Ionization, electrospray

Electrospray ionization (ESI) is a soft ionization technique that accomplishes the transfer of ions from solution to the gas phase. The technique is extremely useful for the analysis of large, non-volatile, chargeable molecules such as proteins and nucleic acid polymers. [1] Different from fast atom bombardment (FAB, Chap. 9) the solution is composed of a volatile solvent and the ionic analyte at very low concentration, typically M. In addition, the transfer of ions 

Nowadays, ESI is the leading member of the group of atmospheric pressure ionization (API) methods and the method of choice for liquid chromatography-mass spectrometry coupling (LC-MS, Chap. 12). [10-13] Currently, ESI and MALDI (Chap. 10) are the most commonly employed ionization methods and they opened doors to the widespread biological and biomedical application of mass spectrometry. [5,10,11,13-17] Moreover, ESI serves well for the analysis of ionic metal complexes [18,19] and other inorganic analytes. [20-22] 

Note Although the range up to m/z 3000 is normally employed for the detection of ions generated by ESI, ions of much higher m/z can be formed. [23,24] Even ions at m/z 85,000 have been observed. [25] 

Electrospray Ionization. Electrospray is an especially soft ionization method, capable of generating molecular ions (without fragmentation) from biological macromolecules present in aqueous solution. For this reason, it has received increased attention in terms of its applicability to protein and DNA analysis. 

Short pulses of laser radiation are used, typically at 337 nm, (nitrogen laser) but UV or infrared (IR) lasers can also be employed, depending on the matrix compound selected. Common matrix compounds are 2,5-dihydroxybenzoic acid, nicotinic acid, sinapinic acid, and a-cyanocarboxylic acid.6 

Electrospray ionization is a process by which ions of an analyte are formed in the liquid state and then quickly transferred to the gas phase of a mass spectrometer. This is particularly useful for high molecular weight substances and for analyzing the eluant from a liquid chromatograph. 

Since ionization occurs in the liquid state, the specific ion formed is highly dependent on not only the analyte but also the solvent and other species present in the liquid. It must be remembered that the solvent will be ionized as well as any analyte that is present. 

Electrospray ionization (ESI) is also an API technique that is applicable to a wide range of liquid-phase samples. In particular, it has made an enormous impact in 

Optimum operation of a normal ESI source is achieved at flow rates of 2 to 10 ixLmin. For stable operation at higher flow rates (0.2 to 1.0 mLmin e.g., effluents from narrow- and wide-bore analytical HPLC columns), some form of an additional source of energy, such as heat or a high-velocity annular flow of gas, is supplemented to disperse the liquid into fine droplets. 

Electrospray analysis can be performed in positive and negative ionization modes. The polarity of the ions to be analyzed is selected by the capillary voltage bias. A novel feature of the ESI mass spectrum is the formation of intact molecular ions of the analyte. Fragmentation, if desired, can be induced in the ion-transport region of the ESI source by increasing the sampling cone voltage. This process is known as in-source collision-induced dissociation (CID) or nozzle-skimmer (NS) dissociation. 

By solving those two simultaneous equations, one can derive Eqs. (2.31) and (2.32), which can be used to calculate the charge state of a particular peak and the molecular mass of a biopolymer, respectively. In favorable cases, the molecular mass of macromolecules can be calculated with a precision of within 0.005%. 

The coupling of an ESI source with a quadrupole mass analyzer is the most successful ESI-MS combination. Since the development of the orthogonal ion 

FAB and PD have been replaced by electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) in the analytical mass spectrometry laboratory, because both of these newer techniques have a wider mass range of analysis and have lower detection limits. ESI and MALDI have become invaluable ionization techniques for nonvolatile components. This is particularly true for a wide range of biological molecules including proteins, peptides, nucleic acids, etc. Samples can be analyzed by ESI using either direct injection or introduction through liquid chromatography. 

In the case of a protein of unknown molecular weight, the molecular weight can be determined by using equations (4) and (5) and knowing the m/z values of two adjacent multiply-chaiged ions. 

Matrix-assisted laser desorption ionization Atmospheric pressure electrospray ionization Atmospheric pressure chemical ionization Atmospheric pressure photoionization Inductively coupled plasma Direct analysis in real time Desorption electrospray ionization 

ESI is used at low flow rates ( 50 pi min ), even down to the nl min level. In the latter case, this is referred to as nanospray. The ionization rate increases with reduced flow, which is the reason for using narrow-bore columns with nanospray ionization. 

Sample preparation requires only dissolution of the sample to a suitable concentration in a mixture of water and organic solvent, commonly methanol, isopropanol, or acetonitrile. A trace of formic acid or acetic acid is often added to aid protonation of the analyte molecules in the positive ionization mode. In negative ionization mode ammonia solution or a volatile amine is added to aid deprotonation of the analyte molecules. 

Different types of instrumentation have been developed to introduce Hquid samples into the MS. Since Fenn has shown that molecular ions can be formed from liquids sprayed at atmospheric pressure in high electric fields, electrospray ionization (ESI) MS has gained increasing popularity for the analysis of biological samples [56]. In an electrospray inlet, the liquid sample is usually emitted as a spray from a capillary at a high potential compared to the mass analyzer into the electric field in front of the mass analyzer (Fig. 8). 

In ESI, the endergonic transfer of ions from solution into the gas phase is accomplished by desolvation. The electric field penetrates the analyte solution and separates positive and negative ions in an electrophoresis-like process. The positive charges accumulate on the surface of the droplets when the surface tension is exceeded, the characteristic Taylor cone is formed and the microspray occurs (Wihn and Mann, 1994). At this point, the droplets are close to their stability 

In comparison with MALDI, ESI represents an even softer ionization technique and causes no fragmentation of analyte ions. 

ESI takes place at atmospheric pressure. The ions produced are efficiently transmitted through a small orifice into the ion optics of the mass spectrometer. 

Mitulovic etal., 2003). An additional benefit of online HPLC/ESI-MS is that sample cleanup, analyte concentration and separation are accomplished in an automated process. However, polypeptides of high hydrophobicity are often retained on reversed-phase columns using a standard gradient and will therefore not be transferred into the MS device for mass analysis. Additionally, very hydrophobic peptides and proteins (e.g., membrane proteins) tend to precipitate in the small glass capillaries during the electrospray process, which leads to the breakup of the spray. 

Another problem concerns the suppression of ion formation from an analyte caused by another analyte or by the presence of another constituent (e.g., buffer) in the solution. Since the MS response significantly depends on solvent and sample composition, ion signal intensities of a given analyte do not necessarily correlate with its concentration in the sample. For the quantitative analysis of an analyte of interest by ESI-MS (the same is true for MALDI-MS), the use of an adequate internal standard is therefore mandatory. 

Considering the wide, positive impact that these techniques had with the grape and wine chemistry in past years (as will be described in detail in Part II) we focus now on the in-depth description of these new methods, in order to give the reader a useful background for critical evaluation of results obtained with their use. 

About one century later, Lord Kelvin studied the charging between water dripping from two different liquid nozzles, which leads to electrospray phenomena at the nozzles themselves (Smith, 2000). In the last century, a series of systematic studies on electrospray were carried out by Zeleny (Zeleny, 1917) and Taylor (Taylor, 1964a and b) allowing a detailed description of the phenomenon. In the middle of the century, electrospray started to be used on the industrial scale, in the application of paints and coatings to metal surfaces. The fine spray results in very smooth even films, with the paint actually attracted to 

This chapter aims to offer a concise description of chemical-physical phenomena that are at the base of the ESI. 

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A connnon feature of all mass spectrometers is the need to generate ions. Over the years a variety of ion sources have been developed. The physical chemistry and chemical physics communities have generally worked on gaseous and/or relatively volatile samples and thus have relied extensively on the two traditional ionization methods, electron ionization (El) and photoionization (PI). Other ionization sources, developed principally for analytical work, have recently started to be used in physical chemistry research. These include fast-atom bombardment (FAB), matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ES). 

FigureBl.7.2. Schematic representations of alternative ionization methods to El and PI (a) fast-atom bombardment in which a beam of keV atoms desorbs solute from a matrix (b) matrix-assisted laser desorption ionization and (c) electrospray ionization.

Fenn J B, Mann M, Meng C K, Wong S F and Whitehouse C M 1989 Electrospray ionization for mass spectrometry of large bio molecules Science 246 64... 

The advent of atmospheric-pressure ionization (API) provided a method of ionizing labile and nonvolatile substances so that they could be examined by mass spectrometry. API has become strongly linked to HPLC as a basis for ionizing the eluant on its way into the mass spectrometer, although it is also used as a stand-alone inlet for introduction of samples. API is important in thermospray, plasmaspray, and electrospray ionization (see Chapters 8 and 11). 

Aerosols can be produced as a spray of droplets by various means. A good example of a nebulizer is the common household hair spray, which produces fine droplets of a solution of hair lacquer by using a gas to blow the lacquer solution through a fine nozzle so that it emerges as a spray of small droplets. In use, the droplets strike the hair and settle, and the solvent evaporates to leave behind the nonvolatile lacquer. For mass spectrometry, a spray of a solution of analyte can be produced similarly or by a wide variety of other methods, many of which are discussed here. Chapters 8 ( Electrospray Ionization ) and 11 ( Thermospray and Plasmaspray Interfaces ) also contain details of droplet evaporation and formation of ions that are relevant to the discussion in this chapter. Aerosols are also produced by laser ablation for more information on this topic, see Chapters 17 and 18. 

Schematic diagram of an orthogonal Q/TOF instrument. In this example, an ion beam is produced by electrospray ionization. The solution can be an effluent from a liquid chromatography column or simply a solution of an analyte. The sampling cone and the skimmer help to separate analyte ions from solvent, The RF hexapoles cannot separate ions according to m/z values and are instead used to help confine the ions into a narrow beam. The quadrupole can be made to operate in two modes. In one (wide band-pass mode), all of the ion beam passes through. In the other (narrow band-pass mode), only ions selected according to m/z value are allowed through. In narrow band-pass mode, the gas pressure in the middle hexapole is increased so that ions selected in the quadrupole are caused to fragment following collisions with gas molecules. In both modes, the TOF analyzer is used to produce the final mass spectrum.

Electrospray Ionization (ES) and Atmospheric Pressure Chemical Ionization (APCI)... 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

Z-spray is a novel kind of electrospray that functions as a combined inlet and ion source. Chapter 8 ( Electrospray Ionization ) should be consulted for comparison. 

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. 

The ion guides are frequently used to transmit ions from an atmospheric-pressure inlet/source system (electrospray ionization, atmospheric-pressure chemical ionization) into the vacuum region of an m/z analyzer. 

Z-spray. Z refers to the approximate shape of the trajectory of particles formed by electrospray ionization... 

Cole, R.B., Electrospray Ionization Mass Spectrometry Fundamentals, Instrumentation and Applications, Wiley, Chichester, U.K., 1997. 

Smith, R.M., Gas and Liquid Chromatography in Analytical Chemistry, Wiley, Chichester, U.K., 1988. Smith, R.M. and Busch, K.L., Understanding Mass Spectra A Basic Approach, Wiley, Chichester, U.K., 1998. Snyder, A.R, Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry, Oxford University Press, Oxford, 1998. 

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. 

ESI (Section 12.4) Electrospray ionization, a mild method for ionizing a molecule so that fragmentation is minimized during mass spectrometry. 

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. 

Electrospray ionization mass spectrometry (ESI-MS) is an analytical method for mass determination of ionized molecules. It is a commonly used method for soft ionization of peptides and proteins in quadmpole, ion-trap, or time-of-flight mass spectrometers. The ionization is performed by application of a high voltage to a stream of liquid emitted from a capillaty. The highly charged droplets are shrunk and the resulting peptide or protein ions are sampled and separated by the mass spectrometer. 

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)... 

Electrospray Ionization Mass Spectrometry (ESI-MS) Elimination Half-life Elimination of Drugs EM-800... 

Two relatively new techniques, matrix assisted laser desorption ionization-lime of flight mass spectrometry (MALDI-TOF) and electrospray ionization (FS1), offer new possibilities for analysis of polymers with molecular weights in the tens of thousands. PS molecular weights as high as 1.5 million have been determined by MALDI-TOF. Recent reviews on the application of these techniques to synthetic polymers include those by Ilantoif54 and Nielen.555 The methods have been much used to provide evidence for initiation and termination mechanisms in various forms of living and controlled radical polymerization.550 Some examples of the application of MALDI-TOF and ESI in end group determination are provided in Table 3.12. The table is not intended to be a comprehensive survey. 

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