A Method for Chiral Separation of Amino Acids by Using a Pectinase-Modified Capillary Silica Monolithic Column

Abstract

The invention discloses a method for chiral separation of amino acids by a pectinase-modified capillary silica monolithic column, belonging to the field of bioanalysis. First, a graphene oxide-modified silica monolithic column is prepared, and then pectinase is selected as a bonding ligand to prepare a novel graphene oxide-modified affinity capillary silica monolithic column. The obtained pectinase-modified capillary silica monolithic column is applied to the separation of various amino acid enantiomers. The above technical solution is simple to operate and establishes a novel high-selectivity separation and detection technology for amino acid enantiomers.

Description

Technical Field

[0001] The present invention belongs to the field of bioanalysis, and particularly relates to a method for chiral separation of amino acids by using a pectinase-modified capillary silica monolithic column. Background Art

[0002] Enzymes are a class of chiral selectors in protein molecules and possess the chiral recognition ability of general protein molecules. Since there are different physiological effects and pharmacological toxicities between a pair of enantiomers, chiral resolution has important practical significance. There is a specific recognition between chiral compounds and biological macromolecules such as enzyme molecules, so affinity analysis based on their specific interactions plays a key role in chiral resolution. Currently, the chromatographic analysis method commonly used for chiral separation is high performance liquid chromatography, but it consumes a large amount of reagents and samples, and the chiral column is expensive, which has great limitations in the detection of trace samples.

[0003] Capillary electrochromatography (CEC) technology combines the high selectivity of high performance liquid chromatography and the high efficiency of capillary electrophoresis. It can achieve rapid separation of various samples by using microliter-level reagents, so CEC is more environmentally friendly and economical. In addition, CEC uses an externally applied voltage as the electroosmotic flow driving force, eliminating the influence of the radial diffusion of the parabolic flow pattern in liquid chromatography driven by pressure on the column efficiency of the finally obtained chromatographic peak, and has higher separation column efficiency. As a rapid, efficient and low-sample-consumption technology, CEC has broad application prospects in the field of chiral separation. At the same time, combining enzyme molecules with a CEC system based on a silica monolithic column innovatively expands the application scope of CEC. Summary of the Invention

[0004] The purpose of the present invention is to broaden the currently available range of chiral selectors, use pectinase produced by the Aspergillus niger strain preserved in this laboratory as a chiral selector, covalently bond pectinase to a graphene oxide-functionalized silica monolithic column, and use the obtained pectinase-modified capillary silica monolithic column for chiral separation of amino acids.

[0005] The technical solution adopted by the present invention to solve its technical problems is:

[0006] (1) First, pretreat the capillary with alkali and acid, then mix polyethylene glycol, urea, acetic acid and tetramethoxysilane, and stir in an ice bath to form a homogeneous sol; ultrasonically degas the obtained sol, inject it into the pretreated capillary column, seal the end, place it in a water bath at 40 °C, take it out after 40 hours, 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 a silica monolithic column;

[0007] The relative dosage relationship of raw materials is as follows: polyethylene glycol: urea: acetic acid (0.01 mol / L): tetramethoxysilane is 0.22 g: 0.225 g: 2.5 mL: 0.9 mL.

[0008] (2) After acidifying the silica gel monolithic column, inject a toluene solution of 3-(aminopropyl)triethoxysilane into the acidified silica gel monolithic column, react after capping, and rinse with toluene and anhydrous methanol to remove impurities to obtain an aminated silica gel monolithic column;

[0009] Furthermore, the acidification treatment of the silica gel monolithic column in step (2) is as follows: inject 1 mol / L hydrochloric acid into the silica gel monolithic column for 3 hours, rinse with pure water until neutral, and rinse with anhydrous methanol for 30 minutes. Furthermore, in step (2), inject a 10% (v / v) toluene solution of 3-(aminopropyl)triethoxysilane into the acidified silica gel monolithic column and rinse for 1 hour, and react at 110 °C for 6 hours after capping.

[0010] (3) After ultrasonic dispersion of the 1 mg / mL graphene oxide aqueous solution, dilute it with a potassium hydroxide solution. After dilution, the concentration of graphene oxide is 0.05 mg / mL, and the concentration of the potassium hydroxide solution is 0.1 mg / mL. Inject it into the aminated silica gel monolithic column, react at 40 °C for 2 hours after capping, rinse with water until neutral, and rinse with anhydrous methanol to remove impurities to obtain a graphene oxide-modified silica gel monolithic column;

[0011] (4) Inject a phosphate solution of 20 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride into the silica gel monolithic column for 1 hour to functionalize the graphene oxide-modified silica gel monolithic column. Inject the 2 mg / mL pectinase solution into the functionalized monolithic column and react for 10 hours. Rinse with a citric acid-sodium citrate buffer solution to remove the unreacted residues to obtain a pectinase-based functionalized graphene oxide silica gel monolithic column.

[0012] (5) Prepare a citric acid-sodium citrate buffer solution as the electrophoresis buffer solution, dissolve the amino acid in the electrophoresis buffer solution, and prepare a 1 mg / mL sample solution at room temperature. After the monolithic column is equilibrated with the electrophoresis buffer solution for 5 minutes, inject the sample at 10 kV for 1 - 3 seconds, and separate the histidine enantiomers under the electrophoresis separation conditions of 5 - 15 kV.

[0013] Furthermore, the buffer concentration is 20 mM and the pH value is 4.5.

[0014] The pectinase-based functionalized graphene oxide silica gel monolithic column can achieve good separation effects on histidine, tryptophan, and arginine enantiomers, and has good separation stability.

[0015] After adopting the above technical solution, the beneficial effects achieved by the present invention are as follows: The method for chiral separation of amino acids using a pectinase-modified capillary silica monolithic column provided by the present invention is simple to operate, and further expands the application of capillary electrochromatography technology in the field of chiral separation. Brief Description of the Drawings

[0016] Figure 1 : Scanning electron micrograph of the silica monolithic column.

[0017] Figure 2 : Scanning electron micrograph of the pectinase-modified silica monolithic column.

[0018] Figure 3 : Chiral separation electrochromatogram of histidine.

[0019] Figure 4 : Influence of separation voltage on the separation of histidine enantiomers. Among them, the separation voltages are a, 5 kV; b, 10 kV; c, 15 kV.

[0020] Figure 5 : Chiral separation electrochromatogram of tryptophan.

[0021] Figure 6 : Chiral separation electrochromatogram of arginine.

[0022] Figure 7 : Chiral separation electrochromatogram of isoleucine.

[0023] Figure 8 : Chiral separation electrochromatogram of alanine.

[0024] Figure 9 : Chiral separation electrochromatogram of methionine.

[0025] Figure 10 : Chiral separation electrochromatogram of chlorpheniramine maleate.

[0026] Figure 11 : Chiral separation electrochromatogram of propranolol hydrochloride.

[0027] Figure 12 : Chiral separation electrochromatogram of nefopam hydrochloride. Detailed Embodiments

[0028] The present invention will be further described with reference to the following examples. However, it should be understood that these examples are only for illustrative purposes and should not be construed as limiting the implementation of the present invention. The pectinase in the examples was obtained by fermentation and cultivation of the Aspergillus niger strain preserved in the laboratory (the preservation number of strain CM3 is: CGMCC No, 23060), and the fermentation method is the same as that in CN202111062610.0.

[0029] Example 1

[0030] Preparation and Characterization of Capillary Silica Monolithic Column

[0031] 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, 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, stir for 45 minutes and then slowly stop, inject it into the pretreated capillary for 15 minutes, seal the end, keep it in a water bath at 40 °C for 40 hours, 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 the silica monolithic column. The structure of the silica column was characterized by scanning electron microscopy( Figure 1 ).

[0032] Preparation and Characterization of Pectinase-Modified Capillary Silica Monolithic Column

[0033] Inject 1 mol / L hydrochloric acid into the silica monolithic column for 3 hours, rinse with pure water until neutral, rinse with anhydrous methanol for 30 minutes, rinse with a toluene solution of 10% (v / v) 3-(aminopropyl)triethoxysilane for 1 hour, seal the end, react at 110 °C for 6 hours, then rinse with toluene and anhydrous methanol for 30 minutes respectively, and dry with nitrogen to obtain the aminated silica monolithic column; dilute the graphene oxide aqueous dispersion with potassium hydroxide solution, and the concentration of graphene oxide after dilution is 0.05 mg / mL and the concentration of potassium hydroxide is 0.1 mg / mL. Inject it into the aminated silica monolithic column, react at 40 °C for 2 hours, and rinse with anhydrous methanol for 30 minutes to obtain the graphene oxide-modified silica monolithic column; inject a phosphate solution (pH = 5.0) of 20 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride into the graphene oxide-modified silica monolithic column for 1 hour to functionalize the graphene oxide, and then inject 2 mg / mL pectinase solution for 10 hours, and rinse with citrate-sodium citrate buffer (pH = 4.5) to obtain the pectinase-based functionalized graphene oxide silica monolithic column.

[0034] The structure of the silica column was characterized by scanning electron microscopy( Figure 2 ).

[0035] Figure 2 Compared with Figure 1 its surface is rougher and the pore size is smaller, which is the result of the bonding of pectinase and others.

[0036] Example 2: Application of Pectinase-Modified Silica Monolithic Column

[0037] According to the method of Example 1, the prepared pectinase-modified capillary silica monolithic column was used for the separation of histidine enantiomers.

[0038] Prepare 200 mL of 20 mM citric acid-sodium citrate buffer (pH = 4.5) as the electrophoresis buffer. Dissolve the purchased histidine (Macklin (Shanghai, China)) in the electrophoresis buffer to prepare a 1 mg / mL sample solution at room temperature. By optimizing the electrophoresis separation conditions, the separation voltage (5 - 15 kV) was investigated to examine the effect of electrophoresis conditions on the separation of histidine enantiomers. As Figure 3 shown, at 25 °C, after the monolithic column was equilibrated with the electrophoresis buffer for 5 minutes, injection was performed at 5 kV for 1 second, and electrophoresis was carried out at 15 kV. This separation system had good chiral separation efficiency for histidine and the separation effect was stable, with a resolution R = 1.01.

[0039] Resolution R = 2(t R2 -t R1 ) / (W1 + W2)

[0040] Example 3

[0041] According to the method of Example 2, the separation voltage was explored. Since an increase in the operating voltage would cause an increase in electroosmotic flow, which would thus play an inhibitory role in the retention of histidine molecules by the capillary column, the column efficiency of the separation changed, further affecting the magnitude of the resolution. Therefore, for this experiment, the effect of voltage changes on its separation performance was further investigated. From Figure 4 it can be seen that as the voltage increased, the retention time of the histidine enantiomers gradually decreased. It was speculated that due to the increase in voltage, the electroosmotic flow in the column increased, resulting in a decrease in the retention of histidine. Therefore, its peak emergence time gradually shortened. As the voltage increased, the column efficiency of the peak showed a gradually increasing trend.

[0042] Example 4

[0043] Prepare a pectinase-modified capillary silica monolithic column according to the method of Example 1. The prepared pectinase-modified capillary silica monolithic column was used for the separation of tryptophan enantiomers. Prepare 200 mL of 20 mM citric acid-sodium citrate buffer (pH = 4.0) as the electrophoresis buffer. Dissolve the purchased tryptophan (Macklin (Shanghai, China)) in the electrophoresis buffer to prepare a 1 mg / mL sample solution. As Figure 5 shown, at 25 °C, after the monolithic column was equilibrated with the electrophoresis buffer for 5 minutes, injection was performed at 10 kV for 3 seconds, and electrophoresis was carried out at 15 kV. This separation system had good chiral separation efficiency for tryptophan and the separation effect was stable, with a resolution R = 1.18.

[0044] Example 5

[0045] Prepare a pectinase-modified capillary silica monolithic column according to the method of Example 1, and use the prepared pectinase-modified capillary silica monolithic column to separate arginine enantiomers. Prepare 200 mL of 20 mM citric acid-sodium citrate buffer (pH = 4.0) electrophoresis buffer. Dissolve the purchased arginine (Macklin (Shanghai, China)) in the electrophoresis buffer to prepare a 1 mg / mL sample solution. As Figure 6 shown, at 25 °C, after the monolithic column was equilibrated with the electrophoresis buffer for 5 minutes, injection was carried out at 10 kV for 3 seconds, and electrophoresis was carried out under the condition of 15 kV. This separation system has good chiral separation efficiency for arginine and the separation effect is stable, with a resolution R = 1.50.

[0046] Comparative Example 1

[0047] Prepare a pectinase-modified capillary silica monolithic column according to the method of Example 1, and use the prepared pectinase-modified capillary silica monolithic column to separate isoleucine, alanine, and methionine enantiomers respectively. Prepare 200 mL of 20 mM citric acid-sodium citrate buffer (pH = 4.0) electrophoresis buffer. Dissolve the purchased isoleucine, alanine, and methionine (Macklin (Shanghai, China)) in the electrophoresis buffer respectively to prepare a 1 mg / mL sample solution. From Figure 7 , Figure 8 , Figure 9 it can be seen that chiral separation cannot be achieved for isoleucine, alanine, and methionine.

[0048] Comparative Example 2

[0049] Prepare a pectinase-modified capillary silica monolithic column according to the method of Example 1, and use the prepared pectinase-modified capillary silica monolithic column to separate chlorpheniramine maleate, propranolol hydrochloride, and nefopam hydrochloride enantiomers respectively. Prepare 200 mL of 20 mM citric acid-sodium citrate buffer (pH = 4.0) electrophoresis buffer. Dissolve the purchased chlorpheniramine maleate, propranolol hydrochloride, and nefopam hydrochloride (Macklin (Shanghai, China)) in the electrophoresis buffer respectively to prepare a 1 mg / mL sample solution. From Figure 10 , Figure 11 , Figure 12 it can be seen that chiral separation cannot be achieved for chlorpheniramine maleate, propranolol hydrochloride, and nefopam hydrochloride.

[0050] It can be seen from the above examples that the capillary electrochromatography technology based on silica monolithic columns provides a rapid and efficient analysis platform for the separation of histidine, tryptophan, and arginine amino acid enantiomers, but the separation effects on other amino acids such as isoleucine, alanine, and methionine, as well as drugs such as chlorpheniramine maleate, propranolol hydrochloride, and nefopam hydrochloride are not obvious.

[0051] Inspired by the above-described ideal embodiments of the present invention, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of this invention. The technical scope of this 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 method for chiral separation of amino acids by a pectinase-modified capillary silica monolithic column, characterized in that: The method includes the following steps: Step 1, preparation of pectinase-modified capillary silica monolithic column: Prepare a silica monolithic column. After acidifying the silica monolithic column, inject a toluene solution of 3-(aminopropyl)triethoxysilane into the acidified silica monolithic column. After capping, react at 110°C for 6 hours, and rinse with toluene and anhydrous methanol to remove impurities to obtain an aminated silica monolithic column; ultrasonically disperse the graphene oxide aqueous solution and dilute it with a potassium hydroxide solution, inject it into the aminated silica monolithic column, react at 40°C for 2 hours after capping, wash with water until neutral, and rinse to remove impurities to obtain a graphene oxide-modified silica monolithic column; then functionalize it with a coupling agent, inject a pectinase solution into the functionalized monolithic column for reaction to obtain a pectinase-modified capillary silica monolithic column; the coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; Step 2, capillary electrophoresis detection: Apply the pectinase-modified capillary silica monolithic column obtained in Step 1 to the separation of amino acid enantiomers; The amino acid is one of histidine, tryptophan, and arginine.

2. The method for chiral separation of amino acids by the pectinase-modified capillary silica monolithic column according to claim 1, characterized in that: The injection of the pectinase solution into the functionalized monolithic column for reaction is specifically as follows: Inject a 2 mg / mL pectinase solution into the functionalized monolithic column and react for 10 hours.

3. The method for chiral separation of amino acids by the pectinase-modified capillary silica monolithic column according to claim 1, wherein: The capillary electrophoresis detection in Step 2 is specifically as follows: Prepare a citric acid-sodium citrate buffer solution as the electrophoresis buffer. Dissolve the amino acid in the electrophoresis buffer and prepare a 1 mg / mL sample solution at room temperature. After the monolithic column is equilibrated with the electrophoresis buffer for 5 minutes, inject the sample at 10 kV for 1 - 3 seconds, and separate the amino acid enantiomers under the electrophoresis separation conditions of 5 - 15 kV.

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