APPARATUS FOR COUNTERCURRENT DISTRIBUTION/MULTIPLE SEDIMENTATION

Abstract: An apparatus for countercurrent distribution in a liquid two-phase system or for multiple sedimentation of suspended particles from a liquid has a rotary device (6) for, respectively, distribution and sedimentation, said device consisting of two parts, i.e. an annular housing (7) and a cylindrical body (8) disposed concentrically therein, said parts being rotatable relative to one another and having opposed working faces (30, 31) forming a number of working cells (32) uniformly distributed about the axis of rotation of said device for, respectively, distribution and sedimentation, at least one of said working cells containing, respectively, the substance to be distributed and the particles suspended in the liquid, while the other working cells merely contain the two-phase system and the liquid, respectively. One part (7) is non-rotatably connected with a rotatable shaft (3), and the other part (8) is coupled to a turning device (10) which, during rotation of said device (6) for, respectively, distribution and sedimentation, is adapted to turn the other part (8) relative to said first part (7) through an angle corresponding to the spacing between two working cells (32).

Description: APPARATUS FOR COUNTERCURRENT DISTRIBUTION/MULTIPLE SEDIMENTATION The present invention relates to an apparatus for coun ercurrent distribution in a liquid two-phase system or for multiple sedimentation of suspended particles from a liquid, said apparatus comprising a rotary de- vice for, respectively, distribution and sedimentation, said device consisting of two parts, i.e. an annular housing and a cylindrical body dispos-ed concentrically therein, said parts being rotatable relative to one another and having opposed ' working faces forming a num- ber of work.ing cells uniformly distributed about the axis of rotation of said device for, respectively, distri¬ bution and sedimentation, at least one of said working cells conta.ining, respectively, the substance to be di¬ stributed and the particles suspended in the liquid, while the other working cells merely contain the two- phase system and the liquid, respectively. The introduction of polymeric liquid two-phase sys¬ tems opened entirely new prospects for the fractiona- tion of biological materials. For separation, one pro- ceeded from the surface characteristics of the material, and the separation method therefore formed a complement to various sedimentation techniques. However, countercurrent distribution cannot be di¬ rectly achieved with this two-phase system technique because these polymeric phase systems have comparatively long separation times. This is due to the small density differences that exist between the phases, and to their high viscosity. The long separation times are an obvious disadvantage in countercurrent distribution effected with an unstable substance, especially if many trans¬ fers are required for efficient separation. Professor Per Ake Albertsson of the university of Lund has con¬ structed an apparatus which, by using thin layers of OMPl of the two-phase system, reduces the separation time by a factor of about 10 as compared with prior art tech¬ nique. For some applications, however, the time needed for a countercurrent distribution experiment will still be inconveniently long. A first object of this invention is to provide an apparatus for countercurrent distribution which reduces the separation time as compared with the above-mentioned apparatus. A second object of the invention is to provide an apparatus for multiple sedimentation which may be used for sedaimentation of various materials, such as size screening of packing material for high-pressure chroma- tography. These objects are achieved by means of the apparatus described .in the characterising clause of claim 1. The apparatus according to the present invention obviates the problem encountered with the -prior art ap¬ paratus and combines the high resolution potential of the countercurrent distribution principle with the in¬ creased phase separation rate which is achieved by cen- trifugation. The crucial point of the present invention is that the transf-er of the lighter phase of the two-phase system between different working cells can be accomplished during centrifugation. By employing this technique, the separation time of polymeric two-phase systems can be reduced by a factor greater than five, as compared with the countercurrent distribution apparatus utilizing thin layers- of two-phase systems. The apparatus may also be used for multiple sedi¬ mentation. Particles of different size, density or shape can often be separated by different sedimentation tech¬ niques. However, to achieve a more efficient separation, a multi-stage method, so-called multiple sedimentation, should be utilized. So far, this method could be made automatic only at 1 x g, which restricts the method to large particles, such as whole cells. Since the apparatus according to the invention makes it possible to perform sedimentation at much higher g values, a much broader range of particle sizes can be fractionated. An embodiment of the invention will be described in more detail below, reference being had to the accom¬ panying drawings. Fig. 1 is, partly in section, a lateral view of an apparatus for countercurrent distribution or multiple sedimentation. Fig. 2 is an exploded view of a rotary distribution or sedimentation device com- prised by the apparatus according to the .invention. Fig. 3 is a top plan view of the device shown in Fig. 2, a cover plate having been removed 'to show working cells. Figs. 4a-4c are longitudinal sections of a part of the device and illustrate the relative positions of two-phase sys- terns in the working cells in different stages of a coun¬ tercurrent distribution method. Fig. 5 illustrates coun¬ tercurrent distribution experiments with the substances 3SA (Bovine Serum Albumin) , .DCIP (2,5-dichlorophenol- indophenol) and cytochrome C. Fig. 6 illustrates a counter current distribution method with chloroplast-thylakoid vesicles. Fig. 7 shows a multiple sedimentation of poly¬ styrene latex particles. Fig. 1 illustrates an apparatus for countercurrent distribution or multiple sedimentation, comprising a centrifugation motor 3, a separation unit 6, a turning motor unit 10 and a shak.ing unit 28, 29. The separation unit 6 which constitutes the principal part of the pre¬ sent invention, comprises an annular housing 7, a cy¬ lindrical body 8 turnable within said housing, and a cover plate 9. The separation unit will be.described .in detail in the follow.ing. The separation unit 6 is non-rotatably connected with a connection plate 1 with axially directed p.ins 2 engaging with corresponding recesses 34 in the housing 7. (Fig. 2) . The connection plate 1 is fixedly connected with a shaft 4 of the centri uga ion motor 3 which is adapted to rotate the separation unit β at a speed of up to 3000 rpm. The shaft 4 is mounted in a bearing 5. The turning or transfer motor unit 10 is non-rotatably connected with the connection plate 1 via the separation unit 6 and comprises a base plate 11 screwed into the housing 7 and firmly urging the cover plate against the housing 7/the body 8 to provide a reliable seal between the cover plate and the housing/body, a lower casing 12 mounted on said base plate, and an upper casing 13 mounted on said lower casing. A turning or transfer motor 15 is mounted within the upper cas.ing 13 on a t r ^g motor bracket 14 fixedly connected with the upper and lower cas.ing 12 and 13, respectively. The shaft 16 of the motor 15 is coupled with the input shaft 19 of a transmission 18 via a break disk 17. The transm.ission output shaft is connected to the cylindrical body 8 of the separation unit 6 and adapted, upon one revolution of the turning motor 15, to turn the cylindrical body 18 through a predetermined angle, relative to the housing 7. Th break disk 17 is essentially circul-ar, but has a discontinuity at its peripheral edge, such as a recess or a detent. The arm of a icroswitch 21 is provided opposite the peripheral edge of the break disk, said microswitch being fixedly mounted in the turning motor unit 10. A shaft unit 24 connected with the upper casing 13 extends axially through a frame 22 surrounding the separation unit and the turning motor unit, and comprises sliding contact terminals 36. A casing 23 carries slid¬ ding contacts 27 in connection with a current supply and control unit (not shown) for the apparatus according to the invention, and supplies current to the turning motor 15 -and the microswitch 21 via the sliding contact terminals 36 and leads (not shown) . Turning of the cylindrical body 3 relative to the housing 7 is effected, in the manner described below, during rotation of the separation unit 6. A control pulse of about 1 second is supplied to the motor which turns the break disk to such an .extent that the arm of the microswitch which, when the apparatus is started, lies opposite the discontinuity, is disengaged from said dis¬ continuity and closes the microswitch, whereby the motor at the end of the control pulse continues to turn the break disk and thereby to turn the cyl.indrical body 8 via the transmission 18. When the break disk has made a full revolution, the discontinuity will actuate the arm of the microswitch such that the switch is discon¬ nected and the motor 15 is inactivated. The relation- ship between the turning of the motor 15 and the turning of the cylindrical body 8 will be explained in detail below. The motor 15, the break disk 17, the transmission 18 and the microswitch 21 are located eccentrically on the separation unit 6, for which reason balance weights (not shown) must be provided on the turning motor unit 10 to properly balance the apparatus. The connection plate 1 is mounted on a shaking table 28 which, by means of a connection 29 to a shak.ing motor (not shown), imparts a shaking motion to the separation unit in the horizontal plane, the movement of the shak¬ ing table during shaking being circular. Fig. 2 is an exploded sectional view of the separa-. tion unit 6 in Fig. 1. The lower side of the housing 7 shows the recesses 34 for engagement with the pins . 2 projecting from the connection plate 1 in Fig. 1. The inner circumference of the annular housing 7 has a number of semicylindrical work-ing faces 30 which are best seen in the left-hand part of Fig. 3 and are uniformly distri- buted around the housing. Furthermore, the upper side of the housing has an annular groove 33 in which an O-ring is placed to constitute a seal between the housing 7 and the cover plate 9 which, in some suitable manner, is detachably mounted on said housing and the cylindrical body 8 disposed therein. The cylindrical body 8 may have a close fit in the housing 7, although permitting rela¬ tive movement between the housing and the body. The peri- phery of the cylindrical body 8 is provided with radial recesses or working faces 31, the width of which at the boundary surface between the housing and the body cor¬ responds to the diameter of the semicylindrical recesses or working faces 30 in the housing 7. These working faces 31 are best shown in the right-hand part of Fig. 3. The central part of Fig. 3 shows how the work.ing faces 30 and 31 together constitute work-ing ceils 32 adapted to receive a two-phase system for countercurrent distribution, or a liquid for multiple sedimentation. It is po.inted out that the long sides of the working cells are directed rad.ially, whereby the cell width will be slightly increased in a radial outward direction. This constitutes an advantage during centrifugation be- cause heavy particles will not impinge upon the long sides of the working cells when thrown radially outwardly by the centrifugal force. In the embodiment here described, the work-ing cells are thirty in number, which means that the angular spac- -ing between two working cells 32 is 12°. The working cells are so closed by the cover plate 9 that their contents are safely retained therein during centrifugation. The housing 7, the body 8 and the cover plate 9 are preferably made of Plexiglass. The function of the apparatus will now be described in more detail in the following, with reference to Figs. 4a-c. The housing and the cylindrical body are first assembled and so adjusted that the working faces 30 of the housing are coincident with the working faces 31 of the body to form working cells 32. For counter¬ current distribution, a sample is placed in a polymer mixture (for multiple sedimentation, in a suitable liquid) .in one or a small number of cells. A suitable volume of top phase and bottom phase of a two-phase system (li¬ quid when multiple sedimentation is carried out) is added to each of the remaining cells. The cover plate 9 is placed upon the housing and the body dispose therein and is screwed in position. The entire separation unit 6 is then mounted on the connection plate 1 (Fig. 1) , and the countercurrent distribution method can be start- ed. Each cycle of this countercurrent distribution method comprises the following steps: 1) The separation unit is shaken at 1 x g to ensure efficient mixing of the phases contained in the working cells in the manner shown in Fig. 4b. 2) When thoroughly mixed, the phases are separated by centrifugation. During testing of the apparatus according to the invention, the centrifugation ve¬ locity amounted to 600 r.p. . , corresponding to an acceleration field of 30 x g. 3) While the separation unit is still rotat.ing at full speed (600 r.p.m.) the lighter phase contained in that part of the working cell which belongs to the body 8 is transferred to a position opposite the next working face 30 in the housing 7 by turning the body 8 relative to the housing 7. 4) After this transfer, the centrifugation motor 3 is decelerated, and a new cycle can be started. 5) The method is continued until the desired number of transfers of the lighter phase has been carried out, whereupon the working cells are emptied for subsequent analysis or processing. The entire method is automated, and the centrifugation motor, the turning motor and the shaking table are controlled by a control unit (not shown) . Fig. 5 illustrates three examples of a countercur¬ rent distribution of pure substances. Curve a shows the countercurrent distribution of BSA, curve b the counter¬ current distribution of DC P, and curve c the counter- current distribution of cytochrome C. The experiments were carried out at room temperature (22°C) with ' a phase system having the following composition: 5% by weight of dextran T 500, 4% by weight of polyethylene glycol 6000, 0.1 M KI, 10 mM sodium phosphate buffer, pH 6.8. Each cycle comprised ' 30 seconds of mixing and 90 seconds of centrifugation (including acceleration and decelera- tion) . The number of transfers was 40. The distribution curves have peak values reflecting the tendency of the material to divide itself into one or the other phase. BSA (curve a) which shows a peak value at a low tube number tends to seek out the heavy phase which is rich in dextran. The countercurrent distribution principle is advantageous in that theoretical curves are readily calculated The theoretical curve of DCIP has been cal¬ culated on the basis of the peak value and follows the experimental curve fairly well. Deviations from the theo- retical curve may be due either to heterogeneities within the distributed material or to leakage between the work¬ ing cells. .As will appear from the following, leakage is .probably not the cause of the peak broadening in this instance. The result of countercurrent distribution of chloro¬ plast-thylakoid vesicles is shown in Fig. 6. Countercur¬ rent distribution was carried out on chloroplast-thylakoid vesicles at 4°C with a phase system having the following composition: 5.7% by weight of dextran T 500, 5.7% by weight of polyethylene glycol 4000, 10 mM of sodium phos¬ phate buffer, pH 7.4, 5 mM of NaCl, 20 mM of saccharose. The number of transfers was 29. The remaining conditions of the experiment are accounted for in connection with Fig. 5.- The material is distributed among all of the working cells and exhibits a considerable heterogeneity of particles with respect to surface properties. The chlorophyll a/b ratios reveal that a partial separation of photo system II (to the left) and photo system I (to the right) has been achieved. Preliminary experiments suggest that the material to the left represents everted thylakoids, while the material to the right represents stroma lamellae vesicles with their right side turned out. Fig. 1 shows sedimentation curves of well defined polystyrene latex particles. These particles have been selected as test material since they can be obtained with rather uniform size. On multiple sedimentation of polystyrene latex particles, the mixing time amounted to 30 seconds in all four cases. The particle size and the centrifugation time of each cycle are shown in the Figure. The filled squares represent an experiment using 0.25 M of saccharose. In all other cases, the medium used was distilled water. A drastic difference in sedi¬ mentation is seen for 7.6 and 2.2 um particles. For the 7.6 ym particles, the shortest centrifugation time used (1 minute in each step) was sufficient to obtain an al¬ most complete sedimentation (open squares in Fig. 7) , while the 2.2 u particles hardly sed.imented at all (open circles in Fig. 7). By increasing th time for each cen¬ trifugation, it was possible to demonstrate sedimentation also of the 2.2 * particles. The theoretical processing of. the sedimentation curves is largely identical with the theoretical process- .ing of the countercurrent distribution curves. The theo¬ retical curve for the 2.2 um particles at a centrifuga¬ tion time of 1 minute well agrees with the experimental curve, and this suggests that no leakage occurs between the working cells. The above results show that the countercurrent di¬ stribution apparatus according to the invention can be used for fractionation. The main advantage of the ap¬ paratus ' according to the invention is that the separation time for the phase system can be reduced. In the above experiments, the separation time was reduced by a factor of 5, compared to the time required for corresponding experiments by means of the countercurrent distribution technique utilizing thin layers. Compared to prior art technique, the apparatus ac¬ cording to the present invention brings the advantage that larger volumes can be processed, and that the theo- retical calculations of distribution coefficients are easily accomplished. These calculations are of essential importance to binding studies utiliz.ing two-phase systems. Using the apparatus for multiple sedimentation offers new possibilities of fractionation of, for example, cells and cells organelles. The multiple sedimentation method should be useful for both preparative and analytical purposes. The acceleration field and the sedimentation time are readily varied during a multiple sedimentation experiment, such that particles within a wide range of sedimentation rates can be analyzed in the same experiment< The present invention provides an apparatus which combaines the high resolving potential of multi-stage methods with the advantage of centrifugal acceleration fields for decreasing the time of each step. This prin¬ ciple therefore should be useful for the fractionation of various biological and non-biological materials, espe¬ cially where the retention of high activities is impor¬ tant, or where the time required for each step is too long.

Claims: CLAIMS 1. An apparatus for countercurrent distribution in a liquid two-phase system or for multiple sedimenta¬ tion of suspended particles from a liquid, said apparatus comprising a rotary device (6) for, respectively, distri- bution and sedimentation, said device consisting of two parts, i.e. an annular housing (7) and a cylin-drical body (8) disposed concentrically therein, said parts being rotatable relative to one another and having op¬ posed working faces (30, 31) forming a number of working cells (32) unifo.rmly distributed about the axis of rota¬ tion of said device for, respectively, distribution and sedimentation, at least one of said working cells, con¬ taining, respectively, the substance to be distribut¬ ed and the particles suspended in the liquid, while the other working ceils merely contain the two-phase system and the liquid, respectively, c h a r a c e r t e r i s - e d in that one part (7) is non-rotatably connected with a rotatably shaft (^, and that the other part (8) is coupled to a turning device (10) which, during rota- tion of said device (6) for, respectively, distribution and sedimentation, is adapted to turn the other part (8) relative to said first part (7) through an angle correspond.ing to the spacing between two work.ing cells (32). 2. An apparatus as claimed in claim 1, c h a r a c ¬ t e r i s e d in that the working cells (32) are formed as axially extending cavities, whereby the two-phase system and the liquid in the working cells (32) , respec¬ tively, are layered along a plane parallel to the axis of rotation during rotation of said device for, respec¬ tively, distribution and sedimentation. 3. An apparatus as claimed in claim 1 or 2, c h a ¬ r a c t e r i s e d in that the device for, respective¬ ly, distribution and sedimentation (6), has an annular sealing plate (9) for sealing the work.ing cells (32) , and that an O-ring (35) is adapted to seal between the sealing plate and the device for, respectively, distri¬ bution and sedimentation. 4. An apparatus as claimed in claim 1, 2 or 3, c h a r a c e r i s e d in that the device (6) for, respective¬ ly, distribution and sedimentation is coupled to a shak¬ ing device (28) adapted to shake said device for, respec¬ tively, distribution and sedimentation in a plane sub¬ stantially perpendicular to the axis of rotation.

Inventors: AKERLUND HANS ERIK
Applicants: AKERLUND HANS ERIK
Application year: 1982
Main classification: B04B5
IPC codes: B04B5, G01N33