Introduction to Light Scattering
Light scattering has become one of the most important techniques for the characterization of macromolecules and nanoparticles in solution. Depending on the analytes, different light scattering techniques can or have to be used. When compared to many other characterization methods, the light scattering techniques show a high precision, have a fast response time and are noninvasive. Once the light scattering technique is set-up properly, absolute measurements of molar mass and particles size can be performed without the use of standards. Beside Laser Diffraction, the most commonly used light scattering techniques are Static Light Scattering (SLS) and Dynamic Light Scattering (DLS):
- Static Light Scattering: This light scattering techniques is also called Classical Light Scattering or Rayleigh Scattering. Static Light Scattering is used for the determination of Molar Mass, Radius of Gyration and the Second Virial coefficient (A2). In Static Light Scattering, the absolute intensity of the scattered light is detected at different scattering angles and the molar mass and radius is calculated from this information. From the relation of molar mass and radius, the so-called conformation plot, additional information about the shape and structure can be extracted. Once this light scattering technique is set-up properly, absolute measurements of molar mass and particles size can be performed without the use of standards.
- Dynamic Light Scattering: This light scattering technique is also called Photon Correlation Spectroscopy (PCS) or Quasi Elastic Light Scattering (QELS). The fluctuation of the scattered light at a certain angle as the effect of the Brownian Motion of the molecules and particles is detected in Dynamic Light Scattering. A correlation function is established using this light scattering data and from that the Hydrodynamic Radius can be calculated from the Dynamic Light Scattering data.
Light scattering can be done in Batch Mode and or in Flow Mode. Batch Mode analysis is easy to perform, but in case of crude complex sample mixtures with a broad and or multi-modal distribution, this approach has significant limitations, as it is difficult to calculate the correct molar mass and particle size distribution with light scattering only. A solution to overcome this limitation is the combination of a separation technique, such as Field-Flow Fractionation or Chromatography, with online light scattering detection (Flow Mode). Field-Flow Fractionation and Size Exclusion Chromatography provide high resolution fractions of the polymers/particles under investigation. After the separation, the eluting monodisperse sample fractions can be ideally sized online by light scattering. Thus the combination of a separation technique and a light scattering detection technique provides a very powerful characterization tool, which exceeds the performance of the single separation or the single detection technology when used alone. An additionally advantage is that sample fractions can be recovered for further subsequent analysis with other techniques after leaving the detector.
The scientific attractiveness of light scattering is based on the unique combination of high sensitivity, high resolution, easy handling and fast analysis times. Other techniques, which can be seen more as complementary than competing, are Analytical Ultracentrifugation, Mass Spectroscopy and membrane Osmometry. Analytical Ultracentrifugation for example shows high resolution, but is much more expensive and complicated and has longer run times. Mass Spectroscopy has become very famous in the last decade, but this technique also requires a big investment and does not reach the same upper molar mass range as light scattering does. Membrane Osmometry is another suitable technique, but it is not possible to use this technology as online detector and the resolution and molar mass range is much more restricted than what can be expected from light scattering.