In Situ Online Spectroscopy and Light Scattering Techniques

Within this chapter, we focus on explaining and introducing latest light scattering techniques as dynamic light scattering (DLS) applied to score and optimize biomolecular solutions prior to crystallization experiments. DLS is today the most common and noninvasive method used to obtain information about the dimensions of monomeric or oligomeric macromolecules by observing their Brownian motion. It requires only a relatively small amount of sample and can be done in various environments. Analyzing the homogeneity of a biomolecular solution at different physiochemical conditions, such as concentrations and pH values, and also over time, before outlining crystallization experiments provides most valuable information to increase the success rate of such experiments to obtain X-ray-suitable crystals. Further, light scattering methods can analyze particle size distributions in real time and optionally also in flow mode. The intensity pattern of light scattered by particles in solution is correlated with itself within short time intervals to determine the autocorrelation function, which allows a calculation of the particle diffusion constant, providing the value of the hydrodynamic radii (R_h) via the Stokes-Einstein equation. In this context, DLS is today most widely used for characterizing biomolecular suspensions in different fields of life sciences and biotechnology. In the following, we introduce light scattering techniques and explain the application of light scattering techniques using selected examples.