REFERENCE LIQUID COMPRISING CSCL AND METHOD OF USE

Abstract: There is disclosed a reference liquid for use with a) a sample liquid to determine ionic activity of analytes, using differential analysis, and b) two substantially identical dry-­ operative, ion-selective electrodes, the reference liquid com­ prising a solution mixture of 1) CsCl, and 2) an anion suitable for poising one of the electrodes for the analysis of the activity of CO₂. A method of use is also described.

Description: REFERENCE LIQUID COMPRISING CsCl AND METHOD OF USE This invention relates to a reference liquid used in a differential mode ion analysis requiring two substantially identical dry-operative, ion-selective electrodes. The ion activity measurements conducted in the assays described in U.S. Patent No. 4,c,pS3,381, issued October 11, 1977, are obtained by using a differential potential measurement on two substantially identical strips of an ion-selective electrode (here inafter, "ISE"), responsive to a particular ion. As used herein, "dry-operative" refers to the ability of the ISE to be used within minutes, without delays for equilibration with H20. This is a property that appears to be peculiar to the ISE's described in the aforesaid patent. Aliquots of a sample, e.g., biological fluid, and a socalled reference liquid", are both deposited. Each aliquot is depositqd so that it contacts one of the two ISE's and one end of an ion bridge that allows the sample and reference solution to make liquid, and thus electrical, contact between the ends of the bridge. The voltage difference between the two electrodes is noted, and this voltage difference is converted by a calibration technique to ion concentration for that particular ion. The reference liquid has several critical functions. For any given ion test, and therefore, for any given kind of ISE, that liquid must set a stable electrode potential (called "poising" the electrode) that is essentially constant from ISE to ISE for that type. That is, the method of calibration is based on the assumption that such potential will be essentially constant within a manufacturing lot. A typical manufacturing lot will contain up to several hundred thousand ISE's, and the goal is for each ISE within the lot to be identical if tested with similar fluids. In addition, it :tust reduce any variation in liquid junction potential when the reference liquid contacts the sample. For this treason, the reference liquid is analogous, not to calibrators, but to saltbridge fluids such as saturated KC1, used in single or double junction reference electrodes such as the saturated calomel electrode. Prior to this invention, commercial reference liquids for systems, such as that provided by Eastman Kodak Company for use with the Ektachem 400to or 700to Analyzer of Eastman Kodak Company, have been successful in most respects. However, they lacked equal ion transference that provides sufficient equal solution mobility for both the cations and anions. lhe chloride ISE for this assay is preferaLly that described in US Patent o. 4,199,412, wherein a cellulose acetate polymer overcoat is disposed on layers of silver chloride and silver. As a result, the ion transference at the liquid junction with the patient sample lacked the ability to dominate a bias that is otherwise created wher the sample ion content changes drastically (such as from patient to patient.) What was observed in practice is that part of the change in the patient's ISE potential was created by 8 change in liquid junction potential rather than solely by a true change in ion activity. Because such changes in junction potential are not a true measure of the changed ion content, there is thus introduced a random bias. Such a bias makes proper operation difficult, since it is not amenable to correction by calibration. For obvious reasons of simplicity, such Eastman Kodak Company reference liquid has a single composition that contains all the ions necessary for use in all the ion tests of interest, namely No , Cle and CO2. That is, it is obviously too cumbersome to switch to a different reference fluid just because a different ion is being tested in the next ISE test. Thus, a typical composition for such a conventional commercial reference liquid is a solution comprising Na+, Cl-, small amounts of K- (0.0045 M), acetate#, and HCO3, with a total ionic strength of 0.15 M. The HCO3 anion is used to poise the ISE for the analysis of CO2. The 0.0045 M amount of is less than that conventionally considered adequate (1 M) to provide equal ion solution transference. It has also been known prior to this invention, that certain ions do provide equitransference, or sufficient equal solution mobility, such as will dominate the junction potential noted above created by varying concentrations of the ion of choice. For the anion of the equitransferant salt, it has been assumed prior to this invention that anions other than chloride can be used to overcome the liquid junction potential. And for the chloride anion, it has been known that the following cat ions have approximate equal solution mobility: potassium, ammonium, cesium and rubidium. This is apparent from the approximately equal equivs- lent ionic conductivity values given for these and for C1 in, e.g., pages D-171 and 172 of the CRC Handbook of Chemistry & Physics, 65th Ed., 1984. (These have not been described, however, for use with an anion used t poise an electrode to assay for CO2.) Because the commercial reference liquid included the bicarbonate anion to allow usage with the C02 ISE, it is readily evident that the ammonium cation is unacceptable in such a combination. That is, there is no pH that will keep the bicarbonate from converting into CO2 gas that escapes from the liquid (which occurs st a pH < 8.0) and at the same time keeps the ammonium from converting into NH3 that escapes from the liquid (at a pH > 8.0). * Tl# also is shown with sufficient solution mobility, but it is known to be unacceptable because of its toxicity and expense. Thus, the problem to be solved by this invention was to find 8 cation having approximately equal solution mobility with C10 and which does not create an unacceptable imprecison when testing for other ions. In accord with one aspect of the invention, there is provided a reference liquid for use with a) 8 sample liquid to determine ionic activity of analytes, using differential analysis, and b) two substantially identical dry-operative, ion-selective electrodes, the reference liquid comprising a solution mixture of 1) CsCl, and 2) an anion suitable for poising one of the electrodes for the analysis of the activity of CO2. In accord with another aspect of the invention, there is provided a method for determining ionic activity of analytes, using the reference liquid set forth in the previous paragraph. Thus, it is an advantageous feature of the invention that an equitransferent reference liquid is provided that overcomes the random bias due to junction potentials created by the sample liquid, without creating imprecision when used to test for other ions such as Na# and HCO#3. The subject invention is described in connection with a reference liquid tested with certain specific ISE's. In addition, it is useful with any ISE construction that analyzes for ions using the differential mode requiring two substantially identical dry-operative ISE's. As noted, the preferred construction of the test element containing the ISE's is that taught in the aforesaid Patent No. 4,053,381. The reference liquid of this invention comprises a solution mixture of CsCl, and an anion that poises one of the paired ISE' for the analysis of CO Most preferably, such poising anion is HC, in an amount of from 0.2 to 0.4 . The equitransferant anion needs to be chloride, because other anions fail to give a sufficiently rapid equilibrium potential when used with a chloride ISE comprising silver and silver chloride layers overcoated with a cellulose acetate polymer. Regarding the Cs of the CsC1, this is used in an amount sufficient to provide equal solution transference with Cl#. Such amounts are preferably 1 to 4 M, and most preferably 1 to 2 M. For reasons not well understood, the use of amounts of potassium > 1 M for equal solution transference, results in unacceptable precision errors when testing for Na with the NaO ISE. Optionally, the solution mixture also includes 0-0.3 21 Na#, 0-0.1 M O F 0.2 M K2S04 or Cs2S04, 0-5 g/L of a thickener such as poly(vinyl pyrrolidone), or 0-120 mg/L of a bactericide such as thiomercurisalicyclic acid svailable under the trade name thimerosal. In any event, such sdditives and composition of the reference liquid are preferably sdjusted so that the pH of the reference liquid is between about 9 and 10. Below 9, the HCO# a tends to decompose to release C02 gas. Above 10, there is enough or9 to possibly interfere with the carbonate in the setting of the CO2 potential. The following Table 1 lists representative useful preparations of reference liquid compositions of the invention. Table 1 ** Useful ComPositions (Molar Concentration) Prep. * * * No. CsCl KHCO3 MHCO3 M'OH tS 1 1.2 - 0.2(Na) 0.08(Na) 9.5 2 1.4 - 0.2(Na) 0.07(K) 9.5 3 1.2 - 0.2(Na) 0.07(K) 9.5 4 1.4 0.2 - 0.07(Na) 9.5 5 1.2 0.2 - 0.07(Na) 9.5 6 1.6 - 0.25(Na) 0.09(K) 9.5 7 1.2 - - 0.005(K) 9.5 8 1.2 - 0.2(Cs) 0.08(Na) 9.5 * M or M' are noted in parentheses for each example. All also contained 3 g/L of poly(vinyl pyrrolidone) thickener. For reasons that are not understood, was found not to give improved precision compared to K. The method of the invention is practiced as described in the aforesaid U.S. Patent No. 4,053,381, except using the reference liquid described above. The same reference liquid is used in all ionic analyte tests using the paired ISE format. Because the ISE's currently available are for Na+, K+, Cl # and HCO#3(also described as CO2), it is for these ISE's that the reference liquid must provide precision. It has been found that, when comprised as described above, the reference liquid gives the neces sary precision. As noted in the following examples, precision is considered to be acceptable if the standard deviation, sigma, is < 0.17 millivolts (hereinafter mV) for Na and C1, 0.27 for K+ and 0.35 for CO2. Because of the use of significant amounts of the Cs cation and C1 anion, this reference liquid also has the necessary equal solution transference of its ions to dominate liquid junction potentials created at the interface of the reference liquid with the sample liquid. Examples The following examples further illustrate the scope of the invention. Examples 1-4: Precision When Used With a Na ISE The purpose of this experiment was to determine the precision of Na+ ISE's using the reference liquid of the present invention. 48 total replicates were made for each reference liquid tested, (12 replicates each of 4 patient blood serum specimens), to determine the pooled standard deviation of the test (designated as amV). The acceptable level is a standard deviation no greater than 0.17 mV, a value that has been easily achieved by the commercial Kodak reference liquid already commercialized. amV was calculated using the standard formula EMI8.1 wherein Vi a individual millivolt readings for each test of 1 to n, n is the number of replicates, and V is the mean of the values Vi. The ISE test elements were the Na ISE test elements available from Eastman Kodak Company, tested on an "Ektachem 70011Th Analyzer of Eastman Kodak by the differential method described in the aforesaid U.S. Patent No. 4,053,381. The following reference liquids were tested: Control = Solution containing 103.5 millimoles Nail, 4.5 millimoles KC1, 11.5 millimoles sodium acetate, 25 millimoles NaHCO3, about 5 g/L poly (vinyl pyrrolidone), 60 mg/L thimerasol ( preservative), 0.145 milli- moles of total ionic strength, and a pH of 9.0. Example 1 - Preparation 2 above Example 2 - Preparation 3 above Example 3 1 Preparation 4 above Example 4 e Preparation 5 above Comparative Example 1 - Same as Preparation 1 except that 1.4 M KC1 and 0.2 K2SO4 were used instead of 1.2 M CsCl. Comparative Example 2 - Same as Preparation 1, except that 0.2 21 K2SO4, 0.5 M KC1, and 0.3 M NaCl were used instead of 1.2 M CsCl. The results appear in Table 2 that follows: Table 2 ExamPle Esuitransferent Cation of Liquid Control None 0.13 1 Cs 0.11 2 Cs 0.12 3 Cs 0.14 4 Cs 0.11 Comp. Ex. 1 K 0.21 Comp. Ex. 2 K 0.19 This test demonstrated that Cs+, but not K#, gave a precision within the goal of < 0.17. This was particularly surprising in light of the fact that J would normally be the cation of choice, since one of the assays tested using the reference liquid is K, rather than Cs+. Just why a reference liquid based upon potassium as the equitransferent cation, should produce such sodium imprecision, is not understood. Ten separate repeats were run on the procedure of Example 1, Comparative Example 1, and the control. In all but one of those separate repeats, the use of significant, equitransferent amounts of failed to provide precision toms) equal to or better than the control, whereas the use of did give precision equal to or better than the control. In the one repeat that did not demonstrste this, no statistically significant difference, at the 95'. confidence level, could be found between the control and on the one hand, or the control and Cs on the other hand. The inconsistency of this one repeat is not understood, and because it was only one out of ten, it is disregarded. Example 5: Precision When Used With a The process of Examples 1-4 was repeated, except that the tests were of Preparation 3, on a Cl- ISE, with 15 replicates. chloride ISE prepared as described in the aforesaid US Pat. No. 4,199,412, as layers of silver and silver chloride overcoated with cellulose acetate polymer.) a,V vas found to be 0.12, well within the goal of c0.17. Most of the above-noted Preparations 1-6 were also tested for precision on CO2 ISE's, and on ISE's, in a similar manner. The standard deviations were all within the goals of 0.27 mV for F and 0.35 mV for CO2. Examples 6-8: Improvements in Removal of Liquid Junction Potential To demonstrate that the reference liquid of the invention does indeed remove the liquid junction potential due'two the equal solution transference of its ions, the process of Examples 1-4 was repeated, except that on the patient sample side, first 0.01 M NaCl in H20, and second 0.1 M NaCl in H2O, were tested against the candidate reference liquids noted hereinafter. The temperature of the test was 220C. The theoretical response, based on the Nernstian equation, for two such samples, is #E = 58.56 log[aNa(0.1)/aNa(0.01)]+ #Ej (0.1/0.01). Solving for this equation for a zero bE (no liquid junction potential), one finds that bE should be 54.6 mV. The purpose of these examples then is to measure the actual hE (in mV), and determine to what extent it differs from the predicted 54.6 mV. That difference represents hE. The AE is, of course, the difference in the electrometer reading obtained, first, when testing the 0.01 21 NaCi solution against candidate 1 of the following list, and second, when testing the 0.1 21 NaCl solution against that same candidate. The other candidate liquids were tested in the same manner. (As noted above, the junction potential is a problem if it is large, only when comparing two liquids of considerably different activity.) Candidate Reference Liquids Control " 2 M KC1 in H2O Ex. 6 = Preparation 1 above Ex. 7 = Preparation 2 above Ex. 8 = Preparation 3 above Comparative Ex. 3 a Control for Ex. 14 above. (The control in this instance is the 2 z KC1 solution, because this is known to provide an acceptably small Ej - that is, it has acceptable equal solution transference in its ions.) The results are given in the following Table 4: Table 4 Candidate Observed (Observed AmV Liquid ssmV - 54.6) Control 55.4 0.80 Ex. 6 55.2 0.60 Ex. 7 55.4 0.80 Ex. 8 54.7 0.10 Comparative Ex. 3 61.7 7.10 As expected, Comparative Example 3 failed to provide an acceptably small hEj, particularly com- pared to what was achieved by the control and the examples of the invention. On the other hand, a variation of only +0.8 mV from the predicted 54.6 value is acceptable.

Claims: What is claimed is: 1. A reference liquid for use with a) a sample liquid to determine ionic activity of analytes and for use with a chloride ISE comprising silver-silver chloride layers overcoated with a polymer, using differential analysis, and b) two substantially identical dry-operative, ion-selective electrodes, said reference liquid comprising a solution mixture of 1) CsCl, and 2) an anion suitable for poising one of said electrodes for the analysis of the activity of CO2. 2. A reference liquid as defined in claim 1, wherein CsCl is present in a concentration of from 1.0 to 2.0 M, and HCO3 is said anion for poising and is present in a concentration of from 0.2 to 0.4 M. 3. 3. A reference liquid as defined claim 1 or 2, and further including N , , , or oF'. 4. A method for determining ionic activity of ana lytes in a sample liquid, using dry-operative ion-selective electrodes including a chloride ISE comprising silver-silver chloride layers overcoated with a polymer, the method comprising the steps of a) contacting an aliquot of the sample liquid with one of two substantially identical dry-operative, ion-selective electrodes and one end of an ion bridge, b) contacting an aliquot of a reference liquid with another, substantially identical ion-selective electrode and the other end of said ion bridge, said reference liquid comprising a solution mixture of 1) CsCl, and 2) an anion suitable for poising one of said electrodes for the analysis of the activity of CO2, and c) measuring the voltage difference generated by said liquids on said electrodes.

Inventors: KISSEL, THOMAS ROBERT
Applicants: EASTMAN KODAK COMPANY (A NEW JERSEY CORPORATION)
Application year: 1986
Main classification: G01N27
IPC codes: G01N27