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. What is the fundamental principle of mass spectrometry?
The fundamental principle of mass spectrometry involves converting a sample compound into gaseous ions, which are then separated based on their mass-to-charge ratio (m/z). The instrument then detects these separated ions and records their relative abundance. This process generates a mass spectrum, a plot of ion abundance versus m/z, which allows for the determination of the molecular weight and structural features of the original sample.
2. What are the main components of a mass spectrometer and their functions?
A mass spectrometer is built around three essential components, all maintained under a high vacuum:
- Ion Source: This component converts the sample molecules into gaseous ions. Different methods like Electron Ionisation or Electrospray Ionisation can be used depending on the sample.
- Mass Analyser: It separates the ions based on their unique mass-to-charge (m/z) ratio. The analyser uses magnetic or electric fields to deflect the ions, with lighter ions being deflected more than heavier ones.
- Detector: This part measures the relative abundance of each separated ion. It generates a signal for each ion that strikes it, which is proportional to the number of ions, thereby creating the mass spectrum.
3. What are some common methods for converting a sample into ions in mass spectrometry?
Several ionisation techniques are used in mass spectrometry to suit different types of samples. Four common methods include:
- Electron Ionisation (EI): A high-energy electron beam bombards the sample, knocking an electron off a molecule to create a positive ion (cation).
- Chemical Ionisation (CI): The sample is mixed with an ionised reagent gas. The sample molecules become ionised through collisions with the reagent gas ions.
- Electrospray Ionisation (ESI): The sample is dissolved in a solvent and sprayed through a charged capillary, creating charged droplets. As the solvent evaporates, the charged sample ions are released. This is gentle and suitable for large biomolecules.
- Desorption Ionisation: The sample is mixed in a matrix and a laser pulse is used to desorb and ionise the sample molecules from the matrix.
4. What are the key stages involved in the working of a mass spectrometer?
The process of analysing a sample in a mass spectrometer typically follows four key stages:
- Ionisation: The sample compound is introduced into the ion source and converted into gaseous ions.
- Acceleration: The newly formed ions are accelerated by an electric field so that they all have the same kinetic energy.
- Deflection/Separation: The accelerated ions enter the mass analyser, where they are deflected by a magnetic field. The degree of deflection depends on the ion's mass-to-charge ratio (m/z)—lighter ions are deflected more than heavier ones.
- Detection: The separated ions strike a detector, which records the m/z ratio and relative abundance of each ion type to produce a mass spectrum.
5. What specific information can be obtained from a mass spectrum?
A mass spectrum provides crucial information for identifying a compound. The peak with the highest m/z value typically corresponds to the molecular ion (M+), which directly gives the molecular weight of the compound. Other peaks with lower m/z values represent fragment ions, and their pattern serves as a unique 'fingerprint' that helps in deducing the molecule's structure. The height of each peak indicates the relative abundance of that particular ion.
6. How does mass spectrometry differ from other common spectroscopic techniques like IR or UV-Vis?
The primary difference lies in the interaction with the sample. Most other spectroscopic methods, like IR or UV-Vis spectroscopy, involve measuring the absorption or emission of electromagnetic radiation by a molecule without destroying it. In contrast, mass spectrometry is a destructive technique where the sample molecule is ionised and broken into fragments. It does not measure light interaction but instead analyses the masses of the molecule and its pieces to determine its structure and weight.
7. Why do only charged particles, and not neutral fragments, get detected in a mass spectrometer?
Only charged particles (ions) are detected because the core principle of a mass spectrometer relies on manipulating particles using electric and magnetic fields. Neutral fragments, such as free radicals formed during fragmentation, are unaffected by these fields. Therefore, they cannot be accelerated or deflected by the mass analyser. They simply get removed from the instrument by the high-vacuum system, while the charged ions travel through to the detector to be recorded.
8. In a mass spectrum, what is the significance of the 'base peak' versus the 'molecular ion peak'?
The molecular ion peak (M+) is the peak with the highest mass-to-charge ratio in the spectrum, representing the intact molecule with one electron removed. Its m/z value gives the molecular weight of the compound. The base peak, on the other hand, is the tallest peak in the spectrum, assigned a relative abundance of 100%. It represents the most abundant and, therefore, the most stable fragment ion formed during ionisation. All other peak intensities are measured relative to the base peak.
9. What are some key applications of mass spectrometry in real-world scenarios?
Mass spectrometry is a versatile technique with numerous important applications, including:
- Pharmaceutical Analysis: Used in drug discovery, studying drug metabolism, and ensuring quality control during drug manufacturing.
- Environmental Analysis: To detect and quantify pollutants in water, soil, and air, such as pesticides or heavy metals.
- Clinical Diagnostics: For disease screening, identifying infectious agents, and monitoring drug therapy in patients.
- Forensic Science: To identify unknown substances, analyse trace evidence at crime scenes, and conduct toxicology tests.
- Food Safety: For detecting contaminants like toxins or harmful bacteria and verifying the authenticity of food products.
10. How does the fragmentation pattern help in determining the structure of an unknown compound?
When a molecular ion is formed in the mass spectrometer, it is often energetically unstable and breaks apart into smaller, more stable pieces. This process, called fragmentation, is not random. The molecule tends to break at its weakest bonds, leading to a predictable pattern of fragment ions. By analysing the masses of these fragments, a chemist can piece together the structure of the original molecule, much like solving a puzzle. This fragmentation pattern is a unique characteristic of a molecule and provides powerful clues about its functional groups and connectivity.
