US3810576A

US3810576A - Ultracentrifuge rotor

Info

Publication number: US3810576A
Application number: US00075146A
Authority: US
Inventors: A Polson; K Kaufmann
Original assignee: INVENTIONS DEV CORP
Current assignee: INVENTIONS DEV CORP
Priority date: 1969-09-29
Filing date: 1970-09-24
Publication date: 1974-05-14
Anticipated expiration: 1991-05-14
Also published as: DE2047704A1; DE7035901U; DE2047704B2; DE2047704C3

Abstract

An ultracentrifuge is adapted to provide fractionation of virus particles without packing the virus into a pellet but instead concentrating it into a small volume of fluid in an outer collection groove. Thin-layer centrifugation is achieved by means of a plurality of sedimentation walls giving a large area for sedimentation and a small sedimenting distance. Sedimentation walls are inclined to an axis of rotation of the ultracentrifuge by an angle between 0* and 90*, preferably about 20* to cause outward transference of a sediment to a collection groove. Sedimentation walls parallel to the axis of rotation have valves operable at desired times, during rotation of the ultracentrifuge to permit transferring a supernatent fluid from one chamber outwardly to other chambers. A penetrable mat on the parallel sedimentation walls and a ring in the collection groove inhibit backwashing of sediment during deceleration of the ultracentrifuge. Relatively low centrifuging rotational speeds are used.

Description

United States Patent [191 Polson et al.

- May 14, 1974 ULTRACENTRIFUGE ROTOR [75] Inventors: Alfred Polson, Milnerton, Cape Town; Karl Josef Kaufmann,

Rondebosch, Cape Province, both of South Africa [73] Assignee: South African Inventions Development Corporation, Pretoria, Transvall, South Africa [22] Filed: Sept. 24, 1970 Appl. No.: 75,146

[30] Foreign Application Priority Data Sept. 29, 1969 South Africa 69/6838 Sept. 29, 1969 South Africa 69/6839 Sept. 29, 1969 South Africa 69/6840 Sept. 29, 1969 South Africa 69/6841 52' us. Cl. 233/37 [51] B04b 1/00 [58] Field of Search", 233/2, 1 R, 27, 28, 37, 233/31, 34, 37, 43, 35, 46, 20 R, 20 A, 19 R; 210/361,371,380

[56] References Cited UNITED STATES PATENTS 3,114,655 12/1963 .liskra 233/46 X 3,249,295 5/1966 Childs 233/20 R- 3,586,484 6/1971 Anderson... 233/27 X 2,022,815 12/1935 Strezynski 233/20 R 3,326,458 6/1967 Meryman et a1 233/27 FORElGN PATENTS OR APPLlCATlONS 641,382 22/1950 Great Britain 233 20 R Germany 233/20 R 662,529 3/1929 France 233/46 130,511 1/1951 Sweden..... 233/20 A 581,488 8/1958 ltaly 210/380 1,037,416 8/1958 Germany 210/380 Primary Examiner-George H. Krizmanich Attorney, Agent, or Firm-Connolly and Hutz axis of rotation have valves operable at desired times, I

during rotation of the ultracentrifuge to permit transferring a supernatent fluid from one chamber outwardly to other chambers. A penetrable mat on the parallel sedimentation walls and a ring in the collec-- tion groove inhibit backwashing of sediment during deceleration of the ultracentrifuge. Relatively low centrifuging rotational speeds are used.

7 Claims, 4 Drawing Figures PATENTEU IAN 4 I874 sum 1 OF 3 PAIENTEIIIIIY 14

m4 sum

3 or 3 FIG. 3

J LOGC 0 MINUTES FIG. 4

a b c d z 0 MIN MIN MIN M MIN 8 v JUL A4\ L /p f -9 h 0 MIN 8 MIN 16 MIN 24 MIN a 1 ULTRACENTRIFUGE ROTOR BACKGROUND OF THE INVENTION Conventional methods of virus purification often make use of ultracentrifugation at high rotor velocities to sediment the virus particles into concentrated pellets. For example influenza virus is commonly centrifuged at 105,000 g. for 1 hour or longer. The extent of damage done to virus particles in general as a result of this type of treatment is not always realized.

When dealing with viruses which have delicate structures such as certain members of the arbovirus group, it is often harmful to the virus to centrifuge it into pellets and subsequently redisperse the pellets.

Infective agents which suffer loss of titre through this treatment are for example the neurotropic strain of African Horsesickness, Rift Valley, Yellow Fever and.

Wesselsbron viruses. It is our experience that more than 90 percent of the infectivity of arbovirus may be destroyed during the procedure. The loss of virus titre seems to be directly related to the difficulty of redispersing a packed virus pellet. Even if it is possible to redisperse a virus pellet without disruption of the virus particles there remains the problem of disaggregation of aggregates of the infective particles. These clumps of particles may result in inaccuracy when doing physico chemical measurements on the infective agent.

An object of this invention is to provide a means permitting centrifugation without packing the virus into a pellet but instead concentrating it into a suspension in a small volume of fluid. v

A further object is to obtain an improved uniformity of fraction from a mixture by the use of a thin-layer ultracentrifugation, in which the distance over which particles are sedimented before being concentrated is greatly reduced.

A further object is to require only relatively low rotor velocities to fractionate a virus satisfactorily.

A further object is to provide a relatively large area for sedimentation of particles for a given volume of a fluid to be fractionated.

SUMMARY OF. THE INVENTION An ultracentrifuge rotor adapted for centrifugal fractionation of a fluid in accordance with this invention comprises a plurality of chambers at increasing distances from an axis of rotation of the rotor and passage means for the transfer during centrifugation of at least one fraction from a chamber nearer said axis to a chamber further away from the axis.

An ultracentrifuge rotor adapted for centrifugal fractionation of a fluid in accordance with another aspect of this invention comprises at least one sedimentation surface inclined to an axis of rotation of the rotor by an angle more than and less than 90, adapted for transferring a sediment along the surface towards a region in which the sediment is concentrated.

In accordance with another aspect of this invention, an ultracentrifuge rotor for centrifugal fractionation of virus particles suspended in a fluid comprises a plurality of sedimentation walls in a volume for the fluid separated by a sedimentation distance which is less than one quarter of any one dimension of the surface of walls and adapted for centrifuging at less than 10,000 times the acceleration of gravity.

In accordance with one embodiment of the invention walls are inclined to an axis of rotation so that the sediment may slide outwardly along a wall surface to a concentration region'.

In accordance with another embodiment of the invention the walls are given valves so that a supematent may be drained outwardly through the valves at a desired stage of sedimentation.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an axial cross-sectional view of an ultracentrifuge rotor in accordance with a first preferred embodiment of this invention,

FIG. 2 is an axial cross-sectional view of an ultracentrifuge rotor in accordance with a second preferred embodiment of this invention.

FIG. 3 is a graph of variation of concentration with time, and

FIG. 4 is a copy of a series of Schliering Photographs.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In FIG. 1 the ultracentrifuge rotor adapted for a centrifugal fractionation of fluids, which is normally used with axis 15 vertical, comprises a rotor 1 having provided in it an annular cavity 2 to form the centrifuging chamber. The cavity 2 is provided with two annular concentric baffles 3 which in this example were separately made and secured in place with spacing blocks (not shown) between them to maintain their correct spacing. The baffles 3 have a fixed edge 50 and free edge 51 in each case. The rotor l is provided with a lid 4 adapted to close the chamber 2 when the lid is screwed down by means of screws 5 and seats correctly on

sealing rings

6 and 7. The lid 4 is also provided with.

three annular baffles or serrations of sawtooth section 8.

The baffles 3 provide a series of surfaces all substantially parallel in this example, as also the baffles 8 for sedimentation or precipitation during centrifuging. The surfaces are inclined so,as to all lead to a narrow passage 9 between the ends of the baffles 3 and the ends of the baffles 8 which provides a path for sedimentation of precipitate, which has slid along the surfaces of the

baffles

3 and 8, into a collecting groove 10. The cross sectional area of the path 9 in a plane normal to the direction of sedimentation is reduced to a minimum by making the gap between the ends of the baffles 3 and the ends of the baffles 8 very small. In this example the gap was 0.5 mm.

The angle of the baffles 3 to the axis of rotation 15 in this example is approximately 20; this angle in FIG. 1 must be more than 0 and less than 90, say between 10 and 80, more preferably between 20 and The distances of sedimentation are less than one-fourth the dimension of the bafiles from fixed edge to free edge, the other (circumferential) dimension of the baffles being larger.

It will be appreciated that the distances of the paths of sedimentation on the surfaces of the baffles or equivalent means may be reduced by reducing the spaces between the baffles, and that the rate of sliding of the precipitate along the surfaces of the baffles may be increased by increasing the angle defined above; however, the increasing of the angle defined above at the same spacing of baffles increases the effective length of the paths to precipitation in accordance with the cosecant function of the angle.

bolts .11 to a section 12 adapted for connection to a centrifuging machine.

When to be used the suspension to be centrifuged is inserted into the cavity 2 and the lid- 4 is closed.

A quantity of fluid which just covers the tops of the baffle 3 is optimal. During centrifuging the free surface of the fluid takes up the position indicated by dotted lines 13.

EXAMPLE I The following example is of a test carried out with an experimental model measuring 17 cm in diameter.

A dilute suspension of the giant haemocyanins, i.e.

that from burnupena cincta S =9O was centrifuged at I I2,000 r.p.m. for 3 hours from a solution of 40 ml and this protein concentrated in 1 ml. in the collecting groove 10.

Using these data as standard the conditions were calculated which would be necessary to concentrate other substances or viruses in the same period. Thus if a virus has an estimated S 90 then the velocity necessary to concentrate it to approximately the same degree would be r.p.m. (90/X) I2,000 approximately or r.p.m. (90/X) 12,000 more closely.

As most viruses have sedimentation coefficient appreciably higher than 90 Svedbergs, the velocities necessary to concentrate them in the end cavity may be easily attained.

A second treatment under identical conditions will ensure that the maximum purity of the virus obtainable by differential ultracentrifugation is attained.

The large surface area in the rotor space is responsible for the rapid removal of the particles. The sedimenting particles have to travel the distance of one baffle to the next which is at an angle along which it will slide .to be forced into the narrow space between the lower baffles and upper baffles.

After centrifuging the supernatant fluid is removed from the chamber 2, as well as that fluid above the sediment in the collection groove 10.

By removing the cord 16 with forceps the fluid bebind the cord 16 may be removed with a Pasteur pipette. By gently triturating the small amount of fluid which is of the order of 1 ml any virus remaining on the surface may be removed. As the ratio between the final volume of the virus suspension to that of the original is 1/40 it may be expected that a'certain amount of extraneous protein would be present in the suspension. The centrifugation may be repeated in an appropriate medium and the concentration of the extraneous material may be reduced to a very low level. The second purification centrifugation should be done at the same rotor velocity and duration as the initial; A table of selected rotor velocities for removal of 90 percent of the infectivity of a variety of substances and infectious agents is given in Table 1. This table is of value for assessment of a suitable rotor velocity to deal with the substance at hand.

i TABLE 1 Rpm required for centrifuging substances of difierent sedimentation coefficient from solution to the extent of to 99 percent. Thin layer centrifugation. Volume 35 m1 T- 4C, medium: 10 percent serum saline 520w sed. coeff.; rpm rotor velocity in revs/min; time 2% hours.

10 ro- Substance S ,,(ref) Rpm S Xrpm 5 8 cincta haemucy. 89 & 91, 12500 I I2 I39 Poliovirus' I55 9150 I42 I30 African Horsesickncss 550 4860 268 I30 virus Influenza virus 690 4360 301 I32 or rpm z (If/V8 ploratory runs were made on proteins and viruses of different sedimentation coefficients.

TABLE 2 a. Concentration of Wesselsbron virus by thin layer centrifugation. Volume original 35 ml T 4C time of centrifugation 3 hours; Vc/V0=ratio of volumes of concentrated material to the original; titre (O/C)qatio of titres of original to concentrated material in neg log LD and titre SNF is the titre of the supernatant fluid after centrifugation.

T.C.F. and m are tissue culture fluid and infected mouse brain extracts respectively. rpm= revolutions per minute.

Source Vc/Vo rpm Titre O/C Titre SNF TCF 0.l66 10,000 5.9 l 6.8 3.8 -m 0143 l0',000 6.5 7.5 4.2

b. Concentration of Influenza virus by thin layer centrifugation. t= 3 hrs T 4C All allantoic fluid HA. and HAc haemoagglutination of original and of concentrate orig.

vol. 35 ml Source Vc/Vo rpm HA0 lHAc HA SNF All. fluid 0.043 5,000 2 l0 I6Xl0 0. Concentration of the haemocyanin'of burnupena cincta. Concentration determined by u v absorption at 28 mm. Conditions the same as above (a') and (b) Cone 0.21

Vc/Vo 0.043

O/ConcC 4.70

rpm Gone From the above table it is evident that the virus from mouse brains infected with Wesselsbron virus was recovered approximately quantitatively after centrifugating it into a small volume in which it was not necessary to recover a pellet. The result with Influenza virus is probably the substance best suited to indicate the usefulness of the technique of thin layer ultracentrifugation. This virus was concentrated quantitatively at a relatively low speed of centrifugation in a small volume of fluid and from electron micrographs taken of the purified material there appeared to be no damage done to the virus particle. RATE OF SEDIMENTATION As the process of sedimentation is complicated by the presence of the baffles the rate at which the concentration of substance in the SNF is diminished during centrifugation was determined analytically. For this purpose the haemocyanin of B. cincta was centrifuged at constant rotor velocity and relative protein concentrations in the SNF was determined from its absorption at 280u after different periods of centi ifugation.

Reference may be m ade to the graph FIG. 3'of the accompanying drawings.

It was found that, by plotting log /c against t, a linear function is obtained.

0 concentration t time It is interesting to note the slope of the straight line is proportional to the sedimentation coefficient. Thus it is only necessary to standardize a rotor for one substance of known sedimentation coefficient at a definite rotor velocity to enable the experimenter to determine the sedimentation coefficients of other biologically active substances. The usefulness of the rotor is illustrated here on the determination of the sedimentation coefficient of a virus. For this purpose ECBO S.A.I. virus was selected as this virus has a known S value determined by analytical ultracentrifugation. This virus grows readily in foetal lamb kidney cells and may be assayed by plaque counting using Copper plates. The average value of 160 Svedbergs obtained from two experiments agreed well with the value of 155S obtained in the analytical ultracentrifuge.

It may be noted that all examples quoted in the text are at ranges of between 4,000 and 13,000 RPM. This velocity is substantially less than the rate of velocities common in conventional analytical centrifuges which may for example be in excess of 30,000 Rpm. These very high rotational velocities tend to Concentrate the virus in a pellet.

Example 2 Separation of proteins of different sedimentation coefficient A mixture of two haemocynins abtained from jasus lalandii and burnupera cincta were dissolved in saline and placed in the chamber 2 of the rotor l and centrifuged at 16,000 revs per minute. The centrifuge was stopped periodically after predetermined periods of centrifuging and aliquots of the supernatant fluid were removed for analysis in an analytical ultracentrifuge by centrifuging for a fixed period of time in each case. The diagrams presented in FIG. 4 graphs are copies of Schliering photographs taken of the samples. Graphs a,

, b, c, d and e show the results obtained before centrifuging, after 30 minutes, 45 minutes. 60 minutes and 90 minutes respectively. Peaks 37 and 38 in each case indicate two fractionsof a mixture and it will be observed that after successive periods the component indicated by peak 38 is progressively reduced, till after 90 minhour at 16,000 Rpm. Diagrams f, g, h, and'i were obtained on that material in an analytical centrifuge by centrifuging respectively for 0 minutes, 8 minutes, 16 minutes and 24 minutes.

The components of the synthetic mixture were of known sedimentation coefficient, namely the following: I

jasus lalandii S 16 "burnupera cincta S 89 and 91.

It appears from the graphs that the component of burnupera cincta (peak 38 in graphs a, b, c, d and e) is sedimented out almost completely after 90 minutes at 16,000 rpm. The graphs f, g, h, and 1' indicate that centrifuging in the analytical centrifuge separated the component of 89 and 91 sedimentation coefficient to a limited degree.

The groove 10 is provided with a roughened ring of solid nylon material 16 which inhibits backwash during deceleration of the rotor 1 from redispersing the sediments or precipitate located at the base of the groove 10. The nylon ring 16 has a roughened surface so that the sediment or precipitate can permeate past the ring during centrifuging and become concentratednear the base of the groove 10, the ring 16 nevertheless inhibiting backwashing of the sedimented precipitate over the comparatively short period of deceleration of the rotor 1 when the supernatant fluid in the chamber 2 develops a relative velocity. However, thesurfaces of nylon ring 16 may be cut in a series of transverse grooves or have circumferential grooves and ridges toallow free communication and permeation of material from the chamber 2 into the collecting groove 10.

The ultracentrifuge of FIG. 2 will now be described.

As shown in FIG. 2 the rotor 1 comprises a series of

chambers

22, 23 and 24 radially displaced and of annular shape thusproviding a substantiallybalanced rotor.

A lid 4 is provided which may be closed over the top of the rotor 1 so as to seal off the chamber for centrifuging. Each chamber is provided with a valve between it and the next, being a valve 25 between the

chambers

22 and 23 and valve 26 between the

chambers

23 and 24. Each valve comprises a ball 27, springloaded by spring 28 so as to seat on the annular seat provided by the valve hole 29. The last chamber 24 is provided with baffles 3 and a collection groove or cavity '10 having features substantially as described in FIG. 1.

A rubber or other resilient material annular ring 17 is located on the rotor 1 between the lid 4 and suitable grooves in the rotor l. The r'ing 17 is provided with a lip 18 which is of suitable section and flexibility that during centrifuging at suitable rotational velocities the lip 18 flexes outwards so as to lift off the nose l9 and provide a path for sedimentation or precipitation of heavy material into. the groove or cavity 10. After deceleration of the rotor the lip 18 will flex back to as to sealably contact the nose l9 and thereby exclude the cavity of groove 10 from the effects of backwashing. The design of the lip 18 and the material used for the whichseveral may be dispersed around the circumference if desired, is of sufficiently small cross section to adequately isolate the groove or cavity 10 from the effect of backwashing during deceleration.

As afurther alternative in accordance with this in vention, the groove or cavity 10 may conveniently be provided in the lid 4, which feature may facilitate recovery of the precipitate in certain circumstances.

A further alternative means for inhibiting backwash in accordance with this invention comprises a plurality of radially disposed baffles located in the collection groove or cavity 10 and sufficiently closely spaced together to substantially inhibit the dispersal of the sediment or precipitate located in the groove or cavity 10.

However, in accordance with a further alternative of this invention instead of providing for the lip 18 to lift off the nose 19, the lip 18 may be perforated by a plurality of apertures or may be reduced substantially to the form of a mesh or the like which has apertures of suitable dimension so as to admit sedimentation or precipitation products to permeate into the cavity or groove 10 but being sufficiently small so as to satisfactorily isolate cavity or groove 10 from the effects of 2 screws 35 were provided around the periphery for additional sealing of the lid 4 on the rotor.

When the apparatus is used strips of hardened filter paper are placed on the 'walls of the first two

cavities

22 and 23 in the positions indicated by the letters A and B. A volume of fluid to be centrifuged is then intro- .duced to the first cavity 22. The rotor is accelerated to a velocity at which the first valve still remains closed but which is satisfactory for bringing about the first fractionation of the fluid. The material is centrifuged at thisspeed until the first or coarsest material is spun out and is held on the filter paper. The rotor is then accelerated to a high velocity at which speed the first valve 25 opens to allow the fluid in the cavity 22 to move into the cavity 23. As the diameter of the cavity 23 is greater than that of the cavity 22, the depth of the fluid is less from which it follows that the distance over which a particle must sediment is less. At the higher vein the cavity or chamber 10 in view of the fact that the lip 18 of the ring 17 moves away from the nose 19 under reaction of centripetal acceleration. The venthole 20 provides for venting-of air in the cavity 10 as it is filled up.

Thus when this rotor is employed crude biological material may be subdivided into four fractions. These are, the coarsest material on the filter, paper A, the finer material on the filter paper B, the material in the receiving cavity 10 and the substance of lower sedimentation co-efficient in the receiving cavity 10 above the precipitate. The components with the lowest sedimentation co-efficient remain in the supernatant fluid amongst the baffles.

Example ity 10 and supernatant remaining in the chamber 24 view of their inclination until the material is collected amongst the baffle 3 in the position E.'These materials were photographed in the electron microscope at 4 X 20,000 magnification and the photographs illustrated a very satisfactory fractionation of the component of the sample. The same from the position A comprised broken up cell nucleii and other matter of fibrous nature, all substantially bulky. The precipitate from the wall B comprised particles ranging in transverse dimension from between 0.5 to l by 10 mm. The precipitate at the base of the collecting groove 10 collected from the position C appear to comprise mainly ribosomes of transverse dimensions between 0.2 to 0.25 X 10 The supernatant in the chamber 24 collected from position E appear to comprise ordinary serum components, albumins, haemoglobulins and the like of a substantially smaller particle size. The supernatant above the collection groove 10 collected from the position D was not identified but was clearly of a slightly coarser particle size to the supernatant in the chamber.

Referring to FIG. 2, the

chambers

22 and 23 are each provided with a nylon mesh 40 located on the walls A and B respectively. The nylon mesh 40 is of suitable kind and mesh size to provide unobstructed passage of the sediment onto the walls A and B of the

chambers

22 and 23 respectively during the centrifuging. Thus the sediment is collected behind each nylon mesh 40 and during deceleration of the rotor a mesh protects the sediment from being redispersed into the supernatant. After stopping of the rotor the lid 4 may be removed, the nylon meshes 40 carefully extracted and the sediment removed from the walls A and B by being scraped off with a scalpel. This provides the more advantageous manner of collecting the sediment and for example precipitating it onto a hardened filter paper, in which case the sediment has to be separated from the surface of the filter paper and one tends to find fibres from the filter paper being transferred into the sediment. It is in fact very difficult to avoid certain of these fibres from contaminating the collected sediment.

What I claim is:

1. An ultracentrifugation process which comprises spinning material which is to be separated about a spinning axis, permitting heavier particles to sediment to a plurality of regions in the material, in which the regions are annular and concentric with the spinning axis and the different regions are at different radial distances from the spinning axis, constraining such particles to slide in thin layers directed at angles between 20 and 70 to the spinning axis outwardly, causing the particles in all the thin layers to all slide into an annular layer which is orientated at 90 to the spinning axis the particles joining to form a narrow-width stream in this annular layer, to stream away from the spinning axis and to collect in an outermost annular collection region, in which the rotational speed of spinning is selected at a value which satisfies the condition that a number, N, computed from the formula is less than 3 X 10, i.e. N 3 X 10', where rpm. is the rotational speed of spinning expressed in revolutions per minute,

r is the radical distance of the annular collection region from the spinning axis expressed in centimeters,

S is the Svedburg coefficient of the material to be collected in the collection region, and

G is the standard acceleration of free fall due to gravity recognized by the US. Department of Commerce, equal to 9,80665 metres per second squared, so that the sedimented material is concentrated as a suspension in a small volume of fluid.

2. An ultracentrifugation process as claimed in claim 1, which is performed as a batch process, comprising preliminarily containerizing a material to be separated and applying a rotational acceleration to the material and terminally decelerating the spinning material at a means for deceleration while isolating the rotational backwash of unsedimented material substantially from the sedimented material by an intervening barrier,

in which every vane terminates in a free edge further removed from the axis of rotation than remaining parts of the vane, every vane has an edge which iscloserto the axis of rotation and which is contiguous with the wall defining the chamber and in which every vane is wholly imperforated, in which every vane free edge is aligned on an imaginary plane normal to the axis of rotation, a narrow passage in the rotor defined between said vane free edges and a surface of the rotor on a plane substantially normal to the axis of rotation, in which the narrow passage is terminated by an annular sediment collection groovelocated outwardly of the vanes, concentric therewith and formed by structure which is fixed to the walls forming the inner and outer chambers and in which means for retaining the collected sediment against backwash is located at a sole annular entrance to the annular collection groove, in which the annular anti-backwash structure is concentric with the axis of rotation, is removable from the entrance to the collection groove and nearly closes the entrance to the groove so that at rotational speeds which cause sedimenting the annular structure permits sedimenting material to pass into the collection groove while during deceleration it resists backwashing.

4. An ultracentrifuge rotor as claimed in claim 3, in which the means for retaining the collected sediment against backwash comprises a roughened ring whereby through passages is provided.

5. An ultracentrifuge rotor as claimed in claim 3, in which the annular structure comprises a ring of flexible material having a 'lip which at standstill closes the groove but which is of suitable cross section and flexibility so that at certain rotational velocities the entrance to the groove is opened as a result of flexing of the lip under centrifugal reaction.

6. An ultracentrifuge rotor as claimed in claim 5, in which the lip has vent holes provided in it.

7. An ultracentrifuge rotor as claimed in claim 3 which comprises an annular inner chamber disposed equidistantly around the axis of rotation disposed inan increase to a predetermined rotational speed of the rotor, in which the passage leads to the outer chamber.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. ,810,576 Dated May 3, 197 4 Invent0r(s) Alfred Poison at all It is Certified that error appears in the above-identified patent and that said Letters Patentare hereby corrected as shown below:

jifi'ifl 3, line 31, the equation shoule read as follows:

" 2626 a x W Signed and sealed this 19th day of November 197%;

(SEAL) Attest McCOY My GIBSON JR. c. MARSHALL DANN Attesting Officer Commissioner of Patents po'wso (w'eg) USCOMM-DC 603764 69 U.$ GOVERNMENT PRINTING OFFICE: I969 0-366-31',

Claims

1. An ultracentrifugation process which comprises spinning material which is to be separated about a spinning axis, permitting heavier particles to sediment to a plurality of regions in the material, in which the regions are annular and concentric with the spinning axis and the different regions are at different radial distances from the spinning axis, constraining such particles to slide in thin layers directed at angles between 20* and 70* to the spinning axis outwardly, causing the particles in all the thin layers to all slide into an annular layer which is orientated at 90* to the spinning axis the particles joining to form a narrow-width stream in this annular layer, to stream away from the spinning axis and to collect in an outermost annular collection region, in which the rotational speed of spinning is selected at a value which satisfies the condition that a number, N, computed from the formula

2. An ultracentrifugation process as claimed in claim 1, which is performed as a batch process, comprising preliminarily containerizing a material to be separated and applying a rotational acceleration to the material and terminally decelerating the spinning material at a means for deceleration while isolating the rotational backwash of unsedimented material substantially from the sedimented material by an intervening barrier, bringing the material to standstill, removing the unsedimented material, removing the intervening backwash barrier and taking out the sedimented material.

3. An ultracentrifuge rotor which comprises an annular outer chamber defined by walls which are disposed equidistantly about an axis of rotation, which has a plurality of radially spaced annular vanes in the chamber presenting inwardly directed surfaces which are concentric with the axis of rotation and which are inclined at an angle between 20* and 70* to the axis of rotation, in which every vane terminates in a free edge further removed from the axis of rotation than remaining parts of the vane, every vane has an edge which is closer to the axis of rotation and which is contiguous with the wall defining the chamber and in which every vane is wholly imperforated, in which every vane free edge is aligned on an imaginary plane normal to the axis of rotation, a narrow passage in the rotor defined between said vane free edges and a surface of the rotor on a plane substantially normal to the axis of rotation, in which the narrow passage is terminated by an annular sediment collection groove located outwardly of the vanes, concentric therewith and formed by structure which is fixed to the walls forming the inner and outer chambers and in which means for retaining the collected sediment against backwash is located at a sole annular entrance to the annular collection groove, in which the annular anti-backwash structure is concentric with the axis of rotation, is removable from the entrance to the collection groove and nearly closes the entrance to the groove so that at rotational speeds which cause sedimenting the annular structure permits sedimenting material to pass into the collection groove while during deceleration it resists backwashing.

5. An ultracentrifuge rotor as claimed in claim 3, in which the annular structure comprises a ring of flexible material having a lip which at standstill closes the groove but which is of suitable cross section and flexibility so that at certain rotational velocities the entrance to the groove is opened as a result of flexing of the lip under centrifugal reaction.

7. An ultracentrifuge rotor as claimed in claim 3 which comprises an annular inner chamber disposed equidistantly around the axis of rotation disposed inwardly of the outer chamber and concentric with it the inner chamber having a wall which forms an inwardly directed sedimentation surface in which the inwardly directed sedimentation surface of the inner chamber is at all points equidistant from and parallel to the axis of rotation in which a passage passes through the wall forming the inwardly directed surface, the passage having closure means adapted to be self-opening upon centrifugal reaction of a closure member of the closure means exceeding a predetermined value by reason of an increase to a predetermined rotational speed of the rotor, in which the passage leads to the outer chamber.