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Jan. 29, 1963 N. G. ANDERSON 3,075,694 PARTICLE SEPARATION METHOD Filed Dec. 29, 1958 . 2 Sheets-Sheet 1 INVENTOR. Norman 6. Anderson BY ?M/Q- Mm ATTORNEY Jan. 29, 1963 N. e. ANDERSON 3,075,694 PARTICLE SEPARATION METHOD Filed Dec. 29, 1958 2 Sheets-Sheet 2 INVENTOR. Norman 6. Anderson BY //M/4M~< ATTORNE Y 3,675,5i94 c Fatented Jan. 29, 1953 2 drawings, and the novel features thereof will be particu larly pointed out in the annexed claims. 3,675,694 PARTICLE SEE’ARATION METHOD 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 20 is that of zone ultracentrifugation in continuous density ' gradients. 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 place. v 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 centrifugal ?eld would appear to be governed by (a) the ents of a solution or suspension which is not limited in sedimentation 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 topes. , 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 cient). ~ 1 3,075,694 3 4 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 temperature. 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 8,075,694 5 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: 15 1. A process for increasing the rate of sedimentation UNITED STATES PATENTS 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 868,483 1,605,596 1,648,369 Reid _______________ __ Oct. 15, 1907 Langelier ____________ __ Nov. 2, 1926 Svedberg et al. _______ __ Nov. 8, 1927 1,893,451 Smith _______________ __ Jan. 3, 1933 1,938,894 2,271,501 2,559,453 2,596,082 Darby et al. _________ __ Dec. 12, Scott _______________ .._ Jan. 27, Merrill et al. _________ __ July 3, Stuart ______________ __ May 6, 2,628,021 Staatf ______________ __ Feb. 10, 1953 1933 1942 1951 1952 2,816,660 Bounin _____________ __ Dec. 17, 1957 trifugal force set up by the centrifuging. 2,822,126 Cohn _______________ _.. Feb. 4, 1958 2. A process for increasing the rate of sedimentation 2,854,143 Novak ______________ _.- Sept. 30, 1958 of particles of molecular size in a suspension comprising the steps of positioning finely divided inert material in 30 FOREIGN PATENTS a centrifuge cell, introducing a quantity of a suspension containing the particles of molecular size to be separated ‘68,800 Germany ____________ _.- June 2, 1893 into the cell, centrifuging the cell for a su?icient inter OTHER REFERENCES 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.