Title of Invention

A PROCESS FOR THE PREPARATION OF CONDUCTING POLYMERIC MEMBRANE USEFUL AS A FILTER FOR CAPTUNIG VIMUSES IN POTABLE LIQUIDS

Abstract A process for the preparation of conducting polymeric membrane useful as a filter for capturing viruses in potable liquids A process for the preparation of conducting polymeric membrane useful as a filter for capturing viruses in potable liquids, by mixing 90 to 99.9 vol % distilled pyrrole and 10 to 0.1 vol % distilled N-methyl pyrrole cooling the mixture to a temperature below 4°C; mixing 40 to 60 vol % alkyl acetate to 40 to 60 vol % cooled pyrroles mixture , soaking in the mixture a porous substrate, cooling the said impregnated substrate to a temperature below 4°C and allowing it to stand for at least 1 hr; treating the impregnated substrate with a solution of ferric chloride in an inert atmosphere at a temperature below 5°C for a period of at least 1 hr to obtain a conducting polymeric membrane; washing the polymeric membrane by distilled water or deionized water and followed by drying in oxygen free atmosphere to obtain conducting polymeric membrane.
Full Text The present invention relates to a process for preparation of conducting polymeric membrane useful as a filter for capturing viruses in potable liquids.
Viruses which pollute water are excreted with faeces or urine from human beings and many species of animals. These viruses such as polio virus, coxsackie B virus, echo virus and other entero virus, adeno virus, reo virus, rota virus, hepatitis A virus, etc. can cause acute infectious non-bacterial gastroenteritis. These viruses are present in relatively large numbers in sewage contaminated water or ground water. As per World Health Organization (WHO) recommendations drinking water should be free from any viruses and no virus should be detectable in samples of 100-1000 liters of directly reclaimed drinking water as described in a report of a WHO Scientific Group published in WHO Technical Report Series 639, WHO, Geneva (1979). For public health, the development of reliable, sensitive and practical methods for detecting viruses in large quantities of water is very essential. Most of the methods of virus concentration have been developed for polio virus. Viruses can be concentrated from water samples by viradel (virus adsorption-elution) technique. The virus gets adsorbed presumably by both electrostatic and hydrophobic interactions between the virus and the filter matrix. However, these previously developed methods for concentration of entero viruses from water have proved of limited values when applied to the concentration of rota viruses. Although ultrafilteration is an extremely useful method for concentrating viruses but is only effective when applied to relatively small volumes of water, which have low turbidities. Since ultrafilteration depends on the physical size of the virus particle, the pore size has to be in nanometer range. This type
of filter will rapidly clog, thus limiting the water volume, which can be processed. In addition, the bulky equipment used for concentration of viruses limits sampling to readily accessible areas as described by G.A. Toranzos and C.P. Gerba, in J. Virological Methods, 24, 131 (1989).
The conventional microporous electronegative (having negative charge on the surface) adsorbent filters adsorb viruses more efficiently in the presence of multivalent cations such as A13+ and Mg2+ on/or at low pH usually, 3.5. The efficient virus adsorption occurs only if water is acidified to pH 3.5 and/or multi cation salts are added. The major drawback is the extensive modification of the water sample. The strongly acidic and basic pH levels are utilized for the formation of precipitates during the reconcentration and their susceptibility to the variation in the quality of water sample. The electropositive filters are composed of fibre glass or cellulose acetate and positively charged organic polymer resin as described in 'Standard Methods for the Examination of Water and Waste-Water' edited by Andrew D. Etaon et al., 19th edition, American Public Health Association, Washington D.C., 1995. The microporous filters which are positively charged are advantageous over negatively charged filters as the virus adsorbence occurs for most natural and tap water in the pH range ~ pH 5-9, because they reduce or eliminate the need for either acid or salt addition to obtain virus adsorption as described by M.D. Sobsey and B.L. Jones, in Applied & Environmental Microbiology, 37, 588 (1979).
The conventional microporous filters suffer from three main limitations: (a) sample suspended matter tends to clog the adsorbent filter thereby limiting the volume that can be
processed and possibly interfering with elution process, (b) dissolved and colloidal organic matters in some waters can interfere with virus adsorption to filters presumably by competing with viruses for adsorption sites and they also can interfere with virus elution. (c) viruses adsorbed to suspended matter may be removed in any cleaning process applied before virus adsorption as described in 'Standard Methods for the Examination of Water and Waste-Water' edited by Andrew D. Eaton et al., 19th edition, American Public Health Association, Washington D.C., 1995.
The main objective of the present invention is to provide a process for the preparation of conducting polymeric membrane and a conducting polymeric membrane prepared thereby useful as a filter for capturing viruses in potable liquids, which obviates the drawbacks as mentioned herein.
Another objective of the present invention is to provide a process for the preparation of conducting polymeric membrane which has the unique property of carrying out large number of positive charges in their polyconjugated backbone and could be useful for virus filtration.
Yet another objective is to provide a conducting polymeric membrane prepared thereby useful as a filter for capturing viruses in potable liquids.
Accordingly the present invention provides a process for preparation of conducting polymeric membrane useful as a filter for capturing viruses in potable liquids, which comprises;
(i) mixing 90 to 99.9 vol% distilled pyrrole and 10 to 0.1 vol% distilled N-methyl
pyrrole cooling the mixture to a temperature below 4°C; (ii) mixing 60 to 40 vol% alkyl acetate to 40 to 60 vol% cooled pyrroles mixture
obtained in step(i) above;
(iii) soaking in the mixture obtained in step (ii)above, a porous substratd smle as herein
deciebed this cooling the said impregnated substrate to a temperature below 4 °C and allowing it to
stand for at least 1 hr; (iv) treating the impregnated substrate obtained in step (iii)above with a solution
of oxidising agent in an inert atmosphere at a temperature below 5 °C for a
period of at least 1 hr to obtain a conducting polymeric membrane;

(v) washing the polymeric membrane obtained in step (iv) above followed by
drying in oxygen free atmosphere.
In an embodiment of the present invention the distillation of the pyrroles may be effected using conventional methods such as vapor condensation.
In another embodiment of the present invention the alkyl acetate used may be such as methyl acetate, ethyl acetate and propyl acetate.
In yet another embodiment the porous substrate used may be such as filter papers, fibre glass.
In yet another embodiment the oxidising agent used may be such as ferric chloride. In still another embodiment solution of the oxidising agent used may be prepared using any clean water such as distilled water or deionized water.
In yet another embodiment the washing of conducting polymeric membrane may be done
using any clean water such as distilled water or deionized water.
In still another embodiment the inert atmosphere used may be such as nitrogen, argon and
helium.
Accordingly the present invention provides a conducting polymeric membrane useful as a filter for capturing viruses in potable liquids prepared by the process as described above.
The process of the present invention is described in detail below: Pyrrole monomer was distilled and kept in a refrigerated environment. Also the N-methyl pyrrole monomer was distilled and placed in the refrigerated environment. Both the distilled monomers were kept in the refrigerated environment for 1-6 hrs. Subsequently both the monomers were mixed and their mixing volume ratio was in the range of 90-99.9% for pyrrole and 10-0.1% for N-methyl pyrrole respectively. Then 60 to 40 vol% alkyl acetate such as methyl acetate, ethyl acetate, propyl acetate was mixed with the 40 to 60 vol% resultant monomers mixture. Then the final mixture was rinsed and filter papers of different diameters according to the requirement were put in the mixture for soaking. This was kept in a refrigerated environment for a time period of at least 1 hr. Then the oxidant solution was prepared in the concentration range of 0.4-1.5 Molar using clean water such as distilled water or deionized water. Then the oxidant was placed in the refrigerated environment for the time period in the range of 0.5-3 hrs. An especially designed double walled glass container was used for polymerization. The temperature of the container was maintained by the flow of cooled ethylene glycol liquid from the cold
bath between the walls of the container. The temperature of the bath was maintained in the range of -20 to 3 °C. Then the mixture soaked filter papers were placed in this glass container and the oxidant solution was poured on the soaked filter papers to initiate polymerization. Before the oxidant was poured on the soaked filter papers, the environment in the glass container was made inert using the inert gas such as argon, nitrogen, helium, etc. The polymerization time was kept at least 1 hr and the polymerization was performed at temperatures in the range of -18 to 5 °C under inert atmosphere. During polymerization the copolymer film was deposited in the pores and on to both the surfaces of the filter papers making a composite structure. Then the prepared conducting polymeric membrane filters were washed with any clean water such as distilled water, deionized water, etc. After that the prepared conducting polymeric membrane filters were placed in a vacuum oven in the temperature range of 30-60 °C for the time period 1-12 hrs.
The basic idea of this invention is that in the doped state most of the conducting polymers have the unique property of carrying large number of positive charges which can capture viruses by electrostatic interactions.
Then these conducting polymeric membrane filters were tested for virus retention on it using stock solutions of viruses prepared using polio 1 virus. Other water borne viruses such as coxsackie B virus, rota virus and hepatitis A virus may also be used for preparing stock solution of viruses. The process used for virus filtration and titration is described below.
Virus Filtration and Titration: A known amount of stock virus (as for example polio I virus - 5xl03 to 2xl05 pfu/ml) was diluted up to 100 ml with phosphate buffer saline, pH 7.2 and was passed through polymeric membrane filter contained in a filtration system. The filterate and the elute were obtained by incubating the membrane in 10% of the fetal calf serum (PCS) containing minimum essential medium (MEM) (pH 9.6) at 4 °C overnight. Aliquots of stock, elute and filterate were stored at -70 °C till the quantification of virus by plaque assay technique was done. The plaque assay technique is the most precise method for enumerating virus multiplicities as described by W.F. Hill, Jr.; E.W. Akin and W.H. Benton in Water Research, 5, 961 (1971). The amount of virus present in three fractions namely stock, elute and filterate was determined by plaquing viruses in 24 well plates containing mono layer of Buffalo Green Monkey Kidney (BGMK) cells. Under optimal condition a plaque originates from a single infectious virus particle. First serial log dilutions of the fractions were prepared with diluent (MEM without PCS). Then the 24 well plates containing conflucent mono layer of BGMK cell line was washed twice with phosphate buffer saline (pH 7.4), the dilutions (200 µl) of the three fractions were added to the cell mono layers and incubated at 37 °C for one hour for the adsorption of virus particles to the host cells. After incubation, excess virus suspension was aspirated and the mono layer was overlaid with plaque medium containing agarose (0.8%). Virus particles infecting and replicating in the cells are localized by the solid overlay and the virus spreads from initially infected cell to adjacent cell producing circumscribed foci of cellular degeneration. On 3rd day, solidified medium was scooped out, mono layer was
washed with buffer saline and stained with 0.5% of crystal violet. Virus infected cells appeared as clear unstained areas against a background of stained viable cells.
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention:
Example 1
Pyrrole and N-methyl pyrrole were distilled separately. Both these distillates were cooled at 0 °C for 30 min. Then 1 ml of the cooled N-methyl pyrrole was added to 99 ml of cooled pyrrole and were mixed together. 60 ml of this pyrrole mixture was mixed with 40 ml of ethyl acetate and two filter papers were added to the resultant mixture in a petri dish and this was kept for cooling at -4 °C for 2 hr. 1.1 molar solution of ferric chloride was prepared and cooled at -4 °C for 30 min, meanwhile the temperature of the cold bath was maintained at -5 °C and the liquid of the cold bath was allowed to pass between the walls of the container for maintaining the temperature of the container at -3 °C. The impregnated substrate i.e. pyrroles soaked filter papers were taken out and kept in double walled glass container and the container was closed with airtight lid and dry nitrogen was bubbled out in the container through the opening in the lid to maintain the inert atmosphere. The cooled ferric chloride solution was poured in the container through the opening in the lid of the container and polymerization was allowed to carry on for 5 hrs. After that the prepared membrane was taken out and washed with distilled water and kept in vacuum oven at 39 °C for 5 hrs. The conducting polymeric membrane as prepared herein was tested for virus retention on it by using stock solution of 1x104 PFU of polio I virus by the
process described above. The test results are: Virus in elute - IxlO4 PFU (100%), Virus in the filtrate - 0 PFU (0.0%).
Example 2
Pyrrole and N-methyl pyrrole were distilled separately. Both these distillates were cooled at 0 °C for 15 min. Then 2 ml of the cooled N-methyl pyrrole was added to 98 ml of cooled pyrrole and were mixed together. 33 ml of this pyrrole mixture was mixed with 27 ml of ethyl acetate and two filter papers were added to the resultant mixture in a petri dish and this was kept for cooling at 0 °C for 14 hrs. 0.95 molar solution of ferric chloride was prepared and cooled at -10 °C for 30 min, meanwhile the temperature of the cold bath was maintained at -12 °C and the liquid of the cold bath was allowed to pass between the walls of the container for maintaining temperature of the container at -10 °C. The impregnated substrate i.e. pyrroles soaked filter papers were taken out and kept in double walled glass container and the container was closed with airtight lid and dry nitrogen was bubbled out in the container through the opening in the lid to maintain the inert atmosphere. The cooled ferric chloride solution was poured in the container through the opening in the lid of the container and polymerization was allowed to carry on for 3 hrs. After that the prepared membrane was taken out and washed with distilled water and kept in vacuum oven at 30 °C for 12 hrs. The conducting polymeric membrane as prepared herein was tested for virus retention on it by using stock solution of 2xl05 PFU of polio I virus by the process described above. The test results are: Virus in elute - 2xl05 PFU (100%), Virus in the filtrate - 0 PFU (0.0%).
Example 3
Pyrrole and N-methyl pyrrole were distilled separately. Both these distillates were cooled at -4 °C for 25 min. Then 3 ml of the cooled N-methyl pyrrole was added to 27 ml of cooled pyrrole and were mixed together. 13.5 ml of this pyrrole mixture was mixed with 16.5 ml of methyl acetate and one filter paper was added to the resultant mixture in a petri dish and this was kept for cooling at 0 °C for 6 hrs. 0.8 molar solution of ferric chloride was prepared and cooled at 0 °C for 30 min, meanwhile the temperature of the cold bath was maintained at -1 °C and the liquid of the cold bath was allowed to pass between the walls of the container for maintaining the temperature of the container at 1°C. The impregnated substrate i.e. pyrroles soaked filter paper were taken out and kept in double wall container and the container was closed with airtight lid and dry nitrogen was bubbled out in the container through the opening in the lid to maintain the inert atmosphere. The cooled ferric chloride solution was poured in the container through the opening in the lid of the container and polymerization was allowed to carry on for 4 hrs. After that the prepared membrane was taken out and washed with distilled water and kept in vacuum oven at 43 °C for 5 hrs. The conducting polymeric membrane as prepared herein was tested for virus retention on it by using stock solution of Sxl03 PFU of polio I virus by the process described above. The test results are: Virus in elute - 1xl03 PFU (20%), Virus in the filtrate - 0 PFU (0.0%).
Example 4
Pyrrole and N-methyl pyrrole were distilled separately. Both these distillates were cooled at 0 °C for 45 min. Then 3 ml of the cooled N-methyl pyrrole was added to 47 ml of cooled pyrrole and were mixed together. 25 ml of this pyrrole mixture was mixed with 25 ml of ethyl acetate and two filter papers were added to the resultant mixture in a petri dish and this was kept for cooling at - 4 °C for 16 hrs. 0.9 molar solution of ferric chloride was prepared and cooled at 0 °C for 30 min, meanwhile the temperature of the cold bath was maintained at 0 °C and the liquid of the cold bath was allowed to pass between the walls of the container for maintaining the temperature of the container at 2°C. The impregnated substrate i.e. pyrroles soaked filter papers were taken out and kept in double walled glass container and the container was closed with airtight lid and dry nitrogen was bubbled out in the container through the opening in the lid to maintain the inert atmosphere. The cooled ferric chloride solution was poured in the container through the opening in the lid of the container and the polymerization was allowed to carry on for 14 hrs. After that the prepared membrane was taken out and washed with distilled water and kept in vacuum oven at 40 °C for 4 hrs. The conducting polymeric membrane as prepared herein was tested for virus retention on it by using stock solution of Sxl03 PFU of polio I virus by the process described above. The test results are: Virus in elute -l.Sxl03 PFU (30%), Virus in the filtrate - 0 PFU (0.0%).
Example 5
Pyrrole and N-methyl pyrrole were distilled separately. Both these distillates were cooled at 0 °C for 15 min. Then 2 ml of the cooled N-methyl pyrrole was added to 48 ml of cooled pyrrole and were mixed together. 24 ml of this pyrrole mixture was mixed with 26 ml of ethyl acetate and two filter papers were added to the resultant mixture in a petri dish and this was kept for cooling at 0 °C for 10 hrs. 0.95 molar solution of ferric chloride was prepared and cooled at -2 °C for 30 min, meanwhile the temperature of the cold bath was maintained at - 4 °C and the liquid of the cold bath was allowed to pass between the walls of the container for maintaining the temperature of the container at -2°C. The impregnated substrate i.e. pyrroles soaked filter papers were taken out and kept in double walled glass container and the container was closed with airtight lid and dry nitrogen was bubbled out in the container through the opening in the lid to maintain the inert atmosphere. The cooled ferric chloride solution was poured in the container through the opening in the lid of the container and polymerization was allowed to carry on for 24 hrs. After that the prepared membranes were taken out and washed with distilled water and kept in vacuum oven at 40 °C for 12 hrs. The conducting polymeric membrane as prepared herein was tested for virus retention on it by using stock solution of 5xl03 PFU of polio I virus by the process described above. The test results are: Virus in elute - Sxl03 PFU (100%), Virus in the filtrate - 0 PFU (0.0%).
Using the process of the present invention to prepare conducting polymeric membrane with potassium peroxodisulfate , potassium dichromate and ferric nitrate as the oxidising agents, it was found that the resultant membrane did not give the desired results.
The results of the conducting polymeric membranes given in the above examples have been summarized in Table I.
Table I
Example No. % of virus in the elute % of virus in the filtrate
T 100.0% 0.0 %
2. 100.0 % 0.0 %
3. 20.0 % 0.0 %
4. 30.0 % 0.0 %
5. 100.0 % 0.0 %
The advantages found in this method are: (a) No modification of pH of the water sample is required. Hence no chemical treatment of the water sample is needed, (b) About 100% viruses can be easily trapped and removed by elution process by beef extract, (c) The membranes can be easily prepared in the wide range of temperature.



We claim:
1. A process for the preparation of conducting polymeric membrane
useful as a filter for capturing viruses in potable liquids, which
comprises;
i) mixing 90 to 99.9 vol % distilled pyrrole and 10 to 0.1 vol %
distilled N-methyl pyrrole cooling the mixture to a temperature below
4°C;
ii) mixing 40 to 60 vol % alkyl acetate to 40 to 60 vol % cooled
pyrroles mixture obtained in step (i),
iii) soaking in the mixture obtained in step (ii) a porous substrate much on large
drcufedcooling the said impregnated substrate to a temperature below 4°C and
allowing it to stand for at least 1 hr;
iv) treating the impregnated substrate obtained in step (iii) with a
solution of ferric chloride in an inert atmosphere at a temperature
below 5°C for a period of at least 1 hr to obtain a conducting
polymeric membrane;
v) washing the polymeric membrane obtained in step (iv) by distilled
water or deionized water and followed by drying in oxygen free
atmosphere to obtain conducting polymeric membrane.
2. A process as claimed in claims 1 wherein, the alkyl acetate used is
methyl acetate, ethyl acetate and propyl acetate.
3. A process as claimed in claim 1 to 2 wherein the porous substrate
used is filter paper, fibre glass.
4. A process as claimed in claims 1 wherein the inert atmosphere is
maintained by using nitrogen, argon and helium gas.
5. A process for the preparation of conducting polymeric membrane
useful as a filter for capturing viruses in potable liquids,
substantially as herein described with rseference to examples.


Documents:

1302-del-1998-abstract.pdf

1302-del-1998-claims.pdf

1302-del-1998-correspondence-others.pdf

1302-del-1998-correspondence-po.pdf

1302-del-1998-description (complete).pdf

1302-del-1998-form-1.pdf

1302-del-1998-form-19.pdf

1302-del-1998-form-2.pdf

1302-del-1998-form-3.pdf

1302-del-1998-petition-138.pdf


Patent Number 215049
Indian Patent Application Number 1302/DEL/1998
PG Journal Number 10/2008
Publication Date 07-Mar-2008
Grant Date 20-Feb-2008
Date of Filing 15-May-1998
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001,
Inventors:
# Inventor's Name Inventor's Address
1 AMARJEET KAUR NARULA MAITREYI COLLEGE NEW DELHI
2 RAMADHAR SINGH NATIONAL PHYSICAL LABORATORY NEW DELHI
3 SUBHAS CHANDRA NATIONAL PHYSICAL LABORATORY NEW DELHI
4 HAWA SINGH NATIONAL PHYSICAL LABORATORY NEW DELHI
5 SHOBA BROOR MEDICAL SCIENCES, NEW DELHI
PCT International Classification Number C08J 5/22
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA