RU2451544C2 - Chromatography sorbent - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to materials meant for sorption or chromatographic separation. The nanodiamond sorbent contains detonation synthesis nanodiamonds immobilised on a polysaccharide matrix, an insert and specific ligands. The sorbent is obtained by treating washed sepharose with nanodiamond hydrosol followed by epoxidation, connection of the insert and ligand.

EFFECT: design of a polyfunctional affine-type nanodiamond sorbent for use in adsorption or partition chromatography, including for low-molecular weight molecules.

3 cl, 4 dwg, 2 ex

Description

The invention relates to pharmaceutical chemistry and chemistry of natural compounds and can be used for purification and fractionation of substances that are used in medicine.

Known sorbent for chromatography of proteins, and a method for its preparation using a finished commercial product of Sepharose (Sepharose. RTM. CL-6B, manufacturer Pharmacia). The method of obtaining consists in washing off sepharose, its activation and a series of successive chemical reactions leading to the addition of Ni ²⁺ , at a concentration of about 7.1 micromol / ml gel [US Pat. USA 72452, 1989].

The disadvantage of this sorbent is that it is designed for sorption of proteins having only "anchor" sites, and the method of its production is multi-stage and long.

A known sorbent and a method for its preparation on the basis of a cellulose carrier treated with potassium permanganate to form a layer of manganese dioxide on a carrier with further processing with a solution of potassium ferrocyanide in the presence of acetic acid [p. RF 2172208, IPC B01J 20/02, publ. 2001.08.20].

The disadvantage of this sorbent is that it is not stable at pH values above 8, and the cellulose matrix is unstable in organic solvents.

The closest to the technical characteristics of the prototype is a method for producing nanodiamond sorbent. A known sorbent, including the immobilization of diamond nanoparticles on a polymer matrix, where a polysaccharide matrix is used as a matrix. Sepharose 2B is used as the polysaccharide matrix. The process is carried out in a chromatographic column [RU 2352387 C1, B01J 20/02 B01J 20/281, publ. 04/20/2009 (prototype)].

The disadvantages of the sorbent produced by this method include the instability of the polysaccharide matrix to organic solvents and solutions with a pH above 8 and below 6, leaching of nanodiamond particles as a result of high solubilization with low molecular weight substances, the properties of the sorbent and the polymer matrix allow you to work only with high molecular weight - protein molecules.

The technical result of the invention is the creation of a multifunctional nanodiamond sorbent with fixed nanodiamond particles, capable of more selectively adsorbing molecules, including those with molecular weights below 1000 Da, as well as having different physicochemical properties, to perform work using various organic eluents in wide ranges of pH values .

The technical result is achieved in that in a chromatography sorbent containing diamond nanoparticles immobilized on a polymer polysaccharide matrix, it is new that the sorbent containing diamond nanoparticles is treated with an epoxy reagent (epoxy remixing), which allows the nanoparticles to be fixed and to increase the specificity of the sorbent due to subsequent immobilization at the residual end groups of the spacer (insert) and the desired specific ligand.

The technical result is also achieved by the fact that in a method for producing a sorbent for chromatography, including the immobilization of diamond nanoparticles on a polymer matrix, it is new that detonation nanodiamonds (DNDs) are sequentially immobilized on the cross-linked polysaccharide sorbent used as a matrix, and they are fixed in the matrix structure by processing an epoxy agent and additionally insert a substance and a ligand substance onto residual epoxy groups. As the polysaccharide matrix used: Sepharose Sepharose® CL 4B or Sephadex® LH-20. These matrices are stable over a wide range of pH values, as well as in organic solvents, which allows the use of the indicated type of sorbents for subsequent chemical modification.

The process of producing a sorbent with detonation nanodiamonds consists of several stages and consists in creating a contact between the nanosized hydrosol particles and the matrix, removing the nanodiamonds that are not bound to the matrix, processing the matrix with immobilized nanodiamonds with an epoxidizing reagent, and attaching the insert to residual epoxy groups and attaching a ligand substance.

The difference from the known prototype is the use of an agarose or dextran matrix stable in an environment of organic solvents and a wide range of pH values, followed by treatment with a hydrosol of detonation nanodiamonds with an epoxidizing reagent and subsequent addition of an insert and a specific ligand. A sorbent modified in this way, unlike the prototype, has specificity, and can also be used for chromatographic purification and isolation of low molecular weight organic substances of secondary plant metabolites and drugs.

Comparison of the claimed technical solutions with the prototype made it possible to establish their compliance with the criterion of "novelty."

Obtaining a sorbent with detonation nanodiamonds occurs in the following stages:

Crosslinked agarose (Sepharose® CL 4B) or hydroxypropylated dextran sorbent (Sephadex® LH-20), washed from preservatives (with distilled water at least 3 times), is transferred into a reaction flask in a swollen state, to which a solution of detonation nanodiamonds pretreated with ultrasound to form a hydrosol is added . The reaction mixture was stirred at 60 ° C for 1 hour on a rotary evaporator. After that, the gel is washed from unreacted particles on a glass filter with a three-fold volume of bidistilled water. Transferred to a reaction flask, to which an epoxy reagent, a solution of 0.5 M alkali solution and a surfactant are added, increasing the quality of the crosslinking. Epoxy activation is carried out at a temperature of 50 ° C for 1 h. Depending on the task, the following can be used as epoxidizing reagent: 1-chloro 2,3-epoxypropane (epichlorohydrin (ECG)), or diglycidyl ether 1,2-ethanediol (DHEED) )

In the next step, free epoxy groups are blocked by the addition of an insert substance, which allows increasing the distance between the ligand and the matrix surface.

At the final stage of the modification, the ligand substance is attached to the insert.

Example 1

Getting the sorbent.

A sorbent based on cross-linked agarose, Sepharose® CL 4B, was used as a carrier. The adsorbent was synthesized according to the following procedure: 50 ml of the prepared adsorbent gel was washed from preservatives (at least 3 times with distilled water). The washed gel is transferred to a reaction flask, where a solution of detonation nanodiamonds pre-treated with ultrasound to form a hydrosol is added. The reaction mixture was stirred at 60 ° C for 1 hour on a rotary evaporator. After that, the gel is washed successively from unreacted particles on a glass filter with a three-fold volume of bidistilled water, 100 ml of purified water, 50 ml of a mixture consisting of 0.5 M sodium hydroxide solution and 25% dimethyl sulfoxide (DMSO) solution. The washed gel was transferred to a round bottom flask containing a reaction mixture consisting of a solution of sodium hydroxide 0.5 M in 25% DMSO, 20 ml of diglycidyl ether 1,2-ethanediol (DHED), and 10 ml of an aqueous solution of tetrabutylammonium bromide (the latter is a catalyst for this process). The reaction mixture was stirred on a rotary evaporator at a temperature of 50 ° C for 1 h.

After that, the reaction mixture was transferred to a glass filter and washed with a solution of sodium hydroxide in DMSO, dimethylformamide and at least three times the volume of distilled water. The treated gel was transferred to a glass filter and washed successively with 80 ml of a mixture of a solution of 25% DMSO in a 0.5 M solution of sodium hydroxide, 200 ml of a saturated solution of sodium tetraborate, 40 ml of a 50% aqueous solution of DMSO and finally washed with distilled water to a negative phenolphthalein wash water sample .

The determination of residual epoxy groups was carried out titrimetrically from the formation of Bunte salts and the concentration of epoxy groups was determined at the level of 20-25 μmol / ml of packed adsorbent gel.

Salicylic acid hydrazide was used as a spacer (insert). For this, a weighed portion of salicylic acid hydrazide 0.25 g was dissolved in 8.0 ml of DMSO and 2.0 ml of a saturated sodium tetraborate solution was added. The salicylic acid hydrazide (HSC) solution thus obtained was introduced into epoxy gel. The reaction mixture was stirred on a rotary evaporator at t = 40-45 ° C for 1.5 hours. After that, the gel was washed thoroughly until the washings were negatively reacted with iron (III) ions.

The ligand was immobilized - a compound with an aromatic amino group, the drug procaine (novocaine) was carried out in the following sequence. For this, a gel washed with sodium tetraborate on a glass filter was placed in a reaction flask (cold bath) containing an equal amount of sodium tetraborate. At the same time, 0.9 g of procaine was dissolved in a 0.2 M solution of hydrochloric acid and 15 ml of a cooled solution of 5% sodium nitrite were added dropwise, the obtained procaine azosol in a volume of 25 ml in the cold, combined with stirring with a gel, controlling the pH and maintaining at a level of at least 10. The reaction mixture was left for 30 minutes at 4-5 ° C. Then, the obtained colored sorbent was washed successively with 0.1 M solutions of sodium hydroxide, hydrochloric acid, 50% DMSO solution and 50% dimethylformamide solution. After that, it is finally washed with water purified to pH 7. The synthesized gel reversibly changes its color depending on the pH of the medium. So, in an alkaline medium, the color intensity intensified to a yellow-orange color. In acidic solutions, it acquires a lemon yellow color. The resulting sorbent has the following abbreviation: Sepharose® CL 4B-DNA-GSK-Procain.

The structural properties of the obtained sorbent complement its IR spectra obtained in the diffuse reflection mode (see figure 1). A characteristic feature of the IR spectrum of the obtained sorbent is the presence of absorption bands of low intensity at 815 cm ^-1 , 952 cm ^-1 , as well as absorption bands of medium and strong intensity 1022.9 cm ^-1 , 1738 cm ^-1 . The initial carrier matrix (Sepharose CL-4B) has a different set of absorption bands.

The chromatographic properties of the obtained sorbent are explained by the separation of a model mixture consisting of the following components: hypoxanthine riboside (A), 4-chloro-N- (2 furylmethyl) -5-sulfamoylanthranilic acid (furosemide) (B), 2 [2,6-dichlorophenyl) amino] α-phenylacetic acid, sodium salt (orthophene) (B), 9α fluoro-16α-methylprednisolone (dexamethasone) (G), rutin (D), the content of substances was 0.02 g / ml. A chromatographic column was used for conventional low pressure liquid chromatography (250 × 5 mm LKB Pharmacia, Sweden). Elution was sequentially carried out with bidistilled water and a gradient of isopropyl alcohol in a 0.1 M solution of hydrochloric acid. Fractions were collected using a 0.5 ml Diafrac 002 fraction collector. The analysis of the selected fractions was carried out using an SF-2000 spectrophotometer (Russia) at key wavelengths of 240, 275, 325, 340 nm. Peak fractions were analyzed in the range of 190-410 nm with a resolution of 0.1 nm. The obtained chromatogram is presented in figure 2. The order of release of substances from the column is as follows: 1) hypoxanthine riboside (Inosine); 2) furosemide; 3) ortofen; 4) dexamethasone; 5) routine. In the control experiment, using unmodified sorbent Sepharose CL-4B, separation of the model mixture was not noted.

Example 2

Getting the sorbent.

A Sephadex Sephadex® LH-20 sorbent was used as a carrier. The adsorbent was synthesized according to the following procedure: 50 ml of the prepared adsorbent gel was washed from preservatives (bidistilled water at least 3 times). The washed gel was transferred to a reaction flask, to which a solution of detonation nanodiamonds pretreated with ultrasound to form a hydrosol was added. The reaction mixture was stirred at 50 ° C for 1 hour on a rotary evaporator. After that, the gel was sequentially washed from unreacted particles on a glass filter with a three-fold volume of bidistilled water, 100 ml of purified water, 50 ml of a mixture consisting of 0.5 M sodium hydroxide solution and 25% dimethyl sulfoxide (DMSO) solution. The washed gel was transferred into a round bottom flask containing a reaction mixture consisting of a solution of sodium hydroxide 0.5 M in 25% DMSO, 22 ml of epichlorohydrin (ECG), and 20 ml of an aqueous solution of tetrabutylammonium bromide (the latter is a catalyst for this process). The reaction mixture was stirred on a rotary evaporator at a temperature of 50 ° C for 1 h.

After that, the reaction mixture was transferred to a glass filter and washed with a solution of sodium hydroxide in dimethyl sulfoxide, dimethylformamide and at least three times the volume of distilled water. The activated gel having a yellowish tint was transferred to a glass filter and washed successively with 80 ml of a mixture of a solution of 25% DMSO in a 0.5 M solution of sodium hydroxide, 200 ml of a saturated solution of sodium tetraborate, 40 ml of a 50% aqueous solution of DMSO and finally washed with distilled water to negative phenolphthalein wash water.

Residual epoxy groups were determined titrimetrically from the formation of Bunte salts and the concentration of epoxy groups was determined at the level of 30-35 μmol / ml of packed adsorbent gel.

As a spacer (insert), n-nitrobenzhydrazide (n-NBG) was used. For this, a weighed portion of n-nitrobenzhydrazide 0.3 g was dissolved in 8.0 ml of DMSO and 2.0 ml of a saturated sodium tetraborate solution was added. The n-nitrobenzhydrazide solution thus obtained was introduced into epoxy gel. The reaction mixture was stirred on a rotary evaporator at t = 45 ° C for 1.5 hours. After that, the gel was washed thoroughly until the washings were negative with the Inman-Dintzis reagent.

The chemical modification of the n-nitro groups of the synthesized nitrogel was carried out using dithionite as a sodium reducing agent.

The ligand was immobilized - a compound with free phenolic hydroxyl (for example, a substance of the flavonoid group) dihydroquercetin on an amino gel was carried out in the following sequence. For this, a gel washed with a cooled 0.1 M hydrochloric acid solution on a glass filter was placed in a reaction flask (cold bath) containing an equal amount of a 0.1 M hydrochloric acid solution, and 15 ml of a cooled solution of 5% sodium nitrite was added. At the same time, 2.0 g of dihydroquercetin was dissolved in a 1 M sodium hydroxide solution. The resulting alkaline solution of the ligand with vigorous stirring was simultaneously introduced into a flask with a diazotized gel. The reaction mixture was left for 30 minutes at 4-5 ° C. The obtained colored sorbent was washed successively with 0.1 M solutions of sodium hydroxide, hydrochloric acid, 50% DMSO solution and 50% dimethylformamide solution. After that, it was finally washed with purified water to pH 7. The synthesized azo gel affinity adsorbent with nanodiamonds reversibly changes color depending on the pH of the medium. So, in an alkaline medium, the color intensity intensified to a yellow-orange color. In acidic solutions, it acquires a lemon yellow color. The resulting sorbent has the following abbreviation: Sephadex® LH-20-ECG-n-NBH-dihydroquercetin.

The resulting sorbent is characterized by the following feature of the IR spectra shown in figure 3. The presence of absorption bands of low intensity at 953 cm ^-1 , as well as a characteristic band of 1738 cm ^-1 . The initial carrier matrix (Sephadex LH-20) has a different set of absorption bands.

The chromatographic properties of the obtained sorbent were analyzed by separating a model mixture consisting of the components and the conditions specified in example 1. The order of release of substances was carried out in the following order: in the mode of water elution 1) dexamethasone; 2) inosine; output from the column of substances containing an aromatic moiety; 3) furosemide; 4) ortofen; 5) rutin was carried out only in the gradient elution with isopropyl alcohol. The chromatogram of the separation shown in figure 4.

Claims (3)

1. The chromatographic sorbent containing a cross-linked polysaccharide matrix with nanodiamonds immobilized on it obtained by detonation synthesis, characterized in that it contains Sepharose CL 4B or Sephadex LH-20 as a matrix, while the matrix containing nanodiamonds is epoxidized and free epoxy groups are blocked by the introduction of an insert with a ligand attached to it. 2. The sorbent according to claim 1, characterized in that the insert is made on the basis of salicylic acid hydrazide, while the attached ligand is procaine (novacaine). 3. The sorbent according to claim 1, characterized in that the insert is made on the basis of n-nitrobenzhydrazide, wherein the attached ligand is flavanoids, for example dihydroquercytin.

Images