Introduction
Mass spectrometry is an analytical technique to evaluate known materials and determine unknown compounds. It also helps to determine the structure and chemical properties of several molecules. The food we eat, the water we drink, medicine we consume when we fall ill all are first tested to check the presence of any harmful elements or any contamination with the help of mass spectrometry. Mass spectrometry is also used in isotope determination, carbon dating, identification of protein, etc.
In mass spectrometry, the sample compound is first converted to gaseous ions with or without the fragmentation method, which is further identified by their mass-to-charge ratio and relative abundances (intensity).
Principle of Mass Spectrometry
The principle involved in mass spectrometry is the formation of several ions from the sample. Further, these ions are separated according to their mass to charge ratio, which is also expressed as m/z and then taking a record of the relative abundance of each ion.
In the first step, the sample compound is converted to ions in the gas phase by the electron ionisation method. After that, the molecular ions undergo fragmentation. Each ion is separated from the other in a mass spectrometer depending on their mass-to-charge ratio and identified according to their relative abundance. A mass spectrum is then formed, which shows the spectrum of ion abundance versus mass-to-charge ratio.
The ions present give information about the structure and properties of the compound. In the spectrum, the molecule ion of the pure compound has the highest value of mass to charge ratio, followed by ions of heavier isotopes. By this, the molecular weight of the compound is determined.
Instrumentation of Mass Spectrometry
There are three major components present in mass spectrometry which are discussed below.
Ion Source: It produces gaseous ions from the given sample.
Analyzer: It is used to analyse and separate the ions into their characteristic mass according to their mass-to-charge ratio.
Detector System: Detectors in mass spectrometry detect the ions and maintain their relative abundance.
Apart from these, a sample introduction system is required to add the sample to the ion source. A high vacuum is maintained (10-5-10-8 torr), and a computer system is needed to control the instrument, store the data and compare the spectrum with the references.
Instrumentation of Mass Spectrometry
Working of Mass Spectrometry
In the ion source, the sample molecules are mostly bombarded by electrons from a heated filament. The volatile liquid samples and gases come into the ion source from the reservoir, and the non-volatile solids and liquids are added directly. The cations are pushed away by the charged repeller plate and moved towards other electrodes, and anions are attracted to the plate. The plate has a slit from where the ions pass as a beam.
The perpendicular magnetic field deflects the ion beam into an arc. The lighter ions are deflected higher than, the heavier ions. By analysing the strength of the magnetic field, the ions having different masses are detected by the detector. According to the mass spectrum formed by the charged ions, one can determine the molecule or atom compared with the known molecular masses.
Mass Spectrum
A mass spectrum is a vertical bar graph of mass to charge ratio versus the relative abundance (intensity) of the ions where each bar represents the ion of specific mass to charge ratio and the length of the bar is the relative abundance of ion. The ion with the most intensity has an intensity of 100 and is called the base peak. The mass-to-charge ratio is equal to the mass of the ion as the ion has a single charge.
Modern mass spectrometers easily determine the ions by a single atomic mass unit denoted by amu and give the precise molecular mass value of the chemical compound. In the mass spectrum, the ion with the highest mass is considered the molecular ion, and the ions with lower mass are the fragments of the molecular ion when the sample is a single pure compound.
Fragmentation Pattern in Mass Spectrometry
In a mass spectrometer, when a sample is passed to the ionisation chamber, it is bombarded with electrons which results in the formation of positive ions. This ion is known as the parent ion or the molecular ion. The molecular ion is denoted by M+.
The molecular ion is usually energetically unstable, so it further breaks into fragments; one is another positive ion, and the other is the uncharged free radical. The uncharged free radical does not form any line in the mass spectrum, whereas the charged ion shows the line in the spectrum. The uncharged free radical gets removed from the vacuum. The fragments show a distinct pattern in the mass spectrum.
CH4 + e- → CH4.+ + 2e-
CH4.+ →CH3+ + H•
CH4•+ + CH4 →CH5+ + CH3
CH3+ + CH4 →C2H5+ + H2
M + CH5+ →MH+ + CH4
M + C2H5+ →MH+ + C2H4
(M = Molecule)
The most stable molecular ions are formed of aromatic compounds, compounds having a conjugated pi-electron system, and cycloalkanes. Also, alcohols branched alkanes, and ether show fragmentation. There are different fragmentation rules in mass spectrometry for different chemical compounds, which helps in determining the unknown chemical compound.
High-Resolution Mass Spectrometry
High-resolution mass spectrometry is used to analyse complex chemical compounds, determine isotopes, etc. It has increased resolution, which helps in differentiating the isotopes, generating fragments pattern, and library matching for compound verification. HRMS is not the replacement for low-resolution mass spectrometry, but it is an advanced technique used to determine unknown compounds and also generate their molecular formula. High-resolution mass spectrometry is quite expensive as compared to low resolution.
Advantages and Limitations of Mass Spectrometry
Advantages:
Works with a small sample size
Fast
Can differentiate isotopes
Limitations:
Does not give direct structural information
The requirement of pure samples
Not ideal for non-volatile compounds
Application of Mass Spectrometry
Environmental Analysis: It is used in water testing, soil contamination, analysis of trace elements, carbon content and pollution analysis.
Pharmaceutical Analysis: It is used in producing new drugs, preclinical development, etc.
Clinical Application: It is used in identifying infectious agents, drug therapy monitoring, clinical tests, screening of diseases, etc.
Forensic Analysis: It helps in confirming drug abuse, identifying explosives, arson investigation, etc.
Conclusion
Mass spectrometry is a spectroscopic technique used to determine the nature and structure of unknown inorganic and organic compounds based on their mass-to-charge ratio and relative abundance. This technique is widely used in different industries because of its advanced technology and application. It is used in forensic analysis, analysing different environmental issues, clinical trials, etc. High-resolution mass spectrometry is used to analyse complex chemical compounds which a low resolution is not able to determine accurately.
FAQs on Mass Spectrometry
1. How are Samples Converted to Ions in Mass Spectrometry?
Electron Ionization: A high energy beam of electrons strikes the sample. The collision between an electron and the sample molecule removes an electron from the molecule, creating a cation.
Chemical Ionization: The sample molecules are mixed with an ionized reagent gas. The collision between the ionized reagent gas and the sample molecules ionizes the latter by electron transfer, proton transfer, and adduct formation.
Electrospray Ionization: The sample is dissolved in a volatile, polar solvent and electrostatically dispersed through a narrow capillary, which generates an aerosol of highly positively charged droplets. Due to solvent evaporation, the airborne droplets shrink, and their charge density increases, eventually releasing the charged sample ions.
Desorption Ionization: The sample is dissolved in a suitable matrix. A short pulse of laser light is used to ionize the sample, which is desorbed from the matrix into a vacuum system for further analysis.
2. What is Time - of - Flight, and How is it Calculated?
Time-of-flight is a type of mass analyzer that measures the flight time of ions in a mass spectrometer. The principle is that two ions with the same kinetic energy will have different velocities based on their masses.
When accelerated through an electric potential V, the kinetic energy of an ion is given as:
E = zV = mv2/2........ (1)
Where, E = kinetic energy
V = electric potential
v = velocity of an ion
z = charge on an ion
m = mass of an ion
The velocity of an ion is given by the length of the flight (L) divided by the time (t) for the ion's flight.
v = L/t....... (2)
Replacing the value of 'v' in (1) with that in (2):
zV = mL2/2t2
or, m/z = 2Vt2/L2
m/z is the mass-to-charge ratio, which determines the separation of ions in the mass spectrometer.
* Protonation is defined as the addition of proton to molecules or atoms to form a conjugate acid.
** Deprotonation is defined as the removal of a proton from a Bronsted-Lowry acid in an acid-base reaction.
3. Why is mass spectrometry different from other spectroscopic methods?
Most spectroscopic methods usually involve absorption, emission or scattering of radiation/light from different regions of the electromagnetic spectrum, whereas, in mass spectrometry, the molecule is ionised using a suitable method and fragmented. The fragments are then analysed.
4. What is MRM in mass spectrometry?
MRM denotes multiple reaction monitoring. It is also known as selective reaction monitoring (SRM). It is a highly specific technique that selectively quantifies compounds in a complex mixture. In this technique, triple quadrupole MS targets the ions present in the compound, which need to be detected with subsequent fragmentation of the target ion to form a range of daughter ions. MRM is used for selecting specific analytes, quality checking of proteins, lipids, serum, biological samples, etc.
5. Why do only charged particles give lines in mass spectrometry?
Only charged particles give lines in mass spectrometry because only charged ions are deflected and accelerated by the magnetic strength of the detector, whereas the uncharged ions get removed by the high vacuum. The ion travel through the mass spectrometer like another positive ion. It will produce a line in the mass spectrum, and all the fragmentation of the molecular ion is possible, which forms a whole line in the mass spectrum.