A method for measuring sedimentation and diffusion of macromolecules in capillary tubes by total intensity and quasi-elastic light-scattering techniques

✍ Scribed by M. A. Loewenstein; Meyer H. Birnboim

• Publisher: Wiley (John Wiley & Sons)
• Year: 1975
• Tongue: English
• Weight: 636 KB
• Volume: 14
• Category: Article
• ISSN: 0006-3525
• DOI: 10.1002/bip.1975.360140214

✦ Synopsis

Abstract

Light scattered from a macromolecular solution in a capillary tube is used to determine both the sedimentation and translational diffusion coefficients. The capillary tube is spun in a preparative centrifuge, removed, and placed in a light‐scattering photometer equipped with a scanning mechanism. The intensity distribution of scattered light along the tube represents the concentration profile in the tube and provides the measure of boundary migration. The sedimentation coefficient is determined from this measure and the applied centrifugal field. The diffusion coefficient is obtained from a time‐autocorrelation analysis of fluctuations in intensity of light scattered from any fixed point of the profile. These coefficients were obtained for two monodisperse systems, R17 bacteriophage and 28__s__ ribosomal rat liver RNA. The molecular weights obtained from ratios of these coefficients are in good agreement with literature values. In the sedimentation analysis, deviations from linearity between boundary displacement and applied field were found to be less than 1%. This precision confirms that the boundary is stable for the capillary geometry even in the absence of a preformed density gradient. The sedimentation coefficients of identical samples were also measured with the Spinco Model E analytical ultracentrifuge; results of the two methods agree to within 4%.

As a consequence of the capillary tube geometry and light‐scattering detection, sedimentation coefficients can be obtained from sample volumes of less than 100 μl. This detection techniques is thus far demonstrated to be at least an order of magnitude more sensitive than Schlieren optics, thereby useful when uv absorption is not applicable. For diffusion measurements there are also several inherent advantages. The diffusion coefficient is obtained from the identical sample, and scanning provides the capability to measure D from various parts of the sedimentation profiles and thereby directly explore concentration dependence, homogeneity, and integrity of the sample. The capillary tube with a layer of silicone oil over the sample and centrifugation provides an effective method to cleanse the solution and trap all dust.