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Mass Spectrometry - Why is calibration important?

One of Polymer Factory’s product lines is SpheriCal® - designed to serve as an outstanding calibration standard for MALDI-ToF Mass Spectrometry. Why is this needed and how can SpheriCal® support you to achieve the most reliable and accurate results?

MALDI-TOF MS is an indirect measurement of the mass of ionized molecules. The name of the technique is an abbreviation explaining the mode-of-action – Matrix Assisted Laser Desorption / Ionization – Time of Flight; as the name indicates, the instrument actually measures time, rather than mass. In order to understand how a measured time relates to a mass, one has to understand the whole chain of events.

How MALDI-ToF MS works

In MALDI-MS, the sample is deposited on a so-called target plate, together with another compound – a matrix - that is known to absorb energy from a defined laser wavelength. The target plate is inserted into the instrument, after which the instrument is evacuated to a high vacuum. A laser is shot onto the analyte/matrix mixture and the matrix absorbs the laser’s energy – resulting in a cloud of matrix and analyte molecules (desorption) in a charged (ionized) state. The electric field in the ion source then accelerates these ions towards the time-of-flight tube (drift tube). The transferred energy, now as kinetic energy, is only related to the amount of charge of the molecule. Thus, all singly charged molecules in the created ion cloud hold the same amount of kinetic energy.

Energy-mass relationship

The kinetic energy explained above can be expressed as Ek = m/2 * v2. Since all singly charged ions hold the same amount of kinetic energy, a higher mass (m) results in a lower velocity (v). In other words, heavy molecules fly slower than lighter ones. Within the time-of-flight tube, there is no electric field, but due to the high vacuum, the ions keep traveling in the tube with the velocity they achieved from the electric field. At the other end of the drift tube, the molecules hit a detector. As mentioned earlier, the instrument actually measures the time the ions need to travel (fly) from the original position (the target plate) to the detector on the other end of the time-of-flight tube. From that time, the software can calculate the velocity and with a known kinetic energy, that gives the mass of the ions.

Possible artefacts and why calibration is necessary

The description of MALDI-MS is very simplified but points out that there are many factors that can affect the calculated mass of the analyte(s):

  • Fluctuations in the electronics yielding a varying electric field

  • Uneven target plate (scratches, misalignment, etc.)

  • Laser focus, alignment, timing

  • Temperature induced changes in distance between analyte and detector

Small and continuous changes in the conditions from day to day cause experimental data to exhibit a significant drift over time and between multiple analysis runs, leading to miss-assignment of the peaks and misleading data. As a result, false positives or false negatives may be generated.

To avoid impact of the above-mentioned factors on the result of the analysis and counter-act false results, the system must be calibrated frequently with well-known compounds – calibration standards. Here, the software is told that a certain measured time corresponds to a certain mass that is well known. This underlines why calibration is so important to achieve accurate and trustworthy results.


Calibration involves utilizing a particular set of compounds with precisely known and quantified values. A good calibration standard should cover the mass range of your analytes and contain at least 4 calibration points to enable a good mathematical fit. Also, it should be easy to handle for the end user, with sample preparation, flexibility, assignment, and storage/shelf life in mind.

Polymer Factory’s SpheriCal® is an extremely user-friendly calibration standard, that transforms the calibration routine from a “have to” to a “want to” – for the most accurate and precise MALDI-ToF results possible.

Find out more information about SpheriCal® here


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