Sensitivity of FAB

The techniques described thus far cope well with samples up to 10 kDa. Molecular mass determinations on peptides can be used to identify modifications occurring after the protein has been assembled according to its DNA code (post-translation), to map a protein structure, or simply to confirm the composition of a peptide. For samples with molecular masses in excess of 10 kDa, the sensitivity of FAB is quite low, and such analyses are far from routine. Two new developments have extended the scope of mass spectrometry even further to the analysis of peptides and proteins of high mass. 

Table 6.16 summarises the main characteristics of FI-MS. FT uses high voltages and was once restricted to sensitive double-focusing magnetic sector instruments of relatively high cost. Field ionisation is considered to be the softest ionisation mode. The reproducibility of the non-standard techniques, such as FI-MS and FD-MS, is less well assessed than that of EI-MS. A noticeable drop in FI use occurred after the mid-1980s because of the advent of FAB and other desorption/ionisation methods. FI-MS is only used in a few laboratories worldwide. 

Table 7.83 lists the main characteristics of TLC-FAB-MS/LSIMS. A key difference between EI/CI and FAB/LSIMS/LD is the fact that sampling in FAB and LSIMS is from a specified location that corresponds to the impact footprint of the primary particle beam. The natural compatibility of FAB, LSIMS and LD with the direct mass-spectrometric analysis of TLC plates is readily apparent. Most mass-spectrometric measurements are destructive in nature, but FAB and LSIMS are surface-sensitive techniques in which the material actually consumed in the analysis is sputtered only from the top few microns of the sample spot. The underlying bulk is not affected, and can be used for further probing. The major limitation of TLC-FAB depends on the capability of the compounds to produce a good spectrum. 

The precursor model of FAB applies well to ionic analytes and samples that are easily converted to ionic species within the liquid matrix, e.g., by protonation or deprotonation or due to cationization. Those preformed ions would simply have to be desorbed into the gas phase (Fig. 9.6). The promoting effect of decreasing pH (added acid) on [M+H] ion yield of porphyrins and other analytes supports the precursor ion model. [55,56] The relative intensities of [Mh-H] ions in FAB spectra of aliphatic amine mixtures also do not depend on the partial pressure of the amines in the gas phase, but are sensitive on the acidity of the matrix. [57] Furthermore, incomplete desolvation of preformed ions nicely explains the observation of matrix (Ma) adducts such as [M+Ma+H] ions. The precursor model bears some similarities to ion evaporation in field desorption (Chap. 8.5.1). 

The principle of FAB, less frequently referred to as liquid secondary ionization mass spectrometry (LSIMS), is very similar to secondary-ion mass spectrometry (SIMS). However, FAB utilizes a liquid matrix, such as glycerol, in which a sample is dissolved. The matrix is used to enhance sensitivity and ion current stability. 

The application of FAB to peptide analysis has two general advantages it typically generates accurate molecular weight information due to the mass analyzers to which it is interfaced, and it offers rapid analysis. Given that FAB is not commonly applied to the analysis of proteins due to its limited mass range, routinely of the order of 5000 to 8000 Da, and relatively poor sensitivity (typically requiring >500 pmol), we will only briefly focus on the sample preparation and analysis procedures for peptides. 

Different methods are used to tackle these problems [10-13], Some of these coupling methods, such as moving-belt coupling or the particle beam (PB) interface, are based on the selective vaporization of the elution solvent before it enters the spectrometer source. Other methods such as direct liquid introduction (DLI) [14] or continuous flow FAB (CF-FAB) rely on reducing the flow of the liquid that is introduced into the interface in order to obtain a flow that can be directly pumped into the source. In order to achieve this it must be reduced to one-twentieth of the value calculated above, that is 5 pi min. These flows are obtained from HPLC capillary columns or from a flow split at the outlet of classical HPLC columns. Finally, a series of HPLC/MS coupling methods such as thermospray (TSP), electrospray (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) can tolerate flow rates of about 1 ml min 1 without requiring a flow split. Introducing the eluent entirely into the interface increases the detection sensitivity of these methods. ESI can accept flow rates from 10 nl min-1 levels to... 

Most of the betalain pigments described in the 1960s have not been characterized by mass or NMR spectra. With FAB MS now at hand, the molecular ions of underivatized betalains can be easily determined, and the sensitivity of high-field NMR spectrometers allows the complete structural assignment, even of small samples. For the structural determination of oligosaccharide moieties, modem two-dimensional (2D) NMR techniques are now the method of choice. 

Whilst the object of this chapter has been to show the extent and type of HPLC technique that is used today in today s environmental laboratories, there are a number of less routine techniques that may or may not have an impact on routine environmental monitoring. One of the most potentially important of these is the use of LC-MS. The problems associated with using LC-MS for trace analysis are twofold one is the usual LC-MS problem of interfacing the second is that of sensitivity of detector. The interfacing problem may well continue to have partial (compared with GC-MS interfacing) solutions such as FAB, and thermospray, etc. However, even given the advances arising from electrospray interfaces the answer may well be to move away from LC-MS to supercritical fluids and SFC-MS. 

Tris(pentafluorophenyl)borane, known as "FAB" (structure below), is the most common arylborane used as cocatalyst for single site catalysts. FAB is a strongly Lewis acidic, air-sensitive solid (T 126-131 °C) that is only slightly soluble in hydrocarbon solvents. The structure of FAB is given below. 

S. Suzuki, K. Kakehi, S. Honda, Comparison of the sensitivities of various derivatives of oligosaccharides in LC-MS with FAB and ESI interfaces, Anal. Chem., 68 (1996) 2073. 

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