Since the light scattering and concentration are measured for each eluting fraction, the molar mass and size can be determined independently of the elution position. This type of arrangement has many advantages over traditional column calibration methods. Batch (cuvette) mode MALS, where the measured quantities are averaged over all masses and sizes present in the sample.Standard SEC, which depends on column calibration by reference standards.SEC-MALS provides accurate distributions of molar mass and size (root mean square radius Rg), as opposed to: The most common fractionation method used with MALS is HPLC size exclusion chromatography (SEC). These comparatively short run times coupled with the absolute determination of molar mass, size, and A 2 make light scattering the method of choice for accurate and fast macromolecular characterization.Ī chromatography-mode light scattering experiment couples a Multi-Angle static Light Scattering (MALS) detector, such as the DAWN® or miniDAWN®, with a fractionation device that separates macromolecules based on their physical properties. 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. Most importantly, light scattering permits measurement of the solution properties of macromolecules. Why Light Scattering?Īlthough 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. Read the Solutions section to better understand how different techniques are applied to various characterization challenges. Each technique has its advantages and disadvantages for characterizing different properties of the analyte. Light scattering is a technique that can be applied in either batch or chromatography mode. DLS measurements can determine the diffusion coefficient of macromolecules or particles, from which the hydrodynamic radius is calculated. 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.Via the concentration dependence of the scattered intensity, it is possible to quantify molecular interactions as the second virial coefficient ( A 2, for non-specific interactions) or equilibrium dissociation constant ( K D, for non-specific interactions). For certain classes of particles having sizes in the vicinity of the wavelength of the incident light, MALS can help determine shape and structure. For macromolecules much smaller than the wavelength of the incident light, the data are analyzed to determine the molar mass or molecular weight M w and rms radius R g. ![]() 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.Wyatt instruments make two different types of light scattering measurements for absolute molecular characterization: In this sense it is an absolute measurement. In contrast to most methods for characterization, it does not require outside calibration standards. Light scattering comprises a versatile suite of non-invasive techniques for characterizing macromolecules and a wide range of particles in solution. Dynamic & Electrophoretic Light Scattering.
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