Introduction to Light Scattering Theory
Light scattering comprises a versatile suite of non-invasive techniques for characterizing macromolecules and a wide range of particles in solution. In contrast to most methods for characterization, it does not require outside calibration standards. In this sense it is an absolute measurement.
Wyatt instruments make two different types of light scattering measurements for absolute molecular characterization:
- Multi-Angle Light Scattering (MALS), Static Light Scattering (SLS) or Classical Light Scattering - In MALS the intensity of the scattered light is measured as a function of angle. For macromolecules much smaller than the wavelength of the incident light, the data are analyzed to determine the molar mass or molecular weight Mw and rms radius rg. For certain classes of particles having sizes in the vicinity of the wavelength of the incident light, MALS can help determine shape and structure.
Via the concentration dependence of the scattered intensity, it is possible to quantify molecular interactions as the second virial coefficient (A2, for non-specific interactions) or equilibrium dissociation constant (KD, for non-specific interactions).
- Dynamic Light Scattering (DLS) or Quasi-elastic Light Scattering (QELS) - In a DLS measurement, time-dependent fluctuations in the scattered light signal are measured using a fast photon counter. DLS measurements can determine the diffusion coefficient of macromolecules or particles, from which the hydrodynamic radius is calculated.
Light scattering is a technique that can be applied in either batch or chromatography mode. Each technique has its advantages and disadvantages for characterizing different properties of the analyte. Read the Solutions section to better understand how different techniques are applied to various characterization challenges.
Why Light Scattering?
Although absolute molecular weights can also be determined via mass spectrometry, membrane osmometry, and sedimentation equilibrium (analytical centrifugation), only light scattering covers so broad a range of macromolecules including their oligomeric states. Most importantly, light scattering permits measurement of the solution properties of macromolecules. While a sedimentation equilibrium run may require 72 hours, a size exclusion chromatography/light scattering study may be completed in well under an hour, and a batch mode analysis in a few minutes. These comparatively short run times coupled with the absolute determination of molar mass, size, and A2 make light scattering the method of choice for accurate and fast macromolecular characterization.
Wyatt Technology Corporation is grateful to the following individuals for the use of materials and graphics in this theory section:
- Dr. Ewa Folta-Stogniew at the Yale School of Medicine. For examples of light scattering theory, analysis, applications, and example results, please visit her light scattering website.
- Dr. Eric R. Weeks in the Department of Physics at Emory University. Dr. Weeks provided the Brownian motion animation in the DLS theory section. For information about his research and to view more fascinating pictures, please visit his website.