Method for the production of bead dextran materials for gel chromatography.

0001The invention pertains to a method for the production of bead dextran materials for gel chromatography.

0002Several types of materials for the gel chromatogrophy are known. Materials based on dextran assume an important position among them and are commercially supplied and commonly used already for more than 15 years. Those materials consist of particles of a spherical shape and are supplied in a series, the mombers of which have defined properties of gradually varied quantity. An important fundamental property is, above all, the crosslink density, which affects the size of pores manifested as so called exclusion limit, i.e. the molecular mass of compounds which are already unable to penetrate inside the particles. Swelling capacity, which is also connected with the crosslink density, is defined either as the water regain in ml of water in a centrifugated sample corresponding to 1 g of dry substance or as the bed volume, i.e. the volume occupied by 1 g of dry sample after swelling to equilibrium. These properties are for commonly used and supplied gels in a broad range of molecular masses 700 to 200,000 for the exclusion limits and in the range of 1 to 20 ml/g for the water regain.

0003Another important property of these materials is their chemical purity, in particular the absence of ionogenic groups, particularly the carboxylic groups, which otherway cause unde- si_rable sorptions and thus reduce the efficiency of separation

0004The dextran gels are produced by the method which is known and protected by patents. The basic potent Cer. (FRG) 1,292,883 (1969), application in Sweden 1958, describes application of crosslinked dextran gels formed in the reaction with l-chloro--2,3-epoxypropane in bulk. The final gel is crushed, washed and classified on screens to obtain the required particle size and consist of particles with irregular shape. According to the following patent Brit. 974,054 (1964), application in Sweden 1961, bead dextran gels are prepared in a suspension process where particles of perfect spherical shape are formed. In a typical experiment, dextran is dissolved in water, a selu- tion of sodium hydroxide is added and the mixture is dispersed in a suitable water-immiscible liquid, advantageously in toluene or dichloroethane in the presence of a stabilizer. The particles are crosslinked by heating in the presence of a crosslinking agent and then the formed gel is isolated, washed, deswelled and dried. According to another analogous procedure (D. Cvetkovié, B. Pekié, S. Jovanovié: Hem. Ind. 33 (1979), 139), mineral oil is used as a dispersion medium. Further procedure (Brit. Patent 1,087,964 - 1967) employs as a dispersion medium the excessive 1-chloro-2,3-epoxypropane, which acts as a crosslinking agent at the same time.

0005However, the above mentioned known procedures have certain disadvantages. The use of mineral oil cause difficulties in washing and purification of the product. In crosslinking with. an excess of 1-chloro-2,3-opoxypropane, it is necessary to reduce the one-sided reaction giving rise to pendant glycidyl and glyceroyl groups.In addition, dextran used as the starting material regularly contains lactones in the amount of about 20 to 50 micromolc per gram of dry substance. These lactones are hydrolyzed in an alkaline medium during preparation of the gel so that the resulting gel contains the corresponding amount of free carboxyls.

0006Another disadvantage of the described procedures consists in the difficult attaining of the required crosslink density. It appears in samples prepared under other conditions constant that the dependence of water regain on the ratio of reactants, i.e. of the amount of crosslinking agent to the amount of dextran, has a characteristic parabolic course. The data for the initial concentraticn of dextran 40% given in the following table may serve as an example. <tables id="tabl0001" num="0001"><img file="EP0188084A2_D0001.tif" /></tables>

0007It follows from the data that the said dependence is very steep in the region of low crosslinked gels (water regain 10 to 20 ml/g). Consequently, control of the suspension process is very sensitive to small incidental variations in weight of charges, in keeping the temperature and stirring regime, and also in the quality of the starting material. Irreproducible results often occur, namely concerning the crosslink density of the products.A batch is often spoiled, for example, by incomplete crosslinking after a standard reaction timo, end the raw material is then depreciated in further processing.

0008The preparation according to the known processes is connectal with difficulties also at the other side of practical range of water regain of 1 to 1.5 ml/g. Neither a large excess of 1-chloro-2,3-epoxypropane is sufficient as such and it is necessary to work out highly concentrated dextran solutions, which causes technical obstacles. It is far from easy to prepare a solution of dextran with the concentration of 60 wt.-%. This requires a special equipment of a kneader type, dissolution and homogenization of the solution takes rather long time, the energy consumption increases, and the quality of raw material can be lowered. The concentrated dextran solutions have high viscosity which brings about further problem in their dispergation.

0009It has been surprisingly found that the above shortcomings can be overcome relatively easily by a method according to the invention.

0010The invention relates to a method for the production of bead dextran materials for gel chromatography, wherein dextran is, before or during the suspension process or in both stages, reduced in an alkaline medium, advantageously with sodium borohydride, whereas the density of crosslinking is controlled above all by varying the concentration of the initial dextran solution and the resulting gel may optionally undergo an additional crosslinking in a separate subsequent operation.

0011The invention employs the fact that carboxyls in the form of lactones may be easily reduced with sodium borohydride. It hes been proved experimentally that this reaction is not hindered even in the complicated dispersion system and that the content of carboxyls is reduced in this way to 2 to 6 micromole per gram of dry substance. Naturally, it is also possible to carry out the reduction before starting the suspension process in such a way, that sodium borohydride and sodium hydroxide are added to dissolve dextran and the suspension crosslinking is started first after the reduction is completed, or also so called hydrogenated dextran may be used as the starting material, i.e. the material which was reduced end isolated in advance. Also various combinations of the said procedures lead to the same result. For example, it is also possible to reduce a main portion in tho first stage and to complete the reduction during the suspension crosslinking.

0012To control the crosslinking density in the region of low densities characterized with water regain ranging between 10 and 20 ml/g, it is advantageous to start with diluted solutions of dextran. It turned out that the ratio of crosslinking agent to dextran necessary to attain the same water regain strongly depends on the concentration of dextran in the initial solution. Thus, for example, the gel with water regain 10 ml/g requires, in the initial solution with concentration 40%, 8.6 g of 1-chloro-2,3-epoxypropane per 100 g of dextran, whereas, et concentrations 30 and 25%, requires already 16 and, respecti- voly, 27 g of l-chloro-2,3-epoxypropane per 100 g of dextran. The application of solutions with a lower concentration thus corresponds to a steady region in the formerly described parabolic dependence, where a small change of starting conditions causes only a small variation in the quality of the product. In this way, the above said disadvantages arc avoided and, in addition to this, the yield is somewhat increased by reducing the extractable portion and also the subsequent processing of product, washing, deswelling and drying are facilitated.

0013In the region of high densities of crosslinking, i.e. in the preparation of eels with water regain of 0.8 to 2 ml/g, the disadvantages of the known process are overcome by a two--ctep procedure. The gel of medium crosslinking density, which has water regain between 2 and 7 ml/g, is prepared first and then, in the following operation, it is dispersed again, subjected to additional crosslinking, isolated, washed, deswelled and dried. The advantage of this procedure consists in avoiding thework with highly concentrated solutions of dextran. It also enables to valorize unsuccessful batches, which did not render the expected water regain in the first step. In addition to this, the reaction in the second step has lower demands than that in the first step. Solid particles already formed do not tend to stick together, so that the stabilizers need not to be used, and the work is carried out with a more suitable ratio of the phases. Thorough washing of the product is carried out only once, namely after the second step.

0014The invention is further illustrated in several examples of performance without, however, limiting its scope by any means.

Example 1

0015Dextran (100 g) was dissolved in a solution of 11.8 g of sodium hydroxide in 390 ml of degassed distilled water, 1 g of powdered sodium borohydride was added and the mixture was allowed to stand for 2 hours. Toluene (1000 ml) containing 30 g of poly (vinyl acetate) and 3 g of oleic acid was charged into a reactor provided with a hooting jacket and an anchor stirrer. The solution of dextran was slowly added under stirring at 500 r.p.m. Then, temperature was raised to 50°C within 30 min, 59 g of 1-chloro-2,3-epoxypropane was added, and the mixture was further stirred at 50°C for 16 hours. The mixture was cooled down and the product was separated by decantation, washed successively with toluene, ethanol and water, threetimes with each solvent, neutralized by titration with diluted sulfuric acid, and eventually washed twice with water. After deswelling in ethanol and draing, the bead dextran gel was obtained with particle size 50 to 300 µm and water regain 15 ml/g. The content of carboxylic groups determined by titration was 4.2 µmol/g.

Example 2

0016Dextran (100 g) was worked out in the same way as in example 1. However, additional 0.2 g of sodium borohydride was added to the reduced solution after two hours and dextran was then dispersed and processed in the some way as in example 1, with the distinction that only 30 g of 1-chloro-2,3-epoxypropane was used. The dried bead gel had particle size 50 to 300 µm, water regain 22 ml/g and the content of carboxylic groups 2.5 µmol/g.

Example 3

0017Dextran (100 g) was dissolved in 150 ml of an aqueous solution containing 11.8 g of sodium hydroxide, 0.8 g of sodium borohydride was then added and the solution was dispersed in 500 ml of toluene with 10 g of dissolved poly(vinyl acetate). After one hour, 25 g of 1-chloro-2,3-epoxypropane was added to the stirred mixture at 50°C. Further procedure was the same as in example 1. The dry bead gel had particle size 100 to 1000 µm, water regain 3.3 and the content of carboxylic groups 3.5/µmol/g.

Example 4

0018The preparation was carried out analogously to example 3, but dextran reduced in advance was used. To dissolve 100 g of dextran, 11.8 g of sodium hydroxide in 140 ml of water was used and the solution was dispersed in 500 ml of toluene with 10 g of poly(vinyl acetate) and 1 g of oleic acid added. After 30 minutes at 50°C, 25 g of l-chloro-2,3-epoxypropane was added. Using the procedure described in example 1, dry bead gel was obtained which had the particle size 50 to 600/µm, water regain 3.4 ml/g, and content of carboxylic groups 5.0/µmol/g.

Example 5

0019Dextran (100 g), which was reduced in advance, was dissolved in the solution of 22 g of sodium hydroxide in 380 ml of degassed distilled water. Toluene (1 liter) containing 30 g of poly-(vinyl acetate) a 3 g of oleic acid was used for dispergation and 41 g of 1-chloro-2,3-epoxypropane was used for crosslinking. The procedure was carried out analogously to example 1. The bead gel had particles of a perfectly spherical shape and size 100 to 500_/um, water regain 9.5 ml/g, and the content of carboxylic groups 5.5_/umol/g.

Example 6

0020The experiment was carried out analogously to example 5 with the distinction that the initial dextran solution had lower concentration. To dissolve 100 g of dextran, a solution of 22 g of sodium hydroxide in 540 ml of water was used. Other initial values and the procedure remained the same as in example 5. The dry bead gel had particle size 50 to 300 µm, water regain 23 ml/g and the content of carboxylic groups 6 µmol/g.

Example 7

0021Dextran (100 g), which was reduced in advance, was worked out in the sane way as in example 4. After stopping the heating, the suspension was threctimes decanted with toluene and threetimes with ethanol, the excess of ethanol was removed by suction and the residual part was evaporated in vacuum. The dry primary gel was dispersed in 300 ml of toluene, which contained 0.5 g of p-dodecylbenzenesulfonic acid. Then, 81 ml of 20% sodium hydroxide was added to the mixture and,after 1 hour, 52 g of 1-chloro-2,3-epoxypropane. The mixture was stirred at 50°C for 16 hours, cooled down, separated by suction, washed with ethanol and water to a neutral reaction, deswelled in ethanol and dried. The product had particle size 50 to 300 _/um, water regain 1.1 ml/g, and the content of carboxylic groups 5.5 µmol/g.

Example 8

0022The experiment was carried out analogously to example 7, with the distinction that 51 ml of 35% sodium hydroxide was added to the dispersion of primary gel in the additional crose- linking and the amount of 1-chloro-2,3-epoxypropane was 67 g. The dried product had particle size 50 to 300_/um, water regain 0.8 ml/g, and the content of carboxylic groups 6.5 µmol/g.