Potentiometric ion determinations using enhanced selectivity asymmetric ion-selective membrane electrodes

Abstract: An ion selective electrode (4) employing an asymmetric membrane for the potentiometric determination of ions, e.g. carbonate in biological samples is disclosed. The asymmectric membrane comprises (i) an ion selective layer (8) containing an ion selective agent and a plasticizing agent and (ii) a sample contacting hydrophilic layer (6) fused to the ion selective layer. In a preferred embodiment for determining carbonate, a cellulose triacetate (CTA) hydrophilic layer with free hydroxyl groups is fused to a cellulose triacetate ion selective layer containing trifluoroacetyl-p-butylbenzene (TFABB) as ionophore, tridodecylmethylammonium bromide (TDMAC) as ion-complexing agent, and bis(2-ethylhexyl)adipate (DOA) as plasticizer. The electrode has the advantage of being less sensitive to interferents such as salicylate, gentisate, perchlorate etc.

Description: Field of the Invention This invention relates to a process for performing potentiometric ion determinations and more particularly to a process for performing enhanced selectivity potentiometric determinations using asymmetric ion-selective membranes. Background Art Electrochemical measurements are widely used to determine the concentration of specific substances in fluids. These devices, referred to as ion-selective electrodes (ISEs), can be employed in a wide variety of potentiometric ion determinations, including, for example, the activity of fluoride ion in drinking water, the pH of process streams, and the determination of electrolytes in serum. In the health care field, and in particularly in the area of clinical diagnostics, ISEs are commonly used to measure the activity or concentration of various ions and metabolites present in blood plasma, serum and other biological fluids. For example, ISEs are typically used to determine Na + , Ca ++ , Mg ++ , K + , Cl - , Li + , pH, and carbon dioxide content in such fluids. In general, conventional ISEs contain an ion-selective membrane, an internal filling solution or electrolyte, and an internal reference electrode. An external reference electrode used in conjunction with the ISE is typically a metal/metal halide electrode such as Ag/AgCl. Conventional ISEs are typically bulky, expensive, difficult to clean and maintain, and tend to require an undesirably large volume of biological fluid. For these reasons, much attention has been directed towards developing more reliable ISEs of smaller size. These relatively small ISEs, referred to as ion-selective sensors or biosensors, can be inexpensively mass produced using techniques similar to these employed in the manufacture of electronic components, including for example, photolithography, screen printing, and ion-implantation. Ion-selective sensors and biosensors can be manufactured at much lower production cost than conventional ISEs, making it economically feasible to offer a single-use or limited-use disposable device, thereby eliminating the difficulty of cleaning and maintaining conventional ISEs. The reduced size of ion-selective sensors further serves to reduce the required volume of patient sample. Generally, a sensor can be either a miniature version of a conventional electrode or a device constructed using one or more of the above mentioned techniques. Maximum accuracy of the analytical or diagnostic result is obtained when the sensor responds only to the concentration or activity of the component of interest and has a response independent of the presence of interfering ions and/or underlying membrane matrix effects. The desired selectivity is often achieved by an ion-selective membrane containing an ion-selective agent such as an ionophore. Generally, ion-selective membranes are formed from a heavily plasticized polymer matrix, such as polyvinyl chloride, which contains the ionophore selective for the ion of interest. For example, the ionophore valinomycin has been incorporated into a layer of membrane selective for potassium ions and trifluoroacetyl-p-butylbenzene or other trifluoroacetophenone derivatives has been used as ionophores selective for carbonate ions. Determination of the concentration or activity of ionic species is achieved by measuring the EMF (electrical potential difference between the internal and external reference electrode, the electrodes being electrolytically connected by means of the sample solution at zero or near zero current flow) using a high impedance volt-meter. The reference electrode is electrolytically connected to the sample solution, typically by means of a salt bridge. The accurate and rapid determination of total carbon dioxide species in physiological, industrial and environmental samples present a formidable challenge. Automated clinical analyzers are now routinely used for the determination of total carbon dioxide in biological samples using carbonate ion-selective electrodes or gas sensing electrodes configured as flow through detectors. However, none of the known carbonate selective membrane electrodes offer sufficient specificity or selectivity for unambiguous determination of carbonate ion. This is because ion-selective sensors utilizing carbonate selective ionophores are susceptible to interfering effects from comparatively large, hydrophobic ions such as perchlorate, gentisate, salicylate, p-amino salicylate and even larger species, such as heparin. Such salicylate ions are commonly present in physiological samples taken from patients treated with common analgesics. U.S. Patent No. 3,898,147, discloses a carbonate ion-selective electrode having a hydrogen ion permeable membrane which consists of a hydrophobic elastomer. Measurement of pH of an aqueous electrolyte solution within the hydrogen ion permeable membrane serves as an indirect measurement of bicarbonate. However, this approach assumes absorption of carbon dioxide is the only source of hydrogen ions in the sample. Further, this system is susceptible to interferences from other endogenous acids. U.S. Patent No. 4,810,351, discloses a carbonate ion-selective membrane and electrode utilizing an ionophore consisting of an alkyl substituted fluoroacetophenone. High alkyl substitution of the ionophore is suggested to reduce drift in the electrode'response in very fast flow situations. Further, the addition of a hydrophobic molecule to the ion-selective membrane formulation is suggested to provide for the improved exclusion of interfering anions by repelling charged species from the solution phase. A C7-C9 quaternary ammonium surfactant is disclosed for binding the carbonate ions. U.S. Patent No. 4,272,328, disclose an ion-selective electrode multilayer analytical element which includes an ionophore containing membrane and a buffer zone in an amount sufficient to control the pH of the solution analyzed between about 7.5 to about 9.5. Under these conditions, it is suggested the element is comparatively less sensitive to the interfering effects of gentisate, salicylate and p-amino salicylate. Elimination of interferences from other large anions, such as, heparin and/or endogenous fatty acids is not mentioned. U. S. Patent No. 4,199,411, discloses the structure of a halide ion-selective device having an overcoat made of a cellulose ester containing a polyol of from 2 to 6 hydroxyl groups. When added to the cellulose ester overcoat it was found the polyol "did not adversely affect the interference inhibiting characteristics of the cellulose ester layer". Hydrolysed cellulose acetate butyrate having from 6.4 to 8.3 percent hydroxyl groups is disclosed as one example of a cellulose ester. Said patent does not mention or suggest the use of an asymmetric membrane or layers. Anonymously, Research Disclosure 18730, November 1979 discloses the construction of halide ion-selective devices and the use of a halide-permeable overlayer comprising a cellulose ester to improve shelf life of the device. There is no mention or suggestion of an asymmetric membrane electrode. U. S. Patent No. 4,505,801 disclose an overcoat layer for an ion-selective membrane, the overcoat layer having high selectivity for carbonate ions over potentially interfering ions, such as, salicylate, particularly at high salicylate concentrations. The disclosed overcoat layer has a discontinuous hydrophobic solvent dispersed within a continuous hydrophilic binder, a complexing agent for extracting oleophilic anions, a buffer which provides a pH in the range 7.5 to about 9.5 and a nucleating agent. U. S. Statutory Invention Registration No. H745 disclose the use of a buffered hydrophilic polymer binder layer laminated to a carbonate ion-selective layer for the purpose of eliminating or reducing interferences from salicylate, p-aminosalicylate and gentisate. Cha et al., "Potentiometric Ion- and-Bio Selective Electrodes Based On Asymmetric Cellulose Acetate Membranes", Talanta, vol. 36, pp. 271-278, (1989) disclose a bioselective electrode consisting of a cellulose triacetate membrane having a hydrolyzed first layer fused to a second layer, the second layer containing a neutral carrier and plasticizer, and an enzyme covalently attached to the exposed hydrolyzed surface of the first layer. The potentiometric response of trifluoroacetyl-p-butylbenzene (TFABB)-doped asymmetric cellulose acetate membranes to carbonate and other anions is disclosed (Figure 4 of Cha). It is further disclosed the "asymmetric modification (to form hydroxyl groups) had little or no effect on the response characteristics of the membrane. The response slope and selectivity (or lack of it with respect to salicylate) observed are essentially the same as those found for PVC based membranes previously reported by Greenberg and Meyerhoff." The asymmetric membranes disclosed by Cha et al. were developed for biosensor applications. Cha et al. do not mention or suggest an enhanced selectivity process for potentiometric determinations. Further Cha et al. do not mention or suggest a process for potentiometric ion determinations in which an asymmetric membrane electrode provides for reduced interference from salicylate or such a process in which the activity of the ion of interest is determined kinetically in order to achieve enhanced selectivity, as opposed to statically (at equilibrium). Brief Description of the Figures Figure 1A depicts the dynamic response of carbonate ion-selective electrodes prepared with: (A): polyvinyl chloride (PVC), (B): unmodified cellulose triacetate (CTA), and (C): asymmetric CTA membranes subject to concentration changes of 10 -5 to 10 -4 M (moles/liter) for salicylate. Figure 1B depicts the dynamic response of carbonate ion-selective electrodes prepared with: (A): polyvinyl chloride (PVC), (B): unmodified cellulose triacetate (CTA), and (C): asymmetric CTA membranes subject to concentration changes of 10 -4 to 10 -3 M for CO 3 2- . Figure 2A depicts the potentiometric response of TFABB-doped unmodified CTA (Δ) and asymmetric CTA (o) membrane electrodes to the hydrophobic anion (A): salicylic acid. Potential measurements were made in a solution of 0.1 M Tris-H 2 SO 4 , at pH 8.6. Figure 2B depicts the potentiometric response of TFABB-doped unmodified CTA (Δ) and asymmetric CTA (o) membrane electrodes to the hydrophobic anion:(B): 3-hydroxy-2-naphthoic acid. Potential measurements were made in a solution of 0.1 M Tris-H 2 SO 4 , at pH 8.6. Figure 2C depicts the potentiometric response of TFABB-doped unmodified CTA (Δ) and asymmetric CTA (o) membrane electrodes to the hydrophobic anion (C): 3-hydroxy-2-anthracenecarboxylic acid. Potential measurements were made in a solution of 0.1 M Tris-H 2 SO 4 , at pH 8.6. Figure 3 is a schematic depiction of a membrane electrode-based flow injection analysis (FIA) system with a large volume wall-jet flow cell. Figure 4A depicts the potentiometric flow-injection anion response obtained with TFABB-doped carbonate-selective PVC membrane electrodes as flow-through detectors. Aliquots (150 µl) of anion standard solutions containing 0.1 M Tris-H 2 SO 4 , pH 8.6 buffer, were injected into a background electrolyte stream containing 0.1 M Tris-H 2 SO 4 , pH 8.6, at 7.5 ml/min. Figure 4B depicts the potentiometric flow-injection anion response obtained with unmodified normal CTA membrane electrodes as flow-through detectors. Aliquots (150 µl) of anion standard solutions containing 0.1 M Tris-H 2 SO 4 , pH 8.6 buffer, were injected into a background electrolyte stream containing 0.1 M Tris-H 2 SO 4 , pH 8.6, at 7.5 ml/min. Figure 4C depicts the potentiometric flow-injection anion response obtained with asymmetric CTA membrane electrodes as flow-through detectors. Aliquots (150 µl) of anion standard solutions containing 0.1 M Tris-H 2 SO 4 , pH 8.6 buffer, were injected into a background electrolyte stream containing 0.1 M Tris-H 2 SO 4 , pH 8.6, at 7.5 ml/min. Figure 5A depicts the dynamic response of the TFABB-doped PVC membrane electrodes to added standard solutions as follows: (A): 25 mM carbonate, (B): 1.0 mM salicylate, (C): a mixture containing 25 mM carbonate, 100 mM chloride, and 1.0 mM salicylate. For each measurement, 2 ml of standard solutions were added to 18 ml of background electrolyte solutions containing 0.1 M Tris-H 2 SO 4 , at pH 8.6. Figure 5B depicts the dynamic response of the asymmetric CTA membrane electrodes to added standard solutions as follows: (A): 25 mM carbonate, (B): 1.0 mM salicylate, (C): a mixture containing 25 mM carbonate, 100 mM chloride, and 1.0 mM salicylate. For each measurement, 2 ml of standard solutions were added to 18 ml of background electrolyte solutions containing 0.1 M Tris-H 2 SO 4 , at pH 8.6. Figure 6A depicts the potentiometric response of the PVC and asymmetric carbonate ion-selective membrane electrodes to added serum samples containing varying salicylate concentrations as follows: (A): reconstituted control serum, (B): control serum containing 25 mM CO 3 2- , (C): control serum containing 25 mM CO 3 2- and 0.01 mM salicylate, (D): control serum containing 25 mM CO 3 2- and 0.1 mM salicylate, (E): control serum containing 25 mM CO 3 2- and 1.0 mM CO 3 2- : (G): control serum containing 25 mM CO 3 2- ; (H) 25 mM CO 3 2- in deionized water. Between the measurements, membranes were washed with deionized water, and the serum samples were added in the order shown in the figure. Figure 6B depicts the potentiometric response of the asymmetric CTA ion-selective membrane electrodes to added serum samples containing varying salicylate concentrations as follows: (A): reconstituted control serum, (B): control serum containing 25 mM CO 3 2- , (C): control serum containing 25 mM CO 3 2- and 0.01 mM salicylate, (D): control serum containing 25 mM CO 3 2- and 0.1 mM salicylate, (E): control serum containing 25 mM CO 3 2- and 1.0 mM CO 3 2- : (G): control serum containing 25 mM CO 3 2- ; (H) 25 mM CO 3 2- in deionized water. Between the measurements, membranes were washed with deionized water, and the serum samples were added in the order shown in the figure. Figure 7 schematically depicts preparation of an asymmetric carbonate ion-selective membrane comprising an asymmetric membrane having a hydrophilic layer fused to an ion-selective layer. Figure 8A schematically depicts a solid state planar sensor format for an asymmetric ISE. Figure 8B schematically depicts the hydrophilic and ionselective layers of an asymmetric ISE in a solid state planar sensor format. This invention relates to a process for performing potentiometric determinations of CO 3 2- ions in biological samples using an ion-selective asymmetric membrane electrode and a reference electrode electrolytically connected to the ion-selective asymmetric membrane electrode which comprises: (a) contacting a biological sample suspected of containing CO 3 2- ions with the ion-selective asymmetric membrane electrode, the ion-selective membrane electrode having an asymmetric membrane with an ion-selective layer and a hydrophilic layer fused to the ion-selective layer, wherein (i) the ion-selective layer contains para-butyltrifluoroacetylbenzene as ionophore, tridodecylmethylammonium chloride as a ion-complexing agent and bis(2-ethylhexyl)sebacate or bis(2-ethylhexyl)adipate as plasticizing agent, (ii) the hydrophilic layer contains -OH groups and is in contact with the sample, and (iii) the hydrophilic layer and the ion-selective layer are membranes prepared from cellulose triacetate; (b) kinetically measuring the potential difference between the ion-selective asymmetric membrane electrode and the reference electrode; and (c) relating the potential difference to the concentration of the CO 3 2- ions in the sample, thereby enhancing potentiometric ion selectivity. In a preferred embodiment of the above process (i) prior to step (a) the biological sample suspected of containing CO 3 2- ions and containing an interfering substance is diluted with a sufficient amount of a buffer solution such that the concentration of the interfering substance is less than 2 x 10 -3 M and the pH of the diluted sample is 7.5 to 9.5. Detailed Description of the Invention This invention relates to a process for performing potentiometric ion determinations on biological samples using an ion-selective asymmetric membrane electrode and a reference electrode electrolytically connected to the ion-selective asymmetric membrane electrode. The process comprises contacting a biological sample suspected of containing CO 3 2- ions with the ion-selective asymmetric membrane electrode, the ion-selective asymmetric membrane electrode having an asymmetric membrane with an ion-selective layer and a hydrophilic layer fused to the ion-selective layer, the ion-selective layer containing an ion-selective agent and a plasticizing agent, the hydrophilic layer being in contact with the sample, measuring the potential difference between the ion-selective asymmetric membrane electrode and the reference electrode; and relating the potential difference to the concentration of the CO 3 2- ions in the sample, thereby enhancing potentiometric ion selectivity. By "ion-selective electrode (ISE)" is meant a potentiometric electrochemical sensor, the potential of which is related to the activity of CO 3 2- ions in the sample. Generally, the potential is linearly dependent on the logarithm of the activity of the ion of interest. The activity of the CO 3 2- ions is defined as its concentration multiplied by an activity coefficient, where the activity coefficient is generally known or available in the art. By "biological sample" is meant any fluid of biological origin including fluids of biological origin which have been chemically and/or physically treated, diluted, or concentrated prior to analysis. Examples of biological samples include serum, urine, plasma, whole blood, cerebrospinal fluid, amniotic fluid, saliva and tears. The CO 3 2- ions are hereinafter also referred to as "ions of interest". A general discussion of the principles of potentiometric ion sensors is provided by Oesch et al., "Ion Selective Membranes for Clinical Use," Clinical Chemistry, Vol. 32, No. 8, pp. 1448-1459, (1986). Known conventional ISEs are typically composed of an ion-selective membrane, an internal filling solution, and an internal reference electrode. ISEs can be classified according to the nature of the membrane material, and include solid state membrane electrodes, glass membrane electrodes, liquid membrane electrodes having charged ion-selective agents, and neutral liquid membrane electrodes having membranes formed from an organic solution containing an electrically neutral, ion-selective agent such as an ionophore held in an inert polymer matrix. An ISE in combination with a sample solution, such as a biological sample, and an external reference electrode comprise a potentiometric cell assembly, so that by selectively transferring the CO 3 2- ions from the sample solution to the membrane a potential difference is generated between the internal filling solution and the sample solution which can be mathematically related to the concentration or activity ratio of the CO 3 2- ions in the two solutions contacting the membrane. Under ideal selectivity conditions of the membrane for the ion of interest, the potential difference is a linear function of the logarithm of the activity ratio of the CO 3 2- ions in the two solutions contacting the membrane (Nernst equation). A semi-empirical extension of the Nernst Equation (Nikolskii Eisenmann equation) for EMF may be utilized for non-ideal conditions. By EMF is meant the electrical potential difference between the internal and external reference electrode, the electrodes being electrolytically connected by means of the sample solution at zero or near zero current flow. The ISE utilized in the present invention is an enhanced selectivity asymmetric membrane electrode. The asymmetric membrane has a hydrophilic layer fused to an ion-selective layer. The hydrophilic layer is prepared using a thin polymeric membrane having a hydrophilic side. The polymer utilized to prepare the polymeric membrane is cellulose triacetate (CTA). The hydrophilic layer and ion-selective layer of the asymmetric membrane are formed by fusing a mixture of a CTA containing ion-selective agents and a plasticizing agent to the polymeric membrane having a hydrophilic side. The polymeric membrane having a hydrophilic side can be prepared by first dissolving CTA in an amount of solvent, or mixture of different solvents, sufficient to dissolve CTA, casting the dissolved CTA onto a plate or other surface, and allowing the solvent to evaporate. The amount of CTA used should be sufficient so as to produce a membrane of suitable thickness and structural integrity to withstand the hydrolysis of one of its sides and subsequent fusion to the CTA containing the ion-selective agent and the plasticizing agent. The thickness of the resulting polymeric membrane having a hydrophilic side can be from 1 to 1000 µm in thickness and is preferably 50 µm in thickness. The hydrolysis of one side of the polymeric membrane can be achieved by subjecting it to a hydrolyzing agent to produce a hydrophilic side composed of -OH groups as hydrophilic functional groups. For example the hydrolysis can be easily achieved by floating the the polymeric membrane on a liquid hydrolyzing agent. By "hydrolyzing agent" is meant any substance capable of reacting with the polymeric membrane to produce -OH groups on one side of the membrane. Bases are examples of suitable hydrolyzing agents. A wide variety of bases including, but not limited to, NaOH can be used. The hydrolysis is allowed to proceed for a time sufficient to produce -OH groups on one side of the layer such that the desired maximum selectivity of the asymmetric membrane is achieved (maximum response to the ion of interest and maximum reduction of response to the interfering substance), without destroying the structural integrity of the polymeric membrane during hydrolysis. Extended periods for hydrolysis may result in hydrolysis of the entire polymeric membrane and can result in subsequent difficulties in fusing with the polymer having the ion-selective agent(s) and plasticizing agent:; such a problem may be overcome by employing thicker polymeric membranes or shorter reaction times. Optimized hydrolysis times for particular polymeric: membranes can be determined by subjecting the membranes to hydrolysis for various time periods and comparing the responses of the membranes thus formed. The reaction time for hydrolysis can vary from 30 minutes to several hours, preferably the reaction time is about three to ten hours. The polymeric membrane having a hydrophilic side can then be dried in preparation for the fusion process. The membrane can be dried using any means which allows for the evaporation of solvent without destroying the structural integrity of the membrane. Air drying at room temperature is preferred. The hydrophilic hydrolyzed side of the polymeric membrane is that side of the asymmetric membrane subsequently placed in direct contact with the biological sample. The preferred polymeric membrane for use in the invention is a cellulose triacetate membrane having a hydrophilic side composed of hydroxyl groups. The layer can be simply prepared by dissolving cellulose triacetate in a mixed solvent such as methylene chloride and chloroform, casting the solution as a thin layer onto a flat surface to produce a layer of . 50 µm thickness, and allowing the solvent to evaporate on standing. The cellulose triacetate layer can then be floated on a solution of NaOH for a sufficient period of time such as 4-10 hours, washed with water, and air-dried. A mixture of a CTA layer containing ion-selective agents and plasticizing agent is then fused with the polymeric membrane having a hydrophilic side. First, a solvent is added to the non-hydrophilic side of the polymeric membrane having a hydrophilic side in an amount sufficient to soften or swell the polymeric membrane without completely dissolving the membrane or destroying its structural integrity by disrupting the hydrophilic side. By "fusion" is meant a process whereby a mixture of CTA, ion-selective agents, and plasticizing agent is cast onto the non-hydrophilic side of the polymeric membrane having the hydrophilic side, and the solvent is allowed to evaporate. The hydrophilic side of the polymeric membrane is immiscible with the plasticizing agent in which the ion-selective agent is dissolved. The ion-selective agents present in the cast polymer solution diffuse into and mixes with the polymeric matrix of the polymeric membrane having a hydrophilic side and stop upon reaching the hydrophilic side of the membrane. The amount of the mixture of polymer containing the ion-selective agents and plasticizing agent used should be sufficient to produce an asymmetric membrane of suitable thickness and structural integrity for use in an asymmetric ion-selective electrode. The thickness of the resulting polymeric membrane can be from 1 to 1000 µm in thickness and is preferably 100 µm in thickness. The resulting asymmetric membrane consists of two fused layers, the hydrophilic layer and the ion-selective layer. The hydrophilic layer, upon forming after fusion is preferably 10 µm in thickness. This process is depicted schematically for the preparation of an asymmetric carbonate ion membrane in Figure 7. Initially a polymeric membrane having a hydrophilic side (1) is prepared using the procedure described above. Then a second polymer (2) containing ion-selective agents (TFABB as an ionophore and TDMAC as an ion-complexing agent) and a platicizing agent is cast onto the non-hydrophilic side of the first polymeric membrane. Diffusion or mixing of the ion-selective agents into the first polymeric membrane occurs (across dotted line in Figure 7) to produce an asymmetric membrane having a hydrophilic layer (3) and an ion-selective (4) layer. Alternatively, the polymeric membrane containing the ion-selective agents can be cast first, and a second polymeric membrane having a hydrophilic layer subsequently attached to the polymeric membrane containing the ion-selective agents This method of preparation is especially suited for use in a solid state planar sensor ISE format. The use of an asymmetric membrane in the present invention is significant in that the fused layers allow for the incorporation of both ion-selective agents and a hydrophilic layer in a given ion-selective membrane electrode. Direct hydrolysis of a membrane containing ion-selective agents does not appear to produce a membrane having the enhanced selectivity characteristics of the asymmetric membrane of the present invention. Further, the relatively slow response of an asymmetric CTA membrane in comparison with non-asymmetric membranes (as described in the Examples) towards large, hydrophobic anions may be due to the presence of the hydrophilic layer of the asymmetric membrane. Differences in response with respect to an asymmetric carbonate ion-selective membrane may be further explained in terms of differences in the transport rate across the hydrophilic layer resulting from the rapid reaction of the hydration sphere of the carbonate ion relative to that of a slower, less hydrophilic ion such as salicylate. The same polymer, cellulose triacetate, is used for both the hydrophilic layer and the ion-selective layer of the asymmetric membrane. By "plasticizing agent" is meant a substantially water immiscible organic solvent which serves to solvate (plasticize) the polymer utilized in preparing the ion-selective layer. Further, the plasticizing agent is utilized to dissolve the ion-selective agents prior to casting of the mixture of the polymer containing the ion-selective agents and the plasticizing agent onto the polymeric membrane having a hydrophilic side. Plasticizing agents which are utilized in the present invention are bis(2-ethylhexyl)sebacate (DOS) and bis(2-ethylhexyl)adipate (DOA). The amount of plasticizing agent used is that amount sufficient to maintain a solvated ion-selective layer without destroying the selectivity or structural integrity of the membrane. The amount of plasticizing agent can vary from 45 to 75 weight%. Preferably a concentration of 65 weight% plasticizing agent is preferred. By "ion-selective agents"is meant a combination of substances which provides for the ion-selectivity of the ion-selective layer. The ion-selective agents are trifluoroacetyl-p-butylbenzene (TFABB) as neutral ionophore and a charged ion-complexing agent. The amount of ionophore used is that amount sufficient to provide for the maximum selectivity of the membrane without altering or destroying the structural integrity of the membrane. The amount of ionophore can vary from 0.01 to 65 weight% Preferably a concentration of 1 weight% ion-selective agent is preferred. By "ion-complexing agent" is meant a substance capable of forming a complex with the ion of interest, wherein the formation of the complex is attributable to differences in charge between the ion of interest and the ion-complexing agent. Thus the ion-complexing agent is tridodecylmethylammonium chloride (TDMAC). The amount of ion-complexing agent used is that amount sufficient to provide for the maximum selectivity of the membrane without altering or destroying the structural integrity of the asymmetric membrane. The amount of ion-complexing agent can vary from 0.1 to 5 weight%. Preferably a concentration of 0.5 weight% ion-selective agent is preferred. The asymmetric ion-selective membrane electrode of the present invention comprises an asymmetric membrane and a conductor. By "conductor" is meant any electrically conductive substance which is in contact with the ion-selective layer of the asymmetric membrane. Examples of suitable conductors include metal/metal salts, metals, and carbon. By contact is meant either direct or indirect contact. By indirect contact is meant the addition of an electrolyte (typically as an electrolyte layer) between the ion-selective layer of the asymmetric membrane and an internal reference electrode. For example, in a conventional ISE the conductor can be an internal reference electrode such as a metal/metal halide electrode (for example, an Ag/AgCl electrode) which is in indirect contact with the ion-selective layer of the asymmetric membrane by means of an electrolyte solution which serves as the internal filling solution. The electrolyte contains a common anion with the metal salt and must be of sufficiently high concentration to provide for low resistance and stable potential. Examples of suitable internal filling solution for use with conventional carbonate ISEs include but are not limited to NaH 2 PO 4, and an aqueous mixture of NaH 2 PO 4 and NaCl. Alternatively, the asymmetric membrane can be utilized in a solid-state planar sensor electrode format in which case the electrolyte may or may not be utilized between the conductor and the asymmetric membrane. Solid state ion sensors are typically made by casting ion-selective polymeric membranes directly onto a solid surface, such as a metal conductor. Figure 8A depicts schematically a solid-state planar sensor format having an asymmetric membrane ISE. The sample solution (1) is diluted with a buffer (2) and mixed using any suitable mixing device (3), such as an in-line mixer (for example, a spinning shaft) or an elastomeric element with mutating fingers for mixing. The mixed solution is then contacted with the asymmetric membrane ISE (4), the response determined by measuring the difference in potential between a reference electrode (not shown) in contact with the sample and the carbonate ion-selective electrode, and the remaining waste (5) solution discarded. The asymmetric membrane (Figure 8B), having a hydrophilic layer (6) and an ion-selective layer (8) is directly in contact with a conductor (9), the membrane being at least partially enclosed by a dielectric material (non-conducting substance) such as ceramic or glass. The asymmetric ISE is placed on a suitable substrate for support (10). By "reference electrode" is meant any suitable external reference electrode, such as a metal/metal halide electrode. The reference electrode maintains a virtually invariant potential under the conditions prevailing in an electrochemical measurement and serves to permit the observation, measurement, or control of the potential of the asymmetric membrane electrode. For example, an Orion sleeve-type double junction Ag/AgCl electrode (Model 90-02) can be utilized as a reference electrode. The reference electrode is electrolytically connected (such as through a salt bridge) to the sample which is in contact with the asymmetric membrane such that the potential difference between the ion-selective asymmetric membrane electrode and the reference electrode can be measured. The electrodes can be connected through a high impedance amplifier or by any means capable of measuring a potential difference. The potential difference or EMF is determined through kinetic measurements. Kinetic measurements are obtained by measuring the potential difference with time, prior to equilibrium in a static mode measurement, or by measurements performed in flow injection analysis studies. Such kinetic measurements can be performed using a flow through detector which is fitted to an electrode body, such as a Phillips electrode body; (IS-561), Glasblaserei Moller, Zurich). A kinetic measurement can be performed using such a flow through detector in conjunction with a standard metal/metal halide reference electrode (such as AG/AgCl), an ion sensing electrode having an asymmetric membrane, and a suitable sample flow loop system (as described in the Examples). The EMF or potential difference values can be recorded with time using a high impedance amplifier/analog-to-digital converter. One of the important features of the present invention is the ability to perform enhanced selectivity potentiometric ion determinations by reducing the undesirable effects caused by various interfering substances. By "interfering substance" is meant a substance other than the ion of interest, whose presence in the biological sample affects the measured potential of an ISE for a particular ion of interest. Such interfering substances can result in incorrect potential difference or EMF determinations, such as falsely elevated or falsely depressed activities or concentrations for the ion of interest. Examples of interfering substances include salicylate or p-amino salicylate, other salicylate-like hydrophobic organic acids, and gentisate. A preferred asymmetric membrane for use in the present invention is a carbonate ion-selective membrane consisting of a cellulose-triacetate hydrophilic layer composed of hydroxyl groups, fused to a cellulose triacetate ion-selective layer having TFABB as the ionophore, DOA as the plasticizing agent and contains TDMAC as an ion-complexing agent. The carbonate species (CO 3 2- ) is related to gaseous CO 2 by the following equilibrium relationships: CO 2(g) + H 2 O <-----> H 2 CO 3 <---K 1 --> H + + HCO 3- HCO 3 - <--K 2 ---> H + + CO 3 2- where K 1 and K 2 are the dissociation constants and K 1 = 4.3 X 10 -7 and K 2 = 4.8 X 10 -11 at 25°C. At normal serum pH of 7.0 to 7.5, the majority of the species are in the bicarbonate form. By shifting the pH to a more alkaline pH of 8.5 to 8.7, a siginficantly higher proportion of the species present will be in the carbonate (CO 3 2- ) form. The carbonate asymmetric membrane responds to CO 3 2- because of the TFABB as a result of the carbonate species forming a 1:1 or a 2:1 adduct with the ionophore TFABB in the ion-selective layer of the membrane This is disclosed by Meyerhoff et al., Role of Trifluoroacetophenone Solvents and Quaternary Ammonium Salts In Carbonate-Selective Liquid Membrane Electrodes", Analytical Chemistry, vol. 59, pp. 144-150, (1987). The structure of TFABB and the 1:1 and 2:1 adducts is shown below: TFABB can be prepared using known procedures such as that described by Herman, H. B., et al.,Preparation and Properties of a Carbonate Ion Selective Membrane and Electrode, Analytica Chimica Acta, vol. 76, pp. 155-164, (1975). The following examples serve to illustrate the present invention. EXAMPLE 1 Preparation of An Asymmetric Cellulose Triacetate Carbonate Ion-Selective Membrane A thin cellulose triacetate membrane was prepared by dissolving 100 mg of cellulose triacetate (CTA), (Fluka Chemicals, Ronkonkoma, NY) in 2.5 ml of a mixed, analytical-reagent grade, solvent containing 1.5 ml of methylene chloride (Junsei Chemical Co., Ltd, Tokyo, Japan) and 1.0 ml of chloroform (Junsei Chemical Co., Ltd, Tokyo, Japan). The solution was cast on a flat plate. Spreading of the solution was confined by a 36 mm inside diameter ring. The solvent was allowed to evaporate overnight leaving a membrane approximately 50 µm thick. The acetyl groups on the lower side of the membrane were hydrolyzed by floating the membrane on an excess of 1 M sodium hydroxide (Junsei Chemical Co. Ltd.) solution at room temperature for 4.5 h. This resulted in a polymeric membrane having a thin hydrophilic, hydroxylated side. The membrane was washed by immersing it in 500 ml of distilled water. This process was repeated a second time to insure termination of the hydrolysis reaction and allowed to air dry at room temperature. The dry membrane was placed on a second glass plate with the hydroxylated, hydrophilic side facing down. A 22 mm inside diameter ring was placed on the upper surface of the membrane to confine the spreading of subsequent solutions. 1.2 ml of methylene chloride was applied to the membrane to soften or pretreat the upper surface for 30 min. During this period the membrane was kept inside a desiccator with its vacuum tap open to reduce the rate of evaporation of the methylene chloride. A solution of the components for the carbonate ion-selective membrane was prepared by dissolving 35 mg of CTA; 16.6 mg of the ionophore trifluoroacetyl-p-butylbenzene (TFABB), (Specialty Organics Inc., Irwindale, CA); 100 µl of the plasticizing agent, bis(2-ethylhexyl) sebacate (Fluka Chemicals, Ronkonkoma, NY) and 2.0 mg of the ion-complexing agent tridodecylmethylammonium chloride (Polysciences Inc., Warrington, PA) in a mixed solvent containing 0.8 ml of methylene chloride and 0.8 ml of chloroform. This solution was applied to the membrane area within the 22 mm diameter ring and allowed to evaporate overnight. Evaporation was controlled by keeping the membrane in a vacuum desiccator with the vacuum tap slightly open. The mixture of the polymer containing the ionophore, plasticizing agent, and the ion-complexing agent was thereby fused to the non-hydrophilic side of the polymeric membrane to produce an asymmetric carbonate ion-selective membrane. EXAMPLE 2 Preparation and Use of a Carbonate Ion-Selective Electrode with an Asymmetric Carbonate Ion-selective Membrane Electrochemical properties of the membranes were evaluated in conventional ion-selective electrode devices. Small 5.5 mm diameter disks were cut from the asymmetric carbonate ion-selective membrane prepared as described in Example 1 and were tested using a Phillips electrode body (Glasblaserei Möller, Model IS-561, Zurich, CH). A disk was placed in an electrode body with the hydrophilic layer facing the outer sample solution (electrolyte). The internal filling solution employed contained 0.1 M sodium dihydrogen phosphate, 0.1 M disodium hydrogen phosphate, and 0.01 M sodium chloride. The conductor (internal reference electrode) used was a standard Ag/AgCl electrode. The reference electrode used was a sleeve-type double junction silver/silver chloride electrode (Orion, Model 90-02). The potential of the carbonate ion-selective electrode was measured using a high input impedance electrometer connected to a personal computer (Young Sun Computer Co., Ltd., Seoul, Korea, Model; compatible with an IBM 286 based computer) equipped with an analog-to-digital converter or by using an electrode switch box (Fisher Accumet, Model 157; Romulus, MI) and a pH-millivolt meter (Fisher Accumet, Model 620) All of the data of Figs. 1B, 2A, 2B and 2C and Tables 1 and 2 below was obtained using the electrode switch box (Fisher Accumet, Model 157; Romulus, MI) and a pH-millivolt meter (Fisher Accumet, Model 620) All of the data of Figures 3 through 6 was obtained using a high input impedance electrometer connected to a personal computer (Young Sun Computer Co., Ltd., Seoul, Korea, Model) equipped with an analog-to-digital converter. The response and selectivity characteristics of the asymmetric membrane electrodes were evaluated by comparison with identical assemblies in which the asymmetric CTA membrane was replaced by an ion-selective layer prepared from either unmodified (not subject to hydrolysis) CTA or polyvinyl chloride (PVC). These membrane materials, prepared and testing using known procedures and techniques are disclosed by Cha, G. S. and Meyerhoff, M.E., "Potentiometric Ion- and BioSelective Electrodes Based on Asymmetric Cellulose Acetate Membranes" Talanta, vol. 36, pp. 271-278, (1989). Equilibrium Potentiometric Responses Using Static Mode Measurements (Comparative Example) Potentiometric evaluation of the carbonate ion-selective electrode with the asymmetric CTA membrane described in Example 2 was conducted using static mode measurements in which the potential difference between the electrolyte and the sample solution was allowed to reach equilibrium. Measurement of the electromotive force at 10 minutes after the standard addition of the respective salt was found to be equivalent to equilibrium. The equilibrium potentiometric response characteristics of the asymmetric CTA membrane were evaluated and compared with unmodified (not subject to hydrolysis) CTA membranes. These responses were obtained by adding standard solutions added to a stirred 200 ml volume of background electrolyte (0.1 M 2-amino-2-hydroxymethyl-1,3-propanediol, TRIS-sulfuric acid mixture, pH 8.6). Measurements were taken at room temperature and the equilibrium measurements recorded. Table 1 provides selectivity coefficients calculated using the equilibrium response potential taken 10 minutes after the standard additions. The potentiometric selectivity coefficient (log k pot co 2- / 3, i ) can be calculated by the following: log k pot co 2- 3 , i = E 2 - E 1 2.303 RT / z A F + 1- ( z co 2- 3 ) z i (log a co 2- 3 ) where: a co 2- 3 is the activity of the carbonate ion. z co 2- 3 is an integer with the sign and magnitude corresponding to the charge of the carbonate ion. z i is an integer with the sign and magnitude corresponding to the charge of the interfering ion. E 1 and E 2 are the experimentally observed potentials of the two separate solutions, one solution containing the carbonate ion at an activity a co 2- 3 , the other containing the ion i at the same activity. R is the gas constant and is equal to 8.31441 JK -1 mol -1. T is the thermodynamic temperature (in K). F is the Faraday constant and is equal to (9.648670 ± 0.000054) x 10 4 C mol -1 . The results shown in Table 1, indicate that asymmetric modification of the instant invention had little or no effect on the response to small anions, such as carbonate and chloride. However, the asymmetric carbonate ion-selective membrane exhibited reduced responses towards larger anions, such as salicylate. Example 4 Kinetic Potentiometric Responses A. Dynamic Responses Using Static Mode Measurement's Differences in the quantitative responses are demonstrated by measuring the dynamic response curves of the electrodes to different ions. This was done by recording the change of potential over time after addition of the standard solutions. Figures 1A and B compare the response of carbonate ion-selective electrodes incorporating three membranes, including non-asymmetric polyvinyl chloride (PVC), non-asymmetric (unmodified non hydrophilic) CTA, and asymmetric CTA carbonate ion-selective membranes, to salicylate and carbonate ions. Figures 1A and 1 B indicate all three electrodes showed a similarly fast response upon the addition of carbonate ion to the sample solution in the concentration range 10 -4 to 10 -3 M. However, while the PVC and CTA membranes responded relatively fast to a salicylate concentration change, the asymmetric CTA membrane exhibited an extremely slow response when the salicylate concentration was changed from 10 -5 to 10 -4 M. The response characteristics of the non-asymmetric CTA membrane was very similar to the response of the non-asymmetric PVC membrane. Both exhibited similar kinetic (dynamic) response towards both carbonate and salicylate ions. It is hypothesized the slow response of the asymmetric CTA membrane towards large, hydrophobic anions was due to the presence of the hydrophilic layer of the asymmetric membrane. It is speculated that differences in response are due to differences in the transport rate across the hydrophilic region due to the rapid reaction of the hydration sphere of the carbonate ion relative to that of a slower, less hydrophilic ion such as salicylate. Support for the above was obtained by repeating the procedure described above with larger, even more hydrophobic analogues. Anions from 3-hydroxy-2-naphthoic acid and 3-hydroxy-2-anthranoic acid were compared to salicylate. Electromotive force (EMF) values were taken at 10 minutes after standard addition of the anions. Figures 2A through C show the increased response with larger additions of the more hydrophobic anions. The asymmetric membrane exhibited consistently showed reduced response characteristics toward these hydrophobic anions relative to the non-asymmetric membrane. At higher concentrations of the hydrophobic anions, the asymmetric membranes achieved only a small degree of response reduction as compared with the non-asymmetric CTA membranes. Table 2 shows the selectivity of both the non-asymmetric CTA and asymmetric CTA membrane for carbonate relative to salicylate over a concentration range of 1 x 10 -5 to 1 x 10 -2 M. At lower salicylate concentrations, the asymmetric membrane achieved a greater degree of salicylate response reduction relative to the non-asymmetric CTA membrane. B. Flow Injection Measurements The arrangement shown in Figure 3 was used to evaluate the performance of the asymmetric carbonate ion-selective membrane electrode in a flow injection analysis mode. A Phillips electrode body was fitted with a special flow cap for use as a flow through detector in a large volume wall jet configuration. The construction of this flow through detector is described by Trojanowicz, M. and Meyerhoff, M. E., "Potentiometric pH Detection in Suppressed Ion Chromatography" Anal. Chem., vol. 91, 787-789.(1989). An Orion silver/silver chloride reference electrode together with the working carbonate ion-selective electrode were placed in a large beaker containing the reagent buffer (0.1 M TRIS-sulfuric acid, pH 8.6]. The four-way injection valve (Rheodyne, Model 7125) equipped with a sample loop of 150 µl was connected to the flow cap with a length of polytetrafluoroethylene (PTFE) tubing having an internal diameter of 0.30 mm. The buffer stream (0.1 M Tris-sulfuric acid, pH 8.6) was pumped at a flow rate of 7.5 ml/min using a peristaltic pump (Ismatech, Model 7331-00, Zurich). Standard solutions of anions were prepared in the same buffer. Volumes of 150 µl were introduced for each test. All tests were performed at room temperature. EMF values were recorded every 0.1 second using a high impedance voltmeter equipped with an analog-to-digital converter as described earlier. When the ion-selective electrode was employed directly in a wall-jet configuration, only a fraction of the response to the injected ions was observed because such flow injection systems operate in a non-equilibrium mode. The resulting response signals (peaks) depended on the dynamic response characteristics of the ion-selective detector used. Figures 4A through C provide a comparison of the potentiometric flow injection response to anions obtained using the PVC non-asymmetric, non-asymmetric CTA and asymmetric CTA membranes in electrodes. A significantly reduced response of the asymmetric CTA membrane was observed for large, lipophilic anions, such as iodide, salicylate and perchlorate compared with the responses of the non-asymmetric PVC and non-asymmetric CTA membranes. The non-asymmetric CTA membrane showed response characteristics practically the same as the non-asymmetric PVC membrane, suggesting the reduced response of the asymmetric CTA membrane towards lipophilic anions is due to the presence of the hydrophilic layer. In the static mode experiment of Example 3, the asymmetric CTA membrane and unmodified CTA membrane showed nearly the same equilibrium selectivity for carbonate and iodide. In contrast, the carbonate selectivity compared to that of iodide for CTA the asymmetric membrane was substantially improved in the flow injection detection system. The selectivity of the asymmetric membrane can therefore be most effectively enhanced by employing non-equilibrium flow through arrangements. EXAMPLE 5 Measurement of Carbonate Ion in Serum Samples The effect of serum matrix on the carbonate response of the asymmetric carbonate ion-selective electrode was determined using SeraChem Level 1 Clinical Chemistry Control Human Serum-Assayed serum (Fisher Scientific, Orangeburg, NJ). Non-asymmetric PVC and asymmetric CTA membrane electrodes were prepared and used as described in Examples 1 and 2. The response of the electrodes was first tested with standards in aqueous solution and subsequently compared with responses obtained with serum samples. Two milliliters of an aqueous standard or reconstituted serum sample were added to 18 ml of background electrolyte (0.1 M TRIS-sulfuric acid, pH 8.6). Figures 5A and B show that, unlike the non-asymmetric PVC membrane, the asymmetric CTA membrane eliminated the 1.0 mM salicylate interference when used to determine 25 mM carbonate in the presence of 100 mM chloride. The response differences between the non-asymmetric PVC membrane and the asymmetric membrane are further shown by comparing curve A (response to sample containing 25 mM CO 3 2- ) with curve C (response to a sample containing 25 mM CO 3 2- , 100 mM chloride and 1.0 mM salicylate) of Figures 5A and B. Samples having a serum matrix were also tested using the Seramchem Level 1 Clinical Chemistry Control Human Serum Assayed. The membranes were washed thoroughly between samples with deionized water and samples were added in the order shown in Figures 6A and B. Curve A (Figures 6A and B) was obtained by adding 2 ml of control serum to 18 ml of background buffer in order to determine the amount of carbonate initially present in the reconstituted control serum. The PVC non-asymmetric and asymmetric CTA membranes showed different amounts of CO 3 2- , the PVC membrane yielding the higher level signal. The initial amount of CO 3 2- present was determined from the response from the asymmetric CTA membrane since it was known that the PVC membrane suffered from interferences from organic acids. Sodium bicarbonate was added to adjust the reconstituted serum pool to a final CO 3 2- concentration of 25 mM. For the study, reported in Figures 6A and B, the required amount of sodium bicarbonate was added to a mixed reconstituted control serum samples (from five 5.0 ml bottles) to make a final total carbonate concentration of 25 mM. Portions (5 ml) of this serum were then spiked with varying amounts of salicylate by adding varying amounts of salicylate to make final salicylate concentrations of 0.01, 0.1 and 1.0 mM respectively. The response of the non-asymmetric PVC and asymmetric CTA membranes to the serum samples were compared by using a high impedance electrometer. All tests were performed at: room temperature and the EMF values were recorded every 1 second. The electrodes were washed with deionized water between measurements. The tests were duplicated and the results were found to be reproducible.

Claims: A process for performing potentiometric determinations of CO 3 2- ions in biological samples using an ion-selective asymmetric membrane electrode and a reference electrode electrolytically connected to the ion-selective asymmetric membrane electrode which comprises: (a) contacting a biological sample suspected of containing CO 3 2- ions with the ion-selective asymmetric membrane electrode, the ion-selective membrane electrode having an asymmetric membrane with an ion-selective layer and a hydrophilic layer fused to the ion-selective layer, wherein (i) the ion-selective layer contains para-butyltrifluoroacetylbenzene as ionophore, tridodecylmethylammonium chloride as a ion-complexing agent and bis(2-ethylhexyl)sebacate or bis(2-ethylhexyl)adipate as plasticizing agent, (ii) the hydrophilic layer contains -OH groups and is in contact with the sample, and (iii) the hydrophilic layer and the ion-selective layer are membranes prepared from cellulose triacetate; (b) kinetically measuring the potential difference between the ion-selective asymmetric membrane electrode and the reference electrode; and (c) relating the potential difference to the concentration of the CO 3 2- ions in the sample, thereby enhancing potentiometric ion selectivity. The process of claim 1 wherein the biological sample contains an interfering substance. The process of claim 2 wherein the interfering substance is selected from salicylate, para-amino salicylate, gentisate, and a combination thereof. The process according to claim 1, wherein prior to step (a) the biological sample suspected of containing CO 3 2- ions and containing an interfering substance is diluted with a sufficient amount of a buffer solution such that the concentration of the interfering substance is less than 2 x 10 -3 M and the pH of the diluted sample is 7.5 to 9.5.

Inventors: CHA GEUN SIG, CHA, GEUN SIG
Applicants: DADE BEHRING INC, DADE BEHRING INC.
Application year: 1993
Main classification: G01N27
IPC codes: G01N27