CN110736798A - Separation method of chiral isomers by electrochemically modified liquid chromatography - Google Patents

Separation method of chiral isomers by electrochemically modified liquid chromatography Download PDF

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CN110736798A
CN110736798A CN201910937514.2A CN201910937514A CN110736798A CN 110736798 A CN110736798 A CN 110736798A CN 201910937514 A CN201910937514 A CN 201910937514A CN 110736798 A CN110736798 A CN 110736798A
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solution
potential
mobile phase
stainless steel
liquid chromatography
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何文杰
丁燕雯
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Fonda Medical Technology (suzhou) Co Ltd
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Fonda Medical Technology (suzhou) Co Ltd
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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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • 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
    • G01N30/60—Construction of the column

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Abstract

The invention discloses a separation method of chiral isomers by electrochemically modified liquid chromatographs, which realizes the separation of hexobarbital and mephenytoin chiral isomers by applying a potential to a liquid chromatogram stationary phase and relying on the change of the potential. β -cyclodextrin in a mobile phase interacts with a porous graphene surface layer in a stationary phase, so that the surface of the stationary phase is reversibly changed, the separation capability is enhanced, and the elution sequence of enantiomers is also changed.

Description

Separation method of chiral isomers by electrochemically modified liquid chromatography
FIELD
The invention relates to the field of pharmaceutical analysis, in particular to a separation method of chiral isomers by electrochemically modified liquid chromatographs.
Background
Liquid chromatography is a key technology used to separate mixtures various types of complex mixtures of substances (e.g. serum, pharmaceutical preparations, waste fluids, etc.) can be separated by a combination of mobile and stationary phases, however, because of the different properties of substances, more stationary phases are often developed.
The Electrochemical Modulation Liquid Chromatography (EMLC) technology applies a potential to the stationary phase, thereby changing the properties of the surface of the stationary phase and finally changing the separation capacity of the chromatography. Early EMLC focused primarily on metal electron deposition and redox reactions affecting separation. In recent studies, EMLC has been more applied to separation of chiral isomers of compounds by using porous graphene as a stationary phase. This separation is mainly based on changes in potential, affecting the properties of the donor and the acceptor. That is, if the potential is positive, the porous graphene becomes a strong acceptor, and the compound is a donor and shows strong retention on the system. If the potential is negative, the opposite is true.
SUMMARY
, the disclosure relates to a method for separating chiral isomers by electrochemically modified liquid chromatographs, comprising the steps of:
applying a potential to a liquid chromatography stationary phase, the stationary phase comprising porous graphene and a chiral isomer, and the mobile phase comprising an aqueous acetonitrile solution and a solution of β -cyclodextrin, and
separating the chiral isomers using the liquid chromatography.
In another aspect , the disclosure relates to a method of preparing a chromatography column comprising:
stainless steel tubes were placed in methanol solution and LiClO in this order4Boiling in the solution;
dispersing porous graphene in a dimethylaminomethane/acetonitrile solution to obtain an th mixture;
packing the th mixture was packed into stainless steel tubes.
Brief description of the drawings
Figure 1 illustrates the interaction between donor and acceptor compounds at different potentials according to certain embodiments of the present disclosure.
Figure 2 shows a schematic diagram of a chromatography column structure according to certain embodiments of the present disclosure.
FIG. 3 shows a graph of the effect of hexobarbital chiral isomer of certain embodiments of the present disclosure in the mobile phase without the addition of β -cyclodextrin and with the addition of β -cyclodextrin, and separation at different potentials.
Figure 4 shows a graph of the effect of chiral isomers of mephenytoin without β -cyclodextrin and with β -cyclodextrin in the mobile phase and at different potentials for separation according to certain embodiments of the present disclosure.
Detailed description of the invention
One skilled in the relevant art will recognize, however, that embodiments can be practiced without or more of these specific details, but with other methods, components, materials, and so forth.
Unless otherwise required by the disclosure, throughout the specification and the appended claims, the words "comprise", "comprising", and "have" are to be construed in an open, inclusive sense, i.e., "including but not limited to".
Thus, the appearances of the phrases "in an embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment, and further, particular elements, structures or features may be combined in any suitable manner in or more embodiments.
Definition of
In the present disclosure, β -cyclodextrin is a cyclic structure compound composed of 7 glucopyranose molecules, has a special cylindrical structure, and can be combined with porous inorganic and organic molecules to form a host-guest inclusion complex.
In this disclosure, hexobarbital has the molecular formula C12H16N2O3 barbiturate derivatives having sedative and hypnotic effects.
In the present disclosure, mefenton has the formula C12H14N2O2The chemical name of 5-ethyl is based on-3-methyl-5-phenylhydantoin, and is kinds of antiepileptic and antiarrhythmic drugs.
In the present disclosure, a working electrode refers to an electrode that can cause a significant change in the concentration of a component to be measured in a test solution during a test.
In the present disclosure, reference electrode refers to electrode used as reference comparison when measuring various electrode potentials, electrode reaction carried out on the reference electrode must be single reversible reaction, electrode potential is stable and reproducibility is good, usually, micro-dissolved salt electrode is used as reference electrode, and hydrogen electrode is only ideal but not easy-to-realize reference electrode.
In this disclosure, the counter electrode is a counter electrode, which functions to form a circuit with the working electrode to pass through the electrode.
In the present disclosure, chiral compounds refer to enantiomers of the same molecular weight, molecular structure, but arranged in opposite left and right directions, such as the real entity and its mirror.
Detailed Description
, the disclosure relates to a method for separating chiral isomers by electrochemically modified liquid chromatographs, comprising the steps of:
applying a potential to a liquid chromatography stationary phase, the stationary phase comprising porous graphene and a chiral isomer, and the mobile phase comprising an aqueous acetonitrile solution and a solution of β -cyclodextrin, and
separating the chiral isomers using the liquid chromatography.
In certain embodiments, the mobile phase further comprises LiClO4Solutions and phosphate buffer.
In certain embodiments, the volume fraction concentration of acetonitrile in the mobile phase is from 20% to 30%.
In certain embodiments, the mobile phase has a volume fraction concentration of acetonitrile of 20%.
In certain embodiments, the mobile phase has a concentration of β -cyclodextrin in the range of 10mM to 20 mM.
In certain embodiments, the mobile phase has a concentration of β -cyclodextrin of 15 mM.
Wherein separation is realized through the electro-adsorption of β -cyclodextrin and a Porous Graphene (PGC) surface layer in a chiral recognition stationary phase, and the separation mainly depends on the change of potential to adjust the separation effect.
In certain embodiments, LiClO is in the mobile phase4The concentration is 0.05M to 0.15M.
In certain embodiments, LiClO is in the mobile phase4The concentration was 0.1M.
In certain embodiments, the concentration of phosphate buffer in the mobile phase is from 10mM to 30 mM.
In certain embodiments, the concentration of phosphate buffer in the mobile phase is 20 mM.
In certain embodiments, the mobile phase is adjusted to a PH of 2 to 3.45 with phosphate buffer.
In certain embodiments, the mobile phase is adjusted to PH2 with phosphate buffer.
In certain embodiments, the potential is applied by constructing an electrochemically modified chromatography column.
In certain embodiments, the working electrode is porous graphene particles, the counter electrode is a stainless steel tube, and the reference electrode is an Ag/AgCl electrode containing a saturated NaCl solution.
In certain embodiments, the potential setting ranges from +0.5V to-1.0V.
In certain embodiments, the porous graphene has excellent conductivity as a carbon material having nano-scale pores on a two-dimensional basal plane, and the presence of the pores facilitates the improvement of the substance transport efficiency, and particularly, the atomic-scale pores may function to screen ions/molecules of different sizes.
In certain embodiments, the sample volume for liquid chromatography is 0.5 μ L.
In certain embodiments, the flow rate of liquid chromatography is 0.9 ml/min.
In certain embodiments, the elution time for liquid chromatography is 30 minutes.
In certain embodiments, the column temperature of the liquid chromatography is from 30 ℃ to 40 ℃.
In certain embodiments, the column temperature of the liquid chromatography is 30 ℃.
In another aspect , the disclosure relates to a method of preparing a chromatography column comprising:
stainless steel tubes were placed in methanol solution and LiClO in this order4Boiling in the solution;
dispersing porous graphene in a dimethylaminomethane/acetonitrile solution to obtain an th mixture;
packing the th mixture was packed into stainless steel tubes.
Wherein, the stainless steel pipe is a porous stainless steel pipe attached with a Nafion exchange membrane.
In certain embodiments, the stainless steel tube is placed in a methanol solution and LiClO4The boiling time in the solution is 0 to 30 minutes.
In certain embodiments, the stainless steel tube is placed in a methanol solution and LiClO4The boiling time in the solution was 10 minutes.
In certain embodiments, the volume ratio of dimethylaminomethane to acetonitrile in the dimethylaminomethane/acetonitrile solution is 10: 5 to 10: 9.
In certain embodiments, the volume ratio of dimethylaminomethane to acetonitrile in the dimethylaminomethane/acetonitrile solution is 10: 7.
In certain embodiments, the packing pressure is from 300bar to 400 bar.
In certain embodiments, the packing pressure is 345 bar.
In certain embodiments, the column format for liquid chromatography is 9.2cm long by 0.4cm internal diameter.
Example 1
porous stainless steel tubes (9.2 cm. times.0.4 cm) attached with Nafion exchange membranes were boiled in methanol solution for 10 minutes and then transferred to 1M LiClO4Boiling the solution for 10 min. The porous graphene is dispersed in dimethylaminomethane/acetonitrile (10: 7, v/v) to form a slurry, and then the slurry is filled into a chromatographic column under the pressure of 345bar, wherein conductive graphene particles serve as a working electrode, and the chromatographic column serves as a counter electrode.
Chromatographic conditions the mobile phase was divided into two, species containing β -cyclodextrin, 20% acetonitrile (0.1M LiClO4) and 80% aqueous solution (containing 15mM β -cyclodextrin, 0.1M LiClO4,20mM phosphate buffer, pH2.0)
Voltage setting: the open circuit voltage of 180mV comprising Ag/AgCl/saturated NaCl electrode is adopted, and the working voltage is set in the range of +0.5 to-1.0V.
Solution preparation: a sample solution of 500ppm HE or ME was prepared with the mobile phase.
Sample introduction volume: 0.5. mu.l
Elution time: and (3) 30 min.
As shown in fig. 1, in order to interact between the donor and the acceptor compound at different potentials, that is, if the potential is positive, the porous graphene becomes a strong acceptor, and the compound is a donor and shows strong retention on the system. If the potential is negative, the opposite is true.
As shown in fig. 2, the structure of the chromatographic column comprises: the device comprises an injector 1, an LC pump 2, a solid stainless steel pipe 3, porous graphene particles (working electrodes) 4, an electrolyte glass container 5, an Ag/AgCl electrode (reference electrode) 6 containing a saturated NaCl solution, and a porous stainless steel pipe (counter electrode) 7.
The results of the Hexobarbital (HE) test are shown in FIG. 3, the result of the upper half A is that β -cyclodextrin is not added, chiral isomers are not separated, the result of the lower half B is that 15mM β -cyclodextrin is added into the mobile phase, the separation effect of isomers d and I at negative potential is poor (separation factor 1.08), the separation factors of isomers d and I at positive potential of 0 and +0.5V are respectively 1.12 and 1.28, and the peak appearance sequence of isomers d and I at positive potential and negative potential is opposite.
The results of the Mefenton (ME) test are shown in FIG. 4, where the result of the upper part A is that β -cyclodextrin is not added and chiral isomers are not separated, and the result of the lower part B is that 15mM β -cyclodextrin is added to the mobile phase, isomers d and I are not separated well at negative potential (separation factor 1.06), and at positive potentials of 0 and +0.5V, the separation factors of isomers d and I are at 1.10 and 1.21, respectively, and at positive and negative potentials, the peak appearance of isomers d and I are in opposite order.
The test results show that the β -cyclodextrin is added in the mobile phase to help realize the separation of chiral isomers, and the separation effect can be changed significantly according to the adjustment of the point position, which is similar to the description in the figure 1, wherein the change of the electric potential causes the change of the electron acceptor and the electron donor, thereby enhancing or weakening the retention of certain compounds and finally influencing the elution sequence of the chiral isomers.
From the foregoing it will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications or improvements may be made by those skilled in the art without departing from the spirit and scope of the disclosure, and that such modifications or improvements are intended to be within the scope of the appended claims.

Claims (7)

  1. The method for separating chiral isomers by electrochemically modified liquid chromatographs comprises the following steps:
    applying a potential to a liquid chromatography stationary phase, the stationary phase comprising porous graphene and a chiral isomer, the mobile phase comprising an aqueous acetonitrile solution and β -cyclodextrin solution, and
    separating the chiral isomers using the liquid chromatography.
  2. 2. The separation method of claim 1, wherein the mobile phase further comprises LiClO4Solution and phosphate buffer solution, wherein the volume fraction concentration of the acetonitrile water solution is preferably 10-30 percent, more preferably 20 percent, the concentration of the β -cyclodextrin is preferably 10-20 mM, more preferably 15mM, and the LiClO is preferably selected4A concentration of 0.05M to 0.15M, more preferably 0.1M; preferably the phosphate buffer concentration is from 10mM to 30mM, preferably 20 mM; preferably the mobile phase has a pH of 2 to 3.45, more preferably 2.
  3. 3. The separation method according to claim 1 or 2, wherein the potential is set by constructing an electrochemically modified chromatography column, the working electrode is porous graphene particles, the reference electrode is an Ag/AgCl electrode containing saturated NaCl solution, the counter electrode is a stainless steel tube, and preferably the potential setting range is +0.5V to-1.0V.
  4. 4. The separation method according to of any one of claims 1 to 3, wherein the liquid chromatography has a sample volume of 0.5 μ L, a flow rate of 0.9ml/min, an elution time of 30 minutes, a column temperature of 30 ℃ to 40 ℃, preferably a column temperature of 30 ℃.
  5. 5. A method of preparing a chromatography column comprising:
    stainless steel tubes were placed in methanol solution and LiClO in this order4Boiling in solution；
    Dispersing porous graphene in a dimethylaminomethane/acetonitrile solution to obtain an th mixture;
    packing the th mixture was packed into stainless steel tubes.
  6. 6. The method of preparing a chromatography column of claim 5, wherein:
    the stainless steel tube was immersed in methanol solution and LiClO4The boiling time in the solution is 0 to 30 minutes, preferably 10 minutes;
    in the dimethylamino methane/acetonitrile solution, the volume ratio of the dimethylamino methane to the acetonitrile is 10: 5 to 10:9, preferably 10: 7;
    the packing pressure is from 300bar to 400bar, preferably 345 bar.
  7. 7. A method of preparing a chromatography column as claimed in claim 5 or 6, wherein the chromatography column has a specification of 9.2cm long and 0.4cm internal diameter.
CN201910937514.2A 2019-09-29 2019-09-29 Separation method of chiral isomers by electrochemically modified liquid chromatography Pending CN110736798A (en)

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US20120226054A1 (en) * 2011-03-03 2012-09-06 Board Of Trustees Of The University Of Arkansas Multiple stationary phase matrix and uses thereof
CN106996964A (en) * 2017-04-20 2017-08-01 山东赛托生物科技股份有限公司 Method and the application of hydroxypropyl beta cyclodextrin using reversed-phased high performace liquid chromatographic separating chiral enantiomer

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19905621A1 (en) * 1999-02-11 2000-08-17 Dechema Voltage-controlled chromatographic column comprises fixed bed of conductive material trapping or delaying polarized particles in flowing fluid as a function of applied voltage, with release following retention delay or change in potential
US20080182136A1 (en) * 2007-01-26 2008-07-31 Arnold Don W Microscale Electrochemical Cell And Methods Incorporating The Cell
JP2011038977A (en) * 2009-08-17 2011-02-24 Sumitomo Electric Ind Ltd Separation analysis method and separation analysis apparatus for redox material
US20120226054A1 (en) * 2011-03-03 2012-09-06 Board Of Trustees Of The University Of Arkansas Multiple stationary phase matrix and uses thereof
CN106996964A (en) * 2017-04-20 2017-08-01 山东赛托生物科技股份有限公司 Method and the application of hydroxypropyl beta cyclodextrin using reversed-phased high performace liquid chromatographic separating chiral enantiomer

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