U.S. patent number 3,708,111 [Application Number 05/071,882] was granted by the patent office on 1973-01-02 for apparatus and method for gradient zonal centrifugation.
Invention is credited to Phillip Sheeler, John R. Wells.
United States Patent |
3,708,111 |
Sheeler , et al. |
January 2, 1973 |
APPARATUS AND METHOD FOR GRADIENT ZONAL CENTRIFUGATION
Abstract
A reorienting gradient zonal rotor for separation of cell
components and the like, said rotor comprising a cylindrical
chamber divided into a plurality of sector-shaped compartments by
vertical septa radiating from a central core. Either the annular
floor or ceiling of said chamber is formed with sloping converging
walls intermediate the spaced outer and inner walls of said chamber
to converge either to an annular V-shaped groove or to a plurality
of funnel-shaped recesses terminating, respectively, in an annular
apex or a plurality of apices at which a plurality of spaced apart
gradient removal ports are located, and toward each of which said
converging sloping walls cause the gradient zones to be constricted
and concentrated for improved definition and separation for
subsequent fraction collection and analysis.
Inventors: |
Sheeler; Phillip (Northridge,
CA), Wells; John R. (Los Angeles, CA) |
Family
ID: |
26752772 |
Appl.
No.: |
05/071,882 |
Filed: |
September 14, 1970 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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886720 |
Dec 19, 1969 |
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Current U.S.
Class: |
494/37; 494/38;
494/74; 494/42 |
Current CPC
Class: |
B04B
5/0407 (20130101); B04B 1/00 (20130101); B04B
5/00 (20130101) |
Current International
Class: |
B04B
5/04 (20060101); B04B 5/00 (20060101); B04B
1/00 (20060101) |
Field of
Search: |
;233/27,28,32,1R,45,23R,19R,46,47R,34,44 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Franklin; Jordan
Assistant Examiner: Krizmanich; George H.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
The present application is a continuation-in-part of a co-pending
United States Patent application entitled APPARATUS AND METHOD FOR
GRADIENT ZONAL CENTRIFUGATION, Ser. No. 886,720, filed Dec. 19,
1969, by the same inventors, Phillip Sheeler and John R. Wells, now
abandoned.
Claims
We claim:
1. A gradient zonal centrifuge rotor comprising an annular chamber
in said rotor, said chamber being bounded by coaxial spaced apart
annular inner and outer vertical walls, a floor at the bottom of
said chamber, said floor comprising a V-shaped annular groove, the
inner and outer respective annular upper edges of said V-shaped
groove intersecting the respective bottom edges of said inner and
outer walls, a plurality of removable spaced apart radially arrayed
separate septa in said chamber dividing said chamber into a
plurality of chamber sectors, the respective vertical edges of each
of said septa extending substantially to the respective inner and
outer walls, each of said septa having a profile conforming
substantially in size and shape to the cross-sectional dimensions
of the combined chamber and V-shaped groove, distributor means
secured to said rotor and means interconnecting the distributor
means with said groove for introduction and removal of material
into and from said chamber.
2. A centrifuge rotor according to claim 1 wherein said means
interconnecting the distributor means with said groove comprises a
channel in each of said septa for introducing materials into and
removing materials from said chamber in the area of the apex of
said groove.
3. A centrifuge rotor according to claim 2 wherein said a
distributor means is located axially on top of said rotor through
which said materials are transmitted to and collected from said
septa channels.
4. A centrifuge rotor according to claim 1 wherein the lower
portions of said septa are V-shaped and wherein the apex of the
V-shaped portion of each septum is spaced apart a short distance
from the apex of the V-shaped groove.
5. A centrifuge rotor according to claim 4 wherein the apex of the
V-shaped groove is in the form of a narrow flat annular floor and
wherein the bottom edge of each septum has a flat edge.
6. A gradient zonal centrifuge rotor comprising an annular chamber
in said rotor, said chamber being bounded by coaxial spaced apart
annular inner and outer vertical walls, a floor at the bottom of
said chamber, said floor comprising a V-shaped annular groove, the
inner a nd outer respective annular upper edges of said V-shaped
groove intersecting the respective bottom edges of said inner and
outer walls, a cylindrical core removably mounted within said
chamber adjacent said inner wall, a plurality of spaced apart
radially arrayed separate septa integrally formed on said core,
said septa dividing said chamber into a plurality of sector
chambers, each of said septa having a profile conforming
substantially in size and shape to the cross-sectional dimensions
of the combined chamber and V-shaped groove, distributor means
secured to said rotor and means interconnecting the distributor
means with said groove for introduction and removal of material
into and from said chamber.
7. A rotor according to claim 6 wherein said means interconnecting
the distributor means with said groove comprises a plurality of
vertical channels in said core equal in number to said septa, each
of said channels extending through said core and into a
corresponding septum and terminating in an open port at the bottom
of the V-shaped portion of said septum.
8. A rotor according to claim 7 wherein the bottom of the V-shaped
portion of each septum is slightly displaced above the bottom of
said V-shaped groove, whereby liquid may flow between adjacent
sector chambers.
9. A rotor according to claim 7 wherein said distributor means is
removably mounted axially on the top of said rotor and includes a
plurality of radially extending channels in said distributor equal
in number to said channels in said core, each of said distributor
channels being detachably connectable to a corresponding channel in
said core for introducing materials into and removing said
materials from said sector chambers in the area of the apex of said
V-shaped groove.
10. A rotor according to claim 1 wherein the upper ends of said
distributor channels converge into a single channel at the top of
said distributor.
11. A gradient zonal centrifuge apparatus comprising a rotor, an
annular chamber in said rotor, said chamber being bounded by
coaxially spaced apart inner and outer circular walls and opposite
end walls, a plurality of spaced apart port means arrayed in a
circle in said rotor intermediate said inner and outer side walls
for removing effluents from said chamber, one of said end walls of
the interior of said chamber being formed in sloping surfaces
converging towards each of said removing ports and distributor
means connected to said port means for introduction and removal of
materials into and from said chamber.
12. A rotor according to claim 11 and further comprising a
plurality of removable spaced apart, radially arrayed septa in said
chamber dividing said chamber into a plurality of chamber sectors,
each of said septa having a profile conforming substantially to the
cross-sectional shape of said chamber and of said converging
sloping surfaces where said removing port means are located.
13. Apparatus according to claim 11 wherein said sloping surfaces
comprise an outer annular sloping surface and an inner annular
sloping surface, said annular surfaces converging to form an
annular V-shaped apex in said chamber, said apex being located
coaxially relative to the axis of said rotor and intermediate the
respective radii of said inner and outer walls.
14. Apparatus according to claim 13 wherein said port means are
located in circular array in said annular apex and wherein gradient
zones in said chamber, when removed therefrom, are constricted and
concentrated in both inward and outward radial directions by said
respective annular surfaces relative to said annular apex.
15. Apparatus according to claim 11 wherein said sloping surfaces
comprise a plurality of circularly arrayed funnel-shaped
recesses.
16. Apparatus according to claim 15 wherein said port means
includes a port in the apex of each funnel-shaped recess, said
ports being arrayed in a circle relative to the axis of said rotor
and located radiallyintermediate the respective radii of said inner
and outer circular walls and wherein gradient zones in said
chamber, when being removed therefrom through said ports, are
constricted and concentrated in all angled directions relative to
the apex of each funnel-shaped recess.
17. Apparatus according to claim 15 wherein an outer edge of each
funnel-shaped recess is contiguous to the outer edge of an adjacent
funnel-shaped recess.
18. Apparatus according to claim 15 and further comprising a narrow
annular shelf located adjacent the inner wall of said chamber, the
inner edge of the upper end of said funnel-shaped recess being
spaced apart from said inner wall by the width of said shelf.
19. A gradient zonal centrifuge rotor comprising an annular chamber
in said rotor, said chamber being bounded by coaxially spaced apart
annular inner and outer walls, a floor at the bottom of said
chamber, a removable cover on said rotor enclosing said chamber,
the inner surface of said cover comprising an inverted V-shaped
annular groove, the inner and outer respective annular lower edges
of said groove intersecting the respective upper edges of said
inner and outer walls, distributor means secured to said rotor and
means interconnecting the distributor means with said groove for
introduction and removal of material into and from said
chamber.
20. A rotor according to claim 19 wherein the annular apex of said
groove is disposed concentrically around the axis of said rotor and
located intermediate said inner and outer walls.
21. A rotor according to claim 19 wherein said groove comprises an
outwardly and downwardly sloping annular outer wall, and an
inwardly and downwardly sloping annular inner wall, said sloping
walls converging upwardly toward each other to form the apex of
said V-shaped annular groove, the angle of said outer groove wall
from the horizontal being smaller than the angle of said inner
groove wall from the horizontal.
22. A rotor according to claim 19 wherein said means
interconnecting said distributor means with said groove comprises a
plurality of spaced apart exit ports arranged in circular array in
said cover, said ports each communicating with said chamber in the
region of said annular apex.
23. A rotor according to claim 19 and further comprising a
plurality of removably spaced apart radially arrayed septa in said
chamber dividing said chamber into a plurality of chamber sector,
each of said septa having a profile conforming substantially to a
cross-sectional shape of said chamber and said V-shaped groove.
24. A rotor according to claim 23 wherein said means
interconnecting said distributor means with said groove comprises a
channel in each of said septa for introducing materials into said
chamber.
25. A rotor according to claim 24 wherein said distributor is
removably mounted axially on top of said rotor and includes a
plurality of radially extending channels in said distributor equal
in number to the channels in said septa, each of said distributor
channels being detachably connectable to a corresponding septum
channel.
26. A rotor according to claim 19 and further comprising a
plurality of spaced apart exit ports in said cover communicating
with the apex of said V-shaped annular groove, a collector mounted
on said cover, and a plurality of channels in said collector, each
of said channels communicating with a corresponding port in said
cover for removing materials from said chamber.
27. A gradient zonal centrifuge rotor comprising an annular chamber
in said rotor, said chamber being bounded by coaxial spaced apart
annular inner and outer side walls and opposite end walls, said
chamber being bounded at a selected one of said end walls by a
V-shaped annular groove forming a portion of said chamber, the
inner and outer respective annular edges of said groove
intersecting adjacent respective edges of said inner and outer side
walls, a plurality of spaced apart radially arrayed septa in said
chamber dividing said chamber into a plurality of chamber sectors,
the respective vertical edges of each of said septa extending
substantially to the respective inner and outer side walls, each of
said septa having a profile conforming substantially in size and
shape to the cross-sectional dimensions of the combined chamber and
V-shaped groove, distributor means secured to said rotor and means
interconnecting the distributor means with said groove for
introduction and removal of material into and from said
chamber.
28. The method of separating particles of biological cells or the
like in the annular chamber of a centrifuge rotor which comprises
introducing a quantity of said cells in suspension into said
chamber, introducing a density gradient into said chamber, spinning
said rotor to form vertical cylindrical columns of isodense layers
of density gradient therein and causing said cells to migrate
through said isodense layers by centrifugation until said particles
have formed separate vertical cylindrical zones in accordance with
their respective characteristics, causing said isodense layers and
said particle zones to become reoriented into disc-shaped rings in
spaced apart horizontal array within said chamber during
deceleration of centrifugation, and removing and collecting said
particle zones separately when said rotor is at rest by causing
each particle zone to flow through an annular V-shaped space
forming part of said chamber whereby the surface area of each zone
becomes reduced and the distance between successive sample zones is
increased thereby reducing the possibility of interference of
contamination between particle zones, said removal taking place
substantially at the apex of said V-shaped space.
Description
BACKGROUND OF THE INVENTION
1. FIELD OF THE INVENTION
This invention relates to zonal centrifuge rotors and, more
particularly, to an improved reorienting gradient zonal rotor for
separation of biological cell components and the like. The
apparatus is utilized to provide quantitative separations of tissue
cells or cell particles, or the like, in density gradients for the
purpose of collecting discrete fractions thereof for subsequent
analysis in research and clinical studies.
Zonal centrifuge rotors are used for quantitative density gradient
centrifugation for fractionating various types of biological
materials ranging from cellular constituents of tissues at the
large end of the spectrum to molecular components of those tissues
representing the small end of the spectrum. The rotor can be used
to separate cells of different sizes from one another as, for
example, red from white blood cells, different types or sizes of
cells present in a culture of microorganisms such as bacteria,
algae, protazoa, and the like. When cells of a tissue to be studied
are mechanically broken, the rotor may be used to separate and
isolate the various constituent particles of those cells such as
cell nuclei, mitochondria, chloroplast, membranes, and ribosomes.
Also, various cellular molecules may be isolated by centrifugation,
such as nucleic acids, polysaccharides, and certain proteins.
2. Description of the Prior Art
The prior art relating to the subject of the present invention is
described in a recent treatise entitled, "The Development of Zonal
Centrifuges and Ancillary Systems for Tissue Fractionation and
Analysis," edited by Dr. Norman G. Anderson, National Cancer
Institute Monograph 21, June 1966, U. S. Department of Health,
Education and Welfare, Public Health Service, National Cancer
Institute, Bethesda, Maryland, Superintendent of Documents, U. S.
Government Printing Office, Washington, D. C.
Zonal centrifuges are described therein as comprising a rotor
having a circular chamber with rotating-seal means for introducing
and removing materials from said chamber while the rotor is
spinning. Rotating-seal zonal rotors suffer disadvantages such as
(a) crossing over at the seal when gradient solutions of high
viscosity are pumped into the rotor or when samples are introduced
under high pressure; (b) inadvertently long loading and unloading
times due to the requirement of gravity feed; (c) continued
sedimentation of particles during the unloading period; and (d)
since the rotor is loaded and unloaded while spinning at 3,000 to
5,000 rpm and the seal assembly must be manually removed and later
inserted during this rotation, considerable dexterity is demanded
of the operator. Furthermore, such rotating-seal zonal rotors are
expensive, require special centrifuges or modification of older
models, and demand extensive maintenance and cleaning between
runs.
Where prior art reorienting gradient zonal rotors have a
funnel-shaped sloping floor with a single outlet located at the
axial center thereof, such rotors are usually mounted upon a fly
wheel which, in turn, is mounted on the centrifuge rotor shaft.
This entails time-consuming operational difficulties involving
extra manipulations that may reduce the possibility of obtaining
optimum or desired results.
SUMMARY OF THE INVENTION
The zonal centrifuge rotor of the present invention is loaded when
the rotor is at rest. A manifold distributor is utilized to load
density gradients simultaneously into each of a plurality of sector
compartments at a point of entry at the apex of the annular
V-shaped groove forming the floor of one embodiment of the rotor
chamber, said annular groove apex being concentric with and being
displaced radially from the axis of said rotor. Thereafter, the
rotor is accelerated during which time the density gradient
undergoes reorientation in which the vertically arranged isodense
layers gradually become concentrically arranged about the axis of
rotation.
The sample may be introduced by initially placing it in the bottom
of the V-shaped groove after which it becomes displaced upwardly
upon introduction of the density gradient, with both the gradient
and the sample undergoing reorientation during acceleration.
Alternatively, after preliminary acceleration of the rotor, the
sample may be spread onto the reoriented density gradient through
an opening in the top cover of the rotor by using a syringe, or the
like.
Thereafter, the rotor is accelerated to the higher speed desired
for particle separation during which said particles become arranged
as a series of concentric zones about the axis of rotation. After
separation is achieved, the rotor is decelerated during which time
both the density gradient and the separated particle zones undergo
reorientation. With the rotor once again at rest, the separated
particles are arranged as a series of disc-shaped layers in the now
vertical density gradient. Finally, the density gradient and the
separated particles are pumped from the rotor beginning with the
dense end of the gradient at the bottom of the V-shaped groove and
the material is collected as a series of fractions.
A unique feature of the present invention is the provision of a
circular V-shaped groove forming the floor of the rotor chamber in
which a removable set of radially arranged partitions or septa are
located. This V-shaped groove is one of the important features
which distinguishes the configuration of the new rotor from the
flat-floor rotating-seal rotors or other reorienting gradient
rotors. The septa are integrally formed upon a cylindrical core
which surrounds the interior circular wall of the rotor chamber.
The core has a plurality of channels leading downwardly and
obliquely outwardly into the septa where they terminate at the
V-shaped vertex of each of the septa which correspond in angular
dimension to the V-shaped floor of the rotor. The materials are
introduced into and withdrawn from the respective multiple sectors
of the rotor chamber through said channels and through a removable
distributor having multiple channels which are connectable to
respective channels in the septa core.
The zonal rotor of the present invention, however, not only offers
all of the advantages of the prior art rotating-seal zonal rotors,
but is considerably simpler in design and construction, does not
require a rotating-seal and can be operated with ease by any
laboratory technician. Also, the rotor of the present invention has
an axial recess adaptable for connection to the tapered drive shaft
of a centrifuge such as disclosed in U.S. Pat. No. 2,827,229, Mar.
18, 1958, as embodied in Model RC-2 produced by Ivan Sorvall, Inc.
The reorienting-gradient zonal rotor described herein employs an
annular V-shaped groove as its floor, the annular apex of said
groove being displaced radially from the axis of said rotor, and
this is in contrast to the prior art rotating-seal zonal rotors
which have a solid, flat bottom. The presence of this annular
V-shaped floor also distinguishes the rotor described herein from
that of the prior art which has a funnel-shaped floor whose apex
exit port is at the axis of rotation. Such prior art funnel-shaped
rotors must be mounted on a fly wheel or the like which, in turn,
is connected to the drive shaft of the centrifuge.
One alternative embodiment of the invention comprises the location
of the annular V-shaped groove formed by two converging annular
slopes in the roof of the rotor chamber instead of the floor, and
wherein the removal of the gradient zones is effected through the
top of the rotor. A still further embodiment of the invention
comprises the formation of a plurality of funnel-shaped recesses in
circular array either in the floor or ceiling of the rotor chamber
whereby the removal of the gradient zones is effected through the
apices of said recesses and wherein the advantageous constriction
and concentration of said zones is effected by the walls of the
recesses sloping towards said apices from all directions instead of
from inward and outward radial directions in the case of the
V-shaped annular grooves.
The rotor of the present invention is loaded and unloaded while the
rotor is at rest by means of a manifold distributor which may be
connected to a peristaltic pump or the like. The use of a
peristaltic pump with a rotating-seal zonal rotor may result in
crossing over. Since the gradient in the rotor of the present
invention is eluted with the rotor at rest, not further particle
sedimentation occurs as obtains in rotating-seal zonal rotors where
the particles continue to sediment as the rotor continues to rotate
during the entire unloading procedure. Thus, the reorienting
gradient rotor of the present invention is less complex in its
operation than the rotating-seal zonal rotor and may easily be
operated by the laboratory technician untrained in the complexities
of zonal centrifugation. Finally, the reorienting gradient rotor of
the present invention is simple in design and far less costly both
in construction and operation than the rotating-seal zonal
rotor.
These and other novel features and advantages of the present
invention will be described and defined in the following
specification and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a vertical section view, partly in elevation and partly
broken away, and some parts being omitted;
FIG. 2 is a top view taken on line 2--2 of FIG. 1;
FIG. 3 is an enlarged detail view, in section, of the manner in
which the distributor channels may be connected to the septa
channels;
FIG. 4 is an exploded view, partially in section and partially in
elevation, of the apparatus shown in FIG. 1, some parts being shown
schematically and some parts being omitted;
FIG. 5 is a vertical central section view, partly broken away, some
parts being omitted, illustrating another embodiment of the
invention;
FIG. 6 is a view of the top of the rotor shown in FIG. 5 with the
cover removed, and other parts being omitted;
FIG. 7 is a greatly enlarged fragmentary view of the upper central
portion of the apparatus shown in FIG. 5;
FIG. 8 is an exploded diagrammatical view, in vertical central
section, illustrating another embodiment of the invention; and
FIG. 9 is a view taken on line 9-9 of FIG. 8.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring now to the drawings in detail, there is shown the upper
fragmentary portion of a cone-shaped hub 21 mounted upon the upper
end of a centrifuge shaft, not shown, connected to a suitable
rotating power source such as an electric motor or the like.
Mounted on hub 21 is a centrifuge rotor, generally designated 22,
made of aluminum or the like, having an axial core portion 23
encircling a bottom conically-shaped axial recess or aperture 24
which fits intimately with hub 21. The upper narrowed end of
aperture 24 terminates in an outwardly extending annular shoulder
26 which forms a boundary between aperture 24 and an upwardly
extending axial aperture 27. The upper end of hub 21 terminates in
a threaded stub 28 which extends upwardly into aperture 27 and is
threadably engaged by nut 29 bearing downwardly upon washer 31,
resting on shoulder 26, to secure rotor 22 to hub 21.
The upper outer portion of rotor 22 is annularly threaded at 32 to
accommodate the interior threaded portion 33 of the downwardly
extending rim 34 of circular cover plate 36 made of aluminum or the
like whereby said cover is removably secured upon said rotor. The
top of cover plate 36 may be provided with a plurality of recesses
37 to accommodate a tool for tightening said cover plate upon and
for removing it from said rotor. Cover plate 36 has a central
aperture 38, the purpose of which will be described hereinafter.
The top surface of the outer portion of rotor 22 has an annular
recess 39 which accommodates O-ring 40 made of a suitable resilient
material such as rubber, nylon, or the like, to form a liquid-tight
seal between cover plate 36 and the adjacent portion of said
rotor.
Rotor 22 has an annular centrifugation chamber, generally
designated 41 (FIG. 4) which is formed by vertical circular outer
wall 42 and a spaced apart vertical circular inner wall 43, both of
said walls being concentrically located in respect of the axial
center of said rotor. The lower end of outer wall 42 is intersected
by the upper end of inwardly and downwardly sloping annular groove
wall 44, while the lower end of inner wall 43 is intersected by the
upper end of outwardly and downwardly sloping annular groove wall
46. Groove walls 44 and 46 converge to form a V-shaped annular
groove, generally designated 47, which is disposed concentrically
around the axis of rotor 22. At least a portion of chamber 41
concentrically surrounds recess 24.
In some embodiments, the convergence of sloping groove walls 44 and
46 may form a sharp vertex. In the embodiment shown in the drawings
herein, the bottom of groove 47 is in the form of a narrow annular
floor 48 whose radius is substantially equidistant from the
respective radii of walls 42 and 43. In other embodiments, the
upper edges of the V-shaped groove need not intersect respective
walls 42 and 43 directly but may be joined by intermediate short
annular slopes or curves. Furthermore, it is contemplated that
walls 44 and 46 of the V-shaped groove may be curved in any
suitable manner provided they perform the function of constricting
the radial area of the materials being removed therefrom as will be
described hereinafter.
Removably positioned in annular chamber 41 are radially arrayed
partitions or septa 51 that divide said chamber into a plurality of
substantially equal sector chambers. The inner vertical edges of
septa 51 are integrally formed upon a tubular core 52, the inner
cylindrical surface 53 thereof having upper and lower inwardly
extending annular bosses 54 and 56 which form a smooth, sliding fit
over the circular surface 43 of core portion 23 of rotor 22. On the
upper portion of vertical edge 58 of each septum is an integrally
formed, outwardly extending boss 59 which serves in conjunction
with bosses 54 and 56 to align said septa within chamber 41. The
removal of septa 51 from, and their insertion into, chamber 41 is
accomplished by raising and lowering cylindrical core 52 relative
to core portion 23 of rotor 22. Although six septa 51 are disclosed
in the embodiment shown in the drawings herein, it is understood
that any suitable number such as 4, 8, 12, or the like, can be
provided as may be necessary or desired.
The bottom of each septum 51 is bounded by a pair of converging
edges 61 and 62, the inwardly angled edge 61 matching the angle of
groove wall 44, and the outwardly angled edge 62 matching the angle
of groove wall 46, to form a close fit between said respective
edges and groove walls as shown in FIG. 1. Septa edges 61 and 62
converge to narrow horizontal annular edge 63 which is
substantially parallel to annular floor 48 of groove 47 and is
spaced a short distance apart from said floor to allow liquid
communication between adjacent sectors which would ensure that
isodense layers occupy identical positions in each sector due to
hydrostatic pressure, and to facilitate introducing materials into
and removing the same from the sector chambers by way of septa
channels described hereinafter.
In some embodiments, the septa 51 may comprise separate plates
which are inserted into chamber 41 in suitable radial array whereby
the separate sector chambers may be filled with materials to be
centrifuged and drained thereof after configuration by suitable
means, the profiles of said septa conforming in size and shape
substantially equal to the cross-sectional area of said chamber and
to the particular configuration of walls 44 and 46 of the V-shaped
groove.
In the preferred embodiment illustrated and described herein,
however, septa 51 are formed into a unitary component with
cylindrical core 52 whereby the filling and draining of the sector
chambers within the centrifuging chamber 41 may be accomplished by
a vertical channel 66 for each septa within said core, said
vertical channel communicating with an outwardly and downwardly
inclining channel 67 whose lower outlet port 68 is located in edge
63.
Removably mounted on top of the axial core portion 23 of rotor 22
is a circular distributor block 71 which is located within axial
aperture 38 of cover plate 35. Distributor block 71 may be
removably secured in position upon rotor 22 by means of a plurality
of spaced apart threaded bolts 72. Distributor 71 has an integrally
formed, upwardly extending axial boss 73 which has a vertical axial
channel 74 whose lower end terminates in a plurality of radially
spaced apart downwardly and outwardly extending channels 76 which
are equal in number to the number of septa 51. Each channel 76
communicates with a corresponding vertical channel 66 in a
respective septum 51.
One typical arrangement for connecting each channel 76 with its
respective channel 66 is shown in fragmentary enlarged detail view
in FIG. 3, where core 52 in the region of channel 66 is provided
with a recess 77 in which is secured an O-ring 78. The upper end of
an elongated tubular adapter 79 extends into block 71 by means of a
press fit to join channel 76 while the lower end of said adapter
extends by a loose fit into core 52 to join channel 66 thereby
forming a unitary passage between channel 76 and channel 66. A
liquid-tight juncture is provided by an integrally formed annular
flange 81 on tube 79 bearing against O-ring 78 when block 71 is
urged downwardly and securely upon the core portion 23 of rotor 22.
Other suitable means may be provided for ensuring a leak-tight
juncture between channels 76 in block 71 and corresponding channels
66 in core 52.
When distributor 71 is bolted in position, it secures core 52 and
septa 51 firmly in position. Port 74 at the top of distributor 71
is connected by means of flexible tubing to the pump when the rotor
is being filled and being emptied of the density gradient and
distributed particles, but the tube is disconnected during
centrifugation.
OPERATION
In operation, channel 74 of axial boss 73 is connected to a
suitable source of continuous liquid density gradient which
comprises a stream of fluid whose density progressively changes
from some initial minimum value to some final maximum value. These
gradients are routinely prepared in laboratories engaged in density
gradient centrifugation and are prepared by commercially available
apparatus known as a gradient engine. The density gradient may be
transmitted by way of suitable flexible tubing 83 to port 74 by
means of a peristaltic pump 84 of the like, connected to a source
85 of said gradient. Before centrifugation is begun and while the
rotor is stationary, the density gradient enters by way of channels
76 in distributor 71 in a continuous liquid stream and passes
through channels 66 and 67 of core 52 and septa 51, respectively,
and emerges from the terminal ports 68 at the bottom of said septa
into the groove vertex whereby each of the sector chambers are
filled simultaneously. The liquid is then displaced upwardly by the
denser fluid flowing immediately behind. In this manner, the
centrifugation chamber of the rotor is slowly filled with a
gradient whose most dense region is at the bottom of the V-shaped
groove and which become progressively lighter on rising vertically.
The spaces between bottom edges 63 of septa 51 and annular floor 48
ensure that loading of all chambers is uniform by permitting
equilibration between neighboring sector-shaped compartments.
According to one process in the operation of the apparatus herein,
the sample whose particles are to be separated may be introduced
first by way of channels 66 and 67 into the bottom of V-shaped
groove 47 after which the density gradient is introduced thereby
lifting the sample toward the top of chamber 41. Thereafter, the
flexible tubing to the pump is removed from distributor 71 and the
rotor is rotated to produce reorientation of both the gradient and
the sample simultaneously. Following centrifugation and the
deceleration of the rotor, the flexible tubing from the pump is
connected to the distributor 71 and by reverse pumping the
separated layers of sample particles are sequentially removed from
chamber 41 by way of ports 68 and channels 67 and 66, and
transmitted to a suitable fraction collector 86.
Alternatively, the isodense gradient is introduced first into the
sector compartments of the rotor by the means described
hereinbefore after which distributor 71 is removed and the gradient
is preliminarily reoriented by rotation of the rotor into a
plurality of vertical cylindrical isodense layers, as described in
the National Cancer Monograph 21 cited hereinbefore. Thereafter,
the sample is introduced by spraying or the like on to the
reoriented gradient, after which centrifugation takes place during
which time the sample initially forms a vertical cylindrical zone
at the inner circular wall 43 of the rotor, after which the
particles of said sample migrate or sediment through the isodense
layers and become arranged as a series of concentric zones about
the axis of rotation. After separation is achieved, the rotor is
decelerated during which time both the density gradient and the
separated particle zones undergo reorientation. With the rotor once
again at rest, the separated particles of the sample are arranged
as a series of spaced apart horizontal disc-shaped layers in the
now vertical density gradient. The flexible tubing from the pump is
then attached to port 74 of distributor 71 and the density
gradient, along with the separated sample particles, is pumped from
the rotor chamber 41 beginning with the dense end of the gradient
at the bottom of V-shaped groove 47 and is collected as a series of
fractions.
It is understood that when density gradients and samples are
introduced into annular chamber 41 for centrifugation, the top
surface of these materials will be a suitable distance below the
top of rotor 22 so that when centrifugation takes place and the
mass of liquids rises to impinge upon the bottom surface of cover
plate 35 to form a vertical cylindrical column of liquid within
chamber 41, the diameter of the inner surface of said rotating
cylinder of liquid, at least at the upper portion thereof, will be
somewhat greater than the diameter of aperture 38 so that no
spillage will take place through said aperture.
The events occurring within the reorienting gradient rotor take
place as follows: As the rotor is initially accelerated, the
density gradient and the sample begin to undergo a reorientation
within chamber 41. Each isodense layer bows inwardly to form a
paraboloid of revolution about the axis of rotation. With continued
acceleration, a centrifugal force is attained which is sufficient
to cause the isodense layers to achieve verticality. The sample
zone also has undergone reorientation so that initially it has
formed a single cylindrical inner zone around the axis of rotation.
The sample particles subsequently sediment radially away from the
axis of rotation under the "g" force and their rate of
sedimentation will be determined by their respective sizes and
densities. Accordingly, the sample particles form several separate
spaced apart concentric vertical cylindrical columns throughout the
density gradient. After the desired separation of particles has
been achieved, the rotor is slowly decelerated during which time
the density gradient which now contains the separated particles
again reorients in chamber 41 resulting in a horizontal array
whereby the separate zones of separated particles are present in
the gradient as a plurality of spaced apart horizontal disc-shaped
rings in vertical array within the density gradient.
While the sloping walls 44 and 46 of annular V-shaped groove 47 and
may be symmetrical as to their respective angular arrays from a
horizontal plane, and in such a configuration may provide
satisfactory operating conditions for the apparatus described
herein, a preferred embodiment is shown in the drawings herein
wherein sloping wall 44 is arrayed approximately 30.degree. and
sloping wall 46 is arrayed approximately 45.degree. from the
horizontal plane. With such an asymmetric array, the bottom of the
vertical annular zone formed by the sample during the early stage
of centrifugation will be located substantially at the bottom of
wall 43 which is at a higher level than the bottom of outer wall
42. Thus, during subsequent centrifugation, the initial narrow
cylindrical sample zone around core 23 of the rotor migrates from
inner wall 43 toward outer wall 42 through rotor chamber 41. Since
sedimentation of the sample particles takes place during
centrifugation in a direction perpendicular to the axis of the
initial cylindrical sample zone, those migrating particles that
ultimately reach the outer annular wall 42 do not enter into the
portion of the density gradient that occupies groove 47 defined by
walls 44 and 46. By providing that the bottom end of wall 42 is
lower than the bottom end of wall 43, the latter being the
approximate locus of the bottom end of the initial cylindrical
sample zone, the asymmetric configuration of the V-shaped groove
ensures that uniform sedimentation of the complete sample zone will
take place by virtue of the unimpeded migration of the total
vertical dimension of said sample zone through chamber 41.
The vertical length of the sample zone can be readily determined by
empirical means with the foreknowledge of the dimensions of
V-shaped groove 47, whereby a sufficient amount of density gradient
material is caused to fill said groove so that the lower end of the
sample zone extends to a level above or slightly above the vertex
between the outer vertical wall 42 of the rotor chamber and the
upper end of the outer sloping wall 44 of the V-shaped groove.
Another advantage of the V-shaped groove of the centrifugation
chamber of the apparatus herein lies in the greatly improved
efficiency in recovering the discrete horizontal sample layers
separately from the rotor. This is accomplished by pumping the
density gradient out of rotor chamber 41 from the bottom of the
annular V-shaped groove by means described hereinbefore, beginning
with that portion occupying the bottom of the V-shaped groove (i.e.
the flow being opposite to that which obtains when the density
gradient is loaded into the rotor). As pumping continues, the fluid
contents of the rotor slowly descend toward and through the annular
V-shaped groove. The discrete layers of separated particles are
sequentially channeled by the converging walls of the V-shaped
groove so that they are drawn in the proper sequence through
channels 67 and 66 of the septa-core component of the apparatus and
collected in separate test tubes after passing from the rotor
proper.
Before unloading chamber 41 of the rotor, the sample particle zones
which are to be collected are arranged as a series of horizontal
layers suspended in the gradient and separated by some vertical
distance. As the gradient is being pumped out of the rotor, all of
the sample layers slowly descend toward the rotor floor. Before the
V-shaped groove is reached, the surface area of each sample zone
remains constant and the distances between sample zones also remain
constant. As the fluid descends into the region of the V-shaped
groove, however, the surface area of each sample zone becomes
reduced, and since the fluid volume of each zone must remain
constant, it follows that each zone becomes deeper in vertical
dimension. By the same token, the vertical distance between sample
zone layers also increases and this phenomenon ensures against
mixing that might otherwise occur between sample zone layers. The
surface area of each zone layer reaches a minimum value when the
vertex of the V-shaped groove is reached and at this time each
sample zone is drawn into the exit ports 68 and through channels 67
and 66 for removal from the rotor. Since all of the particles in
one sample zone layer located at the vertex of the V-shaped groove
are closer to an exit port than particles in other zone layers not
yet near the vertex, the possibility of mixing between zone layers
is minimized.
Converging walls of the V-shaped groove ensure that each zone layer
will be focused into the vertex of the groove and pumped in the
proper sequence from the rotor. Thus, the annular V-shaped groove
acts in a manner similar to a funnel; it guarantees that fluid flow
from the rotor will be orderly and that particles suspended in the
separate layers in the density gradient will leave the rotor in
exactly the same order as they were arranged vertically before
unloading was initiated.
If no groove existed in the floor of the rotor, each sample zone
would maintain the same maximum surface area throughout its descent
and would be spread over the entire annular flat rotor floor. Under
such conditions, the removal of a sample particle zone layer at the
rotor floor would be most inefficient, due in large part to the
fact that particles in other zone layers located higher in the
gradient would be physically as close to exit port 68 as particles
lodged in the margins of the sample zone already resting on the
flat chamber floor. Accordingly, mixing between zone layers would
occur and, consequently, the resolution achieved during
centrifugation would be lost during the collecting operation. It
should also be noted that in the absence of a V-shaped groove, the
distance between sample zone layers would remain the same during
their descent to the flat floor of the rotor, whereas the presence
of a V-shaped groove produces an increase in the distance between
the sample zone layers as they descend to the bottom of the chamber
and thereby further reduces the chances for mixing to occur.
The V-shaped groove plays a similarly vital role during the loading
of the rotor although its effectiveness is not immediately obvious.
The liquid density gradient which is to be loaded into the rotor
may be thought of as a sequence of horizontal zones which differ
slightly in their densities. As fluid emerges from the ports at the
bottom of the core-septa section, it slowly fills the V-shaped
groove. The surface area of each zone slowly increases as the fluid
level is elevated in the groove and finally attains a constant
value once above the groove. In other words, a more dense portion
of the gradient is always layered below a lighter portion and there
is no mixing as would occur if the groove were not present. If no
groove were present, fluid emerging at the base of the core would
have to spread instantaneously over the entire floor surface area.
Thus, the V-shaped groove provides for a smooth and orderly
increase in the surface area of each zone entering the rotor.
In the design of the V-shaped groove, it is desirable to keep the
volume of the density gradient occupying said groove to a minimum.
This could be accomplished by providing very shallow angles for the
groove walls, but this would result in less efficient funneling
during the unloading operation since a very shallow angle would be
almost as deficient as no groove at all. As a corollary, steep
angles in the V-shaped groove would result in efficient loading and
unloading but would severely restrict the usable volume of rotor
chamber 41. Any angles between 10.degree. and 75.degree. from the
horizontal plane could be used, but practical angles would be in
the range of 20.degree. - 60.degree..
In one satisfactory embodiment where the V-shaped groove was
symmetrical, the groove walls 44 and 46 were arrayed at
approximately 47.degree. from the horizontal. In other embodiments
where the asymmetrical V-shaped groove is desired, respective
angles of 30.degree. for wall 44, and 45.degree. for wall 46 from
the horizontal plane have been found useful. Other suitable
asymmetric arrays for groove walls 44 and 46 may be arranged to
accomplish successful results with the apparatus herein.
It is to be understood that the provision of the annular V-shaped
groove 47 herein is also useful without employing the structural
features of core 52 and its channels 66 and channels 67 in septa
51. It is possible to utilize separate septa 51 as partitioning
elements and to load and unload the density gradient relative to
the various sector chambers by means of one or more tubular
elements that are inserted into chamber 41 with the lower ends of
said tubes being located at annular floor 48 whereby the
introduction and removal of the gradient can be achieved in a
manner that takes advantage of the V-shaped groove as described
hereinbefore for fractionally collecting each of the separate
ring-disc particle zones without contamination or interference from
other particle zones.
In another embodiment of the invention, the annular V-shaped groove
which has heretofore been illustrated and described, in connection
with FIGS. 1 - 4, as being located in the floor of the rotor
chamber, may instead be located and formed in cover plate 91 as
shown diagrammatically in FIG. 5.
In this embodiment, rotor 92 has an annular chamber 93 having a
flat annular floor 94. Surrounding the central hub 96 of rotor 92
is a tubular core 97, the inner cylindrical surface 98 of which is
slightly spaced apart from said core portion by means of inwardly
extending annular bosses 99 and 101 on the upper and lower ends
thereof which form a smooth, sliding fit over the circular surface
102 of hub 96. Formed integrally with tubular core 97 and extending
radially outwardly therefrom in chamber 93 is a plurality of
partitions or septa 103 which divide said chamber into a plurality
of substantially equal sector chambers. The bottom horizontal edges
104 of septa 103 rest on floor 94 of rotor chamber 93. Formed
intermediate the inner and outer edges of each septum 103 at the
bottom thereof is an upwardly extending recess 105 which permit
hydrostatic equilibration between adjacent compartments of the
rotor.
The upper end of each septum 103 terminates in an asymmetric apex
comprising outer sloping edge 106 and inner sloping edge 107. Said
septa apices extend upwardly into the top of rotor 92.
Mounted on the hub 96 of rotor 92 is a distributor block 108,
secured thereon by means of circularly spaced apart bolts 109.
Integrally formed upon and extending axially upwardly from block
108 is a tubular conduit stem 111 having a vertical axial channel
112, the lower end of which terminates in a plurality of radially
spaced apart downwardly and outwardly extending channels 113 which
are equal in number to the number of septa 103. Each channel 113
communicates directly with a corresponding vertical channel 114 in
core 97, the bottom open end of channel 114 terminating at the
bottom edge of said core slightly spaced apart above floor 94 of
annular chamber 93. Suitable connecting means, as described
hereinbefore, for example, may be provided between channels 113 and
respective channels 114 to serve as leakproof connections
therebetween. Cover plate 91 has a central aperture for freely
accommodating block 108.
Cover plate 91 has an annular V-shaped groove formed in its bottom
surface which comprises an outwardly sloping wall 116 and an
inwardly sloping wall 117 whose angles of inclination conform with
the angles of corresponding edges 106 and 107 of septa 103 and with
which they make a close fit. The apex of each septum 103 terminates
in a narrow, flat, horizontal top edge 118 while sloping walls 116
and 117 of cover plate 91 converge into an annular apex located
above top edges 118 of septa 103.
Located in cover plate 91 in circularly spaced apart array is a
plurality of outlet ports 119, each of which is located directly
opposite a corresponding top edge 118 of a respective septum 103.
Mounted on the top central portion of cover plate 91 by means of
bolts 121 is a circular distributor block 122 whose diameter is
somewhat greater than that of the annular apex in the bottom
surface of cover plate 91. Each outlet port 119 has a corresponding
vertical channel 123 in cover plate 91 which, in turn, is aligned
with and communicates with a corresponding vertical channel 124 in
block 122. Here, also, suitable means are provided to form
leak-tight seals between channels 123 and respective channels
124.
Channels 124 intersect with corresponding horizontal channels 126
in block 122 which extend inwardly and communicate with an annular
channel 127 formed between the central portion of block 122 and
conduit stem 111 extending axially therethrough. As shown in FIG.
7, annular channel 127 terminates in and intersects with channel
128 in a laterally extending tube 129. Both stem 111 and tube 129
are connectable to suitable apparatus for charging the rotor with
materials to be treated therein and for removing materials
therefrom.
Since the vertical channels 112 and 127 are concentrically
oriented, with channel 127 leading to a peripheral port 128, the
leak-tight integrity of the latter is ensured by the provision of a
static O-ring seal 131 below channel 126, and O-ring seal 132 above
and spaced apart from channels 127 and port 128.
Since chamber 93 of rotor 92 is loaded and unloaded under pressure,
said chamber must be completely sealed off. This is accomplished by
the provision of an O-ring seal 133 near the lower peripheral end
of axial core 96 which provides a seal between the tubular septa
core 97 and said axial core 96. Another O-ring seal 134 is provided
to seal the junction between cover plate 91 and tubular septa core
97.
In operating the embodiment of the apparatus shown in FIGS. 5, 6,
and 7, and with the rotor 92 at rest, the sector-shaped
compartments in rotor chamber 93 are filled with a dense cushion
solution introduced through inlet channel 112 in stem 111 and
passed through channels 113 and 114 into said chamber. The density
gradient is then loaded, with the dense end first, through
peripheral channel 128, vertical channel 127, horizontal channels
126, and vertical channels 124, 123, under pressure, thereby
displacing the initial dense cushion solution out through the core
channels 114, 113, and 112. Eventually, the entire rotor chamber is
filled with a density gradient whose lighter region is at the
vertex of the inverted V-shaped groove formed in the interior
surface of cover plate 91, and whose densest region is represented
by a residual volume of the cushion solution at the rotor
floor.
The sample to be fractionated is then introduced through the cover
plate channels by way of the apex of the annular inverted V-shaped
groove, thereby displacing additional cushion solution out of
channels 114, 113 and 111. Connections to channels 111 and 112 are
removed. Rotor 92 is then accelerated to reorient the density
gradient and the sample, and thereafter centrifugation is carried
out as described hereinbefore. After the desired separation has
been achieved, rotor 92 is then slowly decelerated to rest.
Thereafter, a dense solution is pumped through the core channels
112, 113 and 114, to the bottom of rotor chamber 93 to cause
displacement of the gradient and of the separated sample zones
sequentially up and out through the cover plate channels 124, 126,
127 and 128. The fractions are then collected, utilizing a fraction
collector.
One advantage of providing the inverted annular V-shaped recess at
the top of the rotor chamber lies in the ability to introduce the
sample last at the top of the gradient with the rotor at rest. A
further advantage lies in the ability to displace the contents of
the rotor under controlled pressure which ensures an even flow
through all of the inlet and outlet channels. This is in contrast
to the configuration in FIGS. 1 - 4 where the contents of the rotor
chamber were removed by aspiration or by other negative pressure
means.
It will be noted that slopes 116 and 117 forming the annular
V-shaped groove in cover plate 91 in FIG. 5 are asymmetric in a
manner comparable to the asymmetry of slopes 44 and 46 forming the
annular V-shaped groove in rotor 22 of FIG. 1, whereby the same
functional results are obtained in both embodiments.
A still further embodiment of the invention is shown schematically
in rotor 140 in FIGS. 8 and 9 wherein the annular chamber floor of
rotor chamber 141 has a plurality of generally funnel-shaped
recesses 142 in circular array around the axis of said rotor
chamber. Along the radial line between contiguous recesses 142 as
shown at the left side of rotor 140 (FIG. 8) the floor at the
bottom of the rotor chamber 141 is V-shaped in the form of low
outer slope 143 and low inner slope 144. The funnel-shaped recesses
142 may be machined into the floor of the rotor chamber with a
symmetrical sloping conical surface 146, but since slopes 143 and
144 are arrayed at somewhat different angles, the top perimeters of
recesses 142 as shown in FIG. 9 turn out to be asymmetrical instead
of circular in shape if the annular floor of chamber 141 were
otherwise flat.
The conical wall 146 of each recess 142 converges into a downwardly
extending apex 147. Located in chamber 141 would be a tubular core
and radially extending, integrally formed septa similar to core 52
and septa 51, as shown in FIGS. 1 and 2, with the lower portions of
said septa descending into respective funnel shaped recesses 142
and performing the same function as described hereinbefore in
connection with FIGS. 1, 2 and 4. As in the previous embodiments,
rotor 140 would be enclosed by a cover plate 148.
Whereas in the embodiment in FIGS. 1 and 2 the draining of the
materials from the rotor chamber after reorientation caused said
materials to flow toward the apex of the annular V-shaped groove
generally from walls arrayed on opposite sides of the apex of said
groove, the embodiment in FIGS. 8 and 9 permits the materials to
flow toward the apex of each funnel from all directions
substantially evenly, whereby each separated zone of particles is
more efficiently constricted at the point of removal at the apex
147 of each funnel recess and the corresponding adjacent exit port
of the respective septa. Thus, the converging walls in a
funnel-shaped recess may serve to improve the definition and
complete collection of each particle zone over that which is
possible in the two-sided annular V-shaped groove of FIGS. 1 and
2.
The apices 147 of each funnel recess 142 are equidistantly aligned
in circular array from the axial center of rotor 140. It will also
be noted that the upper edge of each funnel-shaped recess 142 is
preferably contiguous to the upper edge of an adjacent
funnel-shaped recess 142 whereby the downward flow of the rotor
contents into each of said funnels is controlled equivalently in
order to catch particles moving inwardly from the outer wall 150 of
chamber 141.
Furthermore, there is provided an annular shelf 149 adjacent the
bottom end of the inner wall 151 which would act to limit the
sample after loading to eliminate wall effects on the centrifugal,
ascending surface of each funnel. This shelf would be sufficiently
wide so that the sample would not move into the funnels during the
initial reorientation.
since the mouths of the respective exhaust ports 67 of septa 51 are
located in respective apices of the funnel recess 142 and since
said apices are in a circular array equidistant from the axial
center of the rotor 140, it is manifest that the introduction and
removal of materials into and from the rotor chamber 141 of rotor
140 of FIG. 8 takes place substantially in the same manner as the
comparable introduction and removal of materials in annular chamber
42 of rotor 22 of FIGS. 1 and 2. Hence, the two embodiments, namely
FIGS. 1 and 2 on the one hand, and FIGS. 8 and 9 on the other hand,
are substantially equivalent in structure and function except for
the exercise of choice where necessary or desired. In collecting
particle zones in the funnel-shaped recesses of FIGS. 8 and 9, the
materials are caused to flow toward the outlet port from all radial
directions around that focal point, namely, apex 147, in funnel
recess 142, rather than from principally opposite or radially
inward and outward directions from slopes 44 and 46 in the annular
V-shaped groove in the embodiment of FIGS. 1 and 2. In the latter
case, this also includes some flow of materials from generally
opposite lateral directions along the arcuate path of the annular
apex 48. Where it is desired or necessary to concentrate the
specimen zones to a higher degree of resolution, it may be useful
to utilize the funnel-shaped recess as embodied in FIGS. 8 and 9,
although a satisfactorily high resolution of concentration is
achieved in the annular V-shaped groove in the embodiment of FIGS.
1 and 2.
The V-shaped array of slopes 143 and 144 of the floor of rotor
chamber 141 between the respective adjacent funnel recesses 142 are
provided in order to cause a suitable flow of materials toward said
funnel recesses in the areas therebetween so that a continuous flow
of materials is caused to flow into said funnels which might
otherwise remain on some portions of said floor if it were
otherwise flat. Thus, the V-shaped configuration of slopes 143 and
144 in FIG. 8 is comparable to the V-shaped array of slopes 44 and
46 in FIG. 1.
It is to be understood that the configuration of the funnel-shaped
recesses 142 and V-shaped array of slopes 143 and 144 in FIGS. 8
and 9, may be transposed and incorporated, instead, in the cover
plate of the rotor in a manner comparable to that shown in FIG. 5,
in which case the floor of the rotor chamber would be flat as shown
in FIG. 5.
In all of the embodiments illustrated and described herein, the
removal of the gradient occurs at a plurality of spaced apart
locations within the rotor chamber intermediate the inner and outer
walls thereof. The points of removal are each located at an apex of
converging floor or ceiling slopes that are inclined either into an
annular V-shaped groove or into a plurality of funnel-shaped
recesses where, in both cases, said inclined walls produce a
desirable constriction and concentration of the gradient so that
superior separation between gradient zones is achieved. The
converging sloping walls may be incorporated either in the floor or
ceiling of the rotor chamber, as may be desired or required.
Although the present invention has been described with reference to
particular embodiments, methods, and examples, it will be apparent
to those skilled in the art that variations and modifications can
be substituted therefor without departing from the principles and
true spirit of the invention. The "Abstract" given above is for the
convenience of technical searchers and is not to be used for
interpreting the scope of the invention or claims.
* * * * *