Why are atoms vaporized in a mass spectrometer

Ionisation - An electron beam knocks electrons off the vaporised particles, producing positive ions. Acceleration - The positive ions are attracted towards negatively charged plates. Deflection - The stream of ions passes through into the magnetic field which deflects them into a curved path. Detection - The magnetic field is varied by the controller and ions with different masses are directed into the detector.

The operating principles are no longer a requirement for core and AHL. The curved path travelled by each ion depends on its mass actually the mass:charge ratio, but as the charge is always the same and equal to the charge on an electron, but positive, then we can talk about the mass alone.

Heavier ions have more Objects with large momentum values need more force for deviation. The magnetic field strength of the deflector coils is varied and allows detection of all ions according to their mass to charge ratio.

The ions arriving at the detector constitute a flow of positive charge and can be recorded electronically. In this way, the relative atomic mass of each 'peak' is recorded on the mass spectrum read-out. Drug companies and researchers are using the technique for drug discovery, for gaining information on drug metabolism, and for pharmacokinetic studies. These uses also make mass spectrometry when coupled with other analytical techniques a powerful tool in forensic analysis.

If you feel that mass spectrometry is something that could be of use in your research, check whether your department or college has mass spectrometers available for use.

Training will always be provided for these services so ask around. While the number of uses is large, the instrument itself is not — normally the machine can fit on a benchtop. The most important factor in getting accurate results is keeping it clean and free of contaminants. As many a chemist will testify, this is often an issue with communal mass spectrometers — especially if the user before you did not clean the column properly.

On the other hand, also note that accidental contamination during sample preparation can mess up your results, and keeping a tab of these mass spec contaminants can help your experiment. There are various ways of producing the required ions, and the method chosen depends on the nature of the sample molecule. You can find out more information in the references below.

Try out the technique and let us know what new applications it finds in your research. Has this helped you? Then please share with your network. I am very glad that you have provided interesting information about the detection of supra molecular compounds through mass spectroscopy.

Originally published February 16, The positive ion mass spectrum of tetraphenylphosphine is shown in Scheme 1. Observed most commonly with electron ionization EI sources, electron ejection is usually performed on relatively nonpolar compounds with low molecular weights and it is also known to generate significant fragment ions. The mass spectrum resulting from electron ejection of anthracene is shown in Scheme 1. With the electron capture ionization method, a net negative charge of 1- is achieved with the absorption or capture of an electron.

It is a mechanism of ion-ization primarily observed for molecules with a high electron affinity, such as halogenated compounds. The electron capture mass spectrum of hexachloro-benzene is shown in Scheme 1. Prior to the s, electron ionization EI was the primary ionization source for mass analysis. However, EI limited chemists and biochemists to small molecules well below the mass range of common bio-organic compounds. This limitation motivated scientists such as John B.

These techniques have revolutionized biomolecular analyses, especially for large molecules. The following section will focus on the principles of ionization sources, providing some details on the practical aspects of their use as well as ionization mechanisms. The idea of electrospray, while not new, has been rejuvenated with its recent application to biomolecules.

The first electrospray experiments were carried out by Chapman in the late s and the practical development of electrospray ionization for mass spectrometry was accomplished by Dole in the late s. Dole also discovered the important phenomenon of multiple charging of molecules. A more physical explanation of ESI is that the needle voltage produces an electrical gradient on the fluid which separates the charges at the surface.

This forces the liquid to emerge from the needle as a Taylor cone. The tip of the Taylor cone protrudes as a filament until the liquid reaches the Rayleigh limit where the surface tension and electrostatic repulsion are equal and the highly charged droplets leave the filament.

The droplets that break away from the filament are attracted to the entrance of the mass spectrometer due to the high opposite voltage at the mass analyzer's entrance. As the droplet moves towards the analyzers, the Coulombic repulsion on the surface exceeds the surface tension, the droplet explodes into smaller droplets ultimately releasing ions.

Electrospray ionization ESI is a method routinely used with peptides, proteins, carbohydrates, small oligonucleotides, synthetic polymers, and lipids. ESI produces gaseous ionized molecules directly from a liquid solution. It operates by creating a fine spray of highly charged droplets in the presence of an electric field.

An illustration of the electrospray ionization process is shown in Figures 1. The sample solution is sprayed from a region of the strong electric field at the tip of a metal nozzle maintained at a potential of anywhere from V to V.

The nozzle or needle to which the potential is applied serves to disperse the solution into a fine spray of charged droplets.

Either dry gas, heat, or both are applied to the droplets at atmospheric pressure thus causing the solvent to evaporate from each droplet.

As the size of the charged droplet decreases, the charge density on its surface increases. Another possibility is that the droplet explodes releasing the ions. In either case, the emerging ions are directed into an orifice through electrostatic lenses leading to the vacuum of the mass analyzer.

Because ESI involves the continuous introduction of solution, it is suitable for using as an interface with HPLC or capillary electrophoresis. Electrospray ionization is conducive to the formation of singly charged small molecules, but is also well-known for producing multiply charged species of larger molecules.

Fortunately, the software available with all electrospray mass spectrometers facilitates the molecular weight calculations necessary to determine the actual mass of the multiply-charged species. Figures 1. Multiple charging has other important advantages in tandem mass spectrometry. One advantage is that upon fragmentation you observe more fragment ions with multiply charged precursor ions than with singly charged precursor ions. Multiple charging: A 10, Da protein and its theoretical mass spectrum with up to five charges are shown in Figure 1.

Protein ionization is usually the result of protonation, which not only adds charge but also increases the mass of the protein by the number of protons added. Multiple positive charges are observed for proteins, while for oligonucleotides negative charging with ESI is typical.

Although electrospray mass spectrometers are equipped with software that will calculate molecular weight, an understanding of how the computer makes such calculations from multiply-charged ions is beneficial.

Equations 1. Many solvents can be used in ESI and are chosen based on the solubility of the compound of interest, the volatility of the solvent and the solvent's ability to donate a proton.

Some compounds require the use of straight chloroform with 0. This approach, while less sensitive, can be effective for otherwise insoluble compounds. Consequently, volatile buffers such as ammonium acetate can be used more effectively. The off-axis ESI configuration now used in many instruments to introduce the ions into the analyzers as shown in Figure 1. The primary advantage of this configuration is that the flow rates can be increased without contaminating or clogging the inlet.

Off-axis spraying is important because the entrance to the analyzer is no longer being saturated by solvent, thus keeping droplets from entering and contaminating the inlet.

Instead, only ions are directed toward the inlet. Low flow electrospray, originally described by Wilm and Mann, has been called nanoelectrospray, nanospray, and micro-electrospray. This ionization source is a variation on ESI, where the spray needle has been made very small and is positioned close to the entrance to the mass analyzer Figure 1.

The end result of this rather simple adjustment is increased efficiency, which includes a reduction in the amount of sample needed. The flow rates for nanoESI sources are on the order of tens to hundreds of nanoliters per minute. Effusing the sample at very low flow rates allows for high sensitivity. Also, the emitters are positioned very close to the entrance of the mass analyzer, therefore ion transmission to the mass analyzer is much more efficient.

The same experiment performed with normal ESI in the same time period would require 5 picomoles, or times more sample than for nanoESI. As a consequence, nanoESI is more tolerant of salts and other impurities because less evaporation means the impurities are not concentrated down as much as they are in ESI. APCI has also become an important ionization source because it generates ions directly from solution and it is capable of analyzing relatively nonpolar compounds.

However, the similarity stops there. Vaporized sample molecules are carried through an ion-molecule reaction region at atmospheric pressure. Because the solvent ions are present at atmospheric pressure conditions, chemical ionization of analyte molecules is very efficient; at atmospheric pressure analyte molecules collide with the reagent ions frequently.

The moderating influence of the solvent clusters on the reagent ions, and of the high gas pressure, reduces fragmentation during ionization and results in primarily intact molecular ions. Multiple charging is typically not observed presumably because the ionization process is more energetic than ESI. Atmospheric pressure photoionization has recently become an important ionization source because it generates ions directly from solution with relatively low background and is capable of analyzing relatively nonpolar compounds.

Lower background signal is largely due to high ionization potential of standard solvents such as methanol and water IP APPI induces ionization via two different mechanisms. The APPI source imparts light energy that is higher than the ionization potentials IPs of most target molecules, but lower than most of the IPs of air and solvent molecules, thus removing them as interferants. In addition, because little excess energy is deposited in the molecules, there is minimal fragmentation.

To initiate chemical ionization, a photoionizable reagent, also called a dopant, is added to the eluant. Upon photoionization of the dopant, charge transfer occurs to the analyte.

Typical dopants in positive mode include acetone and toluene. Acetone also serves as a dopant in negative mode. It has since become a widespread analytical tool for peptides, proteins, and most other biomolecules oligonucleotides, carbohydrates, natural products, and lipids.

The efficient and directed energy transfer during a matrix-assisted laser-induced desorption event provides high ion yields of the intact analyte, and allows for the measurement of compounds with sub-picomole sensitivity.

In addition, the utility of MALDI for the analysis of heterogeneous samples makes it very attractive for the mass analysis of complex biological samples such as proteolytic digests. In MALDI analysis, the analyte is first co-crystallized with a large molar excess of a matrix compound, usually a UV-absorbing weak organic acid.

Irradiation of this analyte-matrix mixture by a laser results in the vaporization of the matrix, which carries the analyte with it. The matrix plays a key role in this technique. The co-crystallized sample molecules also vaporize, but without having to directly absorb energy from the laser. Molecules sensitive to the laser light are therefore protected from direct UV laser excitation.

Once in the gas phase, the desorbed charged molecules are then directed electrostatically from the MALDI ionization source into the mass analyzer. The pulsed nature of MALDI is directly applicable to TOF analyzers since the ion's initial time-of-flight can be started with each pulse of the laser and completed when the ion reaches the detector. The thermal-spike model proposes that the ejection of intact molecules is attributed to poor vibrational coupling between the matrix and analyte, which minimizes vibrational energy transfer from the matrix to the vibrational modes of the analyte molecule, thereby minimizing fragmentation.

The pressure pulse theory proposes that a pressure gradient from the matrix is created normal to the surface and desorption of large molecules is enhanced by momentum transfer from collisions with these fast moving matrix molecules. It is generally thought that ionization occurs through proton transfer or cationization during the desorption process. The utility of MALDI for biomolecule analyses lies in its ability to provide molecular weight information on intact molecules. The ability to generate accurate information can be extremely useful for protein identification and characterization.

For example, a protein can often be unambiguously identified by the accurate mass analysis of its constituent peptides produced by either chemical or enzymatic treatment of the sample.

Among the variety of reported preparation methods, the dried-droplet method is the most frequently used. In this case, a saturated matrix solution is mixed with the analyte solution, giving a matrix-to-sample ratio of about An aliquot 0. Below is an example of how the dried-droplet method is performed:. Alternatively, samples can be prepared in a stepwise manner. In the thin layer method, a homogeneous matrix "film" is formed on the target first, and the sample is then applied and absorbed by the matrix.

This method yields good sensitivity, resolving power, and mass accuracy. Similarly, in the thick-layer method, nitrocellulose NC is used as the matrix additive; once a uniform NC-matrix layer is obtained on the target, the sample is applied. This preparation method suppresses alkali adduct formation and significantly increases the detection sensitivity, especially for peptides and proteins extracted from gels. The sandwich method is another variant in this category.

A thin layer of matrix crystals is prepared as in the thin-layer method, followed by the subsequent addition of droplets of a aqueous 0. This fortuitous combination of characteristics allows DIOS to be useful for a large variety of biomolecules including peptides, carbohydrates, and small organic compounds of various types. While DIOS is comparable to MALDI with respect to its sensitivity, it has several advantages due to the lack of interfering matrix: low background in the low mass range; uniform deposition of aqueous samples; and simplified sample handling.

In addition, the chip-based format can be adapted to automated sample handling, where the laser rapidly scans from spot to spot. Because the masses of many low molecular weight compounds can be measured, DIOS-MS can be applied to the analysis of small molecule transformations, both enzymatic and chemical. In a number of recent advances with DIOS-MS, the modification of the silicon surface with fluorinated silyating reagents have allowed for ultra-high sensitivity in the yoctomole range Figure 1.

Fast atom ion bombardment, or FAB, is an ionization source similar to MALDI in that it uses a matrix and a highly energetic beam of particles to desorb ions from a surface. It is common to detect matrix ions in the FAB spectrum as well as the protonated or cationized i. The fast atoms or ions impinge on or collide with the matrix causing the matrix and analyte to be desorbed into the gas phase.

The sample may already be charged and subsequently transferred into the gas phase by FAB, or it may become charged during FAB desorption through reactions with surrounding molecules or ions. Once in the gas phase, the charged molecules can be propelled electrostatically to the mass analyzer. Electron ionization is one of the most important ionization sources for the routine analysis of small, hydrophobic, thermally stable molecules and is still widely used.

However, the fragmentation information can also be very useful. For example, by employing databases containing over , electron ionization mass spectra, it is possible to identify an unknown compound in seconds provided it exists in the database. These databases, combined with current computer storage capacity and searching algorithms, allow for rapid comparison with these databases such as the NIST database , thus greatly facilitating the identification of small molecules.

The electron ionization source is straightforward in design Figure 1. The sample must be delivered as a gas which is accomplished by either "boiling off" the sample from a probe via thermal desorption, or by introduction of a gas through a capillary.

The capillary is often the output of a capillary column from gas chromatography instrumentation. Desorption of both solid and liquid samples is facilitated by heat as well as the vacuum of the mass spectrometer. Once in the gas phase the compound passes into an electron ionization source, where electrons excite the molecule, thus causing electron ejection ionization and fragmentation. The utility of electron ionization decreases significantly for compounds above a molecular weight of Da because the required thermal desorption of the sample often leads to thermal decomposition before vaporization is able to occur.

The principal problems associated with thermal desorption in electron ionization are 1 involatility of large molecules, 2 thermal decomposition, and 3 excessive fragmentation. Chemical Ionization CI is applied to samples similar to those analyzed by EI and is primarily used to enhance the abundance of the molecular ion.

Chemical ionization uses gas phase ion-molecule reactions within the vacuum of the mass spectrometer to produce ions from the sample molecule. The chemical ionization process is initiated with a reagent gas such as methane, isobutane, or ammonia, which is ionized by electron impact. High gas pressure in the ionization source results in ion-molecule reactions between the reagent gas ions and reagent gas neutrals. Some of the products of the ion-molecule reactions can react with the analyte molecules to produce ions.

In contrast to EI, an analyte is more likely to provide a molecular ion with reduced fragmentation using CI. However, similar to EI, samples must be thermally stable since vaporization within the CI source occurs through heating.

Negative chemical ionization NCI typically requires an analyte that contains electron-capturing moieties e. Such moieties significantly increase the sensitivity of NICI, in some cases to times greater than that of electron ionization EI. NCI is probably one of the most sensitive techniques and is used for a wide variety of small molecules with the caveat that the molecules are often chemically modified with an electron-capturing moiety prior to analysis.

While most compounds will not produce negative ions using EI or CI, many important compounds can produce negative ions and, in some cases, negative EI or CI mass spectrometry is more sensitive and selective than positive ion analysis. In fact, compounds like steroids are modified Figure 1. As mentioned, negative ions can be produced by electron capture, and in negative chemical ionization a buffer gas such as methane can slow down the electrons in the electron beam allowing them to be captured by the analyte molecules.

The buffer gas also stabilizes the excited anions and reduces fragmentation. The mass spectrometer as a whole can be separated into distinct sections that include the sample inlet, ion source, mass analyzer, and detector. A sample is introduced into the mass spectrometer and is then ionized.

The ion source produces ions either by electron ejection, electron capture, cationization, deprotonation or the transfer of a charged molecule from the condensed to the gas phase. Molecular beams of macroions. Journal of Chemical Physics.

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