Analytical Technique: Mass Spectrometry - Essay Example

? Mass spectrometry is a technique used to determine the characteristics of an unknown compound, most notably its mass. Different compounds, no matter how similar in size and chemical formula, have different spectra. In fact, it can be used to differentiate the same compounds made of different isotopes. Theories There are three important steps in mass spectrometry. First, the compound is converted into cations by loss of an electron. These ions are produced by driving a high energy beam of electrons, which displaces the electrons from the compound. Sometimes, cations split further into neutral fragments and smaller cations. The cation resulting from electron bombardment is a radical cation, while the smaller cations can be radical cations or carbocations depending on the nature of the neutral fragment. Because the resulting ions are intermediate, the process should be kept in a vacuum, 10-5 to 10-6 torr (http://v.cem.msu.edu/-reusch/VirtualText/Spectrpy/MassS). These ions are then sorted out and separated according to their mass and charge. These ions are then formed into a beam, and application of magnetic field deflects it in an arc whose radius is inversely proportional to mass of the ions. Thus, heavier cations are deflected nearer than lighter ones. Lastly, the number of ions for each group is recorded into a graph known as mass spectrum (http://v.cem.msu.edu/-reusch/VirtualText/Spectrpy/MassS). After mass spectrometry, a vertical bar graph is produced, showing the concentrations of ions with specific mass-to-charge ratios (m/z). The most abundant ion is referred to as the base peak, and is given the abundance of 100. By convention, the highest-mass ion is considered the molecular ion, and lower mass ions are its fragments. Their presence in the spectrum is important in predicting the molecular structure (http://v.cem.msu.edu/-reusch/VirtualText/Spectrpy/MassS). Features of the Instrument Basically, the test compound is introduced to the machine through a sample inlet. To produce ions from the sample compound, a heated filament is used to produce the high beam of electrons. The formed cations are pushed away by a positively-charged repellor plate, and driven toward other electrodes, with slits through which the beam of cations will pass. It is then deflected by a magnetic field (mass analyzer). The resulting beams are then recorded using a detector (http://v.cem.msu.edu/-reusch/VirtualText/Spectrpy/MassS). Sample Preparation Requirements The samples analyzed using a mass spectrometer is under the premise of 100% purity. The sample can be purified by passing it into chromatography columns. If the sample is a protein, then this can be easily checked using electrophoresis. As well, as mentioned above, the compound should produce molecular ions, along with its smaller cations and neutral fragments, upon analysis using mass spectrometer. Most organic compounds can be split in these elements, although there are compounds that give off molecular ions that do not last long enough to be detected. In such cases, milder ionization conditions are used (http://v.cem.msu.edu/-reusch/VirtualText/Spectrpy/MassS). Types of Detectors There are many types of detectors, each with their own specific uses as discussed by Scripps Center for Metabolomics and Mass Spectrometry. Each is mentioned below. Electron multiplier The most commonly used detector, the electron multiplier is made up of 12 to 24 aluminum dynodes of increasing potentials. Each dynode causes release of electrons from the ions coming from the dynodes before it. The strength of signals depends on the velocity by which the ions are applied on the detector. Sometimes, an accelerating electrostatic field is used to increase signal intensity and sensitivity. Faraday cup On the other hand, the Faraday cup uses a concave dynode on which the ions strike, causing the release of ions much like what happens in the previous detector. The cup then gains a positive charge, thus attracting an electron current toward the detector. Photomultiplier conversion dynode When ions hit the conversion dynode, it releases electrons, which strike a phosphorus screen that emits photons. These then hit a scintillating surface, which releases electrons to amplify the signal. This detector works much like an electron multiplier, although the former is housed in a vacuum, so contamination is prevented. Aside from the assurance of purity, the vacuum improves the lifetime of this detector. One disadvantage of this apparatus is its photosensitivity. Array detector An array detector is a set of detectors in an array format. Its advantages are it can provide fast and sensitive results, although it reduces resolution, and is expensive. Charge detector This detector detects moving particles that pass between plates that induces a current as an ion moves past. This apparatus detects ions independent of its mass and velocity. However, it has limited compatibility with existing instruments. Detection Limits/Resolution Resolution is the ability of a mass spectrometer to differentiate two ions of different mass-to-charge ratios. It is dependent on the mass analyzer of the machine. Lower resolutions are unable to resolve the comprising isotopes of an element present in a compound, and the mass recorded is the average mass. This is seen in the mass spectrum as a broad peak. On the other hand, high resolution mass spectrometer can narrow down this peak, by splitting it into the number corresponding to the isotopes of the element (Scripps Center for Metabolomics and Mass Spectrometry). Advantages and Disadvantages One advantage of using mass spectrometer is that it can provide a lot of information with just a single run of the sample compound onto the machine. As mentioned above, it can give the mass, chemical formula and subsequently the structure of the compound. However, its use is limited by the small number of personnel that can operate the machine. In addition, many factors must be taken into consideration to optimize the results of the mass spectrometry. As detailed by Lubec and Afjehi-Sadat (2007), the steps from as early as sample preparation and calibration of the machine can affect the run, as well as the maintenance and contamination of the machine. To give a glimpse, a single mass spectrometer should be kept in one air-conditioned room. Its parts, most especially the ion source, should be regularly cleaned (not more than 10 uses). Furthermore, no one machine is catered to detect all compounds. Each of the ionizers, analyzers and detectors have their own sets of benefits and limitations. The advantages and disadvantages of the use of mass spectrometer for a certain compound not only depends on the machine, but on the type of the analyte and the data needed to be obtained from the analysis. Definitely, there are other equipments or techniques more fitting on determining certain characteristics of certain compounds. Let us take proteins and nucleic acids as an example. Examples First, let us take a look at the protein conotoxins for example. Briefly, it is a peptide isolated from Conus sp. for its anti-ion channel potential. It is composed of 12 to 30 amino acid residues. The ones isolated by Zugasti-Cruz et al. (2008) weighs around 3 kDa. The study used the MALDI-TOF mass spectrometry to determine the molecular weight of the compound, although the amino acid sequence had been previously identified using the automatic Edman sequencing. Although the weight can be easily calculated from the sequence, it was shown that it is still different from the experimental weight of the compound. Aside from mass spectrometry, protein molecular weights can be identified using electrophoresis and liquid chromatography. Although the weights obtained from these two techniques may not give out a mass as accurate as that obtained from mass spectrometry. Another example of compounds that can be analyzed using mass spectrometry is nucleic acids. According to Chiu and Cantor (1999), nucleic acids having between 2 to 2000 nucleotides can be run through the mass spectrometer to determine their molecular weights. It may also be used to analyze a mixture of different fragments. As well, the apparatus only needs a few nanoliters of the sample to analyze. Despite its adeptness in identifying the mass of nucleic acids, they are most of the times not needed. What is vital in the study of nucleic acids is accurate sequencing, which can be done by sequencing machines. Its weight can be done using agarose gel electrophoresis, which is a lot less tedious to do than mass spectrometry. However, similar to the case of conotoxins, mass spectrometry gives an exact, accurate molecular weight that no other machine can do. Summary In summary, a mass spectrometer is a useful machine that analyzes a compound in order to determine vital characteristics such as molecular weight (most common), chemical formula and structure. It does it by splitting the compound into ions, which, when applied with magnetic or electric field, gets deflected to a detector that records the amount of deflection, which is inversely proportional to the mass of the ion. Although powerful, it has its own limitations, and its use is still dependent on the compound to be analyzed and its characteristics needed to be determined. References Chiu, N. H. L. and Cantor, C. R. 1999. Mass spectrometry of nucleic acids. Clinical Chemistry, 46(9), p. 1578 Lubec, G. an Afjehl-Sadat, L. 2007. Limitations and pitfalls in protein identification by mass spectrometry. Chem. Rev., 107, 3568-3584 Scripps Center for Metabolomics and Mass Spectrometry. What is Mass Spectrometry? [online]. Available at: Zugasti-Cruz, A. Falcon, A. Heimer de la Cotera, E. P. Olivera, B. M., and Agular, M. B. 2008. Two new 4-Cys conotoxins (framework 14) of the vermivorous snail Conus austini from the Gul of Mexico with activity in the central nervous system of mice. Peptides, 29(2), pp. 179-185. Mass Spectrometry. [online]. Available at: Read More