Title of Invention

A PROCESS FOR THE RECOVERY OF POLYETHYLENE GLYCOL FROM SPENT AQUEOUS TWO PHASE SYSTEM

Abstract

This invention relates to a process for the recovery of polyethylene glycol from spent aqueous two-phase systems. Aqueous two-phase system becomes a costly process because of the high cost of the polymers. Therefore, the polymer used has to be recovered once the extraction is complete. An attempt was made to recover the Polyethylene glycol by using a simple method of thermal and organic solvent extraction. The recovered polyethylene glycol shows all the physical properties similar to that of fresh polyethylene glycol. This a new method to recover polyethylene glycol in a powder form and can be carried out in large scale.

Full Text

The present invention relates to a process for the recovery of polyethylene glycol from spent aqueous two phase systems. The present invention more particularly relates to the recovery of polyethylene glycol from the top phase, which is rich in Polyethylene glycol using a combination of thermal and solvent extraction. An aqueous two phase system is formed when a polymer (polyethylene glycol) and salt (Potassium phosphate, Ammonium sulfate) is mixed above its critical concentration (Albertsson). Aqueous two phase system is finding a wide application in separation, concentration and purification of biomolecules like proteins, viruses and other biomaterials from fermentation broth and cell culture media. Major hindrance for the widespread adaptation of aqueous two phase extraction on industrial scale is the high cost of the phase forming polymers and environmental problems arising due to the disposal of phase forming polymers after the extraction of biomolecules. Presently, polymer phase recycling is being employed after the extraction of biomolecules in order to improve the economics of the process. Hence, there is a need for the polymer recovery from aqueous two phase systems after recycling the phases for repeated extractions (till the loading of the impurities increases significantly) of biomolecules. This results in the reduction in desired levels of purity of the biomolecules in the large-scale operations. Conventional methods like evaporation, vacuum drying alone are economically unviable for the polymer recovery from aqueous two phase systems due to high-energy requirements and need of special equipments for the creation and maintenance of required vacuum. Also, these methods take longer duration for the complete recovery of polyethylene glycol due to presence of water along with the polymer. Hence, there exists a need for an economically viable method for the complete recovery of polyethylene glycol from spent two-phase systems. Reference can be made to 'Polymer-ligands used in affinity partitioning and their synthesis' ( Harries and Yalpani, 1985, in "partitioning in aqueous two phase systems" Eds, Walter et al., pp 589-626, Academic Press, New York) recovery of polyethylene glycol in dry form is accomplished by cooling to give recrystallization or by precipitation with ether. Moreover, additional processing steps are essential to remove ether. Another way to obtain dry polyethylene glycol takes long time and the use of vacuum makes it economically unviable in large-scale operations. Reference can be made to 'recovery of copolymers like ethylene oxide and propylene oxide. (Johansson, 1994, Methods in Enzymology, Vol 228 571) these polymers are recovered by increasing the temperature. However, in this method the polymers are recovered as a mixture of both the polymers. Reference can be made to 'process for the separation and recovery of polyethylene glycol (PEG) from spent aqueous two phase systems by Naveen et al. US6863828 wherein microwave field assisted recovery has been attempted to recover the Polyethylene glycol This can be carried out for small amount of samples and cannot be employed in large scale operations. There are no reports available in the literature on the recovery of dry polyethylene glycol by a combination of thermal and solvent extraction. The main objective of the present invention is to provide a process for the recovery of polyethylene glycol from the spent aqueous two phase systems. Another objective of the present invention is to employ a combination of both the thermal and solvent extraction to obtain polyethylene glycol in solid form (powder). Still another objective of the present invention is to provide a faster method for the separation and recovery of polyethylene glycol from spent aqueous two phase systems completely and can be carried out in large scale. Yet another objective of the present invention is to recover polyethylene glycol without changing its physical properties so that it can be reused for aqueous two phase extraction. Accordingly, the present invention provides a process for the recovery of polyethylene glycol from spent aqueous two phase system, which comprises the steps of; a) separating by a known method polyethylene glycol rich phase from spent aqueous two phase systems; b) subjecting polyethylene glycol rich phase to thermal treatment at a temperature in the range of 60-70 deg. C for a period in the range of 4-5 minutes; c) adding chloroform at the ratio of about 1:1 for 4-5 minutes followed by thorough mixing for 4-5 minutes in order to obtain separate organic phase of aqueous phase allowed for equilibration; d) separating the organic phase containing polyethylene glycol from the aqueous phase; e)removing chloroform using flash evaporator; f) incubating polyethylene glycol in hot air oven at a temperature in the range of 70- 80 deg. C in order to obtain chloroform free polyethylene glycol and recovering the powdered polyethylene glycol after drying with a recovery of 92 percent; and g) powdering the clumps of polyethylene glycol in order to obtain final product. In an ermbodiment of the present invention, polyethylene glycol obtained has a molecular weight in the range of 1000-20,000 Da. Novelty Novelty of the present invention is to recover the Polyethylene Glycol from the spent aqueous two-phase by a combination of thermal and solvent extraction. Another novelty of the present invention is that the recovered polymer reduces the process cost since the polymers are expensive. Yet another novelty of the present invention is that the physical properties of the recovered polymer are similar as that of the fresh polymer. The polyethylene glycol rich top phase is kept in a water bath of 70-80°C for about 4-5 minutes. The top phase is allowed to cool wherein it forms two phases, top phase rich in polyethylene glycol and bottom phase containing water and salts. The bottom phase is removed and the top phase is subjected for heating again. This is continued until no phase formation was observed. To the top phase an equal volume of chloroform was added and mixed well for 5 minutes. This resulted in the formation of two phases; bottom organic phase containing polyethylene glycol and top aqueous phase having salt. The bottom phase was separated and passed through flash evaporator to remove chloroform. The polyethylene glycol obtained was kept in hot air oven for 1 hour to remove the traces of chloroform present. The powder polyethylene glycol obtained was checked for its physical properties. The recovered polyethylene glycol was used as a phase forming polymer in aqueous two phase system in place of fresh polyethylene glycol and the partitioning behavior of proteins was compared and the results was found to be similar as that of fresh polyethylene glycol. Accordingly the present invention provides a process for the separation and recovery of polyethylene glycol from spent aqueous two phase system, which comprises the steps of; a) The polyethylene glycol rich (top) phase is kept in water bath of 60-70 degree C for 4-5 minutes, wherein it forms polyethylene glycol rich top phase and bottom aqueous phase containing salts. The polyethylene glycol rich Top phase is separated and in water-bath heated until no phase separation was observed. b) To the top phase chloroform was added in equal volume and mixed for 5 minutes wherein it forms two phases. The top phase is an aqueous phase containing salts and the bottom phase is an organic phase containing polyethylene glycol. c) The two phases are separated and the bottom phase containing polyethylene glycol was subjected to fractional distillation to obtain the chloroform. d) The polyethylene glycol is kept in hot air oven at a temperature of 60- 70 degree C to remove the chloroform completely. e) Polyethylene glycol is cooled and white powder is obtained. In an embodiment of the present invention, separation and recovery by the above process can be employed for polyethylene glycof having a molecular weight varying from 1000-20,000 The following examples are given by way of illustration of the current invention and should not be construed to limit the scope of the present invention. Example 1 50 ml of polyethylene glycol rich top phase, which is separated from the phase system of phase composition 15.30 grams of polyethylene glycol and 12 grams of ammonium sulfate salt. The top phase was kept in water-bath of temperature 60-70 degree C for 5 minutes. This resulted in the formation of two phases. The top phase is rich in polyethylene glycol and the bottom phase is rich in salts. The phases were separated and the top phase rich in polyethylene glycol was again subjected to heating until no phase formation was observed. To the polyethylene glycol rich top phase chloroform was added and mixed well for 5 minutes. The mixture is allowed to equilibrate wherein it forms two phases; the lower phase is the organic phase containing polyethylene glycol and the top phase aqueous phase rich in salts. The organic phase containing polyethylene glycol was separated and subjected to flash evaporator to remove the chloroform. The polyethylene glycol obtained was kept in hot air oven for 1 hour to remove the residual chloroform. Powder polyethylene glycol was obtained after cooling. The recovered polyethylene glycol was checked for its physical properties and was observed to be similar to that of fresh polyethylene glycol as shown in Table 1. Table 1: Physical Properties of the Recovered Polyethylene glycol (Table Removed)Example 2 50 ml of polyethylene glycol rich top phase, which is separated from the phase system of phase composition 15.30 grams polyethylene glycol and 12 grams of ammonium sulfate salt. The top phase was kept in water bath of temperature 70 degree C for 5 minutes. This resulted in the formation of two phases. The top phase is rich in polyethylene glycol and the bottom phase is rich in salts. The phases were separated and the top phase rich in polyethylene glycol was subjected again to heating until no phase formation was observed. To the polyethylene glycol rich top phase an equal volume of chloroform was added and mixed well for 8 minutes. When allowed for equilibration it forms two phases; the lower phase is the organic phase containing polyethylene glycol and the top phase is an aqueous phase rich in salts. The organic phase containing polyethylene glycol was separated and subjected for fractional distillation to remove the chloroform which can be reused again for the recovery of polyethylene glycol. The polyethylene glycol obtained was kept in hot air oven for 1 hour to remove the residual chloroform. Powder polyethylene glycol was obtained after cooling. By using the recovered polyethylene glycol, phase systems were prepared and the partitioning of proteins was studied. The results are shown in Table 2. Table 2: Yield of proteins obtained using recovered polyethylene glycol (Table Removed) he main advantages of the present invention are: 1. Easy and efficient method to recover polyethylene glycol from aqueous two phase systems. 2. Enables to have a higher efficiency and faster recovery of polyethylene glycol over the conventional processes of polymer recovery. 3. Enables overcoming the environmental hazards involved in disposing of polyethylene glycol. 4. The recovered polyethylene glycol can be used in place of fresh polyethylene glycol. 5. All the operations are carried out at room temperature and hence more economical. 6. The process is simple and easy to scale-up We claim: 1. A process for the recovery of polyethylene glycol from spent aqueous two- phase systems which comprises the step of: a) separating by a known method polyethylene glycol rich phase from spent aqueous two phase systems; b) subjecting polyethylene glycol rich phase to thermal treatment at a temperature in the range of 60-70 deg. C for a period in the range of 4-5 minutes; c) adding chloroform at the ratio of about 1:1 for 4-5 minutes followed by thorough mixing for 4-5 minutes in order to obtain separate organic phase of aqueous phase allowed for equilibration; d) separating the organic phase containing polyethylene glycol from the aqueous phase; e)removing chloroform using flash evaporator; f) incubating polyethylene glycol in hot air oven at a temperature in the range of 70- 80 deg. C in order to obtain chloroform free polyethylene glycol and recovering the powdered polyethylene glycol after drying with a recovery of 92 percent; and g) powdering the clumps of polyethylene glycol in order to obtain final product. 2. The process as claimed in claim 1, wherein polyethylene glycol obtained has a molecular weight in the range of 1000-20,000 Da.

Documents:

321-del-2006-Abstract-(03-01-2012).pdf

321-del-2006-abstract.pdf

321-del-2006-Claims-(03-01-2012).pdf

321-DEL-2006-Claims-(25-04-2012).pdf

321-del-2006-claims.pdf

321-del-2006-Correspodence Others-(03-01-2012).pdf

321-DEL-2006-Correspondence Others-(25-04-2012).pdf

321-del-2006-correspondence-others-1.pdf

321-del-2006-correspondence-others.pdf

321-del-2006-Description (Complete)-(03-01-2012).pdf

321-del-2006-description (complete).pdf

321-del-2006-description (provisional).pdf

321-del-2006-form-1.pdf

321-del-2006-form-18.pdf

321-del-2006-form-2.pdf

321-del-2006-Form-3-(03-01-2012).pdf

321-del-2006-form-3.pdf

321-del-2006-form-5.pdf