U.S. patent number 3,870,617 [Application Number 05/443,158] was granted by the patent office on 1975-03-11 for apparatus for forced flow electrophoresis.
This patent grant is currently assigned to Rhone-Poulenc S.A.. Invention is credited to Guy Bourat.
United States Patent |
3,870,617 |
Bourat |
March 11, 1975 |
APPARATUS FOR FORCED FLOW ELECTROPHORESIS
Abstract
A continuous forced flow 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.
Inventors: |
Bourat; Guy (Bourg-1a-Reine,
FR) |
Assignee: |
Rhone-Poulenc S.A. (Paris,
FR)
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Family
ID: |
26216298 |
Appl.
No.: |
05/443,158 |
Filed: |
February 15, 1974 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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238764 |
Mar 28, 1972 |
3829370 |
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Foreign Application Priority Data
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Mar 30, 1971 [FR] |
|
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71.11180 |
|
Current U.S.
Class: |
204/633 |
Current CPC
Class: |
B01D
57/02 (20130101); B01J 8/12 (20130101); G01N
27/44756 (20130101); B01D 63/082 (20130101); G01N
27/44769 (20130101); B01D 61/425 (20130101) |
Current International
Class: |
B01J
8/08 (20060101); B01J 8/12 (20060101); B01D
57/02 (20060101); G01N 27/447 (20060101); B01d
013/02 () |
Field of
Search: |
;204/18P,18R,301,299 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Williams; Howard S.
Assistant Examiner: Prescott; A. C.
Attorney, Agent or Firm: Cushman, Darby & Cushman
Parent Case Text
This is a division of application Ser. No. 238,764 filed Mar. 28,
1972, now U.S. Pat. No. 3,829,370.
Claims
I claim:
1. A forced flow electrophoresis cell for the continuous
fractionation of an aqueous liquid containing colloidal compounds
which are mobile in an electric field into one fraction enriched
and one fraction depleted in at least one of the compounds, said
cell comprising an anode and a cathode, a plurality of spaced apart
ion-permeable membranes dividing said cell into six compartments,
including two end compartments containing said anode and cathode
respectively, two intermediate compartments and two central
compartments, the membrane separating said two central compartments
being a microporous membrane the membranes separating said central
compartments from the intermediate compartments being dialysing
membranes, and the membrane separating the intermediate
compartments from the end compartments containing the anode and the
cathode being impermeable respectively to anions and 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 an auxiliary electrolyte to and from the
intermediate compartments, whereby the average pH of one stream of
the auxiliary electrolyte between its introduction and removal from
the cell differs from the said average pH of the second stream of
the auxiliary electrolyte.
2. A cell according to claim 1 including a recycling circuit for at
least one of the electrolytes, a source of reactant connected to
said recycling circuit and means which allow the composition of
said source to be maintained.
Description
The present invention relates to a method and to an apparatus for
forced flow 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 flow (forced flow).
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. No. 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 different 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 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 field 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
difference in mobilities between the various globulins and to
obtain, at the outlet of the other central compartment nearer the
anode end compartment, 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 sodium 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 flow
rates, pHs and buffering abilities chosen in advance, but this
necessitates two installations for providing (and, if necessary,
for regenerating) 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 compartments 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 if 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 work 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, flow rates and potential between the electrodes).
The preparation of the liquids 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 flow 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 function corresponds to the compounds
which filter most easily. The ratio of the flow 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 flow of
liquid across the filtering membrane allows the separation zone of
the two 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.sup.+ ions and an anion-selective membrane on the cathode
side allows only the passage of OH.sup.- ions: the electric field
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. In 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 electrolyte 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 ultrafiltering 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 1,000, 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 low, usually less than 10 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).
Of 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. 1 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 various 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.
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 10;
an intermediate anode compartment 4, through which an auxiliary
electrolyte passes between the opposite orifices 17 and 18;
a membrane 11 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 filtered 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. The 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 can 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 removed 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 valves 31 and 32 are are
measured by flow-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 valve 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 necessary 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 of the fractionation
of other liquids can be deduced 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, not 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 nonthrombogen 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 iso-ionic in Na.sup.+. The migration of the Na.sup.+
and OH.sup.- 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.sup.-)
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 increases to between 8 and 9 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 CO.sub.2 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 purified in an unbuffered medium, the
method according to the invention can still be used; it is
sufficient 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.sup.+ ions which are replaed in part by
H.sup.+ ions, and this prevents the increase in pH in the
unbuffered liquid of the compartment 5. The precise adjustment of
the pH is, however, delicate because of the poor buffering 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 5 is
sufficient 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 carotid-jugular 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 in used.
Two polycarbonate plates of dimensions 19 .times. 6.5 .times. 2 cm
clamp a succession of silicone elastomer frames of dimensions 19
.times. 6.5 .times. 0.15 cm, the centre of which includes an
aperture to form compartments of approximately 13 .times. 3 .times.
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 .mu. supported at the side of 5 by a woven fabric of
nylon monofilaments of diameter 80 .mu., mesh size 40 .mu.,
laminated and then treated with silicone by impregnation with a
solution of 1 percent of elastomer in cyclohexane.
The dialysing membranes 11 and 13 are of regenerated cellulose
weighing 60 g/m.sup.2, stretched on a frame of cellulose acetate in
the case of membrane 13 and of fluorinated resin in the case of
membrane 11.
The ion-selective membranes 10 and 14 consist of ion exchange
resins dispersed in a vinyl chloride/butyl maleate copolymer as
described in French Pat. No. 1,584,187 (60 percent of resin and 40
percent of copolymer containing 4 percent 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.sup.2. The
anion-selective membrane contains a resin with quaternary ammonium
groups, and has a substitution electrical resistance of 6
.OMEGA./cm.sup.2 and a selective permeability of 82 percent; the
cation-selective membrane contains a resin with sulphonic acid
groups, and has a resistance of 8 .OMEGA./cm.sup.2 and a selective
permeability of 80 percent (measured as described in the
abovementioned Patent).
The temperature of the tank 29 is adjusted to 40.degree.C, and the
electrophoresis central compartments 4 and 5 and their pipes
(silicone elastomer) are filled with physiological serum to which
heparin has been added to the extent of 50 units/cm.sup.3, in a
phosphate buffer at a pH of 7.4 (composition: NaCl 8 g, KCl 0.2 g,
Na.sub.2 HPO.sub.4 (anhydrous) 1.15 g, Na H.sub.2 PO.sub.4
(anhydrous) 0.2 g, distilled water to make up 800 cm.sup.3). The
arterial outlet 50 of the shunt is connected to the apparatus at
19, a peristaltic pump 51, delivering 360 cm.sup.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 orifice 20, this tube is connected to the venous
branch of the shunt via a filter for removing bubbles 54. As a
result of the pressure drop in the blood return tube and in the
intravenous catheter, the pressure in the compartment 5 is about
100 mm Hg.
The flow of main electrolyte is started, at an average flow rate of
about 50 l/hour (that is, 25 l/hour per compartment), and then the
flow 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 compartment. 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 2A (that is 70 mA/cm.sup.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 flow rate of 480 cm.sup.3 /hour, a flow
rate which is slightly lower than the theoretical flow 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.sup.3 /hour, also with periodic
correction.
The flow rates in 4 and 7 are adjusted to give pHs of 7.4 and 8.6
respectively.
The outlet 21 of the upper compartment 6 is opened, and the rate of
removal is adjusted to 12 cm.sup.3 /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.sup.3,
in a phosphate buffer of pH 7.4 described previously, is injected
into the blood, upstream from the cell, at the same flow rate (that
is, approximately 8 units/hour/cm.sup.3 of blood) by means of a
pump 52.
The adjustment of the potential applied to the electrodes/flow 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
fibrinogen 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.sup.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 is possible to
work at 37 V with a flow rate of 24 cm.sup.3 /hour. It is then
expedient to increase the auxiliary electrolyte flow rates in the
anode and cathode compartments to 95 and 75 l/hour respectively.
Under these conditions as well, the flow rate of the blood is not
critical and could vary, without disadvantage, between 300 and 900
cm.sup.3 /hour.
EXAMPLE 2
An unbuffered isotonic solution containing 2 g/l of gamma-globulin
which contains 2 g/l 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 effect of bringing its pH
back to 7.4 A gamma-globulin solution free of haemoglobin is then
removed from the electrophoresis cathode compartment.
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 gamm-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 flow of the auxiliary electrolytes
occurs transversely and not parallel to the direction of flow 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 .times. 13 .times. 2 cm
clamp a succession of silicone elastomer frames, the centre of
which is hollowed out to form compartments of 40 .times. 6.5
cm.
The thicknesses of the compartments, taking account of the
thickness of the cellulose triacetate and fluorinated resin joints
added at the places mentioned in Example 1, are:
Electrode compartments 2 and 8 . . . . 3 mm
Auxiliary electrolyte compartments 4 and 7 . . . 3.5 mm
Electrophoresis compartments 5 and 7 . . . 1.5 mm
The other characteristics of 6 . cell are equivalent to those of
the cell described in Example 1, with every proportion being
retained.
The accessories for the flow 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 20 amperes is established in the cell under a
potential difference of 18 volts.
The flow 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.
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 .beta.-globulins IgM.
EXAMPLE 5
A cell 1 possessing the general characteristics of that described
in Example 4, but the plates of which have a dimension of 35
.times. 15 .times. 2 cm corresponding to a usable surface area of
the compartments (hollow of the interposed layers) of 24.6 .times.
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
flow 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 100 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 flow 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 flow 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 mc/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.
* * * * *