Patent Application
20090051068
Method for producing molded bodies as well as molded bodies produced by said method from proteins in ionic liquids, in particular in 1,3-dialkyl-imidazolium-salts as well as the product oriented processing of such solutions by forming processes such as spinning, molding and blow processes for producing molded bodies such as fibers, filaments, fleeces, flat and tubular foils, respectively, membranes and films.
The inventional method for producing molded bodies from proteins is characterized in that the protein is dissolved in the ionic liquid, the solution is formed to molded bodies, the protein being regenerated by precipitation in protide solutions, the solvent being separated by washing and the molded bodies are tried.
Proteins within the scope of the present invention are native, highly molecular fibrous or globular proteins which, due to the formation of strong intra-molecular and inter-molecular hydrogen bridge linkages in their structure, have a poor solubility in aqueous and organic solvents. Fibrous protein preferably is silk fibroin from the mulberry silk moth Bombyx mori freed from sericin. Globular proteins preferably are storage proteins such as maize zein and wheat gluten.
According to the invention solutions produced and processed by the present method may contain the proteins singly as well as in combination with other low molecular and/or high molecular inorganic and/or organic substances dissolved in the ionic liquid and/or fine enough dispersed in the latter.
Ionic liquids are salts melting at low temperatures (<100° C.) which practically have no steam pressure. They have been known, in fact, since 1914, but have only recently found growing importance as a solvent and a reaction medium, respectively, for many syntheses. Compounds having positive nitrogen atoms such as, for example, the ammonium cation, the pyridinium cation and the imidazolium cation are thereby of particular interest. (P. Wasserscheid, W. Keim, Angew. Chem. 2000, 112, 3926-3945; J. Dupont et al. Chem. Rev. 2002, 102, 3667; Schilling G. “Ionische Flüssigkeiten” GIT Labor-Fachzeitschrift 2004(4)-372-373). It is known from the Journal of the American Chemical Society 2004, 126, 14350 that certain ionic liquids which are based on the imidazolium cation and the chloride anion can dissolve silk fibroin of the Bombyx mori in an inert nitrogen atmosphere in the absence of water and at an increased temperature (100° C.) and can regenerate the same by addition of aceto-nitrile or methanol (D. M. Phillips et al. J. Am. Chem. Soc. 2004, 126, 14350-14351). However, answers concerning the characterization and processing of the fibroin solution by forming methods such as spinning, casting and blowing processes are not given. In order to (de)form silk fibroins as a non-meltable polymer to filaments, spinning fibers or foils/membranes, different solution spinning processes have been developed. The solubility of the silk fibroins in sulphuric acid, hydrochloric acid and phosphoric acid, in hot concentrated formic acid and in hot sodium hydroxide solution and in potassium hydroxide solution is known. (H. Zahn, B. Wulfhorst, M. Steffens “Silk (Mulberry silk)-tussah-silk” Fiber Material Tables according to Koch, 1994).
In WO03060207 there is described the dissolution of silk fibroin in formic acid under addition of calcium chloride, and the preparation of the solutions to form fibers (J. P. O'Brien WO03060207).
WO02081793 describes the dissolving of amorphous fibroin powder in a mixture consisting of dichloroacetic acid and chloroform and the spinning of such a solution to fibers (C. J. Stephen WO02081793).
In the solvent systems mentioned the silk fibroin dissolved therein is subject to molecular degradation processes which result in a reduction of the mol mass. A preparation of the proteins from such solutions leads to properties of the products having a low quality level.
There is further described the dissolving of silk fibroin in concentrated aqueous salt solutions of lithium bromide, calcium chloride, magnesium chloride and of thio-cyanates of lithium, sodium, magnesium or zinc and of diverse copper salts (H. Zahn Ullmanns Encyclopedia of Industrial Chemistry, Vol. A24, 95-106, VCH Publishers, Inc., 1993; R. L. Lock, DuPont WO93/15244).
The processing of salinous solutions generally leads to a poor quality of the threads and molded bodies produced therefrom due to the residual of the respective salt in the product. Therefore the removal of the salt before the formation process is vital. DuPont has developed a method in which the salt and the water is removed in two additional method steps from the concentrated lithium-bromide containing fibroin solutions in order to subsequently produce a protein solution in 1,1,1,3,3,3-hexafluoro-2-propanol which may be spun to form fibers. (R. L. Lock WO9315244). Alternatively, hexafluoro-2-propanol can be replaced by hexafluoracetone-hydrate for a repeated dissolving of the silk fibroin after the dialysis of a concentrated lithium bromide containing fibroin solution (A. Tetsuo WO02072931, J. Yao et al. Macromolecules 2002, 35, 6-9). In the case of hexafluoracetone-hydrate and due to the high viscosity only solutions with a protein content<10 mass % can be processed in the spinning process.
In both methods the technological expenditure is considerably increased due to the required dialysis of the salt solution and the subsequent drying for removing the water.
A solution of the silk fibroin which can be processed in a wet spinning procedure can be produced in a mixture of calcium nitrate-tetrahydrate and methanol which, however, has also to be subjected to a dialysis for removing the salt before the spinning process (S.-W. Ha et al. Biomacromolecules 2003. 4,488-496).
DE19841649 describes the dissolving of fibrous proteins such as the silk fibroin in n-methylmorpholin-n-oxide-monohydrate at temperatures of 75-160° C. (K. Heinemann, E. Taeger). There speaks much for a use of ionic solvents for the forming of proteins/silk into filaments, spinning fibers and foils/membranes due to a higher thermal stability compared to the protein solutions in n-methylmorpholin-n-oxide-hydrate which tend to an explosive decomposition at temperatures above 130° C.
There still exists the necessity to find suitable solvent systems which permit a technological simple manufacture of fibroin solutions without a reduction of the mol mass of the protein with favourable properties with respect to the practicability of the product oriented processing of such solutions by forming methods such as spinning procedures, casting procedures and blowing procedures for producing molded bodies such as fibers, filaments, fleeces, plate foils and tubular foils, respectively, membranes and films.
It is an object of the present invention is to provide under substantially maintaining the molecular parameters a method for forming in a simple manner proteins into moulded bodies such as filaments, spinning fibers and foils/membranes at a high reliability of the proceeding and environment friendliness.
The object is realized by the inventional method in that
Proteins preferably used are in particular the silk fibroin of the mulberry silk moth Bombyx mori as well as globular proteins such as maize zein and wheat gluten.
According to the inventional method fibrous protein such as silk fibroin will be disintegrated up to single fibers under strong shearing in water. After squeezing out, swollen fibroin is obtained with about 80-90 mass % water. Globular protein, such as maize zein and wheat gluten, ground up to powder will be dispersed in water and filtered off as swollen material. Surprisingly, the moist protein material in aqueous 1,3-dialkyl-imidazolium salts can easily be transferred into a homogeneous suspension which under shearing, heat supply and vacuum turns into a homogeneous spinning solution after having distilled off the water. Thereby the temperature range lies preferably at 80-95° C. The inventional method does not require to work under an inert nitrogen atmosphere and it generally permits to select a considerably lower temperature for dissolving the protein than will be otherwise necessary to dissolve the dry silk fibroin in an ionic anhydrous liquid (refer to D. M. Phillips et al. J. Am. Chem. Soc. 2004, 126, 14350-14351).
The characterization of the quality of the spinning solution can be carried out by analysing the homogeneity of the solution in a polarisation microscope. Rheologigal measurements of the protein solution supply data about the viscosity which permit an evaluation of the required spinning parameters. The analysis of the water content is carried out over the refractive index.
The particle content and the particle distribution in the spinning solution related to the classification width which can be determined by way of the laser diffraction, permit an additional characterization of the spinning solution quality (refer to B. Kosan, C. Michels Chemical fibers Int. 1999, 48, 4, pg. 50-54).
In order to check the conservation of the molecular parameters of the protein after the dissolution in an ionic liquid and regeneration subsequent to the formation procedure, a characterisation can be carried out by capillary-viscometrical measurements. To this end the limit viscosities is detected, that is, the parameters for the size of the protein molecules in the dissolved state at an infinite dilution of the diluted solutions of the regenerated protein compared to the native employed protein. In the case of the silk fibroin a measurement of the viscosities is carried out in 50%-aqueous lithium bromide solutions.
To obtain a high stability of the mol mass of the protein over a long time at an increased temperature, an addition of anti-oxidants such as hydroquinone, p-phenylene-diamine, gallic-acid-ester, tannins and the like prove a success. As thermo-gravimetric analyses (TGA) and measurements by differential-scanning-calorimetry (DSC) show a good thermal stability of the inventional spinning solution is given up to 220° C. Hence, the solutions according to present invention exhibit a considerably better stability compared to protein solutions in N-methylmorpholin-N-oxide-hydrate (NMO-hydrate) which above 130° C. tend to an explosive decomposition (K. Heinemann, E. Taeger DE19841649). While in the manufacture of the protein solutions in NMO-hydrate a precise distillation has to be kept to until the mono-hydrate has been obtained, the simpler procedure when dissolving in ionic liquids yields considerably shorter processing times for producing the spinning solution. Moreover, compared to the NMO-hydrate solutions, there can be obtained at comparable viscosities which permit a processing by spinning procedures a higher mass percentage of protein dissolved in ionic liquids, in particular in 1,3-dialkyl-imidazolium-acetal, which leads to a more effective process and more stable molded bodies.
According to the invention, in order to produce molded bodies preferably concentrated solutions of the protein are employed of at least 10% up to 50% what depends on the viscosity of the solution which is to be obtained.
Furthermore, it is possible to dissolve proteins together with synthetic and/or native polymers soluble in ionic liquids and to form these into molded bodies.
The inventional forming of the solution to filaments, spinning fibers, plane and tubular foils, respectively and membranes is carried out in a dry-wet-extrusion process in such a manner that the solution is fed into a spin pack, setting the required spinning temperature in a heat exchanger with controllable temperature, forming the solution through a spinneret to filaments and foils, respectively, and direct it under draught through a slot to a coagulating bath. The filament formation can be executed as a two-stage process. In the spinning capillary and under the effect of the shearing tension at a constant temperature the solution jet tapers from the entry cross-section of the spinning capillary towards the exit cross-section of the same.
In the slot of definite length and under the affect of the axial stretch at a decreasing temperature, a further tapering of the solution jet takes place—the spinning draught—in a ratio of doffing speed to extruding speed. The draught of the solution jet in the slot is at the same time accompanied by an increase of the filament surface.
According to the invention and in order to regenerate the protein the oriented solution jets are passed through a coagulating bath which contains a protide solvent, preferably methanol, in which the ionic liquid is soluble but not the protein.
Furthermore, by virtue of the inventionally produced dissolution of the protein in an ionic liquid, foils and membranes may be produced in such a manner that a defined amount of said viscous solution is brushed upon a smooth and clean and tempered area by means of a doctor blade.
Subsequently, the protein will be regenerated in a coagulating bath with a protide solvent, preferably methanol, at a sufficient dwelling time. In a further alternative for forming the inventionally produced solution of the protein, the same can be formed into tubular foils by blowing through an annular slotted jet, with protide internal and external bath.
The properties of the membranes of the inventionally produced foils and membranes are a function of the processing parameters. So the layer thickness of the foils and membranes can be affected by varying the protein concentration in the solution, the slot size and/or the draught speed.
The ionic liquids permit to work in a closed solvent cycle system. The ionic liquid which has been washed out in the coagulating bath can be returned as a solvent into the cycle after the solvent content has been increased by distillation. The method according to the invention will now be explained by virtue of examples.
The silk fibroin of the Bombyx mori which is cut to a length of 3-5 mm length will be dispersed in water, disintegrated in a mixture ratio 1:20 and subjected to swelling for 12 h. Dehydration to 10 mass % fibroin is carried out by a slight squeezing-out. 271.25 g mash are obtained by dispersing 210 g press-moist silk fibroin in 61.25 g 80%-aqueous solution of 1-ethyl-3-methyl-imidazolium-acetate (EMIMAc), having before added 0.5 mass % of propylgallate/sodium hydroxide as a stabilizer. The mash will be transferred under complete dehydration into 70 g of a homogeneous solution after having been fed into a kneader under strong shearing at a temperature of 80-90° C. and under a decreasing pressure of 850 to 5 mbar. The dissolving time is about 160 min. The refractive index of the 30 mass % fibroin solution is 1.5107 at 50° C. The homogeneity of the solution is controlled by taken samples in a polarisation microscope. Rheologigal measurements showed a decreasing viscosity from 1140 Pa·s to 640 Pa·s at a temperature of 50° C. in the range of a shearing gradient of 0.3-4.2 l/s.
After regenerating the silk fibroin with methanol, capillary-viscometrical measurements of diluted solutions of the protein in 50%-aqueous lithium bromide resulted in a limiting viscosity of 20.22 ml/g. The limiting viscosity of the initial silk fibroin has been correspondingly determined and amounted to 22.57 ml/g.
In analogy to Example 1 17.5 g silk fibroin are transferred under dissolution in 65.6 g 80%-aqueous solution of 1-butyl-3-methyl-imidazolium-acetate (BMIMc) at pH 7 into 70 g homogeneous viscous solution with 25 mass % fibroin. The dissolution time is 180 min. The refractive index of the fibroin solution is 1.5015 at 50° C. The homogeneity of the solution is controlled by taken samples in a polarisation microscope. Rheologigal measurements showed a decreasing viscosity from 1070 Pa·s to 698 Pa·s at a temperature of 50° C. in the range of a shearing gradient of 0.3-4.2 l/s. After regenerating the silk fibroin with methanol, capillary-viscometrical measurements of diluted solutions of the protein in 50%-aqueous lithium bromide resulted in a limiting viscosity of 20.42 ml/g. The limiting viscosity of the initial silk fibroin has been correspondingly determined and amounted to 22.57 ml/g.
In analogy to Example 1 14 g silk fibroin are transferred under dissolution in 70 g 80%-aqueous solution of 1-ethyl-3-methyl-imidazolium-chloride (EMIMCl) into 70 g homogeneous viscous solution with 20 mass % fibroin. The dissolution time is 140 min. The refractive index of the fibroin solution is 1.5377 at 50° C. The homogeneity of the solution is controlled by taken samples in a polarisation microscope. Rheologigal measurements showed a linearly decreasing viscosity from 977 Pa·s to 574 Pa·s at a temperature of 50° C. in the range of a shearing gradient of 0.3-4.2 l/s.
After regenerating the silk fibroin with methanol, the capillary-viscometrical measurements of diluted solutions of the protein in 50%-aqueous lithium bromide resulted in a limiting viscosity of 22.39 ml/g. The limiting viscosity of the initial silk fibroin has been correspondingly determined and amounted to 22.57 ml/g.
7.0 g of finely ground maize zein is dispersed in water and removed by filtering. The moist protein is given in portions under stirring into 78.75 g of an 80%-aqueous solution of 1-butyl-3-methyl-imidazolium-chloride (BMIMCl) to which was added before 0.5 mass % propylgallate/sodium. chloride as a stabilizer, so that a homogeneous suspension results. Said suspension is converted, after having been fed into a kneader under strong shearing, at a temperature of 80-90° C. and under a decreasing pressure of 850 to 6 mbar and complete hydration into 70 g of a homogeneous solution. The dissolving time is about 120 min. The refractive index of the 10 mass % zein solution is 1.5229 at 50° C. The homogeneity of the solution is controlled by taken samples in a polarisation microscope. Rheologigal measurements showed a linearly decreasing viscosity of 40 Pa·s to 26 Pa·s at a temperature of 50° C. in the range of a shearing gradient of 0.4-4.2 l/s.
8.38 g of silk fibroin of the Bombyx mori which is cut to a length of 3-5 mm length will be dispersed in water, disintegrated into a mixture ratio of 1:20 and subjected to swelling for 12 h. 930 mg of crushed eucalyptus cellulose (Cuoxam DP 569) are also dispersed in water. Furthermore, sodium hydroxide is added to the cellulose suspension until a pH-value of 10 has been set.
A slight squeezing-out is carried out and the pressure-moist materials are mixed with 75.9 g of an 80%-aqueous solution of 1-butyl-3-methyl-imidazolium-chloride (BMIMCl), to which 0.5 mass % of propylgallate/sodium hydroxide have been added before as a stabilizer. This mash will be converted under complete dehydration into 70 g of a homogeneous solution after having been fed into a kneader under strong shearing at a temperature of 85-95° C. and under a decreasing pressure of 850 to 6 mbar. The dissolving time is about 150 min. The refractive index of the 13.3 mass % solution consisting of 90 parts of silk fibroin and 10 parts of cellulose is 1.5252 at 50° C. The homogeneity of the solution is controlled by taken samples in a polarisation microscope. Rheologigal measurements showed a decreasing viscosity of from 1331 Pa·s to 513 Pa·s at a temperature of 50° C. in the range of a shearing rate of 0.3-4.2 l/s.
A spinning solution produced in analogy to Example 2 with 20 mass % silk fibroin in 1-butyl-3-methyl-imidazolium-acetate (BMIMAc) which at 50° C. exhibits a viscosity of 147-129 Pa·s at a shearing gradient of 0.6-4.3 l/s, is processed to mono-filaments by a piston-type spinning device. Thereby the air-bubble-free fibroin solution, tempered to 35° C. in an internally lapped steel cylinder, is pressed by a motor-driven piston through spinnerets having a diameter of 100 μm or 75 μm (l/d-ratio 3-6) and via an air slit of 15 mm into a coagulating bath consisting. of methanol. The resulting mono-filament is directed via a deviation galette through the coagulating bath of 1 m length to a winding galette where the filament is wound up at a defined doffing speed (for example, 2 m/min). In order to entirely remove the ionic liquid, the mono-filaments are left for 3 hours in the methanol washing bath und subsequently dried at room temperature.
The 13.3 mass % spinning solution produced in 1-butyl-3-methyl-imidazolium-chloride (BMIMCl) in Example 5 will be processed in a piston spinning device in analogy to Example 6 to mono-filaments. In this case the spinning temperature is 65° C. A doffing speed of up to 10 m/min is obtained at the winding galette. Methanol at a temperature of 20° C. is preferably used as a coagulating bath.
5 g of the solution of 10 mass % maize zein produced in Example 4 in 1-butyl-3-methyl-imidazolium-chloride (BMIMCl) will be, for producing a membrane, evenly distributed by a doctor blade upon a glass plate preheated to 80° C. The plate is given into a coagulating bath tub and well covered with methanol to regenerate the protein. After a dwelling time of two hours the plate is taken out, dried at room temperature and the membrane is stripped off.
The 13.3 mass % spinning solution produced in 1-butyl-3-methyl-imidazolium-chloride (BMIMCl) in analogy to Example 5 is processed to tubular foils by a pressure pump spinning device. Thereby the air-bubble-free spinning solution, tempered to 65° C. in a supply vessel, is pressed through an annular slot jet having a slot diameter of 20 mm and a slot width of 9.5 mm and via an air gap of 10 cm into a methanol coagulating bath of 3 m length. The tubular foil is transversally stretched in the air gap by compressed air. A continuously renewed internal coagulating bath consisting of methanol is fed into the interior of the tubular foil. The tubular foils are wound up under draught in a moist condition. The tubular foil is then dried in tempered air between two press rollers.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2006 001 773.0 | Jan 2006 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/000087 | 1/8/2007 | WO | 00 | 10/14/2008 |
