Analytical band centrifugation (ABC) is a powerful tool for the analysis of macromolecules and nanoparticles. Although it offers several advantages over the sedimentation velocity (SV) experiment like a physical separation of the individual components and the possibility to perform chemical reactions, its analysis is still very restricted. Therefore, we investigated the integration of ABC data as an alternative approach, as this results in data similar to SV, which can then be evaluated by many established evaluation programs. We investigated this method using two different test systems, myoglobin as a biopolymer with significant diffusion and 100 nm polystyrene latex as a large particle with negligible diffusion, and found some limiting issues. These are namely, broadening of the initial boundary by diffusion of the sample, which can be taken into account and the dynamic density gradient between the solvent in the sector and the overlaid solution, which deforms the initial band upon movement through the gradient and is currently not taken into account. We show the influence these two factors have on the evaluation and show that it is possible to calculate the time-dependent change in solvent density and viscosity in the AUC cell using the integrated form of Fick's second law. We conclude that taking the dynamic density gradient into account will open ABC for the sophisticated methods based on the analysis of the whole sedimentation boundary and not just the determination of an average sedimentation coefficient.

The band sedimentation experiment in analytical ultracentrifugation (AUC) allows for the performance of a chemical reaction inside the AUC and also offers separation of individual pure components in a sedimentation velocity experiment. Although this experiment offers exciting possibilities for application, it is barely used. This is related to the bad definition of the initial conditions. Both the duration and the time of the solution overlay during rotor acceleration are not known. In this study, we investigate these conditions under the variation of the overlay volume using recording of interference patterns in a continuous mode during the acceleration of the rotor. It was found that the overlay occurs at rotor speeds between 770 and 2000 rpm, which is very low compared to typical experimental rotor speeds from 3 000 to 60 000 rpm and therefore elucidates that the generated reaction products, respectively, overlaid species are subject to the centrifugal force almost from the beginning. Also, the duration of the overlay is less than 1.2 s, which is very fast compared to hours of centrifugation time for an experiment and we demonstrated that the overlay compartment is completely emptied during overlay allowing for the precise calculation of the meniscus using the known sample sector geometry. Our results show that the initial conditions of the experiment are defined and should make an adapted analysis possible if the interdiffusion of the two solvents is taken into account, which lead to a dynamic density gradient.

Although silver particles are used in various applications and a countless amount of synthesis routes exists, their formation mechanism is still poorly understood. Especially the first species formed directly after nucleation challenge analysis methods with their small size and transient nature. Analytical ultracentrifugation (AUC) has already proven to provide high size resolution and therefore enables the characterization of early nucleation species. Herein, we present an experiment of multiwavelength (MWL)-AUC of silver clusters, which revealed seven different cluster species. They consist of less than 10 atoms and therefore represent the first species formed after nucleation. Using MWL-AUC, UV/vis spectra could be allocated to each of them, which is shown for the first time. These findings establish MWL-AUC as a high-resolution tool to investigate a nucleation mechanism for silver and other metal nanoparticles.

Gold nanoclusters consisting of a specific atom number have gained popularity in research in recent years due to their outstanding properties. Due to their molecule-like behavior, their properties depend strongly on their size. Although they represent the link species between atoms and nanoparticles and are the subject of current research, a high-resolution characterization is still missing. Here, we used the band forming experiment in analytical ultracentrifugation (AUC) to characterize the gold nanoclusters in the moment of their generation using thioglycerol as a stabilizer. The concentration variation of the gold precursor, reducing agent, and stabilizer was investigated. The formation of different cluster species from the smallest Au4 up to Au911 could be observed. Very stable clusters of Au55 appear in every experiment and other cluster sizes more rarely. The extracted UV/Vis spectra could additionally be correlated to every cluster. The variation in the concentration of sodium borohydride and the stabilizer did not lead to a clear trend, but the gold ion concentration directed the size of the formed clusters. A decrease seemed to promote the generation of a higher abundance of smaller clusters accompanied by less big clusters, and vice versa. These results present the characterization of the different nanocluster generations directly in the formation process of nanoparticles and therefore are a contribution to the understanding of their formation.