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Theory
Understanding Laser Light Scattering Theory

Light scattering is a non-invasive technique 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 technique. Wyatt Technology instruments make two different types of light scattering measurements for absolute molecular characterization:

  • Classical Light Scattering / Static Light Scattering: Here, the intensity of the scattered light is measured as a function of angle. For the case of macromolecules, this is often called Rayleigh scattering and can yield the molar mass, rms radius, and second virial coefficient (A2). For certain classes of particles, classical light scattering can yield the size, shape, and structure.
  • Quasi-elastic Light Scattering (QELS) or Dynamic Light Scattering (DLS): In a QELS measurement, time-dependent fluctuations in the scattered light signal are measured using a fast photon counter. QELS measurements can determine the hydrodynamic radius of macromolecules or particles.

Light scattering is a technique that can be applied in either batch or chromatography mode. In either instance the sample may be recovered at the end of the measurement. Since light scattering provides the weight-averaged molar mass for all molecules in solution, it is generally more useful to utilize the chromatography mode, though each technique has its advantages.

Although absolute molecular weights can be determined also 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.