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A Beginner’s Guide to Mass Spectrometry

February 9, 2023
by Baljit Bains, Marketing Communications Specialist, ACD/Labs

Do you want to determine the structure of a molecule but don’t know where to start? Mass spectrometry (MS) is a valuable tool that can give you vast amounts of MS data to help you identify and quantify components. MS has been used to discover, determine, and quantify sample compounds in the proteomics, metabolomics, imaging, and glycomics applications across the food, pharmaceutical, and environmental industries. Combining MS with appropriate software and computer technology allows the possibility for more extensive applications. But first, we must understand the basics.

What is Mass Spectrometry?

Mass spectrometry is a complex analytical technique where the components of a sample are separated by their mass and electrical charge. It’s used for both quantitative and qualitative chemical analysis of sample compounds. MS is used to identify unknown compounds by determining molecular weight, quantifying known compounds, and elucidating the structural and chemical properties of the molecular components.

What is the basic principle of Mass Spectrometry?

Mass spectrometry produces a mass spectrum which is a plot of the mass-to-charge (m/z) ratio of compounds present in the sample and the relative abundance of each ion. Ions are accelerated to high speeds and deflected at different angles in an electric or magnetic field depending on their m/z ratio. The m/z ratio can be used to calculate the exact molecular weight of the components in the sample.

What are the components of the Mass Spectrometer?

A mass spectrometer works by producing ions from the sample in the ionization source, separating these ions according to m/z ratio, fragmenting the ions, and analyzing them in the mass analyzer. The detector system then detects and processes the electrical signals and measures their abundance.  The associated components are:

  1. Ion source
    The ion source is often referred to as the heart of the spectrometer. This is where either an electron is removed or proton is added, giving the molecule a positive charge. Molecules are ionized/converted into gas-phase ions so that they can be moved and manipulated by external electric and magnetic fields.
  2. Analyzer
    Once ionized, the ions are separated according to their mass (m/z ratio). There are a variety of mass analyzers available, and they work with the ion detection system. Mass analyzers will vary in their operational requirements such as operating speed and separation resolution.
  3. Detector System
    The detector will detect the separated ions by recording the charge of ions passing through the surface and quantify them. A mass spectrum is created plotting the m/z ratio of ions present in the sample against their intensity (relative abundance). Each peak corresponds to a unique m/z ratio in the sample and the height of the peak shows the relative abundance of the components in the sample.

There are various types of ion detectors including Electron Multipliers (EM), Faraday Cup (FC), Photomultiplier Conversion Dynodes, and Array Detectors. The separated ions are measured, and a chart is created to log their relative abundance.

How does a Mass Spectrometer work?

Mass spectrometry involves five steps:

  1. Ionization
  2. Acceleration
  3. Deflection
  4. Detection
  5. Data processing

1. Ionization

Ionization refers to the production of gas-phase ions to allow for mass analysis. The initial sample may be solid, liquid, or gas. If it is solid or liquid, it is vaporized into a gas and then ionized by the ion source. This usually occurs by losing an electron and becoming a cation with a +1 charge. The ionization chamber is kept in a vacuum to avoid the interaction of ionized molecules with molecules in the air. A positively charged metal plate pushes the samples through to the next part of the machine.

Spectrometers work in either a positive ion or negative ion mode. It is important to know the correct setting to correctly analyze the data.

The ionization method is crucial to interpreting the mass spectra. There are various ionization methods available and these can be divided into two categories: hard ionization and soft ionization. We discuss these in further detail in a previous blog: Common Adduct and Fragment Ions in Mass Spectrometry.

Hard ionization methods use excessive amounts of energy on the sample during ionization, resulting in fragmentation of the sample. Soft ionization methods use less excess energy to ionize the sample, resulting in little fragmentation.

There are a variety of ionization methods available, and the method used depends on the experimental goals and sample characteristics. You can learn more about the different ionization methods in A Beginner’s Guide to Mass Spectrometry: Types of Ionization Techniques.

2. Acceleration

In the mass analyzer, the ions are accelerated through a potential difference and focused into a beam. The purpose of the acceleration of ions is to give all the molecules the same kinetic energy. The ions are between a set of charged parallel plates where they are attracted to one and repelled from the other. Adjusting the charge on the plates controls the acceleration speed.

There are several mass analyzers available, each with their own place within different experiments and applications. It is possible to combine mass analyzers to create tandem or hybrid mass spectrometers. These can improve experimental results and give higher throughput experiments.

3. Deflection

The ion beam will then pass through either a magnetic or electric field, which causes the charged beam to bend. Lighter components or more ionically charged components will deflect in the field more than heavier or lesser ionically charged components.

Magnetic fields deflect ions based on their charge and mass. If an ion is heavy or has 2 or more positive charges, it is deflected the least. Ions that are deflected the most tend to be lighter or have just one positive charge.

4. Detection

There are a variety of detectors available that can measure the time it takes for a particle to reach a detector and how much is deflected by the electric/magnetic field, giving more information in addition to mass. The ions with the correct charge and mass will move to the detector. When the ion hits the surface, the detector records the induced charge/current. The m/z ratio is analyzed through the ions that hit the detector. The number of ions at different detections will be counted by the detector. These signals can be quite small, so an electron amplifier, Faraday cup, or ion-to-photon detector can be used. The data obtained are plotted as a mass spectrum.

If all the particles start with the same charge, lighter components will reach the detector first because velocity is mass dependent.

5. Data Processing

Various types of data are produced with mass spectrometry including molecular determination, structure elucidation, and quantitative analysis. The most common is the mass spectrum plotting relative abundance against the m/z ratio.

Mass spectrometry instruments come with their own software which is often used to perform the necessary data processing and analysis of the vast amounts of MS data that is generated. However, labs often have several instruments from many different vendors. This means scientists must be familiar with multiple different interfaces and face the challenge of bringing together all the information. The choice of MS software used depends on several factors:

  • Mass analyzer used
  • Type of sample analyzed
  • Overall goals of the analysis
  • Integration of all data in one interface

There are numerous software tools available for MS data processing that can be used to help speed up MS data analysis. ACD/Labs’ analytical data processing tools can help process and interpret MS data in a single interface, uniting data from all instruments in the lab.

You can learn more about ACD/Labs mass spectrometry software tools here.

Benefits of Mass Spectrometry

Mass spectrometry is a sensitive technique which is very precise and rapid compared to other techniques. It can be used to identify and quantify known components or confirm component presence in a sample. When mass spectrometry is performed by an expert, it yields results that are highly accurate and reproducible. Mass spectrometry can be used to accurately quantify large numbers of molecules at the same time.

Mass spectrometry can be combined with other techniques like gas chromatography (GC/MS) or high-performance liquid chromatography (HPLC/MS), making it versatile and delivering very precise quantification. Optimization of preparation method for this combined use can significantly increase sensitivity.

Limitations of Mass Spectrometry

Like everything, there are also a few limitations of mass spectrometry. It can be costly as it requires significant materials and equipment. Mass spectrometry is a destructive technique and to get the best results, pure samples are needed.

Mass spectrometry must be performed by a specialized operator and multifunctional systems of mass spectrometers do not exist. Mass spectrometry cannot differentiate among isomers of the molecule with the same m/z ratio. To separate enantiomers, you may need chiral columns. It is also not able to separate optical and geometric isomers, as mass spectrometers can’t recognize hydrocarbons that produce similar ions.

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