Continuous flow FAB

In standard FAB, the surface of the matrix solution is depleted of analyte and suffers from radiational damage during elongated measurements. Refreshment of the surface proceeds by diffusion (limited by the viscosity of the matrix) or evaporation. Continuous-flow fast atom bombardment (CF-FAB) continuously refreshes the surface exposed to the atom beam. [107,108] The same effect is obtained in slightly different way by the frit-fast atom bombardment (frit-FAB) technique. [109,110] In addition, both CF-FAB and frit-FAB can be used for online-coupling of liquid chromatography (LC, Chap. 12) [111] or capillary electrophoresis (CE) to a FAB ion source. [112] 

Example The significant increase in sensitivity and the improvements of sig-nal-to-background ratio (Chap. 5.2.4) in CF-FAB spectra are demonstrated by comparison of the standard FAB and CF-FAB mass spectra of peptides. [108,116] In case of 10 pmol of the peptide des(gln)-substance P (Fig. 9.16) solely CF-FAB reveals the low-intensity signals due to some loss of side chain groups and several a-type fragments. 

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Another development arising from FAB has been its transformation from a static to a dynamic technique, with a continuous flow of a solution traveling from a reservoir through a capillary to the probe tip. Samples are injected either directly or through a liquid chromatography (LC) column. The technique is known as dynamic or continuous flow FAB/LSIMS and provides a convenient direct LC/MS coupling for the on-line analysis of mixtures (Figure 40.2). 

Dynamic/continuous-flow FAB allows a continuous stream of liquid into the FAB source hence it constitutes an LC/MS interface for analyses of peptide mixtures. 

El may be used with the moving-belt and particle-beam interfaces. Cl with the moving-belt, particle-beam and direct-liquid-introduction interfaces, and FAB with the continuous-flow FAB interface. A brief description of these ionization methods will be provided here but for further details the book by Ashcroft [8] is recommended. 

When FAB is utilized for FC-MS, often known as continuous-flow FAB, a matrix material is added to the HPFC eluent, either pre- or post-column, and this mixture continuously flows to the tip of a probe inserted into the source of the mass spectrometer where it is bombarded by the atom beam (Figure 3.3). 

Two forms of interface have been commercially developed [7] which allow analytes in a flowing liquid stream - it has to be pointed out, not necessarily from an HPLC system (see below) - to be ionized by using FAB. These are essentially identical except for that part where the HPLC eluate is bombarded with the heavy-atom/ion beam. Both of these interfaces consist of a probe in the centre of which is a capillary which takes the flowing HPLC eluate. In the continuous-flow FAB interface (Figure 4.3), the column eluate emerges from the end of the capillary and spreads over the probe tip, while in the frit-FAB interface the capillary terminates in a porous frit onto which the atom/ion beam is directed. 

Figure 4.3 Schematic of a continuous-flow FAB LC-MS interface. From applications literature published by Kratos Analytical Ltd, Manchester, UK, and reproduced by permission of Mass Spectrometry International Ltd.

Property Direct Liquid Moving Introduction Belt Thermo MAGIC -spray APCI Continuous -Flow FAB... 

Suter, M.J.F. Caprioli, R.M. An Integral Probe for Capillary Zone Electrophore-sis/Continuous-Flow FAB-MS. J. Am. Soc. Mass Spectrom. 1992, 3, 198-206. 

Caprioli, R.M. Continuous-Flow FAB-MS. Anal. Chem. 1990, 62,477A-485A. 

Continuous-Flow FAB Mass Spectrometry Caprioli, R.M., editor John Wiley Sons Chichester, 1990. 

Many interfaces have been developed to meet these demanding challenges. Some of these coupling methods, such as the moving belt or the particle beam interface, are based on the concomitant elimination of the solvent before it enters the mass spectrometer. Other methods such as direct liquid introduction (DLI) or continuous flow FAB rely on splitting the flow of the liquid that is introduced into the interface in order to obtain a flow that can be directly infused into the ionization source. However, these types of interfaces can only handle a fraction of the liquid flow from the LC. 

Continuous-flow FAB was reported earlier but is of no practical importance nowadays. Only few reports have been published dealing with (offline) coupling of CE to MALDI-MS. Recently, a new ionization approach, coordination ion spray (CIS) MS, has been presented (11) in it, charged coordination compounds are formed on-line. 

Mass Spectrometry of Carotenoids NOTE For LC/MS or flow injection using continuous-flow FAB, the mass spectrometer must be equipped with a continuous-flow ionization source. 

A reversed-phase HPLC column (typically Cl 8 or C30) is required for HPLC separations. Because the flow rate into the continuous-flow FAB-MS or LSIMS source must be <10 pl/min, either a capillary column must be used or else the flow must be split postcolumn. For narrow-bore HPLC columns operated at 200 pl/min, the split ratio would be 30 1. Isocratic or gradient separations may be used. A syringe pump is usually necessary for capillary columns, but standard HPLC pumps are sufficient for applications using narrow-bore columns. 

FAB ionization has been used in combination with LC/MS in a technique called continuous-flow FAB LC/MS (Schmitz et al., 1992 van Breemen et al., 1993). Although any standard HPLC solvent can be used, including methyl-ferf-butyl ether and methanol, the mobile phase should not contain nonvolatile additives such as phosphate or Tris buffers. Volatile buffers such as ammonium acetate are compatible at low concentrations (i.e., <10 mM). Continuous-flow FAB has also been used in combination with MS/MS (van Breemen et al., 1993). The main limitationsof continuous-flow FAB compared to other LC/MS techniques for carotenoids, such as ESI and APCI, are the low flow rates and the high maintenance requirements. During use, the 3-nitrobenzyl alcohol matrix polymerizes on the continuous-flow probe tip causing loss of sample signal. As a result, the continuous-flow probe must be removed and cleaned approximately every 3 hr. 

Figure F2.4.2 Positive ion fast atom bombardment (FAB-MS) mass spectrum of phytofluene isolated from blueberries. The base peak of mlz (mass-to-charge ratio) 542 corresponds to the molecular ion. Characteristic of FAB-MS, background signals are observed at every mlz value. The mass spectrum was obtained during continuous-flow FAB-MS LC/MS using a magnetic sector mass spectrometer. Although the 16-c/s isomer of phytofluene is shown, the FAB mass spectra of the all- trans and other cis isomers are indistinguishable.

S. J. 1992. Applications of fast atom bombardment mass spectrometry (FAB-MS) and continuous-flow FAB-MS to carotenoid analysis. Methods Enzymol. 213 322-336. 

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