Molecular Formula and Molecular Weight: What Each Tells You

A molecular formula states how many atoms of each element one molecule contains. Bradykinin, a nine-residue peptide, is C50H73N15O11: fifty carbons, seventy-three hydrogens, fifteen nitrogens and eleven oxygens. That is a count, and it is exact. A molecular weight is calculated from the formula, by multiplying each element’s count by a mass for that element and summing, so its value depends on which masses are used. Two correct molecular weights for one formula are routine; two correct formulae for one molecule are not.

The dalton (Da) and the unified atomic mass unit (u) are two names for one unit: a twelfth of the mass of a free carbon-12 atom at rest in its ground state, about 1.66 × 10⁻²⁷ kg. A mass in daltons is the mass of one molecule. A molar mass in grams per mole is the mass of one mole of them — exactly 6.022 140 76 × 10²³ molecules. Molar mass is the relative molecular mass, which is what "molecular weight" strictly means, multiplied by the molar mass constant. Under the definition of the mole in force since 2019, that constant is no longer exactly one gram per mole but 1.00000000105 g/mol. For laboratory purposes the numbers coincide: 1060.2 Da per molecule is 1060.2 g/mol.

Most elements are mixtures of isotopes. Carbon is mostly carbon-12, which fixes the scale at exactly 12, with between 0.96% and 1.16% carbon-13 at 13.003. An average molecular weight uses standard atomic weights, which reflect those natural mixtures. A monoisotopic mass uses only the principal, most abundant isotope of each element. The two describe different things — a population of molecules, and one isotopic species within it — and the gap between them grows with size.

With roughly one carbon atom in a hundred being carbon-13, a molecule containing hundreds of carbons is unlikely to consist of principal isotopes alone. The species made only of principal isotopes, which a monoisotopic mass describes, becomes a minority, and for proteins its peak is typically hidden in noise or not detected at all. Its position has to be inferred from the isotope pattern, and inference errs: in one evaluation, off-by-one-dalton errors affected 31.9% of proteins on average. For intact proteins the default reported mass is the average, which can be obtained whether or not the isotope peaks are resolved.

Most disagreements between a database, a certificate and a label over a molecular weight have ordinary causes, each of which can be checked.

• One figure is average and the other monoisotopic; for a peptide of about 1,000 Da they differ by more than half a dalton
• One record describes a salt, such as the separate PubChem record for bradykinin triacetate, and the other the free molecule
• The structural state differs: each disulfide bond removes two hydrogen atoms, and a C-terminal amide replaces a hydroxyl group with an amino group
• The atomic weights differ: carbon’s standard atomic weight is an interval, 12.0096 to 12.0116, and software uses a single value from within it
• One figure is for an ion rather than the neutral molecule

A mass spectrometer measures ions, reported as mass-to-charge ratio, m/z. In electrospray ionisation, peptides and proteins pick up protons — often several — or metal ions in their place, so one molecule can appear as a series of peaks. An ion carrying n protons appears at m/z = (M + n × 1.00728) / n, where M is the neutral mass and 1.00728 Da is the mass of a proton. Software can deconvolute such a series into a single neutral mass.

An observed mass that agrees with the formula, within the accuracy of the instrument, is evidence that the material has that composition. It is not evidence of structure: isomers share a formula, and a peptide with its residues rearranged has exactly the same mass. Nor is it evidence of purity beyond what the spectrum shows of other components, which is why mass results sit beside chromatographic ones rather than replacing them.