SEC-MALS
Determine absolute molecular weight and oligomeric state.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Weighing molecules as they leave a column
Size-exclusion chromatography separates molecules by how large they are in solution, not by how heavy. A column calibrated with globular standards turns an elution volume into a mass only if your protein has the same shape and density as those standards; an elongated monomer, a flexible protein or a detergent-solubilised membrane protein can elute where something twice its mass would.
2Reading the result
- Mass across the peak
- Plotted over the chromatogram. A pure species gives a flat line; a mass that falls from the front to the back of a peak is co-eluting species or an equilibrium mixture; a mass that tilts the same way across every peak is misaligned detectors.
- Weight-average mass (Mw)
- The mass of a peak computed from the summed signals across it. Mw/Mn close to 1.00 means a monodisperse peak.
- dn/dc
- How much the refractive index rises per unit of concentration: about 0.185 mL/g for unmodified proteins, lower for glycans, lipids and detergents, which is why conjugates need a protein-conjugate analysis with UV and RI together.
- Delay volume and band broadening
- Each detector sees the peak a little later and a little wider than the one before. Both are measured with a monodisperse standard (often BSA) and corrected before slices from different detectors are combined.
3How it fails
- A mass that slopes across a symmetric single-species peak: the inter-detector delay or the band-broadening correction is wrong.
- A mass consistently too low or too high by a fixed factor: wrong concentration (extinction coefficient, dn/dc) or calibration constant.
- One peak whose mass falls steadily across it: species co-elute, often because the column's range does not bracket them.
- Ragged LS baseline, spikes and scattered masses: a column still shedding particles, unfiltered buffer, or a sample that was not spun.
- Too little protein: light scattering grows with concentration × mass, so small proteins need more material than large ones.
Key terms
Practise these as flashcards →The normalised intensity of light scattered at an angle; the excess over the solvent is what MALS measures.
Relates the excess Rayleigh ratio to concentration, molar mass, Rg and the second virial coefficient; the basis of all static light-scattering mass determination.
From the angular dependence of the scattering, measurable only for particles above roughly 10 nm with visible light.
Using UV and RI together, with each component's extinction coefficient and dn/dc, to split the mass of a glycoprotein or protein–detergent complex into protein and modifier.
Where everything too large to enter the pores elutes; MALS still weighs what comes out there, so aggregates are quantified rather than lumped together.
Calibrating the detectors at different angles against the 90° detector with an isotropic scatterer, so that they agree for small molecules.
When to use it
- Deciding the oligomeric state of a protein in solution, especially when its elution volume is ambiguous (elongated, flexible, glycosylated or detergent-solubilised).
- Measuring the mass and amount of aggregates, fragments and complexes, for example before structural work or as a quality check of a biologic.
- Following complex formation: injecting partners alone and mixed to see a peak of the expected combined mass.
Compared with alternatives
Preparative SEC purifies and its calibration gives an apparent size; add MALS when you need to know what the peaks actually weigh.
Measures size and heterogeneity without a column matrix, so weak or rapidly exchanging complexes are not pulled apart; slower and needs more expertise.
A quick check for aggregates in a cuvette with no separation; gives a hydrodynamic size, not a mass.
Gives an exact covalent mass and modifications, but not the native oligomeric state in solution.