Capillary electrochromatography monolithic column based on gold nanoflower modification and application of capillary electrochromatography monolithic column in chiral separation

Abstract

The invention discloses a capillary electrochromatography monolithic column based on gold nanoflower modification and application of the capillary electrochromatography monolithic column in chiral separation, and belongs to the field of pharmaceutical analysis. The method comprises the following steps: synthesizing guanylic acid-coated chiral gold nanoflowers in a chiral environment constructed by guanylic acid chiral molecules and an L-ascorbic acid chiral reducing agent, immobilizing the guanylic acid-coated chiral gold nanoflowers on an amino-functionalized capillary silica gel monolithic column as a chiral selective agent, and separating a chiral drug under an electrophoresis condition. And baseline separation of propranolol enantiomers can be realized. The separation method is simple to operate, and a novel high-selectivity chiral separation detection technology is established.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a capillary electrochromatography monolithic column modified with gold nanoflowers and a method for chiral separation thereof. Background Art

[0002] Since a pair of enantiomers of a chiral molecule have different physiological effects and pharmacological toxicities, chiral separation has important practical significance. Compared with high-performance liquid chromatography, capillary electrochromatography (CEC) technology can achieve rapid separation of various samples with micro-liter reagents, which is more environmentally friendly and economical. Moreover, combining the high selectivity of high-performance liquid chromatography and the high efficiency of capillary electrophoresis, CEC has unique advantages in the field of chiral separation. Developing new chiral selectors is an important research direction for constructing a chiral CEC system. Existing studies have selected chiral molecules such as polysaccharides and proteins to construct chiral CEC separation systems. Nanomaterials are beneficial to improving chiral separation performance based on their high specific surface area. The surface of gold nanoflowers is covered with irregular branched structures, resembling flowers. There are few reports in the literature on applying gold nanoflowers as chiral selectors in CEC chiral separation.

[0003] The present invention selects chiral gold nanoflowers as chiral selectors to prepare a new type of gold nanoflower-modified capillary silica monolithic column, and the application of this chiral selector has not been reported in the literature. Summary of the Invention

[0004] The purpose of the present invention is to broaden the current range of available chiral selectors. Gold nanoflowers are selected as chiral selectors, and chiral gold nanoflowers are bonded to a capillary silica monolithic column. The obtained gold nanoflower-modified capillary column is used for chiral separation, and the baseline separation of propranolol is preliminarily achieved. The chiral gold nanoflowers are formed in a chiral environment constructed by guanosine acid chiral molecules and L-ascorbic acid chiral reducing agents. The guanosine acid-coated gold nanoflowers used have good chiral recognition performance. The present invention combines the advantages of chiral gold nanoflowers and capillary silica monolithic columns to construct a new type of CEC chiral separation system.

[0005] To solve its technical problems, the technical solution adopted by the present invention is as follows: The present invention first provides a capillary electrochromatography monolithic column modified with gold nanoflowers. The gold nanoflowers are used as chiral selectors and are prepared by reducing chloroauric acid and 5'-guanosine monophosphate (5'-GMP) under the action of a reducing agent.

[0006] Specifically, the synthesis of gold nanoflowers: Add the solution of chloroauric acid (HAuCl4) to the solution of 5'-guanylic acid (5'-GMP), stir magnetically at room temperature for 10 min, and let it stand for 2 h. Slowly add L-ascorbic acid and stir magnetically for 30 min. Place it at room temperature for reaction for 1 - 5 h. Centrifuge to precipitate, and the obtained gold nanoflowers are rinsed with deionized water and stored at 4 o °C for standby.

[0007] Among them, the molar ratio of chloroauric acid, 5'-GMP and L-ascorbic acid is 1:2:2.

[0008] Furthermore, the capillary electrochromatography monolithic column is an amino-modified silica monolithic column.

[0009] The present invention further provides a preparation method of the above-mentioned capillary electrochromatography monolithic column modified with gold nanoflowers, including the following steps: Preparation of the capillary silica monolithic column: First, pretreat the capillary with alkali and acid; mix polyethylene glycol, urea, acetic acid and tetramethoxysilane, and stir to form a sol in an ice bath; ultrasonically degas the sol formed in the previous step, inject it into the pretreated capillary column, seal the end, place it in a water bath at 40 °C for reaction for 40 hours and then take it out, heat it at 120 °C for 3 hours, let it stand for one week, and program the temperature to 320 °C for calcination for 24 hours to obtain the capillary silica monolithic column.

[0010] Amination modification: Inject the toluene solution of 3-(aminopropyl)triethoxysilane into the acidified silica monolithic column, seal the end, react at 110 °C for 6 hours, and rinse with toluene and anhydrous methanol to remove impurities to obtain the amino-modified silica monolithic column; Modification with gold nanoflowers: Pour the chiral gold nanoflowers into the monolithic column and react for 10 hours. Rinse with buffer solution to remove the unreacted gold nanoflowers to obtain the capillary silica monolithic column modified with gold nanoflowers, and store it in the electrophoresis buffer solution.

[0011] The present invention also provides a method for chiral separation using the above-mentioned capillary electrochromatography monolithic column modified with gold nanoflowers, which is used for chiral separation of enantiomer molecules.

[0012] Specifically, the chiral separation method is: Dissolve the chiral molecule in a phosphate electrophoresis buffer solution with a pH value of 7 and a concentration of 20 mM to prepare a sample solution. Equilibrate the capillary column with the electrophoresis buffer solution for 30 min and then inject the sample. Inject at 10 kV for 1 - 6 seconds, and separate the chiral molecule under the capillary electrophoresis separation voltage condition of 10 - 20 kV.

[0013] In a specific embodiment of the present invention, the capillary electrochromatography monolithic column modified with gold nanoflowers is used to separate propranolol, and baseline separation of propranolol is achieved.

[0014] Compared with conventional gold nanoparticles, the chiral separation mechanism of the chiral gold nanoflowers of the present invention is mainly reflected in that the 5'-guanylic acid (5'-GMP) coated on the surface of the nanoflowers has chirality, and the flower-like structure of the gold nanoparticles also has chirality, which can be used as a chiral selector. A method for chiral separation of a capillary silica monolithic column modified with gold nanoflowers provided by the present invention further expands the application of gold nanomaterials as chiral selectors in the field of chiral separation of capillary electrochromatography. Description of the Drawings

[0015] Figure 1 Scanning electron microscope image of the chiral gold nanoflowers prepared in Example 1.

[0016] Figure 2 Hydrodynamic diameter of the chiral gold nanoflowers prepared in Example 1.

[0017] Figure 3 Effect of separation voltage on the separation of propranolol by a capillary silica monolithic column modified with chiral gold nanoflowers; among them, separation voltage a, 10 kV; b, 15 kV; c, 20 kV.

[0018] Figure 4 Effect of injection volume on the separation of propranolol by a capillary silica monolithic column modified with chiral gold nanoflowers; among them, injection volume a, 5 kPa, 1 s; b, 5 kPa, 3 s; c, 5 kPa, 6 s.

[0019] Figure 5 Effect of buffer concentration on the separation of propranolol by a capillary silica monolithic column modified with chiral gold nanoflowers; among them, buffer concentration a, 10 mM; b, 20 mM; c, 30 mM.

[0020] Figure 6 Separation of propranolol using a gold nanoparticle modified capillary silica monolithic column without GMP. Detailed Description of the Invention

[0021] The present invention will be further described with reference to the following examples, but it should be understood that these examples are only for illustrative purposes and should not be construed as limitations on the implementation of the present invention. Example 1

[0022] Preparation and Characterization of Chiral Gold Nanoflowers 4 mL of chloroauric acid (HAuCl4) (2 mM) solution was added to 4 mL of 5'-guanylic acid (5'-GMP) (4 mM) solution, and magnetically stirred at room temperature for 10 min and then left standing for 2 h. 1.6 mL of L-ascorbic acid (10 mM) was slowly added to this solution, and magnetically stirred for 30 min. It was allowed to react at room temperature for 3 h. The precipitate was centrifuged, and the obtained gold nanoflowers were rinsed with deionized water and stored at 4 o °C for standby.

[0023] The prepared gold nanoflowers were characterized by scanning electron microscopy and laser particle size analyzer respectively. As Figure 1 shown, the prepared gold nanoflowers have a flower-shaped structure. At the same time, the particle size of the gold nanoflowers was analyzed, and its particle size is about 110 nm ( Figure 2 ). The above results indicate the successful synthesis of gold nanoflowers. Example 2

[0024] Preparation of a capillary silica monolithic column modified with gold nanoflowers, including the following steps: Preparation of the capillary silica monolithic column: The quartz capillary was rinsed with pure water for 15 minutes, rinsed with 1 mol / L sodium hydroxide for 45 minutes, rinsed with pure water for 1 hour, then rinsed with 0.1 mol / L hydrochloric acid for 45 minutes, and rinsed with pure water and anhydrous methanol for 30 minutes respectively, and blown with nitrogen for 1 hour for standby. Weigh 0.22 g of polyethylene glycol and 0.225 g of urea, add 2.5 mL of 0.01 mol / L acetic acid, add tetramethoxysilane, and slowly stop stirring after 45 minutes, inject it into the pretreated capillary for 15 minutes, cap it, keep it in a water bath at 40 °C for 40 hours, then heat it in an oven at 120 °C for 3 hours, and after standing for one week, program the temperature in the column oven to 320 °C and calcine for 24 hours to obtain a silica monolithic column.

[0025] The silica monolithic column was filled with 1 mol / L hydrochloric acid for 3 hours, rinsed with pure water until neutral, and rinsed with anhydrous methanol for 30 minutes. Then, it was injected with a toluene solution of 10% (v / v) 3-(aminopropyl)triethoxysilane and rinsed for 1 hour, capped, reacted at 110 °C for 6 hours, then rinsed with toluene and anhydrous methanol for 30 minutes respectively, and dried with nitrogen to obtain an aminated silica monolithic column.

[0026] The aminated silica monolithic column was injected with gold nanoflower solution and reacted for 10 hours, and rinsed with buffer solution to remove unreacted gold nanoflowers to obtain a capillary silica monolithic column modified with gold nanoflowers, which was stored in electrophoresis buffer solution. Example 3

[0027] According to the method of Example 2, the capillary silica monolithic column modified with chiral gold nanoflowers obtained by preparation was used for chiral separation. A 20 mM phosphate buffer solution was prepared and its pH value was adjusted to 7. The commercially purchased propranolol (Macklin (Shanghai, China)) was dissolved in the electrophoresis buffer solution to prepare a sample solution with a concentration of 1 mg / mL at room temperature. The optimal value of the separation voltage was confirmed. Since an increase in the operating voltage would cause an increase in electroosmotic flow, which would play an inhibitory role in the retention of chiral molecules in the capillary column, the column efficiency of the separation would change, further affecting the magnitude of the resolution.

[0028] From Figure 3 the preferred results, it can be seen that as the voltage increases, the retention times of the propranolol enantiomers gradually decrease. This is because as the voltage increases, the electroosmotic flow in the column increases, and the retention of the sample molecules decreases. Therefore, the peak elution time gradually shortens. As the voltage increases, the column efficiency of the peaks shows a gradually increasing trend. As the voltage increases, the resolution shows a decreasing trend (the resolution Rs value decreases from 9.499 to 7.465). When the voltage is 10 kV, the electroosmotic flow is small, the retention time is long, and the column efficiency is low. When the voltage is 20 kV, the column efficiency increases, but the resolution decreases. Finally, 15 kV is determined as the optimal voltage value. Example 4

[0029] According to the method of Example 2, the capillary silica monolithic column modified with chiral gold nanoflowers obtained by preparation was used for chiral separation. The optimal value of the injection volume was confirmed. In this example, the effect of the injection volume on the separation of propranolol enantiomers was investigated.

[0030] From Figure 4 the preferred test results, it can be seen that as the injection volume increases, it will cause peak tailing and a decrease in column efficiency. The theoretical plate number of the electrophoretogram of chiral molecules shows a decreasing trend as the injection volume increases. When the injection volume is 5 kPa for 1 s, the resolution Rs value is 6.936. When the injection volume is 5 kPa for 6 s, the resolution Rs value is 5.686. Finally, the optimal value of the injection volume is determined to be 5 kPa for 3 s (Rs value is 7.495). Example 5

[0031] According to the method of Example 2, the capillary silica monolithic column modified with chiral gold nanoflowers obtained by preparation was used for chiral separation. The optimal value of the concentration of the phosphate electrophoresis buffer solution was confirmed.

[0032] From Figure 5 the preferred value results, it can be seen that as the concentration of the electrophoresis buffer solution increases, the retention time of the chiral molecules gradually increases. This is because an increase in the concentration of the electrophoresis buffer solution will cause a decrease in electroosmotic flow, and the retention time of the propranolol enantiomers shows an increasing trend. When the buffer solution concentration is 10 mmol L -1When the concentration is [concentration value], the resolution Rs value is 4.880. When the buffer concentration is 30 mmol / L -1 When the concentration is [concentration value], the resolution Rs value is 7.169. When the buffer concentration is 20 mmol / L -1 When the concentration is [concentration value], the resolution is the largest (Rs value is 7.495). This is because when the concentration of the phosphate electrophoresis buffer is too small or too large, the change in the ionic strength of the solution will affect the electroosmotic flow in the column. When the electrophoresis buffer with the optimal concentration is used, the separation ability of the chiral column is the best.

[0033] For comparison, the present invention also uses a gold nanoparticle-modified capillary monolithic column without GMP for the separation of propranolol enantiomers, which cannot achieve chiral separation of the template molecule ( Figure 6 ), indicating that the gold nanoflowers prepared by the present invention have good chiral recognition performance.

[0034] It can be seen from the above examples that the capillary electrochromatography technology based on the chiral gold nanoflower-modified silica monolithic column provides a fast and efficient analysis platform for the separation of enantiomers.

[0035] Taking the above ideal embodiments of the present invention as an inspiration, through the above description, relevant staff can completely make various changes and modifications within the scope not deviating from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A capillary electrochromatography monolithic column modified with gold nanoflowers, characterized in that, The gold nanoflowers described above are used as chiral selectors, and the gold nanoflowers are prepared by reducing chloroauric acid and 5'-guanylic acid under the action of L-ascorbic acid.

2. The monolithic column for capillary electrochromatography modified with gold nanoflowers according to claim 1, characterized in that, The molar ratio of chloroauric acid, 5'-guanylic acid and L-ascorbic acid is 1:2:

2.

3. The monolithic column for capillary electrochromatography modified with gold nanoflowers according to claim 1, wherein The preparation method of the gold nanoflowers described above: Add the chloroauric acid solution to the 5'-guanylic acid solution, stir evenly and then let it stand; then add L-ascorbic acid, stir evenly, and place it at room temperature for reaction for 1-5 h; centrifuge and precipitate, wash to obtain the gold nanoflowers described above.

4. The monolithic column for capillary electrochromatography modified with gold nanoflowers according to claim 1, wherein The monolithic column for capillary electrochromatography described above is an amino-modified silica monolithic column.

5. The preparation method of the capillary electrochromatography monolithic column modified with gold nanoflowers according to any one of claims 1-4, characterized in that, It includes the following steps: Provide a capillary silica monolithic column and perform acidification treatment; Inject 3-aminopropyltrimethoxysilane into the acidified capillary silica monolithic column to obtain an amino-modified capillary silica monolithic column; Inject the gold nanoflowers into the amino-modified capillary silica monolithic column and react for more than 10 hours; wash away the unreacted gold nanoflowers to obtain a monolithic column for capillary electrochromatography modified with gold nanoflowers.

6. The application of the monolithic column for capillary electrochromatography modified with gold nanoflowers according to any one of claims 1-4 in the separation of enantiomers.

7. The application according to claim 6, characterized in that, The enantiomers described above are propranolol enantiomers.

8. The application according to claim 7, wherein The application method: Dissolve the enantiomer molecules in a phosphate electrophoresis buffer solution with a pH value of 7 and a concentration of 20 mM to prepare a sample solution; After the monolithic column for capillary electrochromatography modified with gold nanoflowers is equilibrated with the electrophoresis buffer solution for 30 min, inject the sample, inject for 1-6 seconds at 10 kV, and perform separation under the condition of a capillary electrophoresis separation voltage of 10-20 kV.

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