Themolecular mass (m) is the mass of a givenmolecule, often expressed inunits ofdaltons (Da).[1] Different molecules of the same compound may have different molecular masses because they contain differentisotopes of an element. The derived quantityrelative molecular mass is theunitless ratio of the mass of a molecule to theatomic mass constant (which is equal to one dalton).[2]
The molecular mass and relative molecular mass are distinct from but related to themolar mass. Themolar mass is defined as the mass of a given substance divided by theamount of the substance, and is expressed ingrams permole (g/mol). That makes the molar mass anaverage of many particles or molecules (weighted by abundance of theisotopes), and the molecular mass the mass of one specific particle or molecule. The molar mass is usually the more appropriate quantity when dealing with macroscopic (weigh-able) quantities of a substance.
The definition ofmolecular weight is most authoritatively synonymous withrelative molecular mass, which is dimensionless; however, in common practice, use of this terminology is highly variable. When the molecular weight is given with the unit Da, it is frequently as a weighted average (by abundance) similar to the molar mass but with different units. Inmolecular biology andbiochemistry, the mass of macromolecules is referred to as their molecular weight and is expressed inkilodaltons (kDa), although the numerical value is often approximate and representative of an average.
The terms "molecular mass", "molecular weight", and "molar mass" may be used interchangeably in less formal contexts where unit- and quantity-correctness is not needed. The molecular mass is more commonly used when referring to the mass of a single or specific well-defined molecule and less commonly than molecular weight when referring to a weighted average of a sample. Prior to the2019 revision of the SI, quantities expressed in daltons (Da) were by definition numerically equivalent to molar mass expressed in the units g/mol and were thus strictly numerically interchangeable. After the 2019 revision, this relationship is only approximate, but the equivalence may still be assumed for all practical purposes.
The molecular mass of small to medium size molecules, measured by mass spectrometry, can be used to determine thecomposition of elements in the molecule. The molecular masses of macromolecules, such as proteins, can also be determined by mass spectrometry; however, methods based onviscosity and light-scattering are also used to determine molecular mass whencrystallographic or mass spectrometric data are not available.
Molecular masses are calculated from theatomic masses of eachnuclide present in the molecule, whilemolar masses and relative molecular masses (molecular weights) are calculated from thestandard atomic weights[3] of eachelement. The standard atomic weight takes into account theisotopic distribution of the element in a given sample (usually assumed to be "normal"). For example,water has a molar mass of 18.0153(3) g/mol, but individual water molecules have molecular masses which range between 18.010 564 6863(15) Da (1H 216O) and 22.027 7364(9) Da (2H 218O).
Atomic and molecular masses are usually reported indaltons, which is defined in terms of the mass of theisotope12C (carbon-12). However, the nameunified atomic mass unit (u) is still used in common practice. Relative atomic and molecular masses as defined aredimensionless. Molar masses when expressed ing/mol have almost identical numerical values as relative atomic and molecular masses. For example, the molar mass and molecular mass ofmethane, whose molecular formula is CH4, are calculated respectively as follows:
Molar mass of CH4
Standard atomic weight
Number of atoms
Total molar mass (g/mol) or molecular weight (unitless)
C
12.011
1
12.011
H
1.008
4
4.032
CH4
16.043
Molecular mass of12C1H4
Nuclide mass (Da or u)
Number of atoms
Total molecular mass (Da or u)
12C
12.0000
1
12.0000
1H
1.007825
4
4.0313
CH4
16.0313
The uncertainty in molecular mass reflects variance (error) in measurement not the natural variance in isotopic abundances across the globe. In high-resolutionmass spectrometry the mass isotopomers12C1H4 and13C1H4 are observed as distinct molecules, with molecular masses of approximately 16.031 Da and 17.035 Da, respectively. The intensity of the mass-spectrometry peaks is proportional to the isotopic abundances in the molecular species.12C2H1H3 can also be observed with molecular mass of 17 Da.
In mass spectrometry, the molecular mass of a small molecule is usually reported as themonoisotopic mass: that is, the mass of the molecule containing only the most common isotope of each element. This also differs subtly from the molecular mass in that the choice of isotopes is defined and thus is a single specific molecular mass out of the (perhaps many) possibilities. The masses used to compute the monoisotopic molecular mass are found in a table of isotopic masses and are not found in a typical periodic table. The average molecular mass is often used for larger molecules, since molecules with many atoms are often unlikely to be composed exclusively of the most abundant isotope of each element. A theoretical average molecular mass can be calculated using thestandard atomic weights found in a typical periodic table. The average molecular mass of a very small sample, however, might differ substantially from this since a single sample average is not the same as the average of many geographically distributed samples.
Mass photometry (MP) is a rapid, in-solution, label-free method of obtaining the molecular mass of proteins, lipids, sugars and nucleic acids at the single-molecule level. The technique is based on interferometric scattered light microscopy.[4] Contrast from scattered light by a single binding event at the interface between the protein solution and glass slide is detected and is linearly proportional to the mass of the molecule. This technique can also be used to measure sample homogeneity,[5] to detect proteinoligomerisation states, and to identify complex macromolecular assemblies (ribosomes,GroEL,AAV) and protein interactions such as protein-protein interactions.[6] Mass photometry can accurately measure molecular mass over a wide range of molecular masses (40 kDa – 5 MDa).
To a first approximation, the basis for determination of molecular mass according toMark–Houwink relations[7] is the fact that theintrinsic viscosity ofsolutions (orsuspensions) of macromolecules depends on volumetric proportion of the dispersed particles in a particular solvent. Specifically, the hydrodynamic size as related to molecular mass depends on a conversion factor, describing the shape of a particular molecule. This allows the apparent molecular mass to be described from a range of techniques sensitive to hydrodynamic effects, includingDLS,SEC (also known asGPC when the eluent is an organic solvent),viscometry, and diffusion orderednuclear magnetic resonance spectroscopy (DOSY).[8] The apparenthydrodynamic size can then be used to approximate molecular mass using a series of macromolecule-specific standards.[9] As this requires calibration, it's frequently described as a "relative" molecular mass determination method.
It is also possible to determine absolute molecular mass directly from light scattering, traditionally using theZimm method. This can be accomplished either via classicalstatic light scattering or viamulti-angle light scattering detectors. Molecular masses determined by this method do not require calibration, hence the term "absolute". The only external measurement required isrefractive index increment, which describes the change in refractive index with concentration.
^IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). Online version (2019-) created by S. J. Chalk. ISBN 0-9678550-9-8.https://doi.org/10.1351/goldbook.
^Sonn-Segev, A., Belacic, K., Bodrug, T. et al. Quantifying the heterogeneity of macromolecular machines by mass photometry. Nat Commun 11, 1772 (2020).https://doi.org/10.1038/s41467-020-15642-w
^Soltermman et al. Quantifying protein-protein interactions by molecular counting using mass photometry. Angew. Chem Int Ed, 2020, 59(27), 10774-10779
^Paul, Hiemenz C., and Lodge P. Timothy. Polymer Chemistry. Second ed. Boca Raton: CRC P, 2007. 336, 338–339.
