CN104707358A - Novel double water phase high-speed countercurrent chromatography - Google Patents
Novel double water phase high-speed countercurrent chromatography Download PDFInfo
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Abstract
The present invention relates to a new high-speed countercurrent chromatography method using a novel double water phase solvent system, wherein the method is used for separation preparation of strongly polar natural products and chemical industry products. According to the method, (1) according to the novel double water phase solvent system, a hydrophilic alcohol, an inorganic salt and water form a double-phase system, the double phase solution contains a certain amount of water, and the polarity of the alcohol water top phase is much greater than the polarity of the organic top phase in the organic/water double-phase system; (2) compared with the conventional double water phase system, the system of the present invention has characteristics of low viscosity and high stationary phase retention, and can achieve multiple sample injection stable operation; (3) the used equipment and the used supplies have the low cost, and the subsequent treatment of the distillate is simple; and (4) the method can be coupled and combined with other chromatographic methods. According to the present invention, the alcohol-water double phase solvent system is applied for the J-type high speed countercurrent chromatograph, such that the important complement is provided for the existing high-speed countercurrent chromatography solvent system, and the significant advantages are provided in the field of separation of highly polar natural products and chemical industry products, particularly separation preparation of strongly polar traditional Chinese medicine efficacy components and unstable components.
Description
Technical field
The present invention is a kind of novel aqueous two-phase high speed adverse current chromatogram, using the aqueous two-phase dicyandiamide solution of hydrophily alcohol or ketone, inorganic salts and water composition one as Stationary liquid, another is as mobile phase, is realized the separation of strong polar compound by J-type helix tube high-speed counter-current chromatograph.
Background technology
As everyone knows, decocting decoction is Chinese medicine clinical practice one of formulation the most widely, and the main body of its effective substance must be made up of a large amount of strong polar compound, and the fact also demonstrates the health of strong polar compound to human body and plays an important role.But material foundation of tcm research is more pays close attention to low pole and middle polarity compound, sets foot in strong polar compound is less.Will open up the new traditional Chinese medicine research field of a slice to the material base of these strong polar compounds and pharmacologic agent research, wherein material base research is again the necessary guarantee of follow-up study.But the separation of strong polarity traditional Chinese medicinal components is the difficult point of material foundation of tcm research always, this is mainly because there are problems in the separation preparation of strong polarity Chinese drugs from the extracting and enriching in early stage to monomeric compound, first, traditional pretreatment process of most of Chinese medicine is organic solvent extraction, to reach the object of effective substance preliminary concentration, but organic solvent polarity is limited, a large amount of strong polar compounds is caused partially or completely to lose in extraction step; Moreover, mostly containing carboxyl, sulfonic group, amino, glycosyl isopolarity group in the molecular structure of strong polar compound, the existence of these groups considerably increases the hydrophily of compound and unstability, and to make it retain on the reverse-phase chromatographic column of routine very weak, even do not retain.Current chromatography separating method can't realize the good separation of strong polar compound.
High speed adverse current chromatogram (High-speed countercurrent chromatography, HSCCC) is a kind of successfully liquid/liquid partition chromatography, is widely used in the separation preparation of Chinese drugs.But all the time, be subject to the restriction of organic/water two phase solvent system polarity, high speed adverse current chromatogram is more applicable to nonpolar being separated with middle polarity compound, even the strongest n-butanol-acetic acid-water solution of polarity still can not meet the separation needs of the strong polarity Chinese drugs of great majority.For meeting the needs of strong polarity Chinese drugs separation and purification, at present both at home and abroad, except development of new counter-current chromatograph, what research was the most active has both direction, one be tradition organic/new elution process is developed on basis to water two phase solvent system, as positive and anti-phase wash-out, double steering wash-out, gradient elution (pH gradient, polarity gradient) etc., to some extent solve the separation problem of strong polarity Chinese drugs.Two is propositions of novel dissolvent system, comprise pH zone system (pH-Zone system), ion pair system (Ion pair system), double-aqueous phase system (Aqueous two-phase system), three-phase system (Three phase system) Gu, liquid/liquid/three-phase system (Liquid/liquid/solid three-phase system) with hydrophilic solvent systems such as ion liquid systems (Ionic liquid system) to be applicable to being separated of strong polarity Chinese drugs.But the novel dissolvent system great majority of development at present all need to run in special counter-current chromatograph, and the method for there is no realizes popularization and application.
Nineteen ninety, Greve and the Kula reported first novel double-aqueous phase system be made up of hydrophily alcohol, water and minerals, this system, compared with traditional double-aqueous phase system, has polarity stronger, the advantages such as viscosity is lower and cheap, environmental protection.Although the research at home and abroad of novel aqueous two-phase dicyandiamide solution is just at the early-stage, in strong polarity Chinese drugs and the extracting and enriching of chemical products and the removing of the impurity such as protein, polysaccharide, wide researching value and application space are shown.Academician as big in Tan Tian first reported the dipotassium glycyrrhizinate (biotechnology Letter24 (2002) 1417-1420) in application ethanol/phosphate system extraction Radix Glycyrrhizae at home; Repair the extract and separate (Separation and Purification Technology66 (2009) 472-478) that this system to be successfully applied to the chemical products such as 1,3-PD in zymotic fluid by will dragon professor.But in the face of the complicated Chinese medical extract of composition, only utilize single-stage, even multitple extraction all cannot the monomer preparation of realize target compound, is applied to by novel double-aqueous phase system in the high-speed countercurrent chromatography of current extensive use and is expected to solve this difficult problem.
Summary of the invention
The object of the invention is to break through organic/water two phase solvent system high-speed countercurrent chromatography when being separated strong polarity Chinese drugs, dicyandiamide solution polarity is low, the preparation problems such as separating degree is poor, the rate of recovery is low, retention time is long, provide a kind of high speed adverse current chromatogram new method adopting novel aqueous two-phase dicyandiamide solution.
Technical scheme of the present invention:
A kind of novel aqueous two-phase high speed adverse current chromatogram: be as Stationary liquid using one of novel for alcohol water two-phase aqueous two-phase dicyandiamide solution, another is applied to high speed adverse current chromatogram as mobile phase, and described novel aqueous two-phase dicyandiamide solution is made up of middle polarity organic solvent, hydrophily alcohol or ketone, inorganic salts and water.
Described high-speed counter-current chromatograph is that J-type partly prepares high-speed counter-current chromatograph, is equipped with the spiral tube of three series connection, column volume 210mL, ratio (β value) 0.5-0.8 of helix tube rotation radius and revolution-radius.
Middle polarity organic solvent in described novel aqueous two-phase dicyandiamide solution is n-butanol, and hydrophily alcohol or ketone are ethanol, inorganic salts be saturated ammonium sulfate and water composition, their volume ratio is at (0-10): (1-10): 10:10(V:V) latitude of formulation.
It is consistent that described novel double-aqueous phase system configuration temperature and distribution coefficient measure temperature, is 30-40 DEG C.
Described J-type helix tube high-speed counter-current chromatograph and described two groups of novel aqueous two-phase dicyandiamide solution preferred types of elution are mobile phase mutually under employing by head end caudad wash-out or upper mutually for mobile phase by tail end to head end type of elution.
In described type of elution, the operational factor of high-speed counter-current chromatograph is: temperature is at 30-40 DEG C, open high-speed counter-current chromatograph, main frame forward or reverse, engine speed is 700-900rpm, and with the flow velocity of 0.6-1.5mL/min, mobile phase being pumped in splitter, whole Establishing dynamic equilibrium, injects sample by sampling valve.
The invention provides and a kind ofly use novel aqueous two-phase high-speed countercurrent chromatography to be separated the new method of strong polar compound, comprise the steps:
The mensuration of step 1) strong polar compound distribution coefficient: adopt high effective liquid chromatography for measuring compound concentration in upper and lower phase system, calculation of distribution coefficient;
Step 2) prepare the dicyandiamide solution forming Stationary liquid, mobile phase: according to step 1) deterministic compound polarity, choose the novel aqueous two-phase organic solvent system being applicable to target compound and being separated, described dicyandiamide solution is made up of Four composition: component A can be selected from the hydrophobic organic solvents such as n-butanol, isobutanol, isoamyl alcohol, ethyl acetate, propyl acetate; B component can be selected from the hydrophilic organic solvents such as methyl alcohol, ethanol, normal propyl alcohol and isopropyl alcohol; Component C can be selected from sodium chloride, ammonium sulfate, sodium carbonate, dipotassium hydrogen phosphate, sodium hydrogen phosphate and the inorganic salts such as potassium phosphate, sodium phosphate; D component is water.Preferred n-butanol-ethanol-ammonium sulfate-aqueous solvent.
In n-butanol-ethanol-saturated ammonium sulfate-aqueous solvent, their volume ratio can at (0-10): the latitude of formulation of (10-0): 10:10, such as 10:0:10:10; 2:8:10:10; 5:5:10:10; 8:2:10:10; 0:10:10:10 etc.When n-butanol ratio wherein becomes 0, system becomes ethanol-ammonium sulfate-aqueous systems.
Step 3) selects high speed adverse current chromatogram type of elution: according to step 2) composition of selected dicyandiamide solution and ratio-dependent type of elution, retain with the Stationary liquid obtaining more than 50%, described type of elution mainly contains four kinds: the first, upper is Stationary liquid mutually, lower be mutually mobile phase by tail end to head end wash-out; The second, upper is Stationary liquid mutually, and lower is that mobile phase is by head end caudad wash-out mutually.The third, lower is Stationary liquid mutually, upper be mutually mobile phase by tail end to head end wash-out; 4th kind, lower is Stationary liquid mutually, and upper is that mobile phase is by head end caudad wash-out mutually; For dicyandiamide solution composition, adopt the second and the third type of elution, it will be higher that Stationary liquid retains.
Step 4) uses novel aqueous two-phase high speed adverse current chromatogram to be separated strong polar compound: by step 2) selected dicyandiamide solution is according to the type of elution described in step 3), make to fill Stationary liquid in counter-current chromatograph, then make its main frame rotate, mobile phase is pumped in post; By sampling valve sample introduction; According to detector spectrogram receiving target composition.
The beneficial effect of this technology is: this method have employed the J-type spiral tube high-speed counter-current chromatograph generally applied, equipment and consumptive material price economy, and loses with sex change can not adsorbing of sample because system avoids without solid support; Solvent for use system utilizes methyl alcohol, ethanol, acetone, n-butanol isopolarity alcohol and inorganic salts to form aqueous two-phase, price is lower, environmental protection more, in addition, the boiling point of most hydrophilic organic solvent is lower than water, it effectively reclaims by the mode by distillation or rectifying, thus eliminates the stripping steps of traditional double aqueous phase system, reduces the loss of extractant and the running cost of system significantly.The most important thing is such system organic with traditional/water two-phase system compared with, polarity is stronger, and viscosity is lower, and separation time significantly shortens, and effectively can avoid emulsion, and the Stationary liquid be increased in counter-current chromatograph retains, and is conducive to the separation of polar compound.Preferred type of elution makes dicyandiamide solution in high-speed counter-current chromatograph splitter, have higher Stationary liquid to retain, and effectively can increase the separating degree of sample room.This method is applicable to the separation preparation of strong polar compound in all kinds of Chinese medicine runic thing, chemical industry product.
Accompanying drawing explanation
Fig. 1 n-butanol-ethanol-saturated ammonium sulfate-water (0.6:0.4:1:1) system is separated the adverse current chromatogram figure of caffeoyl guinic acid isomer.
Fig. 2 n-butanol-ethanol-saturated ammonium sulfate-water (0.6:0.4:1:1) system sub-sampling is separated the adverse current chromatogram figure of caffeoyl guinic acid isomer.
Fig. 3 n-butanol-water (1:1) system is separated the contrast figure of the adverse current chromatogram figure of caffeoyl guinic acid isomer.
Detailed description of the invention
Embodiment 1
Adopt the spiral tube of supporting three series connection of J-type high-speed counter-current chromatograph, choose n-butanol-ethanol-ammonium sulfate-aqueous systems, above is that mobile phase is separated caffeoyl guinic acid (Fig. 1) by tail end to head end wash-out mutually.
First with deionized water preparation ammonium sulfate saturated solution, afterwards with 0.6:0.4:1:1 system than n-butanol, ethanol, saturated ammonium sulfate solution and water are formulated in separatory funnel, shake up rear static layering.Upper and lower phase, after 2 hours, is separated by ready to balance, gets as mobile phase, lower to Stationary liquid.
Take 1-caffeoyl guinic acid, CA, 5-CQ, each 2mg of 1,3-DCQA be dissolved in 5mL mobile phase.
Adopt the semi-preparative high-speed counter-current chromatograph of J-type, and be furnished with pump, 20mL sampling valve, UV-detector and chromatographic work station.The multi-layer helix-tube column capacity of its three series connection is 210mL.
First use Stationary liquid (lower phase) to be full of whole tubing string, adjustment engine speed is 850rpm, column temperature 40 DEG C, pumps in post with the flow velocity of 1.0mL/min by mobile phase (upper phase) by tail end to head end; After whole Establishing dynamic equilibrium, by sampling valve sample introduction; Then according to detector uv atlas receiving target compound.
Embodiment 2
Adopt the spiral tube of supporting three series connection of J-type high-speed counter-current chromatograph, choose n-butanol-ethanol-ammonium sulfate-aqueous systems, sub-sampling wash-out is separated caffeoyl guinic acid (Fig. 2).
First with deionized water preparation ammonium sulfate saturated solution, afterwards with 0.6:0.4:1:1 system than n-butanol, ethanol, saturated ammonium sulfate solution and water are formulated in separatory funnel, shake up rear static layering.Upper and lower phase, after 2 hours, is separated by ready to balance, gets as mobile phase, lower to Stationary liquid.
Take 1-caffeoyl guinic acid, CA, 5-CQ, each 4mg of 1,3-DCQA be dissolved in 10mL mobile phase, be divided into two equal portions.
Adopt the semi-preparative high-speed counter-current chromatograph of J-type, and be furnished with pump, 20mL sampling valve, UV-detector and chromatographic work station.The multi-layer helix-tube column capacity of its three series connection is 210mL.
First use Stationary liquid (lower phase) to be full of whole tubing string, adjustment engine speed is 850rpm, column temperature 40 DEG C, pumps in post with the flow velocity of 1.0mL/min by mobile phase (upper phase) by tail end to head end; After whole Establishing dynamic equilibrium, by sampling valve sample introduction; Then according to detector uv atlas receiving target compound; During system running 220min, by sampling valve sub-sampling, according to detector receiving target compound.
Embodiment 3
Adopt the spiral tube of supporting three series connection of J-type high-speed counter-current chromatograph, choose n-butanol-ethanol-ammonium sulfate-aqueous systems, above is that mobile phase is separated caffeoyl guinic acid (Fig. 3) by tail end to head end wash-out mutually.
First with deionized water preparation ammonium sulfate saturated solution, afterwards with 1:0:0:1 system than n-butanol, ethanol, saturated ammonium sulfate solution and water are formulated in separatory funnel, shake up rear static layering.Upper and lower phase, after 2 hours, is separated by ready to balance, gets as mobile phase, lower to Stationary liquid.
Take 1-caffeoyl guinic acid, CA, 5-CQ, each 2mg of 1,3-DCQA be dissolved in 5mL mobile phase.
Adopt the semi-preparative high-speed counter-current chromatograph of J-type, and be furnished with pump, 20mL sampling valve, UV-detector and chromatographic work station.The multi-layer helix-tube column capacity of its three series connection is 210mL.
First use Stationary liquid (upper phase) to be full of whole tubing string, adjustment engine speed is 850rpm, column temperature 40 DEG C, pumps in post with the flow velocity of 1.0mL/min by mobile phase (lower phase) by head end to tail end; After whole Establishing dynamic equilibrium, by sampling valve sample introduction; Then according to detector uv atlas receiving target compound.
Claims (6)
1. a novel aqueous two-phase high speed adverse current chromatogram, as Stationary liquid using one of novel for alcohol water two-phase aqueous two-phase dicyandiamide solution, another is applied to high speed adverse current chromatogram as mobile phase, it is characterized in that: described novel aqueous two-phase dicyandiamide solution is made up of middle polarity organic solvent, hydrophily alcohol or ketone, inorganic salts and water.
2. novel aqueous two-phase high speed adverse current chromatogram according to claim 1, it is characterized in that: described high-speed counter-current chromatograph is that J-type partly prepares high-speed counter-current chromatograph, be equipped with the spiral tube of three series connection, column volume 210mL, ratio (β value) 0.5-0.8 of helix tube rotation radius and revolution-radius.
3. novel aqueous two-phase high speed adverse current chromatogram according to claim 1, it is characterized in that: the middle polarity organic solvent in described novel aqueous two-phase dicyandiamide solution is n-butanol, hydrophily alcohol or ketone are ethanol, inorganic salts be saturated ammonium sulfate and water composition, their volume ratio is at (0-10): (1-10): 10:10(V:V) latitude of formulation.
4. novel aqueous two-phase high speed adverse current chromatogram according to claim 3, is characterized in that: it is consistent that described novel double-aqueous phase system configuration temperature and distribution coefficient measure temperature, is 30-40 DEG C.
5. according to the novel aqueous two-phase high speed adverse current chromatogram described in Claims 2 or 3, it is characterized in that: described J-type helix tube high-speed counter-current chromatograph and described two groups of preferred types of elution of novel aqueous two-phase dicyandiamide solution, be mobile phase mutually under employing by head end caudad wash-out or upper mutually for mobile phase by tail end to head end type of elution.
6. novel aqueous two-phase high speed adverse current chromatogram according to claim 5, it is characterized in that: in described type of elution, the operational factor of high-speed counter-current chromatograph is: temperature is at 30-40 DEG C, open high-speed counter-current chromatograph, main frame forward or reverse, engine speed is 700-900rpm, and pumps in splitter with the flow velocity of 0.6-1.5mL/min by mobile phase, whole Establishing dynamic equilibrium, injects sample by sampling valve.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112973189A (en) * | 2019-12-13 | 2021-06-18 | 中国科学院大连化学物理研究所 | Method for preparing Chinese medicinal extract absorption component group |
| CN114632349A (en) * | 2017-06-01 | 2022-06-17 | 相达生物科技美国有限公司 | Phase separation behavior modifier for aqueous two-phase separation in porous materials |
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Application publication date: 20150617 |