Absolute molar mass, size and conformation of peptides, proteins and polymers

SEC-MALS combines size-exclusion chromatography with multi-angle light scattering to overcome the limitations of analytical SEC and column calibration. It characterises all types of macromolecules from peptides and small oligomers, to proteins and polymers, limited only by column resolution.

Traditional analytical SEC is a relative method: it depends on the assumption that the elution properties of the reference molecules are identical to those of the analyte. Even under ideal conditions, those properties depend on the conformation and specific volume of the molecule so the assumption is, at best, questionable. In addition, non-ideal column interactions may also lead to significant discrepancies in molar mass.

That’s why an absolute method like SEC-MALS - pioneered by Wyatt Technology nearly 40 year ago - is essential for accurate determination of molar mass and size. With the addition of one of Wyatt’s DAWN®, miniDAWN® or microDAWN® MALS instruments downstream of the column, a standard HP-SEC systems becomes a SEC-MALS system – now capable of much more than just relative molar mass analysis! In fact, SEC-MALS is the foundation for determining multiple molecular properties, and Wyatt’s MALS detectors are widely recognised as the gold standard for reliable characterisation of macromolecules in solution.

Molar mass: The MALS detector analyses each fraction independently of elution volume. Wyatt’s ASTRA® chromatography software combines MALS signals with those from a concentration detector such as UV/Vis or differential refractometer (dRI) to calculate molar mass from first principles. The results include differential and cumulative distributions, moments and polydispersity. Unlike conventional SEC, SEC-MALS will immediately reveal if a chromatographic peak is homogeneous or polydisperse, regardless of peak width, since it determines molar mass absolutely, every second or so of elution time.

Wyatt’s flagship DAWN MALS instrument measures molar mass from 200 g/mol to 10⁹ g/mol with extraordinary sensitivity: it requires as little as 10 nanograms of 100 kDa polystyrene injected on a standard SEC column.

Size: MALS also measures rms radius (a.k.a. radius of gyration), from 10 nm and up to 500 nm. It accomplishes this even without a concentration detector, since size is determined solely from the angular variation of the scattering.

When the analytes are below 10 nm in radius, Wyatt offers other options. One is online dynamic light scattering (DLS), embedded in the MALS instrument, which can measure hydrodynamic radii from 0.5 nm to 50 nm and beyond. The other is differential viscometry using Wyatt’s ViscoStar detector, which is often added for polymer analysis. ASTRA determines hydrodynamic/viscometric radii of polymers by combining molar mass from MALS with specific viscosity from the ViscoStar.

Conformation: ASTRA combines molar mass and size information to evaluate the conformation of polydisperse macromolecules such as polymers or polypeptides.

Conjugation: Combining MALS with two concentration detectors – UV and dRI – enables analysis of binary conjugates such as glycoproteins or block co-polymers. The molar mass of each constituent is determined along with the total molar mass for each eluting fraction.

To learn more about what SEC-MALS can quantify and how it is used in different fields of R&D, visit Wyatt SEC-MALS and Wyatt Solutions.