Title of Invention	"A NOVEL MOLECULARLY IMPRINTED POLYMER (MIP)-IMMOBILISED SILICA GEL SORBENT PREPARATION AND A PROCESS FOR DETERMINATION OF BARBITURIC ACID USING THE SAME MIP-BASED SORBENT"
Abstract	The present invention refers to a novei molecularly imprinte to lamer (MIP) obtainable by polymerisation of monomers in the presence of a template molecule barbituric acid (BA). The obtained linear MIP may be used for selective separation of the template analyte from blood samples in aqueous samples, effluents, pharmaceutical preparations in the presence of other structurally related interferents. Present invention further led refers to the preparation of new a silica gel bound molecularly imprinted polymer-based sorbent of column chromatography for the selective recognition of barbituric acid (BA). The optimised operaţional conditions for 100% retention of the barbituric acid on a chromatographic column (13.0 x 1.4 cm), packed with adsorbent bed 1.0 g, is realised at pH 7.0 and a flow rate of 5.0 mL min"1. The MlP-based adsorbent can be instantaneously regenerated for reuse after washing with hoţ water within 15 min.

Title of Invention

"A NOVEL MOLECULARLY IMPRINTED POLYMER (MIP)-IMMOBILISED SILICA GEL SORBENT PREPARATION AND A PROCESS FOR DETERMINATION OF BARBITURIC ACID USING THE SAME MIP-BASED SORBENT"

Abstract

The present invention refers to a novei molecularly imprinte to lamer (MIP) obtainable by polymerisation of monomers in the presence of a template molecule barbituric acid (BA). The obtained linear MIP may be used for selective separation of the template analyte from blood samples in aqueous samples, effluents, pharmaceutical preparations in the presence of other structurally related interferents. Present invention further led refers to the preparation of new a silica gel bound molecularly imprinted polymer-based sorbent of column chromatography for the selective recognition of barbituric acid (BA). The optimised operaţional conditions for 100% retention of the barbituric acid on a chromatographic column (13.0 x 1.4 cm), packed with adsorbent bed 1.0 g, is realised at pH 7.0 and a flow rate of 5.0 mL min"1. The MlP-based adsorbent can be instantaneously regenerated for reuse after washing with hoţ water within 15 min.

Full Text	Field of the invention The present invention, relates to a novei molecularly imprinted polymer (MIP) for specific recognition of pharmaceutically and industrially relevant compound i.e., barbituric acid (BA) and preparation of the ne w chromatographic column based on the same MIP or more particularly, to a method for removing exclusively barbituric acid from aquatic effluents, body fluids and medicinal preparations by exposure thereof to a molecularly imprinted polymer. The invention also relates to the use ofsaid polymers. Background of the invention and Description of Prior Art Molecular imprinting involves pre-arrangement of funcţional monomers around a template molecule (analyte) through covalent or non-covalent interactions followed by polymerisation. After the template removal, the remaining molecularly imprinted polymer (MIP) possesses a permanent memory of the original template in terms of its complementary shape and chemical functionality. These materials can be synthesised in any standard equipped laboratory in relatively short time and MIPs exhibit binding affinities and selectivity's in the order of those exhibited by antibodies towards their antigens. US Patent No. 6, 870, 021 refers to new classes of polymerisable monomers, to molecularly imprinted polymers obtainable by polymerisation of at least one of the monomers and cross-linking monomers in the presence of a template molecule. It alo refers to two classes of funcţional monomers based on the amidine-functional group, formamidine monomers etc. and their application in molecular recognition catalysis. Unlike the above-mentioned work, we have prepared linear polymers in order to avoid steric overcrowding by the template molecules and facilitated mass-transport. US Patent No. 6, 783, 686 describes a method for removing gasoline additive MTBE (Methyl tertiary butyl ether) from water using molecularly imprinted polymer (MIPs); by exposing the water to a MIP having an imprint on a surface thereof that corresponds to at least a portion of the shape of an MTBE molecules which shows the versatility of application areas of MIPs. Imprinted polymers have been used in several types of chromatographic application (Anal. Chem., News Features, 248A (1999)). These materials after grinding and sieving are of a heterogeneous and polydisperse nature with linii ted efficiency in the column, du e to slow mass transfer (Anal. Chem., 71, 928(1999)). To mitigate this problem, a layer of MIP has been grafted onto derivatized macroporous - silica and packed in HPLC columns. However, this type of coating leads to an apparent reduction in the area of MIP available for interactions owing to interfacial overlapping of MIP and silica. Furthermore, MIPs alone may undergo swelling and shrinkage cycles under the eluent effect and causing irreversible deformations of imprinted cavities resulting in the loss of selectivity. As MIPs are usually prepared and evaluated in organic solvents, their performance in an aqueous and body fluids are considered uncertain. To overcome and address all these problems, the present invention describes a typical silica-immobilised MIP accompanying in Fig. l, as recognition matrix in column chromatographic system for the selective enrichment of BA from dilute aqueous samples. The BA and its derivatives constitutes one of the most vulnerable family of sedative hypnagouge and also ascribe as poisonous, irritating toxicant, intermediate of vitamin B12, acts as a polymerisation catalyst and it is pollutants in aquatic environment (Talanta, 51, 1213 (2000)). Therefore, the determination of barbiturates in aquatic and body fluids, with a narrow therapeutic concentraţiei! index and variable rates of elimination requires a sensitive method development. Several procedures are been reported in this direction which include capillary electrophoresis (Talanta, 51, 1213 (2000)), micro-extraction technique (Anal. Chem. 71, 2146 (1999)) also discloses a procedure. But unlike our present invention they are time consuming, costly and need complicated experimental design. Our present invention deals with the silica-MIP system which is found suitable to overcome all the problems related with swelling and shrinkage effect, slow mass-transfer, column clogging, steric overcrowding and found to be stable to chemical, mechanical and thermal impacts with no sign of deterioration of imprinting sites and work better as truly robust and rigid stationary support. for selective recognition of small as well as bulkier molecules of target analytes. Qbjecf and statement of the invention It is an object of the present invention to prepare a molecularly imprinted polymer for BA, which is economical, comprising of simple steps and safer to use; can be effective for commercial and industrial purposes. It comprises the following steps: (A) Equimolar DMF solution of melamine (formula I) monomer and chloranil monomer (formula II) were mixed together, (B) Volume content of the mixture was reduced to half by evaporating the solvent (C) Added to it equimolar DMF solution of the template BA (formula III) into the reaction mixture, which was refluxed and kept overnight, (D) A brownish black coloured product was obtained with the help of filtering and after complete removal of the solvent which results melamine-chloranil-barbituric acid complex (Mel-Chl-BA)adduct (formula IV), dried in hoţ air oven. (E) Removal of the BA was made by hoţ water with the help of multiple extraction technique, which give rise to molecularly imprinted polymer (MIP) for BA (formula V). It is further object of the present invention to provide a method for removing BA from aquatic and body fluids. Determination of BA in real world samples viz., blood plasma as well as in the presence of interfering substance were made using the MlP-modified silica-gel sorbent in column chromatography which comprises the following steps (1) In the loading step, a known volume of test solution of BA was passed through the sorbent.. The column effluent was directed as waste, (2) The elution step consisting of removal of BA from column using hoţ water, (3) The eluate was then subjected to a suitable technique. It is further object of the invention is to provide a reusable method for removing BA from aqueous medium and blood samples using molecularly imprinted polymers (MIP) which is effective for conimercial as well as industrial purposes. Also an important object of the present invention is to prepare a new chromatographic column using the above mentioned MIP for BA as sorbent that is highly selective in removing the BA molecules while leaving other molecules unaffected comprising the following steps (1) For immobilisation of molecularly imprinted polymer over silica gel, silica gel was shaken with DMF solution adduct polymer of formula IV, (2) AII the DMF content was evaporated off to get a brown coloured coating of polymer on the surface of silica gel, (3) After washing with DMF, the resultant product dried in hoţ air oven, (4) The modified silica-gel ,so produced was packed into the column, (5) Removal of template BA was made using multiple extraction. To assist with understanding the invention, reference will now be made to the accompanying drawings, which shows example of the invention. Bricf description of the accompanying clrawing Accompanying drawing Fig. l describes the preconcentration chromatographic column (13.0 x 1.4 cm). Detailed Description of the Invention The invention pertains, to method and a process for preparation of novei molecularly imprinted polymer (MIP). In one embodiment, the invention pertains to a detection method that can detect barbituric acid (BA) in aquatic samples and body fluids. The invention includes method for preparation of new silica gel- modified molecularly imprinted polymer based sorbent for column chromatography. The obtained MlP-based sorbent can be used for separation of BA from structurally related compounds. In one embodiment of the present invention and contrast to earlier prior art (US Patent 6, 310, 110, and US Patent 6, 852, 818 & US Patent 6, 759, 488) instead of mixing the template with funcţional monomers prior to polymerisation, linear polymeric chains are mixed with the template and the pre-existing polymer chains are further polymerised. However, in the present instance, we preferred enlarged polymer chain rather to cross-linking in order to avoid steric overcrowding and to facilitate unhindered mass-transfer for the template diffusion across the polymeric membrane. The polymerisation mixture includes formula I melamine and formula II chloranil i.e. (funcţional monomers). The template BA in formula III added to the polymerisation mixture, after polymerisation the mixture for 5 h, leading to an adduct compound depicted in formula IV. The bound template molecules in the adduct can be completely removed by multiple extraction technique using hoţ water, leading to formula V (MIP). (Figure Removed) For preparation of linear novei molecularly imprinted polymer in a single batch, the equimolar DMF solution of melamine (formula 1) monomer and chloranil monomer (formula II) were mixed together and volume content was reduced to half by evaporating the solvent at 160°C. Then, after adding an equimolar DMF solution of the template (formula III) into it, the reaction mixture was refluxed for 5 h and kept overnight. A brownish black coloured product was obtained after complete removal of the solvent. This resulting mel-chl-BA (BA complex with the monomers)adducts shown in formula IV was dried in hot-air oven. Removal of the template was made by hoţ water at temperature 80-100°C using multiple extraction technique (n = 6, shaking time = 10 min) with 5.0 mL portions of hoţ water, which gives rise to formula V. In a further embodiment of the present invention, molecular imprinting and its immobilisation over silica-gel is carried out simultaneously in one pot synthesis in order to obtain a uniformly coated adsorbent. The support used in the present invention is preferably selected from the group consisting of porous and non-porous, planar and non-planar support. An example of such support material may be silica-gel. In the process for preparing novei MlP-based column sorbent 2.0 g of silica gel was shaken with DMF solution of mel-chl-BA adduct (formula IV) for 2 h. AII the DMF content are evaporated off to get a brown coloured coating of polymer on the surface of silica-gel. After washing with 10% DMF the resultant product dried in hoţ air oven. The modified silica-gel, so produced was packed into the column and the template BA was removed from the column with the help of hoţ water (80°-90°C temperature, using multiple extraction technique). Accompanying drawing (Fig. 1) showed the chromatographic column with corning glass tube, which is 13.0 cm long and 1.4 cm in internai diameter. Such a gravity-fed bore column is utilised in either preparative chromatography to increase the yield of a purification step or for the isolation and estimation of analyte from a large sample volume. The prepared sorbent was packed into the column to a height of 1.3 cm supported by a G-1 sintered coarse glass frit fixed 2.0 cm above the drainage Teflon ţap in the bottom of the column. In the loading step, a known volume of appropriate (pH 7.0) is passed through the sorbent at optimised flow rate of 5.0 mL/min using peristaltic pump. Finally, in the elution step, removal of BA from the column after 15 min equilibration time using hoţ water (80-100°C). The eluate BA was subjected to any suitable method (We can opt differential puise, cathodic stripping voltammetry (DPCSV) measurement ;Instrument : PAR model 264 A Voltammetric analyzer, Standard addition method), optimum conditions: accumulation potenţial +0.3 V vs. Ag/AgCl, pH 7.0. At optimised flow rate, pH, elution volume determination of BA can be made using any of the suitable analytical method. Once the integrity of the method is developed with known concentration of analyte BA, the method can also be applied for real sample analyses, in the presence of complex matrices of natural samples/biological fluids/ pharmaceutical preparations. The technique is not only effective for clean up of the samples but is also for enrichment of the sample at micro molar level. The present invention also describes the specificity and selectivity of MIP by testing the efficiency with reference polymer. It may be further noted in this context that the silica-reference polymer absorbed is found to be totally ineffective toward BA binding during column operation at pH ~7.0 and a flow of 5.0 mL min" . The following examples are given for the purposes of illustrating the present invention Example l Novei Molecularly imprinted polymer preparation For preparation of linear novei molecularly imprinted polymer in a single batch, the equimolar DMF solution of melamine (formula I) monomer (1.26 g/10 mL) and chloranil monomer (formula II) (2.46 g/10 mL) were mixed together and volume content was reduced to half by evaporating the solvent at 160°C. Then, after adding an equimolar DMF solution of the template (formula II1)(1.30 g BA/10 mL) into it, the reaction mixture was refluxed for 5 h and kept ovemight. A brownish black coloured product was obtained after complete removal of the solvent. This resulting mel-chl-BA adduct as shown in formula IV was dried in hot-air oven. Removal of template was niade by hoţ water using multiple extraction technique (n = 6, shaking time =10 min) with 5.0 mL portions of hoţ water, which gives rise to formula V. Example II Procesş for preparing novei MlP-based column şorbent. In this process, molecular imprinting and its immobilization over silica gel is carried out siimiltaneously together in one-pot synthesis to get a uniformly coated sorbent. For this, 2.0 g of silica gel is shalcen for 2 h with 10 mL DMF solution of 0.0165 g of formula IV (mel-chl-BA adduct) as prepared in example 1. AII the DMF content are evaporated off to get a brown coloured coating of polymer on the surface of silica gel. After washing with 10% DMF, the resultant product dried in hoţ air oven. The modified- silica-gel (1.0 g), so produced is packed into column and template BA is removed from the column using multiple extractions (n = 6, equilibration time 15 min) with 5.0 mL portions of hoţ water. Example III Detection of BA (ppm level) in pharmaceutical preparations and blood samples using novei MlP-based sorbent in column chromatography. Once the integrity of method is established with known concentration of analyte, the method proposed here is also applied for real analysis, in the presence of complex matrices of natural samples/biological fluids (Tables I & II). The present invention is not only effective for clean up of the BA sample but it also for an effective enrichment of the test sample at micro molar level.Table l Sample enrichment of barbituric acid on Silica-MIP tnodified sorbent (Table Removed) Concentration of BA in at 10.0 mL min~ and pH 7.0 b Concentration of desorbed BA ((j.g/mL) detected by differential puise, cathodic stripping voltammetry (DPCSV) with RSD (n = 3) desoiption by 10 mL hoţ water. c Enrichment factor [BA]out/[BA]in d Recovery(amount of desorbed BA)/(amount of BA in sample) Table II Selectivitv test of MlP-based sorbent (Table Removed) In equimolar binary mixture of BA and its interfering substances at optimum pH 7.0 and flow rate 5.0 mL min"1. CLAIMS We claim, Claim 1. A MIP of formula V, prepared by comprising the steps: a) Mixing the equimolar DMF solution of melamine (formula I) monomer and chloranil monomer (formula II). b) Deducing the volume of mixture to the half by evaporating the solvent c) Adding an equimolar solution of the template BA (formula III) to the mixture and refluxing the reaction mixture for 5 h which was kept for ovemight. d) Separating the reaction product by filtering. e) Extraction of the template BA by hoţ water which give rise to MIP (formula V), f) Dried the MIP using hoţ air oven. (Figure Removed) Claim 2. A process for preparing MIP for BA comprising the steps a) Mixing the equimolar DMF solutions of monomers, melamine (formula I) and chloranil (formula II). b) Reducing the volume of the mixture to half by evaporation. c) Addition of the template molecule (BA) in DMF (formula III) to the mixture followed by refluxing the mixture and it was kept for overnight. d) Separating the reaction product by filtration. e) Extraction of the template (BA) by hoţ water. f) Dried the MIP (formula V) in hoţ air oven. Claim 3: A MlP-modified silica gel sorbent is prepared by a) Mixing the dimethyl formamide solution (DMF) of compound (formula IV) with the silica gel. b) Evaporation of the DMF content to get a brown coloured MIP- modified silica-gel. d) Washing the MlP-modified silica gel with DMF solution. e) Removal of BA using hoţ water at temperature by multiple extraction technique. Claim 4: A chromatographic column prepared by packing the MIP modified silica-gel of claim 3 in a column. Claim 5: A process for determinaţion/ removal of barbituric acid (BA) comprising the steps a) Passing a test solution which includes blood solution, aqueous solution and pharmaceutical preparation solution through the MIP-modified silica-gel packed chromatographic column. b) The column effluent is directed as waste. c) In the elution step, the bound BA is be completely stripped off from the column by hoţ water (n = 6, multiple extraction technique). d) The eluate is then subjected to any suitable detection technique.

Full Text

Field of the invention
The present invention, relates to a novei molecularly imprinted polymer (MIP) for specific recognition of pharmaceutically and industrially relevant compound i.e., barbituric acid (BA) and preparation of the ne w chromatographic column based on the same MIP or more particularly, to a method for removing exclusively barbituric acid from aquatic effluents, body fluids and medicinal preparations by exposure thereof to a molecularly imprinted polymer. The invention also relates to the use ofsaid polymers.
Background of the invention and Description of Prior Art
Molecular imprinting involves pre-arrangement of funcţional monomers around a template molecule (analyte) through covalent or non-covalent interactions followed by polymerisation. After the template removal, the remaining molecularly imprinted polymer (MIP) possesses a permanent memory of the original template in terms of its complementary shape and chemical functionality.
These materials can be synthesised in any standard equipped laboratory in relatively short time and MIPs exhibit binding affinities and selectivity's in the order of those exhibited by antibodies towards their antigens.
US Patent No. 6, 870, 021 refers to new classes of polymerisable monomers, to molecularly imprinted polymers obtainable by polymerisation of at least one of the monomers and cross-linking monomers in the presence of a template molecule. It alo refers to two classes of funcţional monomers based on the amidine-functional group, formamidine monomers etc. and their application in molecular recognition catalysis. Unlike the above-mentioned work, we have prepared linear polymers in order to avoid steric overcrowding by the template molecules and facilitated mass-transport. US Patent No. 6, 783, 686 describes a method for removing gasoline additive MTBE (Methyl tertiary butyl ether) from water using molecularly imprinted polymer (MIPs); by
exposing the water to a MIP having an imprint on a surface thereof that corresponds to at least a portion of the shape of an MTBE molecules which shows the versatility of application areas of MIPs.
Imprinted polymers have been used in several types of chromatographic application (Anal. Chem., News Features, 248A (1999)). These materials after grinding and sieving are of a heterogeneous and polydisperse nature with linii ted efficiency in the column, du e to slow mass transfer (Anal. Chem., 71, 928(1999)). To mitigate this problem, a layer of MIP has been grafted onto derivatized macroporous - silica and packed in HPLC columns. However, this type of coating leads to an apparent reduction in the area of MIP available for interactions owing to interfacial overlapping of MIP and silica. Furthermore, MIPs alone may undergo swelling and shrinkage cycles under the eluent effect and causing irreversible deformations of imprinted cavities resulting in the loss of selectivity. As MIPs are usually prepared and evaluated in organic solvents, their performance in an aqueous and body fluids are considered uncertain.
To overcome and address all these problems, the present invention describes a typical silica-immobilised MIP accompanying in Fig. l, as recognition matrix in column chromatographic system for the selective enrichment of BA from dilute aqueous samples. The BA and its derivatives constitutes one of the most vulnerable family of sedative hypnagouge and also ascribe as poisonous, irritating toxicant, intermediate of vitamin B12, acts as a polymerisation catalyst and it is pollutants in aquatic environment (Talanta, 51, 1213 (2000)). Therefore, the determination of barbiturates in aquatic and body fluids, with a narrow therapeutic concentraţiei! index and variable rates of elimination requires a sensitive method development.
Several procedures are been reported in this direction which include capillary electrophoresis (Talanta, 51, 1213 (2000)), micro-extraction technique (Anal. Chem. 71, 2146 (1999)) also discloses a procedure. But unlike our present invention they are time consuming, costly and need complicated experimental design.
Our present invention deals with the silica-MIP system which is found suitable to overcome all the problems related with swelling and shrinkage effect, slow mass-transfer, column clogging, steric overcrowding and found to be stable to chemical, mechanical and thermal impacts with no sign of deterioration of imprinting sites and work better as truly robust and rigid stationary support. for selective recognition of small as well as bulkier molecules of target analytes.
Qbjecf and statement of the invention
It is an object of the present invention to prepare a molecularly imprinted polymer for BA, which is economical, comprising of simple steps and safer to use; can be effective for commercial and industrial purposes. It comprises the following steps: (A) Equimolar DMF solution of melamine (formula I) monomer and chloranil monomer (formula II) were mixed together, (B) Volume content of the mixture was reduced to half by evaporating the solvent (C) Added to it equimolar DMF solution of the template BA (formula III) into the reaction mixture, which was refluxed and kept overnight, (D) A brownish black coloured product was obtained with the help of filtering and after complete removal of the solvent which results melamine-chloranil-barbituric acid complex (Mel-Chl-BA)adduct (formula IV), dried in hoţ air oven. (E) Removal of the BA was made by hoţ water with the help of multiple extraction technique, which give rise to molecularly imprinted polymer (MIP) for BA (formula V).
It is further object of the present invention to provide a method for removing BA from aquatic and body fluids. Determination of BA in real world samples viz., blood plasma as well as in the presence of interfering substance were made using the MlP-modified silica-gel sorbent in column chromatography which comprises the following steps (1) In the loading step, a known volume of test solution of BA was passed through the sorbent.. The
column effluent was directed as waste, (2) The elution step consisting of removal of BA from column using hoţ water, (3) The eluate was then subjected to a suitable technique.
It is further object of the invention is to provide a reusable method for removing BA from aqueous medium and blood samples using molecularly imprinted polymers (MIP) which is effective for conimercial as well as industrial purposes.
Also an important object of the present invention is to prepare a new chromatographic column using the above mentioned MIP for BA as sorbent that is highly selective in removing the BA molecules while leaving other molecules unaffected comprising the following steps (1) For immobilisation of molecularly imprinted polymer over silica gel, silica gel was shaken with DMF solution adduct polymer of formula IV, (2) AII the DMF content was evaporated off to get a brown coloured coating of polymer on the surface of silica gel, (3) After washing with DMF, the resultant product dried in hoţ air oven, (4) The modified silica-gel ,so produced was packed into the column, (5) Removal of template BA was made using multiple extraction. To assist with understanding the invention, reference will now be made to the accompanying drawings, which shows example of the invention.
Bricf description of the accompanying clrawing
Accompanying drawing Fig. l describes the preconcentration chromatographic column (13.0 x 1.4 cm).

Detailed Description of the Invention
The invention pertains, to method and a process for preparation of novei molecularly imprinted polymer (MIP). In one embodiment, the invention pertains to a detection method that can detect barbituric acid (BA) in aquatic samples and body fluids. The invention includes method for preparation of new silica gel- modified molecularly imprinted polymer based sorbent for column chromatography. The obtained MlP-based sorbent can be used for separation of BA from structurally related compounds.
In one embodiment of the present invention and contrast to earlier prior art (US Patent 6, 310, 110, and US Patent 6, 852, 818 & US Patent 6, 759, 488) instead of mixing the template with funcţional monomers prior to polymerisation, linear polymeric chains are mixed with the template and the pre-existing polymer chains are further polymerised. However, in the present instance, we preferred enlarged polymer chain rather to cross-linking in order to avoid steric overcrowding and to facilitate unhindered mass-transfer for the template diffusion across the polymeric membrane.
The polymerisation mixture includes formula I melamine and formula II chloranil i.e. (funcţional monomers). The template BA in formula III added to the polymerisation mixture, after polymerisation the mixture for 5 h, leading to an adduct compound depicted in formula IV.
The bound template molecules in the adduct can be completely removed by multiple extraction technique using hoţ water, leading to formula V (MIP).

(Figure Removed)
For preparation of linear novei molecularly imprinted polymer in a single batch, the equimolar DMF solution of melamine (formula 1) monomer and chloranil monomer (formula II) were mixed together and volume content was reduced to half by evaporating the solvent at 160°C. Then, after adding an equimolar DMF solution of the template (formula III) into it, the reaction

mixture was refluxed for 5 h and kept overnight. A brownish black coloured product was obtained after complete removal of the solvent. This resulting mel-chl-BA (BA complex with the monomers)adducts shown in formula IV was dried in hot-air oven. Removal of the template was made by hoţ water at temperature 80-100°C using multiple extraction technique (n = 6, shaking time = 10 min) with 5.0 mL portions of hoţ water, which gives rise to formula V.
In a further embodiment of the present invention, molecular imprinting and its immobilisation over silica-gel is carried out simultaneously in one pot synthesis in order to obtain a uniformly coated adsorbent. The support used in the present invention is preferably selected from the group consisting of porous and non-porous, planar and non-planar support. An example of such support material may be silica-gel. In the process for preparing novei MlP-based column sorbent 2.0 g of silica gel was shaken with DMF solution of mel-chl-BA adduct (formula IV) for 2 h. AII the DMF content are evaporated off to get a brown coloured coating of polymer on the surface of silica-gel. After washing with 10% DMF the resultant product dried in hoţ air oven. The modified silica-gel, so produced was packed into the column and the template BA was removed from the column with the help of hoţ water (80°-90°C temperature, using multiple extraction technique).
Accompanying drawing (Fig. 1) showed the chromatographic column with corning glass tube, which is 13.0 cm long and 1.4 cm in internai diameter. Such a gravity-fed bore column is utilised in either preparative chromatography to increase the yield of a purification step or for the isolation and estimation of analyte from a large sample volume. The prepared sorbent was packed into the column to a height of 1.3 cm supported by a G-1 sintered coarse glass frit fixed 2.0 cm above the drainage Teflon ţap in the bottom of the column. In the loading step, a known volume of appropriate (pH 7.0) is passed through the sorbent at optimised flow rate of 5.0 mL/min using peristaltic pump. Finally, in the elution step, removal of BA from the column after 15 min equilibration time using hoţ water (80-100°C). The eluate BA was subjected to any suitable method (We can opt differential puise, cathodic stripping voltammetry
(DPCSV) measurement ;Instrument : PAR model 264 A Voltammetric analyzer, Standard addition method), optimum conditions: accumulation potenţial +0.3 V vs. Ag/AgCl, pH 7.0. At optimised flow rate, pH, elution volume determination of BA can be made using any of the suitable analytical method.
Once the integrity of the method is developed with known concentration of analyte BA, the method can also be applied for real sample analyses, in the presence of complex matrices of natural samples/biological fluids/ pharmaceutical preparations.
The technique is not only effective for clean up of the samples but is also for enrichment of the sample at micro molar level.
The present invention also describes the specificity and selectivity of MIP by testing the efficiency with reference polymer. It may be further noted in this context that the silica-reference polymer absorbed is found to be totally ineffective toward BA binding during column operation at pH ~7.0 and a flow of 5.0 mL min" .
The following examples are given for the purposes of illustrating the present invention
Example l
Novei Molecularly imprinted polymer preparation
For preparation of linear novei molecularly imprinted polymer in a single batch, the equimolar DMF solution of melamine (formula I) monomer (1.26 g/10 mL) and chloranil monomer (formula II) (2.46 g/10 mL) were mixed together and volume content was reduced to half by evaporating the solvent at 160°C. Then, after adding an equimolar DMF solution of the template (formula II1)(1.30 g BA/10 mL) into it, the reaction mixture was refluxed for 5 h and kept ovemight. A brownish black coloured product was obtained after complete removal of the solvent. This resulting mel-chl-BA adduct as shown in
formula IV was dried in hot-air oven. Removal of template was niade by hoţ water using multiple extraction technique (n = 6, shaking time =10 min) with 5.0 mL portions of hoţ water, which gives rise to formula V.
Example II
Procesş for preparing novei MlP-based column şorbent.
In this process, molecular imprinting and its immobilization over silica gel is carried out siimiltaneously together in one-pot synthesis to get a uniformly coated sorbent. For this, 2.0 g of silica gel is shalcen for 2 h with 10 mL DMF solution of 0.0165 g of formula IV (mel-chl-BA adduct) as prepared in example 1. AII the DMF content are evaporated off to get a brown coloured coating of polymer on the surface of silica gel. After washing with 10% DMF, the resultant product dried in hoţ air oven. The modified- silica-gel (1.0 g), so produced is packed into column and template BA is removed from the column using multiple extractions (n = 6, equilibration time 15 min) with 5.0 mL portions of hoţ water.
Example III
Detection of BA (ppm level) in pharmaceutical preparations and blood samples using novei MlP-based sorbent in column chromatography.
Once the integrity of method is established with known concentration of analyte, the method proposed here is also applied for real analysis, in the presence of complex matrices of natural samples/biological fluids (Tables I & II). The present invention is not only effective for clean up of the BA sample but it also for an effective enrichment of the test sample at micro molar level.Table l Sample enrichment of barbituric acid on Silica-MIP tnodified sorbent
(Table Removed)

Concentration of BA in at 10.0 mL min~ and pH 7.0
b Concentration of desorbed BA ((j.g/mL) detected by differential puise, cathodic stripping voltammetry (DPCSV) with RSD (n = 3) desoiption by 10 mL hoţ water. c Enrichment factor [BA]out/[BA]in d Recovery(amount of desorbed BA)/(amount of BA in sample)

Table II Selectivitv test of MlP-based sorbent

(Table Removed)
In equimolar binary mixture of BA and its interfering substances at optimum pH 7.0 and flow rate 5.0 mL min"1.

CLAIMS
We claim,
Claim 1.
A MIP of formula V, prepared by comprising the steps: a) Mixing
the equimolar DMF solution of melamine (formula I) monomer and chloranil monomer (formula II). b) Deducing the volume of mixture to the half by evaporating the solvent c) Adding an equimolar solution of the template BA (formula III) to the mixture and refluxing the reaction mixture for 5 h which was kept for ovemight. d) Separating the reaction product by filtering. e) Extraction of the template BA by hoţ water which give rise to MIP (formula V), f) Dried the MIP using hoţ air oven.
(Figure Removed)
Claim 2. A process for preparing MIP for BA comprising the steps a) Mixing the equimolar DMF solutions of monomers, melamine (formula I) and chloranil (formula II). b) Reducing the volume of the mixture to half by evaporation. c) Addition of the template molecule (BA) in DMF (formula III) to the mixture followed by refluxing the mixture and it was kept for overnight. d) Separating the reaction product by filtration. e) Extraction of the template (BA) by hoţ water. f) Dried the MIP (formula V) in hoţ air oven.
Claim 3: A MlP-modified silica gel sorbent is prepared by a) Mixing the dimethyl formamide solution (DMF) of compound (formula IV) with the silica gel. b) Evaporation of the DMF content to get a brown coloured MIP- modified silica-gel. d) Washing the MlP-modified silica gel with DMF solution. e) Removal of BA using hoţ water at temperature by multiple extraction technique.
Claim 4: A chromatographic column prepared by packing the MIP modified silica-gel of claim 3 in a column.
Claim 5: A process for determinaţion/ removal of barbituric acid (BA) comprising the steps a) Passing a test solution which includes blood solution, aqueous solution and pharmaceutical preparation solution through the MIP-modified silica-gel packed chromatographic column. b) The column effluent is directed as waste. c) In the elution step, the bound BA is be completely stripped off from the column by hoţ water (n = 6, multiple extraction technique). d) The eluate is then subjected to any suitable detection technique.

Documents:

2614-DEL-2005-Abstract (21-11-2007).pdf

2614-del-2005-abstract.pdf

2614-DEL-2005-Claims (21-11-2007).pdf

2614-DEL-2005-Claims-(13-02-2008).pdf

2614-del-2005-claims.pdf

2614-DEL-2005-Correspondence-Others (21-11-2007).pdf

2614-DEL-2005-Correspondence-Others-(13-02-2008).pdf

2614-del-2005-correspondence-others.pdf

2614-del-2005-correspondence-po.pdf

2614-DEL-2005-Description (Complete) (21-11-2007).pdf

2614-DEL-2005-Description (Complete)-(13-02-2008).pdf

2614-del-2005-description (complete).pdf

2614-DEL-2005-Drawings (29-9-2005).pdf

2614-del-2005-drawings.pdf

2614-del-2005-form-1.pdf

2614-del-2005-form-18.pdf

2614-DEL-2005-Form-2 (21-11-2007).pdf

2614-DEL-2005-Form-2-(13-02-2008).pdf

2614-del-2005-form-2.pdf

2614-del-2005-form-9.pdf

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Patent Number

215355

Indian Patent Application Number

2614/DEL/2005

PG Journal Number

11/2008

Publication Date

14-Mar-2008

Grant Date

26-Feb-2008

Date of Filing

29-Sep-2005

Name of Patentee

DR. PRASAD, BHIM BALI

Applicant Address

CHEMISTRY DEPARTMENT, FACULTY OF SCIENCE, BANARAS HINDU UNIVERSITY, VARANASI - 221005, U.P., INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. PRASAD, BHIM BALI	CHEMISTRY DEPARTMENT, FACULTY OF SCIENCE, BANARAS HINDU UNIVERSITY, VARANASI - 221005, U.P., INDIA.
2	DHANA LAKSHMI	CHEMISTRY DEPARTMENT, FACULTY OF SCIENCE, BANARAS HINDU UNIVERSITY, VARANASI - 221005, U.P., INDIA.
3	SHARMA, PIYUSH SINDHU	CHEMISTRY DEPARTMENT, FACULTY OF SCIENCE, BANARAS HINDU UNIVERSITY, VARANASI - 221005, U.P., INDIA.

PCT International Classification Number

B01D 71/02

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA