U.S. patent number 3,810,576 [Application Number 05/075,146] was granted by the patent office on 1974-05-14 for ultracentrifuge rotor.
This patent grant is currently assigned to South African Inventions Development Corporation. Invention is credited to Karl Josef Kaufmann, Alfred Polson.
United States Patent |
3,810,576 |
Polson , et al. |
May 14, 1974 |
**Please see images for:
( Certificate of Correction ) ** |
ULTRACENTRIFUGE ROTOR
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.degree. and 90.degree., preferably about 20.degree.
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.
Inventors: |
Polson; Alfred (Milnerton, Cape
Town, ZA), Kaufmann; Karl Josef (Rondebosch, Cape
Province, ZA) |
Assignee: |
South African Inventions
Development Corporation (Pretoria, Transvall,
ZA)
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Family
ID: |
27506062 |
Appl.
No.: |
05/075,146 |
Filed: |
September 24, 1970 |
Foreign Application Priority Data
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Sep 29, 1969 [ZA] |
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69/6838 |
Sep 29, 1969 [ZA] |
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69/6839 |
Sep 29, 1969 [ZA] |
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69/6840 |
Sep 29, 1969 [ZA] |
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69/6841 |
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Current U.S.
Class: |
494/4; 494/36;
494/38; 494/37 |
Current CPC
Class: |
B04B
5/0407 (20130101) |
Current International
Class: |
B04B
5/04 (20060101); B04B 5/00 (20060101); B04b
001/00 () |
Field of
Search: |
;233/2,1R,27,28,37,31,34,37,43,35,46,2R,2A,19R
;210/361,371,380 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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641,382 |
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Aug 1950 |
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GB |
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1,014,348 |
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Jan 1958 |
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DT |
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662,529 |
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Mar 1929 |
|
FR |
|
130,511 |
|
Jan 1951 |
|
SW |
|
581,488 |
|
Aug 1958 |
|
IT |
|
1,037,416 |
|
Aug 1958 |
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DT |
|
Primary Examiner: Krizmanich; George H.
Attorney, Agent or Firm: Connolly and Hutz
Claims
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.degree. and 70.degree. 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.degree. 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 ##SPC1##
is less than 3 .times. 10.sup.6, i.e. N < 3 .times. 10.sup.6,
where r.p.m. 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.sub.20w 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 U.S. 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, 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.degree. and 70.degree. 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.
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 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.
Description
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.
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 0.degree. and less than
90.degree., 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 supernatent 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 1
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.degree.; this angle in FIG. 1 must be
more than 0.degree. and less than 90.degree., say between
10.degree. and 80.degree., more preferably between 20.degree. and
70.degree.. The distances of sedimentation are less than one-fourth
the dimension of the baffles 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.
Thus where the angle is increased it may be desirable to reduce the
distance or spacing between adjacent baffles until the extreme
application of the invention may be a very large number of thin
baffles very closely spaced and inclined at an angle to the axis of
rotation near the upper limits specified above.
Preferably the distance from the axis of rotation to the baffles is
at least as large or substantially larger than the radial width of
the chamber.
The rotor 1 is adapted for attachment by means of 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 1
The following example is of a test carried out with an experimental
model measuring 171/2 cm in diameter.
A dilute suspension of the giant haemocyanins, i.e. that from
burnupena cincta S.sub.20w =90 was centrifuged at 12,000 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.sub.20w = 90
then the velocity necessary to concentrate it to approximately the
same degree would be
r.p.m. = (90/X) .sup.. 12,000 approximately
or r.p.m. = (90/X) .sup.. 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 behind 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.
TABLE 1
Rpm required for centrifuging substances of different sedimentation
coefficient from solution to the extent of 90 to 99 percent. Thin
layer centrifugation. Volume 35 ml T = 4.degree.C, medium: 10
percent serum saline S.sub.20w = sed. coeff.; rpm rotor velocity in
revs/min; time 21/2 hours.
10.sup.-.sup.4 10.sup.-.sup.8 Substance S.sub.20w (ref) Rpm
S.sub.20w .times.rpm S.sub.20w(rpm ).sup.2 B cincta haemocy. 89
& 91 12500 112 139 Poliovirus 155 9150 142 130 African
Horsesickness 550 4860 268 130 virus Influenza virus 690 4360 301
132
or rpm .apprxeq. (K/VS.sub.20w)
In a similar manner the velocity for removal of more than 90
percent of low molecular material may be estimated. A nylon rotor
would not be suitable for this purpose as the tensile strength of
this material is too low to resist very high centrifugal forces.
Rotors made of materials with much higher tensile strengths would
be suitable for this purpose. Duraluminium or even titanium could
be machined into suitable shape.
To indicate the usefulness of the rotor a series of exploratory
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 = 4.degree.C time of centrifugation 3
hours; Vc/Vo=ratio of volumes of concentrated material to the
original; titre (O/C)=ratio of titres of original to concentrated
material in neg log LD.sub.50 ; 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 0.166 10,000 5.9 / 6.8 3.8
m 0.143 10,000 6.5 / 7.5 4.2 m 0.10 12,500 5.6 / 6.5 3.5 m 0.05
10,000 6.4 / 7.8 4.5 m 0.043 12,500 5.5 / 7.0 3.8 m 0.043 12,500
6.5 / 8.6 5.1
b. Concentration of Influenza virus by thin layer
centrifugation.
t = 3 hrs T = 4.degree.C All allantoic fluid HA. and HAc
haemoagglutination of original and of concentrate orig. vol. 35
ml
Source Vc/Vo rpm HAo / HAc HA SNF All. fluid 0.043 5,000
2.times.10.sup.4 / 6.times.10.sup.5 1 .times. 10.sup.3
c. Concentration of the haemocyanin of burnupena cincta.
Concentration determined by u v absorption at 28 mm. Conditions the
same as above (a) and (b)
Vc/Vo rpm Conc O/ConcC Conc SNF 0.043 15,000 0.21 / 4.70 0.015
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 centrifugation.
Reference may be made to the graph FIG. 3 of the accompanying
drawings.
It was found that, by plotting log .sup.1 /c against t, a linear
function is obtained.
c = 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.sub.20w 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 1 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
fractions of a mixture and it will be observed that after
successive periods the component indicated by peak 38 is
progressively reduced, till after 90 minutes the component
indicated by the peak 38 appears to be practically completely
eliminated.
The precipitate or sediment which collected in the end cavity 10 of
the ultracentrifuge 1 after 90 minutes was diluted 1-in-5 and then
ultracentrifuged for analytical purposes in the analytical
ultracentrifuge. The material contained a portion of the smaller
component and in order to get rid of that, was centrifuged for 1
hour 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:
jasus lalandii S.sub.20w = 16
burnupera cincta S.sub.20w = 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
i 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 concentrated near 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, the surfaces of
nylon ring 16 may be cut in a series of transverse grooves or have
circumferential grooves and ridges to allow 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 thus providing a
substantially balanced 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 1.
The ring 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 19
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
19 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
ring 17 must therefore be correctly chosen so that satisfactory
operation occurs at the suitable velocities of rotation. A
vent-hole 20 is provided in the lip 18 so as to provide for escape
of gases as the groove or cavity 10 is filled up during
centrifuging. The venthole 20 of which several 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 a further alternative in accordance with this invention, 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 backwash during deceleration.
The rotor 1 is mountable on a shaft 30 and is tapered substantially
as shown for increased resistance to the stresses generated by
centripetal acceleration during rotation. A screw 31 screws into a
nut 32, which holds the rotor 1 on the shaft 30, so as to hold down
the lid 4. A series of seals 33 is provided to achieve satisfactory
sealing of the rotor during operation.
The tension of the springs 28 are controllable by screw adjusting
the hollow backing-up screws 34. Eight 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 introduced 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 this
speed 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 velocity and greater
radius and also the shorter distance of sedimentation the
components of low sedimentation rates are removed and trapped in a
filter paper trap in position B. The acceleration is then repeated
until the valve 26 opens to admit the fluid into the last chamber
24. This may commonly be the maximum attainable speed with the
rotor. Material in the fluid in the chamber 24 then sediments onto
the surfaces of the baffles 3 and the surface 14 and moves along
the surfaces by view of their inclination until the material is
collected in 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
A mixture of Burnupena cincta and Jasus lalandii was centrifuged in
saline, beginning at the innermost chamber 22. The centrifugation
was continued until the substance passed into the chamber 23 and
then into the chamber 24 and was continued at maximum rotor speed
for final fractionation in the last chamber 24. The rotor was then
opened and the sediment or precipitate collected off the walls A
and B of the chambers 22 and 23, and from the cavity 10 in the
position C as well as supernatant collected from the position D in
the cavity 10 and supernatant remaining in the chamber 24 amongst
the baffle 3 in the position E. These materials were photographed
in the electron microscope at 4 .times. 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 1 by
10.sup.-.sup.4 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 .times.
10.sup.-.sup.4 mm. 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.
* * * * *