Mass Spectrometry Roundup: Ion Sources – Electrospray Ionization (ESI)

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Chances are if you are not a Mass Spectrometry (MS) jockey, or should I say junkie, you’ve at least encountered or occasionally use the technique. Perhaps you just want some background to feed your thirst for knowledge or to help you in conversation at meetings etc. We’ve put together a series of topical articles on the various components and applications in the MS world. Hopefully this series of primers will offer some educational value. Beyond this, we are always looking for interesting suggestions for future posts on this or other subjects – so leave your comments please.

We should start with ionization sources, as this is the initial stage at which the sample is effectively ionized for entry, analysis, and detection in the MS. It’s a critically important process. If the sample is not amenable to ionization due to contaminants such as salts, detergent, lipids, etc., or if the ion source is ineffective for whatever reason, the downstream data (and the researcher) will suffer at the expense of time and resources.

In the majority of cases, the sample will dictate the type of ionization source that will be most effective. Of course, one may not be aware of the nature and behavior of experimental samples and the analytes therein. Thus, an empirical approach might be needed to arrive at the best conditions for ionization and analysis.

Over time, ionization sources have undergone significant refinement. Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption Ionization (MALDI) have emerged arguably as the most successful and versatile methods employed in biological MS.

Electrospray Ionization - ESI is simply the evaporation of charged sample droplets. In more detailed terms, it is the method to produce gaseous ionized particles from a liquid solution such that they can be guided through MS analysis and detection. ESI entails creating a fine spray of highly charged droplets by simultaneous elution from a capillary tube (e.g. liquid chromatography (LC)) and the induction of a strong electric field. The sample spray emanates from a typically steel capillary nozzle charged at approximately 4000 V.

The droplets are then electrostatically attracted to the inlet of the MS analysis chamber. Either inert gas, heat, or both are applied to the droplets prior to entry into the vacuum thereby leading to evaporation. As the droplet size decreases, the electric field density on it’s surface increases. The repulsive forces of the droplets effectively battle the liquid surface tension and the phase change to gas ensues.

Electrospray ionization is associated with the formation of multiply charged species of molecules, which is important as MS resolution is based on mass to charge ratio (m/z). Hence, very large molecules, provided they are ionized, can be resolved using an instrument having a relatively small mass range. Molecules 10 kDa and above in size are routinely analyzed, giving rise to a rich spectrum of peaks based on m/z and any fragmentation that has occurred. Computer programs and databases are used to deconvolute the spectra and assign identity to the peaks, thereby facilitating identification and characterization of the analytes(s) of interest.

Ionization mechanisms include protonation, cationization, deprotonization, electron ejection, and electron capture. The details of these chemical processes are beyond the scope of this post and are perhaps a subject for a future article.

We should, however, briefly discuss the pros and cons of the electrospray ionization method:


  • Practical mass range of up to 70kDa
  • Femtomole to low picomole sensitivity
  • Soft ionization and capability of observing native non-covalent interactions
  • Easily adaptable to capillary liquid chromatography (LC) effluent
  • No matrix interference
  • Easy adaptability to triple quadrupole analysis
  • Multiple charging to allow for better range and accuracy


  • Low salt tolerance
  • Difficulty in cleaning
  • Low tolerance for mixtures and necessity for relatively pure samples
  • Multiple charging can lead to overly convoluted data

See the article entitled Mass Spectrometry Roundup: Ion Sources – Matrix-Assisted Laser Desorption Ionization (MALDI) for review of this method.