EP1235634A1 - Separation et detection bidimensionnelles en solution de substances amphoteres - Google Patents

Separation et detection bidimensionnelles en solution de substances amphoteres

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Publication number
EP1235634A1
EP1235634A1 EP00978541A EP00978541A EP1235634A1 EP 1235634 A1 EP1235634 A1 EP 1235634A1 EP 00978541 A EP00978541 A EP 00978541A EP 00978541 A EP00978541 A EP 00978541A EP 1235634 A1 EP1235634 A1 EP 1235634A1
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EP
European Patent Office
Prior art keywords
analyte
cathode
anode
transverse
plate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00978541A
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German (de)
English (en)
Other versions
EP1235634A4 (fr
Inventor
James T. Champagne
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PROTEOTOOLS LLC
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Individual
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Publication of EP1235634A1 publication Critical patent/EP1235634A1/fr
Publication of EP1235634A4 publication Critical patent/EP1235634A4/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
    • 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
    • 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
    • G01N27/44704—Details; Accessories
    • G01N27/44743—Introducing samples
    • 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]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
    • G01N30/463—Flow patterns using more than one column with serial coupling of separation columns for multidimensional chromatography

Definitions

  • This invention relates to a method and apparatus to separate and detect amphoteric substances, such as proteins, in complex mixtures in two or more dimensions such that all component analytes are segregated to a predetermined level of resolution in multiple channels.
  • Amphoteric molecules are defined by a plurality of positive and negative functional chemical groups located on an accessible surface that can be titrated or buffered by changes in the pH of the solution environment in which they find themselves. Amphoteric entities lose negative and/or gain positive charges in a more acidic environment. Conversely, amphoteric entities lose positive and/or gain negative charges in a more basic environment. When placed in an externally applied direct current electrical field, within a pH gradient environment, in which the acidic pH region is aligned with the anode and the basic region with the cathode, amphoteric entities will migrate, based on their net charge, towards one of the electrodes.
  • This denaturation is provided by rapidly diffusing a detergent, typically sodium dodecyl sulfate (SDS) into the gel matrix, such that all amphoteric molecules are coated with an overwhelming negative charge before they can diffuse away from their isoelectric position in the gel, thus rendering them non-amphoteric.
  • SDS sodium dodecyl sulfate
  • the SDS treated isoelectric focusing acrylamide matrix typically in the shape of a tube or strip, is quickly brought in contact with a second acrylamide gel matrix, typically in the shape of a slab, by application along one side.
  • An electrical potential is applied to the second acrylamide gel perpendicular to the isoelectric focusing gel strip so as to transfer and further separate the denatured molecules.
  • the native charges of the molecule masked by a constant amount of negatively charged SDS per weight of protein, the molecules move through the second gel matrix according to their molecular weight alone, towards the anode.
  • the technique of 2D-gel electrophoresis is difficult to perform in a reproducible manner (see Corbett et al., Positional reproducibility of protein spots in two-dimensional polyacrylamide gel electrophoresis using immobilized pH gradient isoelectric focusing in the first dimension: an inter-laboratory comparison, Electrophoresis 1994, 15 1205-1211) and requires much manual manipulation in equilibrating and transferring the first dimension isoelectric focusing strip or tube gel onto the second dimension gel within a narrow time span. Since the separated proteins are embedded in the separating matrix at the end of the process, the available detection methods are limited to those methods that provide a captured image in two dimensions corresponding to the separation pattern of the proteins.
  • the current invention achieves this goal of an on-line solution based multidimensional separation system. Furthermore, if the separation or detection methodology in this new method included the same two physio-chemical parameters as 2D-gel electrophoresis, i.e. isoelectric point and molecular weight, then a large body of existing archived data could be more easily correlated to any subsequent data. Under these conditions, the data derived from the current invention can be correlated to the large body of existing archived 2D gel data using computational reconstruction.
  • the general method of separation of molecules using various septa or membranes to divide an analyte stream into a series of parallel laminar flow channels with a typically transverse applied electrical field in order to transfer these molecules between channels is well established.
  • these methods of separation make no attempt to optimize the width or geometry of the channels containing the analyte molecules in order to achieve rapid separation equilibrium.
  • the typical widths of the laminar channels and the distances between the anode and the cathode are substantial.
  • the IsoPrimeTM multi-compartment electrofocusing unit (PI 8) manufactured by Hoefer Scientific Instruments, 654 Minnesota St., San Francisco, CA and based on US Patent No. 4,971 ,670 (Faupel et al.), provides a minimum of three laminar channels with a minimum volume of 8 mis within each chamber.
  • the ratio of separating membrane surface area to volume is 1 cm 2 / ml or less.
  • these existing separation methods typically utilize strategies of gradual equilibrium with re-circulation in which the sample is recycled through the separating apparatus in many passes obtaining partial separation with each pass. This is sometimes referred to as interative purification.
  • amphoteric analyte molecules When a transverse electrical field is applied, amphoteric analyte molecules will move through such membranes towards the electrode having the opposite charge from the charge on the amphoteric molecule. This electrophoretic movement will continue until the buffering pH of an incident membrane modifies the charge on the amphoteric molecule such that it no longer has an opposite charge from the charge of electrode to which it had been moving. The amphoteric molecule will then remain in one of the laminar spaces between membranes. The method recycles the sample through each channel many times and achieves gradual equilibrium over time scales of hours with a single stage of separation.
  • any given analyte molecule if randomly located in a laminar space far removed from the laminar space containing its isoelectric point, must travel a considerable distance through two or more membranes and perhaps through all the membranes by electromotive force.
  • the topographic arrangement and geometry are optimized to provide the minimum transfer distance and minimum number of membrane traverses for all the analytes in a complex mixture.
  • the maximum number of membrane traverses for any analyte molecule using the current invention is equal to the number of stages of segregation, which for the example of 2048 discrete channels is 11 , most analyte molecules will make fewer membrane traverses and some analyte molecules will, by chance, make none.
  • the current invention differs from the prior art additionally in the manner in which coulombic heating is dissipated.
  • U.S. Patent No. 5,160,594(Huff et al.) for instance, analyte streams are typically continuously and separately re-circulated into and out of each separation channel by hydraulic means while its composition slowly changes by lateral movement of analytes through the membranes.
  • the analyte stream itself provides heat dissipation by means of radiative or active external cooling during the time it is not within the electrophoretic field.
  • each buffering membrane and electrode is very large relative to the volume of the individual channels (typically 100 cm 2 / ml) and the distance that heat must flow to exit an individual channel is very short.
  • there is only a single separating membrane between each pair of electrodes and the analyte stream reaches isoelectric equilibrium across this buffering membrane within a single pass through the particular stage without recirculation, as in the prior art.
  • each electrode is provided with a separate cooling fluid flow to dissipate coulombic heat and to remove small molecules such as salts. The advantage of removing salts in isoelectric focusing is well established. For instance, In U.S. Patent No.
  • the invention comprises a method and an apparatus for the separation of amphoteric substances, such as proteins in two dimensions in a continuously flowing mobile phase in which the outflow is segregated into as many pH range sub-fractions as is desired.
  • Complex mixtures of soluble amphoteric substances represent a preferred sample to be analyzed by the present invention.
  • Said samples are applied to a first dimension separation means using a sample injection apparatus of the type typically used for sample application in chromatographic workstations.
  • sample injection apparatus are of the type having a sample loop or chamber pre-filled with the analyte sample such that it can be incorporated into the mobile phase stream by mechanical means.
  • a first dimension separation method can partition the various analytes between a stationary phase matrix and a liquid mobile phase using some physio-chemical parameter that varies among the various analytes. This provides a continuously outflowing stream in which the various analytes are distributed in retention times within the apparatus, thus having differential elution times.
  • the first dimension method for separation of the complex mixture is high-resolution ion exchange chromatography.
  • This separation method can include strong ion exchange matrices that can operate at extremes of pH such quaternary ammonium or sulfo-propyl ion exchange or weak ion exchange matrices such as diethylaminoethyl or carboxymethyl ion exchange.
  • the first dimension method for separation is high-resolution size exclusion chromatography, wherein the fractional volume of a stationary phase matrix accessible to a particular molecule is inversely related to the Stokes radius of said molecule that, in turn, is directly related to the molecular weight of said molecule.
  • the first dimension size exclusion separation method provides for a continuously outflowing mobile phase containing soluble analytes of comparable molecular size and molecular weight at any given moment.
  • the first dimensional separation provides a means of high resolution partitioning of the analyte molecules to achieve a maximum variation in elution times of said analyte molecules.
  • a further means is provided to introduce the partitioned outflowing mobile phase of the first dimensional separation means into a second dimensional segregating apparatus wherein a serial and parallel arrangement of a plurality of laminar manifolds is provided in a plurality of stages such that the continuously flowing mobile phase is subdivided into two discrete isoelectric point (pH) ranges of amphoteric molecules in two channels within each stage.
  • the outflow from each said channel is connected to the inlets of two or more channels of a subsequent stage manifold.
  • This configuration provides for further binary separations, subdividing the first two discrete isoelectric point pH ranges into four pH ranges. Further stages generate a "power of two" increase in the number of ever narrower isoelectric point pH ranges (i.e. 8 pH ranges, 16, 32, 64 etc.)
  • a preferred embodiment of the current invention provides for a concomitant "power of two" decrease in the number or volume of laminar channels per stage dedicated to a particular pH segregation (see table 1 ).
  • This preferred embodiment has the advantage that as the isoelectric point range narrows with each stage and the approximate number of species of amphoteric molecules within each range decreases concomitantly; the apparatus provides roughly equal amounts of analyte per unit of laminar manifold capacity.
  • the analyte molecules will be distributed by decreasing molecular weight with time and by isoelectric point in discrete pH range channels.
  • the outflowing streams of amphoteric molecules will be distributed by isoelectric point in discrete pH range channels and one other physio-chemical separating parameter.
  • a means is provided to detect and collect said analyte molecules from all the multiple discrete pH range channels simultaneously.
  • Said means of detection can operate continuously at the time of outflow from the second dimension separation means. It can also be delayed in time or rate of detection from the rate of outflow of the analyte stream.
  • Said means of detection includes all of the established methods of flow cell detection or immobilized sample detection including but not limited to; optical density detection, dynamic and static fluorescence detection, conductivity detection, electrochemical detection, dynamic and static light scattering detection and mass spectroscopic detection.
  • the data can be plotted to create a minimum of a two-dimensional map of the analytes.
  • this two- dimensional map can be comparable to the two-dimensional maps of 2D-gel electrophoresis except that the samples are not embedded in a matrix.
  • TABLE 1 is a representative example of a preferred arrangement of stages and the replicate number of manifolds for each stage resulting in a 2048 channel pH separation.
  • Stage # pH # Replicate # Outflow Total ⁇ total n membrane manifold plates pH ranges manifold plates manifold plates types /stage /stage /stage / stage by stage
  • FIG. 1 is a schematic representation of the various components of the method showing the overall relationship between the major components and the direction of flow of the continuous stream of analytes wherein the first dimension solution based separation method maintains a non-varying (isocratic) mobile phase composition.
  • FIG. 2 is a schematic representation of the various components of the method showing the overall relationship between the major components and the direction of flow of the continuous stream of analytes wherein the first dimension solution based separation method uses a time variant (gradient) mobile phase composition.
  • FIG. 3A is a schematic cross sectional representation of a preferred arrangement of a single cathode plate assembly showing channel and fluid connections.
  • FIG. 3B is a schematic vertical cross section through one typical cathode plate showing the relative position in the vertical dimension of transverse channels for the collection of outflowing analyte streams.
  • FIG. 3C is a schematic cross sectional view cut vertically and horizontally through a single manifold assembly showing the outflowing cathodic analyte stream bypassing an end joint through a short transverse channel and emptying into a pooling cathodic analyte stream transverse channel.
  • FIG. 4 is a schematic cross sectional representation of a preferred arrangement of a single anode plate assembly showing channel and fluid connections.
  • FIG. 5 is a schematic cross sectional representation of a preferred arrangement of a single semi-permeable buffering membrane support assembly showing a plurality of semi-permeable buffering membranes and fluid connections.
  • FIG. 6A is a schematic cross sectional representation of a preferred arrangement of a single spacer gasket used to form and maintain the laminar analyte flow channels on the anodic side of a buffering membrane wherein said channels are in the higher of two positions as aligned with channels formed into said anode.
  • FIG. 6B is a schematic cross sectional representation of a preferred arrangement of a single spacer gasket used to form and maintain the laminar analyte flow channels on the cathodic side of a buffering membrane wherein said channels are in a lower of two positions as aligned with channels formed into said cathode.
  • FIG. 7A is a schematic cross sectional representation of a preferred arrangement of a single spacer gasket used to form and maintain the laminar analyte flow channels on the anodic side of a buffering membrane wherein said channels are in the lower of two positions as aligned with channels formed into said anode.
  • FIG. 7B is a schematic cross sectional representation of a preferred arrangement of a single spacer gasket used to form and maintain the laminar analyte flow channels on the cathodic side of a buffering membrane wherein said channels are in the higher of two positions as aligned with channels formed into said cathode.
  • FIG. 8 is a cross sectional representation of a preferred arrangement of a single semi-permeable buffering membrane manifold assembly; comprised of a cathode plate assembly (FIG. 3), a low molecular weight cutoff semi-permeable membrane (13b), a cathode side spacer gasket (FIG. 7B), a semi-permeable buffering membrane assembly (FIG. 5), an anode side spacer gasket (FIG. 6A), a second low molecular weight cutoff semi-permeable membrane (13a) and an anode plate assembly (FIG. 4).
  • a single manifold assembly shares a cathode plate on one side and an anode plate on the other side with adjacent manifold assemblies.
  • FIG. 9 is a schematic representation of a preferred arrangement of a further assembly of individual manifold assemblies (11) into two representative stages of binary segregating manifolds; wherein individual manifold assemblies are overlapped between stages to provide connecting channels between stages.
  • FIG. 10 is a schematic representation of a preferred arrangement of the overall assembly of individual binary pH segregating stages into a continuous flow multichannel second dimension separating means comprised of a inflow pressure cap, a plurality of individual separating stages, a outflow pressure cap, a means of simultaneous collection of fractions from each channel and a means of immobilizing part of the analyte stream for detection.
  • first and second dimensional separation means are provided in the current invention to partition a complex mixture of analyte substances.
  • the first dimensional separation means segregates the analyte stream relative to some physio-chemical parameter in order to vary the elution times of said analyte molecules.
  • the second dimensional separation means further segregates the outflowing first dimensional analyte stream into a predetermined number of isoelectric point (pH) ranges.
  • pH isoelectric point
  • a means of delivering a mobile phase fluid is provided.
  • Said mobile phase fluid possesses suitable buffering and other physio-chemical characteristics, of the type typically found in biological purification media, and is delivered at a pressure suitable to overcome the pressure drop during flow through a first and second dimension separation apparatus.
  • the corrosive nature of said biological purification media on metal parts is such that a means of delivering the mobile phase without contact to the precision moving parts of a high pressure pumping means is desirable, but optional.
  • FIG.1 illustrates the relationship between the various components of the overall method in one embodiment in which the mobile phase used for the method maintains a non-varying composition with time and isolates said mobile phase from the high pressure pumping means.
  • a syringe-like cylindrical reservoir assembly (4) is provided to encapsulate the full volume of mobile phase that will be needed for the complete separation.
  • Said assembly is comprised of a cylindrical reservoir(5) and is divided into two chambers (7a, 7b) by a piston (6).
  • a high pressure pumping means (3) is attached to the upstream chamber (7a) of said reservoir with a high pressure tube (2b) in order to deliver non-corrosive drive fluid under high pressure to said chamber from a drive fluid storage reservoir (1) through tube (2a).
  • Such a non- corrosive drive fluid is typically pure water without buffers or other solutes.
  • Said mobile phase fluid, containing solutes for biological media is filled into the downstream chamber (7b) of said reservoir for delivery, under pressure, to the first dimension separating means (10) through a sample introduction apparatus (8) described in detail below.
  • the high pressure mobile phase pumping means (3) can be a bio- compatible type pump in which the moving parts of the pumping mechanism are designed to withstand the corrosive nature of typical biological buffer mobile phases.
  • the mobile phase reservoir assembly (4) is directly connected to the upstream side of a bio-compatible pumping means (3) and the outflow of said pumping means is directly connected to a sample introduction apparatus (8) described in detail below.
  • a means for the introduction of an analyte sample having an equivalent volume or a smaller volume than the volume of the first dimension stationary phase matrix is provided (8).
  • said means of sample introduction is provided by a sample reservoir or sample loop (9) that can be incorporated into the mobile phase fluid stream, downstream from said mobile phase reservoir by a mechanical means equivalent to the existing art of two position four- way valves, such that the sample reservoir or sample loop in one said position is fluidly connected to an inlet and outlet port for sample loading into said sample reservoir or sample loop.
  • the mobile phase stream is fluidly connected on the upstream side to the downstream side of the reservoir or pumping means with tube (2c), and is connected on the downstream side to the first dimension separating means with tube (2d).
  • a means is provided to contain a first dimension partitioning stationary phase matrix in a cylindrical chamber or column (10) of the type typically used for chromatography, so as to allow the passage of the mobile phase over said partitioning matrix in a controlled manner and to retain said matrix within said cylindrical chamber or column while allowing for outflow on the downstream side of said cylindrical chamber.
  • Said cylindrical chamber or column (10) is provided with sufficient strength and fluid integrity so as to resist the level of pressure needed to move the mobile phase through both the first and second dimension separating means, while maintaining the partitioning matrix against pressure compression.
  • the first dimension partitioning matrix is a packed microporous or nanoporous granular size exclusion medium of the type typically used for high resolution separation of biological macromolecules, such as proteins
  • an analyte molecule having a large stokes radius corresponding to a high molecular weight will be excluded from all or most of the fractional volume of the interior of said nanoporous granule by steric hindrance.
  • Analyte molecules with smaller stokes radii will be excluded from a diminishing fraction of the interior volume of said nanoporous granule. The result is that the total internal and external volume that a given analyte molecule can access determines the time required to traverse said column. This is known as the art of size exclusion chromatography.
  • a single mobile phase reservoir provides a non-varying mobile phase composition.
  • FIG. 1 further depicts, in a schematic fashion, the arrangement and functioning of three stages of binary pH range segregation within the second dimension separation means.
  • the analyte stream of the first dimension separation means located within high pressure connecting tube (2e), is delivered to the first stage (11) of the second dimension separation means so as to enter the laminar analyte flow channel on the anodic side (16a), and the laminar analyte flow channel on the cathodic side (16b) of a semi-permeable pH buffering membrane (15).
  • Said anodic side laminar flow channel (16a) is bounded on the anode (12) face by a different type of semi- permeable membrane (14a) that allows the passage of low molecular weight solutes having molecular weights below the molecular weight of any of the analyte molecules to be analyzed, typically about 500-2000 Daltons.
  • the cathodic side laminar flow channel (16b) is bounded on the cathode (13) face by another low molecular weight cut-off (about 500-2000 Daltons) semi-permeable membrane (14b).
  • a direct current electrical field potential is applied between the anode(12) and the cathode (13) so as to induce electrophoretic migration of the analyte molecules within said channels.
  • the semi-permeable pH buffering membrane (15), having immobilized buffering moieties with a specific aggregate pK value, provides a barrier to said electrophoretic migration (pH 7 for example in the first stage as depicted in FIG.1).
  • Analyte molecules that start out in the anodic side laminar flow channel (16a) will move through said semi-permeable membrane into the cathodic side laminar flow channel (16b) only if they retain a net positive charge in the buffering zone of said membrane (15) and will continue to migrate toward the cathode (13).
  • analyte molecules that start out in the cathodic side laminar flow channel (16b) will move through said semi-permeable membrane into the anodic side laminar flow channel (16a) only if they retain a net negative charge in the buffering zone of said membrane (15) and will continue to migrate toward the anode (12).
  • Analyte molecules that start out in a laminar space pH range that contains their isoelectric point will develop a net repulsive charge within the buffering zone of the semi-permeable membrane and will be prevented from passing through the membrane.
  • the migrating analyte molecules will either lose their net charge when outside of the buffering zone of said buffering membrane (15) or will be prevented from further migration towards the electrodes by the low molecular weight cut-off membranes (14a) and (14b).
  • the result of the said electrophoretic migration is a segregation of the entire analyte stream into binary pH range fractions during a single passage through a manifold assembly.
  • non-amphoteric ionic solutes within the analyte stream will electrophoretically migrate through said low molecular weight cutoff membranes (14a) and (14b) into a space between said low molecular weight cutoff membranes and the electrodes (12) and (13), thus segregating said non- amphoteric ionic solutes from the analyte stream in a process analogous to the art of electrodialysis.
  • These analyte streams are further segregated into binary pH range fractions during a single passage through the second stage (18) in the same fashion as in the first stage.
  • the outflowing analyte laminar flow stream (16e) will contain only analyte molecules with an isoelectric point below pH 4.5.
  • the analyte laminar flow stream (16f) will contain only analyte molecules with an isoelectric point between pH 4.5 and pH 7.
  • analyte laminar flow stream (16d) will contain only analyte molecules with an isoelectric point between pH 7 and pH 9.5
  • analyte laminar flow stream (16c) will contain only analyte molecules with an isoelectric point above pH 9.5.
  • the two binary segregating manifolds shown in the second stage (18) share a common cathode, thus have reversed polarities but function in exactly the same manner.
  • FIG.2 illustrates the relationship between the various components of the overall method in another embodiment; wherein the mobile phase the first dimension solution based separation method uses a time variant (gradient) mobile phase composition and the mobile phase fluid is isolated from the pumping means in the same fashion as is shown in FIG.1.
  • a first dimensional separating means using such a time variant mobile phase composition includes but is not limited to ion exchange chromatography; wherein the mobile phase composition would increase in soluble ionic strength with time, forming a gradient, in order to elute ionically bound analyte molecules from a stationary phase within the first dimensional separating means.
  • two syringe-like reservoir assemblies (4, 4") are provided to encapsulate the full volume of mobile phase that will be needed for the complete separation.
  • the composition of the mobile phase fluid in each said reservoir would vary and represent two extremes of composition that can be combined in a controlled fashion by varying the flow rate of delivery of each said mobile phase fluid.
  • Said reservoir assemblies (4, 4') are each comprised of cylindrical reservoirs (5, 5') divided by pistons (6, 6') into two chambers (7a, 7b) and (7c, 7d) respectively.
  • High pressure pumping means (3,3') withdraw a non-corrosive drive fluid from a drive fluid storage reservoir (1) through tubes (2a, 2f).
  • Said high pressure pumping means are attached to the upstream chambers (7a, 7c) of each cylindrical reservoir, and use high pressure tubes (2b, 2g) to deliver said non-corrosive drive fluid at a high pressure to said upstream chambers.
  • a non-corrosive drive fluid is typically pure water without buffers or other solutes.
  • the downstream chambers (7b, 7d) of each cylindrical reservoir are filled with said mobile phase fluids containing solutes for biological media.
  • the mobile phase fluids are delivered, under pressure, to a mobile phase mixing means (20) through high pressure tubes (2h, 2i), wherein the mobile phase fluids from each reservoir are combined in a predetermined time varying ratio.
  • each reservoir assembly (4, 4') to the upstream side of bio-compatible versions of each said high pressure pumping means (3, 3') and connect the downstream side of each said pumping means (3, 3") to the aforementioned mobile phase mixing means (20) wherein the mobile phase fluids from each reservoir are combined in a predetermined time varying ratio in a similar fashion to the arrangement depicted in FIG. 2.
  • said combined mobile phase fluid is delivered through a high pressure tube (2c) to a sample introduction means (8) that is identical to that depicted in FIG. 1, having a sample reservoir or sample loop (9) that can be incorporated into the mobile phase fluid stream in the fashion described in FIG.1.
  • High pressure tube (2d) delivers the mixed mobile phase fluids containing the incorporated sample to the upstream side of the first dimensional separation means (10).
  • the configuration of the first and second dimension separation means depicted in FIG.2 is identical to that described for the arrangement depicted in FIG. 1.
  • the first dimension partitioning matrix is a packed microporous granular media using ionic, hydrophobic or other physio-chemical differences in the analyte molecules for partitioning
  • said analyte molecules will be distributed in retention time in the first dimensional separating means in accordance with the partition coefficient of each analyte molecule between the stationary phase matrix and the mobile phase either under non- varying mobile phase composition (isocratic conditions) as in FIG. 1 or under varying mobile phase composition (gradient conditions) FIG. 2.
  • FIG. 2 further depicts (in a schematic fashion identical to the arrangement and functioning of three stages of binary pH range segregation shown in FIG. 1) a second dimension separation means. It is likewise to be understood that, in FIG. 2, as in FIG.
  • FIG.3 illustrates a preferred configuration of fluid channels, electrical connections and overall shape for a single cathode plate assembly (21).
  • said cathode plate assembly would be about 2-20 cm in height and about 2-20 cm in width overall.
  • Each said cathode plate assembly consists of a rigid electrically conductive cathode plate (13) and a rigid non-electrically conductive support plate (22).
  • the thickness of said cathode plate (13) and said support plate (22) are identical and typically would be about 0.05-0.2 cm.
  • said cathode plate (13) is molded from a rigid electrically conductive polymer composite so as to provide a series of channels on each side when assembled with various other components into a manifold assembly.
  • Said molding material should provide for a high modulus of elasticity in bending, a low coefficient of thermal expansion and a conducting surface that is resistant to electrochemical corrosion. It is desirable that such electrode material be easily formed into relatively complex shapes.
  • Such rigid electrically conductive polymeric molding materials include but are not limited to compression molded high performance composite thermoplastics such as polyimide or polyetherimide resins with graphite fiber reinforcement and with or without a corrosion resistant conducting surface coating of stainless steel, platinum or gold.
  • the corrosion resistant conducting surface coating can be limited in area to the plurality of areas in the electrode that correspond in lateral dimension to the channels.
  • said rigid support plate (22) is molded from similar resins formulated and reinforced so as to be non-electrically conductive.
  • Said cathode plate (13) is further provided on each side with a plurality of horizontal depressions or grooves (17), typically about 0.05-0.5 cm in height and slightly less in width than the width of said cathode plate.
  • the depth of said horizontal depressions or grooves (17) would typically be about 10% to 30% of the thickness of said cathode plate (13).
  • the horizontal depressions or grooves (17) on opposite faces of said cathode plate are offset in vertical position relative to one another. The amount of said offset is such that the overall plurality of horizontal depressions or grooves on one side of said cathode plate is shifted relative to the horizontal depressions or grooves on the opposite face by an amount equivalent to one half the typical on center vertical spacing of said horizontal depressions or grooves.
  • Said cathode plate (13) provides for a means of electrical connection to other cathode plates within a single stack and a means of supply and return of cooling fluid to each face of said cathode plate.
  • Said means are provided by two extension tabs of the cathode plate on the upper right corner and on the lower left corner beyond the body of the cathode plate with corresponding slots in the non-electrically conductive support plate (22).
  • a transverse hole (29) in the upper right corner tab provides for a supply of cathode cooling fluid to be delivered through a series of vertical depressions or grooves (26) to the plurality of cooling fluid channels formed from said horizontal depressions or grooves (17) upon assembly of the various components into a manifold assembly (11).
  • a transverse hole is defined here as a hole perpendicular to the face of a plate that penetrates from on side to the other. Such transverse holes can align along an axis so as to form transverse channels.
  • a transverse hole (27) in the lower left corner tab provides for the return of cathode cooling fluid to be collected from the same said series of cooling fluid channels connected through additional vertical depressions or grooves (26).
  • the aforementioned means of electrical connection for said cathode plate (13) is provided by an additional transverse hole (31) in the upper right corner extension tab above transverse hole (29).
  • the transverse hole (31) aligns with other transverse holes on adjacent cathodes to provide for the insertion of a conducting rod, such that a plurality of cathodes are connected to each other and to a source of electrical potential upon assembly of a complete stack of manifolds into a single stage of binary separation (see FIG. 9).
  • the rigid non-electrically conductive support plate (22) fits tightly around said cathode plate (13) so as to support and electrically isolate said cathode plate from other components of the apparatus.
  • Transverse holes (28), (30) and (32) in said support plate (22) align, on assembly, with matching transverse holes in anode plate (12) as shown in FIG. 4, thus providing equivalent cooling fluid supply, return and electrical connections for said anode plate (12).
  • the transverse holes (28), (30) and (32) in said support plate (22) ensure electrical and fluid isolation of said anode plate
  • FIG. 3 further illustrates a plurality of transverse holes (23) in a vertical column on the left end of support plate (22). Each hole (23) is aligned vertically with the upper half of each horizontal depression or groove (17) on the front face of cathode plate
  • Said holes (23), on assembly with the various other components of the apparatus, provide uninterrupted transverse channels through all or some portion of a complete stack of manifolds in a single stage of binary separation.
  • Said transverse channels, formed in part by holes (23), provide a means of integrating and combining the outflowing analyte streams from all equivalent cathodic side laminar flow channels (16b) as shown in FIG. 1 from all previous stage binary segregating manifold assemblies.
  • transverse channels formed on assembly are referred to as "cathodic analyte stream collection and distribution manifolds" and represent, in all cases, the more basic pH range analyte stream from each given segregating manifold.
  • Transverse holes located in various other components of the apparatus that are contiguous with transverse holes (23) and provide for transverse channels which will also be labeled (23) in this description.
  • the plurality of transverse holes (24) in a vertical column located to the right of and above each concomitant transverse hole (23), are vertically aligned with the lower half of each horizontal depression or groove (17) on the front face of cathode plate (13) and the upper half of each horizontal depression (17) on the back face of cathode plate (13) (see FIG. 3B).
  • Said holes (24) form a related set of transverse integrating channels on assembly with various other components of the apparatus.
  • Said formed channels are referred to as "anodic analyte stream collection and distribution manifolds" and by contrast collect in all cases the more acidic pH range analyte stream from each given segregating manifold.
  • FIG. 3 also illustrates a plurality of transverse holes (25) in a vertical column on the right end of support plate (22). Said plurality of transverse holes (25) provide fluid channels connecting the equivalent outflowing cathodic analyte streams on both sides of said cathode plate assembly (21) (see FIG. 3C) for subsequent connection to the "cathodic analyte stream collection and distribution manifolds" of the downstream binary pH range separation stage.
  • cathode support plate (22) also provides a means of connection of equivalent fluid channels in some adjacent columns or rows of transverse channels as is required to integrate equivalent outflowing pH range analyte streams.
  • Said means of connection is provided by non- penetrating short vertical or horizontal hole to hole depressions or grooves in said cathode support plate (22) similar to horizontal depressions or grooves (17) or vertical depressions or grooves (26).
  • Said hole to hole connecting depressions or grooves are formed in said cathode support plate (22) only as required in occasional positions and thus are not depicted in FIG. 3.
  • FIG. 3B illustrates a vertical cross sectional view of cathode plate (13).
  • This diagram depicts how transverse channel (23) for collecting the "cathodic (more basic) analyte stream” and transverse channel (24) for collecting the “anodic (more acidic) analyte stream” are aligned in the vertical direction with grooves (17) in the lower position on the front side of cathode plate (13) and with grooves (17) in the upper position on the back side of cathode plate (13).
  • FIG. 3C illustrates a cut away isometric cross sectional view of cathode plate (13), buffering membrane support plate (36) and anode plate (12) and their respective support plates showing how they assemble to provide collection channels for the cathodic analyte stream that route said streams around the end joints of the cathode support plate (22) where it butts against the cathode support plate (22) of the downstream stage.
  • This downstream cathode support plate is shown by a dotted line.
  • Spacer gaskets (40) see FIG. 6A) and (47) (see FIG. 7B) are also depicted.
  • This diagram depicts how the outflowing cathodic (more basic) analyte stream in the channel aligned with front side groove (17) in the cathode plate (13) passes through short transverse channel (25) and connects to the outflowing cathodic analyte stream in slot (48) of spacer gasket (47).
  • the said combined cathodic analyte streams continue through aligned transverse hole (39) in the buffering membrane support plate (36) and aligned transverse hole (42) in spacer gasket (40) into a short connecting groove (65) in the face of anode support plate (24).
  • FIG. 4 illustrates a preferred configuration of fluid channels, electrical connections and overall shape for a single anode plate assembly (33).
  • Anode plate assembly (33) is of identical size, shape and composition as cathode plate assembly (21) shown in FIG. 3.
  • Anode plate assembly (33) consists of anode plate (12) and anode support plate (34).
  • transverse hole (31) on cathode plate (13) a transverse hole (32) located on an extension tab on the upper left side of anode plate (12) provides a means of electrical connection, on assembly, between all said anode plates within a single stage of binary pH range segregation and electrical connection to a source of electrical potential.
  • transverse hole (28) located on an extension tab on the lower right corner of anode plate (12) provides for a supply of anode cooling fluid to be delivered through a series of vertical depressions or grooves(35) to the plurality of cooling fluid channels formed from the horizontal depressions or grooves (17).
  • transverse hole (30) located on the upper left corner below hole (32) provides an outlet channel for anode cooling fluid to be collected from said plurality of cooling fluid channels formed from the horizontal depressions or grooves (17).
  • Non-conducting anode support plate (34) fits tightly around said anode plate (12) so as to support and electrically isolate said anode plate (12) from other components of the apparatus.
  • Transverse holes (27) and (29) in the lower left and upper right corners of anode support plate (34) align with transverse holes (27) and (29) in cathode plate (12) so as to provide, on assembly, cathode cooling fluid supply and outlet channels.
  • FIG. 4 further illustrates a plurality of transverse holes (23) and (24) on the right side of anode support plate in vertical rows that align with similarly named transverse holes (23) and (24) in cathode support plate (22).
  • Said transverse holes (23) and (24) form, on assembly, the aforementioned outflowing analyte stream transverse integrating channels referred to as "cathodic analyte stream collection and distribution manifolds" and “anodic analyte stream collection and distribution manifolds"
  • anode support plate (34) also provides a means of connection of equivalent fluid channels in some adjacent columns or rows of transverse channels as is required to integrate equivalent outflowing pH range analyte streams.
  • Said means of connection is likewise provided by non-penetrating short vertical or horizontal hole to hole depressions or grooves in said anode support plate (34) similar to horizontal depressions or grooves (17) or vertical depressions or grooves (35).
  • Said hole to hole connecting depressions or grooves are formed in said anode support plate (34) only as required in occasional positions and thus, as in FIG. 3, are not depicted in FIG. 4.
  • Said reinforcement screen (38) consists of an inert open weave fabric screen or net with at least 90% open cross-sectional area that is embedded into said molded support plate (36) during molding to provide dimensional stability without substantially reducing the cross-sectional area of the openings provided for the buffering membranes.
  • said molded support plate (36) would be about 2-20 cm in height and about 2-20 cm in width overall and is provided with a plurality of horizontal transverse slots and a plurality of transverse holes that correspond in size and position with the matching plurality of horizontal depressions or grooves (17) and plurality of transverse holes (23) and (24) located on cathode plate (13) and anode plate (12).
  • the thickness of said molded support plate (36) typically would be about 0.05-0.2 cm.
  • said support plate (36) is molded from elastomeric or semi-elastomeric polymers to include but not limited to the classes; Polysiloxane, Polyisoprene, Polyisobutylene and Polysulfide. Said elastomeric polymers or semi-elastomeric polymers have physical properties that provide for the easy and tight sealing of molded support plate (36) against various other components of the apparatus.
  • molded support plate (36) provides for fluid connection channels across the thickness of said molded support plate using a plurality of transverse holes (39) located inboard on the right and left side of molded support plate (36).
  • Said transverse holes (39) are, in some instances, aligned with similar transverse holes, such as the plurality of transverse holes (36), in various other components of the apparatus.
  • Said transverse holes (39) provide fluid channels that distribute analyte streams only from one side of said molded support plate (36) or cathode support plate (22) to another within a single segregating manifold assembly.
  • Said fluid channels so formed by the plurality of transverse holes (39) do not provide a means of integrating multiple equivalent analyte streams as do transverse holes (23) and (24).
  • FIG. 5 further illustrates a plurality of semi-permeable buffering membranes (37) that are cast into the pre-formed plurality of horizontal slots in support plate(36).
  • Each said semi-permeable buffering membrane (37) is cast into an aforementioned slot so as to have the exact thickness of support plate (36), to have a smooth surface flush with said support plate (36) and to have macroporous reinforcement screen (38) embedded within said semi-permeable buffering membrane (37) so as to provide reinforcement such that the dimensional stability of the porous matrix is maintained without substantially reducing the porosity or capacity of the matrix to provide an immobilized buffering means.
  • Each said semi-permeable membrane (37) consists of a porous matrix having multiple buffering chemical moieties immobilized in specific titrated ratios within said matrix such that a particular aggregate pK value and buffering capacity is established in each said membrane (37).
  • said buffering chemical moieties are of the general class of acrylamide derivatives known as acrylamido-buffers and are co-polymerized into said semi-permeable membrane along with acrylamide and bis-acrylamide or other acrylamido derivatives of acrylamide during polymerization.
  • Said semi-permeable membrane (37) establishes the primary means of binary pH range segregation of the analyte streams.
  • Each aggregate pK formulation of a specific semi-permeable membrane (37) comprises a different pre-determined composition of acrylamido-buffers combined with acrylamide, bis-acrylamide and or acrylamido derivatives of acrylamide. It is a desired feature of the present invention that equivalent semi-permeable membranes (37) having the exact same aggregate pK value be cast from the same formulation batch of monomeric precursors at the same time in order to maintain uniformity in the subsequent semi-permeable membranes (37) and resultant pH range analyte fractions.
  • FIG. 6A illustrates a spacer gasket (40) to be located between the back side of molded support plate (36) and low molecular weight membrane (14a) that is adjacent to the front side of anode plate assembly (33)
  • Said spacer gasket (40) is comprised of a thin, semi-rigid sheet of non-conducting polymeric material of uniform thickness, wherein said thickness varies by not more than 5 % over the entire surface of said spacer gasket.
  • An example of such a material includes but is not limited to polyethylene terephthalate sheet.
  • said spacer gasket (40) would be about 2-20 cm in height and about 2-20 cm in width overall.
  • Said spacer gasket (40) is also provided with a plurality of horizontal transverse slots (41) and a plurality of transverse holes (23) and (24) that correspond in size and position with the matching plurality of horizontal slots (containing semi-permeable membranes (37)) and the plurality of transverse holes (23) and (24) in molded support plate (36) (see FIG. 5), anode support plate (34) (see FIG. 4) and cathode support plate (22).
  • the thickness of said molded support plate (36) typically would be about 0.01-0.1 cm.
  • Spacer gasket (40) provides a means of forming and maintaining the laminar flow channels, (16a) for example, on an anodic side of semi-permeable membrane (37) as illustrated schematically in FIG. 1.
  • Said horizontal transverse slots (41) are generally aligned with said semi-permeable membranes (37) so as to bring the analyte stream located in said transverse slots (41) in very close contact with the entire surface of semi-permeable membrane (37), anode plate (12) and cathode plate (13) thereby optimizing electrophoretic migration of amphoteric analyte molecules across said semi-permeable membrane (37).
  • Each individual horizontal transverse slot (41) provides a means of connecting one "cathodic analyte stream collection and distribution manifold" from a previous stage to said horizontal transverse slot (41) by a narrow extension of said slot so as to fluidly connect each said horizontal transverse slot (41) with one of the plurality of transverse holes (39) in the upstream side of molded support plate (36) (FIG. 5), thus providing inflowing cathodic analyte streams to said horizontal transverse slot (41).
  • a narrow extension of said slot (41) on the downstream side provides a means of connecting the acidic pH segregated outflowing analyte stream in said slot (41) to a subsequent stage of segregation by fluidly connecting each said horizontal transverse slot (41) with one "anodic analyte stream collection and distribution manifold" located on the downstream side of the manifold assembly through a horizontal groove in anode support plate (34) thus establishing a continuous flowing path for said analyte stream on the anodic side of semi-permeable membrane (37).
  • Spacer gasket (40) further provides a transverse hole (42) that is not fluidly connected directly or indirectly to transverse slot (41) and said anodic side laminar flow channels.
  • Said transverse hole (42) aligns with some transverse holes (39) to provide a means of connecting certain unrelated cathodic side laminar flow channels to the "cathodic analyte stream collection and distribution manifolds"
  • FIG. 6B illustrates a spacer gasket (43) be located between the back side of molded support plate (36) and the low molecular weight membrane (14b) that is adjacent to the front side of cathode plate assembly (21).
  • Said spacer gasket (43) is likewise about 2-20 cm wide and about 2-20 cm high and comprised of a thin, semi-rigid sheet of non-conducting polymeric material of uniform thickness, wherein said thickness [does not vary by greater] varies by no more than 5 % over the entire surface of said spacer gasket.
  • Spacer gasket (43) with slots (44) provides a means of forming and maintaining the laminar flow channels, (16b) for example, on a cathodic side of semi-permeable membrane (37) as illustrated schematically in FIG. 1.
  • Spacer gasket (43) provides a means for the inflow of an anodic analyte stream and an outflow of the basic pH segregated outflowing analyte stream in said slot (44) to a "cathodic analyte stream collection and distribution manifold" in the downstream side of the separation manifold in the same manner as described in FIG. 6A for spacer gasket (40).
  • FIG. 7A illustrates a spacer gasket (45) with a plurality of transverse slots (46) to be located between the front side of molded support plate (36) as shown in FIG.5 and the back side of anode plate assembly (33) as shown in FIG. 4.
  • Spacer gasket(45) of the same dimensions as spacer gasket (40) (FIG. 6A), provides a means for the inflow of an anodic analyte stream and an outflow of the acidic pH segregated analyte stream in each said slot (46) to an "anodic analyte stream collection and distribution manifold" in the downstream side of the separation manifold in the same manner as described in FIG. 6A for spacer gasket (40).
  • FIG. 7B illustrates a spacer gasket (47) with a plurality of transverse slots
  • Spacer gasket (47) of the same dimensions as spacer gasket (40) (FIG. 6A), provides a means for the inflow of a cathodic analyte stream and an outflow of the basic pH segregated analyte stream in each said slot (48) to a "cathodic analyte stream collection and distribution manifold" in the downstream side of the separation manifold in the same manner as described in FIG. 6A for spacer gasket (40).
  • FIG. 8 illustrates the relationship between the various components of one binary segregating manifold assembly (11) in an exploded view.
  • a binary segregating manifold assembly with the cathode on the front side and the anode on the back side is shown.
  • the binary segregating manifolds on each side of the one shown will have a reverse polarity because they share electrodes with this binary segregating manifold.
  • the arrangement of components in these reversed polarity assemblies is a mirror of what is depicted in FIG. 8.
  • a single binary segregating manifold assembly such as (11) is comprised of a cathode plate assembly (21) at the front of the overall exploded view depicted in FIG. 8. Said cathode plate assembly (21) is illustrated in detail in FIG. 3.
  • FIG. 7B This is followed (in order from front to rear in FIG. 8) by a low molecular weight cutoff semi-permeable membrane (14b), and spacer gasket (47) shown in detail in FIG. 7B.
  • Said spacer gasket (47) is positioned with respect to the cathode plate assembly (21) such that slots (48) (FIG. 7B) are aligned with the corresponding grooves (17) in cathode plate (13) (FIG. 3), which is a part of said cathode plate assembly (21).
  • a semi-permeable buffering membrane assembly illustrated in detail in FIG.
  • a spacer gasket (40) shown in detail in FIG. 6A is positioned against said semi-permeable buffering membrane assembly (15) with slots (48) (FIG. 6A) in spacer gasket (40) aligned with said individual buffering membranes (37) in said semi-permeable buffering membrane assembly (15).
  • a second low molecular weight cutoff semi-permeable membrane (14a) is then positioned between said anode side spacer gasket (40) and the anode plate assembly (33) shown in detail in FIG. 4.
  • the horizontal depressions or grooves (17) in anode plate assembly (33) are likewise aligned with said slots (48) in anode side spacer gasket (40).
  • a binary segregating manifold assembly such as (11), illustrated in FIG. 8
  • the cathode plate assembly (21) and the anode plate assembly (33) provide for transverse fluid channels (27), (28), (29), and (30) (that have been previously described in FIG.3 and FIG. 4).
  • Said fluid channels pass through the entire binary segregating manifold assembly (11).
  • Said transverse fluid channels are further aligned with similar transverse fluid channels in other binary segregating manifold assemblies stacked within a single stage as shown in FIG. 9.
  • the purpose of the transverse fluid channels (27), (28), (29), and (30) is to provide a path of delivery and removal of electrode cooling fluid to the cathode plate (13) and the anode plate (12).
  • the binary segregating manifold assembly (11), illustrated in FIG. 8, provides a transverse hole (31) passing completely through said manifold assembly for the purpose of connecting the cathode plate (13) using an electrical conductor rod (51) shown in FIG.9 with other cathode plates within a stacked manifold plate assembly and to a source of negative electrical potential.
  • a transverse hole (32) passing completely through said manifold assembly (11) provides an electrical connecting means for anode plate (12) using an electrical conductor rod (50), also shown in FIG.9, connected to other anode plates within a stacked manifold plate assembly and to a source of positive electrical potential.
  • Each binary segregating manifold assembly (11), is provided with a plurality of semi-permeable membranes, located in semi-permeable buffering membrane assembly (15) described in detail in FIG.5, that on assembly with said spacer gasket (47) and spacer gasket (40), divide said manifold assembly into two sets of laminar flow channels such as (16a) and (16b), for instance, as shown in FIG.1.
  • Said laminar flow channels run parallel to the direction of the continuously flowing mobile phase.
  • the inflowing mobile phase enters both sets of laminar analyte flow channels simultaneously on each side of said semi-permeable buffering membrane through the series of transverse supply holes previously described in FIGS.3-7B.
  • the outflowing pH segregated mobile phases exit separately from each laminar flow channel of said semi-permeable buffering membrane through a series of transverse outlet holes also previously described in FIGS. 3-7B.
  • All said transverse supply and outlet holes connect to a limited number of other lateral or transverse supply and collection channels such as "cathodic analyte stream collection and distribution manifolds" and “anodic analyte stream collection and distribution manifolds" both located between stages in a pattern that is designed to provide for the distribution of the various analyte stream pH range fractions to the required zones of equivalent binary segregating manifold assemblies in subsequent separation stages as described in general in FIG. 1.
  • the low molecular weight cutoff semi-permeable membranes (14a) and (14b), shown in FIG. 8, provide for the containment of analyte molecules (having molecular weights above the cutoff of about 500-2000 Daltons) within the laminar analyte flow channels while allowing the free flow of small charged and uncharged molecules below the cutoff of about 500-2000 Daltons through said low molecular weight cutoff semi-permeable membranes (14a) and (14b) into the spaces that contain the electrodes.
  • Said low-molecular weight cutoff semi-permeable membranes (14a) and (14b) typically have a minimal thickness as required to provide for the semi- permeable containment. Examples of such a material include but are not limited to the type of cellulose membrane known as low molecular weight cutoff benzoylated cellulose dialysis tubing.
  • equilibration of the analyte stream by isoelectric point segregation occurs entirely within the residence time of said analyte stream in a given pH segregation stage.
  • Said segregation can be achieved by a process of rap/of equilibration in order to maintain overall high flow rates through all stages of separation.
  • the physical dimensions of the current invention are such that rapid equilibration is achieved by the combination of; (1 ) a large surface area for the semi-permeable buffering membrane relative to the volume of the laminar flow channels, (2) a short transfer distance and (3) a maximum potential electrical field gradient provided over (4) a small electrode spacing.
  • FIG.9 illustrates a further assembly of individual manifold assemblies into a plurality of separation stages. Two such separation stages (49) and (49") are shown. It is to be understood that the number of separation stages, such as (49) and (49'), that can be combined end to end, as depicted in FIG.9, is theoretically unlimited. Although there are no intrinsic limits to the number of stages that can be used, depending on the technology, practical or physical limitations may serve to limit the number of stages.
  • individual manifold assemblies such as (11), shown in FIG.8 that are located within a single separation stage such as (49) or (49'), as illustrated in FIG.9, are further assembled into a compressed stack.
  • Said individual manifold assemblies, in the current embodiment are depicted as (11) in FIG. 9 and are assembled as described in FIG.8 except that every other individual manifold assembly (11) is reversed in polarity and shares an electrode with the adjacent individual manifold assembly.
  • an individual manifold assembly (11) can be construed to run from the vertical centerline of one anode plate to the vertical centerline of the next cathode plate.
  • the adjacent individual manifold assembly then runs from the same said cathode plate centerline to the next anode plate centerline.
  • This arrangement is also depicted schematically in FIG.1.
  • the result of this arrangement is that the anodic side laminar flow analyte streams, shown as (16a), (16d), (16e) and (16g) in FIG.1 , and the cathodic side laminar flow analyte streams shown as (16b), (16c), (16f), (16h) and (16i) in FIG.1, are located on different sides of the semi-permeable buffering membrane assembly (15) in adjacent individual manifold assemblies.
  • FIG.9 further illustrates for each separation stage (49) and (49'), two electrical conductor rods (50), (51) and (50'), (51') respectively.
  • Said electrical conductor rods (50) and (50') run through all of the transverse holes (32) (FIG.4) in all the anode plates a single separation stage such as (49) or (49').
  • Said electrical conductor rods (50) and (50') provide a means of electrical of connection of all said anode plates in a single separation stage to a source of positive electrical potential.
  • said electrical conductor rods (51) and (51") run through all of the transverse holes (31) (FIG.3) in all the cathode plates a single separation stage such as (49) or (49"). Said electrical conductor rods (51) and (51') provide a means of electrical of connection of all said cathode plates in a single separation stage to a source of negative electrical potential.
  • FIG.9 further illustrates for each separation stage, (49) for instance, two transverse channels (28) and (29), that run through all individual manifold assemblies in said single separation stage.
  • Said transverse channels (28) and (29) provide a means of supply and delivery of cooling fluid to each anode plate and cathode plate respectively in said separation stage.
  • said transverse channels (28) and (29) are physically and electrically isolated from one another.
  • FIG.9 illustrates for each separation stage, (49) for instance, two transverse channels (27) and (30), that run through all individual manifold assemblies in said single separation stage.
  • Said transverse channels (27) and (30) provide a means of return and collection of cooling fluid from each cathode plate and anode plate respectively in said separation stage.
  • said transverse channels (27) and (30) are physically and electrically isolated from one another.
  • Said anode and cathode cooling fluid streams also provide for the removal of low molecular weight (about 500-2000 Daltons) charged and uncharged molecules that pass through the semi-permeable low molecular weight membranes (14a) and (14b).
  • This design provides for exceptionally high electrical fields by eliminating the resultant high current and concomitant coulombic heating caused by small molecule electrolytes in the analyte stream that can reduce the effective electrical field strength. Exceptionally high electrical fields minimize the equilibration time.
  • FIG. 10 illustrates in an exploded view, an overall configuration of the second dimension separation means.
  • said overall second dimension separation means provides for an upstream pressure cap (53), and a combined downstream pressure cap and outlet nozzle array plate (54), and a means (not shown) of enclosing all said separation stages between said pressure caps (53) and (54) so as to maintain said separation stages at a pressure or pressures above atmospheric pressure as required to move said analyte stream through said overall second dimension separation means.
  • said overall second dimension separation means shown in FIG.
  • (10) is comprised of a plurality of movable fraction collection plates (55) for collecting simultaneous fractions from each final outlet nozzle and, finally, a plurality of solid phase extraction array assemblies (56) for immobilizing some part of the outlet stream analyte molecules for detection.
  • Said overall analyte stream has a general direction of flow from each said upstream separation stage into subsequent downstream separation stages. Said general direction of flow of the analyte stream from stage to stage is depicted by arrows (52, 52" and 52' ⁇ .
  • the combination of a plurality of binary pH segregating channels distributed vertically within a single manifold assembly and a plurality of said manifold assemblies stacked horizontally to form a binary separation stage provides for an array of channels in two dimensions as viewed from the inlet or outlet faces of said binary separation stage.
  • the pH range of each binary analyte stream narrows by stages, the number of analyte species (isoelectric point values) within each pH range is reduced in a binary fashion.
  • a preferred configuration of said channels is such thata// channels in the initial segregating stage provide for a single initial buffering membrane aggregate pK value, pH 7 for instance as shown in FIG. (1).
  • Said anodic and cathodic distribution manifolds are unconnected.
  • downstream side transverse holes (23 and 24) in the particular anode support plate (34) located between zone (B) and zone(C) are not formed.
  • Subsequent stages subdivide the 32 x 32 array into zones, each containing fewer and fewer equivalent binary pH segregating membranes (37) in a similar fashion (i.e. 8 wide by 16 high, 8 wide by 8 high, 4 wide by 8 high, etc.)
  • the arrays of interconnected collecting channels in the periphery of each stage have limiting 5 barriers between corresponding zones on the upstream face of the subsequent separation stage, thus limiting distribution of analyte streams to single downstream zones.
  • n 10 (49')
  • TABLE 2 illustrates the number and size of zones within a 32 by 32 array of individual channels (in the current example of 2048 channels per stage) for each stage of separation. 5
  • the aforementioned downstream pressure cap and outlet nozzle array plate (54) provides an array of 64 vertical columns (about 0.6-6mm on center) of outlet nozzles (57) and 32 horizontal rows (about 1.2-12mm on center) of outlet nozzles(57), i.e. the spacing between the rows of nozzles, typically is twice that between columns of nozzles.
  • Said outlet nozzle array plate (54) is additionally provided with 32 horizontal 0 surface grooves (59) located between each row of horizontal outlet nozzles (57), running the full width of said end cap and extending to each edge of said end cap.
  • Said horizontal surface grooves (59) are spaced equally between each said row of horizontal outlet nozzles (57) to provide an open channel on the face of outlet nozzle array plate (54) to the atmosphere.
  • Detail A of FIG. 10 illustrates a close up view of the outlet nozzle array plate (54), a single movable fraction collection plate (55) and a single solid phase extraction array (56).
  • the aforementioned plurality of movable fraction collection plates (55) have a width and height corresponding to the width and height of the outlet nozzle array in outlet nozzle array plate (54).
  • Said movable fraction collection plates (55) are provided with an array of transverse channels or micro-capillary tubes (58) corresponding in number and position with the array of nozzles on said outlet nozzle array plate (54).
  • Said channels or micro-capillary tubes (58) being normal to the surface of said movable fraction collection plates (55) and passing completely through said movable fraction collection plates (55) are thus parallel and aligned with said outlet nozzles (57) in outlet nozzle array plate (54).
  • Detail A of FIG. 10 further illustrates a close up view showing said transverse channels or micro-capillary tubes (58) in said movable fraction collection plate (55).
  • the diameter of each said transverse channel or micro-capillary tube (58) is comparable but not necessarily identical to the diameter of said outlet nozzle (57).
  • Each said transverse channel or micro-capillary tube (58) may be a separate removable micro-capillary tube held in place within said movable fraction collection plate (55) or a transverse channel permanently molded into said movable fraction collection plate (55).
  • the thickness of said movable fraction collection plate (55) is variable as required to provide transverse channels or micro-capillary tubes (58) of variable internal volume.
  • said movable fraction collection plate (55) is about 0.2-2.0 cm in thickness.
  • a means is provided of rapidly transporting and positioning said movable fraction collection plate (55) directly against the outlet nozzle array plate (54) such that each of the 2048 outlet nozzles (57) in the example, is aligned with one of the 2048 transverse channels or micro-capillary tubes (58) in said movable fraction collection plate (55).
  • a further means is provided to bring said movable fraction collection plate (55) into close contact with said outlet nozzle array plate (54).
  • Said close contact forms a tight seal between each transverse channel or micro-capillary tube (58) and each outlet nozzle (57) such that the outflowing analyte stream from each said outlet nozzle (57) collects in a corresponding transverse channel or micro-capillary tube (58), thus collecting a single fraction of the outflowing stream from each of the outlet nozzles (2048 in the example) simultaneously.
  • the time of contact between said movable fraction collection plate (55) and outlet nozzle array plate (54) determines the fraction collection time of the analyte streams and therefore the fraction volume in each said transverse channel or micro-capillary tube (58).
  • Said time of contact can be varied from less than the time required for filling the transverse channels or micro-capillary tubes (58) to more than the time required for filling said transverse channels or micro-capillary tubes (58).
  • said time of contact is greater than the time required for filling said transverse channels or micro-capillary tubes (58)
  • a portion of the analyte streams in each said transverse channel or micro-capillary tube (58) will pass completely through the said movable fraction collection plate (55).
  • a means is provided to rapidly shift the position of said movable fraction collection plate (55) vertically relative to said outlet nozzle array plate (54) along the face of said array plate (54) and without losing contact with said array plate (54).
  • Said rapid vertical shift unseals said transverse channels or micro-capillary tubes (58) in movable fraction collection plate (55) from said corresponding outlet nozzles (57) in outlet nozzle array plate (54).
  • Said rapid vertical shift further aligns the transverse channels or micro-capillary tubes (58) with said horizontal surface grooves (59) located between rows of nozzles on said outlet nozzle array plate (54).
  • Said horizontal surface grooves (59) provide a means venting said transverse channels or micro-capillary tubes (58) to the atmosphere at the point of contact of each said transverse channel or micro-capillary tube (58) and each said outlet nozzle (57), thus isolating that portion of the analyte stream within each said transverse channel or micro-capillary tube (58).
  • a further means is provided to rapidly move said movable fraction collection plate (55) in a horizontal direction parallel to said horizontal surface grooves (59) as shown by arrow (63) in FIG. 10, thus maintaining venting for all said channels or tubes in said fraction collection plate until said fraction collection plate is completely clear of said outlet nozzle array plate (54). This arrangement prevents the loss or cross contamination of analyte fractions between channels or tubes.
  • a second, empty movable fraction collection plate (55) is positioned in it's place, such that the transverse channel or micro- capillary tubes (58) in said empty plate (55) are initially aligned with said horizontal surface grooves (59).
  • the empty movable fraction collection plate (55) is in place horizontally, it is rapidly shifted vertically so as to align the transverse channels or micro-capillary tubes (58) in said empty movable fraction collection plate (55) with outlet nozzles (57) on said outlet nozzle array plate (54) to form a tight seal, between each transverse channel or micro-capillary tube (58) and each outlet nozzle (57) thus filling the empty fraction collection plate in the current example, with an array of 2048 second fractions from each analyte stream.
  • a plurality of filled and empty movable fraction collection plates (55) are provided with a means of stacking and de-stacking said movable fraction collection plates (55) in order to place said plurality of movable fraction collection plates (55) in position for fraction collection and storage over the full time required for a complete sample analysis.
  • Said simultaneous means of detection can be continuous at the time of analyte stream outflow from the second dimension separation means or said means of detection can be delayed in time or in rate of detection thus differing from the rate of outflow of said analyte stream.
  • such a means of detection must achieve several requirements; (1 ) said means of detection must detect analytes in all channels essentially at the same time or serially within a short time, (2) said means of detection must detect analytes non-destructively or with a minimum of sample in order to provide, for further analysis, the aforementioned fractions in said channels or micro-capillary tubes (58) and (3) a means of detection must detect all analytes equally and with a high degree of sensitivity.
  • each said solid phase extraction array assembly (56) provides a means of immobilizing some portion of the outlet stream analyte molecules for detection.
  • Each said solid phase extraction array assembly (56) consists of a rigid support plate (60) with a plurality of transverse channels (61) normal to the surface of said rigid support plate (60), forming a horizontal and vertical array (64 columns by 32 rows in the current example).
  • Solid phase extraction array assembly (56) also consists of a membrane (62) bonded to a face of said rigid support plate (60) such that said membrane (62) closes off one end of each said transverse channel (61). Said membrane (62) provides a means of extracting and immobilizing analyte molecules simultaneously from each said analyte stream as said analyte streams pass through said transverse channels (61).
  • said membrane (62) is comprised in whole or part of one of several membrane materials known to bind protein or peptide molecules such as polyvinylidene difluoride and nitrocellulose.
  • the width and height of said solid phase extraction array assembly (56) is about 2-20cm.
  • the thickness of said rigid support plate (60) of solid phase extraction array assembly (56) is about 0.01 -0.1 cm.
  • the thickness of said membrane (62) of solid phase extraction array assembly (56) is about 0.001 -0.01 cm.
  • said membrane (62) of said solid phase extraction array assembly (56) be so formed as to have a minimal and exceptionally uniform thickness while still providing immobilizing capacity sufficient to immobilize the entirety of said analyte molecules in each said analyte stream over the time interval selected for said analyte molecule immobilization.
  • Said desired feature provides for concentrating and placing said extracted and immobilized analyte molecules into a single plane within the area of said membrane (62) that covers said transverse channel (61), thus enhancing subsequent detection by those methods that utilize the art of time of flight mass spectroscopy.
  • Said transverse channels (61) correspond in position and alignment with said channels or micro-capillary tubes (58) in said movable fraction collection plate (55).
  • a means is provided of vertically transporting and positioning said solid phase extraction array assembly (56) directly against the downstream face of movable fraction collection plate (55) such that each of the 2048 transverse channels (61) for instance, in the example shown in FIG. 10, is aligned with one of the 2048 transverse channels or micro-capillary tubes (58) in said movable fraction collection plate (55).
  • said solid phase extraction array assembly (56) is tightly sealed against said movable fraction collection plate (55) so as to allow the analyte stream in each channel or micro-capillary tube (58) in movable fraction collection plate (55) to flow into and through each corresponding transverse channel (61) impinging and immobilizing said analyte molecules through chemical adsorption onto the area of said membrane (62) that covers said transverse channel (61).
  • a means is provided to rapidly shift the position of the fully adsorbed solid phase extraction array assembly (56) vertically relative to said movable fraction collection plate (55) and replace said fully adsorbed solid phase extraction array assembly (56) with a new solid phase extraction array assembly (56) as shown in FIG. 10 by arrow (64).
  • the selected sampling and immobilization period for the analyte streams in said solid phase extraction array assemblies (56) can be chosen to be less than, equal to, or greater than, the selected period of fraction collection in said movable fraction collection plate (55) as desired. This provision allows maximum flexibility in allocating analyte streams between the detection means and the fraction collection means as required.
  • a plurality of fully adsorbed and new solid phase extraction array assemblies (56) are provided with a means of stacking and de-stacking said solid phase extraction array assemblies (56) in order to place a sufficient number of said solid phase extraction array assemblies (56) in position for sample extraction and immobilization during the full time required for a complete first and second dimension sample analysis.
  • the same said means of stacking and de-stacking said solid phase extraction array assemblies (56) can be used as a means of positioning said fully adsorbed solid phase extraction array assembly (56) in position, as required for the chosen method of subsequent detection.
  • Said immobilized analytes can then be analyzed by several methods.
  • a preferred method of detection and analysis of macromolecular analytes such as proteins extracted onto a solid phase matrix is matrix assisted laser desorption ionization mass spectroscopy, in particular infrared laser desorption mediated time of flight mass spectroscopy (IR-MALDI-TOF).
  • IR-MALDI-TOF matrix assisted laser desorption ionization mass spectroscopy
  • Said infrared laser desorption mediated time of flight mass spectroscopy (IR-MALDI-TOF) has been described in the prior art.
  • the aforementioned means of placing said fully adsorbed solid phase extraction array assembly (56) into position for infrared laser desorption mediated time of flight mass spectroscopy can further provide a means for placing said fully adsorbed solid phase extraction array assembly (56) in contact with a means of cooling said fully adsorbed solid phase extraction array assembly (56).
  • the purpose of said means of cooling said fully adsorbed solid phase extraction array assembly (56) is to freeze the residual analyte mobile phase, or otherwise absorbed solutions, in membrane 62) that covers each said transverse channel (61).
  • Said freezing of said absorbed solutions in membrane 62) occurs before entry of said fully adsorbed solid phase extraction array assembly (56) into the required vacuum chamber means of the infrared laser desorption mediated time of flight mass spectroscopy (IR-MALDI-TOF) apparatus.
  • Said freezing provides a required means of embedding said analyte molecules into a energy absorbing matrix as required for infrared laser desorption.
  • the energy-absorbing matrix can be water ice, ethanol ice, glycerol or other media that are readily compatible with a protein analyte, thereby greatly simplifying the subsequent analysis.
  • Said prior art of laser desorption time of flight mass spectroscopy provides for the pulsed desorption of matrix and analyte in a very short time frame (between 200 picoseconds and several nanoseconds) followed by an electrostatic acceleration of the ionized analyte molecules through a vacuum drift tube into a detector with typical total flight times for macromolecules in the range of 10-1000 microseconds. Also, a single pulse can be focused to an area of the target matrix of about 200 microns in diameter and can be subsequently redirected onto other target locations at high speed.
  • the output signal for time of flight mass spectroscopy provides a measure of the mass (molecular weight) of the intact analyte molecule with a resolution typically of one part in 300 to 500 (full width half maximum) and a measure of the relative intensity of the mass peak corresponding to the amount of analyte.
  • Analyte peaks of a few femtograms can be detected and the outline of potentially overlapping or hidden analyte "spots" can be elucidated by the appearance of additional peaks in the mass spectrogram, when scanning an area of a two dimensional map.
  • Additional means of detection include all the established methods of immobilized sample detection to include but not limited to; optical density detection, dynamic and static fluorescence detection, conductivity detection, electrochemical detection, dynamic and static light scattering detection and other forms of mass spectroscopic detection.
  • the solid phase extraction array assemblies (56) are replaced with a second movable fraction collection plate (55) so as to collect the analytes over a period of time in a compact array of corresponding analyte stream sampling volumes.
  • analyte stream sampling volumes collected and stored in a plurality of transverse channels (61) within second movable fraction collection plate (55), can be analyzed in real time or delayed time using a means of moving and positioning said second movable fraction collection plate (55) into a position for transferring said sampling volumes into a detector micro- flow cell or an electrospray ionization means for the introduction of analyte molecules into one of several alternate forms of mass spectroscopy.
  • Said alternate forms of mass spectroscopy include but are not limited to, the prior arts of tandem quadrapole mass spectroscopy and Fourier transform ion cyclotron resonance mass spectroscopy.

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  • Separation Using Semi-Permeable Membranes (AREA)

Abstract

Un procédé et un appareil de séparation de mélanges de substances amphotères utilisent deux dimensions de séparation. Une soupape (8, 9) à échantillons est utilisée pour introduire un échantillon dans la phase mobile (7b) destiné à entrer dans la première dimension de séparation, typiquement une colonne de chromatographie (10). L'échantillon séparé est passé dans la seconde dimension, laquelle est formée d'une série d'étages (11, 18, 19). Le premier étage (11) présente un collecteur ayant une anode (13), une cathode (12) ainsi qu'une membrane (15) d'un pK spécifique, définissant deux voies (16a, 16b). Les molécules amphotères sont séparées entre les deux voies et les molécules séparées sont transmises à un second étage dans lequel chaque voie (16a, 16b) est à nouveau divisée en deux voies (16e, 16f et 16c, 16d), respectivement. Dans chaque étage suivant (19), le nombre de voies est doublé par rapport à l'étage précédent, augmentant le degré de séparation par pH.
EP00978541A 1999-11-16 2000-11-14 Separation et detection bidimensionnelles en solution de substances amphoteres Withdrawn EP1235634A4 (fr)

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