Size Exclusion Chromatography – Multi-Angle Light Scattering

Room 607A

Contact: Krzysztof Skowronek

Access: collaboration/contracted research, experienced users



  •          Binary HPLC Pump 1525 (Waters)
  •          Inline Prep Degasser (Waters)
  •           Manual Injection module Flexinject (100 µl and 5 ml loops) (Waters)
  •          UV/VIS Detector 2489 (Waters)
  •          Column oven (for up to 30 cm columns) (Waters)
  •          Dawn 8+, 8 angles MALS detector (Wyatt)
  •          Optilab T-rEX refractive index detector (Wyatt)
  •          Fraction Collector III (Waters)

General description

This instrument allows calibration-independent measurement of molecular mass in each fraction of SEC. UV/VIS, MALS and refractive index (RI) detectors are in-line with outflow from HPLC system. UV/VIS and RI detectors are used for independent concentration quantification while signal from 8 MALS detectors provides information on light scattering at different angles. MALS data are analysed by selected mathematical formula (by default it is Zimm eqution) to obtain calibration-independent time-resolved molecular mass measurement.

NOTE: In contrast to analytical ultracentrifugation[L] MALS directly measures radius of gyration (not hydrodynamic radius). SEC fractions can be collected by fraction collector for further analysis. Currently we offer two SEC columns:

  • BioSuite 450 Å (8 µm, 7.8 x 300 mm) with resolution range 20 kDa – 1 MDa (for globular proteins)
  • XBridge Protein BEH SEC 200 Å (3.5 µm, 7.8 x 300 mm) with resolution range 10 kDa – 450 kDa (for globular proteins)

SEC-MALS of Waters molecular weight standards resolved on BioSuite 450 column. Peaks: 1 – dimeric thyroglobulin (1.4 MDa), 2 – monomeric thyroglobulin (660 kDa), 3 – IgG (150 kDa), 4 – BSA (66.4 kDa)



  •  Measurement of molecular mass of various biological macromolecules (polysaccharides [using RI detector], proteins, nucleic acids and their complexes)
  • Estimating homogeneity of macromolecule preparation
  • Estimating monodispersity of macromolecules in each fraction
  • Assisting in the last step of macromolecular complex purification (using fraction collector)
  • Determination of stoichiometry of macromolecular complexes

Sample requirements

At least 120 µl at concentration adequate for proper estimation of concentration from relative RI (at least 1-2 mg/ml) equilibrated in chromatography buffer. Sample should be filtered through 0.2 µm filter.


Further reading


2. Mogridge J. Using light scattering to determine the stoichiometry of protein complexes. Methods Mol Biol. 2015;1278:233-8. doi: 10.1007/978-1-4939-2425-7_14. PubMed PMID: 25859953.