Title of Invention	"A METHOD FOR PREPARATION OF A SENSOR MATERIAL FOR DETECTION, DISCRIMINATION AND ESTIMATION OF AMINES"
Abstract	A method for preparation of a sensor material for visual detection, discrimination and estimation of amines is described. The detection and discrimination of monoamines and diamines is used as indirect detection of freshness of meat particularly sea food and specifically fish or shell fish. The method comprising condensation of para tertiarybutyl phenol with formaldehyde, then subjecting to cyclisation and dealkylation, followed by coupling, in any known manner, with diazotised substituted compounds selected from diazotised substituted aryl amines or amino acids or substituted biphenyls to yield a novel calyx(n)arenes, optionally purifying by conventional methods, and absorbing on a suitable article/medium to yield sensor material.

Title of Invention

"A METHOD FOR PREPARATION OF A SENSOR MATERIAL FOR DETECTION, DISCRIMINATION AND ESTIMATION OF AMINES"

Abstract

A method for preparation of a sensor material for visual detection, discrimination and estimation of amines is described. The detection and discrimination of monoamines and diamines is used as indirect detection of freshness of meat particularly sea food and specifically fish or shell fish. The method comprising condensation of para tertiarybutyl phenol with formaldehyde, then subjecting to cyclisation and dealkylation, followed by coupling, in any known manner, with diazotised substituted compounds selected from diazotised substituted aryl amines or amino acids or substituted biphenyls to yield a novel calyx(n)arenes, optionally purifying by conventional methods, and absorbing on a suitable article/medium to yield sensor material.

Full Text	FIELD OF INVENTION: The present invention relates to method for preparation of a sensor material for detection, discrimination and estimation of amines. Particularly the present invention relates to a sensor material for visual detection and discrimination of amines, more particularly the present invention relates to a sensor material for visual detection and discrimination of bio-amines and aliphatic amines, even more particularly it relates to a sensor material for visual detection and discrimination of bio-amines, mono-amines, di-amines and poly-amines and to the method of preparation of the said sensor material. The present invention also relates to a sensor material for detection, discrimination and estimation of amines, wherein said sensor amine is suitable for making test kits and paper strips testing and discrimination of amines. BACKGROUND OF THE INVENTION: It is known in the art that, food, such as meat, fish, shellfish and milk obtained from the infected animal source is a primary source of some notable helminthic and bacterial diseases, which get transmitted to a person who eat such food in raw form or partially cocked or treated form. The heminthic diseases include those caused by pork, cattle and fish tapeworm ingested as larval forms encysted in the muscles of these animals and the trematode infestations acquired by eating fish and water plants infected with larvae. Although the pathogenic symptoms of these diseases differ in many respects, their epidemiology is similar with regard to sources and routes of infection. Further, it is also known in the art that, the contaminated food is a potential occupational health hazard for housewives and cooks, if they taste such food in raw form or in partially cooked or treated form. Still further, the contaminated food, like old meat, fish, shellfish etc. frequently becomes a source of primary or a secondary reservoir of serious infections and a source of dangerous consequences due to pathogenic microorganisms, which grow due to release of amines from inside of body onto the skin of the food, like meat; fish, shellfish etc. which in-turn enhances the growth of microorganisms and in-turn food gets decomposed and cause the helminthic disease. Still serious problems occurs, when contamination of original food by pathogenic microorganisms can assume dangerous secondary reservoirs of infectious diseases or production centers for poisonous bacteria toxins, particularly when such food is improperly preserved, handled or prepared, which in-turn may result in microbial multiplication before it is eaten. It is known that, greater the time has elapsed after food or contaminated food, particularly fish or shellfish is in air, the greater is the chance of its getting infected and consequent out break of helminthic diseases. Particularly in the case food, like fish or shellfish etc., as soon as fish or shellfish is taken out of water, it is susceptible to putrefaction, if not immediately preserved under low temperatures and other treatments. It has been observed that, fish or shellfish starts releasing a mixture of amines, particularly lower amines, and more particularly aliphatic amines. The concentration of amines released in the atmosphere is directly related to the time the fish or shellfish etc. has been out of the water and not properly preserved. It has been further observed that the greater the concentration of released specific ratio of particular amines, the greater is the fish being with pathogenic bacteria. The microbes multiply faster in the environment of these amines. Generally visual examination of food, particularly offish etc. is considered satisfactory for quick results. The major drawback of such method is that, the person testing the food, particularly the fish etc. should be experienced and expert in the area. Generally such experts and experienced persons are housewives or cooks, who normally do not purchase the food. The purchasers are generally un-experienced and will have to depend on the seller's statement. There is no easy method, particularly non-instrumental for determining fish freshness. Therefore, the need was felt to develop a scientific method, particularly a sensor material to detect, discriminate and estimate the released amines from the food or contaminated food, particularly from fish or shell fish etc. with the growing scientific procedures of preservation and transportation of food, particularly in the area of fisheries, which has attracted considerable attention due to its availability in abundance and short life and high food value. One such known method involve olfactory analysis, or gas-chromatographic coupled with mass spectrophotometric (GC-MS) analysis. The major drawback of such known methods is that, these are time consuming and require costly equipment. Therefore, such known method do not meet the requirement of quicker and economical method of testing and estimating the freshness of food, particularly of fish. Further, limitation of such known methods is that, the released amines are difficult to be detected chemically. Another such known methods are based on radio-immuno assay and fluorescence techniques. Such methods also suffer drawback of being time consuming and requiring costly instrumentation for performing such methods, which limit their use only in the research laboratories. Still another known method to test and estimate the freshness of the food, particularly of fish is the use synthetic chemicals, which can form complexes with released amines and such complexes can be detected by change in color and intensity of changed color. Meeting this requirement, some of the chemical reagents have been made available for the said purpose. Some of the presently available reagents are Bratton-Marshall reagent, Paulis reagent and Ninhydrin reagent. Despite extensive usefulness, the unambiguous detection and discrimination of bio-amines, particularly of aliphatic amines have been scantly studied. The methods made available, as describe herein above, generally employ sophisticated instruments or expensive reagents and further such known methods are difficult to operate due to want of skilled manpower. Further, if operated, the results may be misleading due to reasons explained above. Still further, such methods can not be employed in the mobile form or in the remote areas. Recently, there has been another set of chemicals reported in the literature for the said purpose, which are particularly derived from organic meta-cyclopharenes. Such chemicals are known in the prior art as functionalised calixarenes in the contemporary literature. The limitation of such calixareness is that, no derivative of known calixareness show a marked changed in color on interaction with the released amines for clear and easy testing and estimation of freshness of food, particularly offish. Further, such known calixarenes do not differentiate between monoamines, diamines and polyamines, which is the need of the present art. Further known calixarenes exist as calix (n) areness (value of n=4-8) and one has to synthesize a particular calixarenes which can be employed as a reagent for detection and discrimination of amines. Still further, the confrontation of the synthesized calixarene based reagents has not been reported, which is important for using such known reagents, particularly in case of calixarenes. A small change in the conformation of calixarene backbone may lead to false signals, because calixarenes are flexible and their conformation is difficult to freeze without functionalization. Consequently, the use of calixarenes based reagents becomes difficult for discriminating different types of aliphatic amines and the very purpose of the development is forfeited. Further no method is available which freezes the calixarenes with predetermined conformation. Still further no knowledge about a particular conformation of calixarenes exist which is known to change color on interaction with aliphatic amines, Therefore, it is obvious from the forgoing description, that the currently known methods or reagents or chemical reagents are not sufficient to allow exact determination of food, particularly fish freshness. The presently known reagents are therefore not only limited to their use for determination of food, particularly fish freshness but also the concepts where biological amines are to be detected and estimated. Need of the Invention: Therefore, there is a need to have a method, preferably to have a reagent, more preferably to have a chemical reagent herein after as a sensor material, which can overcome some of the drawbacks and disadvantages of the known methods and reagents, as described herein above, and is suitable to detect the released amines and also to discriminate between various released amines, such as bio-amines, mono-amines, di-amines and poly amines and to estimate the amount of released amine. Further, the need is to have a sensor material, which can be used universally, even in remote areas. Still further there is a need for a method which can freeze the conformation of the calixarenes. Yet further there is a need for obtaining calixarenes which change color on interaction with amines. Objects of the Invention: The main object of the present invention is to disclose a method, preferably to disclose a sensor material, which can over some of the drawbacks and disadvantages of such known methods and reagents, as described herein above, and is suitable to detect the released amines and also to discriminate between various released amines, such as bio-amines, mono-amines, di-amines and poly-amines and to estimate the amount of released amine. Another object of the present invention is to propose for a sensor material, which can be used universally, even in remote areas. This is another an object of the present invention to propose for a sensor material the use of which does not require experienced and expert person in the area at least for detection of released amines and estimating the freshness of food, particularly offish etc. when the presently disclosed sensor material is used in the form of a paper strip, standard indicator strip. Still another an object of the present invention is to propose for a sensor material, the use of which is neither time consuming nor involve costly equipment. Therefore, meeting the requirement of quicker and economical method of testing and estimation of freshness of food, particularly offish. Yet another an object of the present invention is to achieve a sensor material, which can eliminate the use of olfactory analysis, and gas-chromatographic coupled with mass spectrophotometric (GC-MS) analysis, and of methods based on radio-immuno assay and fluorescence techniques for discrimination of aliphatic amines, hence is highly economical and easy to use, and is not limited to use in the research laboratories. This is yet an object of the present invention to disclose a sensor material, which is stable and can be easily synthesized and purified in good yield. This is yet another an object of the present invention to disclose a sensor material, which can form stable complexes with released amines and such complexes can be easily detected by a change in color and released amines can be easily estimated by comparing intensity of changed color with the standard paper strips of presently disclosed sensor material or by measuring intensity of changed color by any such known method. This is still an object of the present invention to disclose a sensor material, which does not have a limited utility since the presently disclosed sensor material, which does not react with other chemicals present in the vicinity of the food, particularly fish being tested, that is with amino acids, phenols and sugars. Therefore, the change in color and intensity of changed color is not misleading about the freshness of food, particularly fish etc., when the presently disclosed sensor material is used. This still another an object of the present invention to disclose a sensor material, which can be employed even in the mobile form or in the remote areas. Still further an object of the present invention is to disclose a sensor material, which can differentiate between monoamines, di-amines and polyamines. This is still further an object of the present invention to disclose a sensor material, wherein the conformation of the synthesized sensor material has been established. Other objects of the present invention will become obvious from the following description. Detailed description of the Invention: Accordingly the present invention completely discloses a sensor material, which is suitable to detect the released amines and also to discriminate between various released amines, such as mono-amines, di-amines and polyamines and to estimate the amount of released amines. In accordance to the present invention, the presently disclosed sensor material has been developed by using the principle that, fresh food, particularly fish etc., starts decaying and putrefying soon after being taken out of its natural habitat. Between the time of its proper preservation and being taken out of the natural habitat, particularly water in case of fish, the fresh food, particularly fish starts releasing specific lower amines, particularly aliphatic amines, that is of a particular released amine is the function of food freshness, particularly offish freshness. The presently disclosed sensor material has the advantage that, it changes color from yellow to orange to blue depending upon the concentration of a particular amine in the food sample, particularly offish sample, when kept in air tight container. This change in color can be easily compared with the already reacted paper strips for detection and discrimination of released amine and for estimation of released amine for determination of freshness of food, particularly offish. In accordance to one of the preferred embodiments of the present invention, the change in color can also be measured by digitization of the color so that food freshness, particularly fish freshness can be directly read on an optical scale, however the use of such a system will increase the cost of the use of the presently disclosed sensor material, but such increase in cost will still remain much below the cost of use of similar methods, such as olfactory analytical, and gas-chromatographic coupled with mass spectrophotometric (GC-MS) analytical, and the methods based on radio-immuno assay and fluorescence techniques. In accordance to one of the preferred embodiments of the presently disclosed invention p-tertiarybutyl phenol is condensed with formaldehyde in the presence of a base followed by its cyclisation to yield calix [n] arenas, wherein n may vary from 4 to 8. These are dealkylated and then reacted with diazotised substituted aryl amines or amino acids or 4, 4'-di-aminobiphenyls to yield a sensor material of the present invention. In accordance to the preferred embodiment of the presently disclosed invention, the sensor material of this invention is synthesised by coupling of diazotized para substituted aryl amines or amino acids or 4,4'- di-aminobiphenyls with debutylated calix [4\| arenes in tetrahydro furan (THF)/ dimethyl formamide (DMF)/ methanol (MeOI I) or DMF/MeOH solution respectively. The synthesised sensor material is purified by dissolution preferably in pyridine followed by precipitation preferably with dilute hydrochloric acid (HCI) and washing preferably with sodium bicarbonate (NaHCOs) and water or preferably by coloumn chromatography preferably using silica gel, alumina etc as solid medium and preferably using ethyl acetate as solvent medium. The synthesised and purified sensor material changes color upon interaction with aliphatic amines. The aromatic amines do not show any color change on reaction with the presently disclosed sensor material. The sensor material disclosed in the present invention are synthesised in accordance to the following particularly described method, which is not intended to limit the scope of the present invention. In accordance to one of the preferred embodiments of the present invention about 25g of para-tertiary butyl phenol is treated with about 0.04g of potassium, lithium or sodium hydroxide and formaldehyde solution, preferably about 15ml of about 40% of formalin solution preferably under constant stirring with a magnetic bar. The contents of the flask are heated preferably on a heating mental at a temperature of about 120-125 degree centigrade for a period of about two to four hours. The color of the reacting mixture is changed from yellow and good frothing till observed. The reaction mixture is subsequently changed to a thick yellow viscous mass. The mixture is cooled to room temperature and about 250ml of xylene, toluene or diphenyi ether is added to it preferably with vigorous stirring when thick mass is dissolved. The flask is then heated to boiling preferably in presence of inert, preferably nitrogen atmosphere for a period of about two to three hours when the color of the solution changed to light brown. The flask is fitted with a condenser and the mixture is refluxed preferably under quick stream of inert gas, preferably nitrogen when a small amount of solid started separating which gets dissolved to yield a dark brown liquid. The mixture is cooled preferably to room temperature and about 500ml of ethyl acetate is added preferably when a pale yellow solid separate on cooling is filtered and dried. The crude mass obtained in this step is recrystalized from toluene to give shining rhomboidal crystals with a melting point higher than 320 degree centigrade. About 5g of the product obtained in above step, herein after as step-1 is reacted with about 3g of anisole or phenol and about 13ml of a Lewis acid, like boron solution. The solution is stirred at room temperature for about one hour under inert atmosphere, preferably under nitrogen atmosphere and then poured in to about 250ml of about 0.2N aqueous HC1. The organic phase is separated and the solvent is distilled preferably under reduced pressure of about lOmmHg. Addition of methanol to the residue yielded a percipitate which was crystallized from methanol-chloroform, preferably 1:1 methanol-chloroform to give a product melting at about 315 to 317 Degree Centigrade. The IR spectra shows peaks at 3225, 1600 , 1440, 1305, 1050, 920. The UV spectra shows 290 (CHC13), NMR shows peaks at 10.02, 6.89, 7.45(AR-H), 3.95 (Ar-CH2). About 2g of the product obtained in above step, herein after referred to as step 2, about 0.5 to 0.8 ml of ethyl bromoacetate and about 0.5g of sodium carbonate or calcium carbonate or K2CO3 in about 150ml of acetonitrile is refluxed for about 20hr. The reaction mixture is cooled to room temperature, filtered and the solvent preferably in chloroform or toluene and washed preferably with water, dilute hydrochloric acid and water successively. The organic layer is dried over dehydrating agent, preferably over anhydrous mangnesium sulphate or sodium sulphate and the solution is concentrated when the product is seperated out as colourless solid on addition of organic solvent preferably methanol. The product is identified to have melting point of about 170-I71°C. The IR in KBr, vmax , cm") spectra shows peaks at 3427(bs), 1484(s), 1467(w), 1!94(s), 1095(s), 764(s), 586(m). The 'H-NMR in CDC13, (δ, ppm) spectra shows peaks at 7060 (2H, s, exchangeable with D2O, OH); 7.04, 6.89 (8H., d (J = 13.16Hz), Aril), 6.73 (4H, t(j = 7.84Hz)Arh) 4.72 (4H, s, ArOCH2), 4,48, 3,38 (8H, d (J=13.16Hz), ArCH2Ar), 4.33 (4H, q (J =7.14Hz), OCH2C), 1.35 (6H, t (J =7.15Hz), CM3). About 0.4g of the product obtained in above step, herein after referred to as step-3, is taken in about 25ml of pyridine, collodine or quinoline solution and is maintained at about 0 to 15°C and preferably ice cold solution of about 0.3g of diazotized biphenyls, preferably benzidine, semidine etc. with about 0.5g of NaNO2 and about 25ml of acid, preferably HCI is allowed to give a sensor material which is dissolved on a solvent preferably chloroform, THF, pyridine, TMF or DMSO. In the last step, the sensor material synthesized in the above step, herein after reffered to as step-4, is purified by dissolution in pyridine followed by percipitation with dilute acid, preferably HCI and washed with sodium carbonate and water or by coloumn chromatography over SiO2 gel, A12O3 or cellulose. The synthesized and purified sensor material changes colour upon interaction with aliphatic amines using ethyl acetate as the eluent. The aromatic amines do not show any colour change on reaction with the presently disclosed sensor material. The sensor materials synthesized in accordance to the presently disclosed method invention have definite conformation of calixarenes used. He sensor material disclosed in the present invention on interaction with monoamines, diamines and polyamines give different value of λmax, hence is able to distinguish monoamines, diamines and' polyamines from each other. The sensor materials disclosed in the present invention on interaction with amines give two inflexion point in potentiometry with diamines and one inflexion point in potcntiometry with monoamines . The presently disclosed sensor materials do not give colour on interaction with amino acids and sugars. The presently disclosed invention has been described with a set of specific reaction for synthesis of sensor material of the presently disclosed invention, which is not intended to limit the scope of the present invention. It is obvious that, a minor modification in the reaction conditions of the presently disclosed set of specific reactions for the synthesis of sensor material of the presently disclosed invention is possible without deviating from the scope of the present invention. We Claim: 1. A method for preparation of a sensor material for detection, discrimination and estimation of amines comprising (i) condensation of para tertiarybutyl phenol with formaldehyde, then (ii) subjecting to cyclisation and dealkylation, followed by (iii) coupling, in manner such as herein described, with diazotised substituted compounds wherein dia/otised substituted compounds are diazotised substituted aryl amines or amino acids or substituted biphenyls to yield a novel calyx (n)arenes, and (iv) optionally purifying the said calyx(n)arenes by conventional methods such as herein described, and absorbing on a suitable article/medium to yield sensor material, 2. A method is claimed in claim 1, wherein said condensation of p-tertiarybutyl phenol with formaldehyde is carried out in the presence of a known base such as herein described. 3. A method as claimed in claim 1, wherein the diazotizcd substituted aryl amines arc dia/.oti/ed para substituted aryl amines and substituted biphenyls are 4,4"-di- aminobiphenyls preferably benzidine or semidine. 4. A method as claimed in claim 1, wherein the value of 'n' in said calix[n\|arencs vary from 4 to 8. 5. A method as claimed in claim 1, wherein the coupling of diazotized substituted compounds is carried out in tetrahydro furan (THF)/dimcthyl formamidc (DMlO/mcthanol (McOII) or DMF/MeOH solution. 6. A method as claimed in claim 1, wherein the coupling of diazotized substituted compounds is performed in pyridine, collodine or quinoline solution. 7. A method as claimed in claim 1, wherein the coupling of diazotized substituted compounds is carried out in presence of NaNO2 and acid, preferably MCI are added to the said solution of said diazotizcd compound wherein the diazotized compound is aminobiphenyls. 8. A method as claimed in claim 1, wherein the coupling of diazotized substituted compounds is carried out at about 0 to 15°C. 9. A method as claimed in claim 1, wherein the purification is effected by dissolution of calyx(n)arenes in pyridine followed by precipitation with dilute hydrochloric acid and washing with sodium bicarbonate and water. 10. A method as claimed in claim 1, wherein said purification is conducted by column chromatography. 11. A method as claimed in claim 7, wherein said column chromatography is performed preferably using silica gel, alumina or cellulose as solid medium and preferably ethyl acetate as solvent medium. 12. A method for preparation of a sensor material for detection, discrimination and estimation of amines substantially as claimed and described herein above.

Full Text

FIELD OF INVENTION:
The present invention relates to method for preparation of a sensor material for detection, discrimination and estimation of amines.
Particularly the present invention relates to a sensor material for visual detection and discrimination of amines, more particularly the present invention relates to a sensor material for visual detection and discrimination of bio-amines and aliphatic amines, even more particularly it relates to a sensor material for visual detection and discrimination of bio-amines, mono-amines, di-amines and poly-amines and to the method of preparation of the said sensor material. The present invention also relates to a sensor material for detection, discrimination and estimation of amines, wherein said sensor amine is suitable for making test kits and paper strips testing and discrimination of amines.
BACKGROUND OF THE INVENTION:
It is known in the art that, food, such as meat, fish, shellfish and milk obtained from the infected animal source is a primary source of some notable helminthic and bacterial diseases, which get transmitted to a person who eat such food in raw form or partially cocked or treated form. The heminthic diseases include those caused by pork, cattle and fish tapeworm ingested as larval forms encysted in the muscles of these animals and the trematode infestations acquired by eating fish and water plants infected with larvae. Although the pathogenic symptoms of these diseases differ in many respects, their epidemiology is similar with regard to sources and routes of infection.
Further, it is also known in the art that, the contaminated food is a potential occupational health hazard for housewives and cooks, if they taste such food in raw form or in partially cooked or treated form.
Still further, the contaminated food, like old meat, fish, shellfish etc. frequently becomes a source of primary or a secondary reservoir of serious infections and a source of dangerous consequences due to pathogenic microorganisms, which grow due to release of amines from inside of body onto the skin of the food, like meat; fish,
shellfish etc. which in-turn enhances the growth of microorganisms and in-turn food gets decomposed and cause the helminthic disease.
Still serious problems occurs, when contamination of original food by pathogenic microorganisms can assume dangerous secondary reservoirs of infectious diseases or production centers for poisonous bacteria toxins, particularly when such food is improperly preserved, handled or prepared, which in-turn may result in microbial multiplication before it is eaten.
It is known that, greater the time has elapsed after food or contaminated food, particularly fish or shellfish is in air, the greater is the chance of its getting infected and consequent out break of helminthic diseases. Particularly in the case food, like fish or shellfish etc., as soon as fish or shellfish is taken out of water, it is susceptible to putrefaction, if not immediately preserved under low temperatures and other treatments. It has been observed that, fish or shellfish starts releasing a mixture of amines, particularly lower amines, and more particularly aliphatic amines. The concentration of amines released in the atmosphere is directly related to the time the fish or shellfish etc. has been out of the water and not properly preserved.
It has been further observed that the greater the concentration of released specific ratio of particular amines, the greater is the fish being with pathogenic bacteria. The microbes multiply faster in the environment of these amines.
Generally visual examination of food, particularly offish etc. is considered satisfactory for quick results. The major drawback of such method is that, the person testing the food, particularly the fish etc. should be experienced and expert in the area. Generally such experts and experienced persons are housewives or cooks, who normally do not purchase the food. The purchasers are generally un-experienced and will have to depend on the seller's statement.
There is no easy method, particularly non-instrumental for determining fish freshness. Therefore, the need was felt to develop a scientific method, particularly a sensor material to detect, discriminate and estimate the released amines from the food or contaminated food, particularly from fish or shell fish etc. with the growing scientific
procedures of preservation and transportation of food, particularly in the area of fisheries, which has attracted considerable attention due to its availability in abundance and short life and high food value.
One such known method involve olfactory analysis, or gas-chromatographic coupled with mass spectrophotometric (GC-MS) analysis.
The major drawback of such known methods is that, these are time consuming and require costly equipment. Therefore, such known method do not meet the requirement of quicker and economical method of testing and estimating the freshness of food, particularly of fish.
Further, limitation of such known methods is that, the released amines are difficult to be detected chemically.
Another such known methods are based on radio-immuno assay and fluorescence techniques. Such methods also suffer drawback of being time consuming and requiring costly instrumentation for performing such methods, which limit their use only in the research laboratories.
Still another known method to test and estimate the freshness of the food, particularly of fish is the use synthetic chemicals, which can form complexes with released amines and such complexes can be detected by change in color and intensity of changed color.
Meeting this requirement, some of the chemical reagents have been made available for the said purpose. Some of the presently available reagents are Bratton-Marshall reagent, Paulis reagent and Ninhydrin reagent.
Despite extensive usefulness, the unambiguous detection and discrimination of bio-amines, particularly of aliphatic amines have been scantly studied. The methods made available, as describe herein above, generally employ sophisticated instruments or expensive reagents and further such known methods are difficult to operate due to want of skilled manpower. Further, if operated, the results may be misleading due to reasons
explained above. Still further, such methods can not be employed in the mobile form or in the remote areas.
Recently, there has been another set of chemicals reported in the literature for the said purpose, which are particularly derived from organic meta-cyclopharenes. Such chemicals are known in the prior art as functionalised calixarenes in the contemporary literature.
The limitation of such calixareness is that, no derivative of known calixareness show a marked changed in color on interaction with the released amines for clear and easy testing and estimation of freshness of food, particularly offish. Further, such known calixarenes do not differentiate between monoamines, diamines and polyamines, which is the need of the present art.
Further known calixarenes exist as calix (n) areness (value of n=4-8) and one has to synthesize a particular calixarenes which can be employed as a reagent for detection and discrimination of amines.
Still further, the confrontation of the synthesized calixarene based reagents has not been reported, which is important for using such known reagents, particularly in case of calixarenes. A small change in the conformation of calixarene backbone may lead to false signals, because calixarenes are flexible and their conformation is difficult to freeze without functionalization. Consequently, the use of calixarenes based reagents becomes difficult for discriminating different types of aliphatic amines and the very purpose of the development is forfeited.
Further no method is available which freezes the calixarenes with predetermined conformation. Still further no knowledge about a particular conformation of calixarenes exist which is known to change color on interaction with aliphatic amines,
Therefore, it is obvious from the forgoing description, that the currently known methods or reagents or chemical reagents are not sufficient to allow exact determination of food, particularly fish freshness. The presently known reagents are therefore not only limited to their use for determination of food, particularly fish
freshness but also the concepts where biological amines are to be detected and
estimated.
Need of the Invention:
Therefore, there is a need to have a method, preferably to have a reagent, more preferably to have a chemical reagent herein after as a sensor material, which can overcome some of the drawbacks and disadvantages of the known methods and reagents, as described herein above, and is suitable to detect the released amines and also to discriminate between various released amines, such as bio-amines, mono-amines, di-amines and poly amines and to estimate the amount of released amine.
Further, the need is to have a sensor material, which can be used universally, even in
remote areas.
Still further there is a need for a method which can freeze the conformation of the
calixarenes.
Yet further there is a need for obtaining calixarenes which change color on interaction with amines.
Objects of the Invention:
The main object of the present invention is to disclose a method, preferably to disclose a sensor material, which can over some of the drawbacks and disadvantages of such known methods and reagents, as described herein above, and is suitable to detect the released amines and also to discriminate between various released amines, such as bio-amines, mono-amines, di-amines and poly-amines and to estimate the amount of released amine.
Another object of the present invention is to propose for a sensor material, which can be used universally, even in remote areas.
This is another an object of the present invention to propose for a sensor material the use of which does not require experienced and expert person in the area at least for
detection of released amines and estimating the freshness of food, particularly offish etc. when the presently disclosed sensor material is used in the form of a paper strip, standard indicator strip.
Still another an object of the present invention is to propose for a sensor material, the use of which is neither time consuming nor involve costly equipment. Therefore, meeting the requirement of quicker and economical method of testing and estimation of freshness of food, particularly offish.
Yet another an object of the present invention is to achieve a sensor material, which can eliminate the use of olfactory analysis, and gas-chromatographic coupled with mass spectrophotometric (GC-MS) analysis, and of methods based on radio-immuno assay and fluorescence techniques for discrimination of aliphatic amines, hence is highly economical and easy to use, and is not limited to use in the research laboratories.
This is yet an object of the present invention to disclose a sensor material, which is stable and can be easily synthesized and purified in good yield.
This is yet another an object of the present invention to disclose a sensor material, which can form stable complexes with released amines and such complexes can be easily detected by a change in color and released amines can be easily estimated by comparing intensity of changed color with the standard paper strips of presently disclosed sensor material or by measuring intensity of changed color by any such known method.
This is still an object of the present invention to disclose a sensor material, which does not have a limited utility since the presently disclosed sensor material, which does not react with other chemicals present in the vicinity of the food, particularly fish being tested, that is with amino acids, phenols and sugars. Therefore, the change in color and intensity of changed color is not misleading about the freshness of food, particularly fish etc., when the presently disclosed sensor material is used.
This still another an object of the present invention to disclose a sensor material, which can be employed even in the mobile form or in the remote areas.
Still further an object of the present invention is to disclose a sensor material, which can differentiate between monoamines, di-amines and polyamines.
This is still further an object of the present invention to disclose a sensor material, wherein the conformation of the synthesized sensor material has been established.
Other objects of the present invention will become obvious from the following description.
Detailed description of the Invention:
Accordingly the present invention completely discloses a sensor material, which is suitable to detect the released amines and also to discriminate between various released amines, such as mono-amines, di-amines and polyamines and to estimate the amount of released amines.
In accordance to the present invention, the presently disclosed sensor material has been developed by using the principle that, fresh food, particularly fish etc., starts decaying and putrefying soon after being taken out of its natural habitat. Between the time of its proper preservation and being taken out of the natural habitat, particularly water in case of fish, the fresh food, particularly fish starts releasing specific lower amines, particularly aliphatic amines, that is of a particular released amine is the function of food freshness, particularly offish freshness.
The presently disclosed sensor material has the advantage that, it changes color from yellow to orange to blue depending upon the concentration of a particular amine in the food sample, particularly offish sample, when kept in air tight container. This change in color can be easily compared with the already reacted paper strips for detection and discrimination of released amine and for estimation of released amine for determination of freshness of food, particularly offish.
In accordance to one of the preferred embodiments of the present invention, the change in color can also be measured by digitization of the color so that food freshness,
particularly fish freshness can be directly read on an optical scale, however the use of such a system will increase the cost of the use of the presently disclosed sensor material, but such increase in cost will still remain much below the cost of use of similar methods, such as olfactory analytical, and gas-chromatographic coupled with mass spectrophotometric (GC-MS) analytical, and the methods based on radio-immuno assay and fluorescence techniques.
In accordance to one of the preferred embodiments of the presently disclosed invention p-tertiarybutyl phenol is condensed with formaldehyde in the presence of a base followed by its cyclisation to yield calix [n] arenas, wherein n may vary from 4 to 8. These are dealkylated and then reacted with diazotised substituted aryl amines or amino acids or 4, 4'-di-aminobiphenyls to yield a sensor material of the present invention.
In accordance to the preferred embodiment of the presently disclosed invention, the sensor material of this invention is synthesised by coupling of diazotized para substituted aryl amines or amino acids or 4,4'- di-aminobiphenyls with debutylated calix [4| arenes in tetrahydro furan (THF)/ dimethyl formamide (DMF)/ methanol (MeOI I) or DMF/MeOH solution respectively.
The synthesised sensor material is purified by dissolution preferably in pyridine followed by precipitation preferably with dilute hydrochloric acid (HCI) and washing preferably with sodium bicarbonate (NaHCOs) and water or preferably by coloumn chromatography preferably using silica gel, alumina etc as solid medium and preferably using ethyl acetate as solvent medium. The synthesised and purified sensor material changes color upon interaction with aliphatic amines. The aromatic amines do not show any color change on reaction with the presently disclosed sensor material.
The sensor material disclosed in the present invention are synthesised in accordance to the following particularly described method, which is not intended to limit the scope of the present invention.
In accordance to one of the preferred embodiments of the present invention about 25g of para-tertiary butyl phenol is treated with about 0.04g of potassium, lithium or sodium hydroxide and formaldehyde solution, preferably about 15ml of about 40% of
formalin solution preferably under constant stirring with a magnetic bar. The contents of the flask are heated preferably on a heating mental at a temperature of about 120-125 degree centigrade for a period of about two to four hours. The color of the reacting mixture is changed from yellow and good frothing till observed. The reaction mixture is subsequently changed to a thick yellow viscous mass. The mixture is cooled to room temperature and about 250ml of xylene, toluene or diphenyi ether is added to it preferably with vigorous stirring when thick mass is dissolved. The flask is then heated to boiling preferably in presence of inert, preferably nitrogen atmosphere for a period of about two to three hours when the color of the solution changed to light brown. The flask is fitted with a condenser and the mixture is refluxed preferably under quick stream of inert gas, preferably nitrogen when a small amount of solid started separating which gets dissolved to yield a dark brown liquid. The mixture is cooled preferably to room temperature and about 500ml of ethyl acetate is added preferably when a pale yellow solid separate on cooling is filtered and dried. The crude mass obtained in this step is recrystalized from toluene to give shining rhomboidal crystals with a melting point higher than 320 degree centigrade.
About 5g of the product obtained in above step, herein after as step-1 is reacted with about 3g of anisole or phenol and about 13ml of a Lewis acid, like boron solution. The solution is stirred at room temperature for about one hour under inert atmosphere, preferably under nitrogen atmosphere and then poured in to about 250ml of about 0.2N aqueous HC1. The organic phase is separated and the solvent is distilled preferably under reduced pressure of about lOmmHg. Addition of methanol to the residue yielded a percipitate which was crystallized from methanol-chloroform, preferably 1:1 methanol-chloroform to give a product melting at about 315 to 317 Degree Centigrade. The IR spectra shows peaks at 3225, 1600 , 1440, 1305, 1050, 920. The UV spectra shows 290 (CHC13), NMR shows peaks at 10.02, 6.89, 7.45(AR-H), 3.95 (Ar-CH2).
About 2g of the product obtained in above step, herein after referred to as step 2, about 0.5 to 0.8 ml of ethyl bromoacetate and about 0.5g of sodium carbonate or calcium carbonate or K2CO3 in about 150ml of acetonitrile is refluxed for about 20hr. The reaction mixture is cooled to room temperature, filtered and the solvent preferably in chloroform or toluene and washed preferably with water, dilute hydrochloric acid and water successively. The organic layer is dried over dehydrating agent, preferably over
anhydrous mangnesium sulphate or sodium sulphate and the solution is concentrated when the product is seperated out as colourless solid on addition of organic solvent preferably methanol. The product is identified to have melting point of about 170-I71°C. The IR in KBr, vmax , cm") spectra shows peaks at 3427(bs), 1484(s), 1467(w), 1!94(s), 1095(s), 764(s), 586(m). The 'H-NMR in CDC13, (δ, ppm) spectra shows peaks at 7060 (2H, s, exchangeable with D2O, OH); 7.04, 6.89 (8H., d (J = 13.16Hz), Aril), 6.73 (4H, t(j = 7.84Hz)Arh) 4.72 (4H, s, ArOCH2), 4,48, 3,38 (8H, d (J=13.16Hz), ArCH2Ar), 4.33 (4H, q (J =7.14Hz), OCH2C), 1.35 (6H, t (J =7.15Hz), CM3).
About 0.4g of the product obtained in above step, herein after referred to as step-3, is taken in about 25ml of pyridine, collodine or quinoline solution and is maintained at about 0 to 15°C and preferably ice cold solution of about 0.3g of diazotized biphenyls, preferably benzidine, semidine etc. with about 0.5g of NaNO2 and about 25ml of acid, preferably HCI is allowed to give a sensor material which is dissolved on a solvent preferably chloroform, THF, pyridine, TMF or DMSO.
In the last step, the sensor material synthesized in the above step, herein after reffered to as step-4, is purified by dissolution in pyridine followed by percipitation with dilute acid, preferably HCI and washed with sodium carbonate and water or by coloumn chromatography over SiO2 gel, A12O3 or cellulose. The synthesized and purified sensor material changes colour upon interaction with aliphatic amines using ethyl acetate as the eluent. The aromatic amines do not show any colour change on reaction with the presently disclosed sensor material.
The sensor materials synthesized in accordance to the presently disclosed method invention have definite conformation of calixarenes used. He sensor material disclosed in the present invention on interaction with monoamines, diamines and polyamines give different value of λmax, hence is able to distinguish monoamines, diamines and' polyamines from each other.
The sensor materials disclosed in the present invention on interaction with amines give two inflexion point in potentiometry with diamines and one inflexion point in
potcntiometry with monoamines . The presently disclosed sensor materials do not give colour on interaction with amino acids and sugars.
The presently disclosed invention has been described with a set of specific reaction for synthesis of sensor material of the presently disclosed invention, which is not intended to limit the scope of the present invention. It is obvious that, a minor modification in the reaction conditions of the presently disclosed set of specific reactions for the synthesis of sensor material of the presently disclosed invention is possible without deviating from the scope of the present invention.

We Claim:
1. A method for preparation of a sensor material for detection, discrimination and
estimation of amines comprising (i) condensation of para tertiarybutyl phenol with
formaldehyde, then (ii) subjecting to cyclisation and dealkylation, followed by (iii)
coupling, in manner such as herein described, with diazotised substituted compounds
wherein dia/otised substituted compounds are diazotised substituted aryl amines or
amino acids or substituted biphenyls to yield a novel calyx (n)arenes, and (iv)
optionally purifying the said calyx(n)arenes by conventional methods such as herein
described, and absorbing on a suitable article/medium to yield sensor material,
2. A method is claimed in claim 1, wherein said condensation of p-tertiarybutyl
phenol with formaldehyde is carried out in the presence of a known base such as
herein described.
3. A method as claimed in claim 1, wherein the diazotizcd substituted aryl amines
arc dia/.oti/ed para substituted aryl amines and substituted biphenyls are 4,4"-di-
aminobiphenyls preferably benzidine or semidine.
4. A method as claimed in claim 1, wherein the value of 'n' in said calix[n|arencs
vary from 4 to 8.
5. A method as claimed in claim 1, wherein the coupling of diazotized substituted
compounds is carried out in tetrahydro furan (THF)/dimcthyl formamidc
(DMlO/mcthanol (McOII) or DMF/MeOH solution.
6. A method as claimed in claim 1, wherein the coupling of diazotized substituted
compounds is performed in pyridine, collodine or quinoline solution.
7. A method as claimed in claim 1, wherein the coupling of diazotized substituted
compounds is carried out in presence of NaNO2 and acid, preferably MCI are added to
the said solution of said diazotizcd compound wherein the diazotized compound is
aminobiphenyls.
8. A method as claimed in claim 1, wherein the coupling of diazotized substituted
compounds is carried out at about 0 to 15°C.

9. A method as claimed in claim 1, wherein the purification is effected by
dissolution of calyx(n)arenes in pyridine followed by precipitation with dilute
hydrochloric acid and washing with sodium bicarbonate and water.
10. A method as claimed in claim 1, wherein said purification is conducted by
column chromatography.
11. A method as claimed in claim 7, wherein said column chromatography is
performed preferably using silica gel, alumina or cellulose as solid medium and
preferably ethyl acetate as solvent medium.
12. A method for preparation of a sensor material for detection, discrimination and
estimation of amines substantially as claimed and described herein above.

Documents:

710-del-1999-abstract.pdf

710-del-1999-claims.pdf

710-del-1999-correspondence-others.pdf

710-del-1999-correspondence-po.pdf

710-del-1999-description (complete).pdf

710-del-1999-form-1.pdf

710-del-1999-form-13.pdf

710-del-1999-form-19.pdf

710-del-1999-form-3.pdf

710-del-1999-form-5.pdf

710-del-1999-gpa.pdf

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Patent Number

219870

Indian Patent Application Number

710/DEL/1999

PG Journal Number

28/2008

Publication Date

11-Jul-2008

Grant Date

13-May-2008

Date of Filing

11-May-1999

Name of Patentee

DEAN INDUSTRIAL RESEARCH AND DEVELOPMENT (IRD)

Applicant Address

INDIAN INSTITUTE OF TECHNOLOGY DELHI HOUZ KHAS, NEW DELHI-110016.

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. CHAWLA HAR MOHINDRA

PCT International Classification Number

C08G 83/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA