US3237855A

US3237855A - Centrifuge apparatus

Info

Publication number: US3237855A
Application number: US289974A
Authority: US
Inventors: Norman G Anderson
Original assignee: Union Carbide Corp
Current assignee: Biochem Immunosystems US Inc
Priority date: 1963-06-24
Filing date: 1963-06-24
Publication date: 1966-03-01
Anticipated expiration: 1983-03-01

Description

March 1, 1966 N. G. ANDERSON CENTRIFUGE APPARATUS 2 Sheets-Sheet 1 Filed June 24, 1963 I NVEN TOR. NORMfl/V 6. ANDER 5011/ BY 64W! 47 4,1

March 1, 1966 N. e. ANDERSON 3,237,855

AAAAAAAAAAAAAAAAA US United States Patent 3,237,855 CENTRIFUGE APPARATUS Norman G. Anderson, Oak Ridge, Tenn., assignor to Union Carbide Corporation, a corporation of New York Filed June 24, 1963, Ser. No. 289,974 3 Claims. (Cl. 233-28) The present invention relates in general to centrifugation and more specifically to an improved centrifuge rotor apparatus combination which permits liquid transfer under dynamic conditions. The apparatus of this invention is especially applicable to studies of biological solutions, as for example breis, in which their extremely minute particles such as nuclei, microsomes and mitochrodria represent only a small percentage of the total mass, and which differ only slightly with respect to density.

In density gradient centrifugation using swinging bucket rotors, sedimentation should ideally occur in sectorshaped tubes or compartments to avoid undesirable wall effects.- Early low-speed studies in glass centrifuge tubes of modified sector shape demonstrated that adequate separations of liver cell components could be obtained; however operation periods as long as 18 hours were required, and only 2 ml., of sample could be separated per tube. To eliminate the long interval required for filling with the density gradient, at least one method has been proposed for loading tubes while the centrifuge was running, using centrifugal force to prevent mixing. It was still necessary, however, to recover the separated zones with the tubes at rest.

Since most difiiculties encountered in the aforesaid method are associated with the use of tubes, it would be highly desirable to be able to achieve the requisite zonal separation of biological particles without resort to apparatus employing such tubes.

It is therefore the general object of the present invention to provide a preparative ultracentrifuge capable of achieving zonal separations with a resolution approaching that of an analytical ultracentrifuge for the ultimate isolation of subcellular particles and viruses, and for separation of proteins and other macromolecules which differ in sedimentation rate or density.

It is a more particular object to provide a hollow centrifuge rotor apparatus combination which permits liquids transfer, i.e., introduction of a density gradient and sample into the rotor and recovery of the separated Zones, while the rotor is spinning.

Other and more particular objects will be obvious from the specification appearing hereinafter and from the appended claims.

The specific nature of this invention will be apparent to one skilled in the art from the following detailed description taken in conjunction with the annexed set of drawings in which, by way of preferred example only, is illustrated one embodiment of this invention.

In the accompanying drawings:

FIG. 1 is a side elevation partly in section of the rotor and skimmer apparatus embodying this invention.

FIG. 2 is a top plan view of the apparatus of FIG. 1 taken along line 2-2.

FIG. 3 is a plan view of FIG. 1 taken along line 33 showing the lower section of the skimmer in enlarged detail.

Referring now to the drawings, the apparatus shown in FIG. 1 consists of a hollow rotor 10, stationary skimmer, denoted generally by numeral 30, axially positioned within the core of rotor 10, and means 40 for rotating said rotor.

Rotor It} comprises a gene-rally hollow cylindrical body ice having a top closure 11 and a bottom closure 12, an internally contained axially disposed column 13, said column 13 containing an axially disposed internal bore 14 having a top annular groove 15 and a bottom annular groove 16 adapted to receive in concentric alignment the lower portion of an axially disposed skimmer denote-d generally by numeral 36. Advantageously a plurality of at least two but preferably 31) or more vertically disposed septa or vanes 21 are carried by vertical column 13. Such septa ensure the elimination of tangential flow in the rotor. Density gradient chamber 17 bounded by the inner walls of rotor 10 and the outer surface of column 13 is in hydraulic communication with internal bore 14 of column 13 through opening 18 into lower annular groove 16 and opening 19 into upper annular groove 15. Advantageously opening 19 is formed between the internal wall of top closure 11 and a flange 20 which extends from the top portion of column 13 toward the vertical side wall of rotor 10, thereby forming a flow conduit by means of which liquidus material can be directed to the close vicinity of the inner surface of the said vertical side wall.

Skimmer 30 comprises an entrance conduit 31 communicating an external source of liquidus material through opening 22 in upper closure 11 of rotor 10 to the upper annular groove 15 of bore 14, terminating within said annular groove 15 with a flange 32 extending horizontally to the entrance of or into opening 19. Flange 32 in conjunction with an opposing flange 34 forms a passage through which liquidus material forced through entrance conduit 31 is directed to opening 19 and thence into density gradient cavity 17 of rotor '10. Disposed concentrically within entrance conduit 31 and extending downwardly into lower annular groove 16 is exit conduit 33, said exit conduit terminating with radially disposed tubular members 35 having openings communicating with lower annular groove 16. Skimmer 30 is held stationary with respect to rotor 10 by mounting support 36, which can also serve as an enclosure for the rotor.

Numerous design modifications of skimmer 30 will be readily apparent to those skilled in the art, and are considered to be within the scope of the present invention. For example, in the introduction of liquidus material into the rotor, it is not essential that such material be directed to fluid passage 19 by means of

opposed flange members

32 and 34. Such flanges can be replaced by one or more radially disposed tubular members similar to lower skimmer tubes 35. Further by providing a fluid passage 19 which extends to the vicinity of the vertical rotor walls across the entire cross-section of the rotor cavity, such tubular members can be extended into said fluid passage 19 in order to prevent the possibility of the charged liquidus material entering the lower portions of internal bore 14.

The tubular members 35 shown in enlarged detail in FIG. 3 are preferably of a general S-shape configuration to facilitate admittance of the rotor contents to exit tube 33 during unloading of the rotor. This configuration is not essential however, nor is it essential that there be two of such tubes 35. In view of the extremely high rotationat speeds of the rotor during operation, however, symmetrical configurations of all elements of the over-all apparatus facilitates the attainment of the weight balance so essential to centrifuge apparatus.

In typical operation the empty rotor is accelerated by means of rotor 40 to a relatively low rotational speed, i.e. of the order of about 5000 r.p.m. and filled completely at this speed. Loading and unloading speeds as well as operational speeds are of course dependent of the precise design of the rotor selected and are readily determined by routine experimentation. Loading is accomplished by pumping a density gradient, light end first into the density aaazsss gradient chamber 17 through entrance conduit 31 and thence through opening 19 to the inner vertical wall of the rotor. Gradients typically used range from 17 and 55 percent by weight sucrose in aqueous solution for tissue fractionation, and 12 to 30 weight percent sucrose in aqueous solution for viral and ribosomal separations. After the gradient is in the rotor, a cushion of concentrated sucrose solution, typically from about 55 to 66 weight percent sucrose, is pumped to the edge of the rotor until the initially charged light end of the gradient begins to flow out through opening 18, through the lower skimmer members 35, and out exit conduit 33, indicating that the rotor is completely filled.

The sample to be separated (contained in a short density gradient) is then pumped into the rotor through conduit 33 emerging from skimmer members 35 and forming a sample zone centripetal to the density gradient. This is followed by an overlay solution having a density less than that of the sample layer, thus displacing the sample zone away from the internal core (column 13), but leaving the rotor filled.

The rotor is then accelerated to operating speed. After the desired separation has been achieved, the rotor is decelerated to an unloading speed and the density gradient displaced out through the skimmer in the lower annular groove 16 by pumping a very dense solution to the rotor edge through opening 19. As the gradient flows out of the rotor it can be analyzed continuously using conventional biological analytical techniques. Separations can be made in density gradients on the basis of sedimentation rate or, if the equilibrium or isopycnic position is reached, on the basis of density alone. The apparatus of this invention can also be utilized as a continuous flow centrifuge to concentrate such materials as bacteriophages, and thereafter purify same by zonal sedimentation in a density gradient.

As is well known to those skilled in the art optimum rotor dimensions, rotational speeds, loading and unloading speeds, and materials of construction are to a large degree interdependent factors. In cylindrical rotors spinning about a vertical axis, the rotor length is limited only by total rotor weight, rotor stability at high speeds, and by the loading and unloading speeds which in turn is dependent upon the specific design embodiment of the center core utilized. A typical rotor embodiment has a rotor chamber about 26 cm. long, with an inside diameter of 10.16 cm. and a wall thickness of 1.27 cm., and is constructed of steel or aluminum. The physical properties of the materials of construction in large measure determine such factors as maximum operational speeds, wall thickness, and the like. i i i Although the novel apparatus of this invention has been described herein generally as a biophysical tool of principal utility in the separation of biological fluid systems, it is to be understood that the basic design is equally adaptable to conventional uses which ordinarily require only moderate rotational speeds to concentrate relatively gross particle suspensions.

What is claimed is:

1. A zonal centrifuge comprising in combination a stationary skimmer, a rotor, and means for rotating said rotor around a vertical axis, said rotor comprising a substantially cylindrical body having a top and bottom closure, the density gradient cavity defined by the inner walls of said rotor and an internally contained axially disposed column therein, said column having a plurality of vertical septa and containing an axially disposed internal bore having an upper annular groove and a lower annular groove, said upper and lower annular grooves each being in hydraulic communication with the density gradient cavity of said rotor through separate passages, said internal bore being adapted to receive in concentric alignment an axially disposed skimmer, said skimmer comprising a first conduit having one end external of said rotor and the other end terminating with an opening within the upper annular groove and the rotor column bore and a second conduit having one end external of the said rotor and the other end terminating with at least one radially disposed tubular member within the lower annular groove.

2. A zonal centrifuge of claim 1 wherein the second conduit of the stationary skimmer element terminates within the lower annular groove of the internal rotor column with a radially disposed substantially S-shaped tubular member, each of the two extremities thereof terminating with an orifice the radial plane of which is substantially in longitudinal plane of said internal rotor column.

3. A zonal centrifuge of claim -1 wherein the passage communicating the upper annular groove of the internal rotor column with the density gradient cavity of said rotor terminates adjacent an outer wall of said density gradient cavity and the passage communicating the lower annular groove terminates at the outer wall of said internal axially disposed rotor column.

References Cited by the Examiner UNITED STATES PATENTS 2,495,950 1/1950 Van Der Werlf 23328 X 3,103,489 9/1963 Pickels 233-28 X 3,168,474 2/1965 Stallman et a1 233-33 M. CARY NELSON, Primary Examiner,

Claims

1. A ZONAL CENTRIFUGE COMPRISING IN COMBINATION A STATIONARY SKIMMER, A ROTOR, AND MEANS FOR ROTATING SAID ROTOR AROUND A VERTICAL AXIS, SAID ROTOR COMPRISING A SUBSTANTIALLY CYLINDRICAL BODY HAVING A TOP AND BOTTOM CLOSURE, THE DENSITY GRADIENT CAVITY DEFINED BY THE INNER WALLS OF SAID ROTOR AND AN INTERNALLY CONTAINED AXIALLY DISPOSED COLUMN THEREIN, SAID COLUMN HAVING A PLURALITY OF VERTICAL SEPTA AND CONTAINING AN AXIALLY DISPOSED INTERNAL BORE HAVING AN UPPER ANNULAR GROOVE AND A LOWER ANNULAR GROOVE, SAID UPPER AND LOWER ANNULAR GROOVES EACH BEING IN HYDRAULIC COMMUNICATION WITH THE DENSITY GRADIENT CAVITY OF SAID ROTOR THROUGH SEPARATE PASSAGES, SAID INTERNAL BORE BEING ADAPTED TO RECEIVE IN CONCENTRIC ALIGNMENT AN AXIALLY DISPOSED SKIMMER, SAID SKIMMER COMPRISING A FIRST CONDUIT HAVIG ONE END EXTERNAL OF SAID ROTOR AND THE OTHER END TERMINATIG WITH AN OPENING WITHIN THE UPPER ANNULAR GROOVE AND THE ROTOR COLUMN BORE AND A SECOND CONDUIT HAVING ONE END EXTERNAL OF THE SAID ROTOR AND THE OTHER END TERMINATING WITH AT LEAST ONE RADIALLY DISPOSED TUBULAR MEMBER WITHIN THE LOWER ANNULAR GROOVE.