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Патент USA US3075702

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Jan. 29, 1963
Filed Dec. 29, 1958
2 Sheets-Sheet 1
Norman 6. Anderson
?M/Q- Mm
Jan. 29, 1963
Filed Dec. 29, 1958
2 Sheets-Sheet 2
Norman 6. Anderson
Fatented Jan. 29, 1953
drawings, and the novel features thereof will be particu
larly pointed out in the annexed claims.
In the drawings, FIG. 1 is a schematic of particles of
a liquid, exaggerated in size to indicate the influence of
a centrifugal ?eld in the presence of my improved separat
Norman G. Anderson, Oak Ridge, Tenn, assignor to the
United States of‘ America as represented by the United
States Atomic Energy Commission
ing agent.
Fiied Dec. 29, 1958, Ser. No. 783,628
3 Claims. (Cl. 233-40)
FIG. 2 is an elevation of samples of various liquids
positioned adjacent an indicator chart to indicate the
performance of my improved separating agent.
This invention relates to the separation of particles
of different sizes or densities, and more particularly to a 10
gal force, and for more effectively separating substances
with differing rates of sedimentation.
FIG. 3 is an elevation of a sample tube having a plural
ity of vertical rods with spaced projecting elements hav
ing surfaces to induce greater sedimentation.
method of increasing the rate of sedimentation of dis—
solved or suspended substances which are under the in?u
ence of a centrifugal ?eld without increasing the centrifu
FIG. 4 is a fragmental detail of a portion of one of
said rods showing one form the projecting elements may
15 take.
In the ?eld of separation and fractionation of proteins
. FIG. 5 is a-perspeotive of a rod having spaced disk like
and other colloids, four general methods are'available for
projections that present substantially ?at surfaces that
analytical or preparative application.
facilitate and direct ?ow.
These are elec
trophoresis, precipitation, chromatography and adsorp-v
tion, chromatography and adsorption, and ultracentrifu
gation. A method complementary to ultracentrifugation
FIG. 6 is an elevation of a fragmental portion of a
is that of zone ultracentrifugation in continuous density '
In the prior art of ul‘tracentrifugation it has been
sample tube employing a single vertical rod having spaced
plates for presenting surfaces over which flow may take
FIG. 7 is a schematic of a vessel for separating sedi
mented material from my improved sedimenting agent.
Applicant has discovered that when particles, or
the practice to provide a free solution in which the sub 25
molecules, ‘suspended in a liquid, are sedimented in a
stance to be sedimented is dissolved.‘ The resulting solu
centrifugal ?eld the distribution in ‘the centrifuge tube is
tion or suspension is placed in a centrifuge ‘and sedimen
altered so that the particle concentration increases in
tation is brought about by the application of centrifugal
the direction of the centrifugal force. The presence of
force by t e operation of the centrifuge, generally over
long periods. With available ultracentrifuge rotors sedi 30 a small stationary ‘object oifering a surface substantially
perpendicular to the centrifugal ?eld will cause a local
mentation of proteins requires many hours to several days.
pile-up of sedimenting particles. The liquid containing
Particles of smaller dimensions than colloids have also
been sedimented but special high speed equipment is
the higher concentration of particles at this surface will,
by virtue of the higher density, ?ow off the surface and
required, and a proportionately longer time.
move as a discrete mass further in the direction of the
Applicant with a knowledge of this problem of the
increasing centrifugal force until the stream is ran
prior art has for ‘an object of his invention the provision
domized by thermal agitation. The net result is accel
of an improved method for fractionating colloidal mix
tures and separating their components.
erated sedimentation.
Applicant has as another object of his invention the
The effect is illustrated in FIG. 1 where larger particles,
provision of a method for increasing the rate of sedimen 40 such as protein molecules, are separated from the smaller
tation of dissolved or suspended substances during ultra
molecules or particles of a liquid, such as .1 molar NaCl
centrifugation while stabilizing the boundaries and mak
solution or other dilute salt solution, or distilled water, in
ing recovery‘of the fractions less di?icult.
which they have been dissolved, by sedimentation through
Applicant has as another object of his invention the
the application of centrifugal force, in a direction as
provision of a method for more rapid separation of dis— 45 indicated by the arrow. Small stationary objects, such as
solved or suspended components by sedimentation result
starch granules, also occupy the space and are preferably
ing from centrifuging a liquid having an agent that will
packed therein so that the liquid occupies the interstices.
result in selective transpart of the more rapidly sediment
At level a is illustrated particles 1 of differing masses
ing particles to the bottom While the lighter particles are
which are sedimenting in an unobstructed centrifugal ?eld
diffused out of the descending streams.
50 according to their respective sedimentation rates. At
Applicant has as a further object of his invention the
level I) the sedimenting particles collide with the surface
provision of a method for separating components of a
of a stationary object, such as the starch particles 2, at
solution or suspension by centrifuging and sedimenta
which level a local concentration and density increase
tion wherein the descending streams result in the trans
occurs. The denser ?uid does not adhere to the stationary
port of lighter particles which are displaced upward by 55 object but tends to flow off somewhat like a stream, over
the down streaming heavy fluid.
the surface and to move in the direction of increasing
Applicant has as 1a still further object of his invention
the provision of a method for the selective removal of un
centrifugal ?eld.
As the denser fluid streams through
region 0 randomization occurs as a result of thermal agi
desirable protein materials in hypodermlioally admin
tation and the smaller more rapidly diffusing molecules
istered immunizing and therapeutic agents such as the 60 drift out of the stream in accordance with the Brownian
serurns and vaccines produced in animal media, by em
effect. If the liquid is ?lled with many insoluble stationary
ploying his improved separating agent during the applica
objects, the processes occurring at levels b and 0 will be
tion of centrifuging and sedimentation to the ?uid.
repeated many times, as represented at levels d and e.
Applicant has as a still further object of his invention
The rate of transportation of the particles through the
the provision of a method for separation of the compo
?eld would appear to be governed by (a) the
ents of a solution or suspension which is not limited in
rate of the particles, (b) their concentra
its application to the sedimentation of large molecules,
tion, (0) the size and shape of the solid stationary objects,
but is applicable in principle to solutions of small
(d) the centrifugal force, (e) the temperature, (1‘) the
molecules and ions, and to the separation of various iso
viscosity of the liquid, and‘ (g) the rate at which stream
70 ing material becomes randomized (the diffusion coeffi
Other objects and advantages of my invention will ap
pear from the following speci?cation and accompanying
The sedimenting action is enhanced by the packing of
powder in the sample tube. This is done by feeding a
slurry containing a starch to‘the sample tube, then centri~
fuging to pack the powder, then removing the overlying
liquid, and adding more slurry, and repeating the cycle.
Example 11
The advantages of a centrifugation system which utilizes
a packing of stationary objects in the centrifugal ?eld,
over a system having an unobstructed liquid ?eld are
In one experiment, 5 percent stained bovine serum
albumin (BSA) in a 0.1 ionic strength phosphate-sodium
chloride buffer (pH 7.5) was centrifuged in starch, and
in free solution under otherwise identical conditions. Cen
trifugation was done in a Spinco Model L machine, using
21 SW 391 swinging-bucket rotor at approximately 20°.
After centrifugal acceleration brie?y to 20,000 r.p.m. the
(l) more rapid sedimentation of all components, (2)
was stopped and the excess clear ?uid was re
selective transport of the more rapidly sedimenting parti 10 machine
moved and more starch slurry added. This procedure was
cles to the bottom, (3) additional selectivity based on the
repeated once. These preparations were centrifuged 5
diffusion of the lighter particles out of the descending
hours at 37,000 r.p.m._, and deceleratcd without braking,
streams, and (4) further separation due to the transport
and the tubes removed.
of lighter particles in the ?uid which are displaced upward
The distance the protein sedimented in free solution
by the downstreaming heavy ?uid.
15 was estimated by a visual optical refraction method (FIG.
It is to be noted that the method functions only when
2). It. may be noted that the card in FIG. 2 has a series
the sedimenting material is of such 'a size that thermal
of parallel angular lines. When a lusteriod centrifuge
agitation prevents the particles from being compacted
against the surface which is substantially perpendicular
sample tube, indicated at a, ,is placed against this card,
the angular straight lines will be abruptly bent or dis
to the direction of the centrifugal ?eld. While this‘ sur 20 torted. to provide a sharp substantially horizontal peak
face is shown as spherical, in practice it may be any
at the. inter surfaces. of two liquids which have different
con?guration that does not have occluded surfaces. ‘It
indices of refraction, as indicated by the upper arrow
is therefore best suited to the sedimentation and separa
which intersects sample tube a in FIG. 2. This gives
tion of particles of molecular size, including large organic
an indication of thepoint of separation of the two liquids
molecules and proteins of molecular weight of one thou 25 where. no dye has been provided for this purpose, as in
sand to a million, for example, nucleic acids, and poly
the case of sample tube ’a of FIG. 2. A comparison of
saccharides, and possibly viruses.
tube a, ?lled with 5 percent BSA, and tube b, ?lled with
‘The present development has been found especially
5 percent BSA stained with bromphenol blue in starch,
useful in studies of biological materials such as blood
centrifuged for ?ve hours at. 38,000 r.p.m., reveals
serum and hemoglobin. Potato starch granules are rela 30 both
that the protein moves through starch at about twice the
tively inexpensive and have proven to be satisfactory sta
rate observed in free solution. Tubes 0 and d contain
tionary bodies. Particles of Lucite or other plastics are
stained BSA in starch, and starch in buffer, respectively,
also satisfactory, and although glass beads may be used,
for comparison with tube b. The former is centrifuged
protein particles tend to stick to their surfaces until the
brie?y for packing the starch, and the latter is uncentri
beads become covered. In short, most any powder with 35 fuged. No .color'was observed in the upper fourth of
acceptable surfaces may be employed.
The process is generally carried out by placing the
tube .b. Staining the BSA does not alter its sedimenta
tion noticeably, as is shown by the stained and clear
samples of the desired liquid in sample tubes such as
samples in tubes 2 and j, which. have been centrifuged
shown in FIG. 2. These sample tubes are then placed in
for 6 hours at 37,000 rpm. Tube g contains water and
a conventional centrifuge, such as the “Spinco,” Model L, 40 indicates the pattern of bands seen when no refractive
preparative ultracentrifuge made by Beckman Instrument
index gradient is present.
‘Company, and employing an SW 391 swinging-bucket
It should be appreciated that in carrying out this proc
rotor. Rates of sedimentation for certain materials in free
.655 the supernatent liquid and the sedimented material
solution have been compared with the rates for the same
may be removed from the sample tube after the centri
materials, in starch slurries. Determinations were also 45 fuging step, and the resulting cake containing the sedi
made using an analytical centrifuge, Spinco Model B,
ments-d materials in the lower portion thereof are sepa
under precisely controlled conditions of rotor speed and
rated by placing the cake in a vessel 7 on a fritted or sin
tered glass ?lter disk 8 as shown in FIG. 7. Water is
added to form a concentrated slurry, and to displace
Example I
50 the sedirnented material from between the powder grains,
so that it drips out through the funnel end 9 of the ves
In one experiment, two analytical cells were used in
sel and is collected.
the same rotor at the same time in the Spinco Model E
analytical ultracentrifuge. One contained 1.5 percent
While a powder has been employed in the foregoing
stained bovine serum albumin in 0.1 ionic strength sodium
treatment, it is apparent ‘that other sedimenting agents
chloride-phosphate buffer. The second cell contained the 55 may also be used. In FIGS. 3, 4, and 5, a series of
‘same solution plus suspended potato starch. During cen
spaced ?at surfaces are presented by utilizing spaced ver
trifugation the starch packed tightly leaving a ?uid layer
tically extending rods 3 upon which are formed a plural
.over the starch.
ity of spaced projections which preferably take the form
of disks 4. These rods 3 are suspended in spaced rela
A Schlieren method for determining ?uid boundaries by
refractive index variation was used to determine the 60 ,tion from a plate or hanger 5 carried or supported by
protein boundaries in both tubes during centrifugation,
and in the control tube until the end of centrifugation.
The distance the protein boundary sedimented in starch
was determined by densitometric measurements on photo
graphs made at the end of the run.
After acceleration to 56,100 revolutions per minute
(r.p.m.) two boundaries were observed above the starch
boundary, which moved at the same rate. The boundary
peaks reached the upper level of the starch after 48 min
utes of centrifugation at this operating speed. Decelera
tion was begun 13 minutes later. The protein boundary
in the cell containing starch was observed to move ap
proximately three times as fast as that in the cell
containing no starch,
the upper extremity of the sample tube 6. In practice,
the vertical rods 3 are preferably numerous and closely
spaced, and may be suspended so that the projections or
disks, from red to rod may be staggered and not coin~
- .cide, and in this way the streams ?owing from these pro
jections on one rod may be conveniently directed down
wardly to those on the next adjacent rod. The plates
may be relatively small, with radii of the range of 1A5",
though not limited to this size. The rod and plates may
70 be of metal, plastic or any other suitable material.
In another modi?cation, the sedimenting agent takes
the form of a series of larger diameter plates or disks 4’
mounted on a single center staff or rod, preferably ex
tending axially of the sample tube 6', as shown in FIG. 6.
75 These plates which cover a substantial part of the di
ameter of the tube serve to direct the streams of mole
cules in their movement in the direction of increasing
centrifugal force.
Thus, it is important to maintain
?ow over the surfaces of the plates to realize the de
?uid richer in the particles in the direction of increased
centrifugal force to increase the rate of sedimentation.
3. A process for increasing the rate of sedimentation
sired result of increasing the sedimentation.
of proteins in a liquid comprising the step of position
ing powdered starch in a centrifugal cell, introducing a
While the above method has been described as a batch
process, it might easily be adapted to a continuous proc
liquid containing the proteins to be separated, centrifug
ess. With such an adaptation a solution is continuously
introduced via a suitable tube to the bottom of any of
starch and repeating the step to produce a mass of packed
starch, then centrifuging at a speed that will produce a
ing the cell to pack the starch, then adding additional
the devices described, and the solution is caused to flow 10 centrifugal ?eld that will direct the particles of protein
slowly to the top and is thence collected or removed.
against the surfaces of the relatively ?xed large starch
The rate of ?ow of fresh liquid may be adjusted so that
particles and concentrate the denser protein molecules
downward sedimentation of particles in solution is faster
and complexes into streams and increase sedimentation.
than the upward ?ow of solvent.
References Cited in the ?le of this patent
Having thus described my invention, I claim:
1. A process for increasing the rate of sedimentation
of particles of molecular size in a suspension compris
ing the steps of positioning insoluble powdered material
in a centrifuge cell, introducing the suspension having
the particles of molecular size to be separated into the 20
cell, centrifuging the cell for a period sufficiently long
to pack the powdered material, then adding additional
powdered material and again centrifuging the cell to
cause the particles to impinge upon the surfaces of
the powdered material and induce preferential flow of 25
?uid richer in said particles in the direction of the cen
Reid _______________ __ Oct. 15, 1907
Langelier ____________ __ Nov. 2, 1926
Svedberg et al. _______ __ Nov. 8, 1927
Smith _______________ __ Jan. 3, 1933
Darby et al. _________ __ Dec. 12,
Scott _______________ .._ Jan. 27,
Merrill et al. _________ __ July 3,
Stuart ______________ __ May 6,
Staatf ______________ __ Feb. 10, 1953
Bounin _____________ __ Dec. 17, 1957
trifugal force set up by the centrifuging.
Cohn _______________ _.. Feb. 4, 1958
2. A process for increasing the rate of sedimentation
Novak ______________ _.- Sept. 30, 1958
of particles of molecular size in a suspension comprising
the steps of positioning finely divided inert material in 30
a centrifuge cell, introducing a quantity of a suspension
containing the particles of molecular size to be separated
Germany ____________ _.- June 2, 1893
into the cell, centrifuging the cell for a su?icient inter
val to pack the inert material into a relatively stationary
mass, and continuing the centrifuging at sufficient speed 35 Encyclopedia Britannica, vol. 5, copyright 1957, copy
to set up a centrifugal ?eld in a direction to bring the
received in Scienti?c Library May 2, 1957, page 146,
particles progressively into contact with the surfaces
col. 2, lines 6 to 16, published by Encyclopedia Britan
of the inert material and introduce preferential ?ow of
nica, Inc.
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