US 3829370 A
Description (OCR text may contain errors)
Allg. 13, 1974 G, BOURAT METHOD FOR FORCED FLOW ELECTROPHORESIS Filed March 28, 1972 4 Sheets-Sheet 1 AU8- 13, 1974 G. BouRA'r METHOD FOR FORCED FLOW ELECTROPHORESIS Filed Ilarch 28, 1972 4 Sheets-Sheet 2 NN vd..-
Allg. 13, 1914 C.;- BOURAT 3,829,370
mamon Fon FORCED FLow ELECTROPHORESIS Filed Ilarch 28, 1972 4 Sheets-Sheet 3 Aug. 13, 1974 Gh BQURAT 3,829,370
METHOD Fon FORCED FLow ELECTROPHORESIS Filed March 28, 1972 4 Sheets-Sheet 4 United States Patent O 3,829,370 METHOD FOR FORCED FLOW ELECTROPHORESIS Guy Bourat, Bourg-La-Rcine, France, assignor to Rhone-Poulenc S.A., Paris, France Filed Mar. 28, 1972, Ser. No. 238,764
Claims priority, application France, Mar. 30, 1971,
7111180 Int. Cl. B01d 13/02 U.S. Cl. 204-180 P 8 Claims ABSTRACT OF THE DISCLOSURE A continuous forced fiow electrophoresis cell and a method of operation for the fractionation of an aqueous liquid, such as blood, containing at least two compounds, the relative mobilities of which in an electric field vary as a function of the pH, in order to obtain one fraction enriched and one depleted in one of the compounds, the cell having six compartments divided by ion permeable membranes, the end cell containing an anode and cathode respectively, the central cells being separated' by a microporous membrane. The liquid is fed to one of the central cells the filtered fraction being removed, after passage through the microporous membrane, from the other. A main electrolyte is fed to and from the end cells and an auxiliary electrolyte to the intermediate cells such that the pH in one intermediate cell differs from that of the other.
The present invention relates to a method and to an apparatus for forced ow electrophoresis.
Forced flow electrophoresis allows the separation, in an aqueous medium, of compounds which are mobile in an electric field. It is known that the separating power of an electrophoresis or electrolysis cell can be improved by dividing the cell by means of a filtering component (made of non-conducting material) transversely to the electric field, the filtering component having a porosity chosen to allow the compounds (ions, micelles or molecules) which are mobile in the electric field to pass at least in one direction but to check the passage of these compounds in the reverse direction under the effect of simple diffusion and natural movements of the treated liquid.
In electrophoresis, it has been found that it is advantageous to superimpose, on the electric force produced by the potential at the cell terminals, a hydrodynamic force which is a function of the viscosity of the liquid subjected to electrophoresis and of its direction of fiow (forced ow).
Under these conditions, the separating power is a function of the electric charge of the compounds to be separated, but can be made independent of their molecular weight.
Various improvements in this technique have been proposed. For example, Bier (U.S. Pat. 3,079,318 and later Trans. A.S.A.I.O. XVI (1970), 325-334) proposes especially:
The immersion of the electrodes in an independent electrolyte, separated from the liquid to be treated by a dialysing membrane;
The removal of the heat produced by the passage of the current by means of a cooled electrolyte, separated from the liquid to be treated by dialysing membranes;
The reduction of variations in ionic concentration by equilibrating the composition of the diierent electrolytes;
The immersion of the electrodes in a common electrolyte flowing in parallel in their two compartments, the two streams of electrolyte being combined before being recycled; and
The flow of an auxiliary electrolyte on both sides of the electrophoresis compartments with combination of the different streams of this electrolyte, followed by recycling.
These improvements are however insufficient, and do not allow, for example, a sharp separation of similar products such as the beta and gamma globulins.
According to the present invention there is provided a method for the continuous fractionation by forced flow electrophoresis of an aqueous liquid containing at least two compounds, the relative mobilities of which in an electric field vary as a function of the pH, in order to obtain one fraction enriched and one fraction depleted in one of these compounds, said method comprising the simultaneous steps of: introducing the liquid to be treated into an electrophoresis cell in contact with one face of a filtering component which is permeable to at least one of the said compounds; applying an electric field between two electrodes located on either side of the filtering component; forcing a fraction of the liquid to pass across the filtering component to provide a filtered portion and of removing separately the filtered portion and the unfiltered remaining portion of the liquid; causing a stream of main electrolyte, independent of the liquid to be fractionated, to flow in contact with each electrode; and causing two streams of auxiliary electrolyte to flow between membranes which are permeable to ions, said membranes separating each auxiliary electrolyte stream firstly from the stream of main electrolyte and secondly from the filtered portion of liquid and from the unfiltered remaining portion of the liquid respectively, said two streams of auxiliary electrolytes have different average pHs between their introduction and removal from the cell.
The invention also relates to a forced flow electrophoresis cell for the continuous fractionation of an aqueous liquid containing colloidal compounds which are mobile in an electric yfield into one fraction enriched and one fraction depleted in at least one of the said compounds, the cell comprising, from the anode to the cathode, six compartments separated by ion-permeable membranes, the two central compartments being separated by a microporous membrane which is permeable to at least one of the constituents of the said liquid, and separated from the intermediate compartments by dialysing membranes, the end compartments containing the anode and the cathode being separated from the intermediate compartments by ion-selective membranes which are impermeable respectively to anions and to cations, means for feeding the liquid to be fractionated under pressure to one of said central compartments, means to withdraw a filtered portion of the liquid from the other central compartment and the remaining unfiltered portion from said one central compartment, means for feeding a main electrolyte to and from the end compartments and means for feeding auxiliary electrolyte to and from the intermediate compartments whereby the pH in one intermediate compartment differs from that of the other.
The method according to the invention allows products to be obtained which are considerably purer than those obtained with the prior techniques. 'In effect, it allows a pH corresponding to the mobility maximum of one compound to be separated to be established in at least one of the electrophoresis compartments, whilst the pH of the other compartment is either not modified or ensures the movement of at least one other product in the reverse direction. Thus, in the examples described later in detail to illustrate the invention, the electrophoresis anode compartment receives blood which must return to the donor with a minimum of modification, and in particular the pH of which must remain constant. The hydrodynamic component directed towards the cathode carries the non-ionic compounds and the less mobile of the anions (especially the globulins) as well as cations into the central electrophoresis compartment nearer the cathode end compartment. 'By increasing the pH only in this central compartment it is possible to increase the diiference in mobilities between the various globulins and to obtain, at the outlet of the other central compartment nearer the anode end component, blood which can be reinjected, and, at the outlet of the first central compartment, a solution of gamma globulins which is free of alpha and beta globulins.
Although the nature of the main electrolyte in which the electrodes are immersed is not generally critical, a base such as sodium hydroxide in aqueous solution is preferably used; this allows stainless steel electrodes to be used in place of platinum electrodes. Furthermore, where the treated liquid is blood, the neutralisation of the vsodium hydroxide with hydrochloric acid in the auxiliary electrolyte does not introduce any harmful ions.
The difference in average pHs in the compartments adjacent to the electrophoresis compartments can be produced by feeding each intermediate compartment with its own auxiliary electrolyte at iiow rates, pHs and buffering abilities chosen in advance, but this necessitates two installations for providing (and, if necessary, for regeneration) electrolyte.
It is therefore preferred to use auxiliary electrolytes of the same pH and the same buffering ability, and to make them flow in their respective intermediate cornpartments at average speeds chosen so that the optimum pH is established in at least one of the compartments under the effect of ion transfer. It is then convenient to use a common source of auxiliary electrolyte and to divide it into two streams, each one feeding a particular compartment. If desired, the two streams can then be combined and recycled after correcting their composition is necessary.
Although it is not essential, it is advantageous for the streams of auxiliary electrolyte or liquid to be fractionated to have a buffering ability in at least one of the valuable pH zones. Of course, it is possible to wonk with Unbuffered solutions, but the stabilisation of the pHs is more delicate and requires more precise control of the interdependent parameters (initial composition of the liquids, ow rates and potential between the electrodes).
The preparation of the liquid to be fractionated, its flow rate at the inlet and outlet of the cell and the potential between the electrodes do not call for special comment and are determined, case by case, in the same way as for the previous processes. Thus, the potential at the electrodes is usually adjusted to the ow rate of the fraction of liquid extracted after it has passed across the filtering membrane, the direction of polarity being preferably chosen in order that this fraction corresponds to the compounds which filter most easily. The ratio of the -ow rates of the two fractions is chosen as a function of the desired purity or of the extraction yield, and it depends on the difference in pressure on either side of the membrane and on the porosity of the latter.
The control of the potential as a function of the rate of ow of liquid across the ltering membrane allows the separation zone of the t'wo fractions to be kept at the level of the membrane. At the same time, the potential depends on the linear speeds of the electrolytes in contact with the separating membranes; speeds which are too slow cause an immobile limiting layer to appear there, in which the ions are not renewed; this phenomenon leads to an increase in the potential at the electrodes, and thus to an increased consumption of power.
The apparatus according to the invention allows the process to be put into practice in a remarkably simple way, and it avoids the diffusion of harmful products away from the electrodes.
Thus, if the main electrolyte is a sodium hydroxide solution, a cation-selective membrane on the anode side allows only the passage of Na+ ions and an anionselective membrane on the cathode side allows only the passage of OH'- ions: the electric iield thus has the effect of injecting sodium hydroxide into the auxiliary electrolyte and the only corrections which it can be advantageous or necessary to carry out, depending on the particular cases, are firstly for the main electrolyte, the addition of sodium hydroxide, in an amount corresponding to the electric current intensity across the cell, and secondly for the auxiliary electrolyte, the neutralisation of the sodium hydroxide by the equivalent amount of a suitable acid, for example hydrochloric acid, if the treated liquid contains or can accept chlorides. Furthermore, the presence of the cation-selective membrane prevents the chloride ions from forming chlorine at the anode, as such a formation would -be harmful for a steel anode as well as for the majority of the liquids treated.
If it is desired to use an. acid main electrolyte, an Oxy-acid such as sulphuric acid, which is regenerated at the anode with simple loss of oxygen, is preferably chosen. If the presence of ions from this Oxy-acid is harmful in the liquid to be fractionated, it is possible to feed only the anode compartment with this acid, the cathode being disposed in another, more suitable acid, for example, hydrochloric acid. 4In all cases, the anode ion-selective membrane stops the anions coming from the cathode compartment and keeps them in the auxiliary electrolyte.
The suppression of the formation of chlorine usually allows the auxiliary electrolye to be kept with a minimum of treatment for equilibrating its composition, and it then suffices to neutralise the main electrolyte component carried away by electrolysis and then to recycle the auxiliary electrolyte.
Suitable ion-selective membranes are well known and are available commercially.
The filtering component is of the microporous type, with an average diameter of holes allowing the passage of the elements carried by the hydrodynamic component. The membranes separating the electrophoresis compartments from the auxiliary electrolyte compartments are usually of the dialysing or ultraltering type, with a stoppage threshold corresponding to the ions or molecules, the migration of which it is desired to avoid; the stoppage threshold generally corresponds to a low molecular weight, for example 1000, 500 or even less. Membranes of regenerated cellulose are usually suitable.
In accordance with the usual techniques, the cell can consist of concentric annular components or of superimposed flat components. In the latter case the height of the compartments is preferably greater than their width, preferably in a ratio of 3/ 1 to 5/1. The thickness of the compartments is 10W, usually less than l0 mm. and preferably between 1 and 4 mm. The different compartments of one and the same cell advantageously have usable cross-sections which can be substantially superimposed.
Also according to the usual techniques, several cells can be grouped together as batteries (in series, in parallel or in series-parallel) 0f course, the different parameters and characteristics of the cell can be adapted by the technician according to each particular case.
In order that the invention will be more fully understood, the following description is given, merely by way of example, reference being made to the accompanying drawings, in which:
FIG. l is a schematic longitudinal cross-section of one embodiment of electrophoresis cell according to the invention, with its attachments;
FIG. 2 is an exploded view of one half of the cell, showing one form of its rVarious components;
FIGS. 3 and 5 are assembly diagrams showing how the invention can be put into practice in the laboratory; and
FIG. 4 shows a special form of compartment.
v According to FIG. 1, the electrophoresis cell 1 is divided into six compartments by five membranes, and, from left to right, there is:
An anode electrode end compartment 2 containing the anode 3, across which the principal electrolyte passes between the opposite orifices 15 and 16;
a cation-selective membrane an intermediate anode compartment 4, through which an auxiliary electrolyte passes between the opposite orifices 17 and 18;
a membrane 1.1 which is permeable to ions and molecules of molecular weight less than 500;
an anode side electrophoresis central compartment 5 through which the liquid to be treated passes between the opposite orifices 19 and 20;
a microporous membrane 12 which is permeable to the constituents to be extracted;
a cathode side electrophoresis central compartment 6 through which the ltered liquid passes across 12 up to the orifice 21;
a membrane 13 analogous to 11;
an intermediate cathode compartment 7 through which an auxiliary electrolyte passes between the opposite orifices 22 and 23;
an anion-selective membrane 14;
a cathode electrode end compartment 8 containing the cathode 9 and through which the main electrolyte passes between the opposite orifices 24 and 25.
The liquid to be treated is introduced into the central compartment 5 via the orifice 19. It passes in contact with the filtering component consisting of the microporous membrane 12. By establishing a hydrostatic pressure difference on either side of 12, a portion of the liquid and its filterable constituents are caused to pass from compartment 5 to compartment 6. 'I'he filtered and unfiltered fractions of the treated liquid leave respectively via the orifices 21 and 20.
The main electrolyte is introduced simultaneously via the orifices 15 and 24. into the anode and cathode end compartments, and is removed, along with the gases which may be formed at the electrodes, via the orifices 16 and 25. The two portions of main electrolyte are combined in a tank 26 and recycled by means of a pump 27. The gases 4can escape at the surface of the tank 26 into which the additives 41 of electrolyte necessary to give a continuous operation can be introduced.
The auxiliary electrolyte is introduced simultaneously via the orifices 17 and 22 into the intermediate compartments 4 and 7; it passes across these compartments and is then remo-ved via the orifices 18 and 23. The two portions of auxiliary electrolyte are combined in a tank 29 and recycled by means of a pump 30.
The flow rates of electrolyte in the intermediate compartments 4 and 7 are controlled respectively by Ivalves 31 and 32 and are measured by ow-meters 33 and 34. A heater 35, controlled by a thermostat 45, is immersed in the tank 29 and ensures that suitable temperatures are maintained in the cell. The reactant 42 necessary to keep the value of the pH, controlled by the apparatus 28, constant, and to give a continuous operation, can be introduced into the tank 29.
The electrodes 3 and 9 are connected to a direct current generator of a known type, comprising, for example, a rectifier and a variable transformer. The current intensity provided and the applied potential can be measured at each instant by means of an ammeter A and a voltmeter V (see FIG. 3).
A partial exploded view of one embodiment of the apparatus is shown in FIG. 2. The three compartments 6, 7 and 8 corresponding to the cathode half of the apparatus have been shown therein. The other half is located symmetrically relative to the plane of the microporous membrane 12. The electrophoresis cell consists of a stack of membranes and plane interposed frames, clamped between two rigid plates such as 36 by a system of threaded rods passing through holes such as 43 and nuts (not shown).
The interposed frames 37, 38 and 39 are open in the centre to form the various compartments. They comprise,
as well as the membranes, holes such as 44, which, when they are lined up, form the distribution channels for the fluids between the various compartments and the external faces of the plates such as 36. The frames are made of any suitable material which is insulating and compatible with the fluids which will come into contact with it. For example, in the case of the treatment of blood, they can be made of or covered with fluorinated polymers or silicone elastomers.
The electrodes can each consist of a stainless steel grid to which feed tubes 40 pass in the thickness of the interposed layer. Grids consisting of, for example, two webs of crossed and heat-sealed yarns, made of polyethylene or an equivalent inert plastic, or in some cases a silicone material, are located advantageously in the other compartments. They serve the purpose of supporting the membranes, of separating the electrolyte flows uniformly in each compartment and of causing turbulences which are necesssary for good exchange.
The method according to the invention will be explained in greater detail by referring to the apparatus of FIG. 1 and by illustrating the isolation of gamma-globulins from animal blood circulating outside the body. This treatment requires a maximum of precautions and selectivity. The application of the method to the fractionation of other liquids can be deducted from it by modifying the various parameters in the necessary manner.
The conditions which must be fulfilled in the case illustrated are that, since the blood must be restored to the animal, it is important that neither its temperature, nor its pH, nor its ionic concentration, nor its osmotic pressure, nor the content of the elements which it contains are modified, and that it must be possible to sterilise at least the blood compartment which must be made of non-thrombogen materials.
The auxiliary electrolyte consists of an aqueous solution containing 9 g./l. of sodium chloride, in ionic equilibrium with the blood and at the same pH (7.4) and containing 0 to 5 g./l. of sodium citrate. The main electrolyte is a 0.154 N sodium hydroxide solution, also isoionic in Na+. 'Ihe migration of the Na+ and OH- ions under the effect of the electric field is compensated for by a continuous addition of concentrated sodium hydroxide to the main electrolyte in the tank 26, at a flow rate which is proportional to the current intensity in the cell. In the case of the auxiliary electrolyte, the enrichment in sodium hydroxide is compensated for in the tank 29 by a corresponding addition of 0.154 N hydrochloric acid (isoionic in Cl) containing an amount of citric acid equivalent to that of the auxiliary electrolyte. The flow rate of acid is controlled by testing the pH, which must remain between 7.35 and 7.45. The citric acid provides the electrolyte with buffering ability and, by complexing the calcium, serves as an anti-coagulant in the apparatus. The citrate ions which are drawn into the electrophoresis anode compartment across the membranes 13 and then 12 are not dangerous for the animal because they are rapidly metabolised.
The flow rate of cathode auxiliary electrolyte is adjusted so that its pH yincreases to between 8 and v9 at the outlet of the compartment 7. In the anode compartment 4, the flow rate of auxiliary electrolyte is adjusted so that the pH remains between 7.35 and 7.45. This disparity in pH is made possible because of the differences in buffering ability in the two electrophoresis compartments; the first electrophoresis compartment (nearer the anode) contains the elements which occur in blood and the majority of the soluble proteins, and thus benefits from the buffering ability of the CO2 held by the haemoglobin and (although to a lesser extent) of that of the soluble proteins. On the other hand, the other electrophoresis compartment (nearer the cathode) receives only the neutral molecules (glucose, salts) and the globulins which have practically no buffering ability.
If gamma-globulins must be puried in an unbuffered medium, the method according to the invention can still be used; it is suicient to reduce the flow rate of the auxiliary anode electrolyte to a value such that a practically immobile limiting layer appears in contact with the cation-selective membrane. This layer is depleted in Na+ ions which are replaced in part by H+ ions, and this prevents the increase in pH in the unbutfered liquid of the compartment 5. The precise adjustment of the pH is, however, delicate because of the poor buiering ability of the electrolyte and of the solution to be fractionated.
It is simpler to add albumin to the solution, and the buffering effect produced in the upper electrophoresis compartment is suficient to stabilise the pH.
The examples which follow specify the experimental operation conditions for the extraction of gamma-globulins from animals provided with an arterio-venous carotidjugular shunt or from an aqueous solution.
EXAMPLE 1 An electrophoresis cell according to FIGS. 1 and 2 assembled as indicated below and fed with rabbit blood and electrolytes as indicated above is used.
Two polycarbonate plates of dimensions 19 X 6.5 X 2 cm. clamp a succession of silicone elastomer frames of dimensions 19 x 6.5 X 0.15 cm., the centre of which includes an aperture to form compartments of approximately 13 X 13 x 0.15 cm. The end compartments 1 and 8 each contain an electrode of stainless steel gauze connected to a terminal of a current rectifier supplied by a variable transformer.
The central and intermediate compartments 4, 5, 6, 7 are each equipped with an ethylene-vinyl acetate copolymer grid of the same dimensions, the grid of the compartment 5 being coated with silicone elastomer.
The microporous membrane 12 is a sheet of cellulose triacetate of porosity 0.45,:1. supported at the side of 5 by a Woven fabric of nylon monolaments of diameter 80g, mesh size 40a, laminated and then treated with silicone by impregnation with a solution of 1% strength of elastomer in cyclohexane.
The dialysing membranes 11 and 13 are of regenerated cellulose weighing 60 g./m.2, stretched on a frame of cellulose acetate in the case of membrane 13 and of iluorinated resin in the case of membrane 11.
The ion-selective membranes and 14 consist of ion exchange resins dispersed in a vinyl chloride/butyl maleate copolymer as described in French Pat. 1,584,187 (60% of resin and 40% of copolymer containing 4% by Weight of maleate units) and supported by a polypropylene woven fabric with 24 meshes to the centimetre (0.25 mm. orifice) weighing 77 g./m.-"'. The anion-selective membrane contains a resin with quatennary ammonium groups, and has a substitution electrical resistance of 6S2/cm.2 and a selective permeability of 82%; the cation-selective membrane contains a resin with sulphonic acid groups, and has a resistance of 8tl/cm.2 and a selective permeability of 80% (measured as described in the above-mentioned patent).
The temperature of the tank 29 is adjusted to 40 C., and the electrophoresis central compartments 4 and 5 and their pipes (silicone elastomer) are tilled with physiological serum to which heparin has been added to the extent of 50 units/cm3, in a phosphate butter at a pH of 7.4 (composition: NaCl 8 g., KCl 0.2 g., Na2HPO4 (anhydrous) 1.15 g., NaH2PO4 (anhydrous) 0.2 g., distilled Water to make up 800 cm). The arterial outlet 50 of the shunt is connected to the apparatus at 19, a peristaltic pump 51, delivering 360 cm.3/hour, taking the place of the arterial pressure, the outlet 21 being shut.
When the blood appears at the outlet of the tube 53 which is an extension of the orice 20, this tube is connected to the venous branch of the shunt via a lter for removing bubbles 54. As a result of the pressure drop in the blood return tube and in the intravenous catheter,
8 the pressure in the compartment 5 is about 100 mm. Hg.
The flow of main electrolyte is started, at an average ow rate of about 50 l./hour (that is, 25 l./hour per compartment), an then the ow of auxiliary electrolyte is started, adjusting the flow rates to 65 l./ hour for the anode compartment and to 50-55 l./hour for the cathode cornpartment. It is advantageous to maintain the same pressure in the four electrolyte compartments 2, 4, 7, 8 as in compartment 5, which restricts the deformation of the various membranes.
A potential difference of 14 v. is established at the electrodes, and this gives a current intensity of 2 a. (that is 70 ma./cm.2), and the re-equilibration of the electrolytes is begun: by the addition of 0.154 N hydrochloric acid, to which citrate has been added, in the tank 29, at a ow rate of 480 cm/ hour, a ow rate which is slightly lower than the theoretical ow rate, with periodic compensation according to the readings on the pH-meter; the excess volume corresponding to this addition is removed from the tank by overflow; by the addition of 7.7 N sodium hydroxide in the tank 26 at a flow rate of 10 cm/ hour, also with periodic correction.
The ow rates in 4 and 7 are adjusted to give pHs of 7.4 and 8.6 respectively.
The outlets 21 of the upper compartment 6 is opened, and the rate of removal is adjusted to 12 cm/hour. In order to keep the overall concentration constant, a solution of 1 g./l. of glucose, to which heparin has been added to the extent of 200 units/cm.3, in a phosphate buier of pH 7.4 described previously, is injected into the blood, upstream from the cell, at the same llow rate (that is, approximately 8 units/hour/cm.3 of blood) by means of a pump 52.
The adjustment of the potential applied to the electrodes/ow rate at 21 is chosen so that the gamma-globulin concentration in the extracted solution is about half their concentration in treated blood.
A solution containing approximately 2.5 g./l. of gamma-globulins free of beta-globulins but containing (a) a small amount (0.8 g./l.) of brinogen which can easily be separated by precipitation by means of thrombin, and (b) the small molecules (glucose, urea, inorganic salts) which can be removed by dialysis, is thus recovered in 55.
After 18 hours of such a treatment, spread over a period of 3 days in 6 hour sessions, 200 cm.3 of solution, that is, 500 mg. of immunoelectrophoretically pure gamma-globulins (immunoglobulins G free of immunoglobulins M by the Ouchterlony technique) has been obtained from one and the same rabbit.
An increase in the potential applied to the electrodes improves the separating ability and thus allows the flow rate across the microporous membrane 12 to be increased. Thus it s possible to work at 37 v. with a ow rate of 24 cm3/hour. It is then expedient to increase the auxiliary electrolyte ilow rates in the anode and cathode compartments to and 75 l./hour respectively. Under these conditions as well, the ow rate of the blood is not critical and could vary, without disadvantage, between 300 and 900 cm3/hour.
EXAMPLE 2 An unbufr'ered isotonic solution containing 2 g./1. garnma-globulin which contains 2 g./1. of haemoglobin is treated in the same cell and with the same parameters as in Example 1.
After a few minutes, an increase in pH in the electrophoresis anode compartment is observed, and the fractionation is no longer satisfactory.
The flow rate of anode auxiliary electrolyte is then reduced from 65 L/hour to 55 l./hour, and this has the etect of bringing its pH back to 7.4 a. gamma-globulin solution free of haemoglobin is then removed from the electrophoresis cathode compartment.
9 EXAMPLE 3 The procedure of Example 2 is carried out but, instead of altering the flow rate of anode auxiliary electrolyte, albumin is added to the gamma-globulin solution up to a content of 25 g./l. The pH of the anode solution remains stable, and a gamma-globulin solution free of haemoglobin is removed from the electrophoresis cathode compartment.
EXAMPLE 4 An electrophoresis cell according to FIGS. 1 and 2 is used, but with the difference that the fiow of the auxiliary electrolytes occurs transversely and not parallel to the direction of fiow of the liquid subjected to the electrophoresis, the said electrolytes being distributed at the inlet and removed at the outlet of each compartment by an assembly of 14 pairs of orifices equally spaced along the entire height of the plate 38 (FIG. 4).
Two polycarbonate plates of dimensions 50 x 13 x 2 cm. clamp a succession of silicone elastomer frames, the centre of which is hollowed out to form compartments of 40 x 6.5 cm.
The thicknesses of the compartments, taking account of the thickness of the cellulose triacetate and uorinated resin joints added at the places mentioned in Example 1, are:
Mm. Electrode compartments 2 and 8 3 Auxiliary electrolyte compartments 4 and 7 3.5 Electrophoresis compartments 5 and 6 1.5
The other characteristics of the cell are equivalent to those of the cell described in Example 1, with every proportion being retained.
The accessories for the fiow outside the body are identical to those described in FIG. 3.
The treated animal, which carries a carotid-jugular shunt, is a ewe. If the circulation in the body is established, with the precautions described, at the speed of 60 ml./minute, a current intensity of amperes is established in the cell under a potential difference of 18 volts.
The fiow rate of the anode auxiliary electrolyte is adjusted to 380 l./hour.
The flow rate of the cathode auxiliary electrolyte is adjusted to 900 l./hour. l
The liquid containing the gamma-globulins is removed from the compartment 21 at a speed of 1.6 mL/minute; it has a pH of 8.5.
Phosphate buffer, to which heparin has been added to the extent of 50 units/ml. and glucose to the extent of 1 g./l., is injected by means of the pump 52 at the same speed of 1.6 mL/minute.
The operation lasts for 7 hours and 3.3 g. of electrophoretically pure gamma-globulins, consisting solely of Ig G according to the Ouchterlony method, are collected.
Everything else being equal, if the flow rate of the anode auxiliary electrolyte is brought to 250 l./hour and that of the cathode auxiliary electrolyte is brought to 1200 l./ hour, the pH of the globulin solution removed at 21 is 7.5 and the immunological analysis reveals the presence of -globulins Ig M.
EXAMPLE 5 A cell 1 possessing the general characteristics of that described in Example 4, but the plates of which have a dimension of x 15 x 2 cm. corresponding to a usable surface area of the compartments (hollow of the interposed layers) of 24.6 x 5.5 cm. is shunted into the circulation outside the body of a rabbit according to the diagram of FIG. 5.
The pump 51, of adjustable flow rate, causes the arterial blood to fiow at a rate of 6 mL/minute and sends the latter to a reservoir 57 of 3 ml. capacity. The pump 58 takes up the blood in reservoir 57 again and delivers it into the electrophoresis cell at the rate of 30 ml./
minute. The pump 52. injects a phosphate buffer solution' (heparin content of units/ml.) at the rate of 0.2 mL/minute. The manometer 59 indicates a pressure of 2 cm. of Hg. The pump 46, the tube of which is mounted on the same rotor as the pump 51 to give the same fiow rate, delivers the blood into the jugular vein of the rabbit via a bubble trap 54. The pump 47 injects a solution, which is isotonic and isoionic with blood in Ca, Mg, K and Na chlorides, to which glucose has been added to the extent of 1 g./l., from a reserve 48, at the rate of 0.5 ml./ minute. The heater 56 allows heat losses in the complete circuit to be compensated for, and brings the reinjected blood back to the temperature of the rabbit. The manometer 49 indicates the pressure of reinjection of the blood and controls the clogging of the filter for removing bubbles.
The ow outside the body being got ready with the precautions described above, a current intensity of 10 amperes under 18 volts across the cell is ensured. The flow rate of the anode auxiliary electrolyte is brought to 280 L/hour, and that of the cathode auxiliary electrolyte is brought to 400 L/hour.
The gamma-globulin solution is removed from the cell in 55 at a speed of 0.7 ml./minute; its pH is 7.6. The average content of globulins is 3 mg./ml. so that a rabbit with an initial plasma content of Ig G of 6.4 mg. has this lowered to 1.1 mg./ml. after 7 hours of treatment. The gamma-globulins recovered comprise the Igs G, the Igs M `and the Igs A.
1. A method for the continuous fractionating by forced flow electrophoresis of an aqueous liquid containing at least two compounds, the relative mobilities of which in an electric field vary as a function of the pH, in order to obtain one fraction enriched and one fraction depleted in one of these compounds, said method comprising the simultaneous steps of: introducing the liquid to be treated into an electrophoresis cell in contact with one face of filtering component which is permeable to at least one of the said compounds; applying an electric field between two electrodes located on either side of the filtering component; forcing a fraction of the liquid to pass across the filtering component to provide a filtered portion and of removing separately the filtered portion and the unfiltered remaining portion of the liquid; causing a stream of main electrolyte, independent of the liquid to be fractionated, to ow in contact with each electrode; yand causing two streams of auxiliary electrolyte to flow between membranes which are permeable to ions, said membranes separating each auxiliary electrolyte stream firstly from the stream of main electrolyte and secondly from the filtered portion of liquid and from the unfiltered remaining portion of the liquid respectively, the average pH of one stream of auxiliary electrolyte between its introduction and removal from the cell differing from the said average pH of the second stream of auxiliary electrolyte.
2. A method according to claim 1, wherein the auxiliary electrolyte streams enter the cell with different pHs.
3. A method according to claim-z 1, wherein the auxiliary electrolyte streams enter the cell with identical pHs and fiow in the cell at different average speeds.
4. A method according to claim 3, wherein a single stream of auxiliary electrolyte is divided into two streams which circulate independently in the cell, are combined at the outlet of the cell and are recycled.
5. A method according to claim 1, wherein the main electrolyte and the auxiliary electrolyte are recycled whilst keeping their average compositions constant.
6. A method according to claim 1, wherein the main electrolyte is a base in aqueous solution and the auxiliary 1 1 electrolyte is -a solution buffered at the pI-I of the liquid to be fractionated.
7. A method according to claim 1, wherein the aqueous liquid is blood, and the enriched fraction includes gamma-globulins, and wherein the blood ows in the anode side of the filtering component and the average pHs of the streams of auxiliary electrolytes are adjusted to between 8 and 9 for the cathode compartment and to between 7.35 and 7.45 for the anode compartment.
8.. A method according to claim 7, wherein the blood of a living animal is treated continuously and the treated blood is restored to the animal.
References Cited UNITED STATES PATENTS 2,880,501 4/1959 Metz 204-301 X 3,051,640 8/1962 Traxler 204-180 P 3,617,545 11/1971 Dubois et al 204-180 P X HOWARD S. WILLIAMS, Primary Examiner A. C. PRESCOTT, Assistant Examiner U.S. C1. X.R.