A comparison of data quality using quartz vs. sapphire cell windows in analytical ultracentrifugation
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ORIGINAL ARTICLE
A comparison of data quality using quartz vs. sapphire cell windows in analytical ultracentrifugation Akash Bhattacharya1 · Eric Von Seggern1 Received: 2 June 2020 / Revised: 31 July 2020 / Accepted: 3 August 2020 © European Biophysical Societies’ Association 2020
Abstract Analytical ultracentrifugation (AUC) cells use either quartz or sapphire windows as end caps for the cell housing. Current generation sapphire windows are not recommended for absorbance data collection below 235 nm, because the window material shows a precipitous drop in transmittance at low wavelengths due to impurities in the sapphire. Quartz windows can be used below 235 nm as they do not exhibit adverse transmittance at low wavelengths. In this study, we demonstrate the optical properties of new generation sapphire windows and compare them to those of quartz windows across a wide range of wavelengths and present the results of sedimentation velocity experiments on BSA using both types of windows using data collected at both the 280 nm absorbance maxima as well as the 230–240 nm (closer to the peptide bond maximum). Our results show that the quartz and new generation sapphire windows deliver identical results in absorbance mode. We also demonstrate that quartz windows suffer significant mechanical deformation while spinning at very high speeds, while sapphire windows do not. This renders Rayleigh interference mode data collected at high speeds using quartz windows much noisier than with sapphire windows—which we have quantified by measuring how the signal to noise ratio of Fourier transformed Rayleigh interference scans degrades at high speed. Thus, we conclude that new-generation sapphire windows can be used for all AUC experiments through almost the entire mid UV range—obviating the need for quartz windows, unless wavelengths below 220 nm must be accessed. Keywords Optima AUC · Analytical ultracentrifugation · Rayleigh interference · Sedimentation coefficient
Introduction The analytical ultracentrifuge (Cole et al. 2008) (Cole et al. 2013) tracks the sedimentation of analytes under strong rotational acceleration and provides mass, size, and shape information. These fundamental physical parameters can then be further analyzed to assess aggregation, degradation, complex stoichiometry, binding affinities and more (van Holde 2004) (Stafford, 2003) (Howlett, Minton, and Rivas 2006). AUC experiments are conducted by loading the sample and buffer in separate channels of a metal or epoxy composite centerpiece, which is sandwiched between optical endpieces. This Special Issue: Analytical Ultracentrifugation 2019. * Akash Bhattacharya [email protected] Eric Von Seggern [email protected] 1
Beckman Coulter Life Sciences, Loveland, CO, USA
arrangement of endpiece–centerpiece–endpiece is assembled inside a metal housing and torque sealed (BeckmanCoulter 2016). Detection of the sample as it moves radially outwards while sedimenting is accomplished by either UV–visible absorbance or via Rayleigh interference. The
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