SU1736982A1 - Method of sorbent production - Google Patents

Abstract

The invention relates to the preparation of specific sorbents or carriers for biotechnology. The method includes processing the carrier in a medium with carbohydrates, while the carrier is silicon-containing fine silica aerosil, and the treatment is carried out in vacuum at a ratio of silica to 100 parts 1-1.5 parts carbohydrate with gradual heating from 20 to 240-250 ° C for 3-4 hours. 1 Il.

Description

The invention relates to chemistry, in particular to methods for modifying silicon-containing materials, for example silica, by linking them with carbohydrates, and can be used to obtain specific adsorbents or carriers, in particular for affinity chromatography, in biotechnology.

At present, due to the development of biotechnology, the need for specific adsorbents with selective affinity for biomolecules of various sizes is increasing, which makes it possible to separate a mixture of biomaterials that are complex in composition.

However, the methods for obtaining specific adsorbents suitable for working with biological media are rather complicated, and the resulting sorbents are fragile, short-lived and inefficient in operation.

A known method for producing polyol-modified silochrome sorbents. with grafted carbohydrates. To obtain a carbohydrate-modified sorbent, it is necessary to pre-aminate the silicon-containing material — silochromic 2-amino-4,6-dichlorotriazine, then in the solution containing 10% carbohydrate (for example, xylitol, glucite, lures, glucose), the particles of the aminated silochrome are mixed. In this case, the chlorine atoms are replaced by polyols due to covalent bonds. The sorbent thus obtained is used to isolate and purify glucose isomerase using affinity chromatography.

However, this modification method allows to obtain a highly specific sorbent, which breaks down in aqueous media and under mechanical effects. In addition, the process of obtaining it is multistage and rather laborious.

The aim of the invention is to provide the possibility of obtaining a biospecific adsorbent for cryopreservation of sperm.

This goal is achieved by that the modification of silicon-containing materials with carbohydrates involves treating the substrate in an environment containing, for example, a polyol, while using fine-dispersed silica as the basis with a specific surface area of 270–390 m / g, and

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the treatment is carried out in vacuum at a ratio of 100 parts of silica to 1-1.5 parts of carbohydrate with gradual heating to 240-250 ° C for 3-4 hours.

Mannitol and inositol are used as polyols.

The drawing shows spectrophotometric analysis data confirming the preparation of modified fine silica.

PRI me R 1. Modified silica was obtained by gas-phase modification of non-porous pyrogenic silica - Aerosil - A-300 grade with a specific surface area for nitrogen of 300 ± 30 m / g sorbitol.

For this, the organic impurities are removed from the surface of the fumed silica by calcining in air at 300 ° C for two hours. The surface cleanliness was determined by the absence of absorption in the region 2800-3200 in the l / IK spectrum of the sample.

Then, the thermal vacuum was continued at 700 ° C, cooled for 1 hour and up to 2 0-2 5 ° C. and then the grafting modifier (sorbitol) was injected in an amount of 1.0 g per 100 g of base and the process of modification was carried out from 20 to 250 ° C, increasing the temperature for 3-4 hours. Excess modifier and reaction by-products were removed, followed by hauling at 300-350 ° C.

Graft monitoring was performed by IR spectrometry. The basic information of the grafted sorbitol was given by the part of the spectrum of 3650-3500, where the valence vibration bands of OH groups of polyhydric alcohols are found and by reducing the 3750 cm band belonging to the valent vibrations of the OH groups of the surface of Aerosil, and the bands in the region of 3100-2800, corresponding to the valence vibrations of the CH and CH2 groups of alcohols.

The amount of sorbitol on the surface was 0.3 mmol / g.

EXAMPLE 2 According to the procedure described in Example 1, after the preparation of fine silica, a graftable modifier was injected into the reactor in an amount of 1.50 parts of mannitol per 100 parts of base. The temperature was raised to 250 ° C over 3.5 hours. The resulting product is indented on an IR spectrophotometer,

PRI me R 3. Inositol grafting was carried out according to the procedure described in Example 1. The product obtained was identified on an IR spectrophotometer. The reaction was introduced raw materials at a ratio of

100 parts of the base 1.25 parts of inositol, the temperature was raised to 250 ° C for 3 hours. The resulting modified products are applicable not only in affinity

chromatography due to the fact that they retain their sorption properties after modification, but also acquire the ability to transport carbohydrates through biological membranes, which allows them to successfully

to use in the practice of cryopreservation of semen of animals on the example of semen of sheep.

Application. The medium for cryoconservation of semen of sheep was prepared

in the following way. Lactose, Tris- (hydroxymethyl) -aminomethane, citric acid are thoroughly mixed in a sterile flask and filled with distilled water, sterilized for half an hour. After growing & yen, yolk is added, then glycerin and fine silica with grafted carbohydrate-sorbitol, prepared as in Example 1.

For dilution and freezing, sperm with a mobility of at least 8 points and a concentration of at least 2.5 billion sperm per cm were used. The sperm was diluted with the prepared medium, cooled to HCL (parallel to the sperm was diluted with medium

without modified silica) for 5 hours (equilibration), and then frozen in the form of pellets with a volume of 0.2 mm on a fluoroplastic plate in nitrogen vapor. Frozen sperm was stored in liquid.

nitrogen at -196 ° C.

The viability of thawed sperm was evaluated by survival rates. The sperm survival time increased by 1-2 hours. The introduction of fine silica, modified by carbohydrates, provides a more complete physiological nutrition of cells, lengthens their lifespan, and consequently, increases fertility

animals.

The proposed method provides the modification of fine silica with carbohydrates, which allows to obtain

carbohydrate specific carrier for sperm cryopreservation; the method does not require prior activation (amination) of the base; the modification process takes place under conditions

which provide the complete adsorption of carbohydrates on the developed surface of fine silica; the resulting product can be used as a biospecific sorbent.

Claims (2)

Claim 1. Method for producing sorbent based on silicon-containing material, including treatment of the support with carbohydrates, characterized in that. In order to ensure the possibility of obtaining a biospecific sorbent for the cryopreservation of sperm, aerosil with a specific surface area of 270–330 m2 / g is used as a carrier, while the treatment is carried out in vacuum with gradual heating from 20 to 240–250 ° С for 3 -4 h at a flow rate of 1-1.5 wt.h. carbohydrate per 100 mah. aerosil 2. Method pop. 1, characterized in that for the treatment of the carrier, a carbohydrate selected from the group of sorbitol, mannitol, and inositol is used. ten 2700 3300 2900 - / 2UO 1700 1300 S, cn