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WO2006021465A1 - Séparation électrophorétique dans un fluide en mouvement - Google Patents

Séparation électrophorétique dans un fluide en mouvement Download PDF

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Publication number
WO2006021465A1
WO2006021465A1 PCT/EP2005/051092 EP2005051092W WO2006021465A1 WO 2006021465 A1 WO2006021465 A1 WO 2006021465A1 EP 2005051092 W EP2005051092 W EP 2005051092W WO 2006021465 A1 WO2006021465 A1 WO 2006021465A1
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WO
WIPO (PCT)
Prior art keywords
compounds
flow chamber
analyte solution
electrical field
electrodes
Prior art date
Application number
PCT/EP2005/051092
Other languages
English (en)
Inventor
Christian A. Heid
Klaus Witt
Original Assignee
Agilent Technologies, Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Agilent Technologies, Inc. filed Critical Agilent Technologies, Inc.
Priority to DE212005000044U priority Critical patent/DE212005000044U1/de
Publication of WO2006021465A1 publication Critical patent/WO2006021465A1/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
    • G01N27/44769—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
    • 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

Definitions

  • the present invention relates generally to the field of separation devices for conducting fractionation of organic molecules.
  • IEF iso-electric focusing
  • Iso-electric focusing systems are usually free flowing buffered systems or immobilized buffered systems.
  • Iso-electric focusing systems are usually free flowing buffered systems or immobilized buffered systems.
  • Iso-electric focusing systems are usually free flowing buffered systems or immobilized buffered systems.
  • pi iso-electric point
  • Iso-electric focusing systems are usually free flowing buffered systems or immobilized buffered systems.
  • To conduct efficient and successful scientific experiments a minimum volume of the compound of interest is needed.
  • the separation takes hours to receive the needed amount of substance and frequently additional steps such as removal of carrier ampholytes have to be performed when - as usual - the compound of interest has to be separated from an educt solution containing several compounds having close pi values.
  • Embodiments of the present invention address the aforementioned needs in the art to separate reasonable amounts of a desired compound contained in an analyte solution which is comprised of a diversity of i.e. organic molecules; some of which having a very close pi therefore being difficult to separate.
  • the desired compound shall furthermore be obtained in a way that makes it easy to immediately conduct experiments, which may have an analytical as well as a preparative character.
  • the second object has been described in former patents not been answered in an adequate manner since using the separated compounds for preparative applications requires an excellent purity and therefore quality of the educt.
  • the present invention provides a device consisting mainly of a flow chamber wherein the analyte solution containing the compound of interest is introduced in order to carry out the separation by means of electrophoresis.
  • the central improvement in comparison to the separation systems known in the art is the superimposition of hydraulic forces and electronic forces, both acting upon the analysis solution inside the flow chamber. This is achieved by generating a homogeneous electrical field inside the flow chamber while at the same time the analyte solution is moved, or pumped, respectively, by an upstream pump, from an inlet end to an outlet end across the cell.
  • This mechanism offers a brilliant possibility to enrich compounds such as organic molecules with a definite charge or pi, respectively, at definite places of the inner surface of the flow chamber, and, if once enough material has been enriched, reversing the electronic attraction in a controlled manner, therefore setting free only those compounds which are repulsed by the used voltage. Once set free and being resolubilized, the compounds of interest exit the flow chamber, too. Therefore, the device offers an option for separating volumes of a compound of interest in order to use it in subsequent preparative application within an agreeable limit of time.
  • the flow cell is shaped cylindrically.
  • the electrical field created herein is a homogeneous radial field, generated by axially located electrodes in the centre of the cylinder while the wall of the cylinder is or comprises a second electrode, the electrodes being of opposite polarity.
  • the inner surface of the above flow cell is coated with an appropriate substance for taking up the charged compounds being deflected in direction to the cylinder wall.
  • the flow cell is shaped cylindrically, too, but the electrical field created therein is a homogeneous axial field.
  • the cylinder is arranged in an inclined position.
  • An additional embodiment of the invention depicts the flow cell of the third embodiment, wherein the inner surface of the above flow cell is coated, too, in order to provide an optimal uptake of the enriched compounds.
  • FIG. 1a is a schematic over all front view of a device for electraphoretic separation and purification which can be used to carry out the process according to the present invention, the flow chamber having a cylindrical design, arranged in upright position, designed to create a radial electrical field,
  • FIG. 1b is a cross section of the flow chamber of FIG.1a
  • FIG. 2a is a schematic over all front view of the device of FIG. 1 , showing the beginning of a separation cycle, initial charging with analyte solution, electrical current flowing and therefore creating the radial field,
  • FIG. 2b is a cross section of the flow chamber of FIG. 2a
  • FIG. 3a is a schematic over all front view of the device of FIG.2a during a separation cycle, initial charging with analyze solution, electrical current flowing,
  • FIG. 3b is a cross section of the flow chamber of FIG. 3a
  • FIG. 4a is a schematic over all front view of a device of FIG. 3a, during a separation cycle, charged with analyze solution, electrical current flowing, coated with a gel at the inner surface inside the flow chamber,
  • FIG. 4b is a view from above into the flow chamber of in Fig.4a
  • FIG. 5 is a schematic over all front view of a device for electrophoretic separation and purification which can be used to carry out the process according to the present invention during a separation cycle, charged with analyze solution, electrical current flowing, creating an axial electrical field, the flow chamber being in a tilted position
  • FIG. 6 is a schematic over all front view of the device of FIG. 5 during a separation cycle, charged with analyze solution, electrical current flowing, coated with a gel (16) at the inner surface inside of the flow chamber.
  • a flow chamber is a reactor that is used for processes in chemistry requiring a continuous flow of liquid. This reactor is designed to permit continuous flowing of the liquid along a definite flow path.
  • Process is generally the substance that is obtained, when the educt has been exposed to at least one chemical reaction.
  • a liquid such as an analyte solution can be forced to move through a chemical reactor such as a flow chamber by means of a "motive source".
  • a chemical reactor such as a flow chamber
  • gravity is the source that leads to a movement of a liquid along a flow chamber, which is arranged vertically.
  • pumps are used as "motive sources” in order to achieve an economic flow-through. It has to be understood, that in the present invention there is no restriction for the use of any pumps; some of the embodiments of the present invention require the use of pumps creating a pH-gradient.
  • a "collector” is generally a collecting means suitable for recovery of liquids or compounds dissolved in liquids.
  • a “recycling device” is generally a device suitable for transferring product back to the chemical process.
  • the device of the present invention for electrophoretic separation and purification of analyte solution and subsequent recovery of the product comprises a motive source 12 for moving the analyte solution, which is a conventional pump 12a in this embodiment, one flow chamber 2 which is of cylindrical geometry, having one inlet 3 at the top of the cylinder and one outlet 4 at the bottom of the cylinder. Furthermore, a power source 7 is comprised in order to generate an electrical field by usage of two electrodes that are cathode and, respectively, anode.
  • the electrical field of this embodiment is a radial field 6a, since the wall of the cylindrical flow chamber 2 is providing the one electrode element 5b and an axial element 5a in congruence with the longitudinal axis of the flow chamber 2, which is located upright, is the second electrode.
  • the flow chamber 2 can be generally designed in another way, as long as a homogeneous, or at lest a predictable electrical field is provided, which allows for good controllability of the separation process.
  • a capillary is another example for a cylindrically shaped flow chamber 2 that fulfills the requirements of the present invention.
  • a homogeneous electrical field is only partly existing within the flow chamber. It has to be understood, that the device of the present invention, and therefore the components of which it is built, is not restricted to a definite size; it can be realized in lab size as well as in miniaturized size in order to be integrated in existing apparatus or analysis-lines.
  • any other type of pump can be used.
  • FIG. 1 b shows clearly the cylindrical diameter of the flow chamber 2, one electrode 5a is positioned exactly in the center of the cylinder while the other electrode 5b is integrated in the wall of the flow chamber 2.
  • the other electrode has not necessarily to be integrated into the wall, it can also be close to the wall or to a part of it of to the complete circumference of the cylinder, as long as a radial electrical field is at least partly generated inside the chamber.
  • FIG. 2a the device of FIG.1 a is shown, indicating now the first step of the separating process: An analyte solution stream 1 is entering the flow chamber 2 via the inlet 3 and an electrical current is flowing, therefore initiating a separation cycle. A collecting vessel 18 is placed below the outlet 4 of the flow chamber 2 to receive the product.
  • FIG. 2b shows a cross section of the flow chamber 2 of FIG. 2a, pointing out clearly by arrows that a radial electrical field 6a is created.
  • the cathode is provided by the axial element 5a in the center of the cylinder while the anode is provided by the wall.
  • the polarity can be reversed.
  • the separation process is running, indicated by the continuously flowing analyte stream 1, whose compounds start being separated after the stream has entered the flow chamber 2, which has been shown in FIGS. 1 and 2.
  • the negatively charged compounds 5b leave their flow path directing them from the inlet 3 to the outlet 4 and move to the chamber wall, being the anode herein, while the positively charged compounds 8 move to the cathode, which is provided by the axial element 5a; the deflection being caused by the electrical field 7, causing movement according to the mobility.
  • the electrical field acts in parallel to the hydraulic forces that are generated by pumping the analyte solution.
  • the neutral compounds 10 flow along the flow path, therefore leaving the flow chamber 2 when they have reached the outlet 4 and "falling" then into a colleting vessel 18.
  • FIG. 3b is another view of the situation indicated in FIG. 3a. It shows the flow chamber 2 in cross section and the settling of charged compounds at anode and cathode while neutral compounds 10 are not deflected.
  • the coating of gel or any other suitable material is not restricted to the inside surface of the chamberwall, which makes sense in the above embodiment, thus offering a large surface area for the uptake of separated compounds, but can just as well be applied on an axial element 5a, or in case of more than one axial elements 5a acting as electrode, on all of them.
  • FIG. 4b shows the cross section of the flow chamber 2 of FIG. 4a, showing in detail the settling of charged compounds 8,9 at anode and cathode while neutral compounds 10 are not deflected. It can be seen how negatively charged compounds 9 are trapped in the gel coating the inner surface of the chamber wall 2, which is the anode.
  • the basic components are a motive source 12 for moving the analyte solution, herein not a conventional pump but a pump providing a pH-gradient, one flow chamber 2 of cylindrical geometry, having one inlet 3 at the top of the cylinder and one outlet 4 at the bottom of the cylinder, and a power source 7 in order to generate an electrical field by use of cathode and anode. But in comparison to the above embodiments, the electrical field depicted in FIG.
  • the inlet 3 is a central opening in the anode, the outlet 4 is located at the lowest point of the flow chamber 2.
  • FIG. 6 shows the device of FIG. 5, wherein the inner surface of the top and the bottom are coated with gel.
  • Versions of any of the above embodiments facilitate the enrichment of desired compounds, when at least parts of the inner surface of the flow chamber 2 being an electrode are coated with a gel or an other matrix suitable for the uptake of the preferred compound.
  • the electrical field can be designed in a way that only the negatively charged compounds are kept in the flow chamber 2, while the positively charged compounds 8 exit together with the neutral ones.
  • the device of the present invention can be coupled to downstream separation and analytical devices.
  • the analyte solution which is exposed to the process performed in the device of the present invention, contains a sample that is constituted preferably of biological compounds, more preferably organic compounds, and most preferably, proteins, protein derivatives, protein isoforms, enzymes, antigens, antibodies, peptides or nucleic acids, lipids or carbohydrates.
  • Proteins and their derivatives are the most interesting compounds discussed in the present document; they have a net neutral charge at a defined pH value, which is defined as their iso-electric point. Due to the extraordinary property of becoming iso-electric, the proteins are predestined for the method explained below.
  • a mixture of proteins can be separated into pure fractions by buffering the mixture in order to obtain a definite pH value, wherein only proteins having this very pH value as their pi become iso-electric while the other components of the mixture remain charged.
  • the analyte solution referred to in the present invention contains furthermore buffer solution and reagents or other additives.
  • the educt is introduced into a device of the present invention, in particularly into one of the embodiments shown in FIGS. 1 to 6.
  • the educt is pumped via an inlet 3 into a flow chamber 2, whereby a hydraulic flow is caused.
  • the analyte solution is moved through the flow chamber 2 being driven by hydraulic forces while in parallel electrical forces resulting from an electrical field, which is generated by a power source 7, a cathode and an anode, act upon the analyte solution stream 1 inside the flow chamber 2.
  • any voltage may be used as far as it's in the range which is tolerable for the device of the present invention, e.g. 10 to 10 000 volts. Heat resulting from high voltage is suggested to be dissipated by proper cooling.
  • proteins due to their electrical ambivalence: Proteins have an isoelectric point (pi), defined as the pH at which the protein has a net neutral charge. Therefore, proteins can be fractionated using a pH gradient combined with an electrical field. A pH gradient can be created by using a buffer at a definite pH value. Then, a mixture of proteins can be separated into two fractions as described in the steps above: The "mixture" is pumped through a separation device according the present invention, preferably according to one of the embodiments shown in FIGS. 1 to 6. Those proteins whose iso-electric point is obtained at the given pH-value are neutral then, thus they pass the flow chamber 2 without being deflected and get collected in a collection vessel 18. After a time, all negatively charged proteins/compounds become embedded in the gel or matrix while all positively charged proteins/particles may be attached to the cathode and the neutral proteins/particles remain in the liquid phase.
  • pi isoelectric point
  • the compounds that have been trapped in the flow chamber 2 can be set free in a second step by reversing the polarity of the electrodes; they exit the flow chamber 2 via outlet 4 and get collected in a second collection vessel 18.
  • Both fractions, the one that has been collected in the first collection vessel and the one that has been collected in the second collection vessel, can be further fractionated by using the separation method again at a different pH value.
  • additional fractionation rounds, each at different pH it is possible to use this invention to generate many fractions based on the iso ⁇ electric points. For instance, fractioning of iso-electric points every 0.5 pH (or even narrower) is possible.
  • proteins or compounds types may have properties that permanently embed them in the gel or matrix; these properties give this invention another means for fractionation/separation.
  • the components which are needed to realize the present invention are already existing in technology, e.g. pumps, capillaries, collection vessels, etc., thus, they are well known instruments which can be combined to a controllable, precise device according to this invention.
  • the separation device described inhere is an easy to operate system, which may be integrated as a module in an existing LC-system. Furthermore, this technique can be performed on a small scale relative to FFE; which can lead to a significantly lower cost and reagent consumption. A further advantage is, that this invention could be automated.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Electrostatic Separation (AREA)

Abstract

L’invention porte sur un dispositif de séparation électrophorétique et de purification de composés à charge positive et à charge négative (8, 9) et de composés neutres (10) dans une solution analytique à motivation hydraulique, à l’aide d’un champ électrique radial ou axial qui est généré à l’intérieur d’une chambre d’écoulement (2) ayant de préférence une géométrie cylindrique. Des forces d’écoulement hydrauliques et des forces électriques agissent sur la solution analytique se traduisant par la déviation (13) des composés chargés et neutres (8, 9, 10) de la solution analytique, la déviation étant fonction du champ électrique et des forces hydrauliques appliqués sur la solution analytique et de la charge du composé, respectivement son point isoélectrique, les deux éléments dépendant de la valeur pH de la solution, qui peut être prédéterminée, provoquant ainsi la séparation. Ce dispositif permet de réaliser un procédé de séparation et de purification de composés à charge positive et à charge négative (8, 9) et de composés neutres (10).
PCT/EP2005/051092 2004-08-25 2005-03-10 Séparation électrophorétique dans un fluide en mouvement WO2006021465A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DE212005000044U DE212005000044U1 (de) 2004-08-25 2005-03-10 Elektrophoretische Separation in einem bewegten Fluid

Applications Claiming Priority (2)

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US60461604P 2004-08-25 2004-08-25
US60/604,616 2004-08-25

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WO2006021465A1 true WO2006021465A1 (fr) 2006-03-02

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020104575A1 (fr) * 2018-11-24 2020-05-28 Selectrion GmbH Procédé et dispositif pour séparer des ions de même polarité de charge dans un champ électrique
CN111560063A (zh) * 2020-05-12 2020-08-21 蚌埠医学院 一种动物胰脏来源胰岛素原料药纯化装置及使用方法
CN111973737A (zh) * 2019-05-23 2020-11-24 比欧泰克生物技术服务(北京)有限公司 一种荚膜多糖疫苗蛋白核酸去除装置及去除工艺

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0323948A2 (fr) * 1988-01-05 1989-07-12 Monsanto Company Séparation isoélectrique continue de protéines
US4971670A (en) * 1987-04-11 1990-11-20 Ciba-Geigy Corporation Isoelectric focusing process and a means for carrying out said process
WO1998013689A1 (fr) * 1996-09-28 1998-04-02 Fuhr Guenter Procede et dispositif pour la separation isoelectrique de particules
EP1217367A1 (fr) * 2000-12-21 2002-06-26 Gradipore Limited Dispositif et procédé d'électrophorèse radiale
US20030104449A1 (en) * 2000-05-05 2003-06-05 Faupel Michel D. Electrophoretic separation of compounds

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4971670A (en) * 1987-04-11 1990-11-20 Ciba-Geigy Corporation Isoelectric focusing process and a means for carrying out said process
EP0323948A2 (fr) * 1988-01-05 1989-07-12 Monsanto Company Séparation isoélectrique continue de protéines
WO1998013689A1 (fr) * 1996-09-28 1998-04-02 Fuhr Guenter Procede et dispositif pour la separation isoelectrique de particules
US20030104449A1 (en) * 2000-05-05 2003-06-05 Faupel Michel D. Electrophoretic separation of compounds
EP1217367A1 (fr) * 2000-12-21 2002-06-26 Gradipore Limited Dispositif et procédé d'électrophorèse radiale

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020104575A1 (fr) * 2018-11-24 2020-05-28 Selectrion GmbH Procédé et dispositif pour séparer des ions de même polarité de charge dans un champ électrique
CN111973737A (zh) * 2019-05-23 2020-11-24 比欧泰克生物技术服务(北京)有限公司 一种荚膜多糖疫苗蛋白核酸去除装置及去除工艺
CN111973737B (zh) * 2019-05-23 2022-08-26 比欧泰克生物技术服务(北京)有限公司 一种荚膜多糖疫苗蛋白核酸去除装置及去除工艺
CN111560063A (zh) * 2020-05-12 2020-08-21 蚌埠医学院 一种动物胰脏来源胰岛素原料药纯化装置及使用方法
CN111560063B (zh) * 2020-05-12 2022-11-01 蚌埠医学院 一种动物胰脏来源胰岛素原料药纯化装置及使用方法

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