Title of Invention	" A PROCESS FOR THE PREPARATION OF ION CONDUCTING POLYMER BLEND USEFUL FOR SEPARATION OF PROTEINS
Abstract	A process for the preparation of ion conducting polymer blend useful for separation of proteins by reacting an oligomer chosen from low molecular weight alkyl ether and alcohols in the range of 60% to 80% w/w with a cross-linking agent (1 to 4% w/w) chosen from di or tri substituted alcohols, amines or anhydrides in presence of a catalyst such as ferric chloride (0.5 to 1.0% w/w), drying and powdering the resulting mass and blending the same with another high molecular weight polymer having hydroxyl, acetate or ester groups, soaking the blend in an acid for 1-2 hours to form the ion conducting polymer blend.

Title of Invention

" A PROCESS FOR THE PREPARATION OF ION CONDUCTING POLYMER BLEND USEFUL FOR SEPARATION OF PROTEINS

Abstract

A process for the preparation of ion conducting polymer blend useful for separation of proteins by reacting an oligomer chosen from low molecular weight alkyl ether and alcohols in the range of 60% to 80% w/w with a cross-linking agent (1 to 4% w/w) chosen from di or tri substituted alcohols, amines or anhydrides in presence of a catalyst such as ferric chloride (0.5 to 1.0% w/w), drying and powdering the resulting mass and blending the same with another high molecular weight polymer having hydroxyl, acetate or ester groups, soaking the blend in an acid for 1-2 hours to form the ion conducting polymer blend.

Full Text	This invention relates to a process for the preparation of ion conducting polymer blend useful for separation of proteins. Separation of proteins in a gradient column using isoelectric technique is a well known method used regularly in the laboratory and there are a variety of instruments reported in literature. This consists of placing the mixture of proteins in an electrolyte (cathode compartment) which is separated from the anode solution through a gradient column and complex designed tube in which the electrodes are placed and a high electric field is applied for more than 24 hours in order to obtain separate fractions of each. The main drawbacks of such commercially available apparatus are that (i) it has very complex design, (ii) large size and (iii) requirements of high voltage or power applications. These factors render the instrument costly, cumbersome to operate and hazardous because of high voltages involved. The whole process of separation as such takes long time with these units. Typically, voltages in excess of 1000 V are required and the isoelectric focusing is obtained only after 24 hours. There have been several attempts in the past to modify the design of the electrofucussing units and various shapes and sizes have been reported. However, these have not yielded much improvement on the time duration or the electrical requirements. The main reason for this is the improper transport of ions across the boundary between the two electrolytes used for the formation of the gradient column. The main object of the present invention is to provide a process for the preparation of ion conducting polymer blend useful for separation of proteins using an isoelectric focusing unit. Another object of the present invention is to provide a process for the preparation of ion conducting polymer blend which gives large improvement in the efficiency of the isoelectric focusing of proteins using a miniscale apparatus. Accordingly, the present invention provides a process for the preparation of ion conducting polymer blend useful for separation of proteins which comprises of reacting an oligomer chosen from low molecular weight alkyl ether and alcohols such as herein described in the range of 60% to 80% w/w with a cross-linking agent (1 to 4% w/w) chosen from di or tri substituted alcohols, amines or anhydrides in presence of a catalyst such as ferric chloride (0.5 to 1.0% w/w), drying and powdering the resulting mass and blending the same with another high molecular weight polymer having hydroxyl, acetate or ester groups, soaking the blend in an acid such as herein described for 1-2 hours to form the ion conducting polymer blend. In the preferred range of composition, the ion conducting polymer exhibits high conductivity for certain type of ions and gives high separation efficiency for proteins in a isoelectric focus- ing unit at an applied potential of 400 V within 3 to 5 hrs as compared to large applied' potentials in excess of 500 V for long durations more than 20 hrs required for conventional units without the above polymer. The invention can be worked with small variations of parameters described hereinunder and should not be regarded as departure from the spirit and scope of the invention. The invention is illustrated with examples hereinunder which should not however be construed to limit the scope of the invention . EXAMPLE 1 A prepolymer was prepared by the addition of medium- molecular weight ethylene glycol (4.0 gm), 1,2,6 hexane triol and FeClg (10mg) for 4 hr at 90 C and precipitating the mass in acetone to obtain a powder. This powder was mixed with high molecular weight ( MW more than 10^) polyvinyl alcohol in the proportion of 30% w/w and blended thoroughly. Two grams of resulting blend were mixed with concentrated phosphoric acid (0.5 ml) and allowed to soak for 1 hr to obtain a paste of the ion conducting polymer blend. EXAMPLE 2 Polyvinyl alcohol having high molecular weight (MW 50000) was mixed with polyethylene oxide (MW 105) in the proportion of 1:1 by weight and allowed to dry. This powder ( 1 gm) was soaked in 0.IM phosphoric acid (0.7 ml) for 1 hr to make a paste of the blend. EXAMPLE 3 Styrene monomer was1copolymerised with divinylbenzene (50% in ethyl benzene) (2% w/w) at 60 C for 24 hrs and the mass precipitated in methanol and powdered. The powder was digested in concentrated sulfuric acid with silver sulfate (1%) as catalyst for 4 to 8 hrs. The granules so obtained (1 gm) were mixed with water and high molecular weight (105M.W.) polyehtylene oxide to form a paste of the blend. The blends prepared as in above examples were applied at the end of an isoelectric focusing tube. The apparatus when operated at 400V, 2 raA gave in the case of example (1) sharp distinct bands of proteins placed at 7.5 era within 5 hrs and which could be collected in clear fractions of individual components. Under similar conditions of operation, for the polymer from example (2) the time required for the protein band formation was 10 hrs and the bands gave mixed fractions containing both components. For the polymer blend as in example (3), there w.as no separation of proteins observed even after 24 hrs and the polymer escaped out of the tube causing contamination of the electrolyte. By using suitable combination of ion conducting polymer and another bulk polymer such as polyvinyl acetate, polyvinyl alcohol, polyacrylamide, nylon etc. it is possible to obtain a blend which is useful for the isoelectric separation of proteins. main advantages of the invention are: It is possible to reduce the isoelectric focusing time as well as the applied voltage using the ion conducting polymer prepared by the present invention, It is possible to simplify the design of the apparatus by using ion conducting polymer in a straight tube rather than tubes having complex shapes and designs,/ The apparatus employing the ion conducting polymer blend pre pared by the present invention can yield more pure fractions even at small scales. We claim : 1. A process for the preparation of ion conducting polymer blend useful for separation of proteins which comprises of reacting an oligomer chosen from low molecular weight alkyl ether and alcohols such as herein described in the range of 60% to 80% w/w with a cross-linking agent (1 to 4% w/w) chosen from di or tri substituted alcohols, amines or anhydrides in presence of a catalyst such as ferric chloride (0.5 to 1.0% w/w), drying and powdering the resulting mass and biending at what ratio the same with another high molecular weight polymer having hydroxyl, acetate or ester groups in the probahim 30% W/W soaking the blend in an acid such as herein described for 1-2 hours to form the ion conducting polymer blend. 2. A process as claimed in claim 1 wherein the second component in the polymer blend is having hydroxy or ether groups in the main chain or side chain. 3. A process as claimed in claims 1-2 wherein the acid for soaking used is sulfuric, phosphoric, fluoroboric acids or mixtures thereof. 4. A process for the preparation of ion conducting polymer blend useful for separation of proteins substantially as herein described with reference to the examples.

Full Text

This invention relates to a process for the preparation of ion conducting polymer blend useful for separation of proteins.
Separation of proteins in a gradient column using isoelectric technique is a well known method used regularly in the laboratory and there are a variety of instruments reported in literature. This consists of placing the mixture of proteins in an electrolyte (cathode compartment) which is separated from the anode solution through a gradient column and complex designed tube in which the electrodes are placed and a high electric field is applied for more than 24 hours in order to obtain separate fractions of each.
The main drawbacks of such commercially available apparatus are that (i) it has very complex design, (ii) large size and (iii) requirements of high voltage or power applications. These factors render the instrument costly, cumbersome to operate and hazardous because of high voltages involved. The whole process of separation as such takes long time with these units. Typically, voltages in excess of 1000 V are required and the isoelectric focusing is obtained only after 24 hours.
There have been several attempts in the past to modify the design of the electrofucussing units and various shapes and sizes have been reported. However, these have not yielded much improvement on the time duration or the electrical requirements. The main reason for this is the improper transport of ions across the boundary between the two electrolytes used for the formation of the gradient column.
The main object of the present invention is to provide a process for the preparation of ion conducting polymer blend useful for separation of proteins using an isoelectric focusing unit. Another object of the present invention is to provide a process for the preparation of ion conducting polymer blend which gives large improvement in the efficiency of the isoelectric focusing of proteins using a miniscale apparatus.
Accordingly, the present invention provides a process for the preparation of ion conducting polymer blend useful for separation of proteins which comprises of reacting an oligomer chosen from low molecular weight alkyl ether and alcohols such as herein described in the range of 60% to 80% w/w with a cross-linking agent (1 to 4% w/w) chosen from di or tri substituted alcohols, amines or anhydrides in presence of a catalyst such as ferric chloride (0.5 to 1.0% w/w), drying and powdering the resulting mass and blending the same with another high molecular weight polymer having hydroxyl, acetate or ester groups, soaking the blend in an acid such as herein described for 1-2 hours to form the ion conducting polymer blend.
In the preferred range of composition, the ion conducting polymer exhibits high conductivity for certain type of ions and gives high separation efficiency for proteins in a isoelectric focus-
ing unit at an applied potential of 400 V within 3 to 5 hrs as compared to large applied' potentials in excess of 500 V for long durations more than 20 hrs required for conventional units without the above polymer. The invention can be worked with small variations of parameters described hereinunder and should not be regarded as departure from the spirit and scope of the invention.
The invention is illustrated with examples hereinunder which should not however be construed to limit the scope of the invention .
EXAMPLE 1
A prepolymer was prepared by the addition of medium- molecular weight ethylene glycol (4.0 gm), 1,2,6 hexane triol and FeClg (10mg) for 4 hr at 90 C and precipitating the mass in acetone to obtain a powder. This powder was mixed with high molecular weight ( MW more than 10^) polyvinyl alcohol in the proportion of 30% w/w and blended thoroughly. Two grams of resulting blend were mixed with concentrated phosphoric acid (0.5 ml) and allowed to soak for 1 hr to obtain a paste of the ion conducting polymer blend.
EXAMPLE 2 Polyvinyl alcohol having high molecular weight (MW 50000) was mixed with polyethylene oxide (MW 105) in the proportion of 1:1 by weight and allowed to dry. This powder ( 1 gm) was soaked in 0.IM phosphoric acid (0.7 ml) for 1 hr to make a paste of the blend.
EXAMPLE 3
Styrene monomer was1copolymerised with divinylbenzene (50% in ethyl benzene) (2% w/w) at 60 C for 24 hrs and the mass precipitated in methanol and powdered. The powder was digested in concentrated sulfuric acid with silver sulfate (1%) as catalyst for 4 to 8 hrs. The granules so obtained (1 gm) were mixed with water and high molecular weight (105M.W.) polyehtylene oxide to form a paste of the blend.
The blends prepared as in above examples were applied at the end of an isoelectric focusing tube. The apparatus when operated at 400V, 2 raA gave in the case of example (1) sharp distinct bands of proteins placed at 7.5 era within 5 hrs and which could be collected in clear fractions of individual components. Under similar conditions of operation, for the polymer from example (2) the time required for the protein band formation was 10 hrs and the bands gave mixed fractions containing both components. For the polymer blend as in example (3), there w.as no separation of proteins observed even after 24 hrs and the polymer escaped out of the tube causing contamination of the electrolyte.
By using suitable combination of ion conducting polymer and another bulk polymer such as polyvinyl acetate, polyvinyl alcohol, polyacrylamide, nylon etc. it is possible to obtain a blend which is useful for the isoelectric separation of proteins.
main advantages of the invention are:
It is possible to reduce the isoelectric focusing time as
well as the applied voltage using the ion conducting polymer
prepared by the present invention,
It is possible to simplify the design of the apparatus by
using ion conducting polymer in a straight tube rather than
tubes having complex shapes and designs,/
The apparatus employing the ion conducting polymer blend pre
pared by the present invention can yield more pure fractions
even at small scales.

We claim :
1. A process for the preparation of ion conducting polymer blend useful for
separation of proteins which comprises of reacting an oligomer chosen from low
molecular weight alkyl ether and alcohols such as herein described in the range
of 60% to 80% w/w with a cross-linking agent (1 to 4% w/w) chosen from di or tri
substituted alcohols, amines or anhydrides in presence of a catalyst such as
ferric chloride (0.5 to 1.0% w/w), drying and powdering the resulting mass and
biending at what ratio the same with another high molecular weight polymer having hydroxyl,

acetate or ester groups in the probahim 30% W/W soaking the blend in an acid such as herein described

for 1-2 hours to form the ion conducting polymer blend.
2. A process as claimed in claim 1 wherein the second component in the polymer blend is having hydroxy or ether groups in the main chain or side chain.
3. A process as claimed in claims 1-2 wherein the acid for soaking used is sulfuric, phosphoric, fluoroboric acids or mixtures thereof.
4. A process for the preparation of ion conducting polymer blend useful for separation of proteins substantially as herein described with reference to the examples.

Documents:

2453-del-1995-abstract.pdf

2453-del-1995-claims.pdf

2453-del-1995-complete specification (granted).pdf

2453-del-1995-correspondence-others.pdf

2453-del-1995-correspondence-po.pdf

2453-del-1995-description (complete).pdf

2453-del-1995-form-1.pdf

2453-del-1995-form-2.pdf

2453-del-1995-form-3.pdf

2453-del-1995-form-4.pdf

« Previous Patent

Next Patent »

Patent Number

232836

Indian Patent Application Number

2453/DEL/1995

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

21-Mar-2009

Date of Filing

29-Dec-1995

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SATHIVEL CHINNATHAMBI	NATIONAL CHEMICAL LABORATORY, MAHARASHTRA,INDIA.
2	ANIL HARICHCHANDRA LACHKE	NATIONAL CHEMICAL LABORATORY, MAHARASHTRA,INDIA.
3	SUBRAMANIAM RADHAKRISHNAN	NATIONAL CHEMICAL LABORATORY, MAHARASHTRA,INDIA.

PCT International Classification Number

C08L 29/04

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA