Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G18/00—Polymeric products of isocyanates or isothiocyanates
  • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
  • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
  • C08G18/30—Low-molecular-weight compounds
  • C08G18/38—Low-molecular-weight compounds having heteroatoms other than oxygen
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G18/00—Polymeric products of isocyanates or isothiocyanates
  • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
  • C08G18/08—Processes
  • C08G18/0804—Manufacture of polymers containing ionic or ionogenic groups
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
  • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
  • G01N31/221—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators for investigating pH value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
  • G01N33/525—Multi-layer analytical elements
  • G01N33/526—Multi-layer analytical elements the element being adapted for a specific analyte
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G2210/00—Compositions for preparing hydrogels

Definitions

• the present invention relates to new hydrogels, azo dyes, and pH sensitive dye films and sensing elements made therefrom. More particularly, it relates to hydrogel dye film sensing elements capable of detecting pH and pCO 2 and to apparatus useful for in vivo measurement of the pH and pCO 2 of body fluids, such as blood.
• hydrogels into which dyes may be permanently incorporated and which are suitable for use in optical sensing elements. It is further object to provide hydrogel dye films capable of undergoing an optically detectable, reversible color change as a function of changing pH.
• the azo dye indicators undergo reversible color shifts as a function of pH, and apparatus equipped with the sensing elements of the invention can optically detect such color shifts and calibrate the shifts to give accurate information as to the pH and pCO 2 of the fluid contacting the sensing elements.
• the polyurethane or polyacrylamide hydrogels and azo dyes disclosed herein are employed to form the sensing elements of the present invention.
• FIGURE 1 is a schematic diagram of a fluid analyzer apparatus according to the invention.
• FIGURE 2 is a sectional plan view of a possible configuration for a sensing cuvette in accordance with the invention, employing two sensing cells having sensing elements therein for detecting, respectively, pH and pCO 2 of a fluid.
• FIGURE 3 is a sectional plan view of a sensing cell employing a sensing element according to the present invention showing its relationship toward impinging light from the optical components of a fluid analyzer which measures backscattered light.
• the present invention relates to apparatus for analyzing fluids and to dyes, hydrogels, and dye films useful in the sensing elements in such apparatus.
• Preferred sensing elements of the present invention are useful for detecting pH or for detecting pCO 2 .
• Each of the sensing elements comprises one or more thin films.
• the films are most advantageously deposited in layers on an optically clear substrate and disposed within fluid analyzer sensing cells such that the outermost layer is contacted by the fluid to be analyzed.
• the sensing elements depend on a dye film layer, which is normally the first layer of the sensing element (i.e., deposited directly on the optically clear substrate).
• the sensing elements may also include light scattering film layers and light absorbing film layers, to permit the use of optical equipment that measures backscattered light, as opposed to transmitted light.
• a gas permeable layer will also be employed.
• the arrangement of the layers will depend on the configuration of the optical reading elements of the apparatus.
• the order of the layers will be as follows: optically clear substrate, dye film layer, light reflective layer, light absorptive layer, gas permeable layer (pCO 2 -detecting elements only) .
• the optically clear substrate is oriented toward the light source, and the outermost film layer (e.g., the gas permeable layer or the light absorptive layer) communicates with the channel of the sensing cuvette through which the fluid to be measured is made to pass.
• the indicator dyes used to produce the pH- detecting dye films of the present invention are azo dyes of the general formula:
• R 2 represents a phenyl, naphthyl, or a C 2 -C 12 heterocyclic aromatic radical, which may be substituted with one or more groups selected from nitro, cyano, sulfo, carboxy, carboxamido, carboalkoxy, acyl, alkoxy, perfluoroalkyl, or halogen atoms such as bromine, chlorine, fluorine, etc.;
• R 3 represents a suphonated naphthol or sulphonated aminonaphthol radical
• R 4 represents a reactive substituent capable of binding the dye molecule to a polymeric substrate without affecting the pH-indicating character of the dye.
• Preferred dyes have the formula (I):
• Each R 5 is independently selected from hydrogen, halogen (chlorine, bromine, fluorine, etc.), perfluoroalkyl of 1 to 4 carbon atoms, nitro, sulfo, cyano, carboxy, carboalkoxy of 1 to 4 carbon atoms, carboxamido, or acyl, provided that at least one R 5 is nitro or cyano;
• R 6 is amino, carboxamido, acrylamido, -NHCO(CH 3 ) (CH 2 OH) 2 , or -NHCOCH(NR 7 R 8 )CH 2 NR 7 R 8 , wherein R 7 and R 8 are, independently, hydrogen, straight chain or branched alkyl, aminoalkyl, or hydroxyalkyl groups of 1 to 6 carbon atoms (e.g., methyl, ethyl, propyl, t-butyl, isobutyl, isopentyl, aminoethyl, aminopropyl, hydroxyethyl, hydroxypropyl, tris (hydroxymethyl)methyl, and the like), or R 7 and R 8 taken together form the radical -CH 2 CH 2 NR 9 CH 2 CH 2 -, wherein R 9 is hydrogen or any of the aforementioned C 1 -C 6 aminoalykl or hydroxy-alkyl radicals; and E is hydrogen, sodium, lithium, potassium, magnesium or calcium.
• R 6 are explicitly defined above, also contemplated are other groups which will serve to bind the dye molecules to a substrate without interfering with its pH-indicating properties.
• hydrocarbons eg., alkylene
• ether and thioether bridges terminating in a variety of functional groups may also be employed.
• dyes in which R 5 para to the azo group and on R 5 ortho to the azo group are or cyano nitro are or cyano nitro
• R 6 is -NHCO(CH 3 ) (CH 2 OH) 2 , -NHCOCH 2 NR 7 R 8 , or -NHCOCH(NR 7 R 8 )CH 2 NR 7 R 8
• R 7 and R 8 are aminoethyl, aminopropyl, hydroxyethyl, hydroxypropyl, or tris (hydroxymethyl)methyl.
• the reactive azo dyes of the present invention exhibit a pH-dependent color change with absorption maxima at wavelengths longer than 500 nm for both the protonated and deprotonated forms of the dye, and there is nearly complete spectral separation of the protonated and the deprotonated forms.
• a single sharp transition due to a protonated equilibrium is exhibited within the pH-range encountered with physiological fluids such as blood; and the dyes are stable under the variable conditions of the medical environment in which they can be advantageously used (i.e., in terms of stability to heat, light, oxygen, compatability with physiological fluids, stability to sterilization procedures).
• the dyes are conveniently incorporated into the structure of the polyurethane hydrogels and thereby immobilized, preventing them from leaching or bleeding out of the hydrogel substrate during use.
• the dyes having the structure of formula (I) have a red to blue transition with increasing pH.
• the dyes may be advantageously prepared via a coupling reaction between diazonium salts and amides derived from aminonaphthol sulfonic acids.
• the preferred starting materials for the dyes of formula (I) thus include the 2-halo-4, 6-dinitro-phenyldiazonium salts and their analogues (see R 5 in formula (I)).
• the pK a can be lowered and the water- solubility increased by the introduction of a second sulfonic acid group on the naphthalene ring.
• amides derived from 3-amino-5-hydroxy-2,7-naphthalenedisulfonic acid or 4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid (H-Acid) may be used as coupling components.
• substituted diazonium components a variety of substituents may be selected, depending on the properties desired in the final dye. For example, nitro, cyano, sulfo, carbory, carboxamido, carboalkoxy, acyl, methyl- or methoxy-substituted phenyl or naphthyl diazonium reagents may be used. Hydroxy substitution is also possible, however those dyes containing an ortho hydroxy substituent form complexes with heavy metal ions, a characteristic which may render them useless as pH-indicators.
• hydroxy substituents in the meta or para positions have less of a chelating effect, for the purposes described herein, hydroxy substituents on the diazonium coupling component are to be avoided.
• the position and electronegativity of the substituents can be used to adjust the pK a value of the final dye.
• the actual pK a value may also be further modified by the nature of the reactive group (e.g., R 4 , R 6 , supra) and by subsequent binding of the azo dye to a substrate.
• the amide derivatives may be prepared by dropwise addition of the acylating agent to an aqueous solution of the azo dye or the starting amino-naphthol (di) sulfonic acid.
• the pH of the solution can be maintained by addition of a solution of sodium hydroxide or by the presence of sodium hydrogen carbonate resulting in a weakly acidic to weakly alkaline medium.
• the reaction stoichiometry should be carefully controlled to avoid acylation of the hydroxyl group.
• Diazonium coupling is carried out using standard methods (see, for example, H. E. Fierz-David and L. Blangey, "Fundamental Processes of Dye Chemistry", translated by P. W. Vittum, Interscience Publishers, Inc: New York, 1949; p. 239 ff).
• diazotization is advantageously carried out using nitrosyl sulfuric acid in concentrated sulfuric acid (see G. Roebisch et al., J. Prakt. Chem. 320, 1047-54 (1978); cf. U.S. Patent 3,931,142; Japan Kokai JP 83-152056 and JP 83-160357; Chemical Abstracts 100: 69852e and 8724Oh).
• Coupling is generally rapid at 0oC in acidic aqueous solutions or slurries of the coupling components. If necessary, the product dyes are precipitated by addition of salt, and further purification is possible by washing with water followed by washing with organic solvent such as acetone and methanol; recrystallization from methanol may be used to further improve purity.
• organic solvent such as acetone and methanol
• electrophilic reactive substituents such as aromatic sulfonyl halides or isocyanates, are less desirable.
• Suitable acylating reagents that result in reactive groups, R 4 , of electrophilic character are acryloyl chloride, 2,3-dibromopropanoyl chloride, 2,3-dichloropropanoyl chloride, bromoacetyl chloride, etc.
• Reactive dyes obtained using these reagents can be bound directly to a substrate that contains nucleophilic substituents, or they can be readily converted into stable derivatives by treatment with suitable nucleophiles such as methylamine, ethanolamine, N-alkylethanolamines, diethanolamine, tris(hydroxymethyl) aminomethane, ethylenediamine, piperazine, and the like.
• substituents bearing hydroxyl or amino functions is most preferred, as it permits ready binding of the dye into the structure of polyurethane substrates by reaction with electrophilic groups such as isocyanates.
• electrophilic groups such as isocyanates.
• the 2,3-dihalopropanoamide derivatives are readily converted to 2-haloacrylamides at pH 6 or above.
• Dyes bearing acrylamido or 2-haloacrylamido groups may be linked covalently to a matrix by copolymerization with monomers such as acrylamide. This can be accomplished by heat curing which results in some thermal degradation.
• R 4 may be a substituted amido group such as 2, 2-bis(hydroxymethyl) propanoamido.
• R 4 is an amide derived from a succinamido substituent, for example an amide formed between an alkanolamine or diamine and the free carboxyl group of the succinamide.
• a solution of the diazonium salt was obtained by dissolving 2-bromo-4,6-dinitroaniline (6 mmol, 1.57 g) in concentrated sulfuric acid (11 g, 6 ml) followed by addition of nitrosylsulfuric acid (0.76 g, 6 mmol) . The yellow-orange solution was stirred for 1 hour at room temperature.
• ANS-2-bromoacrylamide (2.38 g, 5 mmol) was dissolved in 100 ml water. The solution was cooled to 0oC by the addition of crushed ice. Approximately 0.5 equivalents of diazonium solution (2.5 ml, 2.5 mmol) was added to the stirred solution. After stirring overnight, the solution was acidified to pH 3. The addition of a sufficient 20% aqueous salt solution gave a 10% salt solution and precipitated the product.
• 4-amino-5-hydroxy-1-naphthalenesulfonic acid (210 g, 875 mmol) was dissolved in 2.8 L water containing NaOH (40 g, 1 mol) and the pH adjusted to 6.5 with concentrated HCl; it was then treated with the acid chloride (105 ml, about 895 mmol) , with the simultaneous addition of NaOH pellets to control the pH. The product started to precipitate before the end of acid chloride addition. Once the pH stabilized at 5, NaCl (300 g) was added to complete precipitation of the product, which was washed with 20% aqueous NaCl. It was then taken up in 3 L water containing KOH (pH 10) and heated to 70oC.
• the yellow component was partially purified by evaporation of the methanol and extraction of the residue into tetradydrofuran, followed by precipitation with ether (yield 1/74 g, 3.0 mmol, 12%). It exhibited a yellow/blue transition at pH 8.5.
• 2-chloro-4,6-dinitroaniline (2.18 g, 10 mmol) was diazotized in concentrated sulfuric acid (10 g) containing nitrosyl-sulfuric acid (1.27 g, 10 mmol) at room temperature. After one hour this solution was diluted with ice (30 g) and poured into a well-stirred slurry of the ANS-2-bromoacrylamide (3.94 g, 10 mmol) in a mixture of ice (50 g), water (25 g) and concentrated sulfuric acid (25 g). Coupling proceeded rapidly to give a deep red product which was recovered by filtration after 1.5 hours.
• Diazonium coupling with 2,6-6initro-4-trifluoromethylanilme (A 3 -2-bromoacrylamide); 2,6-dinitroaniline (A 4 -2-bromoacrylamide); 2,4-dinitro-6-trifluoromethylanilme (A 5 -2-bromoacrylamide) and 4-carboxamido-2,6-dinitroaniline (A 6 -2-bromoacrylamide).
• the methods of Examples 4-6 were used to couple these diazonium components with the ANS-2-bromoacrylamide. All of the resulting dyes were useful pH indicating compounds, capable of further reaction at the 2- bromoacrylamido group, permitting their incorporation into polymeric films. The properties of these dyes are summarized in Table 1, infra.
• Diazonium coupling with 2-bromo-4,6-dinitro-aniline Using conditions similar to those described for the corresponding chloro-substituted aniline, two dyes were obtained with nearly identical characteristics as pH indicators.
• Table 4 summarizes examples of pH-indicating dyes prepared from a variety of other coupling components.
• the addition of organic amines to dyes bearing either the 2,3-dihalopropanylamido or 2-haloacrylamide groups is presumed to occur according to the following equation:
• R and R 1 substituents on the amine are chosen to provide covalent or electrostatic binding of the dye to the hydrogel (covalent linkage is preferred); their choice also influences the solubility of the dye, a significant consideration in the preparation of dye-containing polymers.
• N-methylethanolamine 75 mmol, 5.63 g was added to the B 5 -2-bromo-acrylamide (15 mmol, 10.01 g) in methanol (250 ml) at room temperature.
• the resulting blue solution was stirred for 1 hour.
• TLC analysis sica, eluting with BuOH : EtOH: NH 4 OH - - 3 : 2 : 1) showed the reaction to be complete, with a single product spot at Rf 0.40 replacing the starting material at Rf 0.45.
• the solution was filtered through Whatman No. 1 paper, and then reduced in volume to about 35 ml on the rotary evaporator.
• This dye can also be precipitated in two acidified forms. After reaction with N-methylethanolamine as described in Example 20, treatment of the reaction mixture with glacial acetic acid gave a high yield (73%) of a red powder, presumably protonated at the naphthol end but not at the sulfonate end. This form of the dye was poorly soluble in water or organic solvents.
• Diamino compounds including ethylene diamine, piperazine, and N-2-hydroxyethylpiperazine were added to the reactive dye in aqueous or methanol solution at room temperature, using a four to five-fold excess of the diamine. These reactions were completed within 15 minutes, and the adducts were isolated in high yield (>80%). However, TLC analysis (silica, eluting with BuOH : EtOH: NH 4 OH - - 3 : 2 : 1) revealed a mixture of products . Furthermore, these adducts had significantly lower pK a 's ( ⁇ 7), making them less suitable as physiologic pH indicators.
• a slurry of 4-amino-5-hydroxy-1-naphthalenesulfonic acid (S-acid, 50 g, 209 mmol) in 540 ml water was adjusted to pH 6.5 using a 30% solution of sodium hydroxide.
• the solution was cooled to 10oC using an ice bath.
• Bromoacetyl chloride (32.9 g, 209 mmol) was added dropwise over half an hour- with good stirring.
• the pH was maintained at 6.5 by addition of sodium hydroxide solution.
• the pH stabilized 20 minutes after the acid chloride addition was complete, and a heavy precipitate formed.
• the reaction mixture was acidified to pH 3 using concentrated hydrochloric acid, and the precipitate was collected by filtration.
• the solid product was reslurried in salt water (20% aqueous NaCl, 500 ml), and again collected on a filter. The solids were then slurried in acetone (500 ml), collected by filtration, and dried at 60oC under vacuum for four hours. The yield is 42 g (52%). Further purification is accomplished through recrystallization from hot ethanol.
• the diazonium reagent was prepared as in example 2, from 2-bromo-4, 6-dinitroaniline (20 mmol, 5.24 g) and nitrosylsulfuric acid (20 mmol, 2.54 g) in concentrated sulfuric acid (40 g). Before use, the diazonium solution was diluted with 120 g crushed ice. The resulting yellow solution was added in one portion to the coupling component (ANS-2-bromoacetamide, 20 mmol, 7.64 g) in a well stirred slurry containing water (40 ml), crushed ice (80 g) and concentrated sulfuric acid (40 g). After stirring for one half hour, the red-purple solid was collected on filter paper.
• the coupling component ANS-2-bromoacetamide, 20 mmol, 7.64 g
• a diazonium solution was prepared by adding 2-chloro-4, 6-dinitroaniline (2.17 g, 10 mmol) to concentrated sulfuric acid (20 g) containing nitrosylsulfuric acid (10 mmol, 1.27 g) . After stirring for one hour, the dark yellow diazonium solution was diluted with crushed ice (60 g) and poured into a well-stirred slurry containing ANS-2-bromoacetamide (10 mmol, 3.82 g), water (20 ml), crushed ice (40 g), and concentrated sulfuric acid (20 g).
• the solid product was collected by filtration, reslurried in 10% aqueous NaCl (50 ml), again filtered, and washed with water (50 ml).
• the dye was further purified by dissolving it in methanol (1 L) containing 10 mmol sodium hydroxide (10 ml of a 1 N aqueous solution), followed by filtration and acidification with concentrated hydrochloric acid. The solution was concentrated to about 75 ml, and the resulting precipitate was collected on a fritted glass funnel, dispersed in water (25 ml), refiltered, washed with water and methanol, and dried under vacuum at 70oC. The yield was 4.29 g (70%).
• a diazonium solution was prepared by adding 2-trifluoromethyl-4,6-dinitroaniline (5.02 g, 20 mmol) to concentrated sulfuric acid (40 g) containing nitrosylsulfuric acid (20 mmol, 2.54 g). After stirring for one hour, the dark yellow diazonium solution was diluted with crushed ice (120 g) and poured into a well-stirred slurry containing ANS-2- bromoacetamide (20 mmol, 7.64 g), water (40 ml), crushed ice (80 g), and concentrated sulfuric acid (40 g).
• the solid product was collected by filtration, reslurried in water (150 ml) and precipitated by the addition of 20% aqueous NaCl (150 ml), again filtered, and washed with water (50 ml). It was further purified by recrystallization from hot methanol (1 L) to give, after vacuum drying, 1.57 g (12%) of a golden-red solid.
• a diazonium solution was prepared by adding 2-amino-3,5-dinitrobenzenesulfonic acid (5.70 g, 20 mmol as the sodium salt) to concentrated sulfuric acid (40 g) containing nitrosylsulfuric acid (20 mmol, 2.54 g). After stirring for one hour, the dark yellow diazonium solution was diluted with crushed ice (120 g) and poured into a well-stirred slurry containing ANS-2-bromoacetamide (20 mmol, 7.64 g), water (40 ml), crushed ice (80 g), and concentrated sulfuric acid (40 g) .
• the solid product was collected by filtration, reslurried in water (125 ml) and precipitated by the addition of 20% aqueous NaCl (125 ml), again filtered, and washed with water (50 ml).
• the product was further purified by recrystallization from hot methanol (500 ml), the metallic gold-green crystals being collected by filtration after reduction of the volume of the methanol solution to about 50 ml at the rotory evaporator. After drying, the yield was 6.47 g (9.5 mmol, 48%).
• 2-bromoacetamine derivatives were reacted with diethanolaminer (DEA) to give 2-(DEA)-acetamide derivatives.
• DEA diethanolaminer
• the two hydroxyethyl substituents are capable of reaction with isocyanates to form urethane linkages.
• a 8 -2-(DEA)-acetamide A 8 -2-bromoacetamide (5 mmol, 3.39 g) was treated with diethanolamine (25 mmol, 2.63 g) in tetrahydrofuran (125 ml). Reaction was slow on account of the insolubility of the dye in tetrahydrofuran, so methanol (50 ml) was added. The reaction was nearly complete after stirring for two days at room temperature, according to TLC analysis (silica, eluting with BuOH: EtOH : NH 4 OH - - 3 : 2 : 1) . A green-gold precipitate was collected (0.50 g), which proved to be residual starting material (15%).
• the diazoniun solution (from 2-bromo-4,6-dinitroaniline: 20 mmol, 5.24 g) was prepared in the usual manner (Example 29), and coupled with ANS-succinamide (20 mmol, 7.23 g) in a well-stirred mixture of water (40 ml), crushed ice (80 g), and concentrated sulfuric acid (40 g).
• the resulting red precipitate was collected on Whatman #4 paper and washed with water. It was reslurried in water (100 ml) to which was then added 20% aqueous NaCl (100 ml) , and the resulting precipitate collected by filtration and washed with water.
• the product was recrystallized from methanol to give green-gold microcrystals (5.58 g, 44%) after vacuum drying.
• Table 5 summarizes the properties of the dyes prepared according to Examples 29-37.
• the product was recovered by adding salt (100 g) and then acidifying the solution to pH 3 with concentrated hydrochloric acid; the resulting precipitate was collected by filtration on Whatman #5 paper.
• the solid product was washed with 20% agueous NaCl and then a small amount of water. It was then taken up in 1.25 L of hot ethanol, filtered, and the volume reduced to give off-white crystals (total yield 56.02 g, 62%).
• the reaction mixture was then acidified to pH 0.5 with concentrated HCl and heated at 80oC for 2 hours.
• High performance liquid chromatography (HPLC) analysis was used to determine when cleavage of the cyclic sulfite was complete.
• the solution was then filtered while warm using Whatman #54 filter paper and Celite. An HPLC sample was run to determine ANS-BHPA concentration.
• Examples 40-44 disclose the synthesis of dyes bearing the bis (2-hydroxymethyl)propanoamido group.
• the material was then slurried with 250 ml acetone and filtered (Whatman #54 filter paper). Finally, the dye was slurried with 100 mL methanol and filtered (Whatman #54 filter paper) and then vacuum dried at 60oC for 24 hours. The yield was 12.0 g (37%), and the its purity by HPLC was 96%.
• a diazonium solution was prepared by addition of 2-chloro-4,6-dinitroaniline (25 mmol, 5.44 g) to concentrated sulfuric acid (40 g) containing nitrosylsulfuric acid (25 mmol, 3.18 g) . It was diluted with crushed ice (120 g) and added in one portion to a solution of ANS-BHPA (25 mmol in 60 ml water) to which crushed ice (120 g) had been added. The dense red-purple precipitate was collected by filtration overnight on Whatman #54 paper. It was then dispersed in water (300 ml), precipited with 20% aqueous NaCl (300 ml), and filtered on Whatman #54 paper.
• EXAMPLE 43 A 14 -BHPA: The reguired aniline (4,6-dinitro-2-fluoroaniline) was prepared as described by Schiemann and Miou (Ber. 66, 1179-87 (1933)). It was diazotized on the same scale used in the previous example (25 mmol, 5.03 g), the only difference being that the diazonium solution could not be diluted with ice before use. Rather, it was added slowly to the well-stirred ANS-BHPA solution (60 ml, 25 mmol) containing crushed ice (180 g). Work-up and purification were identical to that described in the preceding example. The yield was 3.81 g (26%) after vacuum drying; purity was 92% according to HPLC.
• the aniline (2-bromo-6-cyano-4-nitroaniline) was prepared according to U.S. Patent 3,821,276 (Mrozik and Bochis). Its diazotization on a 40 mmol scale (9.68 g) was carried out in concentrated sulfuric acid (40 g) containing one equivalent of nitrosylsulfuric acid (40 mmol, 5.08 g) at room temperature. Just before use, the diazonium solution was diluted with crushed ice (120 g), and then added to a well-stirred solution of ANS-BHPA (39 mmol in 170 ml water) containing additional crushed ice (100 g). The reaction mixture turned brown.
• ANS-BHPA 39 mmol in 170 ml water
• the film layers utilized in the present invention are comprised of polyurethane or polyacrylamide hydrogels.
• hydrogel is meant a water-swollen (or swellable) three-dimensional matrix or network of crosslinked, hydrophilic macromolecules. Fully swollen hydro-gels will contain 20% to over 90% water.
• the polyurethane hydrogels of the present invention are a copolymerization product of alkylene glycols or thioglycols, organic polyisocyanates, and optionally, ionic diols, such as sulfonate diols, quaternary ammonium diols, carboxylate diols, and phosphate diols, or acids corresponding to such diols.
• Preferred hydrogels according to the present invention have a three-dimensional network structure and include polymeric units of the following general formulas:
• AX represents a urethane derived by the reaction of a polyalkylene glycol or thioglycol (A) of molecular weight 200 to 15,000 (preferably 600 to 3000) with an organic diisocyanate or triisocyanate (X); T represents a trifunctional or tetrafunctional unit derived from an organic polyol or an organic polyisocyanate; BX represents a unit derived by reacting an organic polyisocyanate (X) with a sulfonate diol, phosphate diol, carboxylate diol, or quaternary ammonium diol; and a, b, and c are integers, a and b being greater than zero, and a, b, and c being selected so that the -(BX)- units make up no more than 40% by weight, preferably 0-20% by weight, of the hydrogel and the molar ratios of A:X:T in the final polymer are from 2:2:1 to 12:12
• Preferred dye films used herein to detect changes in pH are hydrogels having the aforementioned polymer units and also units of the formula -(DX) d -, wherein D represents an azo dye reacted with X, an organic polyisocyanate, and d is an integer selected to provide 0.1% to 20% by weight in the hydrogel of the dye (D), preferably from about 1.0% to 10% by weight.
• the structure and chemical composition of the hydrogels of this invention should be adjusted so that the final film is hydrated 20% to 90%, preferably 40% to 80%, with uniform water distribution and elastomeric properties.
• Reactive azo dyes as disclosed herein may also be incorporated into these polymers to produce pH- indicating dye films.
• Suitable polyalkylene glycols and thioglycols for use in preparing the hydrogels of the invention are poly(C 2 -C 6 ) alkylene glycols and thioglycols such as polyethylene glycol, polypropylene glycol, polytetramethylene ether glycols, mixtures of such glycols and their corresponding thioglycols.
• the molecular weight of the polyalkylene glycols or thioglycols will be from 200 to 15,000, preferably from 600 to 3,000, to obtain the desired hydration.
• the percent hydration is defined as the weight percent of water in the completely hydrated film.
• polyisocyanates examples include tetramethylene diisocyanate, hexamethylene diisocyanate, 1,4-cyclohexane diisocyanate, 1,4-pentane diisocyanate, methylene bis(4-isocyanatoethyl cyclohexane), 1-isocyanato-3,3,5-trimethyl-5-iso-cyanato-methyl cyclohexane, 1,4-isocyanatomethyl cyclohexane, 1,4-isocyanatoethyl cyclohexane, 1,3-isocyanatomethyl cyclohexane, 1,3,5-triisocyanato-cyclohexane, diiso-cyanatopentylcyanomethine, 1, 4-diisocanobenzene, 2,6-diisocyanatotoluene, 2,6-diisocyanatototoluene, toluene diisocyan
• heteroorganic diols and carboxylate diols can also be used in preparing the hydrogels of the present invention.
• Urethane hydrogel polymer units may be derived from reacting the aforementioned isocyanates with sulfonate diols, quaternary ammonium diols, carboxylate diols, and phosphate diols in order to give ionic strength to the hydrogel.
• the corresponding acids of such diols i.e., sufonic acid diols, etc.
• the hydrogel may include up to 40% by weight of the diol component, but preferably 20% or less.
• diol reactants readily copolymerize with the isocyanates to produce hydroxy-terminated polyurethanes, which react further with isocyanate-terminated prepolymers. This results in a single polymer population, as determined by gel permeation chromatography, with the diol components being distributed throughout the polyurethane chains.
• Suitable sulfonate/sufonic acid diols include compounds of the formula:
• R 1 may be the same or different bivalent aromatic or aliphatic radical of from 1 to 12 carbon atoms; Y is nitrogen, carbon, sulfur, silicon, or phosphorus, with any free valencies being taken by hydrogen or halogen atoms; Z is a direct bond or R 1 ; and E is hydrogen, sodium, lithium, potassium, magnesium or calcium.
• examples of such compounds include N,N-bis (2-hydroxyethyl)-2-N-methylaminoethane sulfonic acid, 1,4-butanediol-2-sulfonic acid, and salts thereof.
• the polyurethane hydrogels of the present invention are prepared by reacting organic polyisocyanates with polyalkylene glycols and/or triols at an elevated temperature. Either isocyanate- or hydroxy-terminated polyurethane pre-polymers of different molecular weights can be produced in this manner depending on the stoichiometry.
• the polyols and polyfunctional isocyanates are used to control the crosslink density and degree of branching.
• Hydroxy-functional dyes can be easily incorporated into the polyurethane network with either a hydroxy terminated polyurethane and multi-functional isocyanates, or alternatively, with isocyanate-capped polyurethane prepolymers during cure stage, thereby forming a water insoluble, but swellable hydrogel film.
• polyurethane hydrogels can be produced to meet various specifications by varying the stiochiometry of the isocyanate and polyol components as well as by including ionic diol groups and dye groups.
• isocyanates and polyols can be carried out either in bulk or solution.
• Aprotic solvents such as acetone, methyl ethyl ketone, ethyl acetate, cyclohexanone, DMF, dimethyl sulfoxide (DMSO) N-alkyl-pyrrolidone, and butyrolactone are excellent because they provide dissolution power, chemical stability and temperature control.
• the polymerization temperature can be varied from 20oC to 190oC depending on individual preparations and the presence of a solvent.
• the preferred temperature range is from 70oC to 130oC.
• An inert atmosphere is sometimes required to prevent undesirable side reactions.
• the isocyanate and polyol reaction temperature is between 70oC to 140oC, preferably between 90oC to 110oC, for a desirable cure rate and film properties.
• the polyaddition of isocyanate and polyols can also be carried out in the presence of either acidic or basic catalysts.
• Tertiary amines, carboxylic acids and metal salts are commonly used. Suitable tertiary amines include triethylamine, heptamethylisobiguanidine and triethylenediamine (1,4- diaza [2.2.2] bicyclooctane)
• the metal salt catalysts generally have greater catalytic power than the tertiary amines, and preferred examples include tri-n- butyltin acetate, di-n-butyltin diacetate and din- butyltin dilaurate. Formic acid is an excellent acid catalyst.
• PEG Polyethylene glycol
• TMP trimethylolpropane
• the polymer was prepared according to the procedure of Example 45 except a different grade of polyethylene glycol (mol. wt. 600, 84 g, 100 mmol) was used and no DMF was added.
• the polymer was prepared as in Example 45 except no TMP was added.
• the polymer was prepared as in Example 43 except cyclohexyldiisocyanate (CHDI) (29 g, 175 mmol) was used instead of TDI.
• CHDI cyclohexyldiisocyanate
• PEG1000 100 g, 100 mmol
• TMP 3.35 g, 25 mmol
• CHDI 16.60 g, 100 mmol
• the mixture was allowed to react at 90oC for 3 hours. After cooling, a solid polymer was isolated.
• Polyurethane Prepolymer PEG1000/TDI/TMP The polymer was prepared as in Example 49, except TDI was used. Enough DMF was added to make a 70% solution.
• Polyurethane Prepolymer PEG1000/TDI The polymer was prepared as in Example 50, except TMP was not used. Enough DMF was added to make a 70% solution.
• EXAMPLE 52 Polyurethane Prepolymer PEG600/TDI/TMP: The polymer was prepared as in Example 50, except PEG600 was used. Enough DMF was added to make a 70% solution.
• PEG1000 (40.3 g, 40.3 mmol) was charged and dried in a reactor at 100oC vacuum for one hour. The vacuum was broken with dry nitrogen. TDI (9.61 g, 55.89 mmol) was added and the mixture reacted at 100oC for three hours until there was no TDI left, as shown by stabilization of the NCO peak on IR spectrophotometric analysis. 1,4-butanediol-2-sulfonic acid sodium salt (5.163 g, 26.87 mmol) in of DMSO (23.5 g) were added to the isocyanate-terminated prepolymer solution until the isocyanate peak in IR spectrum disappeared. The final polymer product showed one major population distribution with a number average molecular weight at 47,000.
• N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid sodium salt (BES, Aldrich Chem. Co.) was methylated by the following procedure.
• a thin hydrogel film containing one or more of the aforementioned pH indicator azo dyes is prepared.
• thin hydrogel film is meant a layer of about 0.5 to 50 microns thickness. In preferred embodiments, the film layers will be from about 1-10 microns, most preferably about 2-6 microns, in thickness.
• dye film When a thin film compounded with dye (“dye film”) is hydrated it will change color instantaneously as a function of the acidity or basicity of the environment. Accordingly, such films are advantageously used as optical sensor elements for detecting any parameters which are a function of pH.
• the dye film is formulated so as to provide, in thin layers of 0.5 to 50 microns, sufficient optical density and pH sensitivity to permit optical measurement, considering the light source, optical pathway and photodetector elements to be employed.
• suitable amounts of the dye component may range from about 0.1-15% by weight, based on the total weight of the dye-containing hydrogel polymer (non-swollen).
• the amount of dye will be about 1-10% by weight, most preferably about 3-8% by weight.
• optical sensing elements may include other components, depending on the overall configuration of the optical reader apparatus.
• a backscattering optical pathway is employed, and, accordingly, the sensing elements of the analyzer will include a light scattering film adjacent to the dye film.
• the sensing element may also preferably include a light absorbing layer in order to render the optical measuring device insensitive to optical variation in the sensing cuvette interior.
• a gas permeable film is also juxtaposed between the dye film and the solution to be analyzed.
• a dye film having the following formulation is prepared:
• the dye and DMF are mixed in a vial and sonicated for 40 minutes. Although a trace of material remains, the dye dissolves well. After centrifuging for 10 minutes, the solution is drawn off and filtered through a 0.2 micron syringe filter. 97% of the solution is recovered. After adjusting for the material which is lost, polyurethane hydrogel is mixed in by shaking and using a vortex mixer. The solution is centrifuged again for 10 minutes and filtered through a 5 micron syringe filter. 11.043 parts by weight of solution is recovered for an 88% overall recovery. A final centrifugation is performed for 10 minutes to remove any air bubbles.
• Table 8 gives examples of dye-containing hydrogels which were prepared and tested as pH-sensing materials.
• the diazonium sulfate converted from 2-bromo-4,6-dinitroaniline (550 mg) in Example 15 was added to a slurry prepared from 4-amino-5-hydroxy-1-naphthalene-sulfonic acid (500 mg), water (25 ml), ice (10 g) and H 2 SO 4 (1.5 ml). After completion of the reaction at 20oC, the dye was precipitated by the addition of sodium chloride and subsequently dried. Up to 100 mg of dye was dissolved in dry dimethylsulfoxide. After the addition of the isocyanate terminated polyurethane hydrogel, the dye became bound to the polyurethane. A pH-indicating hydrogel was formed.
• a solution of soluble starch (2.0) in 10 ml water containing 2% K 2 HPO 4 and 2.5% sodium borate is stirred with a 2,3-dichloropropano-amido-subsituted azo dye (50-500 mg) for 1 hour at 60o-80oC. Unreacted dye and reagents may be removed by dialysis or gel filtration.
• the resulting polymeric pH-indicating material is lyophilyzed and subsequently incorporated in a variety of hydrogel-forming materials by simply admixing before curing. Examples of hydrogel-forming materials include isocyanate-terminated polyurethane prepolymers.
• the polysaccharide acted as the cross- linking reagent. It is also possible to form a hydrogel by cross-linking the dye-containing polysaccharide using readily available di- or polyfunctional reagents.
• PEG1000 (2.53 g) was dried in a reactor under vacuum at 100oC for one hour and the vacuum was broken with dry nitrogen and TDI (0.551 g) was added. The mixture was allowed to react for two hours at 100oC to produce an isocyanate-terminated prepolymer.
• B 5 MeETA (1g) in dry DMSO (9g) was charged to the reactor. The reaction was completed in thirty minutes as evidenced by IR spectra.
• the number average molecular weight of the hydroxy-terminated dye prepolymer product was at 13,000 with a broad molecular weight distribution and hydroxyl content of 0.32 meq./gm.
• EXAMPLE 61 OH terminated/Dye (5%) /TDI formulation A hydroxy-terminated polyurethane prepared according to the techniques of the foregoing examples, using PEG1000, TDI and TMP (6.17 g, 70%) was mixed under nitrogen atmosphere with B 5 MeETA (0.257 g) and TDI (0.56 g), with enough DMF to make a 55% solution at room temperature. The viscous solution was spin coated on a Mylar disk to form a 10 micron-thick film, followed by curing at 110oC for two hours. Any unreacted or loosely bonded dye was leached out in DMF. The dye-hydrogel film reached equilibrium hydration of 65% in less than one minute. It changed color from red to blue reversibly on contacting different pH solutions. The pK a of the film was 7.05.
• a dye hydrogel was formulated and cured following the procedures of Example 61, except that a trifunctional isocyanate, Desmodur IL (Mobay Chemicals), was used.
• the film had only 31% hydration and a pK a of 6.51.
• a dye hydrogel was prepared following the procedures of Example 61, except the amount of dye was adjusted to make up 8% by weight of the total formulation.
• the film had 56% hydration and a pK a of 6.74.
• Example 50 The hydroxy-terminated polyurethane prepolymer of Example 50, the diol dye B 5 MeETA, and a mixture of TDI-Desmodur IL in a ratio of 30%-70% (mole) were reacted using the same procedures as in Example 60.
• the resultant film had a 72% hydration and a pK a of 6.70.
• OH-terminated ionic/Dye 5%) /Desmodur IL:
• the hydroxy-terminated ionic polyurethane prepolymer from Example 54 (3.30), B 5 MeETA dye (0.119 g), and Desmodur IL (1.479 g) were mixed well and the solution was spin-coated on Mylar to form a 6.5 micron film.
• the film had a 41% hydration and a pK a of 6.80.
• the isocyanate-terminated polyurethane hydrogel (1.17 g) PEG1000 (0.26 g) and B 5 -Tris dye (0.075 g) (5% by weight) are mixed with DMF (1.426 g) .
• the solution is spin-coated and cured on Mylar to form a 5 micron film.
• the film has a 65% hydration and a pK a of 7.07, and also possesses acceptable optical density.
• B 5 -Tris dye 0.3 g, 4% by weight
• the isocyanate-terminated polyurethane hydrogel 7.2 g
• the solution is spin-coated and cured as in Example 48.
• the film had a 43% hydration and a pK a of 7.4.
• B 5 -Tris dye (0.25 g, 8% by weight) and the isocyanateterminated polyurethane hydrogel (2.875 g) are mixed in DMF (2.66 g).
• the solution is coated and cured as in Example 65.
• the film had a 30% hydration and a pK a of 7.3. This is a good film for using as a sensor.
• B 5 -Tris dye (0.25 g, 10% by weight) and the isocyanateterminated polyurethane hydrogel (0.5 g) are mixed in dry DMF (2.25 g) .
• the solution is spin coated as in Example 65, to form a 4 micron film.
• the film had a pK a of 6.72.
• Example 59 The dye-containing hydroxy-terminated prepolymer of Example 59 (0.493 g) TDI (0.068 g) and Desmodur IL (0.052 g) were mixed well. The solution was spin-coated and cured as in Example 65. The film had a 68% hydration and a pK a of 6.72.
• a pigment-containing hydrogel film may be prepared by mixing a white pigment (Rutile, a crystal form of titanium dioxide, 79.2 g), a hydroxy-terminated polyurethane prepolymer (28.3 g) and cyclohexanone (42.5 g), and dispersing the solution in a ball mill to prepare a mill base. The solution is then compounded with polyurethane (40 g), cyclohexanone (64 g) and TDI (5.42 g) along with a small amount of catalyst such as di-n-butyltin dilaurate and triethylenediamine (1,4-diaza[2.2.2]bicyclcooctane). The resultant coating mix is used to deposit a white reflective layer by spin coating.
• a pigment-containing hydrogel film may be pepared by mixing a black pigment (carbon black, 28 g), polyurethane prepolymer (23 g) and cyclohexanone
• the mill base is compounded with polyurethane (55 g), cyclohexanone (120 g) and TDI (7 g), along with a small amount of catalyst as mentioned in Example 71.
• the resultant mix is used to deposit a black absorptive layer by spin coating.
• a pH-indicator dye film and any other necessary layers may be deposited directly on the interior surface of a cuvette cell into which fluids to be analyzed will be introduced; however, for ease of fabrication and replacement, it is much preferred to deposit the layers on an optically-clear support sheet, such as a 5-mil thick polyester film (e.g., Melinex film, ICI Americas Inc., Wilmington, Delaware or Mylar, E. I. DuPont de Nemours, Inc.).
• an optically-clear support sheet such as a 5-mil thick polyester film (e.g., Melinex film, ICI Americas Inc., Wilmington, Delaware or Mylar, E. I. DuPont de Nemours, Inc.).
• hydrogel polymer maintain its structural integrity when it is loaded with indicator dyes, pigments or other substances, so that it possesses the appropriate optical and chemical properties in the pH range of interest.
• a thin reflective film is deposited over the dye film.
• the reflective film comprises a hydrogel and at least one pigment having a higher refractive index than water, preferably titanium dioxide.
• the hydrogel may be the same or different than that of the dye film layer, * and in addition to the polyurethane hydrogels discussed above, special mention is made of hydrogels based on polyacrylamides, many of which maintain especially high water contents even with high pigment loadings.
• a further pigmented film advantageously applied over the reflective film layer is a light absorptive film.
• This film is similar to the reflective film, except that its pigment absorbs electromagnetic radiation rather than reflecting it. Carbon black is the most preferred pigment for the light absorptive film.
• the total percentage of solids in these formulations is usually about 60%, which gives the solution a sufficiently high viscosity for good coatability, but is not so high as to preclude it from spin coating films of only a few microns thickness.
• the prepolymer is mixed in by stirring or shaking.
• the solution is again centrifuged to make subsequent filtration through a 1 micron membrane easier. This step removes precipitated material and solids resulting from introduction of the prepolymer.
• a final centrifugation removes any air bubbles.
• the hydrophilic polyurethane solutions thus formulated e.g., with pigment or dye
• spin coating methods are used.
• Viscosity is a function of polymer molecular weight, total solids, solvent type, temperature, and the presence of other ingredients which may react with the polymer components. The viscosity also varies according to the processing equipment. For thin film spin coating (0.5 to 50 microns), the desired viscosity is generally in the range of about 100 to 1,000 centipoise.
• a small amount of dyepolymer solution is dispensed in the center of a stationary substrate.
• the substrate is rapidly accelerated centrifugally to a final rpm where it is held for several seconds.
• the dye films are cured, e.g., in a forced air oven.
• complete curing is usually achieved in two hours at 90 to 110oC.
• a “reflective” (i.e., backscattering) film formulation may be prepared by blending a pigment such as titanium dioxide (e.g., Kemira rutile) with the polymer used to make the reflective layer, with a solvent if desired. Pigment loading determines how thick the layer needs to be to give satisfactory reflective properties, keeping in mind that thicker layers lead to longer dynamic response times. Generally, however, for thin reflective films of about 3-5 microns thickness, pigment loadings of about 50-60% by weight are sufficient.
• a pigment such as titanium dioxide (e.g., Kemira rutile)
• pigment loading determines how thick the layer needs to be to give satisfactory reflective properties, keeping in mind that thicker layers lead to longer dynamic response times. Generally, however, for thin reflective films of about 3-5 microns thickness, pigment loadings of about 50-60% by weight are sufficient.
• the pigment-containing formulations are milled for a long period, e.g., 1-10 days depending on such factors as pigment loading, type of mill, presence of a solvent, etc., until a mill base having the desired particle size and desired viscosity is achieved.
• the mill base is then formulated with additional prepolymers (including polyfunctional crosslinking components) stirred, and filtered (e.g., 5 microns) before coating over the dye film layer.
• a non-reflective, absorptive film may be prepared in much the same manner as the reflective film.
• the absorptive layer serves an optical decoupling function, making the measurement of the dye color immune to variations in the reflectivity and/or absorptivity of electromagnetic radiation of the fluid sample to be analyzed.
• Carbon black e.g., Raven 1040, Columbian Chemical
• a cured polymer layer can be made which acts as an optical barrier.
• the absorptive nature of carbon black is such that a 1 micron layer with only a 25% pigment loading by weight is sufficiently opaque.
• an additional film is used in the multilayered sensing element.
• a gas- separating membrane is coated over the non-reflective, absorptive film. This allows for effective diffusion rates of water vapor and CO 2 gas, while liquid water and ions are excluded.
• the membrane can be placed between the dye film and the reflective film which results in shorter response times to pCO 2 changes.
• Three additional processing steps distinguish the pCO 2 sensing element from the pH sensing element. These steps all occur between the washing of the dye layer and the coating of the white layer.
• the pCO 2 sensor functions by changes in pH, caused by shifts in the pCO 2 -carbonic acid equilibrium, producing differences in the dye absorbance.
• the population of dye molecules should first be converted to their anionic form before being sealed in their environment with the gas-separating membrane. Soaking the dye-polymer films in aqueous solutions of pH greater than the pKa of the dye can deprotonate the desired amount of dye. Two aspects of this step are important: The percentage of dye converted to its salt must be very reproducible, and the process cannot leave behind any species which interfere with protonation.
• the films are soaked in buffer solution having high pH (e.g., pH 9-10) which has preferably been filtered (e.g., 0.45 micron) for cleanliness. Before the next step, the films are allowed to air dry completely.
• the gas-permeable layer preferably a silicone membrane, isolates the dye layer from ionic species, specifically hydronium. Polydimethylsiloxane is most preferred for the membrane material because of its high permeability to carbon dioxide and water. Physically, the membrane needs to be thin enough for an acceptable response time while having good integrity so that no bulk fluid can pass through.
• the silicone membrane is made by spin coating a solution of silicone polymer in a suitable solvent.
• Silicone elastomers are diluted to an appropriate percent solids level for spin coating thin films.
• Xylene and polydimethylsiloxane solvents with a b.p. between 110-150oC are used.
• the silicone products Petrarch Systems SE and Dow Corning 3140 are especially suitable to fashion membranes. As sold, Petrarch SE is 35% solids in a naphtha solvent. After dilution, the silicone solution is mixed by shaking and then is filtered through at least a 1 micron syringe filter to remove particulates.
• Cured silicone elastomers are known for their inertness and low surface energy. Coating or painting silicone materials is customarily difficult because of this very low surface energy. Since there is no surface pretreatment, the coating quality is very poor when the reflective film is spincoated onto the silicone membrane. The polymer solution dewets to such an extent that the entire surface can easily become uncovered. Plasma activation of the silicone surface may be used to both increase the wettability so that acceptable coatings can be made and to enhance the adhesion to these subsequent layers. Plasma activation by exposure to radio frequency discharge of a selected gas is a chemically complex process and will not be discussed here in detail.
• a preferred body fluid analyzer incorporating the hydrogels and dyes already disclosed, will be described with reference to the drawings. It will be understood that the hydrogels and dyes, dye films and sensing elements prepared therefrom, may be employed for purposes radically different the uses described herein design without departing from the scope of this invention. Similarly, the following description illustrates a preferred embodiment and is not provided to in any way limit the present invention.
• the body fluid analyzer described hereinafter is only one example of a multitude of possible designs which will be apparent to those familiar to this art. Referring to Figure 1, the body fluid analyzing apparatus is comprised of several linked sub-units, shown as separated structures (1, 2, 3, 5). Such sub-units could, of course, also be united in a single housing but are drawn as discrete units for ease of explanation.
• a monitoring unit 1 is provided which is capable of data signal evaluation and data display.
• a signal preamplification unit 2 may be provided to boost the photoreceptor data signal corresponding to the optical measurement of pH in the sensing cells (described infra).
• a cuvette housing 3 is provided incorporating optical measurement components including an electromagnetic radiation source (“light source”) and photodetectors (not shown) aligned so as to obtain color measurements of samples placed in a sensing cuvette 5 and having a slot 4 adapted for receiving the sensing cuvette 5.
• the sensing cuvette 5 is preferably equipped with tubing adapters 6 which permit introduction of a fluid to be measured into an interior channel (not shown) running through the cuvette 5 where the fluid contacts the sensing elements (not shown) of the apparatus.
• tubing adapters 6 which permit introduction of a fluid to be measured into an interior channel (not shown) running through the cuvette 5 where the fluid contacts the sensing elements (not shown) of the apparatus.
• optically clear windows 7 and 8 located on one side of the cuvette 5, color changes reflecting pH and pCO 2 changes can be measured by optical measuring components in cuvette housing 3.
• the cuvette has one or more sensing cells, each in open communicaton at one end with the channel in the cuvette interior and being closed at the other end by optically clear windows 7 and 8, which windows 7 and 8 come into alignment with the measuring optics of the cuvette housing 3 when the cuvette 5 is properly inserted in the slot 4.
• FIG. 2 a cross-sectional view of a possible sensing cuvette 5 is shown.
• Optically clear windows 7 and 8 are shown forming the closed ends of two sensing cells 13 and 13A, into which multilayered sensing elements have been inserted.
• two sensing cells are shown, having sensing elements for optical measurement of pH (13) and pCO 2 (13A) however, it will be seen that numerous additional cells may be fabricated in the body of the cuvette 5 (for pH, pCO 2 , or other measurements) depending on design preferences and the requirements of the practitioner.
• the sensing cuvette 5 is provided with inlet and outlet tubing 6 providing access to a channel 15 which provides a passageway through the cuvette 5.
• Sensing cells 13 and 13A open on the channel 15 to afford contact of the multilayered sensing elements with any fluid samples introduced into the channel 15.
• the sensing elements are comprised of a dye film 9 and other layers which adapt the sensing elements to the particular optics and design of the cuvette 5 and the analyzer as a whole.
• the sensing elements are adapted to permit optical measurement of light transmitted and backscattered through the dye film 9, which dye film changes color as a function of pH.
• the sensing cell 13 adapted to detect pH of a solution passing through the channel 15 features an optically clear window 7 which will come into line with the particular optic components of the cuvette housing (item 3 in Fig. 1).
• the pH-indicator dye film 9 may be coated directly on the window 7, but preferably the film 9 (and other layers) will be deposited on an optically clear substrate 16, such as polypropylene or a polyester film (e.g., 10 mil Mylar). Many suitable substrates will suggest themselves to the practitioner.
• a dye film 9 is deposited (e.g., spin coated) on a clear polymeric substrate 16.
• a reflective film 10 is deposited on the dye film 9, and an absorptive film 11 is deposited over the reflective film 10.
• the optically clear substrate 16 adjacent the optically clear window 8 is coated with a dye film 9, a reflective film 10 and an absorptive film 11, and also a gas permeable silicone layer 12.
• the gas-separating film 12 may be positioned elsewhere, for instance between layers 9 and 10, if desired.
• the inner dimensions of the cuvette 5 are preferably adapted to minimize the volume of fluid needed for analysis while providing sufficient volume to ensure contact with the sensing elements and completion of the protonation reaction with the indicator dyes.
• the cuvette may be about 40 mm high and about 20 mm wide, and the channel 15 may be about 9 mm in diameter.
• water and ions in the fluid diffuse freely through the hydrogel layers of the sensing cell 13 and contact the pH indicator dye in the dye film 9, which undergoes a shift of the protonation equilibrium, resulting in a shift in the wavelength of absorbance of light passing through the dye film.
• water vapor and CO 2 gas from the fluid diffuse through the gas-separating membrane 12. The water vapor hydrates the dye film in the presence of the CO 2 gas, resulting in the formation of carbonic acid which subsequently dissociates, altering the protonation equilibirium of the dye.
• the cuvette 5 is shown in relation to a light source 18 and an optical receptor 20, which would be contained in the analyzer housing (3 in Fig. 1).
• the light source 17 directs light (arrowed lines) toward the sensing cell 13.
• Light passes through the window 7, the clear film substrate 16 and the dye film 9, and then is backscattered off the reflective film 10.
• Photoreceptor 21 is positioned to accept the returned light emanating from the cell 13.
• the backscattered light differs from the source light directed as a function of the color of the dye film, and therefore gives an indication of the pH of the solution passing through the cuvette channel 15 in contact with the cell 13.
• the photoreceptor 20 emits an electronic data signal which is passed along to preamplification and data evaluation equipment (Fig. 1).
• Diazonium Comp. coupling Comp. Yield acid base pka 4-NO 2 -1-naphthyl 4-(NHCOCBrCH 2 ) 67% red- purple 10.0 5-OH-1-SO 3 H purple blue

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