An unknown compound can be established by identifying the molecular ion peak in the mass spectrum. The molecular ion peak is often weak or absent due to the predominance of fragmentation in high-energy electron beams. In such cases, a low-energy electron beam can be used to scan the spectrum to enhance the intensity of the molecular ion peak. Additionally, chemical ionization, field ionization, and desorption ionization spectra are used to obtain a relatively intense molecular ion peak.

To determine the empirical formula from the molecular ion mass, a high-resolution mass spectrometer like a double-focusing mass spectrometer, a time-of-flight mass spectrometer, an orbitrap mass spectrometer, or a Fourier transform ion cyclotron resonance mass spectrometer is required.

Comparing the intensities of isotope peaks (M+1 and M+2), which arise from the natural abundances of higher-mass isotopes, with the molecular ion peak (M⁺) gives a fair idea of the empirical formula of an unknown compound.

The number of carbon atoms present in the unknown compound can be deduced by comparing the M+1 to M⁺ peak intensity ratio to the expected M+1 contribution per carbon atom.

The nitrogen rule, stating that a molecule having an even molecular weight contains zero or even number of nitrogen atoms, predicts the number of nitrogen atoms present in the unknown compound.

Further, the degree of unsaturation predicted from the formula

indicates the presence of multiple bonds or rings in the unknown compound. Lastly, the study of fragmentation patterns for compounds based on a series of guidelines helps identify signature fragments at characteristic m/z values, suggesting the presence of certain structural features.