Electrospray Ionization ESI

ESI (and MALDI Section 2.2.4) has revolutionized mass spectrometry since the 1990s, and it is now the dominant ionization method for polar compounds, from small molecules to biopolymers, opening up vast new areas of research that were hitherto inaccessible to mass spectrometry. ESI is the softest ionization technique the energies involved are barely above those necessary to generate ions. 

ESI has overcome a major problem that frustrated mass spectrometrists for many years how to volatilize polar compounds without also causing decomposition. Samples used to be introduced into the ion source by evaporation from a solid probe. Heating the probe often failed to produce useful spectra for polar compounds, resulting instead in brown residues of decomposed material on the probe tip. The ESI 

The evaporation progressively forms smaller and smaller droplets with the eventual release of ions into the vapor phase. The desolvation causes the density of the electric field of the droplets to increase to the point where disintegration occurs. There are two models for the disintegration, Coulombic and jet fissions. When the repulsive Coulombic forces between the like charges on the surface of the droplets exceed the forces attributable to surface tension (the Rayleigh instability Umit), the 

Two concentric steel tubes carry the liquid and nebulizing gas. 

As the droplets shrink the charge density increases to the point at which the droplets are no longer stable and ions are released from the liquid matrix into the vapor phase. 

Electrospray ionization is an ionization process by which analyte molecules or ions present originally in solution are transferred to the gas phase through either solvent or ion evaporation. Although the experimental setup is relatively simple, the ion-formation mechanisms are still under systematic studies [24-26]. A scheme for an electrospray source is shown in Fig. 9, while a simplified ion-formation mechanism is indicated in Fig. 10. 

In ESI the analyte previously dissolved in a solution is introduced into the ESI source via a needle either by direct infusion or as an eluent flow from an HPLC chromatograph. The most commonly used solvents include water, methanol, and acetonitrile. Their combinations and specific use depend on the solubility of the analyte. For direct infusion, a typical flow rate is in the range of 1-5 xl/min. More 

Spray (analyte-solvent droplets) at atmospheric pressure 

The more or less uniformly sized droplets enter a heated transfer capillary in which the solvent molecules are being further evaporated. As a consequence, the sixrface charge density increases until the droplet size reaches the Rayleigh limit at which the surface tension cannot compensate for the Coulombic repulsion associated with the surface charge. At this point, the droplet explodes ( Coulombic explosion. Fig. 10) and smaller size droplets are formed. This process can continue until virtually no solvent molecules are present, but only 

Atypical ESI spectrum for a protein (lysosyme) is shown in Fig. 11. The multiply charged molecular ion pattern is clearly recognizable. Note that although this ESI spectrum corresponds to only one protein, there is a mixture of ions in the spectrum each of which has a different mass-to-charge ratio (reminder In mass spectrometry, the m z ratio is measured). To calculate the molecular mass (or MW) of the protein, the charge states of the individual ions should also be determined. Thus, we have two unknowns, the MW and the charge state (n). To determine 

Electrospray ionization (ESI) is one of the two newer ionization techniques (the other being MALDI) that has revolutionized the application of mass spectrometry to biochemistry and molecular biology, and may fairly be said to have revolutionized these disciphnes also. This is the result of the ability of ESI-MS to provide molecular mass information of hitherto unthinkable accuracy and precision for fragile biopolymers, particularly proteins, and to provide amino acid sequence information for specific peptides present at trace levels in complex mixtures characteristic of biological extracts. The discipline of proteomics would not exist without the development of ESI and MALDI. However, this book is concerned with quantitation of small molecules present at trace levels in complex matrices, rather than the essentially qualitative data typically acquired in proteomics experiments, although application of the approach to quantitation of proteins and peptides is discussed in Section 11.6. Nonetheless, development of methods to produce and characterize gaseous ions from macromolecules was very important in the development of ESI-MS, and this history will be briefly described here. Excellent reviews of this history as it pertains to macromolecule characterization (Penn 1990 Smith 1991, 1992 Fernandez de la Mora 1992) are available, while 

At its most basic level, ESI involves production of gaseous ions at atmospheric pressure from analytes in hquid solution by nebulizing the solution in such a way as to produce small droplets that carry a net electrical charge. It is convenient to consider the overall process as two steps, the first leading to production of the charged droplets and the second to the formation of gaseous ions from these droplets. 

There is a long history of investigations of production of electrically charged droplets by spraying or shattering liquids in impact with surfaces, including early work on electrically charged spray from waterfalls. This work culminated in pubhcation of a comprehensive book on the subject (Loeb 1958) and some of it is summarized in one of the recent reviews of ESI (Hamdan 1991). 

This excellent work (Hines 1966), in which fundamental principles were applied to improve the understanding and performance of a strictly practical device, 

It is interesting that this paper (Gieniec 1984) mentions that attempts to characterize these ions using a time 

In electrospray ionization (ESI) mass spectrometry, ions are foimed from a fine spray of solution tmder an applied electrical potential it is a soft technique used for both neutral molecules or ionic salts. Singly and multiply charged ions are observed. 

The soft technique of electrospray ionization (ESI) mass spectrometry has widespread applications in chemical and biochemical analysis of high-molecular mass compounds (M, 200 000). In contrast to the methods described above, both singly and multiply charged ions are observed, making the ESI technique valuable for analysis of ionic compounds. 

Decreasmg droplet size dmoogh mass analyser 

As in FAB and MALDl-TOF mass spectrometries, neutral molecules are converted to positive ions by combination with H or Na ions. Aggregation may also occur to produce ions of the type [2M + Na], and combination with the solvent molecules gives ions such as [M + MeCN + H]+. 

The ligand, H2L, reacts with copper(ll) acetate to give a neutral complex. In the ESI mass spectrum of a CH2CI2/ MeOH solution of the product, the base peak is at mjz 494.1 and exhibits an isotope pattern characteristic of one Cu atom. Assign the peak and, assuming no fragmentation occurs, suggest a formula for the complex. 

For a more detailed description of the ionization process inherent in electrospray, please see Chapter 9, which discusses atmospheric pressure ionization (API), The reader also should compare electrospray with thermospray (see Chapter 11). 

One of the first successful techniques for selectively removing solvent from a solution without losing the dissolved solute was to add the solution dropwise to a moving continuous belt. The drops of solution on the belt were heated sufficiently to evaporate the solvent, and the residual solute on the belt was carried into a normal El (electron ionization) or Cl (chemical ionization) ion source, where it was heated more strongly so that it in turn volatilized and could be ionized. However, the moving-belt system had some mechanical problems and could be temperamental. The more recent, less-mechanical inlets such as electrospray have displaced it. The electrospray inlet should be compared with the atmospheric-pressure chemical ionization (APCI) inlet, which is described in Chapter 9. 

In addition, a pulled capillary tip was inserted and glued to the end of a microchannel to be used as a disposable nanoelectrospray emitter. Membrane 

FIGURE 7.30 Schematic of the chip-CE coupled with a sheath flow ESI interface for MS analysis [812], Reprinted with permission from the American Chemical Society. 

To reduce the dead volume at the electrospray tip, it was inserted (then glued) into a specially drilled flat-bottom hole, as opposed to a conical-bottom hole [132]. A similar method was used for analysis of various peptide standards and tryptic digests of lectins from Dolichlos biflorus and Pisum sativum [812]. 

A single microchannel has been used for sequential MS analysis of multiple samples. In this case, no sample cross-contamination has been reported. For instance, no sample crossover was observed for 10 iM cytochrome c versus 10 iM ubiquitin [943], and tryptic digests of [3-lac, CA, and BSA [776]. In another report, sequential infusion of tryptic digests of CA (290 nM) and BSA (130 nM) into ESI-ITMS was achieved using EOF. No cross-contamination resulted when a central flow of buffer confined the other samples in the reservoir and channel by precise voltage control [801]. 

On-line protein digestion has been achieved by trypsin adsorbed in a porous PVDF (poly [vinylidene fluoride]) membrane (0.45-pm pore) coupled in a PDMS chip. In this way, on-chip protein digestion and subsequent MS analysis have been carried out for horse heart cytochrome c [817]. 

A different, but related ionization method is coldspray ionization (CSI) [14]. The ionization occurs in a similar manner to ESI, with the exception that the source housing and drying gas are cooled in order to stabilize weakly-bound ions. The effect is often incomplete desolvation and distributions of ions with different num- 

FIGURE 3.9 Schematic representation of electrospray ionization (ESI) showing both field evaporation and coulombic explosion. (From Gross, J. H., Mass Spectrometry A Textbook, Springer, Berlin, 2004. Reprinted by permission.) 

Copyright 2013 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. 

ESI-MS is not limited to the study of large biomolecules, however. Many small molecules with moleeular weight in the 100-1500 range can be studied by ESI-MS. Compounds that are too nonvolatile to be introduced by direct probe methods or are too polar or thermally labile to be intro-dueed by GC-MS methods are ideal for study by LC-MS using ESI techniques. 

Desorption electrospray ionization (DESI) combines the soft ionization of the electrospray technique with desorption of the sample ions from a surface. Unlike MALDI, however, no matrix is needed. The DESI technique uses electrosprayed aqueous aerosols to ionize and desorb analyte ions. 

There is no doubt that the most important ionization method used for studying CyDs and other complex and heavy biomolecules is electrospray. 

Typical positive and negative ion ESI spectra of commercial j]-CyD are presented in Fig. 10.2.1. In the positive mode, the [M + Na] ion (m/z = 1157.4) is the main one no [M + H] ions are observed in this experiment. However, there are reports in the literature [22] that [M + H] ions can be generated using a MeOH H20 AcOH (48.5 48.5 3) solvent system. In the negative ion mode the most intense peak corresponds to the [M — H] ion (m/z = 1133.3). There is also an m/z =1169.2 peak visible, which corresponds to the [M + Cl] ion. Other peaks correspond to clusters with different anions. For example, the isotope pattern around the m/z = 1182.8 peak corresponds to the doubly charged 

ESI mass spectrometry is used now as a routine tool for identification of modified CyDs. Eor example, Pean et al. [37] determined the molecular mass of some peptidyl-CyDs such as 1 using ESI in both positive and negative ion modes. 

Only the negative ion mode was found suitable for the analysis of strongly acidic sulfated CyDs (e.g. 2) [38] and sulfobutyl ether derivatives of fi-CyD (e.g. 3) [39]. In the latter case, a mixture of differently substituted fi-CyD derivatives was separated on HPLC directly coupled to an ESI-equipped mass spectrometer. The HPLC/MS technique can also be used for quantitative determination of CyDs in complex matrices. Hammes et al. [40] developed a method for quantitative determination of a-CyD in human plasma using fi-CyD as an internal standard. Linear calibration curve was obtained over the concentration range 5-1000 ng mL .  

FIGURE 15.2 Common protein ionization methods used for MS-based proteomics. Two common ionization technologies are currently available for protein analysis. Top ESI volatilizes and ionizes peptides and proteins in solution. Bottom MALDI uses analytes that are co-crystallized in a matrix composed of organic acid on a solid support. A pulse of ultraviolet laser evaporates the matrix and analyte into gas phase, resulting in generation of single charge ions. 

FIGURE 8.6 Common matrices for SIMS and FAB mass spectrometry. 

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Electrospray ionization (ESI) produces a series of multicharged ions that can be transformed into an accurate molecular mass for proteins with masses of tens of thousands. 

Most biochemical analyses by MS use either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALD1), typically linked to a time-of-flight (TOF) mass analyzer. Both ESI and MALDl are "soft" ionization methods that produce charged molecules with little fragmentation, even with biological samples of very high molecular weight. 

DNA sequencing and. 1113 Electrospray ionization (ESI) mass spectrometry, 417-418 Electrostatic potential map, 37 acetaldehyde, 688 acetamide, 791,922 acetate ion. 43. 53, 56, 757 acetic acid. 53. 55 acetic acid dimer, 755 acetic anhydride, 791 acetone, 55, 56. 78 acetone anion, 56 acetyl azide, 830 acetyl chloride, 791 acetylene. 262 acetylide anion, 271 acid anhydride, 791 acid chloride, 791 acyl cation, 558 adenine, 1104 alanine, 1017 alanine zwitterion, 1017 alcohol. 75 alkene, 74, 147 alkyl halide, 75 alkyne. 74... 

An abundant molecular ion may indicate that an aromatic compound or highly unsaturated ring compound is present. If no molecular ion is observed and one cannot be deduced, the use of chemical ionization (ci), negative chemical ionization (nci), fast atom bombardment (FAB), or electrospray ionization (ESI) should provide a molecular ion. 

Matrix-assisted laser desorption mass spectrometry (MALDI-MS) is, after electrospray ionization (ESI), the second most commonly used method for ionization of biomolecules in mass spectrometry. Samples are mixed with a UV-absorbing matrix substance and are air-dried on a metal target. Ionization and desorption of intact molecular ions are performed using a UV laser pulse. 

Peptide mass fingeiprinting (PMF) is a mass spectrometry based method for protein identification. The protein is cleaved by an enzyme with high specificity (trypsin, Lys-C, Asp-N, etc.) or chemical (CNBr). The peptide mixture generated is analyzed by matrix-assisted laser desorp-tion/ionization (MALDI) or electrospray ionization (ESI)... 

A method has been reported for the quantification of five fungicides (shown in Figure 5.39) used to control post-harvest decay in citrus fruits to ensure that unacceptable levels of these are not present in fruit entering the food chain [26]. A survey of the literature showed that previously [27] APCl and electrospray ionization (ESI) had been compared for the analysis of ten pesticides, including two of the five of interest, i.e. carbendazim and thiabendazole, and since it was found that APCl was more sensitive for some of these and had direct flow rate compatibility with the HPLC system being used, APCl was chosen as the basis for method development. 

The most recent progress in MS analysis of chlorophylls has been obtained with the development of atmospheric ionization methods such as atmospheric pressure chemical ionization (APCl) and electrospray ionization (ESI). These techniques have demonstrated much more sensitivity than thermospray ionization, detecting chloro-... 

The chemical compositions of the isolated Au SR clusters were investigated by mass spectrometry [15,16,18, 22,32-35]. TEM was used to confirm that the species detected by the mass spectrometer represents the clusters in the sample. Figure 3a is a schematic representation of the top view of the mass spectrometer, which consists of five stages of differentially pumped vacuum chambers. The apparatus accommodates two t5 pes of ion sources, electrospray ionization (ESI) and laser-desorption ionization (EDI), and a time-of-flight (TOE) mass spectrometer with a reflectron. Details of the apparatus and the measurement protocols are described below. 

LC/MS/MS. LC/MS/MS is used for separation and quantitation of the metabolites. Using multiple reaction monitoring (MRM) in the negative ion electrospray ionization (ESI) mode, LC/MS/MS gives superior specificity and sensitivity to conventional liquid chromatography/mass spectrometry (LC/MS) techniques. The improved specificity eliminates interferences typically found in LC/MS or liquid chro-matography/ultraviolet (LC/UV) analyses. Data acquisition is accomplished with a data system that provides complete instmment control of the mass spectrometer. 

Reversed-phase Cig chromatography column. Keystone Scientific Betasil, 100 x 2.0-mm i.d., 5-pm particle size, 100 A, Part No. 105-701-2-CPF TSQ 7000 LC/MS/MS system with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) interface and gradient high-performance liquid chromatography (HPLC) unit, or equivalent Vacuum manifold for use with SPE cartridges (Varian Vac Elut 10 or equivalent)... 

As with GC/MS, LC/MS offers the possibility of unequivocal confirmation of analyte identity and accurate quantiation. Similarly, both quadrupole and ion-trap instruments are commercially available. However, the responses of different analytes are extremely dependent on the type of interface used to remove the mobile phase and to introduce the target analytes into the mass spectrometer. For pesticide residue analyses, the most popular interfaces are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Both negative and positive ionization can be used as applicable to produce characteristically abundant ions. 

Electrospray ionization (ESI) and APCI are the two popular API techniques that will be discussed here. The applications to the analysis of pesticides that will be discussed include imidazolinone herbicides, phenoxy acid herbicides, and A-methyl carbamate insecticides. Matrix effects with respect to quantitation also will be discussed. Eor the... 

As in HPLC, the coupling of MS detection with CE has provided an excellent opportunity for more selective analysis, but the much reduced flow rates, small injection volumes, limitations in the types of buffers used [since electrospray ionization (ESI) is used in capillary electrophoresis/mass spectrometry (CE/MS)], and need to... 

Confirmatory system Agilent Series 1100 liquid chromatograph Eclipse XDB Cig HPLC columu, 150 x 4.0-mm i.d., 3.5- im particle size MicroMass Quattro II triple-quadrupole mass spectrometer using an electrospray ionization (ESI) interface... 

It is therefore not surprising that the interest in PyMS as a typing tool diminished at the turn of the twenty-first century and hence why taxonomists have turned to MS-based methods that use soft ionization methods such as electrospray ionization (ESI-MS) and matrix-assisted laser desorption ionization (MALDI MS). These methods generate information-rich spectra of metabolites and proteins, and because the molecular ion is seen, the potential for biomarker discovery is being realized. The analyses of ESI-MS and MALDI-MS data will still need chemometric methods, and it is hoped that researchers in these areas can look back and learn from the many PyMS studies where machine learning was absolutely necessary to turn the complex pyrolysis MS data into knowledge of bacterial identities. 

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. 

Detection of the effluent in a 2D system is carried out at the end of the second dimension s column. UVand LIF are the most widely used and the simplest methods of detection for CE separations because they are performed on-column. MS detection, unlike UV and LIF, is carried out on the effluent as it exits the CE column. The direct coupling of CE with mass spectrometry has shown great potential in proteomic research (Janini et al., 2004). The method of choice for detection of peptides is MS-electrospray ionization (ESI). However, ESI requires a special interface between the CE column and the mass spectrometer that has proven not to be a simple matter (Issaq et al., 2004). 

Electrospray ionization (ESI) is ideally suited as a detection technique for the online interfacing of liquid-phase separations (HPLC and CE) to MS, because it facilitates the transfer of analytes from the liquid phase of the HPLC or CE column to the gas phase of the MS. Also, it allows the detection of high molecular weight species, such as peptides. Three interface designs have been developed in the past 18 years for coupling CE with MS. The first CE-MS interface, coaxial sheath flow, was introduced by Smith and his group in 1987 (Olivares et al., 1987) and was improved upon in later work (Smith et al., 1988). Coaxial sheath flow is formed using two concentric metal capillaries, whereby the CE terminus and the makeup flow line are inserted into the... 

Currently, LC-MS is widely used for the analysis of polar compounds, such as medicinal metabolites and bioactive peptides, since the interface has been improved and several new ionization methods have been developed. The sensitivity and reproducibility are sufficient for a daily quantitative analysis. The usefulness of the LC-MS has been demonstrated for studies on Type II pheromones using a time-of-flight MS with electrospray ionization (ESI) [180]. Each epoxydiene derived from the (Z3,Z6,Z9)-triene shows three ion series of [M+NHJ+, [M+H]+, and [M-OH]+ with high resolution and good sensitivity, indicating its molecular formula. In addition to these, characteristic fragment... 

For confirmatory assay, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is becoming more frequently used in the analysis of OTC owing to its high sensitivity and ability. Electrospray ionization (ESI) [55-57] and atmospheric pressure chemical ionization (APCI) [41] methods combined with tandem mass spectrometry are favored because of their higher sensitivity and better reproducibility. Hamscher et al. [58] developed a method for the determination of persistent TC residues in soil fertilized with manure by HPLC tandem mass spectrometry, MS-MS, and confirmation by MS-MS-MS. Zhu et al. [59] developed an LC-tandem mass spectrometry for the analysis of common tetracyclines in water. The detection limit for oxytetracycline was 0.21 pg/L. Lykkeberg et al. [60] used LC-MS/MS for determination of oxytetracycline and its impurities EOTC, TC, ETC, ADOTC, oc-AOTC, and /i-AOTC. 

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