Dynamic range multiwavelength particle characterization using analytical ultracentrifugation

Abstract

We demonstrate how a sophisticated data analysis methodology enables us to perform multiwavelength evaluations of dynamic rotor speed gradient experiments obtained by analytical ultracentrifugation equipped with a multiwavelength detector. Our data evaluation tool HDR-MULTIFIT allows for the accurate analysis of sedimentation coefficient distributions which can be converted to particle size distributions. By means of multiwavelength evaluation, species dependent extinction spectra can be determined even for complex mixtures. Moreover, optical and hydrodynamic properties can be correlated for spherical particles of known optical properties applying multiwavelength evaluation and Mie's theory leading to a significant increase in the dynamic range of the experiment. We provide the theoretical background about the operation principle of our methodology and compare the performance of the multiwavelength analysis to the conventional single wavelength analysis as it is applied in turbidity analysis. We validate our technique using NIST traceable reference particles and show that our technique is universally applicable to materials of known and unknown optical properties, thus clearly extending the possibilities of particle analysis.

A Comprehensive Brownian Dynamics-Based Forward Model for Analytical (Ultra)Centrifugation

“…For all simulations, the rotor speed was set at 2000 rpm for the first 10 s and then gradually increased to 30 000 rpm at a steady acceleration of 10 rpm s −1 . The acceleration was then increased to 30 rpm s −1 until a speed of 40 000 rpm was attained upon which the rotor speed was kept steady for 600 s. This was done to simulate the actual situation as seen in our AUC, where possibly a safety mechanism prevented the continuous acceleration until a rotor speed of 40 000 rpm could be achieved . Synthetic data are obtained at a radial location of 6.5 cm from the axis of rotation by calculating the extinction in the corresponding bin as a function of time.…”

“…It relates the intensities of the incident ( I 0 ) and transmitted light ( I ) beam that gets attenuated by colloidal particles of extinction coefficient (ε) with total concentration over a path length of l at a wavelength λ. Absorbance and scattering contribute to the attenuation of light and the total extinction depends on the refractive indices of the solvent and particles as well as the physical properties of the particles. As explained in prior studies, “extinction” is better suited than “absorbance” as it accounts for the effect of scattering caused by the NPs in suspension . The extinction measured by the detectors is hence dependent on the extinction coefficient which takes into account the contribution of absorbance and scattering.…”

“…A more detailed derivation of these equations can be found in Walter and Peukert . The size parameter which relates the characteristic length of the particle to the wavelength of the incident light is an important quantity in the Mie calculations and is defined as …”

“…In addition to being a noninvasive technique, no reference systems or calibration experiments are required. More powerful data evaluation procedures have been developed recently to extract information from the sedimentation data . With accurate and powerful data evaluation techniques it is possible to gain information on the sedimentation coefficient distribution, shape anisotropy, interaction parameters as well as optical properties of the colloidal particles.…”

“…Although the information on the particle diffusion cannot be captured, this method has been used extensively to characterize colloidal systems with multimodal or broad PSDs. This concept was then adapted to MWL detectors to gain access to the optical properties of the NPs and to further extend the dynamic range of the measurements . The wide distribution of the particle size necessitates the need to correct the effect of size dependent optical properties like the extinction coefficients of the NPs.…”