US3810576A - Ultracentrifuge rotor - Google Patents

Ultracentrifuge rotor Download PDF

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Publication number
US3810576A
US3810576A US00075146A US7514670A US3810576A US 3810576 A US3810576 A US 3810576A US 00075146 A US00075146 A US 00075146A US 7514670 A US7514670 A US 7514670A US 3810576 A US3810576 A US 3810576A
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Prior art keywords
axis
annular
rotation
rotor
sedimentation
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Expired - Lifetime
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US00075146A
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English (en)
Inventor
A Polson
K Kaufmann
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INVENTIONS DEV CORP
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INVENTIONS DEV CORP
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Priority claimed from ZA696841*A external-priority patent/ZA696841B/xx
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
    • B04B—CENTRIFUGES
    • B04B5/00—Other centrifuges
    • B04B5/04—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers
    • B04B5/0407—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles

Definitions

  • 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.
  • Infective agents which suffer loss of titre through this treatment are for example the neurotropic strain of African Horsesickness, Rift Valley, Yellow Fever and.
  • 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.
  • 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.
  • 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.
  • 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.
  • walls are inclined to an axis of rotation so that the sediment may slide outwardly along a wall surface to a concentration region'.
  • the walls are given valves so that a supematent may be drained outwardly through the valves at a desired stage of sedimentation.
  • 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
  • FIG. 4 is a copy of a series of Schliering Photographs.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the depth of the fluid is less from which it follows that the distance over which a particle must sediment is less.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 rotation
  • 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.
  • 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.

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US00075146A 1969-09-29 1970-09-24 Ultracentrifuge rotor Expired - Lifetime US3810576A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
ZA696840 1969-09-29
ZA696841*A ZA696841B (en) 1969-09-29 1969-09-29 Improved precipitation in ultracentrifuges
ZA696839 1969-09-29
ZA696838 1969-09-29

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4007007A (en) * 1974-02-22 1977-02-08 Hans Otto Ernst Gazda Apparatus for decontaminating liquids of bacteria
US4036425A (en) * 1973-02-08 1977-07-19 Mikhail Ivanovich Ilin Rotor for a centrifuge
US4056225A (en) * 1976-04-29 1977-11-01 Norton George Hein Jr Centrifuge rotor for separating phases of a liquid
US4111355A (en) * 1977-06-15 1978-09-05 Beckman Instruments, Inc. Multi-compartment centrifuge rotor liner
US5589400A (en) * 1994-12-14 1996-12-31 Shandon, Inc. Method of distributing material onto a microscope slide of a large cytology sample chamber

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR662529A (fr) * 1928-10-19 1929-08-08 Separator Ab Dispositif pour bols de centrifugeuses
US2022815A (en) * 1933-09-27 1935-12-03 Laval Separator Co De Centrifugal bowl for separating heavy sludge and solids from lighter liquids
GB641382A (en) * 1947-05-05 1950-08-09 Aage Nyrop Improvements in or relating to centrifugal separators
DE1014348B (de) * 1952-01-08 1957-08-22 Anschuetz & Co Gmbh Radialrohrzentrifuge
DE1037416B (de) * 1951-06-07 1958-08-28 Walter Joseph Podbielniak Nach dem Gegenstromprinzip und unter Fliehkrafteinwirkung arbeitende Einrichtung zum innigen gegenseitigen Durchdringen und unmittelbar anschliessenden Trennen von Fluessigkeiten
US3114655A (en) * 1961-11-16 1963-12-17 Buckau Wolf Maschf R Centrifugal separator
US3249295A (en) * 1966-05-03 Method for separating liquid mixtures
US3326458A (en) * 1965-05-28 1967-06-20 Harold T Meryman Container and process of storing blood
US3586484A (en) * 1969-05-23 1971-06-22 Atomic Energy Commission Multistation analytical photometer and method of use

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3249295A (en) * 1966-05-03 Method for separating liquid mixtures
FR662529A (fr) * 1928-10-19 1929-08-08 Separator Ab Dispositif pour bols de centrifugeuses
US2022815A (en) * 1933-09-27 1935-12-03 Laval Separator Co De Centrifugal bowl for separating heavy sludge and solids from lighter liquids
GB641382A (en) * 1947-05-05 1950-08-09 Aage Nyrop Improvements in or relating to centrifugal separators
DE1037416B (de) * 1951-06-07 1958-08-28 Walter Joseph Podbielniak Nach dem Gegenstromprinzip und unter Fliehkrafteinwirkung arbeitende Einrichtung zum innigen gegenseitigen Durchdringen und unmittelbar anschliessenden Trennen von Fluessigkeiten
DE1014348B (de) * 1952-01-08 1957-08-22 Anschuetz & Co Gmbh Radialrohrzentrifuge
US3114655A (en) * 1961-11-16 1963-12-17 Buckau Wolf Maschf R Centrifugal separator
US3326458A (en) * 1965-05-28 1967-06-20 Harold T Meryman Container and process of storing blood
US3586484A (en) * 1969-05-23 1971-06-22 Atomic Energy Commission Multistation analytical photometer and method of use

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4036425A (en) * 1973-02-08 1977-07-19 Mikhail Ivanovich Ilin Rotor for a centrifuge
US4007007A (en) * 1974-02-22 1977-02-08 Hans Otto Ernst Gazda Apparatus for decontaminating liquids of bacteria
US4056225A (en) * 1976-04-29 1977-11-01 Norton George Hein Jr Centrifuge rotor for separating phases of a liquid
US4111355A (en) * 1977-06-15 1978-09-05 Beckman Instruments, Inc. Multi-compartment centrifuge rotor liner
US5589400A (en) * 1994-12-14 1996-12-31 Shandon, Inc. Method of distributing material onto a microscope slide of a large cytology sample chamber

Also Published As

Publication number Publication date
DE2047704B2 (de) 1974-04-04
DE2047704C3 (de) 1974-11-07
DE2047704A1 (de) 1971-04-22
DE7035901U (de) 1973-06-20

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