WO1982003188A1

WO1982003188A1 - Apparatus for counter current distribution/multiple sedimentation

Info

Publication number: WO1982003188A1
Application number: PCT/SE1982/000070
Authority: WO
Inventors: Hans Erik Akerlund
Original assignee: Hans Erik Akerlund
Priority date: 1981-03-16
Filing date: 1982-03-16
Publication date: 1982-09-30
Also published as: SE423969B; SE8101643L; EP0074376A1

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 countercurrent distribution in a liquid two-phase system or for multiple sedimentation of suspended particles from a liquid, said apparatus comprising a rotary device for, respectively, distribution and sedimentation, said device consisting of two parts, i.e. an annular housing and a cylindrical body disposed concentrically therein, said parts being rotatable relative to one another and having opposed working faces forming a number of working cells 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 twophase system and the liquid, respectively.

The introduction of polymeric liquid two-phase systems opened entirely new prospects for the fractionation of biological materials. For separation, one proceeded from the surface characteristics of the material, and the separation method therefore formed a complement to various sedimentation techniques.

However, countercurrent distribution cannot be directly 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 transfers are required for efficient separation. Professor Per Ake Albertsson of the university of Lund has constructed an apparatus which, by using thin layers of of the two-phase system, reduces the separation time by a factor of about 10 as compared with prior art technique. 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 chromatography. 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 apparatus and combines the high resolution potential of the countercurrent distribution principle with the increased phase separation rate which is achieved by centrifugation. The crucial point of the present invention is that the transfer 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 sedimentation. Particles of different size, density or shape can often be separated by different sedimentation techniques. 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 accompanying 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 comprised 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 systerns in the working cells in different stages of a countercurrent distribution method. Fig. 5 illustrates countercurrent distribution experiments with the substances BSA (Bovine Serum Albumin), DCIP (2,5-dichlorophenolindophenol) and cytochrome C. Fig. 6 illustrates a counter current distribution method with chloroplast-thylakoid vesicles. Fig. 7 shows a multiple sedimentation of polystyrene 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 shaking unit 28, 29. The separation unit 6 which constitutes the principal part of the present invention, comprises an annular housing 7, a cylindrical body 8 turnable within said housing, and a cover plate 9. The separation unit will be. described in detail in the following.

The separation unit 6 is non-rotatably connected with a connection plate 1 with axially directed pins 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 centrifugation motor 3 which is adapted to rotate the separation unit 6 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 casing 13 on a turning motor bracket 14 fixedly connected with the upper and lower casing 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 transmission 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. The break disk 17 is essentially circular, but has a discontinuity at its peripheral edge, such as a recess or a detent. The arm of a microswitch 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 slidding 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 discontinuity 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 cylindrical 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 disconnected and the motor 15 is inactivated. The relationship 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 shaking motor (not shown), imparts a shaking motion to the separation unit in the horizontal plane, the movement of the shaking 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 working faces 30 which are best seen in the left-hand part of Fig. 3 and are uniformly distributed 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 relative 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 corresponds 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 working 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 pointed out that the long sides of the working cells are directed radially, whereby the cell width will be slightly increased in a radial outward direction. This constitutes an advantage during centrifugation because 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 working cells are thirty in number, which means that the angular spacing 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 countercurrent 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 (liquid 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 disposed 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 started.

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 velocity amounted to 600 r.p.m., 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 countercurrent distribution of pure substances. Curve a shows the countercurrent distribution of BSA, curve b the countercurrent distribution of DCIP, and curve c the countercurrent 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 deceleration). 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 calculated on the basis of the peak value and follows the experimental curve fairly well. Deviations from the theoretical curve may be due either to heterogeneities within the distributed material or to leakage between the working 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 chloroplast-thylakoid vesicles is shown in Fig. 6. Countercurrent 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 phosphate 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 sedimentation is seen for 7.6 and 2.2 μm particles. For the 7.6 μm particles, the shortest centrifugation time used (1 minute in each step) was sufficient to obtain an almost complete sedimentation (open squares in Fig. 7), while the 2.2 um particles hardly sedimented at all (open circles in Fig. 7). By increasing the time for each centrifugation, it was possible to demonstrate sedimentation also of the 2.2 μm particles.

The theoretical processing of. the sedimentation curves is largely identical with the theoretical process.ing of the countercurrent distribution curves. The theoretical curve for the 2.2 um particles at a centrifugation 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 distribution apparatus according to the invention can be used for fractionation. The main advantage of the apparatus 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 according 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 utilizing 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 principle therefore should be useful for the fractionation of various biological and non-biological materials, especially where the retention of high activities is important, or where the time required for each step is too long.

Claims

1. An apparatus for countercurrent distribution in a liquid two-phase system or for multiple sedimentation of suspended particles from a liquid, said apparatus comprising a rotary device (6) for, respectively, distribution 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 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 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 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 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), respectively, are layered along a plane parallel to the axis of rotation during rotation of said device for, respectively, 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, respectively, 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, distribution 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, respectively, distribution and sedimentation is coupled to a shaking device (28) adapted to shake said device for, respectively, distribution and sedimentation in a plane substantially perpendicular to the axis of rotation.