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Publication numberUS3747843 A
Publication typeGrant
Publication dateJul 24, 1973
Filing dateApr 9, 1971
Priority dateApr 9, 1971
Also published asDE2144070A1, DE2144070B2, DE7133593U
Publication numberUS 3747843 A, US 3747843A, US-A-3747843, US3747843 A, US3747843A
InventorsJ Joyce
Original AssigneeDamon Corp
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Continuous flow zonal rotor
US 3747843 A
Abstract
A method and apparatus for continuous flow centrifugation wherein a sample is introduced through the top surface of a rotor core into a sample zone adjacent a gradient zone so that the sample passes substantially 360 DEG and a sample gradient is formed in said gradient zone. The spent sample is removed through the bottom surface of said rotor core, and the sample gradient is removed after separation by introducing a liquid gradient through the top surface of the rotor core to the gradient zone to displace the sample gradient radially inward. The sample gradient is passed out of the core while the rotor is spinning through a passage at the core axis at a reduced pressure in the passage. Since only one liquid stream enters or exits at or near the core axis, the possibility of sample product contamination is eliminated.
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nite States Patent [1 1 Joyce 51 July 24, 1973 CONTINUOUS FLOW ZONAL ROTOR [75] Inventor: John E. Joyce, South Weymouth,

Mass.

[73] Assignee: Damon Corporation, Needham,

Mass.

[22] Filed: Apr. 9, 1971 [211 App]. No.: 132,663

[52 us. Cl 2213/32, 2233/16, 233 1 D [51] Int. Cl. 1304b 1/00 [58] Field of Search 233/27, 28, l R, 233/1 D, 32, 33, 46, 47 R, 16, 17, 21

[56] References Cited UNITED STATES PATENTS 3,536,253 10/1970 Anderson 233/33 3,430,849 3/1969 Gibson et a1 233/33 X 3,168,474 2/l965 Stallman et al. 233/33 3,073,517 1/1963 Pickels et al....' 233/32 1,795,958 3/1931 McFarlane 233/28 3,291,387 12/1966 Billen 233/28 3/1970 Harbott 233/16 3,519,201 7/1970 Eisel et al. 233/21 Primary Examiner-George H. Krizmanich Attorney-Kenway, Jenney & Hildreth [57] ABSTRACT A method and apparatus for continuous flow centrifugation wherein a sample is introduced through the top surface of a rotor core into a sample zone adjacent a gradient zone so that the sample passes substantially 360 and a sample gradient is formed in said gradient zone. The spent sample is removed through the bottom surface of said rotor core, and the sample gradient is removed after separation by introducing a liquid gradient through thetop surface of the rotor core to the gradient zone to displace the sample gradient radially inward. The sample gradient is passed out of the core while the rotor is spinning through a passage at the core axis at a reduced pressure in the passage. Since only one liquid stream enters or exits at or near the core axis, the possibility of sample product contamination is eliminated.

11 Claims, 7 Drawing Figures PATENIEUJUtZdIBH SHEU 1 OF 3 INVENTOR JOHN E. JOYCE wmzm ATTORNEYS CONTINUOUS FLOW ZONAL ROTOR BACKGROUND OF THE INVENTION This invention relates to continuous flow centrifugation and particularly to a method and apparatus for recovering sample gradients from continuous flow centrifugal rotors.

In continuous flow centrifugation, a sample to be separated is pumped continuously into a rotating centrifuge and into a liquid gradient zone. The gradient zone is formed so that the liquid gradient density, which increases with its radial position away from the rotor center, is regulated to separate sample strata therein. By regulating the liquid gradient density to accommodate the density of the liquid sample, sample separation can be effected at any desired radial position. Generally the desired radial position is chosen for ease of sample recovery.

While the centrifuge is rotating, the gradient liquid remains stationary in the radially outward region or zone, during the treatment of its sample. The density gradient of the gradient liquid usually is a concentration gradient of a relatively non-diffusing suitable solute such as salt or sucrose. The sample liquid is pumped continuously into a zone radially intermediate the gradient liquid and the rotor and is caused to circulate around all or a portion of the rotor circumference. The gradient functions to entrap particles sedimenting from the sample liquid without further radial movement of the particles at that location where their density equals that of the gradient. The travel of particles of greater or lesser density is varied accordingly. As a consequence, there will be in the gradient, particles separated radially as individual populations of different densities that may be recovered separately after the sample flow has been terminated and while the rotor is spinning. Even in the case of particles that cannot be suspended within the gradient, it functions to so curb their radial velocity that, depending on the flow period, their impingement with the outer boundary of the rotor chamber is prevented even though no effective purification is accomplished.

Some zonal rotors have separate sectors and the sample is divided into streams flowing separately through its sectors. Such sectors are of relatively large volume, each sector typically representing 25 percent of the capacity of the rotor. In addition, a zonal rotor is available having a particle collecting sector that extends substantially 360 to permit the sample to travel the same distance within the. rotor. The latter centrifuge is preferable since it provides more effective sample separation. This centrifuge is described in a copending application to John E. Joyce entitled "Rotors and Rotor Cores for Continuous Flow Centrifuges", Ser. No. 129,055 filed Mar. 29, 1971.

In present continuous flow zonal centrifuges, major problems are associated with the sample recovery step. Presently, the sample to be separated is introduced and the recovered sample is removed adjacent the rotor axis and appropriate sealing is effected to prevent mixing of the two. However, these sealing arrangements have proven unreliable under the forces encountered during centrifugation and undesirable mixture of the incoming and recovery streams is commonly encountered.

ln present systems when a sample or gradient liquid is being introduced into the spinning rotor, either during sample separation or sample recovery, a liquid in the rotor is displaced and is removed therefrom and the liquid must be introduced at a pressure above atmospheric whether or not the liquid is being introduced at a radially inward or outward position with respect to the liquid being removed. These increased liquid pressures place additional stress on the seals between the liquid streams thereby making it essential that the sealing surfaces be machined to almost perfect flatness, usually to within 50 to Angstroms. These machine tolerances are difficult to attain thereby making present systems undesirable from the standpoint of high manufacturing expense as well as unreliability.

It would be highly desirable to provide a centrifuge method and apparatus for continuous flow centrifugation wherein the possibility of leakage between liquid streams is eliminated. Furthermore, it would be desirable to provide an inexpensive sealing arrangement which permits liquid recovery from a spinning centrifuge that does not require machining to extremely close tolerances.

SUMMARY OF THE INVENTION The present invention provides a method and apparatus for continuous flow centrifugation of samples and in one aspect provides a novel sealing arrangement. A centrifuge rotor is provided with two circular grooves in its top surface, each groove being adjacent the other and radially separated from each other. First and second passages through the rotor, connect the grooves with either a sample zone or a gradient zone located between the peripheral rotor surface and the housing. The first passages have an outlet radially outward from the second passage outlets. A third passage has an inlet at sample zone and an outlet in the bottom surface of the rotor to remove the spent sample. A fourth passage connects the sample zone with an outlet conduit at the rotor axis which can be connected with a means for reducing pressure below atmospheric. The fourth passage has a seal with a rotating section and a stationary section that permits sample removal from the spinning rotor. The reduced pressure employed is defined by the equation:

wherein d(lbs/cu. in.) is the density of the fluid being removed, N(rpm) is the speed of the rotor, r(in) is the radial position of sample drain outlet and B is a proportionality constant equal to 2.841 X 10"".

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be more fully described with reference to the accompanying drawings.

FIG. 1 is a top view of a centrifuge rotor.

FIG. 2 is an elevation cross-sectional view of the apparatus for'removing separated sample.

FIG. 3 is a partial cross-section view taken along line 3 of FIG. 1.

FIG. 4 is a cross-sectional view taken along line 4 of FIG. I.

FIG. 5 is a cross-sectional view taken along line 5 of FIG. 1.

FIG. 6 is a partial cross-sectional view taken along Line 6 of FIG. 1.

FIG. 7 is a partial cross-sectional view taken along Line 7 of FIG. 1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to FIGS. 1 and 3 through 7, the centrifuge generally indicated at 1 comprises a rotor housing 2 and a rotor 3 having a frusto-conical shape. The rotor housing 2 has a lower axial socket 8 to receive a motor drive (not shown) and an upper axial socket 9 to receive a sealed sample recovery system described hereafter with reference to FIG. 2. The rotor 3 is provided with a plurality of fluid passages for the introduction and exit of liquid gradient and fluid sample in a continuous cycle so that the sample travels about 360 around the rotor 3. The large arrows indicate the direction and path of sample flow. The liquid gradient and introduction of sample is accomplished as follows: While the rotor 3 is spinning, liquid gradient is introduced into circular groove 10 by placing the outlet of a tube adjacent to or into the groove 10 for delivery of fluid thereto. The liquid gradient passes from groove 10 through a plurality of passageways 11 that terminate at the ends of fins 12a, 12b, 12c and 22 which are positioned adjacent the rotor housing 2. Introduction of liquid gradient into groove 10 and passageways 11 is continued until the desired size of the gradient zone 13A is effected. Generally, the gradient zone 13A extends from the inner surface of rotor housing 2 to the radially outermost point of rotor 3. After the gradient zone 13A has been established, sample to be separated is introduced into circular groove 15 which is radially inward from groove 10 and is connected with sample delivery passage 16 having an outlet 17 that is positioned radially inward from the gradient zone 13A. The fluid sample is constantly fed to groove 15 and into passageway 16 so that there is established inside the rotor 3, while it is spinning, a sample zone 18 that travels clockwise around the rotor 3 substantially 360 until it reaches drain passageway 19 and outlet 20 through which spent sample is removed from the rotor 3.

While the sample zone 18 travels around the rotor, liquid gradient zone 13A does not move relative thereto while it is spinning. Bypass of sample around fin 22 is prevented by means of circular plate 23 and bottom plate 24 which are adhered to rotor housing 2 and prevent passage of sample over or under the fin 22. Plates 23 and 24 extend the circumference of rotor housing 2 and plate 24 is provided with an opening to allow spent sample to drain (See FIG. 6). The sample zone 18 passes through opening 21 of fins 12 but does not bypass fin 22 since fin 22 does not have an opening through which the sample gradient can pass. Accordingly, the sample must pass out of outlet 19 and drain 20. While the sample passes through zone 18, particles therein separate therefrom to form strata in the liquid gradient 13A. After the desired amount of sample has passed through the rotor 1, sample delivery in groove 13A and separated sample particles therein toward the inlet 25 of sample removal passageway 26. Prior to introducing the liquid gradient after sample separation and during the introduction of the new liquid gradient, the sealing arrangement shown in FIG. 2 is positioned so that fluid passing from passage 26 through outlet 27 can pass into inlet 28 of a sample removal tube 29. The sealing arrangement shown in FIG. 2 comprises generally two sections; a rotating seal 30 and a stationary seal 31. The rotating seal 30 and stationary seal 31 are maintained in close contact by means of spring 32. The rotating seal housing 33 is held in the main seal housing 34 by means of ball bearings 35 and the seal housing 33 is moved into and out of rotor 3 by means of lever 36 which is pivoted around pin 37 and bears against the top surface 38 of seal housing 34. The lever 36 moves against the action of spring 40. When positioned to remove sample, the extension 30a sealed to rotating seal 30 fits into an arbor 44 which in turn, is fit into socket 9 and sealed therein by means of O ring 45. The extension 30a is sealed in arbor 44 by means of O ring 46. When the rotor housing 2 is rotated, arbor 44, extension 30a, seal 30 seal housing 33 hearing 35 also rotate while seal 31, spring 32, housing 34, tube 29, spring 40 and lever 36 do not rotate. The main seal housing 34.

is fixed to a housing for the centrifuge (not shown) by means of a seal shelf 42 and a safety disc 43 attached to shelf 42. The plate 42 is provided with openings 10a and 15a to accommodate filling tubes that can extend into the grooves 10 and 15. Sample recovery is effected by means of pump 50 which reduces the pressure in collection chamber 51; tube 29, and passage 26 when valve 52 is open and valves 53 and 54 are closed thereby causing gradual withdrawal of the liquid gradient and the sample strata therein while liquid gradient is being introduced into passage 11, as described above. Sample recovery is effected 'while the rotor housing 2 is spinning at moderate speeds usually about 1000 to 2000 rpm so that the sample strata remain separate in the liquid gradient during recovery. The sample strata are recovered sequentially, with the least dense stratium being recovered first. After the desired sample portion is collected in collection chamber 51, valve 52 is closed and valves 53 and 54 are open so that the sample can be recovered through conduit 55.

The drain outlet for spent sample is radially outward from the radially outer most groove to obtain the desired liquid flow. It is preferred that the groove adapted to receive liquid gradient during sample recovery be located radially outward from the groove adapted to receive the sample to attain desired liquid flow. Also, it is preferred that the grooves be inclined or shaped so the lower portion thereof extends radially further outward than the upper portion to reduce spillage from the grooves onto the top surface of the rotor. It is preferred that the outlet for the liquid gradient passage extend to the inner surface of the housing to provide ease in gradient zone formation and separated sample removal. in this respect, the liquid gradient passage need not extend below the openings 21 in fins 12a, 12b and 12c. A

tube through the openings 21 can be employed to connect liquid gradient passages in the rotor body and the radially outermost portion of the f'ins.

While the invention has been described above with reference to a rotor wherein sample travels about 360 and wherein four sample passages and four liquid gradient inlet passages are employed, it is to be understood that variations in the apparatus can be made to attain the same results. Thus, the rotor can be segmented by closing the openings 21 in fins 12a, 12b and 120 and additional sample inlet passages and drains are provided for each rotor segment so that the sample travels only about 90 through the rotor. However, this embodiment is not preferred since sample separation therein is less complete for a given sample flow rate. Furthermore, more or less sample outlet and gradient inlet passages can be employed to attain equivalent sample separation and recovery. Furthermore, any sealing arrangement can be employed so long as it is effective in providing passage for one liquid stream and to retain adequate sealing between mating rotatable and stationary sections.

I claim:

1. A method for forming a sample gradient which comprises forming a gradient zone in a spinning centrifuge, flowing a sample through the top surface of a rotor core into a sample zone adjacent the gradient zone so that the sample passes substantially 360 and a sample gradient is formed in said gradient zone, removing spent sample through the bottom surface of said rotor core, and removing the sample gradient by introducing liquid gradient through the top surface of the rotor core to the gradient zone to displace said sample gradient radially inward and passing said sample gradient out of said core while the rotor is spinning through a passage at the axis of the core at a subatmospheric pressure in said passage.

2. A continuous flow centrifuge rotor comprising a rotor core having means for introducing two separate streams of liquid through circular grooves in the top surface of the rotor so that one stream can be directed into a sample zone and the other stream can be directed into a gradient zone, a means for removing spent sample from said sample zone through an outlet on the bottom surface of said rotor and means for removing separated sample from said rotor through a passage at the axis of said rotor while the rotor is spinning.

3. The centrifuge of claim 2 having means for flowing a sample stream through said sample zone substantially 360 around said rotor core.

4. A continuous flow centrifuge comprising a rotor housing a rotor core within said housing and spaced from said housing to define a gradient zone adjacent said housing and a sample zone between said rotor and said gradient zone, said rotor core having a top surface, a bottom surface and a peripheral surface, said top surface having'a first circular groove and a second circular groove radially spaced from said first circular groove each groove adapted to receive means for delivering a liquid, at least onepassage connecting said first groove with said gradient zone andat least one passage connecting said second groove with said sample zone at least one drain passage in said core connecting said sample zone and said bottom surface, said drain passage having an outlet radially intermediate the radially outermost groove and the peripheral surface, at least one sample recovery passage in said core connecting the sample zone and an axial passage at the axis of said core and said axial passage having means to recover separated sample at subatmospheric pressure in said axial passage pressure while said rotor is spinning.

5. The centrifuge of claim 4 having means for flowing a sample stream through said sample zone substantially 360 around said rotor core.

6. The centrifuge of claim 5, four passages; equally radially spaced for delivery gradient liquid to said gradient zone and four sample recovery passages equally radially spaced.

7. The centrifuge of claim 5 wherein a plurality of first fins extend from said rotor core into the gradient zone adjacent said housing, each of said first fins having an opening to permit the sample zone to pass therethrough and a solid second fin extending from said rotor core into the gradient zone adjacent the housing a sample stream inlet and a sample stream outlet being positioned adjacent opposing vertical surfaces of said second fin and plate means adhered to the bottom and top surfaces of the second fin extending from the gradient zone to the rotor core to prevent by-pass of said sample zone around said second fin.

8. The centrifuge of claim 6 wherein a plurality of first fins extend from said rotor core into the gradient zone adjacent said housing, each of said first fins having an opening to permit the sample zone to pass therethrough and a solid second fin extending from said rotor core into the gradient zone adjacent the housing, a sample stream inlet and a sample stream outlet being positioned adjacent opposing vertical surfaces of said second fin and plate means adhered to the bottom and top surfaces of the second fin extending from the gradient zone to the rotor core to prevent by-pass of said sample zone around said second fin.

9. The centrifuge of claim 7 .wherein the passages connecting the first groove with the gradient zone extend through the second fin and through each opening in the first fins.

10. The centrifuge of claim -8 wherein the passages connecting the first groove with the gradient zone extend through the second fin and through each opening in the first fins.

11. A method for forming a sample gradient which comprises forming a gradient zone in a spinning centrifuge, flowing a sample through the top surface of a rotor core into a sample zone adjacent to the gradient zone so that the sample passes substantially 360 and a sample gradient is formed in said gradient zone, removing spent sample through the bottom surface of said rotor core, and removing the sample gradient by introducing liquid gradient through the top surface of the rotor core to the gradient zone to displace said sample gradient radially inward and passing said sample gradient out of said core while the rotor is spinning through a passage at the axis of the core.

4 t t it t UNITED STATES PATENT oFHcE CERTIFICATE OF CORECHUN Patent'NQ, 3,747,843 Dated July 24, 1973 Inventor(s) JOHN E JOYCE It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 6, line S, cancel "pressure" Signed and sealed this 22nd day of January 1974..

(S-EAL) Attest: 1

EDWARD M.FLETCHER,JR. RENE D. TEGTMEYER Attesting Officer Acting Commissioner of Patents

Patent Citations
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US3073517 *Apr 7, 1959Jan 15, 1963Beckman Instruments IncContinuous flow centrifuge apparatus and rotor therefor
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Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US3858795 *Oct 23, 1973Jan 7, 1975Int Equipment CoMethod for washing blood cells
US3862029 *Oct 1, 1973Jan 21, 1975Joyce John EDensity gradient fractionator
US4036426 *Feb 15, 1974Jul 19, 1977Pennwalt CorporationMethod of cleaning a centrifuge
US5147280 *Mar 30, 1990Sep 15, 1992Alfa-Lavel Separation AbEnergy transformation device
US6736768 *Nov 2, 2001May 18, 2004Gambro IncFluid separation devices, systems and/or methods using a fluid pressure driven and/or balanced approach
US6773389 *Nov 2, 2001Aug 10, 2004Gambro IncFluid separation devices, systems and/or methods using a fluid pressure driven and/or balanced configuration
US7094196Mar 29, 2004Aug 22, 2006Gambro Inc.Fluid separation methods using a fluid pressure driven and/or balanced approach
US7438679Dec 17, 2007Oct 21, 2008Caridianbct Biotechnologies, LlcApparatus and method for separating volumes of a composite liquid with a balancing assembly
US7674221Dec 12, 2007Mar 9, 2010Caridianbct, Inc.Apparatus for separating discrete volumes of a composite liquid with balancing elements
US7766809Dec 12, 2007Aug 3, 2010Caridianbct, Inc.Apparatus for separating discrete volumes of a composite liquid
US8016736Oct 19, 2007Sep 13, 2011Caridianbct Biotechnologies, LlcMethods for washing a red blood cell component and for removing prions therefrom
US8070665Jul 8, 2010Dec 6, 2011CaridianBCT, IncMethod for separating discrete volumes of a composite liquid
US8840535Apr 26, 2011Sep 23, 2014Terumo Bct, Inc.Multi-unit blood processor with temperature sensing
Classifications
U.S. Classification494/27, 494/41, 494/67, 494/37, 494/44
International ClassificationB04B5/00, B04B5/04
Cooperative ClassificationB04B5/0442, B04B5/00, B04B2005/045, B04B2005/0464
European ClassificationB04B5/04C, B04B5/00
Legal Events
DateCodeEventDescription
Apr 2, 1990ASAssignment
Owner name: MITSUBISHI DENKI KABUSHIKI KAISHA, JAPAN
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:MATSUOKA, ATSUKO;IWATA, TOSHIO;REEL/FRAME:005304/0046;SIGNING DATES FROM 19900313 TO 19900319
Apr 2, 1990AS02Assignment of assignor's interest
Owner name: IWATA, TOSHIO
Effective date: 19900319
Owner name: MATSUOKA, ATSUKO
Effective date: 19900313
Owner name: MITSUBISHI DENKI KABUSHIKI KAISHA, 2-3, MARUNOUCHI
Mar 30, 1990AS02Assignment of assignor's interest
Owner name: DAMON CORPORATION, A CORP OF DE
Effective date: 19901005
Owner name: INTERNATIONAL EQUIPMENT COMPANY, 300 SECOND AVENUE
Mar 30, 1990ASAssignment
Owner name: INTERNATIONAL EQUIPMENT COMPANY, A CORP OF DE, MAS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:DAMON CORPORATION, A CORP OF DE;REEL/FRAME:005328/0295
Effective date: 19901005
Oct 10, 1989ASAssignment
Owner name: BANQUE PARIBAS, THE EQUITABLE TOWER 787 SEVENTH AV
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:INTERNATIONAL EQUIPMENT COMPANY;REEL/FRAME:005305/0535