Chitosan 3, 6-di (aryl carbamate)-2-(benzylimine Schiff base) chiral stationary phase as well as preparation method and application of chitosan 3, 6-di (aryl carbamate)-2-(benzylimine Schiff base) chiral stationary phase

Abstract

The invention discloses a chitosan-3, 6-di (aryl carbamate)-2-(benzimine Schiff base) chiral stationary phase as well as a preparation method and application thereof. The prepared chiral stationary column has good chiral separation performance, and also has a good separation effect on chiral compounds which cannot be separated from traditional ADMPC and CDMPC chiral columns and are poorly separated from the traditional ADMPC and CDMPC chiral columns. According to the present invention, the reaction efficiency is high, the process is simple and green, the conditions are easy to control, the environmental protection is easily achieved, the chitosan is subjected to Schiff alkalization and then esterification, the synthesized chitosan-3, 6-bis (aryl carbamate)-2-(benzylamine Schiff base) has characteristics of high substitution degree and regular structure, the chiral recognition performance of the chiral stationary phase is enhanced, and the application prospect is broad. The introduction of the Schiff base group provides a strong hydrogen bond capability, facilitates the coating combination between the chitosan derivative and aminopropyl silica gel, and is beneficial to enhancing the stability of the separation effect of the chiral stationary phase.

Description

Technical Field

[0001] The invention relates to a derivative material for chiral separation, and in particular discloses a chiral stationary phase of chitosan-3,6-di(aryl carbamate)-2-(benzimidamine Schiff base) type, a preparation method and an application thereof. Background Art

[0002] High performance liquid chromatography (HPLC) is an effective tool for batch separation of chiral compound enantiomers in the field of chromatography. It is a chromatographic mode that uses non-polar organic solvents as mobile phases and achieves the separation purpose through multiple interactions between the chromatographic stationary phase and the chiral enantiomers. In addition, the use of various chromatographic techniques, such as thin layer liquid chromatography (TLC), high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), capillary electrophoresis (CE) and capillary electrochromatography (CEC) to separate enantiomers requires a chiral stationary phase with good separation performance. Therefore, the development of new chiral separation materials is of great significance [(1) Francotte ER. Enantioselective chromatography as a powerful alternative for the preparation of drug enantiomers [J]. Journal of Chromatography A, 2001, 906 (1-2): 379-397.].

[0003] Among the existing chiral stationary phases, the CDMPC and ADMPC separation performance of the coated chiral stationary phases prepared by derivatization based on cellulose and amylose is the best. Since this type of stationary phase has a high loading capacity for chiral compounds and the raw materials are cheap and easy to obtain, the stationary phases prepared by cellulose and amylose have been commercialized. However, these two chiral chromatographic columns still have many chiral compounds that cannot be identified and separated. In addition to cellulose and amylose, chitin and chitosan can also be used to prepare chiral stationary phases. Chitin can be converted into chitosan by deacetylation reaction to remove the acetyl group at position 2 on the glucose skeleton. Due to the difference in the reactivity of amino and hydroxyl groups in chitosan, the amino group of chitosan can be selectively modified. Bai team [(2)Chen W, Jiang JZ, Qiu GS, et al. The interactions between chiral analytes and chitosan-based chiral stationary phases during enantioseparation[J]. Journal of Chromatography A, 2021, 1650: 462259.; (3) Francotte E R. Enantioselective chromatography as a powerful alternative for the preparation of drug enantiomers[J].Journal of Chromatography A, 2001,906(1-2):379-397.;(4)Gao YY,Chen W,Bai Z W.Influence of microstructure at 2-position of chitosan derivatives on enantioseparation[J].Reactive and Functional Polymers,2022,176:105298.;(5)Liang S, Huang SH, Chen W, et al. High-performance chiral stationary phases based on chitosan derivatives with abranched-chain alkyl urea[J]. Analytica Chimica Acta, 2017,985:183-193.; (6) WangJ, Xi JB, Chen W, et al.High performance chiral separation materials based on chitosan bis(3,5-dimethylphenylcarbamate)-(alkyl urea)s[J].Carbohydratepolymers,2017,156:481-489.;(7) Zhang J, Zhang GH, Wang XC, et al. Synthesis and evaluation of novel chiral stationary phases based on N-cyclobutylcarbonylchitosan derivatives[J].Microchemical Journal,2019,147:224-231.;] prepared chiral stationary phases with the same substituted aryl carboxylate substituents at positions 3 and 6 on the chitosan skeleton, and amide and urea substituents at position 2. However, it was found that different substituents at position 2 can improve its chiral separation ability, but the separation performance for some chiral compounds is still insufficient, so it is still important to continue to develop new substituents at position 2. .

[0004] Chitosan-3,6-di(phenylcarbamate)-2-(benzimido Schiff base) derivatives are novel polysaccharide derivative materials obtained by derivatizing the amino groups on chitosan with aldehydes or ketones under alcohol and acid conditions, and then esterifying the hydroxyl groups on the chitosan Schiff base. The chitosan-3,6-di(phenylcarbamate)-2-(benzimido Schiff base) derivative structure contains three aromatic rings, and C2 has an imine conjugated structure with chitosan, so the derivative has a strong conjugation effect. At the same time, the combination of the phenylcarbamate groups on C3 and C6 and the Schiff base group on C2 may change the multi-level structure of the chitosan derivative, so it is a good chiral separation material. Chitosan-3,6-bis(phenylcarbamate)-2-(benzimido Schiff base) derivatives are coated on the surface of aminopropyl silica gel. They are insoluble in n-hexane, isopropanol, ethanol, methanol, and dichloromethane. Therefore, a chiral stationary phase for the forward mode is prepared, which has a good separation effect on many chiral compounds. Summary of the invention

[0005] The present invention provides a chitosan-3,6-di(aryl carbamate)-2-(benzimido Schiff base) type chiral stationary phase, a preparation method and an application thereof. The present invention is achieved through the following technical solutions:

[0006] The present invention discloses a chiral stationary phase of chitosan-3,6-di(aromatic carbamate)-2-(benzimido Schiff base), and a chitosan derivative having both aromatic Schiff base and amide structures. The stationary phase is shown in Formula 1:

[0007]

[0008] Where: n≥10; substituent R on the benzene ring 1 and R 2 Are one of the following groups

[0009]

[0010] As a further improvement, the stationary phase described in the present invention is first obtained by Schiff base treatment of chitosan to obtain a chitosan 2-(benzalimidate Schiff base) derivative, and then further esterification is performed to obtain a chitosan-3,6-di(aryl carbamate)-2-(benzalimidate Schiff base) derivative, and the obtained chitosan derivative is coated on a silica gel carrier modified with aminopropylsilane, thereby obtaining a chitosan-3,6-di(aryl carbamate)-2-(benzalimidate Schiff base) type chiral stationary phase shown in Formula 1.

[0011] The present invention also discloses a method for preparing a chitosan-3,6-di(aryl carbamate)-2-(benzimido Schiff base) chiral stationary phase, which is characterized by comprising the following steps:

[0012] 1) Reaction of chitosan with aldehydes and ketones for Schiff base

[0013] Dissolve chitosan with a deacetylation degree of more than 98% in a certain amount of glacial acetic acid solution, then slowly drop a certain amount of aromatic aldehyde dissolved in methanol and mix, then add a certain amount of triethylamine, mix and react at 45-70° C. for 18-24 hours, wash with a large amount of ethanol to remove unreacted aldehyde after the reaction, filter and vacuum dry to obtain chitosan-2-(benzimido Schiff base) product;

[0014] 2) Esterification of chitosan-2-(benzimido Schiff base) derivatives

[0015] The chitosan-2-(benzimido Schiff base) obtained in 1) is dissolved in a certain amount of organic solvent, and then a certain amount of aromatic phenyl isocyanate is added and reacted at 85-95° C. for 30-48 hours. After the reaction is completed, methanol is used for precipitation, and DMF is used for dissolution. The process is repeated 3-5 times until unreacted aldehyde is removed, and vacuum drying is performed to obtain a chitosan-3,6-di(aryl carbamate)-2-(benzimido Schiff base) derivative;

[0016] 3) coating a chitosan-3,6-di(aryl carbamate)-2-(benzimido Schiff base) derivative on the surface of aminopropyl silica gel;

[0017] A certain amount of the chitosan derivative obtained in 2) is dissolved in a strong polar organic solvent, and coated on a certain amount of aminopropyl silica gel three times, the solvent is removed by rotary evaporation under reduced pressure, and vacuum drying is performed to obtain chitosan-3,6-di(aromatic carbamate)-2-(benzimido Schiff base) chiral stationary phase.

[0018] As a further improvement, in step 1) of the present invention, the amount of aromatic aldehyde added is 5-8 times the amount of chitosan, the amount of glacial acetic acid solution is 20 ml of 1%-2% glacial acetic acid per gram of chitosan, and the amount of triethylamine is the same as the molar amount of the added glacial acetic acid.

[0019] As a further improvement, in step 2) of the present invention, a certain amount of organic solvent is 15-20 ml of dry 10% LiCl N,N-dimethylacetamide solution per gram of chitosan-2-(benzalimidate Schiff base) derivative, and a certain amount of aromatic phenyl isocyanate is 2-6 times the amount of aromatic phenyl isocyanate required to react per gram of chitosan-2-(benzalimidate Schiff base) derivative.

[0020] As a further improvement, in step 3) of the present invention, the mass of the certain amount of aminopropyl silica gel is 4 times the mass of chitosan, and the coating amount is 20%; and the strong polar organic solvent is N,N-dimethylformamide.

[0021] As a further improvement, the aromatic aldehyde and aromatic phenyl isocyanate used in steps 1) and 2) of the present invention have structures as follows:

[0022]

[0023] Substituent R on the benzene ring 1 and R 2 Each of the following groups

[0024]

[0025] The reaction process is:

[0026]

[0027] As a further improvement, the aminopropyl silica gel used in step 3) of the present invention is a microsphere type, the silica gel particle size used is 5-7um, and the pore size is The content of aminopropylsilane is 10-12.5%.

[0028] The present invention also discloses an application of chitosan-3,6-di(aromatic carbamate)-2-(benzimido Schiff base) type chiral stationary phase according to claim 1, and an application of the chiral material shown in formula 1 as a stationary phase in normal phase chromatography for various chiral compounds.

[0029] As a further improvement, the various chiral compounds described in the present invention are mostly any one of amides, aromatic ketones, heterocyclic compounds, and epoxy compounds.

[0030] The beneficial effects of the present invention are:

[0031] The chiral immobilization prepared by the present invention has good chiral separation performance, and also has good separation effect on chiral compounds that cannot be separated or separated poorly by traditional ADMPC and CDMPC chiral columns. The main reasons are:

[0032] 1) The present invention has high reaction efficiency, simple and green process, easy control of conditions, and is beneficial to environmental protection.

[0033] 2) The present invention first performs Schiff base treatment on chitosan and then performs esterification, so that the synthesized chitosan-3,6-di(aryl carbamate)-2-(benzimido Schiff base) has a high degree of substitution and a regular structure, thereby enhancing the chiral recognition performance of the chiral stationary phase.

[0034] 3) The introduction of Schiff base groups provides stronger hydrogen bonding ability, which facilitates the coating and bonding between chitosan derivatives and aminopropyl silica gel, and is beneficial to enhancing the stability of the chiral stationary phase separation effect.

[0035] 4) Due to the introduction of Schiff base and amide groups, the π-π interaction and hydrogen bonding forces between chitosan derivatives and enantiomers are enhanced, and the separation of aromatic amides, aromatic ketones, heterocyclic compounds and epoxy compounds on the stationary phase is enhanced.

[0036] 5) The electronic effect and steric barrier effect of the Schiff base part and the carbamate part of the synthesized chitosan-3,6-di(arylcarbamate)-2-(benzimido Schiff base) can complement each other, thereby improving the chiral separation ability of the stationary phase and specifically identifying certain chiral compounds.

[0037] 6) When the amount of aminopropylsilane is about 10%, the chitosan derivative is more tightly bound to the stationary phase, which is beneficial to the stability and durability of the stationary phase.

[0038] 7) The chitosan derivative is applied three times at a 20% amount to form a uniform and fully covered thin layer, thereby providing the best chiral separation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The infrared spectra of the chitosan Schiff base prepared in Example 1 and the chitosan-3,6-di(phenylcarbamate)-2-(benzimidamine Schiff base) prepared in Example 3 were tableted using KBr;

[0040] Figure 2 To implement the chitosan Schiff base prepared in 1 1 HNMR spectrum;

[0041] Figure 3 Chitosan-3,6-di(phenylcarbamate)-2-(benzimido Schiff base) prepared in Example 3 1 HNMR spectrum;

[0042] Figure 4 20 chiral compounds selected to test the separation performance of the chiral stationary phase of chitosan-3,6-di(phenylcarbamate)-2-(benzimidamine Schiff base) of the present invention;

[0043] Figure 5 A statistical graph showing the number of chiral samples identified and baseline separated by the CSPs prepared by the present invention;

[0044] Figure 6 The CSP1 and CSP2 prepared by the present invention have separation spectra for amides, aromatic ketones, heterocyclic compounds and epoxy compounds of chiral compounds. DETAILED DESCRIPTION

[0045] The present invention will be further described below through specific implementation modes in conjunction with the accompanying drawings.

[0046] The chitosan used in the present invention is prepared by deacetylation of chitin and its molecular weight is determined by reference to the literature [(8). Sheng Yiyu, Zhong Huijuan. Determination of molecular weight of chitosan by high performance gel permeation chromatography [D]., 1994.. (9). Bai Zhengwu, Fu Keqin, Bin Qin, et al. Preparation of ultra-high deacetylated chitosan with different molecular weights [J]. Journal of Wuhan University of Technology, 2014, 36 (8): 16-19.], and the average viscosity is 13.6×10 4 .

[0047] The silica gel used in the present invention (spherical, average particle size 7 μm, pore size 1000A) was purchased from Daiso Co., Ltd.

[0048] The solvent N,N-dimethylacetamide (DMAc) used in the present invention is dried three times with molecular sieves before use, and LiCl is calcined at 300° C. for more than 6 hours before use.

[0049] In order to evaluate the separation performance of the prepared chiral stationary phase, 20 chiral compounds were selected and their chiral separation ability was evaluated by high performance liquid chromatography. At the same time, in order to compare the chiral separation performance of the stationary phase prepared in the present invention, the chiral separation data of amylose-tris(3,5-dimethylphenylcarbamate) (CSPa) and cellulose-tris(3,5-dimethylphenylcarbamate) (CSPb) reported in the literature [(6) Wang J, Xi JB, Chen W, et al. High performance chiral separation materials based on chitosan bis(3,5-dimethylphenylcarbamate)-(alkyl urea)s[J]. Carbohydrate polymers, 2017, 156: 481-489] were compared under the same separation conditions.

[0050] The preparation of aminopropyl silica gel was completed according to the reference [(11). Lai XH, Bai ZW, Ng SC, et al. Preparation and enantioseparation characteristics of two chiral stationaryphases based on mono(6A-azido-6A-deoxy)-perphenylcarbamoylated α-and γ-cyclodextrin[J]. Chirality, 2004, 16(9): 592-597.], and the grafting rate was 10%.

[0051] Example 1

[0052] (1) Preparation of chitosan-2-(3,5-dichlorobenzyl imine Schiff base) (CB1):

[0053] 1.5g (9.3mmol repeating units) chitosan (number average molecular weight 130,000, deacetylation degree 98% or more) was added to a 250mL three-necked flask, 1.5ml glacial acetic acid and 105ml distilled water, and stirred for 2h at 55°C to dissolve. Then, nitrogen was introduced, and 13.0g 3,5-dichlorobenzaldehyde (74.5mmol) solution and 1.5ml triethylamine dissolved in 45ml methanol were slowly added dropwise, and reacted at 60°C for 24h. A large amount of precipitate was generated. After the reaction was completed, the precipitate was poured into 200ml hot ethanol and filtered while hot. The filter cake was repeatedly washed with hot ethanol until the unreacted aldehyde was completely removed, and vacuum dried at 60°C to constant weight. Chitosan-2-(3,5-dichlorobenzyl Schiff base) was obtained in an amount of 2.81g, with a yield of 90.0%; the infrared spectrum is shown in the attached figure Figure 1(CB1) as shown: IR (KBr, cm-1), 3455 (-OH), 3077, 1598, 822, 723 (-Ph), 2927-2883 (-CH), 1646 (-N=C-), from Figure 1 It can be seen that the absorption peak of (-N=CH-) observed at 1646cm-1 for CB1 indicates the formation of Schiff base; 1 H NMR spectrum is attached Figure 2 As shown, 1 HNMR (500MHz, 25℃, CF3COOD, δ / ppm): 5.45-3.38 (7H, chitosan skeleton-H), 9.14-8.79 (1H, -N=CH-, 8.13-7.81 (5H, benzene ring-H). From the integrated area ratio, it can be seen that the degree of substitution of chitosan Schiff base is above 90%; Elemental analysis (%): calculated value (C13H13Cl2NO4·0.4H2O)n C 47.99, H 4.35, N 4.30; measured value C47.64, H 4.58, N 5.11; Since the deacetylation degree of chitosan is close to 100%, the theoretical value of elemental analysis of chitosan derivatives is calculated according to the molecular formula of the repeating unit of completely deacetylated chitosan derivatives. Since the prepared chitosan Schiff base contains a large number of hydroxyl groups, it is easy to absorb water, and the theoretical value of elemental analysis needs to be calculated in place of water.

[0054] Example 2

[0055] (2) Preparation of chitosan-2-(4-chlorobenzyl imine Schiff base) (CB2):

[0056] 1.5g (9.3mmol repeating units) chitosan (number average molecular weight 130,000, deacetylation degree 98% or more) was added to a 250mL three-necked flask, 1.5ml glacial acetic acid and 105ml distilled water, and fully stirred at 55°C for 2h to dissolve. Then, nitrogen was introduced, and 10.5g p-chlorobenzaldehyde (74.5mmol) solution and 1.5ml triethylamine dissolved in 45ml methanol were slowly added dropwise, and reacted at 60°C for 24h to generate a large amount of precipitate. After the reaction was completed, the precipitate was poured into 200ml hot ethanol and filtered while hot. The filter cake was repeatedly washed with hot ethanol until the unreacted aldehyde was completely removed, and vacuum dried at 60°C to constant weight. Chitosan 2-(4-chlorobenzyl Schiff base) was obtained in an amount of 2.24g, with a yield of 89%.

[0057] Example 3

[0058] (3) Preparation of chitosan-bis(4-methylphenylcarbamate)-(3,5-dichlorobenzyl Schiff base) (CS1);

[0059] Weigh 1.5g of dry LiCl in a 100mL three-necked flask, add 20mL of dry DMAc, heat to completely dissolve LiCl, keep the temperature at 80°C, add 1.0g of dry chitosan-3,5-dichlorobenzaldehyde shiff base (3.14mmol repeating unit) and stir to dissolve, then add 1.98mL of 4-methylphenyl isocyanate (15.7mmol) and a catalytic amount of DMAP, keep the temperature at 90°C for 30 hours. Add the reaction solution dropwise to 100mL of methanol while hot, precipitate, filter, dissolve the solid in 15mL of DMF, then add the solution dropwise to 100mL of methanol for precipitation, filter, and repeat the reprecipitation until the filtrate has no UV absorption on the thin layer chromatography. Finally, the solid was washed with methanol, filtered, and dried under vacuum at 60°C to obtain 1.52 g of chitosan-bis(4-methylphenylcarbamate)-(3,5-dichlorobenzyl Schiff base), with a yield of 83%. The infrared spectrum was as follows Figure 1 (CS1) shows: IR(KBr,cm-1)υ:IR(KBr,cm –1 )υ:3408,3322(-NH-),3034,1599,819(-Ph),2954-2868(-CH),1720(-COO-),,1526(-CONH-),; 1 H NMR spectrum is attached Figure 3 As shown, 1 HNMR (500 MHz, 90 ° C, DMSO, δ / ppm): 9.21-8.20 (2H, -NH-CO2-), 8.19-7.73 (1H, -CH = N-), 7.57-6.62 (9H, H on the benzene ring), 5.18-2.83 (7H, H on the chitosan skeleton) 2.30-1.92 (6H, methyl on the benzene ring); Elemental analysis (%): estimated value (C 29 H 27 Cl 2 N 3 O 6 0.6H 2 O)n, C 60.74, H 5.38, N 7.33; measured values ​​C 60.81, H 5.49, N 8.24; because the imine part in chitosan-bis(4-methylphenylcarbamate)-(3,5-dichlorobenzaldehyde Schiff base) can form hydrogen bonds and easily absorb water, it is necessary to replace water when calculating the theoretical value of elemental analysis. The above characterization data show that the prepared material is chitosan-bis(4-methylphenylcarbamate)-(3,5-dichlorobenzaldehyde Schiff base).

[0060] Example 4

[0061] (4) Preparation of chitosan-bis(4-methylphenylcarbamate)-(4-chlorobenzyl Schiff base) (CS2)

[0062] Weigh 1.5g of dry LiCl in a 100mL three-necked flask, add 20mL of dry DMAc, heat to completely dissolve LiCl, keep the temperature at 80℃, add 1.0g of dry chitosan-(4-chlorobenzaldehyde shiff base) (3.53mmol repeating unit) and stir to dissolve, then add 2.23mL of 4-methylphenyl isocyanate (17.65mmol) and catalytic amount of DMAP, keep the temperature at 90℃ for 30 hours. Add the reaction solution dropwise to 100mL of methanol while hot, precipitate, filter, dissolve the solid in 15mL of DMF, then add the solution dropwise to 100mL of methanol for precipitation, filter, repeat the reprecipitation until the filtrate has no ultraviolet absorption on thin layer chromatography. Finally, wash the solid with water, filter, and dry to obtain 1.49g of chitosan-bis(4-methylphenylcarbamate)-(4-chlorobenzyl schiff base), yield: 77%.

[0063] Example 5

[0064] (5) Preparation of chitosan-bis(3,5-dimethylphenylcarbamate)-(4-chlorobenzyl Schiff base) (CS3)

[0065] Weigh 1.5g of dry LiCl in a 100mL three-necked flask, add 20mL of dry DMAc, heat to completely dissolve LiCl, keep the temperature at 80℃, add 1.0g of dry chitosan-(4-chlorobenzaldehyde shiff base) (3.53mmol repeating unit) and stir to dissolve, then add 2.48mL of 3,5-dimethylphenyl isocyanate (17.65mmol) and catalytic amount of DMAP, keep the temperature at 90℃ for 30 hours. Add the reaction solution dropwise into 100mL of methanol while hot, precipitate, filter, dissolve the solid in 15mL of LDMF, then add the solution dropwise into 100mL of methanol for precipitation, filter, repeat the reprecipitation until the filtrate has no ultraviolet absorption on thin layer chromatography. Finally, wash the solid with water, filter, and dry to obtain 1.65g of chitosan-di(3,5-dimethylphenylcarbamate)-(4-chlorobenzyl Schiff base), yield: 81%.

[0066] Example 6

[0067] (6) Preparation of chitosan-bis(3,5-dimethylphenylcarbamate)-(3,5-dichlorobenzyl Schiff base) (CS4);

[0068] Weigh 1.5g of dry LiCl in a 100mL three-necked flask, add 20mL of dry DMAc, heat to completely dissolve LiCl, keep the temperature at 80°C, add 1.0g of dry chitosan-3,5-dichlorobenzaldehyde shiff base (3.14mmol repeating unit) and stir to dissolve, then add 2.21mL of 3,5-dimethylphenylisocyanate (15.7mmol) and a catalytic amount of DMAP, keep the temperature at 90°C for 30 hours. Add the reaction solution dropwise to 100mL of methanol while hot, precipitate, filter, dissolve the solid in 15mL of DMF, then add the solution dropwise to 100mL of methanol for precipitation, filter, and repeat the reprecipitation until the filtrate has no UV absorption on the thin layer chromatography. Finally, the solid was washed with methanol, filtered, and dried in vacuo at 60° C. to obtain 1.40 g of chitosan-bis(3,5-dimethylphenylcarbamate)-(3,5-dichlorobenzyl Schiff base), with a yield of 73%.

[0069] Example 7

[0070] 0.8 g of the chitosan derivative obtained in Example 3 was dissolved in DMF, 10 ml of the derivative solution was taken each time and added to 3.2 g of aminopropyl silica gel, the solvent was removed by rotary evaporation under reduced pressure, the above operation was repeated, the derivative solution was coated on the surface of aminopropyl silica gel three times, and vacuum dried to obtain chitosan-3,6-di(aryl carbamate)-2-(benzimido Schiff base) chiral stationary phase. Other chiral stationary phases were prepared in the same way.

[0071] Example 8

[0072] Chiral recognition and separation performance test:

[0073] The chiral stationary phases prepared from the derivatives obtained in Example 3 and Example 4 were named CSP1 and CSP2. The homogenization method was used, and 30 ml of methanol / 1,4-dioxane (90 / 10, v / v) was used as the homogenization liquid. The CSP was evenly dispersed by ultrasound and then poured into a homogenization tube. Methanol was then used as a displacement liquid. The stationary phases were filled into a chromatographic column (250×4.6 mm) using a filling pump at a pressure of 5500-8000 psi to prepare a chromatographic column. Figure 4 The chiral compounds numbered 1-20 were used as samples, and the chiral recognition and separation performance of these stationary phases were tested under the condition of n-hexane-isopropanol (90 / 10, v / v) as the mobile phase. The statistics of the total number of samples separated and the number of baseline separations on CSP1 and CSP2 are shown in the attached figure. Figure 5 In addition, Figure 6The results of separation of chiral compounds 1, 4, 5, 7, and 15 of amides, aromatic ketones, heterocyclic compounds, and epoxy compounds by CSP1 and CSP2 show good separation performance. In order to compare the chiral recognition performance of the chiral stationary phase in the present invention, the separation results of the chiral chromatographic columns of amylose-tris(3,5-dimethylphenylcarbamate) and cellulose-tris(3,5-dimethylphenylcarbamate) that have been reported so far are compared [(10) Wang J, **JB, ChenW, et al, 2017]. The specific separation results are shown in Table 1 below. Since CSP1, CSP2, CSPa and CSPb are subjected to the same conditions, the results are comparable. From these separation results, for chiral compounds with separation results, although the number of chiral recognitions of CSP2 is less than that of CSPb, the number of chiral compounds that can achieve baseline separation is more than that of CSPa and CSPb. In addition, CSP1 and CSP2 can specifically recognize certain compounds. Therefore, in general, the chiral separation performance of the prepared CSP1 and CSP2 is better than that of the currently known best separation performance of amylose-tris(3,5-dimethylphenylcarbamate) and cellulose-tris(3,5-dimethylphenylcarbamate) stationary phases, indicating that the chitosan derivative stationary phase in the present invention has good chiral separation performance.

[0074] S / N: sample number; mobile phase: n-hexane / isopropanol (90 / 10, v / v); flow rate: 1 ml / min; temperature: 25°C, detection wavelength: 254 nm.

[0075] Chromatographic parameters: The three important chromatographic parameters of chiral stationary phase are capacity factor k, distribution factor α and separation Rs, where k = (tR-t0) / t0, tR is the retention time of sample components, t0 is the dead time, measured by biphenyl; α = k2 / k1, k1 and k2 are the capacity factors of two components respectively; Rs = 2(tR2-tR1) / (w1+w2), where tR1 and tR2 are the retention times of two adjacent chromatographic peaks respectively, and w1 and w2 are the peak base widths of the two peaks in the corresponding chromatograms respectively. When α is greater than 1, it means that the sample can be identified, and when Rs>1.5, baseline separation is achieved.

[0076] Table 1 shows the separation results of chiral compounds on CSP1, CS2, CSPad and CSPob

[0077]

[0078] Mobile phase: n-hexane / ethanol (90 / 10, v / v),

[0079] Flow rate: 1ml / min

[0080] Column temperature: 25°C

[0081] Detection wavelength 254nm

[0082] The subscripts '+', '-', 'R', and 'S' on k1 refer to the optical rotation of the first eluting enantiomer; blank spaces indicate that this sample was not tested on this stationary phase.

[0083] The above description is not a limitation of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several changes, modifications, additions or substitutions can be made without departing from the essential scope of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A chiral stationary phase of chitosan-3,6-di(aryl carbamate)-2-(benzimido Schiff base), characterized in that: The chitosan derivative having both aromatic Schiff base and amide structures, the stationary phase is shown in Formula 1: Where: n≥10; the substituents R1 and R2 on the benzene ring are one of the following groups 2. The chiral stationary phase of chitosan-3,6-bis(aryl carbamate)-2-(benzimido Schiff base) according to claim 1, characterized in that: The stationary phase is prepared by firstly subjecting chitosan to Schiff base treatment to obtain a chitosan 2-(benzimido Schiff base) derivative, and then further subjecting chitosan to esterification to obtain a chitosan-3,6-di(aryl carbamate)-2-(benzimido Schiff base) derivative. The obtained chitosan derivative is coated on a silica gel carrier modified with aminopropylsilane, thereby obtaining a chitosan-3,6-di(aryl carbamate)-2-(benzimido Schiff base) type chiral stationary phase shown in Formula 1.

3. A method for preparing a chiral stationary phase of chitosan-3,6-bis(aryl carbamate)-2-(benzimido Schiff base) as claimed in claim 1 or 2, characterized in that: The following steps are involved: 1) Reaction of chitosan with aldehydes and ketones for Schiff base Dissolve chitosan with a deacetylation degree of more than 98% in a certain amount of glacial acetic acid solution, then slowly drop a certain amount of aromatic aldehyde dissolved in methanol and mix, then add a certain amount of triethylamine, mix and react at 45-70° C. for 18-24 hours, wash with a large amount of ethanol to remove unreacted aldehyde after the reaction, filter and vacuum dry to obtain chitosan-2-(benzimido Schiff base) product; 2) Esterification of chitosan-2-(benzimido Schiff base) derivatives The chitosan-2-(benzimido Schiff base) obtained in 1) is dissolved in a certain amount of organic solvent, and then a certain amount of aromatic phenyl isocyanate is added and reacted at 85-95° C. for 30-48 hours. After the reaction is completed, methanol is used for precipitation, and dichloromethane is used for dissolution. The process is repeated 3-5 times until unreacted aldehyde is removed, and vacuum drying is performed to obtain a chitosan-3,6-di(aromatic carbamate)-2-(benzimido Schiff base) derivative; 3) coating a chitosan-3,6-di(aryl carbamate)-2-(benzimido Schiff base) derivative on the surface of aminopropyl silica gel; A certain amount of the chitosan derivative obtained in 2) is dissolved in a strong polar organic solvent, and coated on a certain amount of aminopropyl silica gel three times, the solvent is removed by rotary evaporation under reduced pressure, and vacuum drying is performed to obtain chitosan-3,6-di(aromatic carbamate)-2-(benzimido Schiff base) chiral stationary phase.

4. The method for preparing chitosan-3,6-bis(aryl carbamate)-2-(benzimido Schiff base) chiral stationary phase according to claim 3, characterized in that: In the step 1), the amount of the added aromatic aldehyde is 5-8 times the amount of chitosan, the amount of the glacial acetic acid solution is 20 ml of 1%-2% glacial acetic acid per gram of chitosan, and the amount of the triethylamine is the same as the molar amount of the added glacial acetic acid.

5. The method for preparing chitosan-3,6-bis(aryl carbamate)-2-(benzimido Schiff base) chiral stationary phase according to claim 3, characterized in that: In the step 2), the certain amount of organic solvent is 15-20 ml of dry 10% LiCl N,N-dimethylacetamide solution per gram of chitosan-2-(benzalimidate Schiff base) derivative, and the certain amount of aromatic phenyl isocyanate is 2.5-6 times the amount of aromatic phenyl isocyanate required for the reaction per gram of chitosan-2-(benzalimidate Schiff base) derivative.

6. The method for preparing chitosan-3,6-bis(aryl carbamate)-2-(benzimido Schiff base) chiral stationary phase according to claim 3, characterized in that: In the step 3), the mass of the certain amount of aminopropyl silica gel is 4 times the mass of chitosan, the coating amount is 20%, and the strong polar organic solvent is N,N-dimethylformamide.

7. The method for preparing chitosan-3,6-bis(aryl carbamate)-2-(benzimido Schiff base) chiral stationary phase according to claim 3, characterized in that: The aromatic aldehyde and aromatic phenyl isocyanate used in the steps 1) and 2) have the following structures: The substituents R1 and R2 on the benzene ring are both one of the following groups The reaction process is:

8. The method for preparing the chiral stationary phase of chitosan-3,6-bis(aryl carbamate)-2-(benzimido Schiff base) according to claim 3, characterized in that: The aminopropyl silica gel used in step 3) is a microsphere, the particle size of the silica gel used is 5-7um, and the pore size is The content of aminopropylsilane is 10-12.5%.

9. An application of the chitosan-3,6-bis(aryl carbamate)-2-(benzimido Schiff base) chiral stationary phase as claimed in claim 1, characterized in that: The chiral material represented by formula 1 is used as a stationary phase in normal phase chromatography for a variety of chiral compounds.

10. The use of chitosan-3,6-bis(aryl carbamate)-2-(benzimido Schiff base) chiral stationary phase according to claim 9, characterized in that: The various chiral compounds are mostly any one of amides, aromatic ketones, heterocyclic compounds and epoxy compounds.

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