Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
  • B01D21/28—Mechanical auxiliary equipment for acceleration of sedimentation, e.g. by vibrators or the like
  • B01D21/283—Settling tanks provided with vibrators
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D17/00—Separation of liquids, not provided for elsewhere, e.g. by thermal diffusion
  • B01D17/02—Separation of non-miscible liquids
  • B01D17/04—Breaking emulsions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
  • B01J19/10—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing sonic or ultrasonic vibrations
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/49—Blood
  • G01N33/491—Blood by separating the blood components

Definitions

• This invention relates to a method of phase partition separation of particles.
• particles includes particularly, but not solely, cells, bacteria, viruses, organelles and molecules.
• Efficient separation and concentration techniques are essential in many areas of biology. Such techniques are required both for soluble substances, e.g. proteins and nucleic acids, and for suspended particles e.g. whole cells, organelles, bacteria and viruses.
• One well-known separation method used in chemistry is the differential partition of solutes between two immiscible liquid phases, perhaps the most well-known being the ether extraction of impurities in organic preparations.
• organic solvents are not suitable for most biological applications. Nevertheless it is possible to produce two immiscible phases with aqueous solutions of certain polymers: such phases have a water content typically in the range 85-99% (w/w) and are very mild towards various biological activities.
• Aqueous two-phase partition was developed by P.A.
• Phase separation of biological particles is a two-step process with each step having different characteristic times.
• the first stage is the partition of the particles or solute into drops of one of the phases. This step is rapid.
• the second stage is the separation of the phases into discrete layers allowing collection of the particles or solute. It is this stage which can take a considerable time to occur. It has been known for some time that particles or droplets in a stationary ultrasonic field can experience time-independent forces that move them to preferred regions of the field separated by half wavelength intervals.
• a number of techniques involving single or double transducer systems or stationary or moving reflectors have been proposed for moving ultrasonically concentrated small particles or large single particles in suspension in order to achieve efficient particle harvesting.
• phase partition separation in which a mixture of two immiscible liquid phases and particles is subjected to a standing wave ultrasound field.
• the ultrasound field may extend in any convenient direction through the mixture or phase system.
• the field may extend either axially or transverse of a tube or other elongate container in which the phase system is contained: for example the container may be positioned with its axis generally vertical or generally horizontal, or at an inclined angle between horizontal and vertical if desired.
• an annular or tubular transducer may be used, preferably with the transducer also forming a tubular wall of the container: in this case the ultrasound field is a radial field.
• the applied ultrasound field causes the phase system to form into a series of bands of the two phases, separated by a half wavelength.
• the ultrasound field is pulsed: when the field is interrupted, the bands of the more dense phase fall under gravity: some break-up of the bands occurs before the field is re-applied.
• the method may also be used simply for separating two liquid phases, e.g. separating one liquid from another in an emulsion. Therefore, also in accordance with this invention, there is provided a method of phase partition separation in which a mixture of two immiscible liquid phases is subjected to a standing wave ultrasound field.
• Figure la is a schematic diagram of a single transducer arrangement for carrying out methods in accordance with this invention.
• FIGURE lb is a similar diagram of a dual transducer arrangement
• FIGURE lc is a similar diagram of an annular transducer arrangement
• FIGURE 2 is a graph to compare the times taken for the formation of two continuous bulk phases under gravity and under the influence of ultrasound, for phase systems of varying relative proportions;
• FIGURE 3 is a diagrammatic side view of the arrangement of Figure la when aligned vertically, showing the separation of phases into bands when subjected to an axial ultrasound field;
• FIGURE 4 is a diagrammatic side view of the arrangement of Figure la to show the degree of separation of the mixture into two phases at successive times of a) 0 in, b) l min, c) 2 min and d) 3 min;
• FIGURE 5 is a diagrammatic side view of the arrangement of Figure la when the tubular container is positioned horizontally, showing a) banding of the phases after 30s and b) complete separation of the phases after 90s;
• FIGURE 6a is an end view of the annular transducer of Figure lc, showing formation of annular zones under the influence of a radial ultrasonic field produced by the transducer; and
• FIGURE 6b is a similar view of the annular transducer when positioned with its axis horizontally, at complete phase separation.
• Figure 1 shows schematically three different arrangements which we have used by way of examples for carrying out methods in accordance with this invention.
• a standing wave field is generated in a cylindrical container C using a single transducer T and a reflector R at its opposite ends:
• Figure lb shows the use of two transducers T at the opposite ends of the container C.
• the or each transducer is driven by an amplifier A (Model A150,ENI, Rochester N.Y.), the input for which is provided by a Hewlett Packard 3326A two- channel frequency synthesizer S.
• the transducers T are 40 mm diameter air-backed piezoceramic discs, driven at their fundamental thickness resonance of 1 MHz.
• Ultrasound was generated from a 32 mm internal diameter, 12 mm long tubular air-backed piezoceramic transducer T with a 665 kHz radial thickness resonance.
• the transducer T was enclosed between two discs of perspex having ports made from shortened (3 mm) 19 gauge syringe needles which did not protrude into the sound field: the transducer T and these two discs formed the container for the mixture.
• Cells were incubated overnight at 30°C with shaking at 2Hz.
• Cells were harvested and washed twice in 50 ml quantities of 10 mM Tris.HCl, pH 7.6 followed by fixation in 3% glutaraldehyde for lh.
• Cells were washed in 50 ml of 10 mM Tris.HCl, pH 7.6 and stored in the same buffer.
• Yeast cells were fixed in glutaraldehyde solely to ensure a ready supply of cells.
• phase systems were produced by mixing equal volumes of 10% (w/v) 500 kD dextran (Pharmacia) and 10% 8 kD (w/v) PEG (Sigma Ltd.) to give a final concentration of 5% (w/v) for each polymer. Where yeast or bacteria were used, these were added at approximate concentrations of 10 7 cells ml "1 . 1.0 ml of cell suspension was added to 10 ml of the phase system, resulting in a modified polymer concentration of 4.55% (w/v) for each polymer.
• a standing wave ultrasonic field was applied to freshly mixed samples of the phase system contained in an acoustic chamber C in the arrangement illustrated in Figure la.
• Alternate layers of a dextran-rich and a PEG-rich solution were formed in less than 1 min at transducer voltages in excess of 2 Vp-p and 3 Vp-p for 4 ml and 10 ml containers respectively.
• the layers formed within 3 s at voltages of twice the above threshold values. If these supra-threshold voltages were maintained on the transducer, layers of the separate phases could be maintained for long time periods (> 1 hr) . Settling of some (10-20%) of the heavier phase could occur over a 1 hour time period. When the power was turned off, the heavier dextran layers settled under gravity.
• Figure la We further used the arrangements of Figure la for the phase partition of yeasts and bacteria.
• Saccharomyces cerevisiae partitions into the dextran phase
• Escherichia coli partitions into the PEG phase.
• characteristic bands of PEG-enriched and dextran- enriched layers were formed.
• organisms were also present in the cylindrical acoustic chamber, they appeared in the appropriate bands P or D as shown in Figure 3.
• S . cerevisiae when the voltage was turned off the yeast cells contained in bands and drops of dextran sedimented to the bottom of the acoustic chamber.
• E . coli which partitions into the PEG enriched phase, separated towards the top of the vessel.
• Figure lc For exposing the PEG/dextran system containing S . cerevisiae to a radial ultrasonic field.
• annular transducers generally the pressure maxima and minima produce tubular zones of the two phases rather than bands, as shown in Figure 6a.
• the annular transducer was mounted in a horizontal position (axis vertical) so that the rings of dextran containing S . cerevisiae would drop to the bottom of the vessel when the sound was turned off, with a much reduced remixing of the two phases.
• timings for separation were based on microscopic examination of two samples taken 1 mm from the top and 1 mm from the bottom of the vessel respectively (samples were removed with a syringe attached to a micromanipulator) . Separation was assumed to be complete when the top sample was essentially devoid of cells and the concentration of cells in the bottom phase reached a maximal value. When we exposed the phase system to two 30 s pulses of ultrasound separated by 15 s, separation appeared to be complete within the total period of 90 s.
• the times required for ultrasonic separation of two continuous bulk phases are compared in Figure 2 with the times required for the separation of continuous phases under gravity.
• the times for phase separation (4.55% w/v PEG, dextran system containing cells) under the faster sedimentation rates obtainable in a bench centrifuge (10 ml sample in a test tube exposed to 300 g) were also measured (Table I) .
• the Table also contains the measured partition "yields" of cells in the preferred phase for centrifugation-enhanced and for ultrasound- enhanced systems. The data shows that ultrasound compares well with centrifugation both in terms of timing and of partition "yield".
• the times for partition of E . coli into continuous bulk phases were as the times for S . cerevisiae separation.
• the partition "yields" for E . coli were, on average, 1.6% (+0.25% 95% confidence limits) lower than those for yeast (Table 1) .
• the transducer voltages for the different systems were in the range 2.2 to 2.6 times the threshold voltage for layer formation in 1 min.
• the yields were calculated from the percentage of cells in the PEG phase (based on bacterial counts of 10,000 and yeast counts in the range 10-200) . There was good agreement between triplicated counts. Each experimental arrangement was tested twice. While the bacterial partition "yield” was consistently lower than the yeast yield, the mean of the four values (in the last column) gives summary guidance to the performance of the different systems.
• the radiation force F r exerted by a plane standing wave of peak pressure P 0 and wave number k on a droplet of volume V, density p * and compressibility ⁇ * suspended in a fluid of density p and compressibility ⁇ is given by:
• dextran (yeast containing) phase acts as a dispersed phase in a continuous PEG enriched phase.
• the rapid ( ⁇ 3 s) development of layers of each phase suggests that dextran droplets, growing by coalescence due to inter-droplet acoustic forces (Eqn. 2) , migrate (Eqn. 1) to regions of the standing wave field where droplet concentration occurs and the probability of coalescence therefore increases.
• Table 1 shows that the completion time (ca 70 s in the tubular transducer) for bulk phase separation of S . cerevisiae by ultrasound is faster than that (3.5 min) achieved by centrifugation of the phase systems. Differences between the mechanisms of development of two final bulk phases by ultrasound and by centrifugation include that while the "g" forces exerted by the sound field are generally lower than those achievable by centrifugation, the distances which droplets need to move before forming a layer in a sound field are very short (a quarter wavelength is 0.37 mm at 1.0 MHz) compared with the length of a centrifuge tube. Additionally there is no centrifugation equivalent of the acoustic interdroplet force described by Eqn. 2. __ ____
• the system which offers particularly rapid sedimentation and a simple means of recovering the phase appears to be a tubular transducer with its axis horizontal (Fig 6) .
• aqueous two-phase partition has been employed to fractionate biological preparations through repeated phase separations

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Cited By (9)

Citations (5)

• 1992
  • 1992-06-22 GB GB929213198A patent/GB9213198D0/en active Pending
• 1993
  • 1993-06-22 AU AU43496/93A patent/AU4349693A/en not_active Abandoned
  • 1993-06-22 WO PCT/GB1993/001315 patent/WO1994000757A1/en active Application Filing

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