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Molar mass turns a weighed sample into a count of specified particles. For a pure chemical substance with specified chemical formula and isotopic abundances, its molar mass iswhere $m$ is the sample mass and $n$ its amount of substance. The SI unit is $\mathrm{kg}\mathrm{mo}{\mathrm{l}}^{-1}$; the common laboratory unit is $\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$, with $1\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}=10^{-3}\mathrm{kg}\mathrm{mo}{\mathrm{l}}^{-1}$. Equal values of amount of substance correspond to equal counts of specified entities, but heavier entities give greater masses.

Combining this definition with molecular count and amount of substance givesThe Avogadro constant is exact under the modern definition of the mole. The mass $M/N_{\mathrm{A}}$ is the mean mass per specified entity; if isotopes differ within the sample, individual molecules need not all have that mean mass.^[ref]

Specify what $n$ counts: water molecules, hydrogen chemical atoms, and oxygen chemical atoms give different values of amount of substance for the same sample. For a mixture, dividing total mass by total amount of substance of its chosen entities gives a composition-dependent mean. A polymer sample can contain many chain molar masses; its number-average molar mass and weight-average molar mass answer different averaging questions. Relating either to degree of polymerization also requires a unit-mass convention and end-group accounting.

A SEC detector response must be specified before interpreting a peak area. Ultraviolet absorbance depends on a species' response factor and concentration, as described by the Beer-Lambert law; differential refractive index detection depends on how the dissolved species changes refractive index. Equal amounts need not produce equal signal areas across unlike species.

Even a calibrated mass-proportional trace is not a chain-count probability distribution. In an original two-population example, equal recovered masses of chains with molar masses $20,000$ and $80,000\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$ contain four times as many chains in the lighter population. Thus equal signal areas, assuming equal response per unit mass, correspond to number fractions $4/5$ and $1/5$: their number-average molar mass is $32,000\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$, while their weight-average molar mass is $50,000\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$. Converting a continuous trace additionally requires response correction and the appropriate change of variables between elution and molar mass bins.

SEC light-scattering detection adds information from scattering of light to the separation. Static scattering with a concentration readout can infer a weight-average molar mass for material in an eluting slice without the standard-family retention calibration. Dynamic scattering estimates a diffusion coefficient and hence a hydrodynamic radius. “Absolute” here means independent of a retention-to-molar mass standard curve; it still needs calibrated detectors, optical response parameters, and a valid sample model. Coeluting species produce a mixture-weighted result, rather than individually identified molecules.

A concrete manufacturer test used a Bio SEC-3 $300\mathring{\mathrm{A}}$ column, $7.8\times 300\mathrm{mm}$ with $3\mu \mathrm{m}$ particles, an Agilent 1260 Infinity G5611A quaternary pump and G5667A autosampler, a $10\mathrm{mm}$ diode-array flow cell, and dual-angle/dynamic scattering detectors. A buffer solution at pH $7.4$ flowed at $0.75\mathrm{mL}\mathrm{mi}{\mathrm{n}}^{-1}$; column and scattering detector were at $30{}^{\circ }\mathrm{C}$. Absorbance at $280\mathrm{nm}$ supplied a concentration-sensitive readout. Dilution and loading comparisons tested response: seventeen ovalbumin injections spanning $2$–$960\mu \mathrm{g}$ gave a reported average $47,198\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$ with $2\%$ relative standard deviation. The note reports poorer separation of thyroglobulin from aggregates and an elevated mass estimate, illustrating the coelution limit. Its $0.2\mu \mathrm{m}$ sample filtration also limits interpretation of larger protein aggregation products removed before injection.^[ref]

Suppose a synthetic polymer population contains three chains of molar mass $10000\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$ for every one chain of molar mass $40000\mathrm{g}\mathrm{mo}{\mathrm{l}}^{-1}$. Multiplying both chain counts by any common factor leaves the averages unchanged. Substitution into the existing number-average molar mass and weight-average molar mass definitions givesThe larger chains are only one quarter of the chain count but supply $40000/70000=4/7$ of the mass. Thus a weighted mean that emphasizes mass gives a larger result than one that weights each molecule equally. Total mass divided by total chain amount of substance gives $M_n$, while $M_w/N_{\mathrm{A}}$ is not the mean mass of a randomly chosen chain. Inferring chain counts from size-exclusion chromatography still requires the existing SEC detector response and SEC molar mass calibration assumptions.

The weight-average molar mass weights each population by its contribution to total mass:Here $n_i$ counts molecules with molar mass $M_i$. SEC light-scattering detection can infer this average for an eluting slice under its optical and concentration assumptions; overlapping species need not have the same individual molar mass.