Despite the general assumption that MS is the most sensitive detection method in use, laser-induced fluorescence detection provides lower limits of detection than MS, but not, of course, with the same specificity. As in LC, the sample molecules of interest are not amenable to evaporation, and so ESI is most often used with CE. In fact, the electrical requirements of the capillary electrophoretic separation often dovetail nicely with the requirements for the ESI source (vide infra). As noted, sample peak purity is usually high because of the extraordinary resolution achievable with this method, and detection may be simplified so the solvent (often methanol) background contribution is simple and often suppressed relative to the signal from the sample.

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