Beads of cross-linked glucomannan and production thereof

Abstract: Glucomannan beads which have a cross-linked structure, exhibit a degree of swelling of 1.5 to 15 ml of wet gel bed per g of dry gel, an exclusion limit molecular weight of 200 to 10⁸, and an average bead size of 1 to 500 µm. Such beads can be prepared by a process which comprises a process for the production of glucomannan beads having an average size of 1 to 500 µm and a cross-linked structure, which comprises suspending in an aqueous medium a solution containing 0.2 to 20% by weight of an ester of glucomannan, optionally together with a diluent, in a chlorinated hydrocarbon solvent which has a boiling point lower than that of the aqueous medium and which is immiscible or substantially immiscible with the aqueous medium; agitating the suspension, to cause the formation of droplets of the solution suspended in the aqueous medium; evaporating off the solvent contained in the liquid droplets, to leave spherical beads of the glucomannan ester; saponifying the beads of the glucomannan ester; and cross-linking the saponified beads by adding thereto a cross-linking agent in an amount of at least 0.5 part by weight per part by weight of the glucomannan beads.

Description: The present invention relates to spherical beads of cross-linked glucomannan, in particular, those which are useful for, such as, packing material in the techniques of gel filtration and gel chromatography, and to a process for their production, For isolation or fractionation of substances by making use of the difference in the molecular size, techniques have been developed in, for example, so-called gel filtration and gel chromatography. The gel filtration and gel chromatography employ as the packing material gelled particles of hydrophilic or hydrophobic nature and there have been in practical use, for example, beads of such as dextran, agarose, cellulose, polyvinyl alcohol, polyacrylamide and polystyrene. Important properties for the packing material for gel filtration and gel chromatography are the stability against pressure in water, namely, the strength to maintain spherical shape of beads, and the capability of voluntary selection of the exclusion limit molecular weight, namely the upper permissible limit of molecular weight of solute to penetrate through the beads, among wide distribution range thereof. Such properties are important for isolating the objective substance in a form as purer as possible among those which have molecular weights distributed over a wide range and for maintaining a uniform packing condition without suffering from destruction or deformation of the beads. The conventional gel beads of hydrophilic or hydrophobic nature found in practical use as mentioned above are not only difficult to obtain in a form of fine particles of true sphere but also are inferior in the strength, besides the shortcoming that they are difficult to attain adjustment of the exclusion limit molecular weight over a wide range. In the magazine "Nippon Kagakukaishi" No. 5, p. 732 (1984), there is a report for "Hydrophilic Gels from Mannan Grafted by Vinyl Acetate as Gel Chromatographic Packings". Here, it is reported that "graft copolymers, which contain 54, 71 and 84 mole % of pendant poly(vinyl acetate) and whose degree of polymerisation were about 2,000, were synthesized by grafting vinyl acetate to mannan granules which contained an aqueous solution of potassium peroxodisulfate. Beads of acetylated graft copolymer were prepared by suspending a solution of acetylated graft copolymer in dichloromethane in an aqueous medium. Then, beads of mannan-poly(vinyl alcohol) graft copolymer were prepared from the beads of acetylated graft copolymer by saponification and subsequent cross linking". In the hydrophobic gel obtained herein however, "highly hydrophilic micro mannan gel phases exist in the poly-(vinyl alcohol) gel matrix". Thus, they are not quite excellent for the packing material for gel filtration etc. There were reported in the magazine "Polymer Preprints, Japan", Vol. 36 , No. 3, 876 (1987), 1 ○ that glucomannan beads suitable for aq. base GPC were prepared by forming micro beads of triacetyl glucomannan by suspension evaporation, saponifying them in heterogeneous phase and cross-linking them with epichlorohydrine, 2 ○ that glucomannan beads having exclusion limit molecular weights in the range of 10² - 10⁷ or more were able to obtain by regulating the condition of cross-linking reaction and selecting the diluent and its concentration during the formation of beads and 3 ○ that this gel exhibited quite lower swellability than conventional hydrophilic gel. In this report however, the method of preparation of gel is given only conceptually, and it is not guaranteed that a superior gel product is always obtained. The material properties of the gel are not clear. Brief Summary of the Invention Beads of glucomannan according to the present invention have a cross-linked structure, a degree of swelling at 40°C of 1.5 to 15 (preferably 2 to 8) ml of wet gel bed per g of dry gel, an exclusion limit molecular weight of 10⁶ to 10⁸, and an average bead size of 1 to 500 µm. Such beads may be obtained by a process which comprises: swelling glucomannan; esterifying more than 85% of the OH groups in the swollen glucomannan; preparing a solution of 0.2 to 20% by weight of the glucomannan ester in a chlorinated hydrocarbon solvent, together with 1 to 10 (preferably 1 to 5) parts by weight, per part by weight of the ester, of a diluent; suspending the solution in an aqueous medium containing 0.1 to 10% by weight of a hydrophilic colloid, the chlorinated hydrocarbon solvent having a boiling point lower than that of the aqueous medium and being immiscible or substantially immiscible with the aqueous medium; agitating the suspension, to cause the formation of droplets of the solution suspended in the aqueous medium; evaporating the solvent contained in the liquid droplets and saponifying the glucomannan ester, to give beads of the glucomannan; and cross-linking the beads by adding thereto a cross-linking agent in an amount of at least 0.5 part by weight per part by weight of the glucomannan beads. The accompanying drawing is a plot of flow rate (FR, ml/min.cm²) against pressure drop (PD, kg/cm²) on flowing water through a column packed with Gel A or Gel B as described in Example 3. Detailed Description of the Invention By the "glucomannan" as used in the specification of the present invention is meant a polysaccharide consisting, as the main constituents, of D-glucose and D-mannose. As a commercially available product, there may be employed adequately konjakmannan, which is obtained from the plant Amorphophallus konjak, though other products may of course be used. The glucomannan beads according to the present invention are those obtained from such glucomannan products by incorporating therein a cross-linked structure by rection with cross linking agent. They are suitable especially for use for hydrophilic packing gel bed for gel filtration and gel chromatography. For the cross linking agent, bifunctional compounds, such as epichlorohydrin, diepoxybutane, toluenediisocyanate or hexamethylenediisocyanate, can be employed. It is preferable to adjust the degree of swelling and the exclusion limit molecular weight of the spherical beads of cross-linked glucomannan according to the present invention by preparing them so as to exhibit a porous macrostructure. It is possible, alternatively or concurrently, to incorporate regulation of the degree of cross-linking to adjust the swelling degree and the exclusion limit molecular weight. The spherical beads of cross-linked glucomannan according to the present invention can be prepared as follows: As the starting glucomannan, a commercially available glucomannan product as such or a refined product treated by dissolving a commercial product once in water and precipitating the glucomannan from this aqueous solution by an addition of a non-solvent, in which glucomannan is not or scarcely soluble, such as ethyl alcohol or methyl alcohol, may be employed. The starting glucomannan is swollen with a solvent, such as formamide or dimethylformamide, and thereto is added an acid to cause reaction to esterify the glucomannan using an esterification catalyst, such as pyridine. For the acid, those of organic or inorganic nature, such as acetic acid, acetic anhydride, propionic acid, butyric acid or nitric acid, may be employed. Two or more acids can be used to obtain a mixed ester. In the case of acids having free carboxyl groups, water is formed upon reaction with glucomannan, which may cause the molecular chain of glucomannan to tend to be broken down, so that it is preferable to install a means for dewatering. On the contrary, an acid anhydride, such as acetic anhydride, does not form water of reaction and is advantageous. The esterification is incorporated in order to render the glucomannan once insoluble in water, so as to form suspended particles of glucomannan. More than 85 %, especially more than 90 %, of the OH groups in the glucomannan are esterified. The thus esterified glucomannan is then purified, e.g. using acetone. Here, if the esterification yield is not higher than 85 %, the solubility of the mannan in acetone will be lower, so that the rate of recovery becomes decreased. The esterified glucomannan is thereafter deposited by contacting it with water and is then dried sufficiently. Then, the ester of glucomannan thus obtained is dissolved together with a diluent in a solvent. For the solvent, it is necessary to employ those which have boiling points lower than the aqueous medium, as will be explained later on, and which are not or scarcely miscible with said aqueous medium. For the solvent, chlorinated hydrocarbons, for example, dichloromethane, chloroform, carbon tetrachloride and trichloroethylene, are employed alone or in a mixture. Chlorinated hydrocarbon solvents are suitable for this invention, because they offer sufficient solubility for glucomannan but are not or scarcely missible with water. The concentration of the solution of glucomannan ester prepared as above should be such that spherical beads will be formed after it is introduced into an aqueous medium and the beads may possess sufficient strength for use for packing material for, such as, gel chromatography and gel filtration, after the solvent has been removed by evaporation. The concentration is from 0.2 to 20 %, prepferably from 0.5 to 5 % and, in particular, from 0.5 to 2 % by weight. If the concentration of the glucomannan eater is less than 0.2 % by weight, a finest precipitate in floc form will be formed and beads are not obtained. On the other hand, if it exceeds 20 % by weight, the particle size will become excessively large and the particle will exhibit non-porous surface, what makes the product impossible to employ for a gel packing. The diluent is used in order to attain the adjustment of the degree of swelling and exclusion limit molecular weight by forming a porous macrostructure inside the bead formed, after the diluent has been removed from the bead to leave behind voids or pores. For the diluent, those which have boiling points higher than that of said solvent and which do not dissolve the glucomannan ester. Specific examples include tetrahydronaphthalene, decahydronaphthalene, ethylbenzene, diethylbenzene, methyl dodecanoate, methyl caprinate, toluene, hexyl alcohol, heptyl alcohol and octyl alcohol. If glucomannan is swollen preliminarily with formamide, tetrahydronaphthalene and decahydronaphthalene are preferably chosen as the diluent, to obtain preferable results. The starting process solution containing the ester of glucomannan according to the present invention is prepared as described above. The starting process solution is then introduced into an aqueous medium, in order to cause formation of spherical liquid droplets of the process solution suspended in the aqueous medium as the precursor of the beads of glucomannan ester. The aqueous medium contains 0.1 to 10%, preferably 0.3 to 5%, by weight of a hydrophilic protective colloid, such as partially hydrolyzed polyvinyl acetate, polyvinyl alcohol, carboxymethyl cellulose, ethylcellulose, methylcellulose, soluble starch or gelatine. The use of, above all, partially hydrolyzed polyvinyl acetate and polyvinyl alcohol as the protective colloid is preferable, since they dissolve quite well in water even at lower temperature and, in addition, reveal the ability for dispersing particles and a viscosity increasing effect more easily as compared with other ones. A concentration of protective colloid lower than 0.1 % by weight will tend to form glucomannan particles having excessively large particle size and concentrations outside the range of from 0.1 to 10 % by weight tend to produce glucomannan particles of not spherical form. The aqueous medium may preferably be used at least in the same volume and preferably in an amount of 2 - 20 times of the volume of the glucomannan ester solution. The starting process solution is introduced into the aqueous medium all at once, followed by agitation to disperse to form a suspension or, it may be introduced into the aqueous medium held in an agitated state all at once or dropwise to form a suspension. The intensity of agitation is so regulated, that the average bead size of the final product will be from 1 to 500 µm. Upon introduction of the starting solution into the aqueous medium, the glucomannan ester will be dispersed and suspended finely in the aqueous medium, since esterified glucomannan is insoluble in water. Here, evaporation of the solvent starts concurrently with the formation of beads. In this manner, spherical beads of glucomannan ester from which the solvent has substantially be evaporated off will finally be obtained. For the temperature for evaporating off the organic solvent in the liquid droplets, a temperature above the freezing point of the aqeous medium and below the boiling point of the organic solvent is employed. Here, a temperature which is lower than the boiling point of the organic solvent, in particular by 1 - 15°C, is preferred, in order to keep the spherical form of the bead in a better manner and to facilitate the evaporation of the organic solvent. During the process step for evaporating off the organic solvent from the liquid droplets, the aqueous medium is held under agitation so as to maintain the spherical form of the liquid droplet. After the organic solvent contained in the liquid droplet has been evaporated off, a spherical bead will be left behind. The beads are formed in a gel state and contain internally the diluent within a pumice-like porous matrix and can hold their shape of beads after they have been taken out of the aqueous medium. The diluent contained in the beads of glucomannan ester will be removed partly in this process step and is removed completely in the subsequent process step of saponification. The spherical beads of glucomannan ester are then subjected to a saponification after they have been taken out of the aqueous medium. The saponification is effected for restoring the water soluble nature by re-conversion into free glucomannan. Here, substantially all the esterified glucomannan is saponified. It is necessary to employ a saponification bath which permits to effect saponification without causing any harmful effect to the spherical form of the glucomannan ester beads which have restored their water-solubility after the re-conversion into free glucomannan. As the saponification bath, for instance, an alcohol solution of alkali hydroxide, such as a methanol solution of sodium hydroxide or potassium hydroxide, an aqueous solution of alkali hydroxide, such as an aqueous solution of sodium or potassium hydroxide dissolved in an aqueous solution of salts such as sodium sulfate may be used. One specific manner of effecting the saponification consists in that spherical beads of a glucomannan ester are soaked in an amount of methanol, which may amount to 5 - 10 times of the weight of the beads in accordance with each particular requirement, for 0.1 - 2 hours. The thus soaked beads are then introduced into a saponification bath of a mixture, which consists of an alcohol, such as, methanol etc., and of an aqueous solution of sodium hydroxide in a weight ratio of [alcohol] : [aqueous solution] of [9 ∼ 7] : [1 ∼ 3] and has a concentration of 0.1 - 5 N NaOH, in such an amount that the ratio of [glucomannan] : [NaOH] is 1 : [0.5∼ 10] on the weight basis, and the mixture is agitated at room temperature for 10 - 24 hours to effect the saponification. Here, the sodium hydroxide solution may be incorporated by adding it dropwise so as to reach finally said range. If the concentration of NaOH of the bath is lower than 0.1 N, the saponification does not proceed sufficiently and, on the other hand, if it exceeds 5 N, cracks may occur in the beads. If the content of the alcohol is outside of the above mentioned range, undesirable phenomena, such as, elution or leakage of the saponified glucomannan and abnormal progress of saponification and so on, may appear. During this saponification step, the glucomannan ester in the beads is saponified and the diluent contained in the beads will be extracted into the alcohol, leaving behind many voids or pores inside the bead to form a porous macrostructure. One specific manner of effecting the saponification with an aqueous alkali solution consists in that the spherical beads of glucomannan ester are introduced into an aqueous solution prepared by dissolving sodium hydroxide or potassium hydroxide in an aqueous solution of sodium sulfate and the mixture is agitated for 24 hours at room temperature. The concentration of the alkali hydroxide in this aqueous solution is 10 - 15 % by weight and the amount of the alkali hydroxide may be over 50 %, based on the amount of the glucomannan ester. The amount of sodium sulfate employed may be in the range of 20 to 30 %, based on the total weight of aqueous solution. Among the specific manners of saponification described above, the use of alcohol/aq. NaOH mixture may be preferable for the reason that it realizes simultaneous elution or removal of the residual diluent. The thus obtained beads of glucomannan which have restored their water-solubility by the saponification are then contacted with a cross-linking agent explained previously. The cross-linking agent will penetrate into the inside of the spherical bead of glucomannan to realize cross-linking of the glucomannan molecules with each other. The cross linking agent is employed in a form of a solution in an organic solvent. As the organic solvent for the cross-linking agent, there may be employed kerosene or liquid paraffin or a mixture of them (for example, in a volume ratio of 7 : 3) with addition of a surface active agent (a non-ionic surfactant, such as, sorbitan fatty acid ester) in an amount of 1 - 2 % by weight. Another example of the solvent for the cross-linking agent consists of a mixture of acetone with dimethyl sulfoxide (with a volume ratio of, for example, 6 : 4). The concentration of the solution of cross-linking agent may be at least 0.01 mole per liter of the cross-linking solution and preferably in the range from 0.01 to 15 moles per liter of the cross linking solution. The cross-linking of glucomannan in the spherical beads is attained by introducing 1 - 5 parts by weight of the glucomannan beads into 100 parts by weight of the solution of cross linking agent and agitating the mixture at room temperature for 24 - 36 hours. The thus cross-linked spherical beads of glucomannan are then filtered off and rinsed with acetone and washed then using a neutral surfactant, followed by water wash. If the amount of cross-linking agent used is short of the above mentioned value, the stability against pressure will become inferior. The porous spherical beads of glucomannan thus obtained according to the present invention are easy to handle and are insoluble both in water and in aqueous alkaline or acid solution and are robust, exhibiting high resistance to compression on packing in column in an aqueous medium. The beads of cross-linked glucomannan according to the present invention exhibit a degree of swelling in the range from 1.5 to 15, preferably in the range of 2 - 10. If the degree of swelling is less than 1.5, the amount of retention water becomes very small and the beads can not be used for the gel packing in practice. If, on the other hand, it exceeds the value of 15, the mechanical strength of the beads will be decreased considerably, so that the pressure stability to be attained in accordance with the present invention will not be obtained any more. By the exclusion limit molecular weight is meant such a limiting molecular weight, that particles having molecular weights higher than this limitation will not be allowed to penetrate through the micropores of the packing material. This is represented in practice in terms of the molecular weight determined by the procedure, in which a series of aqueous solutions of a certain high molecular weight substance, such as dextran and polyethylene glycol, of varying known molecular weights, are passed through a column charged with a packing material to be tested, in order to observe each eluted amount using, for example, a differential refractometer or spectrophotometer, the data observed are plotted on a graph with respect to the molecular weight of the substance observed, and the molecular weight value at the break of the curve is read. The beads of the cross-linked glucomannan according to the present invention can be used as the packing material for gel filtration and gel chromatography, beside other uses, such as, for basic materials for ion-exchangers, for packing material in affinity chromatography and for solid carrier for fixed enzyme, so that they are very useful in industry. The spherical beads of glucomannan can be prepared by the process according to the present invention with control of their exclusion limit molecular weight over a quite wide range with superior resistance to compression. In the following, the present invention will be described by way of Examples, in which the degree of swelling and the amount of retained water of the gel, as given, were determined as follows: The gel to be tested was charged in a 10 ml-measuring cylinder in an amount of about 2 - 3 ml and was maintained in a constant temperature bath at 40°C for 4 hours, whereupon the volume V ml of the gel was read. Then, all of the gel in the measuring cylinder was transferred into a basket (with a tare weight of W₁ g) covered with a net of polyvinylidene chloride and the void water was removed by a centrifugal separator at 1,000 r.p.m. for 3 minutes and the basket was then weighed together with the gel contained therein (weighed to W₂ g). The thus dewatered gel was dried in vacuum for 24 hours and was finely weighed together with the basket (weighed to W₃ g). Degree of Swelling = V/(W₃ - W₁) Amount of Retained Water = (W₂ - W₃)/(W₃ - W₁) Example 1 4.0 g of konjakmannan powder were added to 4 liters of deionized water and were dissolved at 60 - 70 °C with agitation. This solution was poured into 6 liters of ethanol dropwise to form a precipitate, which was then filtered off and dried in air and then in vacuum at 20 °C. 30 g of this dried mannan powder were added to 1 liter of formamide and caused to swell for 2 days and thereto were then added 300 ml of pyridine and 150 ml of acetic anhydride and the reaction was continued at 50 °C for 5 days. The reaction mixture was poured into 7 liters of water with agitation and the precipitate formed was filtered off and washed with water. The precipitate was dried in the air and further in vacuum. 3 g of the so dried product were dissolved together with 6 g of tetrahydronaphthalene in 300 ml of chloroform. The so obtained solution was dropped into 3 liters of 2 % aqeous solution of polyvinyl alcohol at 57 °C and, after 24 hours, the mixture was gradually cooled, whereupon the spherical beads formed were filtered and washed with water. The beads were then introduced into 270 ml of methanol and 30 ml of 10 N solution of NaOH and the mixture was stood under agitation for 24 hours to effect saponification and removal of the diluent. The saponified spherical beads were filtered off and were introduced into a cross-linking agent bath prepared by mixing 35 g of epichlorohydrin into 300 ml of a mixed solution of acetone and dimethyl sulfoxide (1 : 1) and were treated at 60 °C for 24 hours. The exclusion limit molecular weight, the degree of swelling, the average bead size and the amount of retained water of the so obtained spherical beads were found to be 2,000,000, 5.1, 50 - 200 µm and 2.5 respectively. Using the beads of glucomannan obtained as above (Gel A) and, as a comparison product, a commercial conventional packing material for gel filtration "Sepharose 4B" (Trade Mark; a product of the firm Pharmacia Fine Chemicals with an exclusion limit molecular weight of 3,000,000 (Gel B) ) , pressure tests were carried out. The tests were performed in such a manner, that a metal column having an inner diameter of 0.4 cm and a length of 15 cm was filled with the gel sample and water is flown through this column using a high pressure pump and the variation of the pressure upon alteration of the flow rate was observed. Results were given in the graph of Fig. 1. As is clear from the graph, Gel A according to the present invention revealed a far higher pressure stability as compared with the prior product (Gel B) which have nearly the same exclusion limit molecular weight. Example 2 In the same manner as in Example 1, a konjakmannan powder was treated by acetylation, precipitation and drying. 3 g of the so treated mannan powder were dissolved together with 15 ml of diethylbenzene in chloroform. The beads formation and the saponification were effected as in Example 1. The so obtained saponified spherical beads were suspended in a cross linking agent bath prepared by adding 53 g of epichlorohydrin to 300 ml of a mixed solvent of acetone and dimethyl sulfoxide (1 : 4) and were treated at 60°C for 24 hours. The exclusion limit molecular weight, the degree of swelling, the average bead size and the amount of retained water of the so obtained cross-linked beads were determined to be 100,000,000, 5.2, 50 - 200 µm and 5.8 respectively.

Claims: Glucomannan beads suitable for use in gel filtration and gel chromatography, which are porous, and which have a cross-linked structure, a degree of swelling at 40°C of 1.5 to 15 ml of wet gel bed per g of dry gel, an exclusion limit molecular weight of 10⁶ to 10⁸, and an average bead size of 1 to 500 µm. Glucomannan beads according to claim 1, wherein the degree of swelling is 2 to 8. Glucomannan beads according to claim 1 or claim 2, wherein the cross-linked structure is derivable from the use of a bifunctional cross-linking agent. Glucomannan beads according to claim 3, wherein the cross-linking agent is epichlorohydrin, diepoxybutane, toluenediisocyanate or hexamethylenediisocyanate. Glucomannan beads according to any preceding claim, wherein the glucomannan is konjakmannan. A process for the production of glucomannan beads according to any preceding claim, which comprises: swelling glucomannan; esterifying more than 85% of the OH groups in the swollen glucomannan; preparing a solution of 0.2 to 20% by weight of the glucomannan ester in a chlorinated hydrocarbon solvent, together with 1 to 10 parts by weight, per part by weight of the ester, of a diluent; suspending the solution in an aqueous medium containing 0.1 to 10% by weight of a hydrophilic colloid, the chlorinated hydrocarbon solvent having a boiling point lower than that of the aqueous medium and being immiscible or substantially immiscible with the aqueous medium; agitating the suspension, to cause the formation of droplets of the solution suspended in the aqueous medium; evaporating the solvent contained in the liquid droplets, to leave spherical beads of the glucomannan ester; saponifying the beads of the glucomannan ester; and cross-linking the saponified beads by adding thereto a cross-linking agent in an amount of at least 0.5 part by weight per part by weight of the glucomannan beads. A process according to claim 6, wherein the chlorinated hydrocarbon is dichloromethane, chloroform, carbon tetrachloride or trichloroethylene. A process according to claim 6 or claim 7, wherein the diluent is tetrahydronaphthalene, decahydronaphthalene, ethylbenzene, diethylbenzene, methyl dodecanoate, methyl caprinate, toluene, hexanol, heptanol or octanol. A process according to any of claims 6 to 8, wherein the glucomannan is esterified by reaction with acetic anhydride. A process according to any of claims 6 to 9, wherein the glucomannan is swollen with formamide and is subjected to the formation of liquid droplets together with a diluent chosen from tetrahydronaphthalene and decahydronaphthalene. A process according to any of claims 6 to 10, wherein the aqueous medium contains, as the hydrophilic protective colloid, 0.5 to 5% by weight of a partially-saponified polyvinyl acetate. A process according to any of claims 6 to 11, wherein the aqueous medium is heated to a temperature 1 to 15°C lower than the boiling point of the organic solvent, to accelerate evaporation of the organic solvent from the liquid droplets.

Inventors: MOTOZATO YOSHIAKI, MOTOZATO, YOSHIAKI
Applicants: KURITA WATER IND LTD, MOTOZATO YOSHIAKI, KURITA WATER INDUSTRIES LTD., MOTOZATO, YOSHIAKI
Application year: 1987
Main classification: C08B37
IPC codes: C08B37, B01J20, G01N30, C08L5, C08J3