Electrospray ionization is a technique used in mass spectrometry to produce ions using
an electrospray in which a high voltage is applied to a liquid to create an aerosol.
It is especially useful in producing ions from macromolecules because it overcomes the
propensity of these molecules to fragment when ionized. ESI is different than other
atmospheric pressure ionization processes since it may produce multiply charged ions,
effectively extending the mass range of the analyser to accommodate the kDa-MDa orders
of magnitude observed in proteins and their associated polypeptide fragments.
Mass spectrometry using ESI is called electrospray ionization mass spectrometry or, less commonly,
electrospray mass spectrometry. ESI is a so-called 'soft ionization' technique, since there is
very little fragmentation. This can be advantageous in the sense that the molecular ion is always
observed, however very little structural information can be gained from the simple mass spectrum
obtained. This disadvantage can be overcome by coupling ESI with tandem mass spectrometry.
Another important advantage of ESI is that solution-phase information can be retained
into the gas-phase. The electrospray ionization technique was
first reported by Masamichi Yamashita and John Fenn in 1984. 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. One of the original instruments
used by Dr. Fenn is on display at the Chemical Heritage Foundation in Philadelphia, Pennsylvania.
History
In 1882, Lord Rayleigh theoretically estimated the maximum amount of charge a liquid droplet
could carry before throwing out fine jets of liquid. This is now known as the Rayleigh
limit. In 1914, John Zeleny published work on the
behaviour of fluid droplets at the end of glass capillaries and presented evidence for
different electrospray modes. Wilson and Taylor and Nolan investigated electrospray in the
1920s and Macky in 1931. The electrospray cone was described by Sir Geoffrey Ingram
Taylor. The first use of electrospray ionization with
mass spectrometry was reported by Malcolm Dole in 1968. John Bennett Fenn was awarded
the 2002 Nobel Prize in Chemistry for the development of electrospray ionization mass
spectrometry in the late 1980s. 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.
To decrease the initial droplet size, compounds that increase the conductivity are customarily
added to the solution. These species also act to provide a source of protons to facilitate
the ionization process. Large-flow electrosprays can benefit from additional nebulization by
an inert gas such as nitrogen or carbon dioxide. The aerosol is sampled into the first vacuum
stage of a mass spectrometer through a capillary carrying a potential difference of approximately
3000V, 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 as the electrostatic repulsion of
like charges, in an ever-decreasing droplet size, becomes more powerful than the surface
tension holding the droplet together. At this point the droplet undergoes Coulomb fission,
whereby the original droplet 'explodes' creating many smaller, more stable droplets. The new
droplets undergo desolvation and subsequently further Coulomb fissions. During the fission,
the droplet loses a small percentage of its mass along with a relatively large percentage
of its charge. There are two major theories that explain
the final production of gas-phase ions: the ion evaporation model and the charge residue
model. The IEM 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 CRM 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. A large body of evidence, which is consider
either direct or indirect that small ions are liberated into the gas phase through the
ion evaporation mechanism, while larger ions form by charged residue mechanism
A third model invoking combined charged residue-field emission has been proposed. Another model
called chain ejection model is proposed for disordered polymers.
The ions observed by mass spectrometry may be quasimolecular ions created by the addition
of a hydrogen cation and denoted [M + H]+, or of another cation such as sodium ion, [M + Na]+,
or the removal of a hydrogen nucleus, [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 even-electron ion species: electrons
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. This effect is demonstrated in the direct ionization of noble metals such
as copper, silver and gold using electrospray. Variants
The electrosprays operated at low flow rates generate much smaller initial droplets, which
ensure improved ionization efficiency. In 1993 Gale and Richard D. Smith reported significant
sensitivity increases could be achieved using lower flow rates, and down to 200 nL/min.
In 1994, two research groups coined the name micro-electrospray 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. 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. The latter was renamed nano-electrospray in 1996.
Currently the name nanospray is also in use for electrosprays fed by pumps at low flow
rates, not only for self-fed electrosprays. Although there may not be a well-defined flow
rate range for electrospray, microspray, and nano-electrospray, studied "changes in analyte
partition during droplet fission prior to ion release" . In this paper, they compare
results obtained by three other groups. and then measure the signal intensity ratio [Ba2+
+ Ba+]/[BaBr+] at different flow rates. Cold spray ionization is a form of electrospray
in which the solution containing the sample is forced through a small cold capillary into
an electric field to create a fine mist of cold charged droplets. Applications of this
method include the analysis of fragile molecules and guest-host interactions that cannot be
studied using regular electrospray ionization. Electrospray ionization has also been achieved
at pressures as low as 25 torr and termed subambient pressure ionization with nanoelectrospray
based upon a two-stage ion funnel interface developed by Richard D. Smith and coworkers.
The SPIN implementation provided increased sensitivity due to the use of ion funnels
that helped confine and transfer ions to the lower pressure region of the mass spectrometer.
Operation at low pressure was particularly effective for low flow rates where the smaller
electrospray droplet size allowed effective desolvation and ion formation to be achieved.
As a result later the researchers were later able to demonstrate achieving in excess of
50% overall ionization utilization efficiency for transfer of ions from the liquid phase,
into the gas phase as ions, and through the dual ion funnel interface to the mass spectrometer.
Ambient ionization
In ambient ionization, the formation of ions occurs outside the mass spectrometer without
sample preparation. Electrospray is used for ion formation is a number of ambient ion sources.
Desorption electrospray ionization is an ambient ionization technique in which a solvent electrospray
is directed at a sample. The electrospray is attracted to the surface by applying a
voltage to the sample. Sample compounds are extracted into the solvent which is again
aerosolized as highly charged droplets that evaporate to form highly charged ions. After
ionization, the ions enter the atmospheric pressure interface of the mass spectrometer.
DESI allows for ambient ionization of samples at atmospheric pressure, with little sample
preparation. Extractive electrospray ionization is an spray-type,
ambient ionization method that uses two merged sprays, one of which is generated by electrospray.
Laser-based electrospray-based ambient ionization is a two-step process in which a pulsed laser
is used to desorb or ablate material from a sample and the plume of material interacts
with an electrospray to create ions. For ambient ionization, the sample material is deposited
on a target near the electrospray. The laser desorbs or ablates material from the sample
which is ejected from the surface and into the electrospray which produces highly charged
ions. Examples are electrospray laser desorption ionization, matrix-assisted laser desorption
electrospray ionization, and laser ablation electrospray ionization
Applications Electrospray is used to study protein folding.
Liquid chromatography–mass spectrometry
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. Among the numerous operating
parameters in ESI-MS, the electrospray voltage has been identified as an important parameter
to consider in ESI LC/MS gradient elution. The effect of various solvent compositions
or spraying conditions on Electrospray-LCMS spectra and/or nanoESI-MS spectra. have been
studied. Capillary electrophoresis-mass spectrometry
Capillary electrophoresis-mass spectrometry was enabled by an ESI interface that was developed
and patented by Richard D. Smith and coworkers at Pacific Northwest National Laboratory,
and shown to have broad utility for the analysis of very small biological and chemical compound
mixtures, and even extending to a single biological cell.
Noncovalent gas phase interactions Electrospray ionization is also utilized in
studying noncovalent gas phase interactions. The electrospray process is thought to be
capable of transferring liquid-phase noncovalent complexes into the gas phase without disrupting
the noncovalent interaction. Problems such as non specific interactions have been identified
when studying ligand substrate complexes by ESI-MS or nanoESI-MS. An interesting example
of this is studying the interactions between enzymes and drugs which are inhibitors of
the enzyme. Competition studies between STAT6 and inhibitors have used ESI as a way to screen
for potential new drug candidates. See also
Laser ablation electrospray ionization Sonic spray ionization
References
Further reading
External links Electrospray Ionization Primer National High
Magnetic Field Laboratory Electrospray Ionization Mass Spectrometry
at the US National Library of Medicine Medical Subject Headings