US3536253A

US3536253A - Zonal centrifuge

Info

Publication number: US3536253A
Application number: US801521A
Authority: US
Inventors: Norman G Anderson; Clifford E Nunley
Original assignee: US Atomic Energy Commission (AEC)
Current assignee: US Atomic Energy Commission (AEC)
Priority date: 1969-02-24
Filing date: 1969-02-24
Publication date: 1970-10-27
Anticipated expiration: 1987-10-27

Description

United States Patent [72] Inventors NormanGAnderson 3,288,360 1 H1966 Babelay et al. 233/33X Oak Ridge, an 3,430,849 3/1969 Gibson et al 233/33X pp No ggfggi Tennessee Primary Examiner-William l. Price A. A Flled Feb. 24 1969 Attorney Roland nderson [45] Patented Oct. 27, 1970 [73] Asslgme 3:ig zgfg ii ggzz gzzzs ABSTRACT: A liquid centrifuge rotor bowl has a peripheral comnfission wall which is internally tapered to form an apex about the ingy ternal circumference of the bowl. A diametrically eccentric groove is provided in the rotor wall along the apex. An integral 54 ZQNAL CENTRIFUGE septum body comprising a central hub and radially extending 7C|aims,3 Drawing Figs. septa s disposed within the rotor bowl, with the radial extremitles of the septa being tapered to conform with the [52] US. Cl 233/33 tapered periphem W3" and having radially extending nipples [51] Int. Cl B04b l/04, which engage the eccemric groove at its radially outermost Bold 21/26 points. Internal conduits extend through the septa and nipples [50] Field ofSearch 233/33, 34, to provide quid communication between the eccentric 44 groove and the central hub. A rotor core with internal con- [56] References Cited duits is disposed within the hub to provide liquid flow communication between external liquid flow means and the internal UNITED STATES PATENTS conduits extending outward through the septa to the eccentric 3,195,809 7/1965 Pickels et al 233/33X groove l I r 1! Patented Oct. 27, 1970 3,536,253

Sheet 1 of 2 INVENTORS, Norman GA BY Clifford E Iey ATTORNEY.

Patehted Oct. 27, 1970 3,536,253

Sheet 2 of 2 3808008 .LNHOHEd .LH9I3M o o o o o o wunv 092 1v xouvauosav INVENTORS.

Norman (iAndersan BY Clifford E. Nunley ATTORNEY.

l ZONAL CENTRIFUGE BACKGROUND OF THE INVENTION The invention described herein relates generally to liquid centrifuges and more particularly to an improved bowl-type zonal centrifuge. It was made in the course of, or under, a contract with the US. Atomic Energy Commission.

In the operation of prior art zonal centrifuges, zonal frac tions have typically been recovered by forcing the gradient inward to the core of the rotor by' displacement with dense displacing liquids at the periphery or edge of the rotor. Rotor designs have, therefore, characteristically incorporated tapered core designs with a conduit opening at the radially innermost point of the tapered core. The funneling effect provided by such an arrangement is known to provide a high degree of resolution between flowable components during the recovery of a liquid density gradient from the rotor bowl. For a detailed description of the design and operation of zonal centrifuges, refer to National Cancer Institute Monograph 21, The Development of Zonal Centrifuges and Ancillary Systems for Tissue Fractionation and Analysis, US. Department of Health, Education and Welfare, public Health Service.

It has been determined that a number of new and very useful separations can be achieved in rotors which can be unloaded from either the center or edge, including sequential removal of subcellular particles which are approaching their isopycnicpoints. For example, liver cell membrane fragments may be removed while mitochondria are still sedimenting. It is also possible to remove a large fraction of the large mitochondria while the lysosomes are still trailing behind them. All of ithese particles have very similar isopycnic positions but different sedimentation rates. Edge unloading facilitates the use of a narrow density gradient band near the edge of the rotor bowl and a large sample volume. Such arrangement increases the rotor capacity, saves on the use of expensive, high density. displacing fluid, and reduces unloading time since the gradient need not be displaced over a large radial distance. Edge or peripheral recovery tends to decrease the resolution between gradient components, however, because of the relatively large circumferential displacement which must occur as the gradient is driven toward collecting conduits spaced about the rotor bowl wall. Axial displacement occurs to substantially the same degree in both center and-edge collectioncentrifuge machines.

It is, accordingly, a general object of the invention to provide a bowl-type zonal centrifuge wherein a high resolution recovery of zonal fractions can be made from the edge of the rotor bowl.

Other objects will be apparent from an examination of the following description and appended drawings.

SUMMARY OF THE INVENTION In accordance with the invention, an improved, bowl-type, zonal centrifuge is provided in which high resolution recovery of zonal fractions can be made from the edge of the rotor bowl. The peripheral walls of the rotor bowl are tapered internally to form an apex about the circumference of therotor bowl and a diametrically eccentric groove ,is provided in the rotor wall along the apex. An integral septum body comprising a central hub and radially extending septa is disposed within the rotor bowl, with the peripheral surfaces of the septa being tapered to mate with the tapered peripheral walls and having radially extending nipples which engage the eccentric groove at its radially outermost points. Internal conduits extend through the septa and nipples to provide liquid communication between the eccentric groove and the central hub. A 'rotor core with internal conduits is disposed within the hub to provide liquid flow communication between external liquid ,flow means and the internal conduits extending outward through the septa to the eccentric groove. Radially outward displacement of the zonal fractions during unloading causes a funneling effect as the fraction reaches the tapered peripheral wall and is driven axially inwardly toward the eccentric ment occurs toward its radially outermost points where the nipples extending from the peripheral surfaces of the septa engage the groove. The circumferential displacement occurs as a result of the funneling effect created by the eccentricity of the groove which causes displacement toward its radially outermost points. Zonal fractions reaching the nipples are displaced inwardly through the conduits extending through the septa and nipples. The combined funneling effects of the tapered rotor bowl wall and eccentric groove facilitate gradient recovery from the edge of the rotor bowl with high resolution being maintained between adjacent zonal fractions.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a vertical sectional view of a rotor bowl assembly made in accordance with the present invention.

FIG. 2 is a horizontal sectional view of the rotor bowl assembly 30 ofFlG. l.

FIG. 3 is a graph of data obtained using a centrifuge rotor assembly made in accordance with the invention to separate a mixture of bovine serum albumen and ragweed pollen.

DESCRIPTION OF THE PREFERRED EMBODIMENT Referring now to FIGS. 1 and 2 where a preferred embodiment of the invention is illustrated, a rotor bowl 1 comprises threadably engaged top and

bottom bowl segments

2 and 3, respectively. Internal peripheral surfaces 4 and 5 of

bowl segments

2 and 3 are tapered outwardly to form an apex at their junction which, in the embodiment shown, lies approximately in the horizontal midplane of the rotor bowl. A diametrically eccentric groove 6 is formed by a recess in the wall of bottom bowl segment 3 where it abuts with top bowl segment 2.

An integral septum body comprising a hub 7 and four tapered septa 8 divides the rotor bowl into four sector-shaped compartments 9. The peripheral surfaces 10 of the septa are tapered to conform with tapered surfaces i and 5 and provided with nipplesll for engaging eccentric groove 6. Internal conduits 12 extend through septa 8 and hub 7 to a central rotor core 13 which contains internal conduits and sealing means providing liquid flow communication with external liquid flow means (not shown). 'An internal conduit 14 opens into groove 15 which encircles core 13 at an axial position so as-to be'in register with the open ends of conduits 12. Thus, zonal fractions moving inward through conduits 12 discharge into groove 15, and then flow outward through conduit 14 to external liquid flow means (not shown). A second conduit 16 extends through core 13 and terminates in a groove 17 adjacent the top end of hub 7. Conduit 16 and groove 17 are used to insert sample material into the rotor bowl and to pump displacing fluid into the rotor bowl when it is desired to recover zonal fractions from the edge of the rotor. Hub 7 is provided with flat faces tapered axially toward the center of the rotor bowl to provide a funneling effect when it is desired to recover zonal fractions from the center of the rotor bowl in accordance with existing practice.

' In a typical operation of the invention, the rotor bowl is first loaded with a liquid of graded density which is pumped into the bowl through conduits 14 in central core 13 and conduits 12 in septa 8. During the loading operation, the rotor bowl is rotated at low speed (500-3Q 00 r.p.m.) causing the liquid to form concentric annular zones of constant density liquid. Septa 8 extend radially through the zones, segmenting them and reducing mixing between adjacent zones of different densities. After the rotor bowl is filled with liquid of graded intensity, a sample to be centrifuged is pumped through conduit 16 into the center of therotor bowl followed by an overlay of low density liquid to move the sample clear of the central hub 7. The rotor core is then capped where it extends through top bowl segment 2 and accelerated for the actual centrifuging operation. When centrifugation has been completed, the rotor bowl is decelerated to unloading speed (5003000 r.p.m. and the rotor contents emptied by pumping low density displacing fluid through conduit 16 to the center of the rotor bowl, thereby displacing the rotor contents from the edge of the bowl out through conduits 12. The funneling effects provided by tapered surfaces 4 and 5 and eccentric groove 6 direct the displaced zonal fractions toward conduits 12 while maintaining a high resolution between adjacent zonal fractions.

EXAMPLE In order to demonstrate the operation of the invention, a rotor was fabricated substantially as shown in the drawings. The internal dimensions of the bowl were 6.2 inches diameter at the inner edge of the tapered surfaces 4 and 5, 6.7 inches at their outer edge or apex, and 3 inches in height. Tapered surfaces 4 and 5 were each inclined about with respect to the axis of rotation of the rotor bowl which was vertically oriented. A 0.125 inch width eccentric groove was milled into the wall of the lower rotor bowl segment to a maximum depth of 0.125 inch at the four points of liquid pickup at the ends of the septa and to zero depth midway between these points on the bowl periphery. The volume of the rotor bowl from the beginning of the tapered walls inward was 1100 ml. and a volume of 350 ml. was included in the tapered region.

A typical functionality test was run in which the rotor was operated at 5C and was loaded from the edge during rotation at 2500 r.p.m. with an unbuffered sucrose gradient (1000 ml. total volume) extending from 17 to 55 weight percent sucrose followed by an underlay of 55 percent sucrose to fill the rotor completely. A sample consisting of 10 ml. of 1 percent bovine serum albumen (BSA) in 8.5 percent sucrose and 2 ml. of alcohol-washed ragweed pollen was introduced through the center of the rotor. The test mixture of BSA and pollen was chosen because it provided two narrow zones, one near the center of the rotor bowl and one near its edge. An overlay of 50 ml. of 4 percent sucrose was added through the center after the sample to move the sample out clear of the core. With the gradient, sample, and overlay in position, the rotor was accelerated to 10,000 rpm. during a 10 minute interval, and then decelerated to 2500 rpm. for unloading.

The gradient was recovered from the rotor by using distilled water pumped to the rotor center in order to displace the gradient out through the edge of the rotor. The rotor contents were monitored using light having a 260 u wave length with a 0.2 cm flow cell and were collected in 40 m1. fractions.

From the experimental data as shown plotted in FIG. 3, the starting sample was calculated to be in a zone 1 mm wide when initially loaded into the rotor. When the rotor was unloaded from its edge, the pollen peak at point A had a calculated width at half peak height of 1.5 mm, whereas the BSA peak at point B was 2.25 mm wide at half height.

Although a specific embodiment has been described as an aid in understanding the invention, that description was for illustrative purposes only and should not be interpreted in a limiting sense. For example, the slope of peripheral surfaces 4 and 5 may be varied from the 10 value mentioned in the Example. Also, eccentric groove 6 and the apex formed by tapered peripheral surfaces 4 and 5 can be at an axial position other than at the horizontal midpoint shown. The midpoint position is preferred, however, since it minimizes the average axial displacement of zonal fractions during an unloading operation. It is intended rather that the invention be limited only by the scope of the appended claims.

We claim:

l. A bowl-type liquid centrifuge rotor assembly for peripherally removing zone fractions of a liquid gradient under dynamic conditions comprising:

a. a rotor bowl having a peripheral wall which is tapered internally to form a circumferentially extending apex, said wall being further recessed along said apex to form a circumferentially extending groove of varying depth; an integral septum body comprising a central hub with radially extendingsepta attached thereto disposed within said rotor bowl, said septa being tapered along their radial extremities to conform with said tapered peripheral wall of said rotor bowl, radially extending nipples being provided on said radial extremities for engaging said groove, said central hub and septa being provided with liquid passageways extending from the interior of said hub to said groove; and

means for providing external liquid flow communication with said liquid passageways.

2. The rotor assembly of claim 1 wherein said peripheral wall is tapered about 10 with respect to the axis of rotation of said rotor bowl.

3. The rotor assembly of claim 1 wherein the radially outermost surface of said groove describes a super elliptical curve.

4. The rotor assembly of claim 1 wherein said circumferentially extending apex lies in the horizontal midplane of said rotor bowl.

5. The rotor assembly of claim 1 wherein said rotor bowl comprises threadably engaged top and bottom bowl segments, said top segment having an internal peripheral surface which extends downwardly and outwardly, said bottom segment having an internal peripheral surface which extends upwardly and outwardly.

6. The rotor assembly of claim 1 wherein said radially extending nipples engage said groove at its points of maximum depth.

7. The rotor assembly of claim 1 wherein said means for providing external liquid flow communication with said liquid passageways comprises a core disposed within said hub, said core being provided with a circumferential groove positioned axially to register with said passageways where they open inside said hub and axially extending passageways with one end opening into said circumferential groove.