CA2037988A1 - Continuous flow method and apparatus for separating substances in solution - Google Patents

Continuous flow method and apparatus for separating substances in solution

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Publication number
CA2037988A1
CA2037988A1 CA 2037988 CA2037988A CA2037988A1 CA 2037988 A1 CA2037988 A1 CA 2037988A1 CA 2037988 CA2037988 CA 2037988 CA 2037988 A CA2037988 A CA 2037988A CA 2037988 A1 CA2037988 A1 CA 2037988A1
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CA
Canada
Prior art keywords
container
cell
solution
continual
fluid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA 2037988
Other languages
French (fr)
Inventor
Otto Sova
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
908098 ONTARIO Inc OPERATING AS MH ENTERPRISE
Original Assignee
908098 ONTARIO INC. OPERATING AS MH ENTERPRISE
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Filing date
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Priority to CA 2037988 priority Critical patent/CA2037988A1/en
Publication of CA2037988A1 publication Critical patent/CA2037988A1/en
Application status is Abandoned legal-status Critical

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Abstract

ABSTRACT

Provided is a continual flow cell for separating materials using electrophoresis. The cell includes a solution inlet container in flow communication with a vertical flow through chamber having a plurality of perforated, vertical partitions positioned therein. The perforations are aligned to give horizontal flow paths between two electrodes located at either end of the chamber. The perforated panels are dimensioned to produce vertical flow passages therebetween. A bias potential is applied between the electrodes which produces an electric field horizontally across the cell which acts to drive charged species horizontally. Outflow ports located horizontally across the top of the flow through chamber provide for outflow of peripheral components which may then be collected or recycled back to the inflow of another cell. The cell may also be utilized to separate materials by isoelectric focusing wherein a pH gradient is established by low molecular weight impurities associated with the material.

Description

The present invention relates generally to the field of separating substances by the technique of electromigration.
The movement of a charged particle in an electric field, known as electrophoresis, forms the basis of several separation techniques. In one such technique, electromigration, oppositely charged species such as inorganic ions migrate under the influence of an electric field toward spaced electrodes of opposite sign to that of the charged species. The velocity of migration depends on the mobility of the species in the electrolyte as well as the electric field strength.
In a related technique, isoelectric focusing, zwitterionic or amphoteric molecules, i.e. molecules possessing both positive and negative charges depending on the solution pH, migrate under the influence of an electric field in a solution containing a pH gradlent. The charged molecules are driven or focused to a point in the cell having a pH corresponding to the pI of the molecule. The pI of a molecule is the pH at which the overall charge on the molecule is zero. At the pI of the molecule, the neutralized molecule is immobile with respect to the electric field and thus remains stationary in the absence of convection currents. In isoelectric focusing the pH
gradient is usually established using buffering systems composed of synthetic, low molecular weight amphoteric materials (polyampholytes) having a wide spread of pI

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values to give a uniform pH gradient. The use of commercial polyamphol~tes for establishing a uniform pH
gradient is well known. A drawback to this method of establishing the pH gradient is the expense of the buffering solutions which places economic limits on the amount of material which can be separated.
Electromigration may be carried out in either a static mode or a continuous flow mode. In the static mode a solution of fixed volume containing the material to be separated is subjected to an electric field wherein oppositely charged components migrate to opposite ends of the cell in electromigration or alternatively are focused in a pH gradient to a point in the cell having a pH
corresponding to the pI of that component. An obvious disadvantage of the static mode is that only a fixed volume of material can be separated and must be removed from the cell in a separate process. In the continuoùs flow mode a solution containing the various components to be separated is continuously fed through an electrochemical cell having an electric field applied thereacross along with a pH
gradient.
In addition to the expense and need for continual replenishment of the polyampholyte solutions, continuous flow cells currently used for large scale separations of amphoteric materials employ expensive multi-channel focusing cells, heat exchangers and multi-channel pumping systems all of which require periodic maintenance and repair. As well, in order to suppress bulk fluid between .
' adjacent channels in the direction in which t~e pH gradient is established, ion non-selective permeable membranes are utilized to separate the adjacent channels. These membranes allow for interchange of fluid constituents between adjacent channels while acting to suppress convection currents. A drawback to this type of arrangement is the fact that over time the membranes will become clogged thereby requiring periodic replacing. Accordingly, there is a need to provide a continuous flow cell for isoelectric focusing which does not require the use of expensive synthetic polyamopholytes, fluid pumping systems and which is constructed of components requiring minimal maintenance.
The present invention provides a method and device for separating large quantities of organic and inorganic materials which overcomes the disadvantages of the prior art.
The subject invention provides a continual flow cell apparatus for separating materials in an electrically conductive fluid, which apparatus includes a first non-conductive fluid container having a solution inflow port.
There is included a second non-conductive fluid container having two or more fluid outflow ports and a passageway for ; gravity flow of the solution from the first container into the bottom of the second container. A pair of spaced electrodes are located in the second container and are ; adapted to be coupled to an external power supply. The electrodes are located so as to contact the fluid flowing , ~
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through the second container. A plurality of adjacent partition members are located in the second container and extend thereacross. The partition members provide substantially convection free fluid and current flow pathways in the horizontal direction and are in a spaced apart relationship in order to provide vertical fluid flow paths therebetween.
In another aspect of the invention a gravity fed, continual flow cell apparatus for separating materials in an electrically conductive fluid includes a first non-conductive fluid container having a fluid inflow port. A
second non-conductive fluid container is provided having a bottom panel, a pair of end walls, a front wall and a back wall and includes two or more fluid outflow ports located on the front wall spaced from the upper edge thereof. The containers are in flow communication through a flow passageway extending between the bottoms thereof. A pair of spaced electrodes are located in the second container, each electrode located at a respective end of said container and extending substantially vertically in the compartment a distance e~ual to most of the height of the container. The electrodes are adapted to be coupled to an external power supply. A plurality of non-conducting, adjacent partition members provided with a plurality of holes therethrough are vertically disposed in the second container between the front and back walls. The partitions extend between the end walls for providing substantially convection free horizontal fluid and current flow paths , therebetween. The partition members are in a spaced apart relationship such that vertical Eluid flow paths are established in the second container from the top to the bottom of the container.
In still another aspect of the invention, a source of liquid containing a material to be separated is provided wherein the solution has a conductivity in the range from 500 to 2000 uS/cm and which contains no carrier ampholytes.
A fluid container is included having two or more fluid outflow port means. A solution inlet means is provided for the container which is adapted to allow gravity flow of the solution from the liquid source into the container. A pair o~ spaced electrodes are located in the container and are adapted to be coupled to an external power supply. A
plurality of adjacent partition members are located in the container and extend thereacross. The partition members provide substantially convection free fluid and current flow pathways in the horizontal direction and are in a spaced apart relationship in order to provide vertical fluid flow paths therebetween.
In yet another aspect of the invention, a method of separating materials in a liquid includes providing a liquid solution containing the substance to be separated, the solution having a conductivity in the range 500-4000 uS/cm. The solution is flowed into a chemically inert non-conducting electrochemical cell container through a bottom opening in said container from a non-conducting storage container in flow communication therewith. The solution is .:

, flowed vertically upwards through a plurality of substantially vertical flow paths formed between a pair of spaced electrodes. A bias voltage is impressed between the - electrodes for causing separation of the charged components substantially horizontally across the cell container. The separated components are collected as they exit through outflow ports located near the top of the cell container.
In this same aspect of the invention, the material to be separated comprises at least one zwitterionic species characterized by a pI value, wherein said species has amphoteric impurities associated therewith, wherein a pH
gradient is formed across the second container by said impurities when the bias potential is applied thereacross.
The zwitterionic species is driven in ~he pH gradient to a point having a pH corresponding to the pI of said species whereupon the material is neutralized and flows vertically therefrom to an outflow port.
In still this same aspect of the invention the material to be separated comprises non-amphoteric components, wherein applying a bias potential between the slectrodes causes oppositely charged components to be driven to oppositely charged electrodes thereby depleting the liquid in substantially the central portion of the electrochemical cell container of said charged components, and wherein the liquid from said central portion is collected at outflow ports located in substantially the central portion of the cell compartment.

The invention will now be described, by way of example only, with reference to the accompanying drawings, in which:
Figure 1 is a perspective view of the continual flow cell embodying the subject invention;
Figure 2 is a vertical front view of the cell;
Figure 3 is a vertical sectional side view of the cell taken along the line 3-3 of Figure 2;
Figure 4 is a plan view of the continual flow cell;
Figure 5 is a vertical side view of a partition member utilized in the continual flow cell;
Figure 6 is a plan view of the continual flow cell showing two partition members permanently mounted therein;
Figure 7 is a plot of the steady state solution conductivity across the width of the continual flow cell;
Figure 8 is a schematic illustration of the system for employing the continual flow cell of the invention; and Figure 9 is a schematic perspective view of another embodiment of a continual flow cell in accordance with the invention.
Reference will now be made to the drawings wherein ` like parts are designated with like numerals. Referring ; first to Figures 1, 2, 3 and 8, a continual flow cell 20 of the present invention is shown wherein cell 20 comprises a generally rectangular electrochemical cell con~ainer 22 having end walls or panels 24, a bottom panel 26, a front wall 28 and a back wall 30 thereby defining an electrochemical cell chamber 32. Cell 20 is provided with :. :
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a container shown generally at 33 comprising a rectangular C-shaped member 34 vertically attached to the outer central portion of back wall 30 and attached to bottom panel 26 thereby enclosing a chamber 36. A portion of back wall 30 adjacent to member 34 enclosing chambe~ 36 extends vertically upwards above the top edge of front wall 28 and vertically downwardly to terminate above bottom panel 26.
A horizontal gap 38 is formed between the bottom edge of back wall 30 and bottom panel 26. The width of panel 26, W2, is grea~er than the width of container 22, W1, for maintaining stability of cell 20, see Figure 2. Chamber 36 is open at the top thereby defining a fluid inlet port 40.
The connection of flange 34 to back wall 30 and panel 26 is a watertight seal. Preferably ~here is a central opening 37 at the top of member 34 for introduction of a feedpipe.
As illustrated in Figure 8, the separation system of the invention employs a source 18 of li~uid solution containing a solution to be separated. This solution preferably has a conductivity in the range from 500 to 2000 uS/cm and, unlike many earlier systems, it contains no carrier ampholytes. The drip flow from source 18 can be regulated by a suitable valve lQ. This flow enters inlet chamber 36 which forms inlet means for container 22.
Chamber 32 is provided with a plurality of outflow ports 42 located along and spaced below the upper edge of front wall 28. Ports 42 are inclined slightly downwardly from the horizontal. Generally, the number of outflow ports 42 utilized in cell 20 will depend on the nature of the . ~

separation process being used, as will be discussed below.
The separated liquid collected from each of the ports 42 is collected in containers 45, one of which is shown in Figure 8, or by other suitable means. The outflow drips into these containers so there is no electrical current loss to the contents of these containers.
Cell 20 includes a pair of levelling screws 44 located on the outer ends of bottom panel 26, wherein screws 44 are threadably insertable therethrough for adjusting the horizontal level of outflow ports 42 located on front wall 28.
Cell 20 is provided with a pair of spaced electrodes 46 located adjacent end walls 24 in compartment 32. Electrodes 46 extend from substantially the bottom of compartment 32 to the top of same and are mounted in such a way as to be easily inserted and removed such as by a plurality of retaining brackets 48. Electrodes 46, which form an anode and a cathode, terminate in electrical connectors or plugs 50 situated at the tops of end walls 24 in order to facilitate coupling electrodes 46 to an external power supply, shown generally as 52 in Figure 2.
Electrodes 46 are preferably chemically inert in the solution of interest as well as being stable against cathodic and anodic dissolution in the same solution under applied bias conditions. Platinum, carbon and nickel have been found to perform satisfactorily.
Referring to Figures 4 and 5, cell 20 is provided with a plurality of liquid permeable, vertical partition :
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members 54 which extend vertically between the bottom of compartment 32 and a point just below the level of outflow ports 42, and extend horizontally between front wall 28 and back wall 30. Partitions 54 and 54' include a plurality of holes 58 having the same diameter D and which are une~ually spaced along the vertical dimension thereof. The spacing between adjacent holes 58 increases from the bottom to the top of partitions 54. Two vertical partition members, shown at 54', are of substantially the same height as compartment 32. The purpose of partitions 54' will be discussed below.
Perforated panels 54 (54') perform several roles.
First, holes 58 in partitions 54 and 54' are bifunctional, one function being to provide horizontal fluid flow channels across electrochemical chamber 32. The more closely spaced holes at the bottom of the panels provides for rapid horizontal displacement of the liquid as it enters chamber 32 from reservoir 36 through gap 38. The second function of holes 58 is to provide pathways for the formation of a plurality of horizontal ionic current flow paths between electrodes 46 when a voltage is impressed therebetween, illustrated by the broken lines shown at 60 in Figure 4. For this reason the holes should preferably extend to the top of partitions 54 in order to allow separation along substantially the full vertical length of partitions 54. Secondly, the rectangular C-shape of partitions 54 facilitates the formation of vertical flow channels 62 in chamber 32 which allow for continuous laminar fluid flow upwards from the bottom to the top of cell 20. Finally, partitions 54 and 54' provide for turbulence and convection suppression horizontally across compartment 32 between electrodes 46.
Referring to Figure 6, cell 80 has the two partitions at 54' sealed along their outer edges to wall 28' and back wall 30' for a distance of approxima-tely 25%
of the height of the cell from the top of cell 20 to form a liquid impermeable seal. Permanently installing the partitions 5~' in this way forms three sub-compartments 84, 86 and 88, wherein outer sub-compartments 84 and 88 are each approximately 25% of the total volume of compartment - 32' while middle sub-compartment 86 is approximately 50% of the total volume. Each of sub-compartm~nts 84, 86 and 88 are provided with a plurality of partitions 54 (Figure 1) and an outflow port 42'.
In operation, a mixture containing the material to be separated is dissolved in purified distilled water and the concentration of the starting material preferably maintained in the range suitable to give a solution with a conductivity in the range of approximately 500-3000 uS/cm.
Cell 20 is then filled with this solution by pouring the solution into inlet port 40 whereupon the solution fills up chamber 32 through gap 38. In this way the solution is circulated between the first and second compartment using gravity. When chamber 32 is filled to a level just below outflow ports 42, the inflow of solution is terminated and levelling screws 44 adjusted so that outflow ports 42 are :

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all horizontally aligned. Electrodes 46 are then connected to external power supply 52 and a direct current (DC) potential drop applied between electrodes 4~ and the solution allowed to sit for 10-15 minutes. After this time, the flow of solution into reservoir 3~ is re commenced, The flow of solution into compartment 36 is drop by drop since a current flow is present between electrodes 46. The solution level in compartment 32 increases until it reaches the outflow ports 42 whereupon it flows downwardly into storage containers, or alternatively it may be recycled back to the first compartment.
There are two distinct but related separation processes in which the continual flow cell of the present invention may be utilized. The first process is separation based on autofocusing wherein a material containing the substance to be separated is dissolved in a solvent, normally purified water. The concentration of the starting material is high enough to give on the one hand a stable pH
gradient in addition to a solution conductivity in the vicinity of 800-2000 uS/cm while on the other hand being low enough to avoid precipitation of the material. Low molecular weight impurities contained within the starting material automatically form a pH gradient horizontally across the cell when a potential drop is applied thereacross, while the various zwitterionic components comprising the starting material are driven or focused to a position in the cell having a pH corresponding to the pI

of that component. The low molecular weight impurities, when present in sufficient quan~ities, functionally take the place of the low molecular weight polyampholytes employed in classical isoelectric focusing for pH gradient formation.
Once the species being separated is neutralized at its pI, it becomes immobile in the electric field in the horizontal direction while still flowing vertically upwards in flow channels 62 to the top of cell 20. Once reaching the top of cell 20 the neutralized material exits cell 20 via outflow ports g2 where it may be collected and isolated from the solvent component, or alternatively, it may be redirected into reservoir chamber 36 of another flow cell placed in series with the first flow cell. The number of outflow ports 42 used on cell 20 will depend on the number of components being separated. When separating a large number of components with differing pI's, using a large number of outflows results in a greater pH resolution of the flow cell. It will be appreciated that cell 20 may be constructed with a large number of outflow ports 42 which may have sliding members mounted on the interior of front wall 28 for opening and closing the outflow passageway through ports 42. In this way the same cell may be efficiently utilized for autofocusing solutions containing an arbitrary number of zwitterionic components.
The second separation process for which the continual flow cell of the present in~ention may be employed is straight using electromigration. In this - ~, . ~'' ' ' : . .

process oppositely charged species are driven by the field to opposite ends of the cell since in general no pH
gradient will be present. Cell 20 would be a suitable cell for electromigration wherein the purified solution component may be collected from the middle outflow port 42, It will be appreciated that other modifications to cell 20 may be required when straight electromigration is being used. Specifically, the charged species being driven to the oppositely charged electrodes may in general undergo electrochemical oxidation or reduction which may produce either precipitates or gaseous components. Depending on the nature of the gaseous components, special collection and storing arrangements may be required.
The cell material used in the construction of the continual flow cells of the present invention will depend on the nature of the material being separated. For continual flow autofocusing applications in the separation of biological materials such as proteins, enzymes and the like, various plastics such as acrylate, or Teflon may be used. For applications with more stringent material requirements such as the purification of organic solvents or the desalination of salt water, the cell components would preferably be fabricated from glass, ceramic or again Teflon .
Typical cell volumes range from approximately 0.50 litres to several litres. The liquid flow rates are dependent on the cell volume, with smaller cell volumes having lower flow rates and conversely higher cell volumes requiring lower flow rates. Normally, the solution flow rate through the cell is typically maintained at a tenth of the cell volume per minute. This interrelation between the solution flow rate and the cell volume holds for cells in which the volume is increased by increasing the cell hei~ht while maintaining the cell width constant and arises due to the time re~uired for migration of charged species in the electric field in the horizontal direction.
For many applications it has been found that to achieve acceptable flow rates with good separation of components, the cell length can be in the range of ~0-50 cm. Increasing the vertical height of the cell therefor permits higher flow rates to be achieved. It will be understood that it may be desirable when utilizing continual flow cell 20 for autofocusing applications to increase the horizontal length of cell 20. Specifically, in applications wherein the pI of the components being separated are very close and it is necessary to increase the resolution of the cell with respect to pH, the hori20ntal length of the cell must be increased. Of course, by increasing this dimension, a longer time for migration of charged species horizontally will result thereby necessitating a lower flow rate. In this case the relation between cell volume and flow rate is reversed to that described above, increasing the cell volume by increasing the cell length is accompanied by a reduction in solution flow rate.
Another factor determining the upper limit on the .~
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solution flow rate through flow cell 20 in autofocusing applications is the establishment and maintenance of a stable pH gradient across the cell. When the amphoteric impurities associated with the material being separated have a much lower molecular weight than the latter, the pH
gradient will be established in a time shorter than the transit time of the species being separated due to a higher mobility of the former over the latter. In this situation the optimum flow rate is such that the migration time across the cell is shorter than the residence time in the cell as discussed above. Where the mobility of the amphoteric impurities is comparable to the mobility of the species being separated, the flow rate must be maintained low enough to permit establishment of a stable pH gradient.
Note that this is not a consideration when separating materials using straight electromigration.
For a cell volume of 800 cc and a maximum cell width of 50 cm, a typical fluid flow rate is approximately 0.08 litres/minute or 5 litres/hour. Flow cells having a volume of the order of 1 litre have solution flow through capacities of approximately 150 litres/day. The flow cells of the prasent invention only utilize electrical energy for establishing an electric field across the cell i.e. no energy is consumed for driving pumping systems or heat exchangers. The continual flow cells of the present invention may therefore be run for days at a time unattended as there are no electromechanical or other devices requiring operator attention.

The power which results in optimum operating characteristics, i.e. most rapid separation time with minimum ohmic heating has been determined to lie in the range 3-30 Watts for continual flow autofocusing applications using a cell having a volume in the range of 0.50 litres up to several litres. The upper limit used in a particular situation will usually be determined by trial and error, being dependent on the efficacy of the material to undergo denaturization under potential biasing conditions. Thus while 30 Watts is an acceptable upper limit for separation of proteins, enzymes and the like, it may be too high for separating foodstuffs such as liquids used in the food industry where undesirable changes in taste and/or chemical composition may result. In such cases a lower power would be employed and the solution flow rate adjusted to compensate.
Higher power levêls can be utiliæed for separations using straight electromigration, such as removing inorganic ions from solvents where chemical denaturization is not an issue. Power levels ranging from 30 to 100 Watts have been found to be quite acceptable for these applications.
Figure 7 illustrates the steady state conductivity profile across continual flow cell 20 for separations using both straight electromigration and autofocusing and shows that the conductivity is a minimum in the middle portion of the cell. This parabolic conductivity curve shows that the steady state conductivity in the central portion of the cell is a minimum and is higher in the outer compartments.

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In autofocusing applications this decrease in conductivitY
arises for two reasons, a decrease in charged amphoteric species being neutralized at the pI and charged non-amphoteric species such as inorganic ions being swept by electromigration to the elec~rodes of opposite sign at the ends of the cell. The achievement of low conductivity in the middle o~ the cell clearly illustrates the utility of flow cell 20 for the separation of charged impurities from neutral liquids such as water, alcohols, organic solvents and the like.
Another alternative embodiment of the continual flow cell of the subject invention is shown at 100 in Figure 9. Cell 100 is provided with a pair of ion selective, waterproof membranes 102 mounted therein which divide container 22' into three working spaces or subcompartments 84', 86' and 88'. Perforated partitions 55', similar to those shown at 54 in Figure 4 extend across subcompartments 84', 86' and 88' thereby creating a ` plurality of vertical flow passageways 62'. Cell 100 includes at least two or more solution drains 104 and associated valves 106 located along the bottom of cell 100.
A source of liquid solution (not shown) may have an outlet port located above cell 100 whereby cell 100 is filled from the top. The liquid input is preferably drop by drop.
Cell 100 is preferably used for desalination of water.
The continual flow cell of the present invention may be utilized in the separation and purification of many materials. Several non-limiting examples of the . :

' ~. - 19 --application of continual flow autofocusing and separation using continual flow electromigration will now presented.

Isolation of alpha-amylase Alpha-amylase is an enzyme utilized in the cleavage of starch and starch-like substances and is widely used in the food industry. The usage of alpha-amylase in beer production and baking products is quite significant. Saline precipitation is a widely known means of isolating alpha-amylase from malt for the beer industry. The isolation of alpha-amylase from bacterial sources is prohibited in many countries since these methods are not able to eliminate all the bacteria. A drawback to these methods, in addition to their individual unique problems, is the expense of the isolation process, environment damage and a high energy requirement.
These disadvantages of the above mentioned methods can largely be eliminated by employing the method and apparatus of the present invention. The alpha-amylase material from various biological sources, after filtration ; for elimination of insoluble particles, by diluting, ultrafiltration or dialysis is prepared so that its conductivity is no greater than 2 mS/cm, preferably about 1 mS/cm. This solution is continuously purified in a continual flow autofocusing cell using a DC power of 20 watts. The alpha-amylase fraction is collected from an outflow(s) located substantially in the central portion of , the flow cell in a pH range from 5-7. The purification process may be repeated by flowing the outflow of the first cell into a second, and subsequently more cells. If a very pure enzyme is required, the next step may involve purification using gel chromatography, or alternatively a combination of con~inual flow autofocusing alternated with gel chromatography may be employed until the required purity of enzyme is obtained.
In another e~pariment to isolate alpha-amylase, a li~uid bread from bacillus subtillis, an alpha-amylase producing strain, is subjected to centrifugation to remove the bacteria, after which the solution was mixed with water until a solution conductivity of 1.5 mS/cm was obtained.
This solution was introduced into a continual flow cell having the same dimensions and using the same parameters of flow rate and applied power. The outflow from the middle ports was introduced into a second continual flow cell for further isolation. The appropriate outflow of this cell was subjected to gel filtration on a Sepharon P-40 column.
The resulting second maior peak portion contained alpha-amylase with a 50 fold higher activity than the initial liquid had. The alpha~amylase so isolated can then be freeze dried and stored.

Purification of Insulin Insulin (manufactured by Nova Works, Denmark) was dissolved into 100 litres of tap water which had 3 M of urea dissolved therein to give a 3% insulin solution with a conductivity of 1 mS/cm. This solution was applied to a 2 litre volume con~inuous flow cell at a flow rate of 200 ml/minute with a DC power of 20 watts applied thereto. The cell was provided with five outflow ports uniformly spaced along the front face thereof and the fractions from the first and fifth outermost outflow ports were discarded.
The outflow from the fourth port was recycled back to the storage reservoir of the first cell while the outflow from the second and third ports relative to the electrode serving as the anode were collected and directed into the input of a second continual flow cell having a cell volume of 0.80 litres. A flow rate of 80.0 ml/minute was used and a power or 10 Watts applied to the cell. The second cell utilized five outflow ports and the outflow from the second and third ports were collected and the outflow from the remaining ports discarded. The collected fractions from the second cell were sub;ected to gel chromatography to separate the isoelectrically homogeneous insulin from the urea. The choice of urea was to provide the required solution conductivity while at the same time being unreactive towards the insulin. It will therefore be appreciated that any other compounds may be utilized in place of urea in so far as they are unreactive towards insulin, results in a solution conductivity in the desirable range and has a sufficiently different molecular weight than the insulin to facilitate separation therefrom after producing isoelectrically homogeneous insulin.

E:XAMPLE 3 Separation_of L-lysine The method and apparatus of the present invention can be used to prepare chemically pure L-lysine from biological sources.
The preparation of the amino acid L-lysine by microbial biosynthesis of corynebacteriacea is well known.
Using a bread source containing L-lysine and employing microorganisms, an impure solution containing the amino acid is obtained from ~hich the amino acid is isolated using saline precipitation. L-lysine isolated in this way can have a purity of up to 86% which may be increased up to 96% by using more complicated and expensive purification procedures.
These shortcomings may be essentially eliminated by use of this invention. After impure L-lysine has been obtained using biosynthesis, the microorganisms used for this process are removed by centrification. The resulting solution is filtered for removal of large particles. The ~0 solution is dissolved in water to give a solution conductivity up to 2 mS/cm, preferably about 1 mS/cm. This solution of no more than 2 mS/cm is introduced to a 1 litre continual flow cell at a flow rate of 100 ml/minute (or maximum rate of one tenth of the total volume of the cell per minute). A DC power of between 20-30 Watts is applied to the cell and the mixture subjected to autofocusing. The solution from the cell outflow ports in the pH range 9-11 contain the separated L-lysine which is then introduced into another continual flow cell and the procedure repeated until the desired purity of L-lysine is achieved. The solution obtained from the outflow ports in the pH range from 9-6 may also be refocused. If the initial cell has three outflows, which can be called a cathode outflow, a middle outflow and an anode outflow, the cathode outflow provides the partially purified L-lysine and the middle outflow is the li~uid that can be returned back to the raw liquid input. The anode outflow is discarded.
The advantages of the isolating L-lysine using the method and apparatus of this invention are the low expense for e~uipment, minimal process costs, energy efficiency and environmentally safe operations.

Isolation of Peroxidase 20 kg of horse radish was homogenized in 100 litres of tap water and the green particles removed. A 25 mM
solution of gluthathion was then added to the solution.
This solution was then subjected to centrifugation to remove large particles. The conductivity of the solution is adjusted if necessary, by dilution so that it does not exceed 2 mS/cm and preferably does not exceed 1 mS/cm.
Also, the contents of the proteins is adjusted if necessary so as not to exceed 30 grams/litre. The supernatant was then poured into the reservoir of a continual flow cell having an internal volume of 1 liter, and provided with 4 outflow ports equally spaced along the front wall of the . . .
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cell. Once the solution filled the electrochemical compartment the flow was stopped, a DC power of 15 Watts was applied and the solution allowed to sit for 15 minutes.
After this time the solution flow was recommenced at a rate of 0.10 litres/minute (one-tenth of the internal volume of the cell per minute). A power of 15 Watts was chosen since it resulted in minimum heat generation. The solution outflows from the two central outflow ports, whose outflows had a pH in the 5.5 to 6.6 range, were collected as these contained the separated peroxidase while the solution outflow from the outer two outflow ports was discarded.
The collec~ed solution was immediately applied to a second continual flow cell placed in series therewith, having an internal volume of 0.80 litres and provided with three outflow ports. A flow rate of 0.080 litres/minute was used and a DC power of 10 Watts applied to the cell. The solution from the middle outflow port was collected and while the outflow of the outer ports was discarded. If even higher purity is desired, gel chromatography is then used as the next step to separate the peroxidase from other molecular weight components.

Separation of Lysozyme Lysozyme is an enzyme which is present in a number of biological materials including egg whites. To date, lysozyme has been isolated using various methods including ion-exchange chromatography and saline precipitation to .
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- 25 - ~, i mention a few. These methods tend to be very expensive, energy inefficient while the latter method of saline precipitation is environmentally unsafe. The inventors have isolated lysozyme from egg whites using the continuous flow method and apparatus of the present invention.
Egg whites were dissolved in tap water and insoluble particles removed b~ filtration. The solution was diluted in tap water to achieve a solution conductivity no higher than 2 mS/cm and preferably around 1 mS/cm. This solution was then poured into the solution inflow container of a continual flow cell having an internal volume of 1 liter and provided with 4 outflow ports equally spaced along the front wall of the cell. Once the solution ~illed the electrochemical compartment the flow was stopped, a DC
power of 15 Watts was applied and the solution allowed to sit for 15 minutes. After this time the solution flow was recommenced at a rate of 0.10 litres/minute (one-tenth of the internal volume of the cell per minute). A power of 15 Watts was chosen since it resulted in minimum heat generation. The solution outflow from the outflow port adjacent the cathode (also termed the cathode outflow) whose outflow had a pH in the range from 8.5 to 11, was collected as this contained the lysozyme. The outflow from the port adjacent the cathode outflow was collected and routed back into the first cell inflow container while the outflow from the cathode outflow was introduced into the inflow container of a second continual flow cell placed in series with the first cell. The above process was repeated i ` ~ '` , '`

: :`
, using five continual flow cells in series. The solution of isolated lysozyme collected from the fifth cell had an activity of 3,000 U/mg. This solution was subjected to gel filtration using a Sephadex G 25 column (a Sepharon P 40 column has also been used in separate experiments) and operated using distilled water. The purified lysozyme is collected from the last fraction leaving the column due to the molecular weight of lysozyme (approximately 150,000 MW). The resulting isolated lysozyme had an activity of between 20,000 to 40,000 U/mg.

Water Desalination The second related process for which the continual flow autofocusing cell of the present invention may ~e utilized for is separation b~ straightforward electromigration. This process differs from the autofocusing application in that no pH gradient is required and is therefore applicable to separating inorganic ions and the like from liquids. Thus the ions, both anions and cations, are swept to the respective ends of the cell containing electrodes of opposite sign to the ions thereby leaving behind a region in the centre of the cell depleted of the charged impurities.
An array of several continual flow cells placed in series has been utilized for the desalination of salt water. In one experiment, a salt solution having a concentration of 15 grams/litre NaCl was made up and ::
.
, applied to a first continual flow cell with a volume of 0.80 litre at a flow rate of 0.080 litres/minute. A power of 50 watts was applied to the cell. The cell comPrised three outflow ports and therefore the most purified fraction was obtained from the central or middle compartment. The outflow from this compartment was then introduced into a second continual flow cell in series with the first cell while the outflow from the outer compartments was recirculated back to the solution reservoir of the original flow cell. The solution outflow from the central outflow port of the second cell was routed into a third flow cell placed in series therewith while the outflow of the outer two outflow ports was fed to another cell also placed in series with the second cell. Employing more cells in series results in higher purity water being obtained.
The continuous flow cell of the present invention may be r~adily adapted to forming a component part in a series of coupled, cascaded continual flow cells. In this arrangement, several flow cells would be stacked in a series configuration wherein the outflow from one cell containing the species being isolated is introduced into the inflow port of the next flow cell in the series. In this way, a solution more concentrated in the material being isolated is output from one cell and introduced into another cell to further concentrate and isolate it. In such a process, the output from the other outflows may either be recycled back to any of the preceding cells in '' ':

- ; '`, ~ ' :

the array or alternatively may be discarded.
The continual flow cell of the present invention may be utilized in conjunction with a static autofocusing cell apparatus similar to the apparatus disclosed in Applicant's copending Canadian Patent Application Serial No. filed on December 14, 1990. In such an arrangement, the partially separated component from one or more compartments of the static cell are drained therefrom at the end of the static autofocusing process and 1~ introduced into a continual flow cell for further separation.
While the present invention has been described and illustrated with respect to the preferred and alternative embodiments, it will be appreciated that numerous variations of these embodiments may be made without departing from the scope of the invention, which is defined in the appended claims.

- . :
.

Claims (40)

1. A continual fluid flow cell for separating materials in solution by electrophoresis comprising:
a) a first fluid container having means defining a solution inflow port;
b) a second fluid container having two or more fluid outflow port means;
c) a passageway for gravity flow of said solution from said first container into the bottom of said second container;
d) a pair of spaced electrodes located in the second container, said electrodes being adapted to be coupled to an external power supply and located so as to contact the fluid flowing through said second container;
e) a plurality of adjacent partition members located in the second container and extending thereacross, wherein the partition members are spaced for establishing a plurality of vertical, separated fluid flow paths therebetween; and f) means for establishing substantially convection free horizontal fluid and current flow paths through the partition members from one electrode to the other electrode.
2. The continual fluid flow cell according to Claim 1 wherein the first and the second containers are adjacent, a waterproof dividing barrier is disposed therebetween, and the dividing barrier has a horizontal bottom edge which is spaced above the bottom of the compartments for providing said passageway from the first container to the second container.
3. The continual fluid flow cell according to Claim 2 wherein the partition members are provided with a plurality of holes vertically spaced and aligned for providing fluid and current flow paths substantially horizontally across the cell.
4. The continual fluid flow cell according to Claim 3 including two or more outflow ports located on the second compartment and in flow communication therewith, wherein the outflow ports are vertically spaced the same distance from the top of the second compartment.
5. The continual fluid flow cell according to Claim 1,2 or 4 further comprising level adjustment means for levelling the cell.
6. The continual fluid flow cell according to Claim 1, 2 or 3 wherein the cell is fabricated from a chemically inert material.
7. The continual flow cell according to Claim 1, 2 or 3 wherein the electrodes are each located adjacent a respective end wall of the second container.
8. The continual flow cell according to any one of Claims 1 to 4 wherein the electrodes extend vertically a distance substantially equal to most of the height of the second container.
9. The continual flow cell according to any one of Claims 1 to 4 wherein the vertical height of the second container is greater than the width of the second container.
10. The continual flow cell according to Claim 1 wherein said cell is operative with a solution flowing therethrough and the solution flow rate is such that the migration time of charged material in an electric field established in said cell is less than the residence time of the charged material in the second compartment.
11. A gravity fed, continual fluid flow cell for separating materials in solution by electrophoresis comprising:
a) a first electrically non-conducting fluid container provided with a fluid inflow port;
b) a second non-conducting fluid container, the second container being generally rectangular and having a bottom panel, a pair of end walls, a front wall and a back wall, two or more horizontally spaced outflow ports located on the front wall spaced from the upper edge thereof;
c) a fluid flow passageway interconnecting the first and second containers, said passageway extending between bottom portions of said containers;
d) a pair of electrodes located in the second container, each electrode being located at a respective end of the second compartment and adjacent an end wall, wherein the electrodes extend substantially vertically in the second container a distance equal to most of the height of the container and are adapted to be coupled to an external power supply; and e) a plurality of parallel and spaced, electrically non-conducting partitions vertically disposed within said second container between said end walls for establishing a plurality of vertical fluid flow paths extending from substantially the bottom to the top of said container, said partition members having a plurality of holes therethrough for providing substantially convection free horizontal fluid and current flows paths therebetween.
12. The continual fluid flow cell according to Claim 11 wherein the first and the second containers are separated by the back wall of the second container, and wherein said back wall terminates at a bottom edge located above the bottom of the containers for providing a horizontal fluid flow passageway between the first and second containers.
13. The continual fluid flow cell according to Claim 12 further comprising level adjustment screws located on the bottom panel for levelling the cell.
14. The continual fluid flow cell according to Claim 12 wherein the cell is fabricated from a chemically inert material.
15. The continual fluid flow cell according to Claim 14 wherein the cell material is teflon.
16. The continual fluid flow cell according to Claim 14 wherein the cell material is acrylate.
17. The continual flow cell according to Claim 14 wherein the cell material is a ceramic material.
18. The continual flow cell according to Claim 14 wherein the cell material is made from glass.
19. The continual flow cell according to Claim 12 or 18 wherein at least two of said partitions are permanently mounted in the second container in spaced apart relationship for forming at least three subcompartments in the second container, and wherein each subcompartment is provided with one of said outflow ports.
20. A continual fluid flow cell for separating materials in solution by electrophoresis, comprising:
a) a source of liquid solution containing a material to be separated, the solution having a conductivity in the range from 500 to 2000 uS/cm and containing substantially no carrier ampholytes;
b) a fluid container having two or more fluid outflow port means;
c) solution inlet means for said container adapted to allow gravity flow of liquid solution from said source into said container;
d) a pair of spaced electrodes located in said container and adapted to be coupled to an external power supply;
e) a plurality of adjacent partition members located in the container and extending thereacross, wherein the partition members are spaced for establishing a plurality of vertical, separated fluid flow paths therebetween; and f) means for establishing substantially convection free horizontal fluid and current flow paths through the partition members from one electrode to the other electrode.
21. The continual fluid flow cell according to Claim 20 wherein said inlet means comprises another fluid container and the solution passing through said inlet means is introduced into the bottom of the first mentioned container.
22. The continual fluid flow cell according to Claim 20 wherein said solution has a conductivity in the range from 500 to 1000 uS/cm.
23. The continual fluid flow cell according to Claim 20, 21 or 22 wherein the partition members are provided with a plurality of holes vertically spaced and aligned for providing fluid and current flow paths substantially horizontally across the cell.
24. The continual fluid flow cell of Claim 20 including at least two waterproof ion-selective membranes mounted in said container and extending thereacross, said membranes dividing said container into at least three working spaces, wherein said container has a fluid outflow port means for each of said working spaces.
25. The continual fluid flow cell according to Claim 24 wherein said membranes are mounted symmetrically in said container on opposited sides of a central vertical plane midway between said electrodes.
26. The continual fluid flow cell according to Claim 24 or 25 wherein most of said liquid solution at said source is water.
27. The continual fluid flow cell according to Claim 24 or 25 wherein the partition members are provided with a plurality of holes vertically spaced and aligned for providing fluid and current flow paths substantially horizontally across the cell.
28. A method for separating materials having charged components in a continually flowing solution, comprising the steps of:
a) providing the liquid solution containing a material to be separated, the solution having a conductivity in the range from 500-4000 uS/cm;
b) flowing the solution into a chemically inert and electrically insulating electrochemical cell container through a bottom opening in said cell from a chemically inert and electrically insulating passageway in flow communication therewith, flowing the solution vertically upwards through a plurality of substantially convection free vertical flow paths formed between a pair of spaced electrodes;
c) impressing a bias voltage between the electrodes for causing separation of the charged components substantially horizontally across the cell container; and d) collecting the separated components as they exit through outflow ports located near the top of the cell container.
29. The method according to Claim 28 wherein the solution is fed into the storage container drop by drop, and wherein the separated solutions exit the electrochemical cell drop by drop.
30. The method according to Claim 29 wherein the solution inflow rate is maintained at a value such that the migration time of the charged components in the electric field is less than the residence time of the charge material in the electrochemical cell container.
31. The method according to Claims 28, 29 or 30 wherein the outflow from the outermost outflow ports is recirculated back to the passageway.
32. The method according to Claims 28, 29 or 30 wherein the material being separated comprises a zwitterionic species dissolved in a liquid, said species characterized by a particular pI value, said material further comprises amphoteric impurities suitable for forming a natural pH
gradient substantially horizontally across the cell container, the zwitterionic species is driven to a point in the pH gradient corresponding to the pI of the species, and said zwitterionic species is neutralized at said pI and flows substantially vertically upwards therefrom to one of said outflow ports.
33. The method according to Claim 30 wherein the material being separated comprises a plurality of zwitterionic species, each of said species having a pI

value, and further comprises amphoteric impurities suitable for forming a natural pH gradient substantially horizontally across the cell container, said species are driven each to a respective position in the pH gradient corresponding to the pI of each species, and each species is neutralized at said pI and flows substantially therefrom each to a separate one of said outflow ports.
34. The method according to Claim 33 wherein the material to be separated is insulin, the liquid solution comprises insulin, and the solution has a conductivity in the range 500-2000 uS/cm.
35. The method according to Claim 33 wherein the substance to be separated is peroxidase, the liquid solution comprises peroxidase, and the solution has a conductivity in the range 500-2000 uS/cm.
36. The method according to Claim 33 wherein the substance to be separated is alpha-amylase, the liquid solution comprises alpha-amylase, and the solution has a conductivity in the range 500-2000 uS/cm.
37. The method according to Claim 28 wherein the material to be separated comprises non-amphoteric charged components, at least one component being positively charged and at least one component being negatively charged, said charged components being dissolved in a neutral liquid, said bias potential between the electrodes causes the charged components to be driven to an oppositely charged electrode of said pair of electrodes thereby depleting the liquid in substantially the central portion of the electrochemical cell container of charged components, and the liquid from said central portion flows upwardly to one of said outflow ports to exit therefrom.
38. The method according to Claim 37 wherein the liquid outflow from at least one of the outflows positioned between the outermost outflows is routed into an inlet of the electrochemical cell container.
39. The method according to Claims 28, 29 or 30 wherein the solution includes water containing metal ion impurities.
40. The method according to Claim 37 further comprising a plurality of electrochemical cell containers coupled in a series combination, wherein the outflow from the central portion of each cell container in said series, except the last cell container, is coupled to an inlet of a cell container which is next in the series combination, the liquid solution is sea water, the power applied to each cell container is at least 20 Watts, and the outflow from central outflow ports of the last cell in the series combination is retained for use as fresh water.
CA 2037988 1991-03-11 1991-03-11 Continuous flow method and apparatus for separating substances in solution Abandoned CA2037988A1 (en)

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Application Number Priority Date Filing Date Title
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Application Number Priority Date Filing Date Title
CA 2037988 CA2037988A1 (en) 1991-03-11 1991-03-11 Continuous flow method and apparatus for separating substances in solution

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US9744617B2 (en) 2014-01-31 2017-08-29 Lockheed Martin Corporation Methods for perforating multi-layer graphene through ion bombardment
US9833748B2 (en) 2010-08-25 2017-12-05 Lockheed Martin Corporation Perforated graphene deionization or desalination
US9834809B2 (en) 2014-02-28 2017-12-05 Lockheed Martin Corporation Syringe for obtaining nano-sized materials for selective assays and related methods of use
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US9870895B2 (en) 2014-01-31 2018-01-16 Lockheed Martin Corporation Methods for perforating two-dimensional materials using a broad ion field
US10005038B2 (en) 2014-09-02 2018-06-26 Lockheed Martin Corporation Hemodialysis and hemofiltration membranes based upon a two-dimensional membrane material and methods employing same
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