Observations on the labeling of fringes for ultracentrifugation with interference optics

Abstract

Molecular weight determinations of protein solutions at sedimentation equilibrium with interference optics often require the identification or “labeling” of a unique fringe. A system for recognizing the zero-order fringe is described which relies on interferograms recorded with light of different wavelengths. Fringe positions on the wavelength dependent interferograms generally do not superimpose for equivalent positions in the centrifuge cell, with the exception of the zero order or a chromatic fringe. Therefore, by applying coincidence tests similar to those used in reading a vernier scale, an unambiguous determination of the zero-order fringe is obtained. With this information the absolute concentrations in the cell are measured by one of the standard methods (e.g., conservation of mass, fringe relaxation) to obtain data in the appropriate form for calculating molecular weights. The operations are readily conducted with the usual green light (λ = 546 nm) and a supplementary wavelength such as blue (λ = 436 nm) and yield excellent molecular weight results in experiments with ribonuclease. White light, i.e., unfiltered emission from the mercury lamp, may also be employed as the supplementary light. In this case, the ambiguities arising from the use of white light to select a unique fringe solely by its appearance (2) can be fully eliminated by applying the coincidence test with interferograms generated with light of 546 nm. Finally, experiments with myglobin demonstrate that, with the proper choice of the monochromatic wavelengths, successful measurements can be performed on materials for which use of white light is precluded due to absorption of light by the material in the visible region of the spectrum.