Optical fine-structure of a meniscus in analytical ultracentrifugation in relation to molecular-weight determinations using the Archibald principle

• Published in: Biochimica et biophysica acta Vol. 28; no. 2; pp. 417 - 431
• Main Author: Trautman, Rodes
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.1958
• Subjects: Centrifugation; Joints; Molecular Weight; Old Medline; Ribonucleases; Ultracentrifugation; CENTRIFUGATION; RIBONUCLEASE
• ISSN: 0006-3002; 1878-2434
• DOI: 10.1016/0006-3002(58)90490-6

1. 1. Ribonuclease sedimenting in 6 M urea is used to show that the apparent molecular weight calculated by the Archibald principle at times less than 3 h at any speed varies about 10% per 0.1 mm error in location of the meniscus on the photographic plate. 2. 2. The main shadow conventionally chosen as the meniscus, instead of being due to the infinite index of refraction gradient casting light out of the system, is probably due to the contact of the meniscus with the top and bottom quartz windows. Since both points cannot be in focus simultaneously, the appearance of their shadows (denoted as the “central shadow”) is extremely dependent upon optical adjustment, especially the position of the light source relative to the optical axis and the camera focus. The central shadow can appear double for off-axis illumination and is a function both of speed and optical properties with pressure of the fluids forming the interface. It is not used in precision location of the meniscus. 3. 3. A double system of diffraction-like bands in the meniscus region is independent of the position of the light source, speed, or the nature of the fluids forming the interface. The bisector between corresponding bands in each half of the double pattern is independent of camera focus, and is chosen to be the location of the interface. The double set of bands are denoted as “flanking fringes” and are probably due to the flat portion of the interface between the windows. The interference causing these fringes may be due to a complex interaction of totally reflecting light and secondary wavelets from the discontinuity. 4. 4. In the absence of detailed optical theory of the fine-structure, the location to + 0.01 mm on the plate of the flanking fringe bisector was indirectly verified as the top of the solution column by test of ribonuclease in acetate buffer. The common intersection of ( ∂c/ ∂/ r)/ rc curves occurred at the location of the bisector. 5. 5. Modification of the Spinco Model E ultracentrifuge to enable direct viewing of the image in the plane of the photographic emulsion is described. 6. 6. Special cells are described which are useful in instrumentation studies. In particular, a mirror quartz facilitates alignment of the light source onto the optical axis so that the central shadow approximates the position of the flanking fringe bisector. 7. 7. A channel mask is described which screens out the meniscus from the solvent side when a double sector centerpiece cell is used. This enables simplication of the complex meniscus region in a separate reacceleration of the cell at the completion of a molecular weight experiment.