Mass Spectrometry

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Basics of MS

Mass spectrometry is a technique that allows us to identify and quantify compounds in our sample. It works by bringing the molecules into a gas phase, ionizing & separating them, and measuring the abundance of each generated ion.

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Ionization Techniques

There are two kinds of ionization: hard ionization and soft ionization. Hard ionization techniques (e.g. EI) result in more fragmentation due to more energy being applied to the analyte of interest. These techniques are useful for determining structural information about an analyte because they can provide more data about the functional groups and bonds present in the molecule. Soft ionization techniques (e.g. CI, electrospray, MALDI) result in less fragmentation because they apply less energy to the analyte; this preserves more of the parent ion, giving you more information about the mass of the ion [1,2].

The fragmentation pattern and theoretical upper limit of detection depend on the ionization source, and the analyte ions need to be in the gas phase in order to be analyzed. Gas-phase sources vaporize the sample before ionizing it. Desorption sources utilize the solid or liquid matrix that the analyte is in to convert it directly to gaseous ions [1].


Electron Ionization (EI)

Electron ionization, also called "electron impact ionization," is a hard ionization method. The molecules are put in the gas phase before being hit with a beam of electrons and broken apart by the high energy [1]. The high energy of the electrons will break bonds in a predictable way across instruments, so libraries of EI spectra of thousands of compounds have been compiled to allow for identification of unknown compounds in a sample [3].

The fragmentation of each individual molecule depends on how the electrons impact it. Fragmentation of the parent ion occurs when electron impacts break bonds within the parent molecule and form daughter ions [1]. One possible ion generated by EI is the molecular ion (M+), which is the radical form of the parent molecule formed when a passing electrons knocks one of the analyte's electrons off [2]. It is possible for ions with a higher m/z (proton adducts) to form from reactions between the parent molecules and H+ ions (MH+). The formation of MH+ can be minimized by maintaining very low pressure in the ionization source [3].

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Advantages: good sensitivity due to high ion currents; unique and extensive fragmentation pattern gives lots of detail & high confidence in identification; lots of reference libraries to compare spectra [1].

Disadvantages: extensive fragmentation may eliminate molecular ion peak; requires volatile sample, which may result in thermal degradation of analyte before ionization; not useful for molecular masses higher than 103 Daltons because spectra get too complicated [1].


Chemical Ionization (CI)

Chemical ionization is a soft ionization method that uses a reagent gas (e.g. methane) to ionize the analyte. The gas is ionized using EI and the pressure is adjusted so it is much larger than that of the sample, causing the ionized gas to collide with the vaporized analyte and transfer the charge. Indirectly ionizing the analyte results in less fragmentation and most ions having m/z values close to the molecular ion M+. The structural information provided by the spectra will depend on the chosen reagent gas [1,3].

Ionized gases can form strong acids that may protonate the analyte and result in a proton adduct (MH+), or perform a hydride transfer (in cases where the analyte is a saturated hydrocarbon). The ionized gas may also attach itself to the analyte and result in a spectrum peak with a m/z higher than the M+ peak; the difference between the peaks is the molar mass of the reagent gas. CI spectra can be complex due to additions to the analyte and peaks from the reagent gas [1].

Negative Chemical Ionization (NCI)

NCI is very similar to CI, but is performed with analytes that contain strongly electronegative atoms or functional groups (e.g. fluorine, nitrobenzyl groups). It produces negatively charged ions by reacting the analyte with a negatively charged reactant [3].


Electrospray Ionization (ESI)

ESI is a soft ionization technique in which the analyte in solution is passed through a charged metal capillary. As it passes through the electric field of the capillary, the solvent forms a mist of charged droplets. As the solvent evaporates off, the charge density of the droplet increases until it gets too high and burts into even smaller droplets. Eventually all the solvent will have evaporated and the charges will be transfered onto the analyte; this results in m/z ratios that are not 1:1, so data analysis becomes much more challenging [1,3].

ESI is often used for mass analysis of drugs of abuse [3] and large biomolecules (e.g. proteins, fatty acids, and other polar molecules) which are thermally unstable and highly non-volatile. It can be paired with LC or HPLC for analysis of analytes in solution [4].

The system has two operating modes: in drug analysis, positive mode is used for basic drugs with a stable HCL salt (e.g. cocaine), and forms MH+ as the primary ion; negative mode is used for acidic drugs with a stable Na salt (e.g. GHB), and forms MH- as the primary ion. [3].


Matrix-Assisted Laser Desorption Ionization (MALDI)

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Direct Analysis in Real Time (DART)

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Fast Atom Bombardment (FAB)

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Hardware

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Mass Analyzers

Detectors



Sample Introduction Methods

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Interpretation of Spectra

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References

  1. Class materials & my notes from CHEM 409 Instrumental Analysis taught by Dr. M. J. Holle (Virginia Commonwealth University, spring 2024).
  2. Gross, J. H. Principles of Ionization and Ion Dissociation. In Mass Spectrometry; 3rd ed.; Springer International Publishing: Gewerbestrasse, Switzerland, 2017; pp 29-84.
  3. Materials from the ABFT board certification prep course (May-Aug 2017) by Fredric Rieders Family Foundation Center for Forensic Science Research & Education.
  4. Ho, C. S.; Lam, C. W.; Chan, M. H.; Cheung, R. C.; Law, L. K.; Lit, L. C.; Ng, K. F.; Suen, M. W.; Tai, H. L. Electrospray ionization mass spectrometry: Principles and clinical applications. Clin. Biochem. Rev. 2003, 24(1), 3-12.

Further Reading



Page created: March 28, 2025
Last updated: March 28, 2025