WO2010010366A1 - Agencement chromatographique à contre-courant - Google Patents

Agencement chromatographique à contre-courant Download PDF

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Publication number
WO2010010366A1
WO2010010366A1 PCT/GB2009/001849 GB2009001849W WO2010010366A1 WO 2010010366 A1 WO2010010366 A1 WO 2010010366A1 GB 2009001849 W GB2009001849 W GB 2009001849W WO 2010010366 A1 WO2010010366 A1 WO 2010010366A1
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WO
WIPO (PCT)
Prior art keywords
phase
column
liquid
mixture
fluid
Prior art date
Application number
PCT/GB2009/001849
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English (en)
Inventor
Peter Hewitson
Svetlana Ignatova
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Brunel University
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 Brunel University filed Critical Brunel University
Publication of WO2010010366A1 publication Critical patent/WO2010010366A1/fr

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Classifications

    • 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
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
    • 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
    • G01N30/42—Flow patterns using counter-current
    • 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
    • G01N2030/381—Flow patterns centrifugal chromatography

Definitions

  • the present invention relates to obtaining a substance, particularly by separation, by means of counter-current chromatography.
  • bioactive compounds are often found in plant extracts in very low concentrations. Thus it is desirable to process a large quantity of crude extract at a high throughput. It is also desirable to provide a process with reduced consumption of organic solvents which are harmful to the environment. It is further desirable to obtain an enriched and/or concentrated target compound.
  • CCC counter-current chromatography
  • ICcE intermittent counter-current extraction
  • DE 19842550A1 discloses a process monitoring method for simulated moving bed chromatography apparatus. Liquid-solid exchange columns are arranged in series and measurements are continuously taken at product extraction points to estimate concentrations within the columns which cannot be directly measured themselves.
  • US 2006/0243665 discloses an arrangement for separating constituents of a liquid feed by liquid-liquid centrifugal chromatography. With constituents having different partition coefficients such that they are respectively carried along at unequal velocities by a light solvent and a heavier solvent, the feed is continuously injected at an intermediate point of a cell assembly. Successive cycles comprising alternately a heavy solvent injection stage and a light solvent injection stage are carried out respectively at the two ends of the column consisting of the interconnection in series of at least one set of separation cells, and the separated constituents are collected during each one of said stages at the column ends opposite the solvent injection points.
  • aspects of the present invention seek to provide an improved method and apparatus for the separation of a target substance from a mixture by means of counter-current chromatography.
  • a method of separating components of a mixture employing fluid-liquid counter-current chromatography in which the mixture is introduced into a central region of a chromatographic column having an upper fluid phase and a lower liquid phase, the fluid and liquid phases alternating in time between a normal phase, in which the less polar phase is mobile in one direction and the more polar phase is substantially stationary, and a reversed phase in which the more polar phase is mobile in the opposite direction and the less polar phase is substantially stationary.
  • the fluid phase may also be a liquid; alternatively it may be a gas or a foam.
  • the mixture is introduced into the less polar phase at the central region, and during reversed phases, the mixture is introduced into the more polar phase at the central region.
  • the arrangement may be the other way round.
  • composition of the eluant at one or both ends of the column is monitored and the flow rates of the fluid and liquid phases and/or the durations of the normal and reversed phase cycles are varied in dependence upon the composition at one or both ends.
  • the durations of, and the flow rates during, one or more initial normal and reversed phases may be predetermined; thereafter these operating parameters are determined in dependence upon the detected composition(s).
  • Alternate pumping of the upper and lower phases may continue. This serves to remove remaining impurities with different distribution ratios from that of the target component.
  • a chromatographic apparatus comprising a fluid-liquid counter-current column having a supply region for a mixture to be separated, first means at end one of the column for intermittently supplying upper phase fluid to the column, second means at the opposite end of the column for intermittently supplying lower phase liquid to the column, detection means at one or both ends of the column for detecting the composition of an eluant, and processor means for controlling the first and second supply means in dependence upon the detected compositions).
  • the first and second supply means are preferably operated alternately, so that one phase moves while the other is stationary.
  • the mixture is preferably supplied to a central region of the column.
  • Embodiments of methods according to the invention may be performed in a centrifugal partition chromatograph with mobile and stationary phases.
  • a dual-flow arrangement may be provided so that the phases flow continuously in opposite directions, and control of the flow rates is used to retain the target within the column.
  • the processor means may control the duration of the periods of operation of the first and second supply means and/or the flow rates produced thereby.
  • the processor means at least during initial phases of operation, may also be controlled by a timer.
  • the processor means may also control the supply of the mixture to the central supply region. Accordingly to a third aspect of the present invention there is provided a method of separating and/or concentrating a target substance including adaptive control to retain the target compound during alternating elution in a liquid-liquid chromatographic apparatus.
  • isocratic involves the use of a constant composition phase.
  • an “isocratic” run is one which resolves a solute using a solvent system that does not change composition during the run.
  • Figure 1 is a schematic view of an apparatus according to a preferred embodiment of the present invention.
  • Figure 2 is a view of the apparatus of Figure 1 operating in a first, normal phase
  • Figure 3 is a view of the apparatus of Figure 1 operating in a second, reversed phase
  • Figure 4 is a chromatogram obtained using a scaled-up apparatus in accordance with a second embodiment of the present invention.
  • the Figures show a counter-current chromatography device or column 10 comprising two coils 11, 12 which rotate in planetary motion. Between the coils is located an intermediate four- way junction 14.
  • the conduits 16 to 19 respectively connect the junction 14 to first coil 11, to a first reservoir 27 from which a sample mixture with upper phase liquid is pumped into the upper liquid phase, to a second reservoir 28 from which a sample mixture with lower phase liquid is pumped into the lower liquid phase, and to second coil 12.
  • a pump 31 is provided at the side of the coil 11 remote from junction 14 for supplying upper phase liquid to the device 10.
  • a pump 32 is provided at the side of coil 12 remote from junction 14 for supplying lower phase liquid to the device 10.
  • the apparatus 10 is broadly similar to that disclosed in the Couillard patent application mentioned above.
  • ultra-violet detectors 35, 36 are respectively provided for monitoring the eluant in the lower phase and the upper phase. Control of the flow and/or timing of the cycles is provided dependant on the monitoring of the eluant by the detectors.
  • a reservoir 45 for receiving lower phase liquid from the column.
  • a reservoir 46 for receiving upper phase liquid from the column.
  • the operation of the pumps 27, 28, 31, 32 of the apparatus 10 is controlled by a processor 40 which is associated with a timer device 41.
  • a hydrodynamic equilibrium is established between two liquid phases within the coils 11, 12 such that one liquid phase (the mobile phase) flows past the other liquid phase (the stationary phase).
  • the stationary phase is held in the column by the force field due to rotation of the coils in planetary motion.
  • a sample solution is pumped into the system at the junction 14 between the coils and the mobile phase is flowed alternately, first in normal phase (upper phase mobile, see Figure 2) and then in opposite direction, reversed phase (lower phase mobile, see Figures 3).
  • the various compounds in the sample have different distribution ratios between the two liquid phases. Accordingly, • they separate with time between those phases and elute from opposite ends of the coils.
  • the operating parameters i.e. the various flow rates, and the duration of the flow in each direction are established such that the target compound is retained within the coils.
  • the upper phase is initially pumped from left to right at a selected flow rate of 40ml/min for a normal phase run of 4 minutes, while an original crude sample solution of the herbal medicine in the upper phase is loaded at junction 14 at a lower flow rate of lOml/min, see the arrows in Figure 2.
  • the target compound flows a similar distance in each alternating step rather than being eluted from either end of the coils with the residual sample.
  • the detectors 35, 36 of the apparatus 10 continuously monitor the eluants from both ends of the column and adjust the operating parameters to ensure as much as possible of the target is retained in the column. The process thus results in the enrichment and concentration of the target compound.
  • the residuals are eluted to leave only the target compound retained within the column. Then the target can be easily (and at low cost) removed from the column by pumping out its content. It is then recovered from solution, e.g. by vacuum evaporation, freeze drying etc.
  • the criteria for terminating the alternate flows may be the monitoring by detectors 35, 36 that the target compound is beginning to elute from one end of the column, or both ends, or that the flow of target compound at one or both ends has exceeded a predetermined threshold value.
  • the alternating flows shown in Figures 2 and 3 continue until target compound is observed by the detection system to elute from one end of column 10.
  • the processing system 40 adjusts the operating parameters (flow rates and/or cycle timings) to ensure as much as possible of the target compound is retained in the column. Slight adjustments to flow rates and timings continue being made until the target compound begins eluting from both sides of the column.
  • the sample injection at junction 14 is stopped and the upper and lower phases are pumped alternately into their respective reservoirs 36, 35 to remove all remaining impurities with different distribution ratios from that of the target compound.
  • the column 10 now contains only the target compound in solution, which is pumped out from one end and recovered.
  • the above-described arrangement has numerous advantages.
  • the target substance is retained within the apparatus during the separation procedure and the crude residual material is washed away.
  • the separated target substance can then be pumped from the column as an enriched solution.
  • This is in contrast to existing chromatographic techniques for such separations which dilute the target substance. It is easier to remove a substance contained in the column in a liquid- liquid arrangement than in other types of liquid chromatography which employ a solid stationary phase.
  • the arrangement has the advantage of providing liquid-liquid extraction in dynamic mode.
  • Methods according to the invention lead to increased throughput for pharmaceutical manufacturing and, as a consequence, reduced consumption of organic solvents.
  • UV detectors 35, 36 for example a photo diode array (PDA), Evaporative Lightscattering Detectors (ELSD) and Mass Spectrometers (MS). Detection may be undertaken intermittently rather than continuously.
  • PDA photo diode array
  • ELSD Evaporative Lightscattering Detectors
  • MS Mass Spectrometers
  • Only one of the detectors may be provided; in this case the processor 40 needs more detailed programming to decide when to terminate flow phases away from the detector. Alternatively this decision may be based partly or wholly on human observation.
  • both detectors are omitted and all decisions are taken by the operator.
  • the necessary input controls are provided to enable the operator to stop the flow, to start the flow in a particular direction, and optionally a timer device to set a particular duration of flow.
  • a control for setting the rates of flow may also be provided.
  • junction 14 may be provided at any convenient location in the central region of the column and there may be separate connections to the main flow path for conduits 17 and 18.
  • a single reservoir can be provided with the upper and lower phases with sample being selectively supplied thereto.
  • the supplies are controlled manually.
  • the method and apparatus can be used to separate a wide range of different substances from mixtures, and from mixtures having 2, 3, 4 or more components. More than one target substance may be separated at the same time by arranging for the system to retain a plurality of substances in the column with similar distribution ratios.
  • a plurality of columns 10 may be provided e.g. to allow different substances to be separately collected.
  • the upper phase may comprise a gas or a foam material.
  • CPC centrifugal partition chromatograph
  • DPF dual-flow counter-current chromatograph
  • any other type of liquid-liquid chromatograph there may be used a hydrostatic toroidal coil arrangement.
  • the introduction point can be a junction near the midpoint of a single bobbin.
  • the separation process was scaled up from the relatively small apparatus (identified below as “Midi”, and typically 912ml) to larger apparatus, e.g. 4.6L, and identified below as “Maxi”.
  • larger apparatus e.g. 4.6L, and identified below as “Maxi”.
  • phase system used was HEMWat-16a (n-heptane, ethyl acetate, methanol and water) in a volume ratio 4:5:4:5.
  • the sample system was based on the GUESSmix proposed by Friesen.
  • the sample was a mix of caffeine (C), vanillin (V), carvone (O) and naringenin (N) dissolved in 50/50 (v/v) of upper and lower phases of the HEMWat-16a phase system.
  • the columns were filled, from empty, with upper phase as follows. All lines in the liquid handling system were initially primed with the appropriate phase system. The columns were filled with upper phase at 2000 niL/min (1000 mL/min through each column) from tail (periphery) to head (centre) with the columns in parallel to maintain balance during rotation. After 3 min, the centrifuge was rotated at 200 rpm to expel trapped air from the system from the "head" end of the column. . Equilibration was performed in reversed phase mode, aiming to fill the columns with a 50/50 mix of upper and lower phase.
  • the centrifuge was rotated at 350 rpm (41 x g) and lower phase was flowed from head (centre) to tail (periphery) at 500 mL/min with the flow path through the bobbins in parallel
  • the centrifuge rotational speed was increased to 600 rpm (121 x g) and then the flow rate was increased to 1200 mL/min to initiate break through.
  • the total volume of upper phase displaced was measured to confirm the initial volume of each phase in the columns was approximately 50%.
  • Figure 4 shows the resulting chromatogram from the Maxi-CCC intermittent counter- current extraction run.
  • the operating conditions were: speed, 600 rpm; upper and lower phase flow, 250 mL/min; sample loading 40.5g in 20 min.
  • Phase system Phase system: HEMWat (4:5:4:5, v/v/v/v).
  • Distribution ratios caffeine (0.09); vanillin (0.55); naringenin (1.25) and carvone (7.39).
  • Area A of the chromatogram represents upper phase elution, area B represents lower phase elution and arrow C indicates the period of coil pumpout.
  • the curves are as follows: squares - naringenin triangles - carvone diamonds - vanillin circles - caffeine.
  • the length of the column can be increased.
  • arrangements according to the present invention can be used to undertake flow chemistry, with appropriate reagents being fed to the apparatus.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

La présente invention concerne un appareil chromatographique de séparation des composants d’un mélange comprenant une colonne d’extraction fluide-liquide à contre-courant (10) présentant une région d’alimentation centrale (14) pour le mélange, des pompes d’alimentation en phase supérieure et en phase inférieure (31, 32) aux extrémités opposées de la colonne et des détecteurs UV (35, 36) permettant de détecter la composition d’un éluant, l’appareil étant surveillé et commandé par un processeur (40). Les pompes d’alimentation (31, 32) fonctionnent alternativement de telle sorte qu’une phase se déplace pendant que l’autre est immobile. Après l’arrêt de l’introduction du mélange dans la région (14), les substances résiduelles sont éluées pour concentrer un composant cible dans la colonne. Le composant cible est alors extrait par pompage et récupéré.
PCT/GB2009/001849 2008-07-24 2009-07-24 Agencement chromatographique à contre-courant WO2010010366A1 (fr)

Applications Claiming Priority (2)

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GB0813579.0 2008-07-24
GB0813579A GB0813579D0 (en) 2008-07-24 2008-07-24 counter-current chromatographic arrangement

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014048469A1 (fr) * 2012-09-26 2014-04-03 Technische Universität Dortmund Extracteur pour extraction de partage centrifuge et procédé de réalisation d'une extraction centrifuge
EP3512616B1 (fr) 2017-11-16 2019-11-20 ChromaCon AG Procédé pour surveiller, évaluer et commander un processus de purification chromatographique cyclique
CN111239310A (zh) * 2018-11-28 2020-06-05 中国科学院大连化学物理研究所 一种交替进样分析的高效液相色谱分离系统及操作方法
US20220016546A1 (en) * 2018-11-14 2022-01-20 Institut National Polytechnique De Toulouse Method and device for continuous countercurrent transfer of material between two fluid phases
US11548864B1 (en) 2022-06-15 2023-01-10 Zaiput Flow Technologies LLC Separation of chemical species using multiple liquid phases and related systems

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4615805A (en) * 1985-09-13 1986-10-07 The United States Of America As Represented By The Department Of Health And Human Services Method for continuous countercurrent foam separation
US4714554A (en) * 1986-10-03 1987-12-22 The United States Of America As Represented By The Department Of Health And Human Services Cross-axis synchronous flow-through coil planet centrifuge free of rotary seals: apparatus and method for performing countercurrent chromatography
FR2856933A1 (fr) * 2003-07-02 2005-01-07 Inst Francais Du Petrole Procede et dispositif de separation des constituants d'une charge liquide par chromatographie liquide-liquide centrifuge

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4615805A (en) * 1985-09-13 1986-10-07 The United States Of America As Represented By The Department Of Health And Human Services Method for continuous countercurrent foam separation
US4714554A (en) * 1986-10-03 1987-12-22 The United States Of America As Represented By The Department Of Health And Human Services Cross-axis synchronous flow-through coil planet centrifuge free of rotary seals: apparatus and method for performing countercurrent chromatography
FR2856933A1 (fr) * 2003-07-02 2005-01-07 Inst Francais Du Petrole Procede et dispositif de separation des constituants d'une charge liquide par chromatographie liquide-liquide centrifuge

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Title
DELANNAY E ET AL: "Multiple dual-mode centrifugal partition chromatography, a semi-continuous development mode for routine laboratory-scale purifications", JOURNAL OF CHROMATOGRAPHY, ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, NL, vol. 1127, no. 1-2, 15 September 2006 (2006-09-15), pages 45 - 51, XP024967628, ISSN: 0021-9673, [retrieved on 20060915] *
HEWITSON P ET AL: "Intermittent counter-current extraction as an alternative approach to purification of Chinese herbal medicine", JOURNAL OF CHROMATOGRAPHY, ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, NL, vol. 1216, no. 19, 8 May 2009 (2009-05-08), pages 4187 - 4192, XP026050507, ISSN: 0021-9673, [retrieved on 20081207] *
ITO ET AL: "Rapid determination of carbamate pesticides in food using dual counter-current chromatography directly interfaced with mass spectrometry", JOURNAL OF CHROMATOGRAPHY, ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, NL, vol. 1187, no. 1-2, 6 February 2008 (2008-02-06), pages 53 - 57, XP022547070, ISSN: 0021-9673 *
RUBIO N ET AL: "Multiple dual-mode countercurrent chromatography applied to chiral separations using a (S)-naproxen derivative as chiral selector", JOURNAL OF CHROMATOGRAPHY, ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, NL, vol. 1216, no. 48, 27 November 2009 (2009-11-27), pages 8505 - 8511, XP026740857, ISSN: 0021-9673, [retrieved on 20091008] *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014048469A1 (fr) * 2012-09-26 2014-04-03 Technische Universität Dortmund Extracteur pour extraction de partage centrifuge et procédé de réalisation d'une extraction centrifuge
EP3512616B1 (fr) 2017-11-16 2019-11-20 ChromaCon AG Procédé pour surveiller, évaluer et commander un processus de purification chromatographique cyclique
US11460454B2 (en) 2017-11-16 2022-10-04 Chromacon Ag Method for monitoring, evaluating, and controlling a cyclic chromatographic purification process
US20220016546A1 (en) * 2018-11-14 2022-01-20 Institut National Polytechnique De Toulouse Method and device for continuous countercurrent transfer of material between two fluid phases
CN111239310A (zh) * 2018-11-28 2020-06-05 中国科学院大连化学物理研究所 一种交替进样分析的高效液相色谱分离系统及操作方法
US11548864B1 (en) 2022-06-15 2023-01-10 Zaiput Flow Technologies LLC Separation of chemical species using multiple liquid phases and related systems

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