# Relative abundance of mixture in mass spectra

In MS, as far as I know, the $$y$$-axis, relative abundance means the number of ions detected. Then does ratio of heights of two peaks stand for molar ratio, not mass ratio

For example, given that I analyze a mixture of two components A and B using MS, and there appears only molecular ion peak (although it is unrealistic), then is the ratio of heights of two peaks equal to molar ratio of A and B?

• No. There are all sorts of ionisation effects that influence the peak heights that have nothing to do with the molar ratios. Phosphine oxides for example ionise very well and a small amount can completely dominate a MS spectrum. Aug 2 '21 at 7:55

There are universal detectors (e.g. CAD) with which you can compare signals of different substances. But MS detectors are not universal — meaning that high intensity of the signal doesn't necessarily mean high concentration. And vice versa.

Whether the signal is high or not will depend on how well the compound is ionized. So in case of you A & B compounds:

• A may be well-ionized and thus the signal will be high even though the concentration is low;
• B is poorly ionized and you may not even see a peak even though the concentration may be high.

To identify how much of analyte you have you first need to run A with a known concentration so that you determine the relationship between area and concentration. Then if you use the same LCMS conditions you can run A with your to-be-determined concentration and by ratios of peak areas you can determine the unknown concentration.

Note, that this calculation depends on the linearity of the signal. Meaning: if the area is smaller by $$x$$ compared to the signal of known concentration, then the concentration will be also smaller by $$x$$. This isn't often the case and you may need to run multiple known concentrations to find out if the signal is actually linear. This allows you to build calibration curve which results in an $$ax + b$$ equation that you can further use to determine the unknown concentration.

To sum up: you can compare the signal of the same compound in different runs (given LCMS conditions and mixture matrix were the same). But not the signal of different compounds.

• Oh.. I forgot that MS detector can detect only charged ion. Thanks a lot. Aug 2 '21 at 9:52
• Minor correction: There are no universal HPLC detectors, even charged aerosol detector will give different peak heights and areas for simple anions. I do not know the exact reason but there are limitations. Aug 2 '21 at 15:39
• @M. Farooq: In 1980s, Waters have made two different detectors with their HPLCs: UV detectors and refractive detectors. The refractive detectors are universal and detect every component in the mixture. However, their accuracy was very poor when compared to UV detectors. Aug 2 '21 at 16:29
• @MathewMahindaratne, You are right that RI and CAD should ideally respond to every analyte "equally" but this is rarely the case. For example, if we have 0.1 mM of analyte A and B, the peak area will not be identical in RI or CAD. The RI has reproducibility problems and it cannot be used with gradient methods. Aug 2 '21 at 16:55