An L-lysine-derived gel factor mixed-mode chromatography stationary phase, its preparation method and application

Abstract

An L-lysine-derived gel factor mixed-mode chromatography stationary phase, wherein the L-lysine-derived gel factor mixed-mode chromatography stationary phase is based on silica gel, and the bonding phase groups on the surface of the silica gel matrix include hydrophilic amide groups, hydrophobic tert-butyl groups and fluorene groups; the preparation method for preparing the stationary phase includes: synthesizing an L-lysine-derived gel factor; preparing amino silica gel; synthesizing an L-lysine-derived gel factor-modified mixed-mode chromatography stationary phase; the application of the stationary phase is for application in multi-chromatographic separation in the same column. The stationary phase synthesized in the present invention contains hydrophilic amide groups, hydrophobic tert-butyl groups, fluorene groups, etc., and can provide various interactions such as hydrophobic interaction, π-π stacking interaction, hydrogen bond interaction and hydrophilicity. The stationary phase can be used as a chromatographic column to realize the application of multi-chromatographic separation mode in the same column during the separation process, making up for the defects and deficiencies shown by a single chromatographic mode when separating complex samples.

Description

Technical Field:

[0001] The present invention relates to the technical field of chromatographic stationary phase preparation, and specifically relates to an L-lysine-derived gelator mixed-mode chromatographic stationary phase, its preparation method and application. Background Art:

[0002] As a multi-component separation and analysis technique, high performance liquid chromatography has become an indispensable part of separation science. The separation performance of a chromatographic stationary phase is mainly determined by the functional groups modified on the stationary phase surface, which is generally considered to be the core element of chromatographic separation. Traditional chromatographic stationary phases are mainly based on a single separation mode, and inevitably show their inherent defects and deficiencies when separating complex samples. For example, for RPLC, it can only provide hydrophobic interaction between the stationary phase and the analyte, which limits its application range for polar or hydrophilic compounds; HILIC with polar functional groups is exactly the opposite of RPLC, and HILIC is only applicable to the separation of polar hydrophilic compounds; IEC only relies on the ion pair interaction between the stationary phase and the analyte, and it is limited to analytes with a charge opposite to that of the stationary phase. Therefore, there is an urgent need to develop a chromatographic stationary phase with multiple different types of interactions between the stationary phase and the analyte. Summary of the Invention:

[0003] In view of the above problems, the present invention provides an L-lysine-derived gelator mixed-mode chromatographic stationary phase, its preparation method and application. The L-lysine-derived gelator mixed-mode chromatographic stationary phase can be used in hydrophilic chromatography, reverse-phase chromatography and ion exchange chromatography modes, and can also be used for the separation of water-rich chromatography and chiral compounds.

[0004] An L-lysine-derived gelator mixed-mode chromatographic stationary phase, with silica gel as the matrix, and the bonding phase groups on the silica gel matrix surface include hydrophilic amide groups, hydrophobic tert-butyl and fluorene groups. The structural formula of the L-lysine-derived gelator mixed-mode chromatographic stationary phase is:

[0005]

[0006] Among them, the structural formulas of R1 and R2 are both (9-fluorenylmethyl) or (tert-butyl) one of them.

[0007] Preferably, the dosage of the bonding phase contained on each gram of silica gel of the L-lysine-derived gelator mixed-mode chromatographic stationary phase is 0.1 - 2 g.

[0008] Preferably, the silica gel is spherical silica gel, with a particle size of 5 - 7 μm and a specific surface area of 300 m 2 g -1 .

[0009] A preparation method of a mixed-mode chromatographic stationary phase derived from L-lysine gelator, specifically comprising the following steps:

[0010] Step 1: Adopt a protection strategy for the main chain and side chain amino groups in L-lysine to synthesize an L-lysine-derived gelator;

[0011] Step 2: Prepare amino silica gel: Disperse spherical silica gel in anhydrous toluene, the dosage of anhydrous toluene required per gram of silica gel is 15 - 25 ml, add a silanization reagent with an amino group, the dosage of the silanization reagent required per gram of silica gel is 1 - 1.5 ml, reflux and stir for 8 - 24 hours under nitrogen protection, then filter with a sintered funnel, wash with toluene, ethanol, and acetone three times each, and dry in a vacuum drying oven at 40 - 80 °C for 8 - 24 h to obtain amino silica gel;

[0012] Step 3: Synthesize a mixed-mode chromatographic stationary phase modified with an L-lysine-derived gelator: Disperse the L-lysine-derived gelator prepared in Step 1 in N,N-dimethylformamide, add a crosslinking agent, and react at room temperature for 8 - 24 h; add a buffer solution and stir, add the amino silica gel prepared in Step 2, and stir at room temperature for 8 - 24 h. The obtained stationary phase is filtered with a sintered funnel, washed with water and ethanol three times each, and dried at 40 - 80 °C for 8 - 24 h to obtain a mixed-mode chromatographic stationary phase modified with an L-lysine-derived gelator.

[0013] Preferably, the silanization reagent with an amino group is (3-aminopropyl)-triethoxysilane.

[0014] Preferably, the dosage of the (3-aminopropyl)-triethoxysilane required per gram of silica gel is 1 - 1.5 ml.

[0015] Preferably, the crosslinking agent is 1:1 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride:N-hydroxysuccinimide.

[0016] Preferably, the dosage of the 1:1 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride:N-hydroxysuccinimide required per gram of silica gel is 0.2 - 4 g.

[0017] Specifically, Step 3 is a two-step reaction. First, activate the carboxyl group in the L-lysine-derived gelator with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to form a stable intermediate; then couple the intermediate with the amino silica gel to obtain a mixed-mode chromatographic stationary phase modified with an L-lysine-derived gelator.

[0018] The application of an L-lysine-derived gel factor mixed-mode chromatographic stationary phase is for application in multi-chromatographic separation in the same column.

[0019] Preferably, the multi-chromatographic separation mode in the same column is HILIC / RPLC / IEC / PALC and chiral compound separation.

[0020] The stationary phase synthesized in the present invention contains hydrophilic amide groups, hydrophobic tert-butyl and fluorene groups, etc., which can provide various interactions such as hydrophobic interaction, π-π stacking interaction, hydrogen bond interaction and hydrophilicity. In addition, L-lysine can also provide a chiral microenvironment. At the same time, the stationary phase synthesized in the present invention can be used as a chromatographic column to realize the application of multi-chromatographic separation mode in the separation process, effectively making up for the defects and deficiencies shown by a single chromatographic mode in separating complex samples. Specifically, for HILIC / PRLC / IEC / PALC and chiral compound separation, sulfonamide compounds (sulfamethazine, sulfamerazine, sulfathiazole, sulfacetamide, sulfisoxazole, sulfabenzamide) and nucleosides, nucleobases can be selectively separated under the hydrophilic interaction chromatography mode; polycyclic aromatic hydrocarbons (biphenyl, fluorene, phenanthrene, fluoranthene, pyrene) can be selectively separated under the reversed-phase chromatography mode; aniline compounds (o-toluidine, N-methylaniline, aniline, N,N-dimethylaniline, p-phenylenediamine) and aromatic acid compounds (nitrophthalic acid, 2-aminophthalic acid, benzoic acid, 2,5-dihydroxyphthalic acid) can be separated under the ion-exchange chromatography mode; nucleosides, nucleobases (thiourea, cytosine, inosine, guanosine, adenosine, adenine) can be separated under the water-rich chromatography mode under the chromatographic conditions with ultrapure water as the mobile phase; even for some chiral compounds (D,L-phenylethanol, D,L-phenylpropanol, 4-chloro-α-methylbenzyl alcohol, ofloxacin, etc.), good separation effects can be achieved. Finally, the chromatographic column prepared in the present invention only needs to adjust the ratio of water and organic solvent in the mobile phase to meet the requirements of various sample analyses and separations. Under the water-rich chromatography mode, the separation of hydrophilic compounds can be achieved only by using ultrapure water, avoiding the use of toxic organic reagents and reducing the harm to the environment and experimental personnel. Description of the drawings:

[0021] Attached Figure 1 is the infrared characterization diagram of the L-lysine-derived gel factor mixed-mode chromatographic stationary phase 1. Attached Figure 2 is the thermogravimetric characterization diagram of the L-lysine-derived gel factor mixed-mode chromatographic stationary phase 1. Attached Figure 3 is the chromatogram of Application Example 1.

[0022] Attached Figure 4 is the chromatogram of Application Example 2.

[0023] Attached Figure 5 is the chromatogram of Application Example 3.

[0024] Attached Figure 6 is the chromatogram of Application Example 3.

[0025] Attached Figure 7 is the chromatogram of Application Example 4.

[0026] Attached Figure 8 is the chromatogram of Application Example 5.

[0027] Attached Figure 9 is the chromatogram of Application Example 6.

[0028] Attached Figure 10 is the chromatogram of Application Example 7. Detailed implementation method:

[0029] In order to make the technical solution of the present invention easier to understand, a gel factor mixed-mode chromatographic stationary phase derived from L-lysine, its preparation method and application disclosed in the present invention will be clearly and completely described in the form of embodiments.

[0030] Embodiment 1:

[0031] A preparation method of a gel factor mixed-mode chromatographic stationary phase derived from L-lysine, the method comprising the following steps:

[0032] Step 1: Adopt a protection strategy for the main chain and side chain amino groups in L-lysine to synthesize a gel factor derived from L-lysine: Dissolve 3.5 g of NaHCO3 in 18 ml of water, add 2 g of L-lysine hydrochloride to its solution, stir to dissolve, add 1.4 g of CuSO4·4H2O in three portions, stir for 1 h, then add 3 g of di-tert-butyl dicarbonate and 12 ml of acetone, and stir at room temperature overnight; add 25 ml of water and 25 ml of acetone, stir for 4 h, then add 25 ml of ethyl acetate, stir to obtain a blue precipitate, filter, wash, and dry to obtain a copper complex with the side chain amino group of L-lysine protected.

[0033] Dissolve 1 g of the copper complex with the side chain amino group of L-lysine protected in a mixed solution of 10 ml of water and 8 ml of acetone, add 1 g of anhydrous sodium carbonate and 0.5 g of 8-hydroxyquinoline, react at room temperature for 1 h, then add 1 g of 9-fluorenylmethyl-N-succinimidyl carbonate, filter to obtain a brown filtrate. Adjust the pH = 2 - 3 with hydrochloric acid in an ice-water bath to obtain a yellow viscous solid, wash, dry, and recrystallize with ethyl acetate / petroleum ether to obtain gel factor 1 derived from L-lysine.

[0034] Step 2: Preparation of amino silica gel

[0035] Dissolve 2 g of SiO2 in 40 ml of 3.0 mol / L HCl solution, reflux at 120 °C for 7 hours, then centrifuge, wash with ultrapure water until neutral, and dry to obtain activated SiO2. Disperse 2 g of activated SiO2 in 40 ml of anhydrous toluene, add 2.2 ml of (3-aminopropyl)-triethoxysilane, stir magnetically for 24 h under a nitrogen atmosphere, and then filter with a sintered funnel. Then, wash three times with toluene, ethanol, and acetone respectively, and dry at 50 °C for 12 h to obtain amino silica gel.

[0036] Step 3: Synthesis of L-lysine-derived gelator mixed-mode chromatographic stationary phase

[0037] Disperse 2 g of the L-lysine-derived gelator 1 prepared in Step 1 in N,N-dimethylformamide, add 2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a ratio of 1:1, react at room temperature for 20 h, add 2 ml of N-methylmorpholine-N-oxide, stir, and then add the above-prepared amino silica gel and stir at room temperature for 24 h. The obtained stationary phase is filtered with a sintered funnel, washed three times with water and ethanol respectively, and dried at 40 °C for 12 hours to obtain the L-lysine-derived gelator mixed-mode chromatographic stationary phase 1, the structure of which is as follows:

[0038]

[0039] Table 1 Elemental analysis table of L-lysine-derived gelator mixed-mode chromatographic stationary phase 1

[0040]

[0041] Example 2:

[0042] A preparation method of an L-lysine-derived gelator mixed-mode chromatographic stationary phase, the method comprising the following steps:

[0043] Step 1: Dissolve 1 g of L-lysine hydrochloride in 20 ml of water, add 20 ml of acetone, 1 g of anhydrous Na2CO3 and 2 g of 9-fluorenylmethyl-N-succinimidyl carbonate, and react overnight. Adjust the pH ≈ 3 in an ice-water bath, white solid precipitates, add ethyl acetate and petroleum ether, separate with a separating funnel, and rotary evaporate the organic phase to obtain white L-lysine-derived gelator 2.

[0044] Step 2: Preparation of amino silica gel

[0045] Dissolve 2 g of SiO2 in 40 ml of 3.0 mol / L HCl solution, reflux at 120 °C for 7 hours, then centrifuge, wash with ultrapure water until neutral, and dry to obtain activated SiO2. Disperse 2 g of activated SiO2 in 40 ml of anhydrous toluene, add 2.2 ml of (3-aminopropyl)-triethoxysilane, stir magnetically for 24 h under a nitrogen atmosphere, and then filter with a fritted funnel. Then, wash three times with toluene, ethanol, and acetone respectively, and dry at 50 °C for 12 h to obtain amino silica gel.

[0046] Step 3: Synthesis of L-lysine-derived gelator mixed-mode chromatographic stationary phase

[0047] Disperse 2 g of the L-lysine-derived gelator 1 prepared in Step 1 in N,N-dimethylformamide, add 2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a 1:1 ratio, react at room temperature for 20 h, add 2 ml of N-methyl morpholine-N-oxide, stir, then add the above-prepared amino silica gel, and stir at room temperature for 24 h. The obtained stationary phase is filtered with a fritted funnel, washed three times with water and ethanol respectively, and dried at 40 °C for 12 hours to obtain the L-lysine-derived gelator mixed-mode chromatographic stationary phase 2, the structure of which is as follows:

[0048]

[0049] Example 3:

[0050] A preparation method of an L-lysine-derived gelator mixed-mode chromatographic stationary phase, the method comprising the following steps:

[0051] Step 1: Dissolve 1 g of L-lysine hydrochloride in 20 ml of water, add 20 ml of acetone, 1 g of anhydrous Na2CO3 and 2 g of di-tert-butyl dicarbonate, and react overnight. Adjust the pH ≈ 3 in an ice-water bath, add ethyl acetate and petroleum ether, separate with a separatory funnel, and rotary evaporate the organic phase to obtain a transparent gel-like L-lysine-derived gelator 3.

[0052] Step 2: Preparation of amino silica gel

[0053] Dissolve 2 g of SiO2 in 40 ml of 3.0 mol / L HCl solution, reflux at 120 °C for 7 hours, then centrifuge, wash with ultrapure water until neutral, and dry to obtain activated SiO2. Disperse 2 g of activated SiO2 in 40 ml of anhydrous toluene, add 2.2 ml of (3-aminopropyl)-triethoxysilane, stir magnetically for 24 h under a nitrogen atmosphere, and then filter with a fritted funnel. Then, wash three times with toluene, ethanol, and acetone respectively, and dry at 50 °C for 12 h to obtain amino silica gel.

[0054] Step 3: Synthesis of L-lysine-derived gelator mixed-mode chromatographic stationary phase

[0055] Disperse 2 g of the L-lysine-derived gelator 1 prepared in Step 1 in N,N-dimethylformamide, then add 2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a 1:1 ratio, react at room temperature for 20 h, add 2 ml of N-methylmorpholine-N-oxide, stir, and then add the prepared amino silica gel above, and stir at room temperature for 24 h. The obtained stationary phase is filtered through a sintered funnel, washed three times with water and ethanol respectively, and dried at 40 °C for 12 hours to obtain the L-lysine-derived gelator mixed-mode chromatographic stationary phase 3, with the structure as follows:

[0056]

[0057] Application Example 1:

[0058] Using the chromatographic column prepared in Example 1, several sulfonamide compounds were separated in the hydrophilic chromatography mode. Figure 3 The chromatographic separation results are as follows: 1 - Sulfadimidine; 2 - Sulfamerazine; 3 - Sulfathiazole; 4 - Sulfacetamide; 5 - Sulfisoxazole; 6 - Sulfabenzamide. The results show that the chromatographic column prepared in Example 1 has good separation effect on sulfonamide compounds; the chromatographic conditions are: acetonitrile: water (60 / 40, v / v); flow rate is 1.0 ml / min; temperature is: 30 °C; detection wavelength is: 270 nm.

[0059] Application Example 2:

[0060] Using the chromatographic column prepared in Example 1, several nucleoside and nucleobase compounds were separated in the hydrophilic chromatography mode. Figure 4 The chromatographic separation results are as follows: 1 - Thiourea; 2 - Cytosine; 3 - Inosine; 4 - Guanosine; 5 - Adenosine; 6 - Adenine. The results show that the chromatographic column prepared in Example 1 has good separation effect on nucleoside and nucleobase compounds; the chromatographic conditions are: ultrapure water; flow rate is 1.0 ml / min; temperature is: 30 °C; detection wavelength is: 260 nm.

[0061] Application Example 3:

[0062] Using the chromatographic column prepared in Example 1, D,L-phenylethyl alcohol and benzoin were separated in the normal-phase chromatography mode. Figure 5, 6 are the separation results of D,L-phenylethyl alcohol and benzoin, respectively. The results show that the chromatographic column prepared in Example 1 has good separation effects on D,L-phenylethyl alcohol and benzoin; the chromatographic conditions are as follows: n-hexane: isopropanol (95 / 5, v / v) and n-hexane: isopropanol (100 / 0, v / v); the flow rate is 1.0 ml / min; the temperature is 30 °C; the detection wavelength is 254 nm.

[0063] Application Example 4:

[0064] Using the chromatographic column prepared in Example 2, several aromatic acid compounds were separated in the ion exchange chromatography mode. Figure 7 The chromatographic separation results are as follows: 1-benzoic acid; 2-nitroterephthalic acid; 3-2-aminoterephthalic acid; 4-2,5-dihydroxyterephthalic acid. The results show that the chromatographic column prepared in Example 2 has good separation effects on aromatic acid compounds; the chromatographic conditions are: the flow rate is 1.0 ml / min; the temperature is 30 °C; the detection wavelength is 254 nm.

[0065] Application Example 5:

[0066] Using the chromatographic column prepared in Example 2, several sulfonamide compounds were separated in the hydrophilic chromatography mode. Figure 8 The chromatographic separation results are as follows: 1-sulfamethazine; 2-sulfamerazine; 3-sulfathiazole; 4-sulfacetamide; 5-sulfisoxazole; 6-sulfabenzamide. The results show that the chromatographic column prepared in Example 2 has good separation effects on sulfonamide compounds; the chromatographic conditions are: acetonitrile: water (40 / 60, v / v); the flow rate is 1.0 ml / min; the temperature is 30 °C; the detection wavelength is 270 nm.

[0067] Application Example 6:

[0068] Using the chromatographic column prepared in Example 3, several polycyclic aromatic hydrocarbons were separated in the reversed-phase chromatography mode. Figure 9 The chromatographic separation results are as follows: 1-biphenyl; 2-fluorene; 3-phenanthrene; 4-fluoranthene; 5-pyrene. The results show that the chromatographic column prepared in Example 3 has good separation effects on polycyclic aromatic hydrocarbons; the chromatographic conditions are: acetonitrile: water (10 / 90, v / v); the flow rate is 1.0 ml / min; the temperature is 30 °C; the detection wavelength is 254 nm.

[0069] Application Example 7

[0070] Using the chromatographic column prepared in Example 3, several aniline compounds were separated in the ion exchange chromatography mode. Figure 10The results of its chromatographic separation are as follows: 1 - aniline; 2 - o - toluidine; 3 - N - methylaniline; 4 - N,N - dimethylaniline; 5 - p - phenylenediamine. The results show that the chromatographic column prepared in Example 3 has good separation effect on aniline compounds; the chromatographic conditions are: ultrapure water; flow rate is 1.0 ml / min; temperature is: 30 °C; detection wavelength is: 254 nm.

[0071] It should be noted that: for those of ordinary skill in the art, without departing from the principles and purposes of the present invention, several improvements, substitutions, variations and retouches can still be made, and these improvements, substitutions, variations and retouches should also be regarded as the protection scope of the present invention.

Claims

1. An L-lysine-derived gel factor mixed-mode chromatographic stationary phase, characterized in that, The L-lysine-derived gelator mixed-mode chromatographic stationary phase uses silica gel as the matrix, and the bonding phase groups on the surface of the silica gel matrix include hydrophilic amide groups, hydrophobic tert-butyl groups, and / or fluorene groups. The structural formula of the L-lysine-derived gelator mixed-mode chromatographic stationary phase is: Among them, the structural formulas of R1 and R2 are both one of the following.

2. The L-lysine-derived gel factor mixed-mode chromatographic stationary phase according to claim 1, characterized in that, The dosage of the bonding phase contained per gram of silica gel in the L-lysine-derived gelator mixed-mode chromatographic stationary phase is 0.1 - 2 g.

3. The L-lysine-derived gel factor mixed-mode chromatographic stationary phase according to claim 2, characterized in that, The silica gel is spherical silica gel with a particle size of 5-7 μm and a specific surface area of 300 m 2 g -1 .

4. A preparation method of the L-lysine-derived gel factor mixed-mode chromatographic stationary phase according to claim 1 or 2, characterized in that, The method specifically includes the following steps: Step 1: Adopt a protection strategy for the main chain and side chain amino groups in L-lysine to synthesize an L-lysine-derived gelator; Step 2: Prepare amino silica gel: Disperse spherical silica gel in anhydrous toluene. The dosage of anhydrous toluene required per gram of silica gel is 15 - 25 mL. Add a silanization reagent with an amino group. The dosage of the silanization reagent required per gram of silica gel is 1 - 1.5 mL. After refluxing and stirring for 8 - 24 hours under nitrogen protection, filter, wash 3 times with toluene, ethanol, and acetone in sequence, and then dry in a vacuum drying oven at 40 - 80 °C for 8 - 24 h to obtain amino silica gel; Step 3: Synthesize an L-lysine-derived gelator-modified mixed-mode chromatographic stationary phase: Disperse the L-lysine-derived gelator prepared in Step 1 in N,N-dimethylformamide, add a crosslinking agent, and react at room temperature for 8 - 24 h; add a buffer solution and stir, add the amino silica gel prepared in Step 2, stir at room temperature for 8 - 24 h, then filter, wash 3 times with water and ethanol respectively, and dry at 40 - 80 °C for 8 - 24 h to obtain an L-lysine-derived gelator-modified mixed-mode chromatographic stationary phase.

5. The preparation method of the L-lysine-derived gel factor mixed-mode chromatographic stationary phase according to claim 4, characterized in that, The silanization reagent with an amino group is (3-aminopropyl)-triethoxysilane.

6. The preparation method of the L-lysine-derived gel factor mixed-mode chromatographic stationary phase according to claim 4, characterized in that, The crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride:N-hydroxysuccinimide at a ratio of 1:

1.

7. The preparation method of the L-lysine-derived gel factor mixed-mode chromatographic stationary phase according to claim 6, characterized in that, The dosage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride:N-hydroxysuccinimide at a ratio of 1:1 required per gram of silica gel is 0.2 - 4 g.

8. An application of the L-lysine-derived gel factor mixed-mode chromatographic stationary phase according to claim 1 or 2, characterized in that, Application of the L-lysine-derived gelator mixed-mode chromatographic stationary phase in multi-chromatographic separation in the same column.

9. An application of the L-lysine-derived gel factor mixed-mode chromatographic stationary phase according to claim 8, characterized in that, The multi-chromatographic separation mode in the same column is HILIC / RPLC / IEC / PALC and chiral compound separation.

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