For example small-angle X-ray scattering (SAXS), NMR spectroscopy and crystallization experiments usually require a high degree of particle size homogeneity, i.e. In this context DLS is frequently used in combination with other scattering techniques or mass spectrometry. Borgstahl ( 2006) in terms of X-ray crystallography or exemplified for RNA-protein complexes (Stetefeld et al. DLS is very often used in preparation and systematic optimization of sample solutions (pH-value, ionic strength, solvent etc.) to meet strict requirements of particle homogeneity and long-term stability in further structural investigation of macromolecules, see e.g. Further, all changes of D over time for given particles, typically due to particle size changes, can also be followed in real time. 10 kDa to several MDa for compact globular macromolecules, can be investigated. This relationship between diffusion and size was established in 1906 and is also useful for complementary methods like Taylor-Dispersion analysis or nano particle tracking analysis ( k B is the Boltzmann constant 1.380648 × 10 −23 J K −1):ĭepending on the implemented auto-correlation unit (and a few other parameters of the setup), particles in a wide range of hydrodynamic radii, typically from below 1 nm up to a few µm, which corresponds to a molecular weight range from approx. As for most measurements the temperature T is kept constant and the viscosity η of the sample solution is known, or can be determined rather easily, this translational diffusion constant D can be used to determine the hydrodynamic radius r h of a corresponding spherical particle, according to the Stokes - Einstein equation (Eq. The ACF can be evaluated by the CONTIN algorithm according to Provencher ( 1982), which allows to fit even a complex decay of correlation in polydisperse solutions to calculate the respective diffusion constants (D) and process the data. Decay time constants of small particles are shorter in agreement with the dependency of diffusion speed on particle mass (Fig. Depending on how fast particles are diffusing, the correlation decay time constants, which are derived from the exponential auto-correlation function (ACF) are individually different. Scattering intensity pattern are correlated with themselves (auto- correlation) after short delay intervals of time (τ) to monitor the continuous decay of correlation. 6.2), which results in characteristic size- dependent fluctuation intervals of the electric field component of the scattered light. This is explained by Brownian motion of particles in and around the sample volume that is in focus of the laser (Fig. The number of photons hitting the detector, which can be a photo-multiplier tube, is fluctuating over time. The laser light that is scattered by the particles inside a small focal volume of a DLS sample is continuously recorded and quantified by a photon-counting detector positioned at a specific angle if not hindered by other geometrical restraints, an angle of 90° might be suitable for the experimental setup. In general, all kinds of particles scatter electromagnetic waves in all directions, depending on their size isotropically or anisotropically.
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