US3498531A - Continuous flow ultracentrifuge - Google Patents

Description

March 3, 1970 c. H. CHERVENKA ETAL 3,498,531

commuous mow ULTRACENTRIFUGE 2 Sheets-Sheet 1 Filed Feb. 16. 1968 INVENTORS CHARLES 1-1 CHERVENKA BY KAREN s. CHERRY HG 4 I ATTORNEY 2 sheets-shjeet '2 m V 5 Y I: %///////////fi//// IN CH ES H. CH VENKA BY K EN s. c RY ATTORNEY M;arch 3, 19-70 c. H. CH EVIEN-KA Filed Feb. 16, v.1968

3,498,531 CONTINUOUS FLOW ULTRACENTRIFUGE Charles H. Chervenka, Sunnyvale, and Karen S. Cherry, Los Altos, Calif., assignors to Beckman Instruments, Inc., a corporation of California Filed Feb. 16, 1968, Ser. No. 706,043 Int. Cl. B04b 11/00, 7/04, N02

US. Cl. 233-12 Claims ABSTRACT OF THE DISCLOSURE A predetermined volume of gas, such as air, is introduced into a rotor assembly of a continuous flow centrifuge immediately prior to the introduction of a high density displacement liquid to prevent mixing between such displacing liquid and the density gradient solution containing the centrifuged particles of interest as the displacing liquid forces the density gradient solution from the rotor cavity. The predetermined air volume lodges in a vertical slot provided in the rotor core which slot communicates by way of a connecting radial slot with the outer surface of the core member disposed in the cavity to prevent the displacing liquid from reaching the rotor cavity through this passageway. As a result, all of the displacing liquid enters the rotor cavity at the outside edge of the cavity, thus preventing mixing of the displacthe liquid and the density gradient solution containing the particles of interest.

BACKGROUND Continuous flow or zonal centrifugation has become an increasingly important analytical tool in the investigation of the nature of numerous biological and other chemical substances. Typically, in continuous flow centrifugation a sample solution is continuously pumped through a rotor cavity which is filled with an appropriate density gradient solution. During centrifugation the particles of interest disperse in a radial direction throughout the density gradient solution and at equilibrium are suspended in the density gradient solution at a location wherein their respective buoyant densities correspond to that of the solution.

To remove the particles of interest the density gradient solution containing the centrifuged particles is displaced from the rotor cavity by means of a liquid having a density greater than the highest density portion of the density gradient solution. Ideally, all of the displacing liquid is fed to the outer edge of the rotor cavity. However, in practice some of the displacing liquid flows through the sample exit passageway provided in the rotor core and enters the rotor cavity at the surface of the rotor core member. Since the lighter density portions of the density gradient solution are located closest to the surface of the core member, mixing between the displacing liquid and the density gradient solution takes place (as the displacing liquid gravitates under centrifugal force toward the outer edge of the rotor cavity) causing substantial dilution of the rotor contents. Such mixing, of course, is highly undesirable and, in fact, limits the usefulness of a continuous flow centrifuge for certain experimental investigations.

One approach to the solution of this mixing problem has been to insert a flexible O-ring in the vertical portion of the sample exit passageway provided in the core, which O-ring acts as a valve to control the flow of fluid through this passageway. The operation of the O-ring valve is governed by the particular rotational speed of the rotor. That is, at low rotational speeds the elastomeric O-ring valve is closed to block the flow of liquids through the sample exit passageway. On the other hand, at high ro- United States Patent 0 3,498,531 Patented Mar. 3, 1970 ICC" tational speeds the O-ring valve is subjected to a suflicient centrifugal force to cause the valve to open and allow the flow of fluid through the passageway. Thus, it is apparent that when a flexible O-ring valve is employed the sample solution may be pumped only at rotor rotational speeds above the minimum required to activate the valve and the heavy unloading solution may be pumped only when the rotor is rotated at speeds below this minimum speed. Of course, the particular speed required to activate the O-ring valve may vary with use as the properties of the O-ring change. Moreover, replacement of the O-ring requires the substitution of a second O-ring of very similar properties and siZe which, from a design standpoint, is extremely diflicult to achieve to say the least.

SUMMARY The present invention contemplates a method for use in a continuous flow ultracentrifuge for preventing the inadvertent mixing of the displacing liquid and the den sity gradient solution containing the centrifuged particles of interest as the displacing liquid forces the density gradient solution out of the rotor cavity. To this end, a predetermined volume of gas, such as air, is introduced into the rotor assembly immediately prior to the introduction of the heavy density displacing liquid. The predetermined volume of air lodges in a portion of the sample exit passageway provided in the top of the rotor core to block the flow of the displacing liquid through this passageway and, thus, prevents the mixing of the displacing liquid with the density gradient solution contained in the rotor cavity. In an alternative embodiment contemplated by the present invention an additional radial slot is provided in the core member to receive the predetermined volum of air which blocks the flow of the displacing liquid through the sample exit passageway.

Accordingly, a primary object of the present invention is a new and novel method for preventing the inadvertent mixing between the displacing liquid and the density gradient solution contained in the rotor cavity of a continuous flow centrifuge.

Another object is the provision of a method for preventing the inadvertent mixture between the displacing liquid and the density gradient solution contained in the rotor cavity, which method is independent to the rotational speed of the rotor.

Another object is a simple and expedient method of preventing the intermixing of a displacing liquid and a density gradient solution contained in the rotor cavity of a continuous flow centrifuge.

These and other objects and advantages of the present invention will become apparent from the following detailed description when read in conjunction with the ac 'co'mpanying drawings in which:

FIG. 1 is an elevation view, partly in section, showing a continuous flow centrifuge apparatus;

FIG. 2 is an enlarged partial sectional view showing a suitable centrifuge rotor;

FIG. 3 is a reduced schematic perspective view of the rotor core;

FIG. 4 is a plan view of the rotor core taken along lines 4-4 of FIG. 3.

With reference now to the drawings and more particularly to FIG. 1 thereof, it will be observed that the continuous flow ultracentrifuge includes an outer housing 10 which serves to enclose a drive means, designated generally by the reference numeral 2, and a rotor housing 3. The top of outer housing 10 is provided with an opening 5 through which a rotor 6 may be passed for mounting within a rotor chamber 7 on a drive shaft or spindle 8. A movable door 9 threadably receives and supports an upper bearing and seal assembly 11. Door 9 is provided with spaced rollers 12 which ride in space channels 13 secured to the sides of housing to provide easy access to chamber 7. A latch mechanism (not shown) cooperates in conjunction with controls (not shown) to releasably lock door 9.

The side walls of rotor chamber 7 include a generally cylindrical steel member 14 which acts as a guard should the rotor core explode under the strain created by the relatively high rotational velocities at which it is operated. The lower end of member 14 includes a bottom wall 15 likewise made of relative strong material. The interior of rotor chamber 7 may be provided with refrigeration means including evaporator coils 16 which serve to control the temperature therein.

A suitable vacuum seal is formed between bottom wall 15 and spindle 8 as it extends upwardly into chamber 7. Preferably spindle 8 is made of flexible material and extends downwardly with its lower end journaled in an oil filled bearing assembly 17. Bearing assembly 17 is supported by space brackets 18 extending downwardly from a resiliently mounted base 19. Base 19 may be supported from a fixed member 2 by springs 21. A driven pulley 22 is carried by spindle 8 and is driven by a belt 23 from a drive pulley 24 and motor 25. Means (not shown) may be disposed in housing 10 for evacuating air from chamber 7.

The rotor assembly 40, as best illustrated in FIG. 2, includes a cylindrical bowl 26, the wall of which is threaded at both ends 27 and 28. End 28 threadably receives a closure member or base 29 which includes a generally cylindrical well 31 adapted to receive the upper end of drive spindle 8. Drive spindle 8 fits tightly into well 30 to provide a positive frictional drive connection between spindle 8 and base member 29 whereby rotor bowl 26 may be driven.

The rotor assembly, designated generally by the reference numeral 40, also includes a rotor core member 33 which fits tightly over a boss 34 provided at the upper end of base 29. Boss 34 is provided with an O-ring 31 to provide a tight frictional interconnection between core member 33 and base 29 so that the core member 33 rotates with base 29.

The upper end of bowl 26 threadably receives a cover or lid member 35. A suitable O-ring 36 is interposed between a shoulder 37 formed on the sides of bowl 26 and the lower edge of lid member 35 while a second O-ring 32 is held in an annular groove formed in the top of core member 33 to provide an effective seal between core 33 and lid member 35. The lid member 35 is positioned with respect to the rotor core 33 to provide a relatively large annular space 38 between the upper end of the rotor core 33 and the lid member 35. The lid member 35 is also provided wtih a plurality of radially extending, downwardly sloping bores or holes 39 which communicate between the annular space 38 and the outer edge 41 of the rotor chamber. In practice, four equally spaced holes 39 are formed in lid member 35. However, it will be appreciated that, due to the nature of the illustrated cross-sectional view, only one such hole (39) is shown.

A feed and shaft assembly 42 extends downwardly through an aperture provided in cover member 35 into a well 43 provided in the top of rotor core member 33. The shaft assembly 42 includes an outer tubular conduit 44 and a concentric inner tubular coduit 45 each of which is adapted to carry fluids into and out of the rotor chamber simultaneously during rotation.

The rotor core 33 is a solid cylinder, formed from a suitable metal, which is tapered slightly in an outward direction from bottom to top and which includes at least four projecting vanes, 46, 47, 48 and 49 (best shown in FIG. 3) that divide the rotor cavity into four sector compartments for minimizing turbulence. Core member 33 is provided with a small centrally located axial hole 50 extending from top to the bottom of the core member 33 and which communicates with the rotor cavity 100 by way of a radial extending, downwardly sloping, hole 51 formed in the bottom of the rotor core 33. In practice, rotor core 33 is provided with four equally spaced holes 51 but, again, due to the nature of the illustrated crosssectional view, only one hole 51 is shown. The inner fluid-carrying tube 45 communicates with the axial hole formed in the center of the rotor core while the outer fluid-carrying tube 44 cooperates with both the annular space 38 provided at the top of the core 33 and the radial holes 39 formed in the cover member 35 which lead to the outer edge 41 of rotor cavity 100.

Core member 33 is also provided with four equally spaced vertical extending holes 53 in the top of the core which cooperate with four radially extending slots 54 extending to the surface 55 of core member 33 disposed in rotor cavity 100. Each radial slot 54 communicates with a vertical hole 53 to form a sample eflluent exit passageway.

The inner fluid-carrying tube 45 cooperates with an outlet tube 56 while the outer fluid-carrying tube 44 is associated with a second outlet tube 57.

To facilitate a complete understanding of the operation of the present invention it is believed it would first be appropriate to discuss briefly the typical operating procedure used in analyzing suitable samples by continuous flow ultracentrifugation techniques. Initially the rotor is loaded by pumping a density gradient solution into the rotor cavity by way of outer tubular line 44, annular space 38 and downwardly sloping radial hole 39 formed in the cover member 35. The lightest density portion of the density gradient solution is pumped into the rotor cavity first and enters at the outer edge 41 of the rotor cavity 100. As successively denser portions of the gradient are pumped into the rotor cavity 100 they in turn force the lighter density portions of the density gradient solutions centripetally toward the surface 55 of the core member 33. After the gradient is in the rotor cavity, a fluid having a density greater than the highest density portion of the density gradient solution, which fluid is generally referred to as a cushion, is introduced into the rotor cavity through outer tubular feed-line 44, annular space 38 and radial holes 39 to completely fill the rotor cavity 100.

After the rotor cavity has been completely filled with the density gradient solution, the fluid flow through the outer tubular conduit 44 is terminated and the sample solution is introduced into the rotor cavity 100 by way of inner tubular conduit 45, centrally located axial hole 50 and downwardly sloping radial holes 51 provided in the bottom of core member 33. The sample solution flows through the rotor cavity 100 from the bottom to the top along the surface of core member 33, and exits from cavity 100 by Way of sample exit passageway comprising radial slot 54, vertical hole 53, and annular space 38. The sample solution then flows out through outer tubular conduit 44 and connecting conduit 56 to a suitable container (not shown).

After centrifugation the density gradient solution containing the centrifuged particles of interest is displaced from the rotor cavity 100 by means of a highly dense liquid. That is to say, a highly dense liquid is fed to the outer edge 41 of the rotor cavity 100 by way of outer tubular conduit 44, annular space 38, and radial holes 39. The displacing liquid being of greater density pushes or forces the lighter density gradient liquid solution containing the particles of interest out of the bottom of the rotor cavity 100 through radial holes 51, centrally located axial extending hole 50, inner tubular conduit 45, and connecting conduit 56 to a suitable fraction collector (not shown). In practice it has been found that some of the displacing liquid flows through the sample exit passageway comprising vertical hole 53 and radial slot 54 provided in the top of the core member 33 and enters the rotor cavity 100 at core surface 55. As previously discussed, inasmuch as the lighter density portions of the density gradient solution are located toward this surface of the rotor core member, the displacing liquid (being of a much greater density) flows in a radial direction outwardly through the density gradient solution and, thus, intermixes with it. This naturally results in a dilution of the density gradient solution containing the particles of interest and detrimentally affects the end resolution of the ultracentrifugation process.

In accordance with the principles of the present invention, to prevent the intermixing of the displacing liquid and the density gradient solution containing the centrifuged particles of interest, a predetermined volume of gas, such as air, is introduced into the rotor assembly immediately prior to the introduction of the displacing liquid. In particular, the predetermined volume of air is fed by way of outer tubular conduit 44 to the top of the rotor assembly 40. The volume of air which is introduced through this passageway completely fills each vertical hole 53 provided in the top of core member 33 and partially fills the annular space 38 between cover member 35 and core 33. It should be noted that the vertical holes 53 are orientated perpendicular to the direction of centrifugal force produced by the rotor assembly and hence the volume of air tends to lodge in these vertical holes 53. The highly dense displacing liquid, which follows the predetermined volume of air down the outer tubular conduit 44, easily passes through the annular space 38 to the radial holes 39 and enters the rotor cavity 100 at the cavitys outer edge 41. Of course, since the displacing liquid is of much higher density than that contained in the rotor, its movement to the outer radius is aided by the centrifugal force field. However, the volume of air lodged in the vertical holes 53 blocks the flow of displacing liquid through the sample exit passageways. It should be noted that relatively high pressures would be required to displace the air out of holes 53, because the only escape passage for this air is by way of radial slots 54, against the centrifugal field. Consequently, none of the displacing liquid may enter the rotor cavity 100 at core surface 55 and henceintermixing between the displacing liquid and the density gradient solution is prevented. Moreover, the flow of the sample solution in the normal direction (from the bottom to the top of the core) is not hindered in the least since the sample flow rate is sufficient tc sweep the air block out of the sample exit passageway.

The volume of air necessary to effectively block the sample exit passageway while at the same time keeping free the radial holes 39 leading to the outer edge 41 of rotor cavity 100 is generally governed by the particular type of rotor assembly used in the continuous flow cen trifuge system. For instance, in a suitable rotor manufactured by the present assignee and designated model B-IX, a volume of around 2.5 to 4.0 milliliters was found to be effective. Of course, it will be appreciated that the necessary volume of air varies according to the circumstances. The volume of air may be conveniently controlled by removing the tubular conduit 57 at the seal housing 11 and flushing it with displacing liquid and then draining the required quantity back out of the line before re-attaching it. The length of empty tubing may then be used to indicate the volume of air contained therein.

In an alternative embodiment commensurate with the principles of the present invention, there is provided in at least one core sector an additional slot or small bore 60 (as indicated by the dotted line) in the top of the core member 33 immediately above the O-ring 32, which bore extends in a radial direction to interconnect vertical hole 53 and annular space 38. That is, bore 60 communicates with a small space (not shown) between cover member 35 and the right side of the top of core member 33 to form a generally U-shaped passageway. After forming this passageway, each of the vertical portions 53 of the sample exit passageways is plugged with a suitable stopper (not shown). As a result, the air being fed into the as sembly flows through this U-shaped passageway.

In operation the predetermined volume of air introduced into the rotor assembly immediately prior to the displacing liquid lodges in each of the additionally formed bores 60 and thus forces the displacing liquid to flow through radial holes 39 to the outer edge 41 of rotor cavity in a manner previously discussed. It will be appreciated that either one or all four of the sample exit passageways may be modified in this just-described manner if desired.

Numerous modifications and departures from the specific apparatus described herein may be made by those skilled in the art without departing from the inventive concept of the invention. Accordingly, the invention is to be construed as limited only by the spirit and scope of the appended claims.

What is claimed is:

1. A method for use with a continuous flow centrifuge to prevent mixing between the displacing liquid and the density gradient solution containing the centrifuged particles of interest as the displacing liquid forces the density gradient solution from the rotor cavity comprising the steps of introducing a predetermined volume of gas into the rotor assembly and then introducing a displacing liquid into the rotor cavity containing the density gradient solution.

2. A method for use with a continuous flow centrifuge as defined in claim 1 wherein the predetermined volume of gas is transmitted to the entrance to the rotor cavity, said predetermined volume of gas entering the lodging in a portion of a sample exit passageway provided in the top of a rotatable core member disposed in the rotor cavity and displacing liquid is blocked from flowing through the sample exit passageway by the predetermined volume of gas lodged therein and thus forced to flow through an alternate slot communicating with the outer edge of the rotor cavity.

3. In a continuous flow centrifuge apparatus for preventing mixing between displacing liquid and the density gradient solution containing the particles of interest as the displacing liquid forces the density gradient solution from the rotor cavity comprising a generally cylindrical bowl, a cover member secured to the open end of said bowl, the interior surface of said bowl member and said cover member defining a rotor cavity, a rotor core member disposed in said cavity, means for driving said bowl and core members together at a predetermined rotational speed, said core member including a small bore communicating with the bottom portion of said rotor cavity, the top surface of-said core member and the interior surface of said cover member defining an annular space, the upper end of said core member including a sample exit passageway connecting said annular space with the edge of said core member, the cover member including a radial extending hole connecting said annular space with the outer edge of said rotor cavity, conduit means passing through an aperture in the cover member, one end of said conduit means communicating with said annular space and means for transmitting a predetermined volume of gas through said conduit means immediately prior to transmitting a displacing liquid through said conduit means, said predetermined volume of gas becoming lodged in said sample exit passageway to block the flow of displacing liquid through the sample passageway and forcing substantially all of the displacing liquid to flow through the radial hole provided in the cover member to the outer edge of said rotor cavity to thereby prevent the intermixing of the displacing liquid and the density gradient solution containing the centrifuged samples of interest.

4. Apparatus as claimed in claim 3 wherein said sample exit passageway includes a vertical orientated portion and a radially extending portion, said predetermined volume of gas being directed to and becoming lodged in said vertical orientated portion.

5. Apparatus as claimed in claim 3 wherein said sample exit passageway includes a vertically orientated portion and a radially extending portion and said core member includes a second passageway including a radially extending portion connecting said vertical portion of said sample exit passageway to said annular space, and means for blocking said vertical portion of said sample exit passageway whereby said predetermined volume of air is directed to and becomes lodged in the radially extending portion of said second passageway.

References Cited UNITED STATES PATENTS 1,634,246 6/1927 Jones et al. 233- 8 1,866,638 7/1932 Fawcett 23347 3,332,614 7/1967 Webster et a1. 233-43 OTHER REFERENCES 370,651 4/1932 Great Britain.

WILLIAM I. PRICE, Primary Examiner US. Cl. X.R. 23316, 28, 46

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,498,531 March 3, 1970 Charles H. Chervenka et a1.

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 28, "the" should read and line 31, after "and" insert the Signed and sealed this 24th day of November 1970.

(SEAL) Attest:

WILLIAM E. SCHUYLER, JR.

Commissioner of Patents Edward M. Fletcher, J r.

Attesting Officer

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