Electron ionization Electron ionization (EI, formerly known as electron impact) is an ionization method in which energetic electrons interact

with gas phase atoms or molecules to produce ions. This technique is widely used in mass spectrometry, particularly for volatile organic molecules. The following gas phase reaction describes the electron ionization process:

where M is the analyte molecule being ionized, e - is the electron and M+ is the resulting ion. In an EI ion source, electrons are produced through thermionic emission by heating a wire filament that has electric current running through it. The electrons are accelerated to 70 eV in the region between the filament and the entrance to the ion source block. The accelerated electrons are then concentrated into a beam by being attracted to the trap electrode. A sample vapour containing the neutral molecules is introduced to the ion source in a perpendicular direction to the e- beam. Upon interaction with the e- beam, the analyte molecules ionize to radical cations which are then directed towards the mass analyzer by a repeller electrode. Due to the high energy electrons and the initial thermal distribution of the neutral molecules, the ionization process frequently causes cleavage reactions that give rise to fragment ions, which can convey structural information about the analyte. The ionization efficiency and production of fragment ions depends strongly on the chemistry of the analyte and the energy of the electrons. At low energies (around 20 eV), the interactions between the electrons and the analyte molecules do not transfer enough energy to cause ionization. At around 70 eV, the de Broglie wavelength of the electrons matches the length of typical bonds in organic molecules (about 0.14 nm) and energy transfer to organic analyte molecules is maximized, leading to the strongest possible ionization and fragmentation. Under these conditions, about 1 in 1000 analyte molecules in the source are ionized. At higher energies, the de Broglie wavelength of the electrons becomes smaller than the bond lengths in typical analytes; the molecules then become "transparent" to the electrons and ionization efficiency decreases.

Chemical ionization Chemical ionization (CI) is an ionization technique used in mass spectrometry. Chemical ionization is a lower energy process than electron ionization. The lower energy yields less fragmentation, and usually a simpler spectrum. A typical CI spectra has an easily identifiable molecular ion. Mechanism In a CI experiment, ions are produced through the collision of the analyte with ions of a reagent gas that are present in the ion source. Some common reagent gases include: methane, ammonia, and isobutane. Inside the ion source, the reagent gas is present in large excess compared to the analyte. Electrons entering the source will preferentially ionize the reagent gas. The resultant collisions with other reagent gas molecules will create an ionization plasma. Positive and negative ions of the analyte are formed by reactions with this plasma. Primary Ion Formation:

Secondary Reagent Ions:

Product Ion Formation: (protonation) (H abstraction) (adduct formation) (charge exchange) Self chemical ionization occurs when the reagent ion is an ionized form of the analyte.[5] Variations Negative chemical ionization (NCI) Chemical ionization for gas phase analysis is either positive or negative. Almost all neutral analytes can form positive ions through the reactions described above. In order to see a response by negative chemical ionization, the analyte must be capable of producing a negative ion (stabilize a negative charge) for example by electron capture ionization. Because not all analytes can do this, using NCI provides a certain degree of selectivity that is not available with other, more universal ionization

techniques (EI, PCI). NCI can be used for the analysis of compounds containing acidic groups or electronegative elements (especially halogens).[4] Because of the high electronegativity of halogen atoms, NCI is a common choice for their analysis. This includes many groups of compounds, such as PCBs[6], pesticides[7], and fire retardants.[8] Most of these compounds are environmental contaminants, thus much of the NCI analysis that takes place is done under the auspices of environmental analysis. Atmospheric pressure chemical ionization (APCI) Chemical ionization in an atmospheric pressure electric discharge is called atmospheric pressure chemical ionization. The analyte is aa gas or liquid spray and ionization is accomplished using an atmospheric pressure corona discharge. This ionization method is often coupled with high performance liquid chromatography where the mobile phase containing eluting analyte sprayed with high flow rates of nitrogen and the aerosol spray is subjected to a corona discharge to create ions. Atmospheric pressure chemical ionization

Atmospheric pressure chemical ionization (APCI) is an ionization method used in mass spectrometry. It is a form of chemical ionization which takes place at atmospheric pressure.[1] How it works APCI allows for the high flow rates typical of standard bore HPLC to be used directly, often without diverting the larger fraction of volume to waste. Typically the mobile phase containing eluting analyte is heated to relatively high temperatures (above 400 degrees Celsius), sprayed with high flow rates of nitrogen and the entire aerosol cloud is subjected to a corona discharge that creates ions. Often APCI can be performed in a modified ESI source. This is basically a gas phase ionisation, unlike ESI which is a liquid phase ionisation process. Also, we can use nonpolar solvent for solution making instead of polar solvent for supporting ions in solution as gaseous state conversion of solvent before reaching to corona discharge pin is carried out here, which well supports the ions formed. Typically, APCI is a less "soft" ionization technique than ESI, i.e. it generates more fragment ions relative to the parent on

Desorption atmospheric pressure photoionization Desorption atmospheric pressure photoionization (DAPPI) is an atmosphericpressure ionization technique for mass spectrometry. DAPPI enables direct analysis of solid samples without pretreatment and analysis of samples deposited on surfaces by means of a jet of hot solvent vapour and vacuum ultraviolet light. The hot jet thermally desorbs the sample from a surface and the vaporized sample is ionized by the vacuum ultraviolet light and consequently sampled into a mass spectrometer. Ionization mechanisms The ionization mechanism depends on the analyte and solvent used and for example the following analyte (M) ions may be formed: [M + H]+, [M - H]-, M+, M-. Applications DAPPI has the potential to analyze both polar (e.g. verapamil) and nonpolar (e.g. anthracene) compounds. Performance of DAPPI has also been demonstrated on direct analysis of illicit drugs

Electrospray ionization Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized. The development of electrospray ionization for the analysis of biological macromolecules[1] was rewarded with the attribution of the Nobel Prize in Chemistry to John Bennett Fenn in 2002.[2] Mass spectrometry using ESI is called electrospray ionization mass spectrometry (ESI-MS) or, less commonly, electrospray mass spectrometry (ES-MS). Ionization mechanism The liquid containing the analyte(s) of interest is dispersed by electrospray into a fine aerosol. Because the ion formation involves extensive solvent evaporation, the typical solvents for electrospray ionization are prepared by mixing water with volatile organic compounds (e.g. methanol, acetonitrile). To decrease the initial droplet size, compounds that increase the conductivity (e.g. acetic acid) are customary added to the solution. Large-flow electrosprays can benefit from additional nebulization by an inert gas such as nitrogen. The aerosol is sampled into the first vacuum stage of a mass spectrometer through a capillary, which can be heated to aid further solvent evaporation from the charged droplets. The solvent evaporates from a charged droplet until it becomes

unstable upon reaching its Rayleigh limit. At this point, the droplet deforms and emits charged jets in a process known as Rayleigh fission. During the fission, the droplet loses a small percentage of its mass along with a relatively large percentage of its charge.[6] There are two major theories that explain the final production of gas-phase ions:

The Ion Evaporation Model (IEM)[7] suggests that as the droplet reaches a certain radius the field strength at the surface of the droplet becomes large enough to assist the field desorption of solvated ions. The Charged Residue Model (CRM)[8] suggests that electrospray droplets undergo evaporation and fission cycles, eventually leading progeny droplets that contain on average one analyte ion or less. The gas-phase ions form after the remaining solvent molecules evaporate, leaving the analyte with the charges that the droplet carried.

While there is no definite scientific proof, a large body of indirect evidence suggests that small ions are liberated into the gas phase through the ion evaporation mechanism, while larger ions form by charged residue mechanism. The ions observed by mass spectrometry may be quasimolecular ions created by the addition of a proton (a hydrogen ion) and denoted [M + H]+, or of another cation such as sodium ion, [M + Na]+, or the removal of a proton, [M H]. Multiply-charged ions such as [M + nH]n+ are often observed. For large macromolecules, there can be many charge states, resulting in a characteristic charge state envelope. All these are evenelectron ion species: electrons (alone) are not added or removed, unlike in some other ionization sources. The analytes are sometimes involved in electrochemical processes, leading to shifts of the corresponding peaks in the mass spectrum. Variants The electrosprays operated a low flow rates generate much smaller initial droplets, which ensure improved ionization efficiency. In 1994, two research groups coined the name micro-electrospray (microspray) for electrosprays working at low flow rates. Emmett and Caprioli demonstrated improved performance for HPLC-MS analyses when the electrospray was operated at 300-800 nL/min.[9] Wilm and Mann demonstrated that a capillary flow of ~ 25 nL/min can sustain an electrospray at the tip of emitters fabricated by pulling glass capillaries to a few micrometers.[10] The latter was renamed nano-electrospray (nanospray) in 1996.[11] Currently the name nanospray is also in use for electrosprays fed by pumps at low flow rates, not only for self-fed electrosprays. Unfortunately, there are no clear flow rate boundaries between electrosprays, microsprays, and nanosprays. Applications Liquid chromatographymass spectrometry (LC-MS) Electrospray ionization is the ion source of choice to couple liquid chromatography with mass spectrometry. The analysis can be performed online, by feeding the liquid eluting from the LC column directly to an electrospray, or offline, by collecting fractions to be later analyzed in a classical nanoelectrospray-mass spectrometry setup.

Noncovalent gas phase interactions Electrospray ionization is also ideal in studying noncovalent gas phase interactions. The electrospray process is capable of transferring liquid-phase noncovalent complexes into the gas phase without disrupting the noncovalent interaction. This means that a cluster of two molecules can be studied in the gas phase by other mass spectrometry techniques. An interesting example of this is studying the interactions between enzymes and drugs which are inhibitors of the enzyme. Because inhibitors generally work by noncovalently binding to its target enzyme with reasonable affinity the noncovalent complex can be studied in this way. Competition studies have been done in this way to screen for potential new drug candidates. Matrix-assisted laser desorption/ionization Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. It is most similar in character to electrospray ionization both in relative softness and the ions produced (although it causes many fewer multiply charged ions). The ionization is triggered by a laser beam (normally a nitrogen laser). A matrix is used to protect the biomolecule from being destroyed by direct laser beam and to facilitate vaporization and ionization. Matrix The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), -cyano-4hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB). A solution of one of these molecules is made, often in a mixture of highly purified water and an organic solvent (normally acetonitrile (ACN) or ethanol). Trifluoroacetic acid (TFA) may also be added. A good example of a matrix-solution would be 20 mg/mL sinapinic acid in ACN:water:TFA (50:50:0.1). The identity of suitable matrix compounds is determined to some extent by trial and error, but they are based on some specific molecular design considerations: They are of a fairly low molecular weight (to allow facile vaporization), but are large enough (with a low enough vapor pressure) not to evaporate during sample preparation or while standing in the spectrometer. They are acidic, therefore act as a proton source to encourage ionization of the analyte.

They have a strong optical absorption in the UV, so that they rapidly and efficiently absorb the laser irradiation. They are functionalized with polar groups, allowing their use in aqueous solutions.

The matrix solution is mixed with the analyte (e.g. protein-sample). The organic solvent allows hydrophobic molecules to dissolve into the solution, while the water allows for water-soluble (hydrophilic) molecules to do the same. This solution is spotted onto a MALDI plate (usually a metal plate designed for this purpose). The solvents vaporize, leaving only the recrystallized matrix, but now with analyte molecules spread throughout the crystals. The matrix and the analyte are said to be co-crystallized in a MALDI spot. Laser The laser is fired at the crystals in the MALDI spot. The matrix absorbs the laser energy and it is thought that primarily the matrix is ionized by this event. The matrix is then thought to transfer part of its charge to the analyte molecules (e.g. protein), thus ionizing them while still protecting them from the disruptive energy of the laser. Ions observed after this process consist of a neutral molecule [M] and an added or removed ion. Together, they form a quasimolecular ion, for example [M+H]+ in the case of an added proton, [M+Na] + in the case of an added sodium ion, or [M-H]- in the case of a removed proton. MALDI is capable of creating singly-charged ions, but multiply charged ions ([M+nH]n+) can also be created, as a function of the matrix, the laser intensity and/or the voltage used. Note that these are all even-electron species. Ion signals of radical cations can be observed eg. in case of matrix molecules and other stable molecules.

Atmospheric pressure matrix-assisted laser desorption/ionization Atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) is an ionization technique (ion source) that in contrast to vacuum MALDI operates at normal atmospheric environment. The main difference between vacuum MALDI and APMALDI is the pressure in which the ions are created. In vacuum MALDI, ions are typically produced at 10 mTorr or less while in AP-MALDI ions are formed in atmospheric pressure. Disadvantage of the AP MALDI source is the limited sensitivity observed and the limited mass range. AP-MALDI is used in mass spectrometry (MS) in a variety of applications ranging from proteomics to drug discovery fields. Popular topics that are addressed by AP-MALDI

mass spectrometry include: proteomics, DNA/RNA/PNA, lipids, oligosaccharides, phosphopeptides, bacteria, small molecules and synthetic polymers, similar applications as available also for vacuum MALDI instruments. The AP-MALDI ion source is easily coupled to an ion trap mass spectrometer [12] or any other MS system equipped with ESI (electrospray ionization) or nanoESI source. Mass spectrometer The type of a mass spectrometer most widely used with MALDI is the TOF (time-offlight mass spectrometer), mainly due to its large mass range. The TOF measurement procedure is also ideally suited to the MALDI ionization process since the pulsed laser takes individual 'shots' rather than working in continuous operation. MALDI-TOF instruments are typically equipped with an "ion mirror", deflecting ions with an electric field, thereby doubling the ion flight path and increasing the resolution. Today, commercial reflectron TOF instruments reach a resolving power m/m of well above 20'000 FWHM (full-width half-maximum, m defined as the peak width at 50% of peak height). History The term matrix-assisted laser desorption ionization (MALDI) was coined in 1985 by Franz Hillenkamp, Michael Karas and their colleagues.[13] These researchers found that the amino acid alanine could be ionized more easily if it was mixed with the amino acid tryptophan and irradiated with a pulsed 266 nm laser. The tryptophan was absorbing the laser energy and helping to ionize the non-absorbing alanine. Peptides up to the 2843 Da peptide melittin could be ionized when mixed with this kind of matrix. [14] The breakthrough for large molecule laser desorption ionization came in 1987 when Koichi Tanaka of Shimadzu Corp. and his co-workers used what they called the ultra fine metal plus liquid matrix method that combined 30 nm cobalt particles in glycerol with a 337 nm nitrogen laser for ionization.[15] Using this laser and matrix combination, Tanaka was able to ionize biomolecules as large as the 34,472 Da protein carboxypeptidase-A. Tanaka received one-quarter of the 2002 Nobel Prize in Chemistry for demonstrating that, with the proper combination of laser wavelength and matrix, a protein can be ionized.[16] Karas and Hillenkamp were subsequently able to ionize the 67 kDa protein albumin using a nicotinic acid matrix and a 266 nm laser. [17] Further improvements were realized through the use of a 355 nm laser and the cinnamic acid derivatives ferulic acid, caffeic acid and sinapinic acid as the matrix.[18] The availability of small and relatively inexpensive nitrogen lasers operating at 337 nm wavelength and the first commercial instruments introduced in the early 1990s brought MALDI to an increasing number of researchers.[19] Today, mostly organic matrices are used for MALDI mass spectrometry. Use In Biochemistry

In proteomics, MALDI is used for the identification of proteins isolated through gel electrophoresis: SDS-PAGE, size exclusion chromatography, and two-dimensional gel electrophoresis. One method used is peptide mass fingerprinting by MALDI-MS, or with post ionisation decay or collision-induced dissociation (further use see mass spectrometry). In Organic Chemistry Some synthetic macromolecules, such as catenanes and rotaxanes, dendrimers and hyperbranched polymers, and other assemblies, have molecular weights extending into the thousands or tens of thousands, where most ionization techniques have difficulty producing molecular ions. MALDI is a simple and rapid analytical method that can allow chemists to analyze the results of such syntheses and verify their results. In polymer chemistry In polymer chemistry MALDI can be used to determine the molar mass distribution.[20] Polymers with polydispersity greater than 1.2 are difficult to characterize with MALDI due to the signal intensity discrimination against higher mass oligomers.[ Reproducibility and performance The sample preparation for MALDI is important for the result. Inorganic salts which are also part of protein extracts interfere with the ionization process. The salts are removed by solid phase extraction or washing the final target spots with water. Both methods can also remove other substances from the sample. The matrix protein mixture is not homogenous because the polarity difference leads to a separation of the two substances during crystallization. The spot diameter of the target is much larger than that of the laser, which makes it necessary to do several laser shots at different places of the target, to get the statistical average of the substance concentration within the target spot. The matrix composition, the addition of trifluoroacetic acid and formic acid, delay between laser pulses, delay time of the acceleration power, laser wavelength, energy density of the laser and the impact angle of the laser on the target are among others the critical values for the quality and reproducibility of the method.

Desorption electrospray ionization Desorption electrospray ionization (DESI) is an ambient ionization technique that can be used in mass spectrometry for chemical analysis. It is an atmospheric pressure ion source that ionizes gases, liquids and solids in open air under ambient conditions. It was developed in 2004 by Professor Graham Cooks et al. from Purdue University and is now available commercially by Prosolia Inc. [1] DESI is a similar ionization technique to Direct Analysis in Real Time (DART) in its versatility, applications and analysis time. It is different in that some sample preparation is required; liquid samples have to be

deposited on a desired surface and allowed to dry and gases have to be adsorbed unto a material. Principle of operation

Schematic diagram of the DESI ion source DESI is a combination of electrospray (ESI) and desorption (DI) ionization methods. Ionization takes place by directing an electrically charged mist to the sample surface that is a few millimeters away. [2] The electrospray mist is attracted to the surface by applying a voltage on the sample holder. After ionization, the ions travel through air into the atmospheric pressure interface which is connected to the mass spectrometer. DESI is a technique that allows for ambient ionization of a trace sample at atmospheric pressure, with little sample preparation. Ionization mechanism In DESI there are two kinds of ionization mechanism, one that applies to low molecular weight molecules and another to high molecular weight molecules. [2] High molecular weight molecules, such as proteins and peptides show electrospray like spectra where multiply charged ions are observed. This suggests desorption of the analyte, where multiple charges in the droplet can easily be transferred to the analyte. The charged droplet hits the sample, spreads over a diameter greater than its original diameter, dissolves the protein and rebounces. The droplets travel to the mass spectrometer inlet and are further desolvated. The solvent typically used for the electrospray is a combination of methanol and water. For the low molecular weight molecules, ionization occurs by charge transfer: an electron or a proton. There are three possibilities for the charge transfer. First, charge transfer between a solvent ion and an analyte on the surface. Second, charge transfer between a gas phase ion and analyte on the surface; in this case the solvent ion is evaporated before reaching the sample suface. This is achieved when the spray to surface distance is large. Third, charge transfer between a gas phase ion and a gas phase analyte molecule. This occurs when a sample has a high vapour pressure.

The ionization mechanism of low molecular weight molecules in DESI is similar to DARTs ionization mechansim, in that there is a charge transfer that occurs in the gas phase. Ionization efficiency The ionization efficiency of DESI is complex and depends on several parameters such as, surface effects, electrospray parameters, chemical parameters and geometric parameters. [2] Surface effects include chemical composition, temperature and electric potential applied. Electrospray parameters include electrospray voltage, gas and liquid flow rates. Chemical parameters refers to the sprayed solvent composition, e.g. addition of NaCl. Geometric parameters are , , d1 and d2 (see figure on the right). Furthermore, and d1 affect the ionization efficiency, while and d2 affect the collection efficiency. Results of a test performed on a variety of molecules to determine optimal and d1 values show that there are two sets of molecules: high molecular weight (proteins, peptides, oligosaccharide etc) and low molecular weight (dizazo dye, stereoids, caffeine, nitroaromatics etc). The optimal conditions for the high molecular weight group are high incident angles (70-90) and short d1 distances (1-3 mm). The optimal conditions for the low molecular weight group are the opposite, low incident angles (35-50) and long d1 distances (7-10 mm). These test results indicate that each group of molecules has a different ionization mechanism; described in detail in the Principle of operation section. The sprayer tip and the surface holder are both attached to a 3D moving stage which allow to select specific values for the four geometric parameters: , , d1 and d2. Sonic spray ionization Sonic spray ionization (SSI) is method for creating ions from a liquid solution, for example, a mixture of methanol and water. A pneumatic nebulizer is used to turn the solution into a supersonic spray of small droplets. Ions are formed when the solvent evaporates and the statistically unbalanced charge distribution on the droplets leads to a net charge. Complete desolvation results in ions that can be detected using mass spectrometry.[1] Applications Sonic spray ionization has been coupled with high performance liquid chromatography for the analysis of drugs. Oligonucleotides have been studied with this method. SSI has

been used in a manner similar to desorption electrospray ionization for ambient ionization and has been couplet with thin layer chromatography in this manner.