WO1991010129A1 - Systeme de purification de proteines base sur la focalisation iso-electrique et l'isotachophorese - Google Patents

Systeme de purification de proteines base sur la focalisation iso-electrique et l'isotachophorese Download PDF

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Publication number
WO1991010129A1
WO1991010129A1 PCT/US1990/007409 US9007409W WO9110129A1 WO 1991010129 A1 WO1991010129 A1 WO 1991010129A1 US 9007409 W US9007409 W US 9007409W WO 9110129 A1 WO9110129 A1 WO 9110129A1
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WIPO (PCT)
Prior art keywords
chamber
fluid flow
individual
fluid
loops
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PCT/US1990/007409
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English (en)
Inventor
Leon E. Barstow
Milan Bier
Glen D. Ward
Garland E. Twitty
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Protein Technologies, Inc.
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Application filed by Protein Technologies, Inc. filed Critical Protein Technologies, Inc.
Publication of WO1991010129A1 publication Critical patent/WO1991010129A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
    • B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—Electrophoresis
    • C07K1/28—Isoelectric focusing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44769—Continuous electrophoresis, i.e. the sample being continuously introduced, e.g. free flow electrophoresis [FFE]

Definitions

  • the invention relates to techniques for the separation, purification, or both, of biological materials and, more specifically, to a method and apparatus for isoelectric focusing and isotachophoresis.
  • Isoelectric focusing, isotachophoresis, and zone electrophoresis are variants of electrophoretic techniques, differing in the buffer system employed and mode of separation achieved. The theoretical distinctions between these three methods has been described in some detail in Bier, 219 Science 1281-87 (1983) .
  • Zone Electrophoresis (“ZE”) , is the oldest of these techniques and most commonly used. Separation is carried out in the presence of a background of homogeneous buffer, and sample components separate according to their mobilities in this buffer. No steady state is ever reached, but migration continues with gradual broadening of sample zones due to diffusion and other effects.
  • Isotachophoresis is a more recent variant of electrophoresis, characterized by the fact that separation is carried out in a discontinuous buffer system.
  • Sample materials to be separated are inserted between a "leading electrolyte” and a “terminating electrolyte", the characteristic of these two buffers being that the leader has to have ions of net mobility higher than those of sample ions, while the terminator must have ions of net mobilities lower than those of sample ions.
  • sample components sort themselves according to decreasing mobilities from leader to terminator, in a complex pattern governed by the so-called Kohlrausch regulating function. The process has been described repeatedly, as for instance, Bier and Allgyer, Electro inetic Separation Methods 443-69 (Elsevier/North-Holland 1979) .
  • ITP Isoelectric focusing
  • electrofocusing is a powerful variant of electrophoresis.
  • the principle of IEF is based on the fact that proteins and peptides, as well as most biomaterials, are amphoteric in nature, i.e., are positively charge in acid media and negatively charged in basic media.
  • PI isoelectric point
  • amphoteric materials are exposed to a d.c. current of proper polarity in a medium exhibiting a pH gradient, they will migrate, i.e., 'focus' toward the pH region of their PI, where they become virtually immobilized. Thus a stationary steady state is generated, where all components of the mixture have focused to their respective Pis.
  • the pH gradient is mostly generated 'naturally' i.e, through the electric current itself.
  • Appropriate buffer systems have been developed for this purpose, containing amphoteric components which themselves focus to their respective PI values, thereby buffering the pH of the medium.
  • Such buffer mixtures are known as 'carrier ampholytes', the best known being "Ampholine", a trademark of the LKB Fetter AB, a Swedish company.
  • Other carrier ampholyte mixtures can be formulated by judicious mixing of suitable ampholytes, as, for example, described in Bier, 211 J. Chromatography 313-35 (1981) .
  • IEF and ITP differ in that IEF attains a stationary steady state whereas in ITP a migrating steady state is obtained.
  • IEF a finite length of migrating channel is always sufficient.
  • complete resolution may require a migrating channel that is longer than is practical.
  • the migrating components can be virtually immobilized by applying a counterflow of leading electrolyte, the rate of counterflow being matched to the rate of frontal migration of the sample ions. This is also known in the art.
  • Isoelectric focusing is an inherently elegant analytical procedure due to its simplicity and high resolution. Because agarose and polyacrylamide IEF gels can resolve proteins differing in isoelectric points by as little as .01 pH units while minimizing fluid flow disturbances, they are routinely used to assay protein purity. But while gels enable such high resolution analytically, solid supports severely complicate both heat removal and product recovery and are thus the major impediment to the scale up of IEF.
  • ITP is most often carried out in capillaries.
  • the sample is inserted at one end of the capillary, at the interface between leader and terminator, and the migration of separated components recorded by appropriate sensors at the other end of the capillary. Both such systems are used mainly for analytical or micro-preparative purposes.
  • the d.c. electric field is applied in a direction perpendicular to buffer and sample flow.
  • the critical feature of such instruments appears to be the dimension of the gap between the two parallel plates, i.e., the thickness of the fluid film. This is usually of. the order of 0.5 to 1.5 mm.
  • the passage of the electric current generates heat, and thus one or both of the parallel plates are cooled.
  • the cooling capacity of these plates sets the limit for power dissipation within the apparatus. It is implicit in such continuous flow devices, whether applied to ZE, IEF, or ITP, that separation of sample functions be obtained in a single pass through the apparatus. This requires slow flow of buffer and long residence time of the sample within the apparatus.
  • the residence time of the fluid in the center of the gap is much shorter than that of the fluid close to the wall; (3) finally, as the fluid at the center of the gap is warmer than at the walls, all electrophoretic parameters (conductivity, viscosity, electrical field, mobility of ions, etc.) are affected.
  • the effects of the three factors are complex and cause the well known 'crescent phenomenon' (Strickler and Sacks, 209 Annals New York Acad. Sci. 497-514 (1973)), i.e., a crescent-like deformation of the migrating sample zones.
  • the crescent deformation is most pronounced closest to the walls of the electrophoretic chamber.
  • the present invention is directed to a method and apparatus for separation and purification of proteins and other biological materials by isoelectric focusing (IEF) or isotachophoresis (ITP) .
  • IEF isoelectric focusing
  • ITP isotachophoresis
  • the invention is based on the discovery that stabilization of fluid flow during preparation IEF or ITP is achievable by rapid recirculation of process fluid through a narrow channel of a continuous flow electrophoresis chamber of limited depth. Thereby are eliminated the previously enumerated causes of fluid flow disturbance due to temperature and density gradients, electroosmosis and parabolic flow.
  • the apparatus suitable for implementation of the invention differs from conventional continuous flow instruments in several respects: (1) there is a matched set of in- and outflow ports at the opposite ends of the electrophoresis chamber; (2) means are provided for rapid recirculation of the process fluid in closed external loops between each matched set of in- and outflow ports; (3) these loops include individual refrigerated heat exchangers.
  • the highly stable hydrodynamic flow achieved by the electrophoresis chamber design and method of operation according to the present invention enables isoelectric focusing to be performed in free solution without solid supports or the screen stabilization systems necessary in early recycling designs.
  • the processing method differs also in a substantial manner from the customary methods of operation of continuous flow electrophoresis chambers: (1) rather than trying to achieve the desired degree of separation in a single pass through the electrophoresis chamber, only a small shift towards the final steady state is achieved in each of several passes through the chamber; (2) rather than having slow flows and long residence times within the chamber, of the order of minutes to fractions of an hour, rapid recycling is established, with residence times of the order of seconds.
  • the stabilizing effect of rapid flow is so striking that cooling of the electrophoresis chamber is no longer necessary.
  • great emphasis is placed on uniformity of temperature within the electrophoresis chamber, to avoid convective disturbances.
  • the Joule heat generated by the applied electric current can be dissipated in heat exchangers external to the electrophoresis chamber. This is a major advantage, greatly simplifying the design of the apparatus.
  • the Joule heat is absorbed by the latent heat capacity of the process fluid during transit through the electrophoresis chamber and released to the external heat exchanger.
  • the allowable heating within the chamber is limited only by the heat-sensitivity of the sample, rather than the stability of liquid flow.
  • the present invention also provides a fluid circulation system coupled to the continuous flow electrophoresis chamber of the invention, to rapidly recirculate fluid from the chamber in a plurality of individual recirculation loops, each one of the recirculation loops coupling one of the out-flow ports to a matched one of the in-flow ports of the electrophoresis chamber.
  • the plurality of individual recirculation loops facilitates and maintains parallel and contiguous flows of individual fluid streams of separated biological materials achieved by the electrophoresis occurring within the continuous flow electrophoresis chamber.
  • the fluid circulation system includes a novel multi-channel peristaltic pump to provide the fluid pressure and velocity required for rapid recirculation of each of the fluid streams, through the electrophoresis chamber, pursuant to the invention and a heat exchanger, external to the electrophoresis chamber, for cooling of the fluid flow in each of the individual recirculation loops.
  • Fig. 1 is a front view of a continuous flow electrophoresis chamber according to the invention.
  • Fig. 2 is a cross-sectional top view of the chamber shown in Fig. 1, taken along plane II - II of Fig. 1.
  • Fig. 3 is a cross-sectional top view of an alternate embodiment of the invention.
  • Fig. 4 is an exploded, perspective view of a further embodiment of a continuous flow electrophoresis chamber assembly according to the present invention.
  • Fig. 4a is a top plan view of a detail of a chamber- forming member including in and out-flow ports.
  • Fig. 4b is a side view of the detail of Fig. 4a.
  • Fig. 5 is a partial perspective, cross-sectional view of a side plate of the continuous flow electrophoresis chamber assembly of Fig. 4.
  • Fig. 6 is an overall schematic view in block diagram form of an exemplary apparatus, as used for recycling IEF according to the present invention.
  • Fig. 7 is a perspective view, partially exploded, of a fluid circulation system to be coupled to an electrophoresis chamber, according to the present invention.
  • Fig. 8 is a front, plan view of the fluid circulation system illustrated in Fig. 7.
  • Fig. 9 is a rear, perspective view, partially exploded, of the fluid circulation system of Fig. 7.
  • Fig. 10 is a front view of the multi-channel peristaltic pump of Fig. 7.
  • Fig. 11 is a top view of the multi-channel peristaltic pump of Fig. 10.
  • Fig. 12 is a side view of the multi-channel peristaltic pump of Fig. 10.
  • Fig. 13 is a front, cross-sectional, view of the multi ⁇ channel, peristaltic pump taken generally along line A-A of Fig. 10.
  • Fig. 14 is a side, cross-sectional view of the multi- channel, peristaltic pump taken generally along line B-B of Fig. 10.
  • Fig. 15 is a top plan view of a tubing set installed * into the peristaltic pump of Figs. 10-14.
  • Fig. 15a is a front view of a male-type connector attached to each end of the tubing set of Fig. 15.
  • Fig. 16 is a detail, in a side cross-sectional view, of the male-type connector of Fig. 15a.
  • Fig. 17 is a detail, in a side cross-sectional view, of a tension bar used in the tubing set of Fig. 15.
  • Fig. 18 is an exploded perspective view of a heat exchanger according to the present invention.
  • Fig. 19 is a side view of a sample collection device according to the present invention.
  • Fig. 20 is a top plan view of the sample collection device of Fig. 19.
  • Fig. 21 is an end, cross-sectional view of the sample collection device of Fig. 19.
  • Fig. 22 is a side view of the tubing set of the sample collection device of Fig. 19.
  • Fig. 23 is a side view, partially in cross-section, of a bubble trap apparatus according to the present invention.
  • Fig. 24 is an end view of the bubble trap apparatus of Fig. 23.
  • Fig. 25 is a side, cross-sectional view of a fill/wash connector according to the present invention.
  • Fig. 26 is a top view of the fill/wash connector of Fig. 25.
  • the process is applicable only to electrophoretic methods resulting in a steady state exhibiting self- stabilizing and self-sharpening sample concentration boundaries, namely IEF and ITP.
  • suitable buffer mixtures appropriate carrier ampholytes capable of generating pH gradients for IEF, and discontinuous buffer systems for ITP. Random selection of homogeneous buffers used for ZE will not result in effective fractionation.
  • Re 2 inerti.a force pU2 here g is the gravitational acceleration, ⁇ Pthe density difference, L the length, and U the through-flow velocity.
  • the convection effects will be negligible if above ratio is much smaller than 1. This requires operation at relatively high Reynolds numbers, incompatible with single pass operation and necessitating recirculation. This, in turn, is compatible only with IEF and ITP modes of operation.
  • Shear stress is defined as the ratio of force, F, and surface, A, and is given by
  • R WLD Q - [5] which allows to define the shear stress in terms of R and D i 12 . * R D 2
  • Equation 6 the shear stress is proportional to L/R and inversely proportional to the square of chamber thickness, being zero in the center of the channel and maximal at the walls. Equation 6 has some interesting implications, for instance, lengthening the chamber length L to increase throughput Q, does not result in increased shear stress, as the ratio L/R remains constant.
  • This power causes a temperature rise of approximately 12°C in a non-refrigerated chamber, easily tolerated by most biological samples, provided the inflowing liquid was cooled in the external heat exchanger to near freezing. Optional cooling of one of the plates of the chamber avoids all temperature rise.
  • Our apparatus differs significantly from other electrophoretic apparatus that utilizes shear for fluid stabilization. See Mattock, Aitchison and Thomson, 9 Separation and Purification Methods 1-68 (1980) . That apparatus carries out separation in an annulus between two cylindrical electrodes, an inner stationary one and an outer rotating one. Sample and carrier buffer are introduced at the bottom of the annulus and the separated fractions withdrawn at the top. The apparatus can be used only for ZE and there is no possibility for fluid recycling. Moreover, liquid flows within a channel with one stationary and one moving boundary, and d ⁇ /dy rather than dp/dL is constant across the depth of the channel. Thus, the shear stress is constant across the whole channel (see equation 1) .
  • shear stress for fluid stabilization in the two instruments: in the prior art apparatus, uniform shear across the channel is used, while in the present invention, shear is maximal at the wall of the chamber and zero at the channel center. This is not a trivial difference: electroosmosis is exclusively a wall effect, and that location is where maximal shear stress is needed.
  • biological materials are sensitive to shear, and thus the invention offers the advantage of minimizing shear in the bulk of the liquid.
  • Maximum flow of course, is in the center of a channel, where in our apparatus there is zero stress.
  • rotationally induced . shear is in a direction perpendicular to the direction of liquid flow through the annulus. In our apparatus, shear stress is in the direction of the liquid flow.
  • the fluids to be processed are circulated through a processing chamber in a first direction, and exposed to a d.c. current in a second direction, roughly perpendicular to the first direction.
  • the chamber itself is constituted by two parallel plates 1 and 2, of an electrically non-conducting material, such as plexiglass, glass, etc., these plates defining a relatively narrow channel for fluid flow.
  • Multiple parallel entry ports 8 for process fluid are provided at one end of the chamber, matched at the opposite end of the chamber by outlet ports 7, located at the bottom and top of the chamber, respectively.
  • Electric current is provided by electrodes 9 and 10, mounted on side plate carriers 3, 4 fitted with connectors 11 and 12.
  • the electrodes are mounted in compartments, usually lateral, of the chamber, as shown, and separated from the main cavity of the chamber by electrically conducting but protein non-permeable membranes 5 and 6.
  • these membranes may be either ion-permselective or electrically neutral, while only the latter are acceptable for ITP.
  • electrically neutral membranes are the various types of dialyzing membranes commercially available, and ion-permselective membranes may be the type used in electrodialysis.
  • the electrode chambers are provided with ports 13 and 15 and 14 and 16 for circulation of electrolytes.
  • All entry and exit ports are individually connected outside of the chamber in a series of closed loops, so that a parallel and contiguous flow of individual fluid streams is established through the chamber cavity.
  • the fact that the fluid loops outside the cavity are closed loops assures the volumetric constancy of in- and outflow through each matched set of ports. Ion transport between adjacent streams in the chamber takes place under the influence of the electric field. Separate electrolyte circulation paths are provided for each of the two electrode chambers.
  • ITP For IEF, there is provided a matched number of entry and exit ports, the number depending on the number of fractions desired. ITP requires counterflow, thus necessitating inflow of buffer at one side of the chamber and outflow at the other side. This flow can be accomplished through inflow and outflow of excess fluid into the recirculation loops, at opposite sides of the chamber, or by provision for separate non-recirculating ports.
  • cooling means may be provided if desired, as shown in Fig. 3. Such means are most conveniently mounted on the chamber back plate, as generally it is desirable to view that chamber through a transparent covering over the front of the apparatus. Also, sealing is facilitated by mounting the electrodes in the front plate. As shown, a metallic plate 32, provided with channels 23, absorbs and transfers heat. An insulating layer 22 electrically isolates the cooling means.
  • Fluid addition or withdrawal during fractionation can be effectuated through any of the external fluid channels. This is necessary for establishing counterflow in ITP, through the input of leading electrolyte at one side of the chamber and withdrawal of excess fluid so introduced at the outer side of the chamber. If desired, this input or withdrawal could also be accomplished through additional ports in the chamber.
  • FIG. 4 there is illustrated an exploded view of an assembly of a further embodiment of a continuous flow electrophoresis chamber assembly 49 according to the present invention.
  • Each one of a pair of opposed separation chamber-forming members 50 is formed to be of a T-shaped cross-section and includes a chamber-forming surface 51.
  • the chamber-forming members 50 are assembled into a confronting relation and mate with complementary T-shaped side plates 52, as illustrated by the dash lines in Fig. 4.
  • the dimensions of the chamber-forming members 50 and side plates 52 are such that the chamber-forming surfaces 51 are spaced from one another by the side plates 52 by a distance of between 0.025 and 0.25 cm and, preferably approximately 0.075 cm, when assembled with the side plates 52.
  • each of the chamber-forming members 50 and side plates 52 is made from clear, optical grade stress-relieved acrylic, polished to an optical clear finish to prevent cracking and assure dimensional stability.
  • Each of the chamber-forming members 50 is formed to include a plurality of threaded openings 54 spaced in linear arrays along each surface 55 of the chamber- forming member 50.
  • each of the complementary side plates 52 is provided with a plurality of openings 56 formed completely through the T portion 57 of the side plate 52.
  • the plurality of openings 56 is arranged to align with the plurality of threaded openings 54 of the chamber-forming members 50 when the chamber-forming members 50 and side plates 52 are assembled together as illustrated in Fig. 4.
  • each one of a plurality of screws 58 can be passed through a respective opening 56 of each side plate 52 and screwed into an aligned threaded opening 54 to securely assemble the chamber-forming members 50 and side plates 52 together.
  • one of the chamber-forming members 50 is provided with alignment pins 65 which are each received in opposed openings (not illustrated, but placed at the end of each dash line 66) of the opposite chamber-forming member 50.
  • side plate assembly illustrated in Fig. 4 is provided by threaded bolts 67 which are each received through a pair of aligned openings 68 formed completely through each of the chamber-forming members 50 and fastened by a nut 69 and washer 70.
  • Each of the side plates 52 includes a channel 59 formed along the longitudinal axis of the respective side plate 52 to house an electrode and to provide an electrolyte flow channel, as will appear.
  • the channels 59 are arranged to extend along the 0.025 to 0.25 cm. spacing formed between the chamber-forming surfaces 51.
  • An electrode membrane 60 preferably made from an ion per selective or an electrically neutral material, is sandwiched between two electrode gaskets 61 and is secured in a covering relation over a respective channel 59.
  • each of the side plates 52 includes a plurality of threaded openings 62 which are arranged to align with respective corner openings 63 formed through the corners of each of the electrode membranes 60 and electrode gaskets 61.
  • Each one of a plurality of screws 64 is passed through a respective set of aligned corner openings 63 and screwed into a respective, aligned threaded opening 62 to secure the membrane 60 and electrode gaskets 61 in the covering relation over the respective channel 59.
  • the electrophoresis chamber is defined by the confronting chamber-forming surfaces 51, the end sealing gaskets 53 that are secured between the chamber-forming surfaces 51 and the electrode membranes 60.
  • Ingress and egress of a solution containing biological materials to be purified and separated by * electrophoresis within the electrophoresis chamber is achieved by means of a plurality of in-flow ports 71 and matching out-flow ports 72 formed at respective ends of one of the chamber-forming members 50.
  • Each of the ports 71, 72 communicates with an individual flow channel 85 (See Figs.
  • the arrays of ports 71, 72 at each respective end of the one chamber- forming member 50 provide female-type connector areas for coupling to external tubing sets for recirculation of individual fluid streams in external loops, as will appear.
  • a pair of threaded openings 73 is arranged, one on each side of each port array 71, 72, for threaded reception of connector studs 74 to mount a male-type tubing connector of a tubing set, as will be described in more detail below.
  • Alignment pins 75 can also be secured within openings 76 formed adjacent the port arrays to provide alignment for such tubing connectors.
  • each channel 59 is provided with a port 77 including a threaded portion of enlarged diameter.
  • An upchurch nut and ferrule 78 is secured into each port 77 to provide fluid communication between the channel 59 and a tube 79 connected to the upchurch nut and ferrule 78.
  • Each of the tubes 79 is connected to an electrolyte circulation system, as will be described in more detail below to provide electrolyte fluid circulation through the channels 59.
  • an opening 80 including a threaded portion of reduced diameter, is arranged adjacent at least one of the ports 77 of each channel 59 for threaded engagement by a respective banana connector 81.
  • a platinum wire 82 is coupled to each banana connector 81 and is arranged to extend through the opening 80 and longitudinally along the axis of the respective channel 59.
  • one of at least a pair of set screws 83 is threadily received within a respective one of at least a pair of threaded openings 84, arranged adjacent to each opening 80, to support and position a respective platinum wire 82.
  • banana connectors 81 can be coupled to positive and negative terminals of a voltage source respectively, to provide an electrical field between the platinum wires 82 that extends across the electrophoresis chamber, transverse to the flow of the solution containing biological materials, between the in and out-flow ports 71, 72, as will appear.
  • a multi-channel, peristaltic pump 500 is coupled to the electrophoresis chamber 100 to provide fluid pressure in the external loops provided by modular tubing sets 1300, where constancy and uniformity of flow through each channel are desirable.
  • a power supply 800 is electrically coupled to the electrophoresis chamber 100 to apply an electric field across the chamber 100, as described above.
  • the individual flows are also channeled through a heat exchanger 1100 (including a source of coolant 900) , to dissipate the Joule heat generated by the electric current.
  • a heat exchanger 1100 including a source of coolant 900
  • a collection valve or apparatus 300 is used to collect samples in an array of collector tubes 400 after biological materials have been separated from one another by isoelectric focusing or isotachophoresis within the electrophoresis chamber 100.
  • the tubing sets 1300 can be coupled to one another or to the various components of the system to provide any sequence of components 100, 200, 300, 500, 600, 700, 1100.
  • the tubing sets 1300 therefor provide a modular construction for the recirculation system according to the present invention.
  • the apparatus of Fig. 6 may be constructed of glass, a variety of machineable plastics or even ceramics. Cooling plates, if desired, can be metallic, provided they are separated from the chamber's interior by electrically insulating layers of suitable material, which could again be either glass or plastic.
  • the front side of the of the apparatus is preferably transparent, to facilitate observation.
  • FIG. 7-9 A detailed illustration of a cabinet 1200 is contained in Figs. 7-9.
  • the cabinet 1200 is divided into a processing compartment 1201 and an electronics compartment 1202 by a wall 1203.
  • the processing and electronic compartments 1201, 1202 are used to mount the electrophoresis chamber assembly 49 of the present invention and to house the components of a fluid recirculation system of the type illustrated schematically in Fig. 6.
  • the cabinet 1200 is provided with a front door panel 1250 hingedly attached to the cabinet 1200 and preferably made from a transparent material.
  • a high- voltage by-pass switch 1251 is mounted on the wall 1203 of the cabinet 1200 such that power to the electrophoresis chamber assembly 49 is interrupted whenever the front door panel 1250 is open.
  • Access to the electronics compartment 1202 is through a removable side panel 1252. Likewise, access to the rear of the cabinet 1200 is through a removable back wall panel 1207. A removable top panel 1253 provides access to the top of the processing compartment 1201. A by ⁇ pass switch (not illustrated) can be mounted adjacent the removable top panel 1253 to interrupt power to electrophoresis chamber whenever the top panel 1253 is removed.
  • An electronic component required to provide the electric field across the electrophoresis chamber is mounted on the wall 1203 within the electronics compartment 1208.
  • the power supply 800 may comprise a Hoefer Scientific Instruments PS 1500 Power Supply, which is a 1500 volt, 400 milliamp, 200 watt unit capable of operating at constant current or power.
  • the power supply 800 is coupled to a control panel 801, mounted at a front wall 1204 of the electronic compartment 1201 of the cabinet 1200.
  • the control panel 801 includes various control switches and output displays, such as a voltage limit control knob 802, a current limit control knob 803 and a power limit control knob 804. These knobs 802-804 can be manipulated by a user to vary and control the voltage, current and power of the output of the power supply 800, that is applied to the platinum wire 82 electrodes of the electrophoresis chamber 49.
  • output lines 1255, 1256 extend from the power supply 800 into the processing compartment 1201 for coupling to the banana connectors 81 mounted by the side plates 52 of the electrophoresis chamber assembly, respectively, to provide an electric field across the electrophoresis chamber via the platinum wires 82 (See Fig. 5) .
  • one of the output lines 1255 is connected to a positive output terminal and the other output line 1256 is connected to a negative output terminal of the power supply 800.
  • LED display panels 805-807 are mounted adjacent the control knobs 802-804, respectively, to provide volt, milliamp and watt readings, respectively, during the operation of the power supply 800.
  • a toggle switch 808 is provided to display on the panels 805-807 either set limits resulting from the manipulation of the control knobs 802-804 or the actual output readings for the power supply 800.
  • Display lights 809-814 are also mounted in the control panel 801 of the wall 1204 to indicate various operating conditions of the power supply 800, such as constant voltage, current or power operation, and open circuit, current leakage or ready to operate conditions.
  • an electrolyte buffer pump motor 815 and a peristaltic pump motor 816 are each powered by an external AC power source (not specifically illustrated) and are controlled by on/off control switches 817, 821, respectively. Additional control over the peristaltic pump motor 816 is provided by pump direction and pump speed control knobs 822, 823, respectively, and an emergency power off switch 824, all mounted on the control panel 801.
  • the control panel 801 is also provided with a main power supply on-off switch 820 and high voltage bypass and high voltage power switches 818, 819 to apply and controllably bypass the high voltage source provided by the power supply 800 to the electrophoresis chamber via the platinum wire electrodes 82.
  • the processing compartment 1201 mounts the electrophoresis chamber assembly 49 and a bubble trap apparatus 700' on a mounting wall 1205.
  • the electrophoresis chamber assembly 49 is mounted so that the in-flow and out-flow ports 71, 72 (See Fig. 4) are horizontally spaced from one another with the in-flow ports 71 arranged at the bottom of the assembly 49. (See Fig. 8) .
  • the electrolyte buffer pump motor 815 is mechanically coupled to a pair of pumps 850, 851 mounted in tandem on the processing compartment 1201 side of the wall 1203, directly opposite to the motor 815 to which the pumps 850, 851 are mechanically coupled in a well known manner.
  • the pumps 850, 851 comprise, e.g., Masterflex brand pump heads, each of which engages one of the tubes 79 of a respective side plate 52 (See Fig. 5) to provide peristaltic pumping action for flow of an electrolyte.
  • a tube 79a extends from the pump 850 to a reservoir 852 mounted on the wall 1203 and containing a first electrolyte, e.g. 0.1 M Na OH.
  • the tube 79a is passed behind the mounting plate 1205 to the upchurch nut 78 of one of the side plates 52 (See Fig. 5) for flow through the channel 59 of the one side plate 52 to an upchurch nut 78 and tube 79b at the opposite end of the channel 59.
  • the tube 79b again passes behind the mounting plate 1205 back to the pump 850.
  • Another electrolyte as, e.g., 0.1 M H-PO., is similarly pumped through the channel 59 of the other side plate 52 via tubes 79c, 79d by the pump 851.
  • a second reservoir containing the 0.1 M H 3 P0 4 supply is not illustrated, but is mounted on the wall 1203 directly behind the first reservoir 852.
  • a multi-channel peristaltic pump 500' and sample collection device 400' are each mounted on a bottom floor panel 1206 of the processing compartment 1201.
  • a heat exchanger 1100' is mounted on the back side of the mounting wall 1205 within a compartment defined by the mounting wall 1205 and the back wall panel 1207 of the cabinet 1200 (See Fig. 9) .
  • Fluid flow of a solution comprising a buffer carrier ampholyte containing biological materials to be purified and separated into individual fluid flows for collection is rapidly recirculated between the electrophoresis chamber assembly 49 and the other components 500', 700', 1100' of the processing system by removable tubing sets, each containing a plurality of individual fluid tubing, e.g. 30.
  • Each tubing set includes a male or female-type connector at each end to provide fluid communication between the tubing sets, as will now be described.
  • flow of the solution to be processed within the electrophoresis chamber by isoelectric focusing or isotachophoresis is from the in-flow ports 71 at the bottom of the assembly 49, upwardly through the electrophoresis chamber to the matching out-flow ports 72 to a first tubing set 1208.
  • the first tubing set 1208 includes a male-type connector 1209 at each end.
  • each male-type connector 1209 includes a plurality of openings 1210 formed therethrough and arranged in a predetermined array corresponding to, e.g., the array of out-flow ports 72 (See Fig. 4) .
  • a gasket element 1211 is received within a first recess 1215 formed in an outer surface of the connector 1209 to cover the outer ends of the openings 1210.
  • the gasket element 1211 defines a plurality of hollow, cylindrical, seal-forming extensions 1216, one over each opening 1210, to provide a fluid-tight seal around each opening 1210 and a complementary opening of a female-type connector of another component of the system, as e.g. , an out-flow port 72 of the female-type array of the electrophoresis chamber (See Fig. 4) .
  • each connector 1209 is provided with connector openings 1212 that align with and receive, e.g., the connector studs 74 adjacent to the array of out-flow ports 72 (See Fig. 4) .
  • the connector 1209 can be placed over the array of out-flow ports 72 and fastened by, e.g., knurled nuts 1213 screwed onto the connector studs 74 (See Fig. 8) , with each one of the openings 1210 in a confronting relation to a corresponding outflow port 72 which is sealed in a fluid-tight relation by the gasket element 1211.
  • the solution flow out of the electrophoresis chamber will be subdivided by the longitudinal channels 85 (See Fig. 4a) , flow through the out-flow ports 72 and into the openings 1210 of the connector 1209.
  • a semi-rigid tubing 1214 made from, e.g. teflon, extends through each opening 1210 to the gasket element 1211 to provide a plurality of individual flow channels or loops.
  • An * epoxy block 1217 is received in a second recess 1218 formed in the connector 1209 to support the tubing 1214.
  • the tubing 1214 is bundled together and received within a cover tubing 1219 which extends within the processing compartment 1201, behind the electrophoresis chamber assembly 49, to the multi-channel, peristaltic pum 500' (See Fig. 8) .
  • the cover tubing 1219 can be made of foamed material and provides thermal insulation for the recirculating materials.
  • each tubing 1214 is fanned out, and each tubing 1214 is inserted into a complementary opening 1220 of a linear array of openings formed completely through and spaced across a first tension bar 1221.
  • each semi-rigid tubing 1214 mates in a telescoping relation with a flexible tubing 1222 made from, e.g. silicon, which extends into similar opening 1220 of a second tension bar 1223 and into telescoping relation with another corresponding semi-rigid tubing 1214.
  • the semi-rigid tubing 1214 extending from the second tension bar 1223 is bundled together and inserted into another cover tubing 1219, which then extends from the multi- channel, peristaltic pump 500', through the mounting wall 1205 (not specifically illustrated) to the other male-type connector 1209 of the first tubing set 1208.
  • the other male-type connector 1209 is, in turn, connected to a female-type connector 1115 of a second tubing set 1224.
  • the second tubing set 1224 is mounted within the heat exchanger 1100', as will be described in more detail below.
  • the peristaltic pump 500' provides an even peristaltic pumping action across the silicon tubing 1222 of the first tubing set 1208 for rapid recirculation of the individual fluid streams from the out-flow ports 72 of the electrophoresis chamber assembly 49 to the matched in-flow ports 71.
  • the peristaltic pump 500' comprises a housing 501 rotatably mounting a rotor assembly 502.
  • the rotor assembly 502 is mechanically coupled to a drive shaft 503 which extends through the wall 1203 to a mechanical coupling with the electric pump motor 816.
  • the housing 501 is mounted onto a support bracket 504 by screws 505 so that the drive shaft 503 is positioned for coupling to the pump motor 816.
  • the support bracket 504 is, in turn, supported on the bottom floor panel 1206 of the cabinet 1200 (See Fig. 8) .
  • the drive shaft 503 extends, longitudinally through the housing 501 and is rotatably supported within shaft openings 506 formed in each of the side walls 501a of the housing 501.
  • the rotor assembly 502 comprises a pair of circular roller support plates 507, mounted at opposed, spaced locations on the drive shaft 503.
  • the drive shaft 503 is provided with pins 508, which are received within complementary slots 509 formed in each of the roller support plates 507 such that the support plates 507 will rotate with the drive shaft 503 when the drive shaft 503 is rotated by the motor 816.
  • Each one of a plurality of pump rollers 510 is rotatably mounted by a pair of aligned, opposed roller support openings 511 formed in the opposed roller support plates 507, respectively.
  • the openings 511 of each roller support plate 507 are arranged in a circular array around the respective roller support plate 507 to provide a generally cylindrical array of rotatable pump rollers 510 extending symmetrically about the drive shaft 503, as clearly illustrated in Fig. 14.
  • Structural support for the roller assembly 502 and mounted pump rollers 510 is provided by a core roller 512 which extends between the circular roller support plates 507 in a co-axial relation to the drive shaft 503.
  • the core roller 512 is secured to each of the roller support plates 507 by threaded bolts 513.
  • rotation of the drive shaft 503 by the pump motor 815 causes the circular roller support plates 507 to rotate, as discussed above, so that the pump rollers 510 revolve about the axis of the drive shaft 503.
  • a ring gear 514 is secured within a recess formed in one of the side walls 501a of the housing 501 by a set of screws 515 to facilitate rotation of each of the pump rollers 510, as they revolve around the axis of the drive shaft 503.
  • a spur gear 516 is pinned 518 to an extension 517 of each pump roller 510 and meshes with the ring gear 514 to positively rotate the respective pump roller 510 as it revolves.
  • the spur gear 516, ring gear 517 arrangement causes each roller 510 to rotate in a direction that is opposite to the direction of revolution of the rotor assembly 502 to facilitate movement of the pump rollers 510 over the tubing 1222, as will appear.
  • the housing 501 includes a base plate 519, a top cover 520 and a thrust plate 521.
  • the screws 505 secure the base plate 519 to each of the support bracket 504 and the side walls 501a.
  • the top cover 520 is secured to the thrust plate 521 by a plurality of screws 522 and the thrust plate 521 is, in turn, secured to the top of the side walls 501a by nut studs 523.
  • the thrust plate 521 rotatably supports a thrust bolt 524 which extends from the exterior of the housing 501 through an opening 523 formed through each of the top cover 520 and thrust plate 521 into the interior of the housing 501.
  • the thrust bolt 524 includes a threaded end portion 531.
  • a sleeve 529 is secured in a friction fit to a mounting plate 525 and the threaded portion of the thrust bolt 524 is threadily received within the sleeve 529, which has a threaded internal surface 530.
  • the mounting plate 525 supports a pump shoe 526 by means of screws 527.
  • the pump shoe 526 includes a curved inner surface 528 that forms an arcuate surface around the upper portion of the roller assembly 502, as illustrated in Fig. 14.
  • the gap distance between the curved inner surface 528 and the pump rollers 510 of the roller assembly 502 that are positioned beneath the curved surface 528 is adjustable by rotation of the thrust bolt 524 inasmuch as the sleeve 529 is movable axially toward and away from the roller assembly 502 due to the thread engagement between the threaded end 531 of the thrust bolt 524 and the threaded internal surface 530 of the sleeve 529.
  • the portion of the thrust bolt 524 that extends to the exterior of the housing 501 mounts a pump knob 533 by a friction fit.
  • the knob 533 can be selectively rotated to rotate the thrust bolt 524 and thereby control the gap distance.
  • the side walls 501a include two pairs of opposed recesses 535 spaced downwardly from and to either side of the roller assembly, adjacent the base plate 519, (only the front pair illustrated) each pair of recesses being arranged to receive and mount opposite end portions 1224 (See Fig. 15), of one of the tension bars 1221, 1223.
  • the flexible tubing 1222 extending between the tension bars 1221, 1223 (See Fig. 15) is arranged to pass around the roller assembly 502 and within the gap defined by the curved inner surface 528 and the pump rollers 510.
  • the gap is dimensioned so that the pump rollers 510 each squeeze each tubing 1222 against the arcuate surface 528 as the pump rollers revolve about the axis of the drive shaft 503 to provide an even peristaltic pumping action on the fluid within the tubing 1222.
  • the amount of pressure applied by the pump rollers 510 can be controlled to a suitable level to achieve rapid recirculation of the solution within the system by adjusting the pump knob 533 to vary the gap distance between the curved inner surface 528 and the pump rollers 510, as discussed above.
  • the heat exchanger comprises a cooling fluid circulation chamber 1101 including a plurality of vertically spaced, parallel tubing support-fluid flow baffle elements 1102 that extend within the chamber 1101 from side walls 1103, 1104 thereof, in an alternating sequence, as illustrated in Fig. 18.
  • a cover panel 1108 and sealing gasket 1109 are mounted over the cooling fluid circulation chamber 1101, by, e.g. screws 1110 screwed into aligned sets of openings 1111, 1112, 1113 formed in each of the cover panel 1108, cooling fluid circulation chamber 1101 and sealing gasket 1109.
  • Each one of the tubing support-fluid flow baffle elements 1102 includes a linear array of tubing-support grooves 1105, each one of which grooves 1105 is longitudinally aligned with corresponding grooves 1105 of adjacent baffle elements 1102 to receive and position a respective individual semi-rigid fluid flow tubing 1106 of the second tubing set 1224.
  • Each individual fluid flow tubing 1106 of the second tubing set 1224 extends from the top of the cooling fluid circulation * chamber 1101 to the bottom thereof, in a spaced, parallel relation to the other individual fluid flow tubing 1106, and is supported by a set of the aligned grooves 1105.
  • Cooling fluid inlet/outlet ports 1107 are arranged at each of the top and bottom of the fluid circulation chamber 1101, respectively and are each coupled to a tube fitting 1108.
  • the tube fittings 1108 provide fluid communication with an external source of cooling fluid (not illustrated) , such as, for example, a NESLAB CFT Series Refrigerated Recirculating Cooler.
  • the source of cooling fluid is operated to continuously circulate a cooling fluid around the baffles 1102 and the individual tubing 1106 to remove, e.g., 250 watts of power from the solution circulating in the individual tubing 1106 of the second tubing set 1224.
  • the tubing 1106 of the second tubing set 1224 is bundled, at each of the top and bottom of the heat exchanger 1100' into cover tubing 1114 and coupled to connectors 1115 arranged at either end of the second tubing set 1224.
  • the cover tubing 1114 can also be made of foamed material for thermal insulation.
  • Each connector 1115 includes an array of openings 1116 that match, e.g., the array of openings of the male-type connector 1209 of the first tubing set 1208.
  • the connectors 1115 can be of a similar construction as the connectors 1209, however, each opening 1116 of each of the connectors 1115 is flush to the connecting surface 1117 of the respective connector 1115, as are the in and out-flow ports 71, 72, to provide a female-type connector.
  • Each connector 1115 is also provided with connector openings 1118 that align with corresponding connector openings, e.g., the openings 1212 of the male-type connector 1209 of the first tubing set 1208 that is not coupled to the out-flow ports 72 of the electrophoresis chamber assembly 49 (See Fig. 15) such that a pair of connectors 1115, 1212 can be coupled together and fastened by, e.g., knurled nuts, to provide fluid communication between the tubing 1214 of the first tubing set 1208 of the peristaltic pump 500' and the tubing 1106 of the second tubing set 1224 of the heat exchanger 1100'. Either one of the connectors 1115 can be paired with the male-type connector 1209.
  • the lower connector 1115 is coupled to the connector 1209 due to the proximity of the lower end of the heat exchanger 1100' to the peristaltic pump 500'.
  • the other female-type connector 1115 of the heat exchanger 1100' is coupled to a male-type connector 701 of a third tubing set 702, which connector 701 is of similar construction as the male-type connector 1209 (See Figs. 15a, 16) .
  • the third tubing set 702 extends from the upper end of the heat exchanger 1100' through an opening in the mounting wall 1205 (not specifically illustrated) to the bubble trap apparatus 700' (See Figs. 8, 23, 24) .
  • the third tubing set 702 comprises a plurality of individual tubing 703, equal in number to the number of tubing in each of the first and second tubing sets 1208, 1224, e.g., 30.
  • Each individual tubing 703 is coupled in a telescoping relation to a tube 704 of a respective one of a plurality of bubble and pulse trap, . reservoir devices 705.
  • each bubble and pulse trap, reservoir device 705 is formed to a tube 706 which fits within an individual tubing 707 of a fourth tubing set 708.
  • the fourth tubing set 708 extends from the bubble and pulse trap apparatus 700' through an opening (not specifically illustrated) in the mounting wall 1205, down the heat - exchange compartment and out an opening 710 of the mounting wall 1205 adjacent the in-flow ports 71 of the electrophoresis chamber assembly 49 (See Fig. 8) .
  • the end of the fourth tubing set 708 adjacent the in-flow ports 71 is provided with a male-type connector 709 adapted to mate with the female-type connector surface defined by the array of in-flow ports 71 of the electrophoresis chamber assembly (See Figs. 4 and 8) .
  • the connector 709 is fastened to the connector studs 74 by knurled nuts 1213 in a similar manner as the male- type connector 1209 is fastened to the out-flow ports 72 (See Fig. 8) .
  • the plurality of bubble and pulse trap, reservoir devices 705 are housed in a linear array within a block housing body 711.
  • the body is provided with side openings 711a, 711b, to permit access by the tubing of the tubing sets 702, 708 for coupling to the inlet and outlet tubes 704, 706, as illustrated in Fig. 24.
  • a plurality of threaded studs 712 extends upwardly from the housing body 711. All of the bubble and pulse trap, reservoir devices 705 are covered by a gasket 713 made from a sel -sealing elastomer such as silicon sheet.
  • the gasket 713 includes openings (not specifically illustrated) arranged to pass over the threaded studs 712.
  • the elastomer gasket 713 defines a plurality of septa, one over an open top 705a of each bubble and pulse trap, reservoir device 705.
  • the gasket 713 therefore permits puncture by, e.g. a syringe for addition or withdrawal of small fluid volumes to or from any one of the devices 705.
  • a cover 714 is placed over the threaded studs and the gasket 713 and fastened by knurled nuts 715 threadily received on the threaded studs 712.
  • the cover 714 can be provided with a plurality of openings (not specifically illustrated) , one over each device 705 for passage of a syringe through the gasket 713, as discussed above.
  • each bubble and pulse trap, reservoir device 705 is governed by the placement of the outlet 704.
  • pulse trapping is obtained to abate pulsations caused by the peristaltic pump 500'.
  • a T-coupling 716 can be inserted into one or more tubing 707 of the fourth tubing set 708 to provide a coupling to an access tubing 717 including a Luer adapter 718.
  • the access tubing 717 permits selective fluid communication with the solution flow within the system for taking of samples for measurements of, e.g., pH levels.
  • the tubing sets 1209, 1224, 702, 708 provide continuous closed individual flow channels or loops from the out-flow ports 72 of the electrophoresis chamber assembly 49, through the peristaltic pump 500', heat exchanger 1100' and bubble trap apparatus 700', and back to the in-flow ports 71 for rapid continuous recirculation of the solution containing biological materials to be separated through the electrophoresis chamber.
  • the sequence of components 500', 1100', 700', 49 can be varied by changing the couplings between the various tubing sets.
  • a fill/wash connector 86 which comprises a male-type connector 87 coupled to a tubing 88.
  • the male-type connector 87 is provided with a gasket 89 received within a groove 90 formed in a connection surface 91 of the connector 87.
  • the gasket 89 circumscribes a fluid cavity 92 also formed within the connection surface 91 and arranged and configured to generally coincide in shape and dimension to the outline of the array of in-flow ports 71 of the electrophoresis chamber assembly 49.
  • the knurled nuts 1213 of the connector 709 are rotated to unfasten and remove the connector 709 from the in-flow ports 71.
  • the connector 87 is then placed over the in-flow ports 71 such that the connector studs 74 of the chamber-forming member 50 (See Figs. 4 and 8) are received through connector openings 93 formed through the connector 87.
  • Knurled nuts 1213 are then used to fasten the connector 87 to the electrophoresis chamber assembly 49.
  • the fluid cavity 92 overlies the in-flow ports 71 when the connector 87 is fastened to the connector studs 74 and the gasket 89 forms a leak-tight seal around the in-flow ports 71 and the fluid cavity 92.
  • An inlet port 94 including a threaded internal surface, is formed from the cavity 92 to the rear surface of the connector 87 and receives a threaded end of a coupling adapter 95.
  • the coupling adapter 95 is provided with a connection tubing 96 that is inserted into the tubing 88 to form a fluid coupling between the tubing 88 and the cavity 92. Accordingly, the solution can be filled into the system through the tubing 88. After the system is filled with the solution, the connector 87 is unfastened and removed, and the connector 709 is again fastened to couple the fourth tubing set 708 in a fluid coupling with the in-flow ports 71.
  • the connector 87, tubing 88 assembly can also be used to flush the system with a cleaning fluid, when desired.
  • Total fluid capacity of the system which may be, e.g. 80-500 ml, is determined by the volume of the electrophoresis chamber and that of the external loops of the tubing sets, which can incorporate variable volume expanders.
  • the bubble traps in the above-identified embodiment are arranged to be of a volume sufficient to provide a reservoir for each loop of the tubing sets.
  • chamber volume is only a minimal faction of total volume.
  • the focusing process can best be visualized using colored samples, such as red hemoglobin, blue-stained albumin, or a suspension of green chloroplasts from plant leaves.
  • colored samples such as red hemoglobin, blue-stained albumin, or a suspension of green chloroplasts from plant leaves.
  • a uniform colored film of flowing liquid is observed.
  • current to the electrodes 82 progressive focusing is seen.
  • the pH gradient originates at the electrodes and progressively moves inwards.
  • initial clearing of color from both sides of the chamber will be seen.
  • the pi of the albumin is lower than that of hemoglobin, a faster blue and slower red band will be seen at the cathodic side of the chamber, the reverse being visible at the anodic side.
  • the advancing bands of the two proteins will merge at their respective pH's in the chamber.
  • each protein In each pass through the chamber, the movement of the protein is imperceptible, as there is only a small shift of protein distribution in the short residence time.
  • each protein When final focusing is achieved, each protein will be confined to a narrow band of colored material in continuous recirculation. Visually, the stability of the fluid is such that the bands appear stationary and their flow is visible only by careful observation.
  • Chamber A 40 matched entry and exit ports, chamber height 30 cm, width 6 cm, and variable depth (gap between front and back plate) between 0.025 and 0.15 cm. Experiments with this chamber have determined that optimal depth is of the order of 0.075 cm.
  • Chamber B 12 matched ports, height 20 cm, width 2.8 cm and depth 0.075 cm.
  • Chamber C 48 matched ports, height 30 cm, width 6 cm and depth 0.050 cm or 0.075 cm. Only chamber C had the external cooling, requiring frontal electrode compartments, as illustrated in Fig. 3.
  • the sample collection device 400' generally comprises two side walls 401 that are held together by a distribution plate 402 and a top plate 403.
  • the sides of the distribution plate 402 are received within recesses 404 formed in each of the side walls 401 and are fastened thereto by a set of screws 405.
  • the top plate 403 is fastened to the top of each side wall 401 by another set of screws 406.
  • a female-type connector 407 is mounted to a lower side of the top plate 403 by screws 408 received within suitable openings (not specifically illustrated) formed in the top plate 403 and threaded into opening 409 formed in the connector 407.
  • the top plate 403 also includes an opening 411 to expose a connection surface 410 of the connector 407.
  • the connection surface 410 includes an array of openings 412 arranged to mate with the openings of the male-type connector 709 (See, as an example, the illustration of Fig. 15a) .
  • Each of the openings 412 communicates with a respective tubing 413 of a fifth tubing set 416.
  • the number of tubing 413 in the fifth tubing set 416 equals the number of tubing in the other tubing sets described above, e.g., 30.
  • the tubing 413 extends to and is received within respective openings 414 formed through the distribution plate 402.
  • the openings 414 of the distribution plate 402 are arranged in a predetermined array, as most clearly illustrated in Fig. 20.
  • the distribution plate 402 and side walls 401 define a chamber 415 of suitable dimensions to receive a test tube rack holding, e.g., 30 individual test tubes (not specifically illustrated) .
  • the predetermined array of openings 414 is arranged so that the end of each tubing 413 is spaced above a complementary test tube for collection of a fluid sample from one individual tubing 413.
  • the connector 709 is unfastened from the in-flow ports 71 (See Fig. 8) and placed in a mating relation to the connector 87 for fluid flow from the individual tubing 707 of the fourth tubing set 708, through the openings 412 and tubing 413 for collection in the individual test tubes.
  • Utilization of the apparatus for ITP is more complicated, as it requires the establishment of a counterflow of, e.g., the leading electrolyte within the electrophosesis chamber 49.
  • a pair of valved input/output tubing 1301, 1302 can be provided at, for example, each of a left-most and right most tubing of one of the tubing sets 1300, for establishing the counterflow (see fig. 6) .
  • the chamber is filled with the leading electrolyte, as is the appropriate electrode compartment — the anodic for separation of negatively charged species and the cathodic for positively charged species.
  • the remaining electrode is filled with the terminating electrolyte.
  • the leader is replaced by the terminator in a few (2-5) of the recirculating channels closest to the terminator.
  • the sample to be separated is then injected into one or more of the circulating loops between leader and terminator and the power is applied. This injection can be in a single bolus, or can be continued for as long as it is desired during the run.
  • the samples will migrate, displacing the leader, and will be followed by the terminator.
  • sample components will sort themselves according to their respective electrophoretic mobilities in the chosen electrolyte system.
  • the advancing band of the fastest sample can be monitored in a variety of ways: color (if visible) , absorption in the ultraviolet, conductivity, or electric potential gradient.
  • the potential is uniform within the leader zone, but has a sharp stepwise increase at every interface between successive zones, such as leader-sample, sample-sample, or sample- terminator interface.
  • Monitoring of ultraviolet absorption is effective, because it is universally applicable.
  • a UV monitor such as an LKB, AB (Sweden) 2138 Uvicord S monitor has been found to be suitable for the monitoring function.
  • a colored sample is very helpful, however, for the initial setting of approximate parameters of flow and electric power.
  • the balancing of the input flow of the leader can be adjusted either manually, to maintain constancy of the position of the leader-sample interface, or can be automated.
  • Electronic circuitry has been constructed which senses the electric potential between each successive pair of the exit ports of the chamber, thus monitoring the whole process. When the chosen position of the critical interface is neared, counterflow is initiated. At that time the circuitry can be employed in two alternate modes: it either controls the applied power at constant leader counterflow, or adjusts the counterflow at constant power. Obviously, both have to be preset manually at approximately proper relation for the system to work effectively. Also proposed is an array of sensors monitoring the ultraviolet absorption in all recirculating channels. The logic of the operation is controlled by a data processor 1000 (Fig. 6) , such as a personal computer.
  • the leading electrolyte contained 5mM Cacodylic acid, adjusted to pH 7.4 using Tris (hydroxymethyl) aminomethane (TRIS) .
  • the terminator was 5mM B-alanine, adjusted to pH 9.2 with barium hydroxide.
  • the sample was a mixture of hemoglobin and albumin, 250 mg each, added with an appropriate spacer, i.e. 500 mg of the dipeptide Gly-Gly. Processing revealed a very sharp recirculating leading zone of albumin, separated from that of hemoglobin by a colorless zone of Gly-Gly. The zone was virtually immobilized by applying counterflow of the leading electrolyte. As in all experiments, collection of separated proteins zones was accomplished by stopping the recirculation and the applied power and collecting the contents of all the external recirculation loops. The contents of the cell itself was discarded.

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Abstract

Un système de purification de protéines basé sur la focalisation iso-électrique et l'isotachophorèse comprend une chambre de séparation (100) présentant des ensembles appariés d'orifices d'admission et de sortie. Les orifices de sortie sont reliés aux orifices d'admission par des modules de tubes (1300) comprenant des ensembles de boucles individuelles d'écoulement de fluide, lesquelles sont couplées sélectivement et de manière amovible les unes aux autres, ainsi qu'aux orifices d'admission et de sortie de la chambre de séparation (100). Les modules de tube (1300) sont agencés pour s'étendre dans une pompe à canaux multiples (500), destinée à produire une pression fluidique dans chacune des boucles individuelles d'écoulement de fluide et dans un échangeur thermique (1100), afin d'extraire de manière régulée de la chaleur de chacune des boucles individuelles d'écoulement de fluide.
PCT/US1990/007409 1989-12-13 1990-12-13 Systeme de purification de proteines base sur la focalisation iso-electrique et l'isotachophorese WO1991010129A1 (fr)

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US7316771B2 (en) 2000-09-21 2008-01-08 Becton, Dickinson And Company Medium for analytic and preparative electrophoresis
US7399394B2 (en) 2000-12-18 2008-07-15 Becton, Dickinson And Company Electrophoresis device, electrophoresis method using an electrophoresis device and use of the electrophoresis device
US7491304B2 (en) 2000-12-18 2009-02-17 Becton, Dickinson And Company Carrierless electrophoresis process and electrophoresis device for carrying out this process
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CN113631518A (zh) * 2018-12-29 2021-11-09 普里斯蒂南有限责任公司 用于通过重结晶净化水的系统以及用于实现其的热交换装置（变形）
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