CN1538171A - Preparation method of chiral ligand exchange chromatographic stationary phase - Google Patents
Preparation method of chiral ligand exchange chromatographic stationary phase Download PDFInfo
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- CN1538171A CN1538171A CNA031114903A CN03111490A CN1538171A CN 1538171 A CN1538171 A CN 1538171A CN A031114903 A CNA031114903 A CN A031114903A CN 03111490 A CN03111490 A CN 03111490A CN 1538171 A CN1538171 A CN 1538171A
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Abstract
A process for using L-isoleucine as the chiral selective agent to prepare the stationary phase for chiral ligand displacement chromatography is disclosed. Its advantages are simple operation (directly splitting the chiral substance in simple experimental condition), high analysis speed, and wide application range.
Description
Technical field
The present invention relates to the fractionation of amino acid enantiomer, specifically a kind of preparation method of chiral ligand exchange chromatograph stationary phase.
Background technology
The fractionation of amino acid enantiomer can be adopted vapor-phase chromatography and liquid phase chromatography, but need carry out the preceding analyte derivativeization of post usually, the not only time-consuming trouble of derivatization process, and might make analyte generation racemization, thus the impact analysis effect.In the chiral ligand exchange chromatograph method (CLEC) that the seventies early-stage development gets up, is a kind of effective ways of resolving chiral compound, particularly amino acid and alcohol acid enantiomorph by people such as Davankov, and its selectivity height does not need to carry out pre-column derivatization.
Is the main flow of chiral ligand exchange chromatograph method development with photolytic activity amino acid or pipecolic acid as the preparation that is coated with stain, bonding phase of chirality chooser.Chinese patent application (application number is 99117305.8) discloses a kind of chiral ligand exchange chromatograph stationary phase and preparation method thereof, specifically be to be raw material with the L-phenylalanine, prepare class novel chiral chooser 2-(2-hydroxyl-3-alkoxy) propyl group-(S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is coated with it and steeps on reverse-phase chromatography bonded stationary phase; Can realize direct fractionation to the amino acid sample; Its weak point is: it is longer to separate required time, and it is more serious that back outflow component is taken off tail; With respect to the bonding stationary phase, the coating-type stationary phase easily runs off, and has limited its serviceable life.Synthetic (the Zhu Xinyi of the silica gel bonded chiral ligand crossover fixation of the L-proline phase of two kinds of different bonded amounts, people such as Han Xiaoqian " chromatogram ", 2002,20 (3): 223-226), utilize ligand exchange chromatography to separate a series of amino acid, the result shows that the different stationary phase of bonded amount is bigger to amino acid whose fractionation ability difference; It has only split a limited number of several seed amino acids, and required time is longer, and it is serious that back component is taken off tail.2-(2-hydroxyl-3-alkoxy) propyl group-(S)-1,2,3, synthetic (the Wang Qunbiao of 4-tetrahydroisoquinoline-3-carboxylic acid chirality chooser, people such as Long Yuande " chromatogram ", 2000,18 (2): 112-114), prepare two kinds of novel stain chiral ligand exchange chromatograph stationary phase that are coated with, split several seed amino acids; Because of component keep stronger, so need analysis time of growing.Propyl group-(S)-1; 2,3,4-tetrahydroisoquinoline-3-carboxylic acid is chirality chooser (Long Yuande; people such as Wang Qunbiao " SCI "; 2001,22 (4): 566-568), synthesized silica gel keys mould assembly ligand exchange chromatograph stationary phase with 2-(2-hydroxyl-3-alkoxy); split some amino acid; though realized amino acid whose chiral separation, operate under the higher temperature and carry out, and analysis time be longer.M.Wachsmann, H.Br ü ckner (Chromatographia, 1998,47 (11/12): 637) after use three chlorotriazines priority and methyl alcohol, L-proline uncle butyl ether (or the N-tertbutyloxycarbonyl-L-lysine uncle butyl ether) reaction, react with amine propyl group silica gel again, the ligand exchange type chiral stationary phase of synthesizing new, detachable amino acid and derivant thereof, but preparation process is more loaded down with trivial details.Other have with the L-proline by different spacerarms be bonded to silica gel (G.G ü bitz, S.Mihellyes, G.Kobinger, A.WutteJChromatogr A, 1994,666:91-97), under higher temperature, split amino acid and derivant thereof; But partial amino-acid is longer analysis time.
In above relevant research document, with the prepared stationary phase of distinct methods (used amino acid mostly is proline and hydroxyproline greatly) when DL-amino acid is separated, general required time long (it is serious that the component hangover is flowed out in the back), perhaps need under higher temperature, to separate, not only make complicated operation, also influenced the serviceable life of chromatographic column to a great extent.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of chiral ligand exchange chromatograph stationary phase, the chiral stationary phase that makes can separate most natural amino acids at normal temperatures, and disengaging time is short, and simple to operate, range of application is wider.
For achieving the above object, the technical solution used in the present invention is: with L-isoleucine (containing two chiral centers) is that chiral selector prepares the chiral ligand exchange chromatograph stationary phase.
Preparation process can be bonding, coating and polymer type; The stationary phase host material can be Al in bonding and the application pattern
2O
3, ZrO
2, TiO
2Or carbosphere;
The alkylating reagent of bonding process can adopt the silylating reagent that contains epoxy radicals; Be specially: with after the stationary phase matrix (silicon ball) of acid treatment and the silylating reagent reaction that contains epoxy radicals, add the L-isoleucine of handling with aqueous sodium carbonate in right amount, (between 15～30 ℃ all can) concussion reaction is after 24～48 hours under the room temperature, washing, drying; With the copper ion complexing, make ligand exchange type chiral stationary phase then; Described and copper ion complexing are specially: it is added copper sulfate solution, and after concussion a few minutes, placement is spent the night, suction filtration, washing, drying.
With the L-isoleucine is that chiral selector prepares coating and the chiral ligand exchange chromatograph stationary phase of polymer type can be operated according to a conventional method; Can be referring to (1) Yuan Zhi, what handle woods, central metallic ions and temperature be to the influence of chirality recognition capability, " ion-exchange and absorption ", 1999,15 (6): 567-571; In this piece document, the author is chiral selector with the hydroxyproline, is carrier with macropore polyvinylamine resin, makes the chiral ligand resin, has split some amino acid.(2) Wang Qunbiao, Long Yuande, yellow Tian Bao, the preparation and the application of novel chiral ligand exchange chromatograph stationary phase, " chromatogram ", 2000,18 (2): 112-114; In this piece document, the author is chiral selector with isoquinolinecarboxylic acid, has prepared two kinds of coating-type ligand exchange type chromatographic stationary phases.
The present invention has following advantage:
1. (during application) simple to operate.The present invention uses the L-isoleucine to be bonded to silica gel as chiral selector by simple method first, and preparation chiral ligand exchange chromatograph stationary phase under simple experiment condition, is realized the direct fractionation to chiral materials such as amino acid, alcohol acids.
2. analysis time is short.Adopt the L-isoleucine chiral ligand exchange chromatograph stationary phase that the present invention produced, can carry out baseline separation to the natural a-amino acid of major part at normal temperatures, with respect to technology described in other document both domestic and external, the amino acid quantity that splits is more, separates required time also short (Most amino-acids is separated required time in 20min).
3. applied range.The bonded amount of chiral selector of the present invention is big, and (chiral ligand is 4-5 μ mol/m in the surface concentration of bonding phase
2), so can adopt short chiral column (10-15cm), at normal temperatures the Most amino-acids enantiomorph is separated or purity analysis, be more suitable in the analysis of asymmetric syntheses research and daily amino acids drug.
Description of drawings
Fig. 1 is the fractionation spectrogram of DOPA on the part post;
Fig. 2 is the fractionation spectrogram of serine on the part post;
Fig. 3 is the fractionation spectrogram of phenylalanine on the part post;
Fig. 4 is the fractionation spectrogram of methionine on the part post;
Fig. 5 is the fractionation spectrogram of asparagine on the part post;
Fig. 6 is the fractionation spectrogram of aspartic acid on the part post;
Fig. 7 is the fractionation spectrogram of threonine on the part post;
Fig. 8 is the fractionation spectrogram of tyrosine on the part post;
Fig. 9 is the fractionation spectrogram of histidine on the part post;
Figure 10 is the fractionation spectrogram of isoleucine on the part post;
Figure 11 is the fractionation spectrogram of ornithine on the part post;
Figure 12 is the fractionation spectrogram of valine on the part post;
Figure 13 is the fractionation spectrogram of tryptophane on the part post;
Figure 14 is the fractionation spectrogram of a-aminophenyl acetic acid on the part post.
Embodiment
Embodiment 1
With the L-isoleucine is the preparation of the chiral ligand exchange chromatograph stationary phase of chiral selector:
Kromasil spherical silica gel: Sweden Eka Nobel company, particle diameter 5 μ m, specific surface 340m
2/ g, aperture 10nm; L-isoleucine: available from Sigma company (USA)
Acid-treated silicon ball 4.0g learns from else's experience, 150 ℃ after vacuum drying 4-6 hour, cooling places the 250mL three-necked round bottom flask, adds 2mL3-diglycidyl propyl trimethoxy silicane and 40ml toluene, under the nitrogen protection in 110 ℃ of following stirring and refluxing 6-7 hours, cooling is filtered, and washs successively for several times with toluene, methyl alcohol, acetone, 50 ℃ of vacuum drying 2 hours, 3-diglycidyl propyl group silica gel;
Get the 3.93gL-isoleucine, add the sodium carbonate (example: the 0.03mol isoleucine is handled with 0.015mol sodium carbonate) and the suitable quantity of water of a great deal of, after the stirring and dissolving, steam near and do, add 40ml methyl alcohol, add and go up the 3-diglycidyl propyl group silica gel that the step makes, concussion reaction 24～48h under the room temperature (is that room temperature is when higher, reaction time can shorten, when room temperature is low, the reaction long period), filter, use methyl alcohol, water washing successively for several times, vacuum drying under the room temperature;
Product is added in 15% copper sulphate (or Schweinfurt green) aqueous solution, and behind the concussion 5min, placement is spent the night; Filter, wash with water 2-3 time, vacuum drying promptly gets the ligand exchange chiral stationary phase under the room temperature after 2 hours.
Synthesizing of chiral stationary phase:
Prepared stationary phase is to the fractionation of common amino acid:
With phenixin: dioxane (2: 1) is a homogenate, and ethanol is displacement fluid.Column size is 150mm * 4.6mmi.d., stainless steel column.
With 0.1 mmol/L CuSO
4Be moving phase, investigated the fractionation of chromatographic column at normal temperatures 16 kinds of common amino acids.Do not have except that alanine and leucine outside the effect of fractionation, most amino acid can be at normal temperatures by fine fractionation.(gained the results are shown in institute's subordinate list one, Figure of description)
The fractionation of 14 kinds of DL-amino acid of table one on L-isoleucine chiral ligand exchange chromatograph stationary phase
| ?Amino?acid | ?k D’ | ?k L’ | a(k L’/k D’) | ??Rs |
| ?Dopa | ?5.10 | ?8.28 | ?1.62 | ?1.42 |
| ?Serine | ?1.97 | ?3.05 | ?1.55 | ?1.78 |
| ?Phenylalanine | ?5.12 | ?8.24 | ?1.61 | ?1.70 |
| ?Methionine | ?3.08 | ?3.85 | ?1.25 | ?0.88 |
| ?Asparagine | ?2.02 | ?2.82 | ?1.40 | ?1.19 |
| ?Aspartic?acid | ?2.73 | ?3.28 | ?1.20 | ?0.44 |
| ?Threonine | ?2.37 | ?3.09 | ?1.30 | ?1.12 |
| ?Tyrosine | ?3.83 | ?8.53 | ?2.23 | ?2.59 |
| ?Histidine | ?8.36 | ?14.87 | ?1.78 | ?1.31 |
| ?Isoleucine | ?2.65 | ?4.22 | ?1.60 | ?1.66 |
| ?Orithine | ?2.64 | ?3.48 | ?1.32 | ?0.98 |
| ?Valine | ?2.43 | ?3.30 | ?1.36 | ?1.22 |
| ?Tryptophan | ?8.64 | ?17.92 | ?2.07 | ?2.02 |
| ?a-aminophenyl ?acetic?acid | ?3.71 | ?4.71 | ?1.27 | ?0.59 |
Moving phase: 10
-4Mol/L CuSO
4Flow velocity: 1mL/min column temperature: 20 ℃, detect wavelength: 223nm
Claims (6)
1. the preparation method of a chiral ligand exchange chromatograph stationary phase, it is characterized in that: with the L-isoleucine is that chiral selector prepares the chiral ligand exchange chromatograph stationary phase.
2. by the described preparation method of claim 1, it is characterized in that: preparation process can be bonding, coating and polymer type.
3. by the described preparation method of claim 2, it is characterized in that: the stationary phase host material can be Al in bonding and the application pattern
2O
3, ZrO
2, TiO
2Or carbosphere.
4. by claim 2 or 3 described preparation methods, it is characterized in that: the alkylating reagent of bonding process can adopt the silylating reagent that contains epoxy radicals.
5. by the described preparation method of claim 4, it is characterized in that:
Specific operation process is as follows: with after the stationary phase matrix of acid treatment and the silylating reagent reaction that contains epoxy radicals, add the L-isoleucine of handling with aqueous sodium carbonate, concussion was reacted after 24～48 hours under the room temperature, washed drying; With the copper ion complexing, make ligand exchange type chiral stationary phase then.
6. by the described preparation method of claim 5, it is characterized in that:
Described and copper ion complexing specific operation process is: the product behind the bonding is added copper salt solution, and after the concussion reaction, placement is spent the night, suction filtration, washing, drying.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102039060A (en) * | 2010-11-09 | 2011-05-04 | 苏州清安生物科技有限公司 | Chiral separation chromatographic column |
| CN102389783A (en) * | 2011-10-19 | 2012-03-28 | 苏州苏凯路化学科技有限公司 | Chiral chromatographic column and preparation method thereof |
| CN109239172A (en) * | 2018-08-06 | 2019-01-18 | 广州百兴网络科技有限公司 | A kind of electrochemistry chiral Recognition detection method of Lorazepam |
| CN111208216A (en) * | 2018-11-21 | 2020-05-29 | 太景生物科技股份有限公司 | Method for separating optical isomers of nemonoxacin or salt thereof |
| CN111323497A (en) * | 2018-12-17 | 2020-06-23 | 武汉武药科技有限公司 | Optical purity analysis method of pasireotide starting material |
-
2003
- 2003-04-18 CN CNA031114903A patent/CN1538171A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102039060A (en) * | 2010-11-09 | 2011-05-04 | 苏州清安生物科技有限公司 | Chiral separation chromatographic column |
| CN102039060B (en) * | 2010-11-09 | 2012-05-02 | 苏州福瑞斯生物科技有限公司 | Chiral separation chromatographic column |
| CN102389783A (en) * | 2011-10-19 | 2012-03-28 | 苏州苏凯路化学科技有限公司 | Chiral chromatographic column and preparation method thereof |
| CN109239172A (en) * | 2018-08-06 | 2019-01-18 | 广州百兴网络科技有限公司 | A kind of electrochemistry chiral Recognition detection method of Lorazepam |
| CN109239172B (en) * | 2018-08-06 | 2021-02-05 | 广州百兴网络科技有限公司 | Electrochemical chiral recognition detection method of lorazepam |
| CN111208216A (en) * | 2018-11-21 | 2020-05-29 | 太景生物科技股份有限公司 | Method for separating optical isomers of nemonoxacin or salt thereof |
| CN111208216B (en) * | 2018-11-21 | 2023-03-14 | 浙江医药股份有限公司新昌制药厂 | Method for separating optical isomers of nemoxacin or salts thereof |
| CN111323497A (en) * | 2018-12-17 | 2020-06-23 | 武汉武药科技有限公司 | Optical purity analysis method of pasireotide starting material |
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