WO2017213528A1 - A method of preparative separation of substances using electrophoresis and chromatography processes conducted orthogonally and simultaneously - Google Patents

A method of preparative separation of substances using electrophoresis and chromatography processes conducted orthogonally and simultaneously Download PDF

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Publication number
WO2017213528A1
WO2017213528A1 PCT/PL2017/000062 PL2017000062W WO2017213528A1 WO 2017213528 A1 WO2017213528 A1 WO 2017213528A1 PL 2017000062 W PL2017000062 W PL 2017000062W WO 2017213528 A1 WO2017213528 A1 WO 2017213528A1
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substances
mixture
separated
separation
mobile phase
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PCT/PL2017/000062
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French (fr)
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Tadeusz H. DZIDO
Rafał GAJOS
Beata POLAK
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Uniwersytet Medyczny W Lublinie
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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
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3861—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36 using an external stimulus
    • B01D15/3885—Using electrical or magnetic means
    • 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
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3833—Chiral 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
    • G01N2030/388—Elution in two different directions on one stationary phase
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8877—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample optical isomers
    • 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
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44739—Collecting the separated zones, e.g. blotting to a membrane or punching of gel spots

Definitions

  • the subject matter of the invention refers to a method of separating the substance components of a multi-component mixture using electrophoresis and orthogonally and simultaneously chromatographic processes.
  • the separation of substance takes place in the adsorbent layer through which the mobile phase solution flows in one direction and an electric field is applied perpendicularly to this direction and the mixture/solution of the separated substances is continuously supplied to a specific location of the separation system.
  • the separated substances form bands deviating at different angles in relation to the flow direction of the mobile phase if their molecules undergo electrolytic dissociation.
  • the device and method are described in the patent specification: Ch. D. Scott, Method and apparatus for continuous annular electrochromatography, United States Patent, 1987, Patent Number 4, 683,042.
  • the method described in the patent specification does not solve the problem of separating two substances that form the common band, i.e. the bands of substances migrating through the separation system and deflecting at the same angle.
  • the invention solves the problem of separation of a substance, especially from a multi- component mixture in which two essential components form a common band as a result of these two components demonstrating equal electrophoretic mobility when conducting an orthogonal pressurized planar electrochromatography process conducted under the conditions of an electric field applied perpendicularly to the flow direction of the mobile phase through the adsorbent layer (stationary phase), where after the separation process the two essential components are collected at the output as separate solutions.
  • the method of preparative separation of two essential substances comprised in a multi-component mixture - the two mentioned essential components which form a band deviated at the same angle with respect to the direction of the flow of the mobile phase in the orthogonal pressurized planar electrochromatography in a not overloaded system, while the direction of the flow of the mobile phase through the adsorbent layer is perpendicular to the applied electric field - consists in the multicomponent mixture of separated substances being introduced with periodic interruptions into the separation system, preferably when the duration of the periodic delivery of the solution/mixture to the separation system and the interruption of delivery of the mixture to the separation system is equal to the difference in the retention time of the two essential substances showing the same deflection of their bands from the direction of the flow of the mobile phase, in the chromatographic system of a two-dimensional separation system; that is, the difference in time needed to elute these substances from the chromatographic system when it functions as an analytical system for these essential substances.
  • the periodic delivery of the separated mixture is reduced by at least the period of time corresponding to the double width of the analytical peak of one of the two essential substances, preferably that of the higher retention, while the duration of periodical interruption in the delivery of the solution/mixture is extended by the period corresponding to the double width of the analytical peak of one of the two essential separation substances, preferably that of the higher retention.
  • the described substance separation method allows continuous and alternate collection from the separation system of each of the two essential components that show the same angle of deflection of their bands from the hydrodynamic flow direction of the mobile phase, even if there are third/other components in the mixture of the separated substances.
  • the conditions for the separation process of the substance mixture are so selected that the bands of the other/third components of this mixture show different angles of deflection from the flow direction of the mobile phase in relation to the angle of deflection of the essential components. They will then flow out of the separation system through other output channels/tubes than the essential substances.
  • the method allows for complete separation of the mixture by properly selecting the composition of the mobile phase and/or the stationary phase and the duration of periodic delivery and the duration of periodic interruption of the delivery of mixture of the separated substances. Due to the increase in the efficiency of the separation process, the solution of the essential components, created at the boundary of their bands and forming a transition zone between the bands of the two essential components at the output of the separation system can be collected in a separate container and recycled for separation as described above.
  • the method of separation particularly refers to those substances which show the same electrophoretic mobility and thus their bands deviate from the direction of the hydrodynamic flow of the mobile phase at an equal angle.
  • Enantiomers and diastereoisomers are substances that fulfill this feature.
  • the separation system has a chiral stationary phase or the mobile phase should contain a chiral additive.
  • the method is schematically presented in the drawing, in which the mobile phase solution flows through the adsorbent layer (1) in the direction indicated by the arrows (2). Perpendicular to the flow of the mobile phase is the electric field generated by the high voltage power supply connected to the electrodes (3).
  • a solution of the mixture of separated substances is supplied to the adsorbent layer at the point indicated by the arrow (4) with periodic interruptions, as in the invention.
  • the substances of hypothetical separation mixture form four bands (5 - 8) that are deviated from the flow direction of the mobile phase at different angles. These bands, actually the solutions of the separated substances, flow out of the separation system (mobile phase - adsorbent) through the tubes/channels (9) into the containers (10 - 14) onto the solutions of the separated substances.
  • the band (6) is formed by two essential components and is presented as a continuous line because these components show the same electrophoretic mobility and their bands deviate in the electric field from the direction of the hydrodynamic flow of the mobile phase at the same angle.
  • the bands (5, 7, 8), in contrast to the band (6), are characterized by interrupted continuity as they relate to individual substances.
  • Two essential separate components, their solutions, are alternately directed into the containers (11) and (13) through a three-way valve that is switched according to their periodic discharge from the separation system accordingly to the interrupted delivery of a solution of separated substances into the separation system.
  • the solution flowing through the output channel via a three-way valve (15), forming a zone of transitional solution between the bands of the two essential components, that contain these components jointly, is collected in the container (12).
  • the collected solution of the two essential components in the container (12) can be returned to the separation system or separated by another method.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
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  • General Physics & Mathematics (AREA)
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  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

A method of preparatively separating two analytes having the same electrophoretic mobility, such as enantiomers, from each other and from a multicomponent sample using orthogonal pressurized planar electrochromatography (OPPEC) is disclosed. The sample (4) is periodically injected into the flow (2) of a mobile phase through an adsorbent layer (1) having an electric field (3) applied perpendicularly to the direction of flow of the mobile phase. The sample undergoes simultaneous 2D separation (chromatographic in the mobile phase flow direction, electrophoretic in the orthogonal direction). The two analytes having the same electrophoretic mobility are deflected at the same angle (6), but are separated in time due to their different retention times. The two analytes may be collected as separate fractions in separate containers (11) and (13).

Description

A method of preparative separation of substances using electrophoresis and chromatography processes conducted orthogonally and simultaneously
The subject matter of the invention refers to a method of separating the substance components of a multi-component mixture using electrophoresis and orthogonally and simultaneously chromatographic processes.
In the prior art a device for orthogonal pressurized planar electrochromatography is known and its method of separation for preparative purposes is described in: T.H. Dzido, E. Lopaciuk, P. Piocharz, A. Chomicki, M. Zemhrzycka, H. Frank, Equipment and preliminary results for orthogonal pressurized planar electrochromatography, J. Chromatogr. A 1334 (2014) 149-155, and a patent application T.H. Dzido, E. Lopaciuk, P. Piocharz, A. Chomicki, A. Torbicz, Chamber for simultaneous conduct of processes of orthogonal pressurized planar electrochromatography and liquid chromatography, Application No. P.403242, filing date 20.03.2013. According to these descriptions, the separation of substance takes place in the adsorbent layer through which the mobile phase solution flows in one direction and an electric field is applied perpendicularly to this direction and the mixture/solution of the separated substances is continuously supplied to a specific location of the separation system. In such an arrangement, the separated substances form bands deviating at different angles in relation to the flow direction of the mobile phase if their molecules undergo electrolytic dissociation.
These bands can be continuously collected at the output of the separation system into the fraction collector containers, which is the greatest advantage of this device and the way of performing the separation of multi-component mixtures of substances. These advantages are not shared by the column chromatography systems to which the mixture of the separated substances is pulse-introduced and the separated components of this mixture are sequentially eluted from the separation system and collected into the fraction collector containers. Typically, the next portion of the mixture of separated substances is introduced/injected into the chromatographic column after eluting the last component from the previous input dose.
Separation of substances by orthogonal pressurized planar electrochromatography has a significant disadvantage when the bands of two substances are deviated at the same angle with respect to the flow direction of the mobile phase. This applies to compounds with equal electrophoretic mobility, e.g. enantiomers. Under such conditions these two substances are not separated from one another (they are collected into one container after leaving the separation system) but can only be separated from other components that show different angles of deviation from the flow direction of the mobile phase.
Also known are the device and method of two-dimensional continuous separation of the substance mixtures with the forced flow of the mobile phase through a rotating bed of the stationary phase (adsorbent) in the annular form and an electric field applied perpendicularly to this flow. During the separation process the substance bands deflect at different angles in the adsorbent bed and the solutions of these components can be collected at the output of the separation system. The device and method are described in the patent specification: Ch. D. Scott, Method and apparatus for continuous annular electrochromatography, United States Patent, 1987, Patent Number 4, 683,042. The method described in the patent specification does not solve the problem of separating two substances that form the common band, i.e. the bands of substances migrating through the separation system and deflecting at the same angle.
A method of continuous separation of substance mixtures using the simulated moving bed/adsorbent process is known, D.B. Broughton, G.G. Gerhold, Continuous sorption process employing fixed bed of sorbent and moving inlets and outlets, US Patent 2,985,589 (1961) and DM. Ruthven, C.B. Counter-current and simulated counter- current adsorption separation processes, Chem. Eng. Sci. 44 (1989) 1011-1038. However, this method uses only 'liquid chromatography', i.e. the partition of substance in two phases, and allows the separation of uncomplicated mixtures of substances, usually two in the separated mixture.
The invention solves the problem of separation of a substance, especially from a multi- component mixture in which two essential components form a common band as a result of these two components demonstrating equal electrophoretic mobility when conducting an orthogonal pressurized planar electrochromatography process conducted under the conditions of an electric field applied perpendicularly to the flow direction of the mobile phase through the adsorbent layer (stationary phase), where after the separation process the two essential components are collected at the output as separate solutions.
According to the invention, the method of preparative separation of two essential substances comprised in a multi-component mixture - the two mentioned essential components which form a band deviated at the same angle with respect to the direction of the flow of the mobile phase in the orthogonal pressurized planar electrochromatography in a not overloaded system, while the direction of the flow of the mobile phase through the adsorbent layer is perpendicular to the applied electric field - consists in the multicomponent mixture of separated substances being introduced with periodic interruptions into the separation system, preferably when the duration of the periodic delivery of the solution/mixture to the separation system and the interruption of delivery of the mixture to the separation system is equal to the difference in the retention time of the two essential substances showing the same deflection of their bands from the direction of the flow of the mobile phase, in the chromatographic system of a two-dimensional separation system; that is, the difference in time needed to elute these substances from the chromatographic system when it functions as an analytical system for these essential substances. Subsequently, two essential components forming the bands deviated at the same angle with respect to the flow direction of the mobile phase are collected alternately as separate solutions into separate containers, where the transition zone solution formed at a merger of the bands of two essential components that form the solution of these two essential ingredients is collected in a separate container and then re-introduced into the separation system and subjected to the separation described above or by other known methods or is removed from the process. The remaining substances that form bands deviated from the hydrodynamic flow direction of the mobile phase at different angles with regard to the band of essential components appearing at other outputs of the separation system are collected separately.
Preferably, if in accordance with the proposed invention the periodic delivery of the separated mixture is reduced by at least the period of time corresponding to the double width of the analytical peak of one of the two essential substances, preferably that of the higher retention, while the duration of periodical interruption in the delivery of the solution/mixture is extended by the period corresponding to the double width of the analytical peak of one of the two essential separation substances, preferably that of the higher retention.
It is advantageous if, in accordance with the proposed invention, the shortening the periodic delivery of the mixture of separated substances and the extension of the duration of periodic interruption of the delivery of the mixture of separated substances is experimentally performed depending on the degree of purity of the substances obtained in the process.
The described substance separation method allows continuous and alternate collection from the separation system of each of the two essential components that show the same angle of deflection of their bands from the hydrodynamic flow direction of the mobile phase, even if there are third/other components in the mixture of the separated substances. The conditions for the separation process of the substance mixture are so selected that the bands of the other/third components of this mixture show different angles of deflection from the flow direction of the mobile phase in relation to the angle of deflection of the essential components. They will then flow out of the separation system through other output channels/tubes than the essential substances.
In addition, the method allows for complete separation of the mixture by properly selecting the composition of the mobile phase and/or the stationary phase and the duration of periodic delivery and the duration of periodic interruption of the delivery of mixture of the separated substances. Due to the increase in the efficiency of the separation process, the solution of the essential components, created at the boundary of their bands and forming a transition zone between the bands of the two essential components at the output of the separation system can be collected in a separate container and recycled for separation as described above.
In accordance with the invention the method of separation particularly refers to those substances which show the same electrophoretic mobility and thus their bands deviate from the direction of the hydrodynamic flow of the mobile phase at an equal angle. Enantiomers and diastereoisomers are substances that fulfill this feature. In the case of the former, the separation system has a chiral stationary phase or the mobile phase should contain a chiral additive. According to the invention the method is schematically presented in the drawing, in which the mobile phase solution flows through the adsorbent layer (1) in the direction indicated by the arrows (2). Perpendicular to the flow of the mobile phase is the electric field generated by the high voltage power supply connected to the electrodes (3). A solution of the mixture of separated substances is supplied to the adsorbent layer at the point indicated by the arrow (4) with periodic interruptions, as in the invention. In the figure, the substances of hypothetical separation mixture form four bands (5 - 8) that are deviated from the flow direction of the mobile phase at different angles. These bands, actually the solutions of the separated substances, flow out of the separation system (mobile phase - adsorbent) through the tubes/channels (9) into the containers (10 - 14) onto the solutions of the separated substances. The band (6) is formed by two essential components and is presented as a continuous line because these components show the same electrophoretic mobility and their bands deviate in the electric field from the direction of the hydrodynamic flow of the mobile phase at the same angle. The bands (5, 7, 8), in contrast to the band (6), are characterized by interrupted continuity as they relate to individual substances. A solution of two essential components separated in the band (6), showing equal electro -osmotic mobility, flows out through one of the four exit tubes (exit channels) (9) via a three-way valve (15) into the containers (11-13). Two essential separate components, their solutions, are alternately directed into the containers (11) and (13) through a three-way valve that is switched according to their periodic discharge from the separation system accordingly to the interrupted delivery of a solution of separated substances into the separation system. Subsequently, the solution flowing through the output channel via a three-way valve (15), forming a zone of transitional solution between the bands of the two essential components, that contain these components jointly, is collected in the container (12). The collected solution of the two essential components in the container (12) can be returned to the separation system or separated by another method.

Claims

Patent Claims
1. A method for preparative separation a multicomponent mixture with the separation of two components having the same electrophoretic mobility, hereinafter referred to as the essential components, comprising continuous delivery of the said mixture to the adsorbent layer through which the solution of the eluent hydrodynamically flows, when applying the electric field perpendicularly to the flow direction of this eluent, where the separated components form bands deviating at different angles relative to the flow direction of the mobile phase, and then collecting the separated substances into separate containers, characterized in that the multicomponent mixture of the separated substances is introduced with periodic interruptions into the separation system; it is advantageous when the duration of periodic delivery of the separated solution/mixture into the separation system and the duration of interruption in delivering the mixture to the separation system are equal to the difference in retention time of the two mentioned essential substances with equal deviation angle of their bands from the direction of the flow of the mobile phase in the two-dimensional chromatographic separation system used, and then two essential components appearing at the output of the separation system and forming the bands deviated at the same angle with respect to the flow direction of the mobile phase are alternately collected into separate containers as separate solutions, where the transition zone solution formed through a combination of bands of two essential components, that contain essential ingredients, is collected in a separate container and then re-introduced into the separation system and subjected to separation or removal from the process, while the remaining components of the mixture that form bands deviated from the direction of the hydrodynamic flow of the mobile phase at different angles relative to the band of essential components that appear at other outputs of the separation system, are collected separately.
2. The method of claim 1, characterized in that the duration of the periodic delivery of the mixture of separated substances is reduced by the period of time corresponding to the double width of the analytical peak of one of the two separated essential substances, preferably the one of higher retention, and the period of interruption in the delivery of the solution/mixture of separated substances is extended by the period of time corresponding to the double width of the analytical peak of one of the two separated essential substances, preferably the one of higher retention.
3. The method of claim 2, characterized in that the shortening of the periodic delivery of the mixture/solution of separated substances and the extension of the periodic interruption in the delivery of the solution/mixture of separated substances are made according to the degree of purity of the substances obtained in the process.
PCT/PL2017/000062 2016-06-09 2017-06-08 A method of preparative separation of substances using electrophoresis and chromatography processes conducted orthogonally and simultaneously WO2017213528A1 (en)

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