CN1664576A - Stationary phase for chiral ligand exchange chromatography and method for making same - Google Patents
Stationary phase for chiral ligand exchange chromatography and method for making same Download PDFInfo
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- CN1664576A CN1664576A CN 200510020617 CN200510020617A CN1664576A CN 1664576 A CN1664576 A CN 1664576A CN 200510020617 CN200510020617 CN 200510020617 CN 200510020617 A CN200510020617 A CN 200510020617A CN 1664576 A CN1664576 A CN 1664576A
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Abstract
The invention relates to a method of making chirality ligand exchange color spectrum fixed phase, which is used for splitting the chirality compounds especially for the DL- amino acid antimer and belongs to analytical chemistry fields. The method coats the optical activity replacing aminothiopropionic acid on the surface of the reverse-phase chromatography and makes the chirality ligand exchange color spectrum fixed phase. The accompanying diagram is the resolution of DL-Phegly in the chromatographic column (I).
Description
Technical field
The present invention relates to a kind of chiral ligand exchange chromatograph stationary phase and preparation method thereof, and this stationary phase is used for high performance liquid chromatography resolving chiral compound, particularly splits the DL-amino acid enantiomer, belongs to the analytical chemistry field.
Background technology
The chromatogram of DL-amino acid enantiomer on chiral stationary phase splits, and can adopt gas chromatography and liquid-phase chromatography method, but needs the preceding analyte derivative program of post usually, the program of deriving is time-consuming and bother, not only need derivative reagent, and racemization might take place, the impact analysis result.The chiral ligand exchange chromatograph method that last century, the seventies grew up is a kind of effective ways of resolving chiral compound, particularly amino acid and alcohol acid enantiomorph, and its selectivity height does not need to carry out the pre-column derivatization program.
Adopting photolytic activity amino acid chiral chooser, perhaps as the moving phase adjuvant, perhaps be used for preparation and be coated with stain or bonded stationary phase, is to realize that chiral ligand exchange chromatograph splits the approach commonly used of enantiomorph.(Science such as Hare, 1979,204:1226) photoactive proline and copper ion are added in the chromatographic eluents, enantiomorphs such as Alpha-Methyl ornithine have been split at the octadecyl bonding on mutually, because the chiral resolution agent consumption that adds is big, the expense height, and detachable object range is narrow, uses limited.With chirality chooser L-proline (Wu Banggui etc., analytical chemistry, 1991,19:291), L-hydroxyproline (Chen Tao etc., Chinese ChemicalLetters, 1995,383), L-isoleucine (Huang Xiaojia etc. 6 (5):, chromatogram, 2003,21:230) with epoxidised silica gel reaction, preparation chiral ligand exchange bonded stationary phase, realized that the amino acid whose chromatogram of DL-splits, but bonded stationary phase post effect is lower, the chromatographic peak hangover is serious.Davankov etc. (Chromatographia, 1980,13:677) photolytic activity proline and the hydroxyproline that adopts chain alkyl to replace respectively made the chirality chooser, is coated with to be stated from the octadecyl bonding and to go up mutually, split enantiomorphs such as DL-amino acid effectively.Wang Qunbiao etc. (Chinese patent ZL 99117305.8) have synthesized 2-(2-hydroxyl-3-alkoxy) propyl group-(S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid chirality chooser, again it is coated with and steeps on reverse-phase chromatography bonded stationary phase, prepared two kinds and be coated with stain chiral ligand exchange chromatograph stationary phase, realized amino acid whose fractionation DL-, but it is longer to separate required time, and it is more serious that the component hangover is flowed out in the back.Oi etc. (J.chromatogr., 1992, be the chirality chooser 592:291) with the Beracilline derivant, prepared to be coated with and steeped chiral ligand crossover fixation phase, split amino acid, alcohol acid, the amino alcohol enantiomorph.
Summary of the invention
The present invention relates to a kind of chiral ligand exchange chromatograph stationary phase and preparation method thereof, can be used for directly splitting the DL-amino acid enantiomer, and does not need the pre-column derivatization program.One end of this chiral stationary phase molecule has amino and the carboxyl that the ligand exchange effect can take place, and the other end is a straight chained alkyl, because hydrophobic effect can be adsorbed on the surface of inverse bonded stationary phase securely.Its preparation method is simple, be used to split the amino acid whose selectivity height of DL-, post imitate high, chromatographic peak profile is symmetrical, fractionation speed is fast.
The present invention is a raw material with photoactive D-or L-halfcystine, and synthetic D-or the L-halfcystine chirality chooser of replacing is coated with it method of steeping on reverse-phase chromatography bonded stationary phase again, makes chiral ligand exchange chromatograph stationary phase of the present invention.The structure of chirality chooser is:
R in the formula
1Be C
4～C
22Alkyl, benzyl, allyl ,-CH
2CH (OH) CH
2R
2, R
2Be C
4～C
30Straight chained alkyl, straight chain alkoxy.
The preparation method of stationary phase of the present invention is as follows: be to be raw material with D-or L-halfcystine, by reacting with straight chain alkane (oxygen) basic ring oxygen compound, perhaps elder generation and halohydrocarbons reaction, make the chirality chooser with the reaction of straight chain alkoxy epoxy compound again, adopt dynamic approach then, it is coated with steeps, promptly obtain ligand exchange chromatograph chiral stationary phase of the present invention in the bonded stationary phase microparticle surfaces.
Description of drawings
Fig. 1, Fig. 2, Fig. 3 and Fig. 4 are respectively DL-Val (valine), DL-Phegly (phenylglycine), DL-Hpg (D-pHPG), the chromatogram broken away view of DL-Norval (norvaline) on chromatographic column (I).Chromatographic condition: moving phase is 0.5mmol/L Cu (Ac)
2Aqueous solution, flow velocity 1.0ml/min, 20 ℃ of column temperatures, the detection wavelength is 254nm.
Fig. 5 and Fig. 6 are respectively DL-Cit (citrulline) and the chromatogram broken away view of DL-Asp (aspartic acid) on chromatographic column (II), and Fig. 7 is DL-Val (valine), DL-Met (methionine) and the mixed sample chromatogram broken away view of DL-Leu (leucine) on chromatographic column (II).Chromatographic condition: moving phase is 0.2mmol/L Cu (Ac)
2Aqueous solution, flow velocity 1.0ml/min, 20 ℃ of column temperatures, the detection wavelength is 254nm.
Fig. 8, Fig. 9 and Figure 10 are respectively DL-Phegly (phenylglycine), DL-Pro (proline) and the chromatogram broken away view of DL-Val (valine) on chromatographic column (III).Chromatographic condition: moving phase is 1.0mmol/L Cu (Ac)
2Aqueous solution, flow velocity 0.8ml/min, 30 ℃ of column temperatures, the detection wavelength is 254nm
Embodiment
The concrete implementation step of stationary phase of the present invention is as follows:
1, in containing the ethanolic solution of sodium alkoxide, adds D-or L-halfcystine and halogenated hydrocarbons (R
4X), at 20～60 ℃ of reaction 2～12h, removal of solvent under reduced pressure then, the gained solid with hydrochloric saturated brine washing, with the ethanol washing, gets S-R earlier again after the drying
4-D-(or L-) halfcystine.At S-R
4In the alcoholic solution of-D-(or L-) halfcystine or sodium salt, add straight chain alkoxy epoxy compound, react 4～60h down in 10～50 ℃, solvent is removed in decompression, wash with containing aqueous acid earlier, and then use the ether washed product, get N-(the positive alkoxy of 2-hydroxyl-3-) propyl group-S-R
4-D-(or L-) halfcystine chirality chooser.
2, in the ethanolic solution that contains sodium alkoxide or NaOH, add D-(or L-) halfcystine and straight chained alkyl epoxy compound or straight chain alkoxy epoxy compound, react 4～60h down in 10～50 ℃, solvent is removed in decompression, get N, S-two (the positive alkyl of 2-hydroxyl-3-) propyl group-D-(or L-) halfcystine chirality chooser or N, S-two (the positive alkoxy of 2-hydroxyl-3-) propyl group-D-(or L-) halfcystine chirality chooser.
3, at room temperature, with the CH of 30～90 times of column volumes
3The OH flushing waits to be coated with the reverse-phase chromatographic column that carries, remove the retention that may exist on the post, cross post with the chirality chooser sodium salt solution of step 1 or step 2 gained then, water with 10～25 times of column volumes washes again, use the copper solution balance chromatographic column of 15～30 times of column volumes again, promptly get chiral stationary phase chromatography post of the present invention.
Embodiment 1
(1) in the 30ml absolute ethyl alcohol, adds 8mmol CH
3ONa, 4mmol L-halfcystine, 4mmol benzyl chloride, behind 40 ℃ of reaction 5h, removal of solvent under reduced pressure successively with saturated aqueous common salt and the ethanol washing of 1mol/LHCl, gets 540mg (2.56mmol) white solid S-benzyl-L-halfcystine after the drying.
(2) in 25ml epichlorokydrin and 0.8g bromination four butylamine catalyst solutions, add the NaOH solution of 30ml 25mol/L, stir, after the frozen water cooling, add the 9.5ml n-octyl alcohol, stir, treat that ice-water bath is warming up to room temperature naturally after, continue to stir 5h again, add 80ml water, pour in the separating funnel, extract with the 150ml isopropyl ether, organic phase is washed secondary respectively with salt solution, water successively, uses Na
2SO
4After the drying,, obtain colourless transparent liquid n-octyl glycidol ether with the solvent evaporate to dryness.
(3) in the methyl alcohol of 30ml, add 1.5mmol S-benzyl-L-halfcystine, 1.5mmol NaOH, be stirred to the solution clarification, add the n-octyl glycidol ether 1.5mmol that step (2) obtains, at 30 ℃ of stirred in water bath 20h, remove solvent then, promptly get N-(2-hydroxyl-3-n-octyloxy) propyl group-S-benzyl-L-halfcystine sodium, it is dissolved in 100ml methyl alcohol, makes and treat coating solution.
(4) at room temperature, with CH
3OH is that the flow velocity flushing of moving phase and 1ml/min waits to be coated with the YWG-C that carries
18Bonded stationary phase chromatographic column (stainless steel column, 300 * 3.9mm I.D., 10 μ m) 3h, the flow velocity with 0.5ml/min pumps into N-(2-hydroxyl-3-n-octyloxy) propyl group-S-benzyl-L-halfcystine sodium methanol solution 2h that step (3) obtains then, and the flow velocity with 0.4ml/min pumps into H again
2O washes chromatographic column 2h, uses the Cu (AC) of 1.0mmol/L at last
2Aqueous solution promptly obtains chiral stationary phase chromatography post of the present invention (I) with 0.5ml/min flow velocity balance chromatographic column 2h.
(1) in the 30ml absolute ethyl alcohol, the n-octyl glycidol ether that adds step (2) gained of 8mmolNaOH, 4mmolL-halfcystine, 8mmol embodiment 1, at 30 ℃ of stirred in water bath 20h, remove solvent then, promptly get N, S-two (2-hydroxyl-3-n-octyloxy) propyl group-L-halfcystine sodium is dissolved in 200ml methyl alcohol with it, makes and treats coating solution.
(2) use N, S-two (2-hydroxyl-3-n-octyloxy) propyl group-L-halfcystine replaces N-(2-hydroxyl-3-normal octane oxygen base) propyl group-S-benzyl-L-halfcystine, is undertaken by the step (4) of embodiment 1, gets chiral stationary phase chromatography post of the present invention (II).
Embodiment 3
(1) in the 30ml absolute ethyl alcohol, add 8mmolNaOH, 4mmol L-halfcystine, 8mmol epoxy hexadecane, at 30 ℃ of stirred in water bath 20h, remove solvent then, get 780mg (2.16mmol) N, S-two (2-hydroxyl-3-cetyl) propyl group-L-halfcystine sodium is dissolved in stain to be coated with in the 200ml methyl alcohol with it.
(2) use N, S-two (2-hydroxyl-3-cetyl) propyl group-L-halfcystine replaces N-(2-hydroxyl-3-normal octane oxygen base) propyl group-S-benzyl-L-halfcystine, is undertaken by the step (4) of embodiment 1, gets chiral stationary phase chromatography post of the present invention (III)
Claims (5)
1, a kind of chiral ligand exchange chromatograph stationary phase, be used for the resolving chiral compound, particularly split the DL-amino acid enantiomer, it is characterized in that: with photoactive replacement halfcystine is the chirality chooser, adopt dynamic coating in the lip-deep method of reverse-phase chromatography bonded stationary phase, preparation chiral ligand exchange chromatograph stationary phase.
2, the described chirality chooser of claim 1, be to be raw material with D-or L-halfcystine, by reacting 4～60h at 10～50 ℃ with straight chain alkane (oxygen) basic ring oxygen compound, perhaps react 2～12h with halogenated hydrocarbons at 20～60 ℃ earlier, make at 10～50 ℃ of reaction 4～60h with straight chain alkane (oxygen) basic ring oxygen compound, its chemical structural formula is again:
R in the formula
1Be C
4～C
22Alkyl, benzyl, allyl ,-CH
2CH (OH) CH
2R
2, R
2Be C
4～C
30Straight chained alkyl, straight chain alkoxy.
3, the described reverse-phase chromatography bonded stationary phase of claim 1 can be represented by the formula:
Silica-Si-CH
2CH
2CH
2-R
3
Silica is silica particles (particle size is 3～30 microns) in the formula, R
3Be C
1～C
20Straight chained alkyl, phenyl, cyano group, amino.
4, the described straight chain alkane of claim 2 (oxygen) basic ring oxygen compound can be represented by the formula:
R in the formula
2Be C
4～C
30Straight chained alkyl, straight chain alkoxy.
5, the described halogenated hydrocarbons of claim 2 can be represented by the formula:
R
4-X
R in the formula
4Be C
4～C
22Alkyl, benzyl, allyl, X is chlorine, bromine, iodine.
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|---|---|---|---|
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Cited By (3)
| 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 |
| CN105080516A (en) * | 2014-05-13 | 2015-11-25 | 中科院大连化学物理研究所淮安化工新材料研究中心 | Bonding type hybrid mesoporous silica gel chiral chromatographic stationary phase and preparation method therefor |
-
2005
- 2005-03-30 CN CN 200510020617 patent/CN1664576A/en active Pending
Cited By (4)
| 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 |
| CN105080516A (en) * | 2014-05-13 | 2015-11-25 | 中科院大连化学物理研究所淮安化工新材料研究中心 | Bonding type hybrid mesoporous silica gel chiral chromatographic stationary phase and preparation method therefor |
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