CN111707771A

CN111707771A - Chiral capillary electrochromatography open tubular column based on gold nano modification, preparation method and application

Info

Publication number: CN111707771A
Application number: CN202010592097.5A
Authority: CN
Inventors: 江沛; 赵世媛; 崔昌萌; 徐鹏飞; 王长水
Original assignee: JINING NO1 PEOPLE'S HOSPITAL
Current assignee: JINING NO1 PEOPLE'S HOSPITAL
Priority date: 2020-06-25
Filing date: 2020-06-25
Publication date: 2020-09-25
Anticipated expiration: 2040-06-25
Also published as: CN111707771B

Abstract

The invention relates to a chiral capillary electrochromatography open tubular column based on gold nano modification, a preparation method and application, wherein Glycidyl Methacrylate (GMA) tentacle type polymer coating is bonded on the inner wall of a capillary, the amino of reduced Glutathione (GSH) is utilized to carry out alkaline ring opening on the epoxy group of GMA under alkaline condition, GSH is successfully synthesized on the capillary column, the property that the mercapto group of the glutathione can generate Au-S bond with gold nano is utilized, the gold nano is combined on the capillary column, the gold nano is successfully introduced into the capillary column and fixed phase, and a chiral resolution nano particle system based on carboxymethyl-beta-cyclodextrin is constructed. Experiments prove that compared with an empty tubular column without modified gold nano, the prepared capillary open tubular column has greatly improved separation degree and selectivity for three rocolol drugs (atenolol, sotalol and bisoprolol), and achieves the purpose of base line separation.

Description

Chiral capillary electrochromatography open tubular column based on gold nano modification, preparation method and application

Technical Field

The invention belongs to the technical field of capillary column preparation, and relates to a gold nano-modification-based chiral capillary electrochromatography open tubular column, a preparation method and application.

Background

More than half of the 1850 drugs currently used in clinic are chiral drugs, and most are put on the market in a racemic form. It has been found that drug enantiomers often have different pharmacological, pharmacokinetic and toxic effects. Such as thalidomide, which has sedative effects, its R form is used to treat pregnancy in pregnant women, while its S form causes fetal abnormalities. Limited to the technical difficulties of separation analysis, most of the drug enantiomers are sold in the form of racemic bodies, which is extremely dangerous.

Chromatography is currently the most efficient method used in chiral separation detection methods. Among them, capillary electrochromatography is used as a high-efficiency and fast chromatographic micro-separation technique emerging in recent years, and has been applied to chiral separation and detection. The capillary electrochromatography separation technique is an electric separation mode in which a capillary is filled or a chromatographic stationary phase is coated and bonded on the inner wall of the capillary, a mobile phase is pushed by electroosmotic flow, and neutral or charged sample molecules are separated and analyzed according to the difference of adsorption, distribution equilibrium constant and electrophoresis rate between the chromatographic stationary phase and the mobile phase. The preparation of the capillary electrochromatography column is an important link for the separation and analysis of the capillary electrochromatography, because the service life, the column efficiency, the column repeatability and the like of the column are the most key and basic indexes in the practical application of the electrochromatography.

However, the application range of the capillary electrochromatography column is limited due to the limitation of the preparation technology of the capillary electrochromatography column (which is divided into a packed column, an integral column and an open tubular column according to different stationary phase forms). The open tubular column has the advantages of simple preparation process, good permeability of the column, difficult generation of bubbles and vortex diffusion, higher column efficiency compared with a packed column, no need of firing a plunger and capability of realizing effective separation and analysis of chiral substances in a relatively simple and economic mode. However, the open tubular column prepared by adopting a common coating mode has the advantages that the stationary phase is easy to fall off and the service life is short; the open tubular column prepared by adopting the bonding mode can overcome the defects and shortcomings in the aspect that the stationary phase is firmly bonded on the inner wall of the capillary tube through a chemical bond. Therefore, the design and preparation of various bonded open tubular columns has become a hotspot explored in the art.

The particle size of the gold nano is mainly concentrated between 1 nm and 100nm, and the gold nano has the quantum effect, the surface effect and the quantum tunnel effect, so that the gold nano is more and more concerned and applied. The gold nano-particles have various forms, such as spherical nano-particles, gold nanorods, gold nanoclusters and the like. The method is mainly applied to the fields of biomedicine, chemical environment, photoelectricity, food safety detection and the like. In a capillary electrophoresis splitting system, gold nanoparticles are commonly used for a capillary stationary phase to improve the separation performance of an analyte due to the advantages of easy modification, good biocompatibility, controllable particle size and form and the like. The Au nano-particles can form Au-NH with amine or thiol₂A bond or an Au-S bond, and thus gold nanoparticles can be easily bonded to the open-tube stationary phase.

Glycidyl methacrylate, a tentacle-type compound with epoxy groups, was grafted mainly onto the inner wall of silanized capillaries to increase phase and binding capacity. The enhanced binding capacity of these grafted polymers apparently comes from tentacles extending away from the inner surface, which provide sufficient distance to allow nanoparticles to bind in multiple layers, thereby increasing the binding capacity of the capillary inner wall stationary phase. The epoxy group carried by the GMA surface also facilitates bonding of a variety of functional groups by simple ring-opening reactions.

Glutathione is an important component of antioxidation in human bodies, is an important antioxidant and free radical scavenger in vivo, is composed of three amino acid micromolecules of glutamic acid, cysteine and glycine, and belongs to chiral short peptide.

At present, no relevant report for introducing gold nano-particles, glycidyl methacrylate and glutathione into a chiral capillary electrochromatography open tubular column for preparation exists.

Disclosure of Invention

In view of the above, the present invention aims to provide a gold nanoparticle modified chiral capillary electrochromatography open tubular column, a preparation method and an application thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a preparation method of a chiral capillary electrochromatography open tubular column based on gold nano modification comprises the following specific steps:

(1) firstly, silanizing the inner surface of a hollow capillary column to obtain a silanized capillary column;

(2) then, glycidyl methacrylate reacts with the inner surface of the silanized capillary column to obtain a GMA column;

(3) reacting the inner surface of the GMA column with glutathione to obtain a GSH-GMA column;

(4) and finally, modifying the GSH-GMA column by using gold nanoparticles to obtain the chiral capillary electrochromatography open tubular column (Au-GSH-GMA column) based on the gold nanoparticles modification.

Preferably, the specific method of step (1) is: activating a capillary column, then flushing a gamma-MAPS methanol solution with the volume concentration of 50% into the activated capillary column, sealing the end, placing the capillary column in a water bath kettle at 55 ℃ for reacting for 10-14 hours, taking out the capillary column, flushing methanol to remove unreacted gamma-MAPS, and flushing nitrogen for later use.

Further preferably, the specific method of the activation treatment is: washing the capillary column with 1mol/L NaOH solution for 1 hour, washing the capillary column with water to be neutral, washing the capillary column with 1mol/L HCl for 30 minutes, washing the capillary column with water to be neutral, washing the capillary column with methanol for 15 minutes, blowing the capillary column with nitrogen for drying, activating the capillary column at a high temperature of 100 ℃ for 1 hour, and placing the capillary column in a refrigerator at a temperature of 4 ℃ for later use.

Preferably, the specific method of step (2) is: dissolving glycidyl methacrylate in toluene to prepare a solution with the mass concentration of 10%, adding 0.75mg/mL AIBN serving as a thermal initiator to obtain a mixed solution, flushing the mixed solution into a silanized capillary column, carrying out end capping, carrying out water bath reaction at 80 ℃ for 10-14 hours, and alternately flushing the column with methanol and water to remove unreacted reactants.

Preferably, the specific method of step (3) is: dissolving glutathione in 50mmol/L phosphate buffer solution to prepare 1mol/L solution, then adjusting pH =8.0 to obtain GSH solution, then flushing the GSH solution into GMA column, sealing the end, reacting for 10-14 hours at 40 ℃, flushing to remove unreacted GSH; finally, the capillary column was washed with 50mmol/L of TCEP solution (tris (2-chloroethyl) phosphate solution) to cleave any disulfide bonds that may have formed, and then washed with water to remove unreacted TCEP.

Preferably, the specific method of step (4) is: and (3) flushing the freshly prepared gold nano-colloid into the GSH-GMA column until the whole capillary column becomes red and red liquid drops emerge from the top of the column, and flushing unreacted gold nano-colloid by using water to obtain the Au-GSH-GMA column.

Further preferably, the preparation method of the gold nanocolloid is as follows: firstly, chloroauric acid (HAuCl) with the mass concentration of 0.01 percent₄) Heating the aqueous solution to boiling, adding a trisodium citrate aqueous solution with the mass concentration of 1% while stirring, continuing to heat and boil for 15 minutes until the color of the reaction solution changes from light yellow to gray, then to black, finally to be stable to red, stopping the reaction, cooling to room temperature (25 ℃), adding distilled water to restore the original volume, transferring to a refrigerator, and keeping at 4 ℃ for later use.

Even more preferably, HAuCl₄The volume ratio of the aqueous solution to the trisodium citrate aqueous solution is 100: 0.7.

2. the chiral capillary electrochromatography open tubular column based on gold nano modification is obtained by the preparation method.

3. The application of the gold nano-modified chiral capillary electrochromatography open tubular column in chiral drug separation is provided.

Preferably, the chiral drug is a rochol drug, and further preferably atenolol, sotalol or bisoprolol.

Preferably, carboxymethyl- β -cyclodextrin is used as chiral selector, with a mass concentration of more preferably 2.0%.

4. The chiral resolution system based on the chiral capillary electrochromatography open tubular column comprises the chiral capillary electrochromatography open tubular column and running buffer solution, wherein the running buffer solution is prepared by taking carboxymethyl-beta-cyclodextrin as a chiral selector, the pH =7, and the mass concentration of the contained carboxymethyl-beta-cyclodextrin is 2.0%.

5. The chiral drug separation method based on the open tubular column of the chiral capillary electrochromatography comprises the following specific steps:

(A) firstly, dissolving a chiral drug by using a solvent to prepare a sample solution with the concentration of 1 mg/mL;

(B) then dissolving a chiral selector in a 40mmol/L Tris solution, and dropwise adding a phosphoric acid solution with the mass concentration of 30% to the required pH value to obtain an operation buffer solution;

(C) and (3) utilizing the chiral capillary electrochromatography open tubular column, adopting the running buffer solution obtained in the step (B), injecting the sample solution obtained in the step (A), and performing capillary electrophoresis to realize the separation of chiral drugs.

Preferably, in step (a), the chiral drug is a rocolol drug, and more preferably atenolol, sotalol or bisoprolol.

Preferably, in step (a), the solvent is water or methanol.

Preferably, thiourea is further added as a neutral marker to the sample solution obtained in the step (A), and the injection concentration is 1 mg/ml.

Preferably, in step (B), the chiral selector is carboxymethyl- β -cyclodextrin, and the concentration by mass is further preferably 2.0%.

Preferably, methanol is also added as an organic additive in step (B), and the volume percentage of methanol in the running buffer is further preferably 20%.

Preferably, in step (B), the pH of the running buffer is adjusted to 7.

Preferably, in step (C), the operating voltage for capillary electrophoresis is 16 kV.

The invention has the beneficial effects that:

according to the invention, a GMA tentacle type polymer coating is bonded on the inner wall of a capillary, the amino group of reduced glutathione is used for carrying out alkaline ring opening on the epoxy group of GMA under alkaline conditions, the reduced glutathione is successfully synthesized on a capillary column, the property that the mercapto group of the glutathione can generate Au-S bond with gold nanoparticles is utilized, the gold nanoparticles are combined on the capillary column, the gold nanoparticles are successfully introduced into a capillary fixed phase, and a chiral resolution system based on carboxymethyl-beta-cyclodextrin is constructed. Experiments prove that compared with an empty tubular column without modified gold nano, the prepared capillary open tubular column has greatly improved separation degree and selectivity for three rocolol drugs (atenolol, sotalol and bisoprolol), and achieves the purpose of base line separation. The experiment also inspects the conditions influencing the resolution, the optimal pH value of the buffer solution is 7.0, the mass fraction of the concentration of the added carboxymethyl-beta-cyclodextrin is 2.0%, the volume ratio of the optimal methanol concentration is 20%, and the optimal operating voltage is 16 kV.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a transmission electron micrograph of gold nanoparticles;

FIG. 2 is a graph of the distribution of the particle size of gold nanoparticles;

FIG. 3 is a gold nano-modified capillary column preparation process;

FIG. 4 is a scanning electron microscope image of capillary columns at different stages of synthesis, wherein a is a bare quartz capillary wall, b is a GMA column, c is a GSH-GMA column, and d is an Au-GSH-GMA column;

FIG. 5 is a graph of the effect of electroosmotic flow on different capillary columns at different buffer pH conditions;

FIG. 6 shows the chemical structures of three drugs of the genus rocolol, wherein a is atenolol, b is sotalol, and c is bisoprolol;

FIG. 7 is an electrophoresis separation diagram of atenolol, sotalol and bisoprolol in an Au-GSH-GMA column or an empty column, wherein a is atenolol in the Au-GSH-GMA column, b is sotalol in the Au-GSH-GMA column, c is bisoprolol in the Au-GSH-GMA column, d is atenolol in the empty column, e is sotalol in the empty column, and f is bisoprolol in the empty column;

FIG. 8 is a graph of the effect of carboxymethyl- β -cyclodextrin concentration on drug separation, where a is atenolol, b is sotalol, and c is bisoprolol;

FIG. 9 is a graph of the effect of methanol concentration on drug separation, where a is atenolol, b is sotalol, and c is bisoprolol;

FIG. 10 is a graph of the effect of operating voltage on the three drug separations and selection factors, where a is the effect of different voltages on the three drug separations and b is the effect of different voltages on the three drug selection factors.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Drugs and reagents

3- (methacryloyloxy) propyltrimethoxysilane (. gamma. -MAPS, 97%), glutathione (reduced), glycidyl methacrylate (GMA, 98%), gold (III) chloride hydrate, 2, 2-azobisisobutyronitrile (AIBN, 98%), Tris (2-chloroethyl) phosphate (TCEP), Tris (hydroxymethyl) aminomethane (Tris) were obtained from Shanghai Arlatin Biotechnology, Inc., trisodium citrate was obtained from Shanghai Mecline Biotechnology, Inc., Carboxymethyl-beta-cyclodextrin (CM-beta-CD, Mw ≈ 1320) was obtained from Shandong Zimbo Bo Qian Fine chemical, Atenolol (ATE), Bisoprolol (BIS), sotalol (SOT) is provided by food and drug testing in Jiangsu province, and all chiral drugs are racemates.

Methanol, phosphoric acid, hydrochloric acid, potassium hydroxide, and sodium hydroxide were purchased from Nanjing chemical reagents, Inc.; the experimental water was double distilled water.

Apparatus and method

Capillary tube: the 50-micron I.D. hollow tube fused quartz capillary tube is purchased from Jiangxi chromatographic device Limited, Yongnian, Hebei province, and has the total length of 33cm and the effective length of 24.5 cm.

The instrument comprises the following steps: agilent^3DCE (G7100A, Agilent Technologies, Waldbronn, Germany), DAD detector (190 nm-600 nm), electrophoresis system control, data acquisition, and spectrogram analysis were all accomplished using the software package AgilentChemStation; pHS-3C type precision pH meter (Shanghai Lei magnetic Instrument factory); electronic balance (sidoris scientific instruments ltd); s-4800 field emission scanning electron microscope (Hitachi High-Technologies corporation, Japan); tecnai 12 transmission electron microscope (Philips company, Holland); malvern laser particle sizer (MLPSA, Zetasizer Nano S).

Pretreatment of the new capillary: according to 1mol/L of Na respectivelyOH、0.1mol/L NaOH、H₂Sequentially washing the quartz capillary column for 30min respectively by using the O; before the experiment every day, H is used₂Flushing for 10 minutes, and then flushing to a stable baseline by using a buffer solution; h is respectively used between two sample introduction₂And flushing the capillary for 2min by O, and flushing the capillary for 3min by buffer until the base line is stable.

Electrophoresis sample introduction conditions are as follows: adopting a pressure sample injection mode (50 mbar multiplied by 2 s), injecting a sample from the positive electrode, and detecting the negative electrode. The operating voltage range is 8-20KV, and the capillary column temperature control device is stabilized at 20 ℃.

Example 1:

preparation of gold nanoparticles

Taking a clean round-bottom flask, preparing 100ml of HAuCl with the mass concentration of 0.01 percent₄Heating the aqueous solution until the solution boils, adding 0.7ml of trisodium citrate aqueous solution with the mass concentration of 1% under the condition of magnetic stirring, continuously heating, boiling and reacting for 15 minutes, wherein the color of the solution can be observed to change from light yellow to gray, then to black, and finally to be stable to red. The reaction was stopped, cooled to room temperature, and distilled water was added to return to the original volume. Placing in a refrigerator, and keeping at 4 ℃ for later use.

The size and shape of the synthesized gold nano-particles are characterized, the particle size of the synthesized gold nano-particles is measured by a Malvern laser particle size analyzer, and the report shows that the average particle size of the synthesized gold nano-particles is about 40nm, and the particle size distribution is concentrated. By using a transmission electron microscope to observe the morphological characteristics, the prepared gold nanoparticles are uniformly dispersed spherical particles, have good shapes and the particle size of about 20nm as can be visually seen from figures 1 and 2.

Example 2:

preparation of gold nano-coating column

Capillary column activation: firstly, washing a capillary column for 1 hour by using 1mol/L NaOH, washing the capillary column to be neutral by water, then washing the capillary column for 30 minutes by using 1mol/L HCl, washing the capillary column to be neutral by water, washing the capillary column for 15 minutes by using methanol, drying the capillary column by using nitrogen, activating the capillary column for 1 hour at the high temperature of 100 ℃, and placing the capillary column in a refrigerator at the temperature of 4 ℃ for later use.

The first step is as follows: silanization of the inner surface of the capillary. Preparing 50% (V/V) gamma-MAPS methanol solution, flushing the solution into the activated capillary column, sealing the end, and placing the capillary column in a 55 ℃ water bath kettle for reaction for 10 to 14 hours. Taken out, flushed with methanol to remove unreacted gamma-MAPS and flushed with nitrogen for use.

The second step is that: and synthesizing a GMA polymerization layer. 10% (w/w) GMA was prepared in toluene, about 0.75mg/ml AIBN was added as a thermal initiator, which was flushed into a silanized capillary column, capped, reacted in a water bath at 80 ℃ for 10-14 hours, and the column was rinsed with methanol and water to remove unreacted reactants.

The third step: and bonding glutathione. Glutathione solution of 1mol/L is prepared and dissolved in phosphate buffer of 50mmol/L, and the pH is adjusted to 8.0. And (3) the prepared GSH solution is flushed into a GMA column, end capping is carried out, and the reaction lasts for 10 to 14 hours at the temperature of 40 ℃. Unreacted GSH was removed by flushing. A50 mmol/L TCEP solution was prepared to wash the capillary column to break potential disulfide bonds and washed to remove unreacted TCEP.

The fourth step: fixing the gold nano-meter. And (3) immediately flushing the prepared gold nano colloid into the capillary column synthesized in the previous step until the whole capillary column becomes red and red liquid drops emerge at the top of the column, flushing unreacted gold nano by using water, and finally obtaining the column which is called as the Au-GSH-GMA column. The specific reaction steps are schematically shown in FIG. 3.

Characterization of the synthesized gold nanopillars, it can be seen visually from a in fig. 4 that the inner surface of the capillary blank after activation is smooth, and that a relatively thick polymer coating (b in fig. 4) is produced on the surface after bonding of the GMA coating, with more pronounced wrinkling. In FIG. 4, c is the coating after glutathione bonding, the magnification is larger than that of the empty column and GMA column, and no significant change is observed at this magnification due to the smaller size of the bonded GSH short peptide, and the polymer has a fine particle polymer on the surface. In fig. 4, d is a scanned image after fixing the gold nanoparticles, and spherical protrusions, which may be the products of gold nanoparticle polymerization, can be clearly seen from the scanned image. The success of the gold nano-coating column preparation can be further illustrated by the result picture of a scanning electron microscope.

The size of capillary electrophoresis electroosmotic flow depends on the dissociation condition of groups carried by the stationary phase of the inner wall of the capillary, and the change of the groups on the inner wall of the capillary can be verified from the side surface by measuring the size of the electroosmotic flow. The pH in the buffer is one of the main factors affecting electroosmotic flow, and the experiment characterizes the synthesis of the column by measuring the electroosmotic flow of the capillary column in the pH range of 4-9. As can be seen in fig. 5, the capillary column electroosmotic flow of empty tubes varies greatly with pH change because of the increasing dissociation of the silicon hydroxyl groups on the inner surface of the column with increasing pH. Capillary electroosmotic flow became small after bonding of the GMA coating because the synthesized GMA coating masked most of the silicon hydroxyl groups, whereas epoxy groups were difficult to dissociate under the conditions tested, so electroosmotic flow was small and not significantly changed in the pH range of 4-9. After bonding glutathione, capillary electroosmosis flow is increased because glutathione contains carboxyl groups, which can dissociate to produce stronger carboxyl negative ions, so electroosmosis flow is increased in the measured range. After gold nano-bonding, the electroosmotic flow of the inner surface of the capillary is reduced, and presumably after the gold nano-particles are introduced, because the diameters of the gold nano-particles are larger, partial carboxyl groups are shielded, so that the electroosmotic flow is reduced. The successful synthesis of Au-GSH-GMA column can be proved by the characterization of electroosmotic flow.

Example 3:

preparation of sample solution and running buffer solution

Sample solution: taking appropriate amount of the above medicines, dissolving with water or methanol to obtain sample solution with concentration of 1mg/ml, wherein the sample injection concentration is 1mg/ml, and thiourea is used as neutral marker.

Preparing a running buffer solution: weighing a proper amount of a chiral selector CM-beta-CD according to required conditions, dissolving the chiral selector CM-beta-CD in a Tris solution of 40mmol/L, dropwise adding phosphoric acid with volume concentration of 30% to adjust to a required pH value, and ultrasonically processing all sample solutions and running buffer solution through a 0.45 mu m microporous filter membrane for use.

Calculating the formula:

the calculation formula of the electroosmotic flow is as follows:

μ_eof= ( L × l ) / ( V × t₀)

wherein mu_eofElectroosmotic flow, L, total length and effective length of capillary; v is an operating voltage; t is t₀Is a neutral marker, i.e. the time of thiourea peak.

The chiral drug separation and selection factor calculation formula is:

R_s= 2 ( t₂- t₁) / ( W₁+ W₂)

α = t₂/ t₁

wherein R is_sFor degree of separation, α is a selection factor, t_1，t₂Represents migration time of two enantiomers of racemate drug; w_1，W₂Peak widths for the two enantiomers respectively.

Example 4:

examination of separation Effect

1. Reproducibility and stability

To investigate the reproducibility and stability of the open columns prepared, we assessed the relative standard deviation of the time to peak of thiourea in an Au-GSH-GMA capillary column by determining it several times. The intra-day precision was evaluated by measuring the RSD of the time of the thiourea peak for six consecutive needles within one day; the precision in the daytime is measured by measuring the RSD of the peak-off time of thiourea in an Au-GSH-GMA capillary column in six days; the inter-column precision was evaluated by measuring the RSD of the thiourea migration time of the different 4 batches of synthesized columns. The experimental result shows that the prepared capillary column has good reproducibility, the daily precision is less than 1.7%, the daytime precision is less than 2.2%, and the inter-column precision is less than 6.3%.

The prepared column is placed at a low temperature of 4 ℃ for 3 months, the migration time of thiourea before and after the placement is measured by using the same electrophoresis condition, and the RSD value of the migration time of the thiourea is found to be less than 3.4% through comparison, which shows that the prepared open tubular column has good stability and the service life is at least 3 months.

2. Construction of Au-GSH-GMA open tubular column chiral resolution system based on carboxymethyl-beta-cyclodextrin

Three rocolol drugs (atenolol, sotalol and bisoprolol, the chemical structural formulas of which are respectively shown as a, b and c in figure 6) are selected as template drugs, and the change of the resolution result is considered to show the modification effect of the prepared open tubular column on the carboxymethyl-beta-cyclodextrin, namely the chiral selective agent. It can be seen from table 3 and fig. 7 that CM- β -CD fails to achieve baseline separation for all three drugs in the activated empty column, while in the prepared gold nano-coated column, the separation degree and selectivity factor of all three drugs are significantly improved, the column efficiency is also significantly improved, and the purpose of baseline separation is achieved. The open tubular column system synthesized by the method can effectively improve the selectivity of CM-beta-CD enantiomer. Experiments a series of investigations were carried out on the conditions affecting the chiral resolution of each drug, including the pH of the buffer, the concentration of the chiral selector, the separation voltage and the concentration of the organic additives, and the optimal separation conditions were found therefrom.

The reason for testing the chiral selectivity of the open tubular column is probably because the gold nanoparticles can generate electrostatic interaction and hydrophobic interaction with the drug molecules and the chiral selector, so that the retention of the drug is enhanced, the action time of the drug and the chiral selector is prolonged, and the separation capability of the chiral selector on the enantiomer is improved.

TABLE 1 resolution results of drugs in empty column and Au-GSH-GMA column

Electrophoresis conditions 40mM Tris-H₃PO₄The buffer solution comprises 20% of methanol by volume, 2.0% of carboxymethyl- β cyclodextrin by mass is added into the buffer solution, the pH =7 of the buffer solution, the application voltage =16KV, the capillary temperature is 20 ℃, and the detection wavelength is 230 nm.

3. Optimizing separation conditions

3.1 optimization of buffer pH

In the process of chiral resolution of enantiomer electrophoresis, pH is an important condition influencing the separation effect. On one hand, the method can influence the dissociation state of the inner wall groups of the capillary, thereby changing the size of electroosmotic flow, further changing the action time of the medicament and the chiral selector and changing the resolution effect; on the other hand, the degree of protonation of the basic drug and the state of dissociation of the carboxyl groups contained in the chiral selector can be influenced, changing electrostatic forces, hydrogen bonds and other interactions between the two, thus changing the separation of the enantiomers.

The separation and selection factor of 3 lols was examined for changes in buffer pH from 6.0 to 8.0, as can be seen in table 2: the time of the drug to peak is gradually shortened along with the increase of the pH; the enantiomeric separation and the selection factor show a tendency to increase first and then decrease; at pH 7, the enantiomeric separation is maximized. Therefore, pH 7 was selected as the optimal pH for the buffer. (electrophoresis conditions: 40mM Tris-H₃PO₄The buffer solution contains 20% of methanol by volume fraction, 2.0% of carboxymethyl- β cyclodextrin by mass fraction is added into the buffer solution, the application voltage =16KV, the capillary temperature is 20 ℃, and the detection wavelength is 230 nm)

TABLE 2 influence of buffer pH on enantiomeric separation and alpha value

3.2 Effect of chiral selector concentration on resolution

The addition amount of carboxymethyl- β -cyclodextrin directly affects the contact probability of the drug enantiomer with the chiral selector in the resolution process, along with the increase of the chiral selector, the contact probability of the enantiomer with the chiral selector is increased, the complex formation trend is also increased, the chiral recognition effect is enhanced, the retention time and the separation degree are naturally increased, the experiment researches the influence of the change of the mass fraction of carboxymethyl- β -cyclodextrin from 1.0-3.0% on the separation degree, as shown in figure 8 (electrophoresis condition: 40mM Tris-H)₃PO₄The buffer solution contains 20% of methanol by volume fraction, the pH =7 of the buffer solution, the application voltage =16KV, the capillary temperature is 20 ℃, the detection wavelength is 230 nm), the separation effect is better and better along with the increase of the concentration of CM- β -CD, when the concentration is 2.0%, the enantiomer separation degree reaches the maximum, when the concentration is further increased, the interaction of CM- β -CD and the medicament is saturated, the migration time is too long, the peak is widened, the column efficiency is reduced, and the separation degree of the medicament shows a trend of reductionAnd (3) a component.

3.3 Effect of organic additive concentration on drug resolution

The research on the concentration of the organic additive has important influence on the improvement of resolution, and the introduction of the organic additive can inhibit electroosmotic flow and increase the retention time of the medicament on one hand, so that the effect of the chiral selector on the medicament is increased; on one hand, the elution capacity of the buffer solution can be increased, the adsorption effect of the tube wall on the alkaline drug can be reduced, and the peak shape can be improved; on the other hand, the effective charges carried by the drug molecules and the chiral selector can be changed by adding the organic reagent, so that the interaction condition of the drug molecules and the chiral selector is changed.

Experimental examination of the effect on separation at different methanol concentrations (0%, 10%, 20%, 30%, 40%) was carried out, and FIG. 9 (electrophoresis conditions: 40mM Tris-H) was observed₃PO₄The buffer solution, wherein carboxymethyl- β cyclodextrin with the mass fraction of 2.0% is added into the buffer solution, the pH =7, the application voltage =16KV, the capillary temperature is 20 ℃, and the detection wavelength is 230 nm), shows that the separation degree and the α value of the chiral resolution of the medicament show the trend of increasing and then decreasing along with the addition of methanol, and the resolution effect is best when the methanol concentration is 20%, so the optimal methanol concentration is selected to be 20%.

3.4 Effect of operating Voltage on chiral separation

The voltage mainly influences the column effect and the migration time in the drug splitting process, the increase of the voltage can improve the column effect, improve the peak pattern and improve the separation degree, but the overhigh voltage can generate larger current, thereby generating joule heat and deteriorating the drug splitting result, so that the satisfactory separation effect can be generated by applying the high voltage as far as possible on the premise of generating less joule heat. The results of experiments in which the drug separation effect was examined under the conditions of operating voltages of 8KV, 12KV, 16KV and 20KV were shown in FIG. 10 (electrophoresis conditions: 40mM Tris-H)₃PO₄The buffer solution contains 20% of methanol by volume fraction, 2.0% of carboxymethyl- β cyclodextrin by mass fraction is added into the buffer solution, the pH =7 of the buffer solution, the capillary temperature is 20 ℃, and the detection wavelength is 230 nm), so that the separation degree of the three rochell drugs reaches the maximum at 16KV, and therefore 16KV is selected as the optimal separation voltage.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A preparation method of a chiral capillary electrochromatography open tubular column based on gold nanometer modification is characterized by comprising the following specific steps:

(4) and finally, modifying the GSH-GMA column by using gold nanoparticles to obtain the chiral capillary electrochromatography open tubular column based on the gold nanoparticles modification.

2. The method for preparing the compound according to claim 1, wherein the specific method of the step (1) is as follows: activating a capillary column, then flushing a gamma-MAPS methanol solution with the volume concentration of 50% into the activated capillary column, sealing the end, placing the capillary column in a water bath kettle at 55 ℃ for reacting for 10 to 14 hours, taking out the capillary column, flushing methanol to remove unreacted gamma-MAPS (3- (methacryloyloxy) propyl trimethoxy silane), and flushing nitrogen for later use.

3. The method for preparing the compound of claim 1, wherein the specific method of the step (2) is as follows: dissolving glycidyl methacrylate in toluene to prepare a solution with the mass concentration of 10%, adding 0.75mg/ml of LAIBN (2, 2-azobisisobutyronitrile) as a thermal initiator to obtain a mixed solution, flushing the mixed solution into a silanized capillary column, sealing, reacting in a water bath at 80 ℃ for 10-14 hours, and alternately flushing the column with methanol and water to remove unreacted reactants.

4. The method for preparing the compound of claim 1, wherein the specific method of the step (3) is as follows: dissolving glutathione in 50mmol/L phosphate buffer solution to prepare 1mol/L solution, adjusting pH to 8.0 to obtain GSH solution, introducing the GSH solution into GMA column, capping, reacting at 40 deg.C for 10-14 hr, and removing unreacted GSH by flushing; finally, the capillary column was washed with 50mmol/L of TCEP solution to break potential disulfide bonds, and then washed with water to remove unreacted TCEP.

5. The method for preparing the compound of claim 1, wherein the specific method of the step (4) is as follows: and (3) flushing the freshly prepared gold nano-colloid into the GSH-GMA column until the whole capillary column becomes red and red liquid drops emerge from the top of the column, and flushing unreacted gold nano-colloid by using water to obtain the Au-GSH-GMA column.

6. A chiral capillary electrochromatography open tubular column based on gold nano-modification, which is obtained by the preparation method of any one of claims 1-5.

7. The use of a gold nanoparticle modified chiral capillary electrochromatography open tubular column of claim 6 for chiral drug separation.

8. A chiral resolution system based on the chiral capillary electrochromatography open column of claim 6, comprising the chiral capillary electrochromatography open column of claim 6 and a running buffer solution, wherein the running buffer solution is prepared by taking carboxymethyl-beta-cyclodextrin as a chiral selector, the pH value of the running buffer solution is 7, and the mass concentration of the contained carboxymethyl-beta-cyclodextrin is 2.0%.

9. A chiral drug separation method realized based on the chiral capillary electrochromatography open tubular column of claim 6 is characterized by comprising the following steps:

(B) then dissolving a chiral selector in 40mmol/L Tris solution, and dropwise adding a phosphoric acid solution with the volume concentration of 30% to obtain an operation buffer solution;

(C) the chiral capillary electrochromatography open tubular column of claim 6, wherein the separation of chiral drugs can be realized by sampling the sample solution obtained in step (A) with the running buffer solution obtained in step (B) and performing capillary electrophoresis.