Beckman Instruments, Inc.

The United States of America As Represented by the Secretary of

The United States of America As Represented by the Department of Health

Allen P Minton

James V Sullivan

Arun K Attri

Robert H Giebeler

Paul J Voelker

Jeffrey J Marque

Steven J Chulay, Deceased

Paul Fitze

A method of controlled centrifugation including automatic determination (before or during a centrifugation run) of the time to reach 'completion' (within specified limits, depending on the criteria adopted) of a centrifugal separation. By simulation, the sample solute concentration distributions are determined and examined periodically. Centrifugation completion is deemed to have been reached based on certain predetermined criteria relating to spatial changes in the sample solute concentration distributions between successive examinations. The positions of the end points of the concentration distributions or band edges of the sample solutes (transitions from regions of sample solutes to regions of no sample solutes) are determined to facilitate resolving spatial changes of the concentration distributions. The position of an end point or band edge is characterized by a specific location along the centrifuge tube where an imaginary boundary exists at which no more than a specific % of the total mass of the solute in the band lies outside said boundary. In order to avoid premature termination of centrifugation before actual equilibrium has been reached as a result of excessive frequent checks of the motion of the concentration distribution because of relatively long time scale in sedimentation, an estimate is provided of a characteristic periodic interval between successive checks of sample solute concentration distributions.

Terminating centrifugation on the basis of the mathematically simulated

A process and apparatus for predicting the time and position dependent concentration of macromolecules of differing species in a gradient forming solution during centrifugation is disclosed. A mathematical model of the time dependent behavior of gradient forming solutes in a centrifugal field, based upon the underlying physical principles governing sedimentation and diffusion is formulated. The simplifying approximation is made that the wall effects may be ignored, and that the transport of solute may be treated as a one dimensional process with forces dependent only upon the radial distance. A sedimentation diffusion (Lamm) equation in one dimension is numerically integrated. The sedimentation diffusion equation (Lamm equation) for each macrosolute is solved concurrently with that for the gradient forming solute, utilizing the experimentally based density dependent sedimentation coefficient. The equations are solved for incremental time periods for the centrifuged samples in successive radial segments. The equations use concentration dependent values of sedimentation and diffusion coefficients of gradient forming solutes. There is obtained the time and position dependence of the concentration of both gradient-forming solutes and macrosolutes at a fixed temperature for an arbitrarily specified starting concentrations of the density forming solute. The distribution of the macromolecule follows from distribution of the gradient forming solute for each time period and each radial slice.

Optimum centrifugal separation of particles by transient analysis and

An apparatus for sequentially fractionating a centrifuge tube includes a capillary tube and a means for applying positive pressure. The capillary tube has an O-ring at the lower end thereof. As the capillary tube is placed within the centrifuge tube, the O-ring forms a seal within the tube. Movement of the capillary tube within the centrifuge tube places the liquid in the centrifuge tube under pressure, thus forcing the liquid to flow up through the capillary tube and into a chamber. A chase fluid is then pumped horizontally through the chamber to force the liquid therein through an exit port and into a fraction collector. The apparatus and method of the present invention may be entirely automated and controlled by a single microprocessor.

Method and apparatus for sequential fractionation

An automated system for measuring the concentration gradient of centrifuged solute and directly calculating the molecular weight and/or sedimentation coefficient of an optically absorbing solute. Monochromatic light passes through a stationary slit and a previously centrifuged sample in a tube while the tube is moved vertically at a uniform rate by a platform which is driven by a motor and gearing, including a gear nut which drives a vertical screw attached to the platform. The transmitted light impinges on a photodetector which provides absorbance readings. This provides data of absorbance vs. time through the scanning cycle, which is processed digitally. A microcomputer is used to calculate the molecular weight and/or sedimentation coefficient from this data. The centrifuge tube is supported in a transparent cuvette containing a black plastic adaptor block which conformably receives the tube and supports it in upright position, allowing the light beam to pass through opposite vertical slot portions in the block. The cuvette rests on the platform, which is moved vertically relative to the slit defining the fixed beam path. A microswitch is operated at the upper limit of travel to stop the drive motor. The motor is external to the housing in which the optical components and the sample tube are located. The external motor is coupled by a flexible shaft to the drive gearing. Alternatively, a stepping motor may be employed instead of a synchronous motor to move the centrifuge tube in an incremental fashion rather than at uniform velocity.

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Bethesda, MD, United States of America

Allen P Minton