Electrosprays components

Electrospray mass spectrometry (ESMS) — The electrospray component of ESMS [i] provides a very soft method of transferring ions from solution to the gas phase with minimal risk of decomposition and fragmentation of ions prior to their determination by mass spectrometry. Ideally suited for the determination of charged solution species which are frequently generated in electrochemical experiments [ii]. 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

It is worth noting that some of these methods are both an inlet system to the mass spectrometer and an ion source at the same time and are not used with conventional ion sources. Thus, with electrospray, the process of removing the liquid phase from the column eluant also produces ions of any emerging mixture components, and these are passed straight to the mass spectrometer analyzer no separate ion source is needed. The particle beam method is different in that the liquid phase is removed, and any residual mixture components are passed into a conventional ion source (often electron ionization). 

The electrospray source can be coupled directly to a liquid chromatographic (LC) column so that, as components of a mixture emerge from the column, they are passed through the source to give accurate mass data. As an example, a mixture of the peptides shown in Figure 40.8(a) was separated by LC and accurately mass-analyzed by ES. 

The suppression effects associated with electrospray ionization have been discussed earlier although if the compounds present are similar in behaviour these may be minimal. The intention, when using chromatography as an introduction device, is to allow individual components to enter the mass spectrometer for analysis. The separation capability of HPLC has been discussed previously and it is not unusual, particularly when complex mixtures are being studied, to encounter electrospray spectra from more than one component. 

This sometimes complicates the extraction of molecular weight data as it is not always immediately clear which ions in the spectrum originate from each component. This can be determined by the use of equation (4.6). An example of this is shown in Figure 4.18, which shows the electrospray spectrum from what is apparently a single chromatographic response, while Table 4.3(a) displays the results of applying equation (4.6) to the major ions found in that spectrum. As... 

Figure 5.6 Positive-ion electrospray spectrum obtained from the major component in the LC-MS analysis of a purified recombinant 62 kDa protein using a Cig microbore 50 X 1 mm column and a flow rate of 50 p.lmin . The starting buffer (buffer A ) was 0.1% TEA in water, while the gradient buffer (buffer B ) consisted of 0.1% TEA in acetonitrile-water (9 1 vol/vol). The running conditions consisted of 0% B for 5 min, followed by a linear gradient of 100% B for 55 min. Reprinted from J. Chromatogr., B, 685, McAtee, C. P., Zhang, Y., Yarbough, P. O., Fuerst, T. R., Stone, K. L., Samander, S. and Williams, K. R., Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry , 91-104, Copyright (1996), with permission from Elsevier Science.

Figure 5.28 LC-electrospray-MS total ion chromatogram of sulfated oligosaccharides from mucins purified from the porcine large intestine, where the annotations indicate the molecular ions observed from each component. Reprinted with permission from Thoms-son, K. A., Karlsson, H. and Hansson, G. C., Anal. Chem., 72, 4543-4549 (2000). Copyright (2000) American Chemical Society.

Hapten density, and also the common positions where haptens are bound, can also be estimated by cyanogen bromide or enzymatic cleavage of the protein and either MALDI-MS or separation of the components by reversed-phase ion-pair chromatography and electrospray or electrospray time-of-flight (TOF) analysis. 

A detailed description of sources used in atmospheric pressure ionization by electrospray or chemical ionization has been compiled.2 Atmospheric pressure has been used in a wide array of applications with electron impact, chemical ionization, pressure spray ionization (ionization when the electrode is below the threshold for corona discharge), electrospray ionization, and sonic spray ionization.3 Interferences potentially include overlap of ions of about the same mass-charge ratio, mobile-phase components, formation of adducts such as alkali metal ions, and suppression of ionization by substances more easily ionized than the analyte.4 A number of applications of mass spectroscopy are given in subsequent chapters. However, this section will serve as a brief synopsis, focusing on key techniques. 

The mass spectra of mixtures are often too complex to be interpreted unambiguously, thus favouring the separation of the components of mixtures before examination by mass spectrometry. Nevertheless, direct polymer/additive mixture analysis has been reported [22,23], which is greatly aided by tandem MS. Coupling of mass spectrometry and a flowing liquid stream involves vaporisation and solvent stripping before introduction of the solute into an ion source for gas-phase ionisation (Section 1.33.2). Widespread LC-MS interfaces are thermospray (TSP), continuous-flow fast atom bombardment (CF-FAB), electrospray (ESP), etc. Also, supercritical fluids have been linked to mass spectrometry (SFE-MS, SFC-MS). A mass spectrometer may have more than one inlet (total inlet systems). 

A limitation in the use of API sources results from the frequent application of mobile-phase composition programming in pSFC. Pinkston el al. [411] have compared electrospray and electron impact for open-tubular and packed-column SFC-MS. Direct on-line coupling of SFC to FAB/MS (as well as SFC-ELSD) is also very promising to detect components which give no response in a UV detector [412]. 

Electrospray has been successful for numerous azo dyes that are not ionic salts. Several anthraquinone dyes have been analysed by LC-ESI-MS [552]. Electrospray achieves the best sensitivity for compounds that are precharged in solution (e.g. ionic species or compounds that can be (de)protonated by pH adjustment). Consequently, LC-ESI-MS has focused on ionic dyes such as sulfonated azo dyes which have eluded analysis by particle-beam or thermospray LC-MS [594,617,618]. Techniques like LC-PB-MS and GC-MS, based on gas-phase ionisation, are not suitable for nonvolatile components such as sulfonated azo dyes. LC-TSP-MS on... 

Wu, Z. Jernstroem, S. Hughey, C. A. Rodgers, R. R Marshall, A. G. Resolution of 10,000 compositionally distinct components in polar coal extracts by negative-ion electrospray ionization Fourier transform ion cyclotron resonance. Mass Spectrom. Ener. Fuels 2003,17, 946-953. 

FIGURE 13.4 Total ion chromatograms from the ID LC/MS analysis of a yeast ribosomal protein fraction separated using 0.1% TFA (Panel a) and 0.1% formic acid (Panel b) as mobile phase modifiers. TFA produced narrower, more concentrated, peaks for mass analysis that did not overcome the significant electrospray ionization suppression associated with using this modifier for LC/MS studies, resulting in an overall reduction in component intensities. 

Although El MS is an efficient way to provide structural information on several molecular constituents of various lipid substances it only provides partial information and it is particularly not suitable for the study of the low volatile components. High molecular weight and nonvolatile compounds are particularly difficult to analyse in this way and it may therefore be interesting to explore the possibilities of other ionisation modes such as electrospray for an accurate structural study of high molecular constituents such as monoester and diester species of beeswax (Gamier et al., 2002) and TAGs of animal fats... 

F.L. Liu, C.Y.W. Ang, T.M. Heinze, J.D. Rankin, R.D. Beger, J.P. Freeman and J.O. Lay Jr, Evaluation of major active components in St. John s Wort dietary supplements by high performance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation, J. Chromatogr. A, 888, 85 92 (2000). 

Mass spectrometry requires that the material being studied be converted into a vapor. Great strides have been taken in recent years to address this problem, especially in enticing large, thermally fragile (bio)molecules into the vapor state. Matrix assisted laser ionization-desorption (MALDI) and electrospray ionization (ESI) are two current forefront methods that accomplish this task. Even components of bacteria and intact viruses are being examined with these approaches. John B. Fenn and Koichi Tanaka shared in the award of a Nobel Prize in 2002 for their respective contributions to development of electrospray ionization and soft laser desorption. 

Zeng H., Deng Y., and Wu J., 2003a. Fast analysis using monolithic columns coupled with high-flow online extraction and electrospray mass spectrometric detection for the direct and simultaneous quantitation of multiple components in plasma. J Chromatogr B 788 331. 

Figure 6.3. Real-life example of a tandem MS experiment in an electrospray ion trap instrument. Top panel a complex peptide mixture. Middle panel ion at 1318.9 m/z was isolated from other sample components. Note the lack of any other peaks and a very low background. Bottom panel fragmentation spectrum of the selected parent ion (1318.9 m/z), note the different scale of the m/z axis. All peaks seen in this mass spectrum are product ions that were formed due to the controlled fragmentation of the parent ion. The main peak at 1300.8 m/z corresponds to the loss of water molecule, a lower intensity parent ion at 1318.9 m/z is also seen.

One problem with GC-MS, in addition to being labor intensive and having particularly long analysis times, was that higher molecular weight (molar mass) components or compounds with preformed cations (such as cholines or carnitine) are easily hydrolyzed and cannot be analyzed effectively using GC-MS. With the advent of new ionization techniques for LC effluents (see Section 4.1.2), such as electrospray ionization (see Section 2.1.15), more volatile and larger molecular mass compounds could be analyzed,... 

In some cases, more than one mass spectrometer may be used to carry out the analysis of a sample. Thus, the components from the first MS analysis may be passed to another MS for further analysis. In this type of analysis, a fragment from the first MS analysis is further broken down and the resulting new fragments are analyzed. This allows for analysis of complex samples. Because there are several types of sample ionization (e.g., El, Cl, and electrospray) and different types of mass spectrometers (e.g., quadrupole, time of flight [TOF], and magnetic sector) there are several different ways an MS-MS analysis can be carried out. 

The qualitative determination of anionic surfactants in environmental samples such as water extracts by flow injection analysis coupled with MS (FIA-MS) applying a screening approach in the negative ionisation mode sometimes may be very effective. Using atmospheric pressure chemical ionisation (APCI) and electrospray ionisation (ESI), coupled with FIA or LC in combination with MS, anionic surfactants are either predominantly or sometimes exclusively ionised in the negative mode. Therefore, overview spectra obtained by FIA—MS(—) often are very clear and free from disturbing matrix components that are ionisable only in the positive mode. However, the advantage of clear... 

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