CZ307111B6 - A sorbent with incorporated hydroxylapatite and a method of its production - Google Patents

Description

Technical field

The invention relates to a process for the production of sorbent and chromatographic carrier of macroporous pearl cellulose spherical particles with hydroxylapatite embedded in cellulose beads, whereby the cellulosic particles are significantly strengthened and can therefore be used for separations in large industrial columns.

BACKGROUND OF THE INVENTION

As a dynamically developing industry, biotechnology separation and chromatography needs chromatographic carriers and sorbents of suitable properties. In addition to the chemical properties that can be modified in various ways by linking suitable functional groups, the specific properties of the carrier itself are important. It is a macroporosity allowing sorption and separation of macromolecular substances such as proteins, enzymes, DNA sequences, etc. Another important parameter is the mechanical strength and elasticity of the sorbent matrix, especially when used in large columns, so as to avoid deterioration of the flow properties of the columns.

The classic sorbent and carrier for gel permeation chromatography is dextran cross-linked with epichlorohydrin. Furthermore, there are a number of chromatographic supports based on styrene-divinylbenzene copolymers, acrylamide, hydroxymethyl methacrylate, but also silica gel and porous glasses.

Agarose is also a frequently used chromatographic carrier. Crosslinked agarose gel has been manufactured for years under the trademark Sepharose.

One of the more recent patented processes is the preparation of an agarose sorbent by injecting drops of agarose gel into an oil dispersion medium [Gazda L, Laramore M, Hamilton T, Smith B, AU 2013/202026] from 2013. A similar patent of the same year also concerns the preparation of agarose microspheres. gel by pearl in oil [Ma Guanghui, Zhao Xi, Wu Xie, Su Zhiguo, Cui Jinmei, Zhou Weiqing, CN 103055773].

Another 2013 patent describes the preparation of cross-linked agarose 4B [Xia Haifeng, Zheng Mengjie, Wu Puqiang, CN 102989400]. Crosslinking takes place in two steps with pentaerythritol-glycidyl ether and epoxychloropropane.

Chromatographic carriers and sorbents also include macromolecules pearl cellulose (Czech Copyright Certificates CS 172640 and CS 210701). Its macroporosity predetermines it for biotechnological separations, purification and separation of proteins, enzymes, etc. Its mechanical strength is better than that of agarose, it is also possible to strengthen it with epichlorohydrin. Macroporous pearl cellulose is produced with different pore volumes and is less rigid at the highest porosity (trade mark MT 500) due to its very high water content (up to 95%), making it difficult to apply in industrial columns due to pressure drop. For the needs of biotechnological separations, separation and purification of proteins, enzymes and similar biological materials, a sorbent seems to be very suitable, which would achieve the required mechanical strength and minimum pressure losses at high macropore volume. The solution according to the invention offers such properties.

SUMMARY OF THE INVENTION

These drawbacks are at least partially overcome by a macroporous bead cellulose sorbent characterized in that it contains incorporated hydroxylapatite particles and is in the form of cellulose beads.

- 1 GB 307111 B6

Preferably, the content of hydroxylapatite particles in the dry matter is from 5 to 40% by weight.

The present invention also provides a process for the preparation of this sorbent, which comprises adding hydroxylapatite, preferably in the form of a powder or an aqueous suspension, to a suspension of viscose in distilled / demineralized water prior to pouring it into dispersed media.

Preferably, the sorbent may also contain a larger pore volume than macroporous pearl cellulose alone. These pores are formed when the hydroxylapatite initially incorporated in the macroporous bead cellulose beads is washed out of the reaction medium after completion of the beading and washing of the cellulose beads with an acid, preferably an inorganic acid, more preferably hydrochloric acid.

According to a particular embodiment of the invention, the preferred sorbent is a sorbent having a pore volume in the beads of from 9 to 12 ml / g.

Clarification of drawings

Giant. 1 is a graphical representation of the pressure versus flow rate of the original cellulose prepared according to Example 3 and the hydroxylapatite cellulose prepared according to Example 2

DETAILED DESCRIPTION OF THE INVENTION

Example 1

For reaction glass PZL 100, non-duplicator, 1000 ml (CJK 032 449 651 940) fitted with lid (CJK 011 426 610 100) with teflon stirrer shaft guide, stirrer (CJK 032 443 116 300), ground-glass thermometer (NZ 14 / 23) 750 ml of white spirit was added to 100 ° C, heated with a laboratory heating nest LTHS 1000. While stirring at 560 rpm, a mixture of 176 grams of viscose containing 7 wt% was slowly added. (Z-cellulose, 1.3 grams of hydroxylapatite powder and 16.10 grams of distilled water. After stirring at room temperature for 5 minutes to stabilize the dispersion, the mixture was slowly heated to 75 ° C. The dispersion liquid was then aspirated with suction pad S2, and the cellulose beads were washed on the suction pad with 800 ml of acetone and then hydrolyzed with distilled water to neutralize the filtrate.

155 ml of macroporous pearl cellulose with a content of 82.67% by weight were obtained. % water and 10.75 wt. hydroxylapatite in dry matter with a particle size of 30 to 800 μιη.

Example 2

To the PZL 100 glass reaction vessel described in Example 1 was charged with 810 mL of toluene. While stirring at 602 rpm, a mixture of 195 grams of viscose containing 7% α-cellulose, 6.8 grams of hydroxylapatite powder and 68.0 grams of distilled water was slowly added. After stirring at room temperature for 5 minutes to stabilize the dispersion, the mixture was slowly heated to 90 ° C. After reaching this temperature, the mixture was stirred for an additional 30 minutes. Further processing is the same as in Example 1.

270 ml of macroporous pearl cellulose containing 84.40 wt. % water and 33.21 wt. hydroxylapatite in dry matter with a particle size of 20 to 500 μιη.

-2GB 307111 B6

From the hydroxylapatite cellulose thus prepared, 25 ml was removed, 50 ml of 2 mol / l hydrochloric acid were poured in, and the suspension was made up to 150 ml with demineralized water, mixed and allowed to stand for 24 hours. The suspension was then filtered and washed with water until neutral.

Table 1 shows specific surface area and pore volume data of several different diameters in the range of 15 to 35 nm for hydroxylapatite cellulose prepared according to this example without further treatment and for this cellulose after treatment with hydrochloric acid. For pore volume and surface area for pore sizes of 15 to 30 nm, the brackets are always 100% for the untreated product, while for hydrochloric acid treated products the percentage is compared to the untreated product. From these data, it can be seen that both the pore volume and specific surface area of the pore size of 15 and 20 nm increased after treatment with HCl to 1.75 to double. For 30 nm pores, both the volume and surface area after HCl treatment decreased to one-third, but larger pores of 35 nm were formed that were not present in the original product at all, and the volume and surface corresponding to these pores was comparable to 30 pore values. nm for untreated samples. Thus, this measurement demonstrates that the additional elution of hydroxylapatite increases the total pore volume and results in larger pore sizes than were present prior to treatment.

Table 1: Effect of hydroxylapatite pearl cellulose treatment on pore volume and surface area

CPV means total pore volume, CSS total specific surface area, SPP mean pore diameter

Pore diameter, nm Pore volume, cm ³ / g Specific surface area, m ² / g Pore volume, cm ³ / g Specific surface area, m ² / g according to Example 2 without modification according to Example 2 with HCl 15 Dec 1,325 (100%) 353.2 (100%) 2,341 (176.8%) 624.2 (176.8%) 20 May 2.884 (100%) 566.8 (100%) 5.589 (197.2%) 1,117.8 (197.2%) 25 3.022 (100%) 483.5 (100%) 1.102 (36.5%) 176.3 (36.5%) 30 1,611 (100%) 214.9 (100%) 0.559 (34.7%) 74.5 (34.7%) 35 0 0 1,744 199.3 CPV 8.792 cm ³ / g (100%) 11.334 cm ³ / g (128.9%) CSS 1,618.4 m ² / g (100%) 2,192.0 m ² / g (135.4%) SPP 22.8 nm 22.3 nm

Example 3: Preparation of parent cellulose without hydroxylapatite

To the PZL 100 glass reaction vessel described in Example 1 was charged with 810 mL of toluene. With stirring at 602 rpm, a mixture of 195 grams of viscose containing 7% T 2 /--cellulose and 68.0 grams of distilled water was slowly added. After stirring at room temperature for 5 minutes to stabilize the dispersion, the mixture was slowly heated to 90 ° C. After reaching this temperature, the mixture was stirred for an additional 30 minutes. Further processing is the same as in Example 1.

260 ml of macroporous pearl cellulose containing 83.50 wt. water and a particle size of 20 to 500 µm.

Table 2: Dependence of pressure on column flow with the original cellulose prepared according to Example 3 and the hydroxylapatite cellulose prepared according to Example 2

with hydroxylapatite original cellulose ml / min pressure (MPa) pressure (MPa) 0.1 0.2 0.42 0.2 0.63 0.93 0.3 1,02 1,875 0.5 1.87 3 0.6 2.09 3.45 0.7 2.25 3.78 1 2.76 4.77 1.5 4.05 6.42

Industrial applicability

A sorbent based on macroporous pearl cellulose with built-in hydroxylapatite is useful for the separation, separation and purification of biological materials on an industrial scale.

Claims (9)

A sorbent consisting of spherical particles of macroporous pearl cellulose, characterized in that it contains incorporated hydroxylapatite particles and is in the form of cellulose beads. Sorbent according to claim 1, characterized in that the particle content of the hydroxylapatite is from 5 to 40% by weight, based on dry matter. Sorbent according to claim 1 or 2, characterized in that the pore volume in the beads of the post-washed hydroxylapatite particles is greater than the pore volume of the macroporous bead cellulose itself. The sorbent of claim 3, wherein the pore volume in the beads is from 9 to 12 ml / g. -4GB 307111 B6 A process for producing a sorbent according to claim 1 by viscose beading in a dispersion medium, comprising the steps of: - an organic solvent based on white spirit, toluene, dimethylbenzene, trimethylbenzene, ethylbenzene or chlorobenzene is introduced into the reaction vessel; mixing with said solvent a viscose mixture containing alpha cellulose, hydroxylapatite and optionally water; said mixture is stirred at room temperature and subsequently at elevated temperature to viscose beading in a dispersed medium and to precipitate hydroxylapatite-containing cellulose beads; - the dispersion liquid is sucked off; - the beads are washed with an organic solvent and then hydrolyzed with water; - the product is dried. A process for the production of a sorbent according to claim 5 by viscose beading in a dispersion medium, characterized in that the hydroxylapatite particles are added to the viscose suspension in the form of a powder or an aqueous suspension. A process for the production of sorbent according to claim 5 or 6, characterized in that the hydroxylapatite initially incorporated in the cellulose beads of the sorbent is additionally washed out with acid to form additional pores in the macroporous pearl cellulose of the sorbent. A process according to claim 7, wherein the acid is an inorganic acid. 9. A process according to claim 8, wherein the inorganic acid is hydrochloric acid.