Combining SEC/GPC with MALS and DLS



Size Exlusion Chromatography (SEC), also known as Gel Permeation Chromatography (GPC), has been widely used to characterize the molar mass and aggregation state of proteins as well as the molar mass distribution of biopolymers and polymers. However, SEC in itself is a relative method, relying on calibration with well known standards.

  • Multi-angle light scattering (MALS) determines the absolute molar mass of proteins, biopolymers and polymers in solution without reference to molar mass standards, column calibration, or molecular conformation. All constants are measure from first principles and do not depend on vague empirical relations.

  • Dynamic Light Scattering (DLS) directly measures diffusion coefficients and thus derives the hydrodynamic radius.

Both MALS and DLS can be readily coupled to an SEC system to provide the absolute molar mass, root-mean square radius, and hydrodynamic radius of individual peaks of interest. In addition, SEC-MALS can be used for the assessment of size heterogeneity (aggregation and fragmentation), molar mass distribution, degree of pegylation or glycosylation of proteins, stoichiometry of complexes, and conformational information.

 

Just by adding a Wyatt Technology MALS detector to your existing chromatographic system you will be determining absolute molar masses and sizes without ever having to calibrate you GPC again!

 


Each Wyatt DAWN® and miniDAWN® MALS detector comes with ASTRA® 6, the most powerful GPC/SEC software package for collecting and analyzing data associated with protein, macromolecule, polymer and nanoparticle characterization. This versatile software package is used for data collection and analysis of either batch (unfractionated) or chromatographically separated samples.

ASTRA® 6 Software includes specialized routines for:

  • Determinaton of absolute molar mass, size, and the second virial coefficient
  • Integrates light scattering (MALS and QELS/ DLS), RI, UV, and viscometry (VI) measurements
  • Calculate Mw based on column or universal calibration using a ViscoStar™ viscometer
  • Measure intrinsic viscosities and Mark-Houwink parameters
  • Determine polydispersity values (Mw/Mn ) and (Mz/Mn )
  • Count particle number densities
  • Measure the molar mass and rms radius for each elution slice (and therefore the conformation)
  • Determine protein mass and fraction for protein conjugates
  • Correct for interdetector band-broadening
  • Calculate the precision of each result it reports
  • Comply with 21 CFR Part II regulations
  • Learn more about ASTRA