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 was rewarded with the attribution of the Nobel Prize in Chemistry to John Bennett Fenn in 2002.

Mass spectrometry using ESI is commonly called electrospray ionization mass spectrometry (ESI-MS) or electrospray mass spectrometry (ES-MS).

How it works
In electrospray ionization, a liquid is pushed through a very small, charged and usually metal, capillary. This liquid contains the substance to be studied, the analyte, dissolved in a large amount of solvent, which is usually much more volatile than the analyte. Volatile acids, bases or buffers are often added to this solution too. The analyte exists as an ion in solution either in its anion or cation form. Because like charges repel, the liquid pushes itself out of the capillary and forms an aerosol, a mist of small droplets about 10 &mu;m across. The aerosol is at least partially produced by a process involving the formation of a Taylor cone and a jet from the tip of this cone. An uncharged carrier gas such as nitrogen is sometimes used to help nebulize the liquid and to help evaporate the neutral solvent in the droplets. As the solvent evaporates, the analyte molecules are forced closer together, repel each other and break up the droplets. This process is called Coulombic fission because it is driven by repulsive Coulombic forces between charged molecules. The process repeats until the analyte is free of solvent and is a lone ion. There is still debate about the exact mechanism of the process, particularly the last stage, when lone ions form. Lone ions move to the mass analyzer of a mass spectrometer.

In electrospray processes, the ions observed 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 $$[M+2H]^{2+}\,$$ are often observed. For large macromolecules, there can be many charge states, occurring with different frequencies; the charge can be as great as $$[M+25H]^{25+}\,$$, for example. All these are even-electron ion species: electrons (alone) are not added or removed, unlike in some other ionizations. The formation of ions in electrospray is somewhat homologous to acid-base reactions. Redox reactions do occur and a circuit with measurable current flow exists, but atomic and molecular ions are the primary carriers of charge in the solution and gas phases.

History
Early researchers:
 * John Strutt, 3rd Baron Rayleigh
 * Zeleny
 * Dole
 * Gall
 * Geoffrey Ingram Taylor

Ionization mechanism
There are two major competing theories about the final production of lone ions, the charged residue model (CRM) and the ion evaporation model (IEM).

Electrospray droplets start off highly charged, and as they shrink through evaporation the Coulomb repulsion forces approach the force of surface tension that holds droplet together. The droplet then becomes unstable and disintegrates into several droplets of smaller radius.
 * The Charged Residue Model suggests that electrospray droplets undergo evaporation and disintegration cycles, with each initial droplet leading to a multitude of much smaller "daughter" droplets. Each final "daughter" droplet contains on average one or less molecule of analyte. When the last solvent molecules evaporate from such droplet the analyte molecule is left with the charges that the droplet carried.


 * The Ion Evaporation (Desorption) Model suggests that as the droplet reaches a certain radius the field strength at the surface of the droplet becomes great enough to push or desorb ions directly out of the droplet. Characteristically, the fission event corresponds to an almost negligible loss in droplet mass, but a significant drop in charge.

It has been suggested that both models probably occur for different analytes/solvents and in the limit of both models they have a tendency to converge. That is to say that the distinction between a droplet containing an analyte molecule and an analyte molecule surrounded by a layer of solvent eventually disappears and coulombic fission looks a lot like ion evaporation. The real question is scale and timing and the techniques to definitively determine this are not yet available.

The use of the word "ionization" in "electrospray ionization" is criticized by some because many of the ions observed are thought to be preformed in solution before the electrospray process or created by simple changes in concentrations as the aerosolized droplets shrink. It is argued that electrospray is simply an interface for transferring ions from the solution phase to the gas phase.

Variants
There are many variations on the basic electrospray technique, that generally offer better sensitivity than it. Two important ones are microspray (µ-spray) and nanospray. The primary difference is in the reduced flow rate of the analyte containing liquid, µLiters/minute and nLiters/minute respectively; this causes many other differences, such as the reduced internal diameter of the tubing or lack of nebulization gas.

Liquid chromatography–mass spectrometry (LC-MS)

 *  see also the main article on liquid chromatography-mass spectrometry

Electrospray ionization is the primary ion source used in liquid chromatography-mass spectrometry because it is a liquid-gas interface capable of coupling liquid chomatography with mass spectrometry.

Static nanospray mass spectrometry
In protein mass spectrometry besides LC-MS often static (or offline) nanospray MS is used. Thereby the analyte solution, usual peptides after in-gel digestion of proteins from electrophoresis gels, is desalted and transferred to an ESI capillary. This capillary consists of a thin, metallised glas tube, which is stretched at one end to form a very fine, closed tip. The tip is broken off at the cone of the ESI source and positioned perpendicular to the front of the electrospray inlet. After application of the source voltage to the capillary the electrospray is formed and the analysis of the peptides by MS or MS/MS starts.

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.