COMPOSITION FOR MEASURING IONIC STRENGTH OR SPECIFIC GRAVITY OF LIQUID SPECIMEN AND TEST PIECE PREPARED FROM SAID COMPOSITION

Abstract: A composition for measuring the ionic strength or specific gravity of a liquid specimen and to a test piece prepared from said composition. Specifically, the invention concerns a composition constituted of, for example, one weight percent of heparin sodium in refined water and 1 mM of Methylene Blue, and a test piece in which test paper consisting of filter paper which is impregnated with a solution of said composition, dried, and cut to size is stuck to a plastic piece. The use of said composition and test piece enables easy, simple, quick but exact measurement of the ionic strength or specific gravity of a liquid specimen without influenced by the characteristics of said specimen, such as pH and temperature.

Description: TECHNICAL FIELD This invention relates to a composition for measuring the ionic strength or specific gravity of a liquid specimen, to a test piece prepared by using the composition and to methods for measuring the ionic strength or specific gravity of liquid specimen using the composition mentioned above. BACKGROUND ART The measurement of specific gravity of a liquid specimen is employed in a wide variety of technical fields. The measurement of specific gravity of urine is one of the important of items of measurement in the field of clinical chemistry. Physical method for measurement using liquid gravimeters, urine gravimeters, pycnometers, refractometers,osmometers,etc. were usually employed for measuring the specific gravity of urine. The instruments used by these methods were, however, expensive and fragile, and their handling called for the utmost care. Moreover, the instruments called for occasional maintenance including cleaning and calibration. Thus, the measurement by any of these physical methods took a great deal of labor and time. Simpler physical methods for measurement have been proposed. For example, Japanese Patent Application Laid-open No. 120843/1984 discloses a sheet for gravimetric examination formed from a porous resinous material, but a method employing this sheet calls for the strict determination by a spectrophotometer, or refractometer of the transmission, or refractive index of the sheet impregnated with a liquid to be examined, such as urine. Japanese Patent Application Laid-open No. 112251/1984 discloses a piece for the gravimetric measurement of urine having a gravimetric measuring chamber which holds a plurality of beads differing from one another is specific gravity, but it is too complicated in construction to be easily manufactured. In the field of clinical chemistry methods relying upon colorimetric analysis by test pieces are employed for carrying out an assay for constituents in a specimen of body fluids, such as urine, at a low cost in a simple and quick operation. The methods relying upon colorimetric analysis are easy to understand and carry out with few errors, insofar as variations in color can be visually recognized. The results are easy to obtain visually, or by a simple device such as a color analyzer. Methods relying upon colorimetric analysis by test pieces for determining the ionic strength, or specific gravity of a liquid specimen have recently been proposed in, for example, Japanese Patent Publication No. 12858/1987, Japanese Patent Laid-open Nos. 160739/1984, 21064/1981, 66451/1990 and 95462/1987, and Japanese Patent Publication No. 46374/1985. The known method for the colorimetric determination of the ionic strength, or specific gravity of a liquid specimen as disclosed in the publications referred to above employed test pieces prepared from, or by using , an electrolytic polymer, a chelate forming agent, a pH buffer agent, a pH indicator, a highly absorbent resin, a microcapsule, etc. It has, however, been difficult to carry out accurate determination by any of the known methods, since the results thereof are easily affected by the pH or temperature of the liquid specimen, the length of time employed for the determination etc. Moreover, many of the test pieces employed by those methods are generally difficult to prepare. This invention is intended for overcoming the drawbacks of the prior art as above pointed out. It is an object of this invention to provide a composition which enables to measurement of ionic strength, or specific gravity of a liquid specimen to be carried out easily, quickly and accurately without being influenced by the characteristics of the specimen, such as its pH and temperature, and a test piece which is easy to prepare by using the composition. DISCLOSURE OF THE INVENTION The present invention provides a composition for measuring the ionic strength, and/or the specific gravity of a liquid specimen, said composition comprising: (i) an ion-associable anion, (ii) an ion-associable basic dye cation, and (iii) optionally a pH buffer agent, wherein the ion-associable anion is able to associate with the basic dye cation to give a colour, which changes when salts are present in the solution. The anionic ion-associable anion reagent in the composition of this invention is preferably a polymer, or chain compound having a sulfonate, sulfate or phosphate ester group. The polymer preferred as the anionic ion-associable anion reagent is selected from the group consisting of sodium dextran sulfate, sodium heparinate, sodium chondroitin sulfate, sodium cellulose sulfate and nucleic acid. The chain compound preferred as the anionic ion-associable reagent is sodium dodecylsulfate or sodium dodecylbenzensulfate. The preferred polymer, or chain compound have always single negative change over a wide pH range, since it is a strong electrolyte. The ion-associable cation in the composition of this invention is a basic dye. It is a dye which does not change color in a pH range of 5 to 7 (the pH range of normal urine). The basic dye is preferably selected from the group consisting of thiazine, oxazine, azine, triphenylmethane, diphenylmethane, ago, indoaniline, and aminoindophenol dyes. The preferred thiazine dye is Methylene Blue. The preferred oxazine dye is Brilliant Cresyl Blue, or Nile Blue. The preferred azine dye is Safranine O. The preferred triphenylmethane dye is Crystal Violet. The preferred indoaniline dye is o-Toluidine Blue. The mixing ratio of the anionic and cationic ion-associable anion and cation reagents depend on the substances used as such and the liquid to be examined. It is possible to add an appropriate amount of, for example a phosphate, nitrate, carbonate, citrate, diethylmalonate, or a derivative of animoalkylsulfonic acid as the buffer, if required. The pH buffer range is preferably in a pH range of 5 to 10. The composition of this invention can conveniently be used to impregnate an absorbent carrier to form a test piece having appropriate properties. The absorbent carrier can be prepared from, for example, paper, cotton, wood a non-woven fabric, a powder, or a hydrophilic polymer. The use of filter paper is, among others, preferred. The absorbent carrier may itself be used as a test piece, or may alternatively be appropriately sized and shaped, and attach to an appropriate support, for example, a plastic sheet, as of polystyrene or polyvinyl chloride, It is also possible to use a hydrophilic polymer as an absorbent carrier and adhesive, apply a mixture thereof with the composition of this invention onto a support such as-a plastic sheet, and dry it to solidify to make a test piece. The support may, for example, be colored, or marked with symbols, letters, figures or patterns as desired to facilitate the identification of the results of measurement. I) Principle of Measurement: The ionic strength, or specific gravity of an aqueous solution is indirectly measured by utilizing the effects which salts exert on the ion-association between ion-associable anion and cation reagents. II) Explanation of the Principle of Measurement: The ion-association is the combination of an anion and cation which does not rely upon any coordinate bond (Bunseki, 1989, 1, p61), and the change in color of a dye by an ion-association is called metachromasy (Bunseki, 1990,3, p82). A dye of ion-associable cation changes its electron state, and its color, too, as a result of its ion-association with an ion-associable anion (Analytical Sciences, vol. 3, 1987, pp.479-488). A typical combination which causes metachromasy is of polyvinyl potassium sulfate as anion type (PVSK) and Toluidine Blue (TB) as cation type in colloidal titration. TB changes its color from blue to red violet upon reacting with PVSK. This phenomenon is, however, greatly affected by salts, though not affected by sugar or glycerol ( Shinkagaku Jikkenkouza 9, Bunseki Kagaku (II) 1977). Under the circumstances, we, the inventors of this invention, have done a great deal of research work and found that the change in color of dye which results from metachromasy under the influence of salts can be utilized for determining indirectly the ionic strength, or specific gravity of an aqueous solution containing it. The change in color of a dye which results from metachromasy is not due to a change in pH of an aqueous solution containing it. Therefore, the measurement of ionic strength, or specific gravity of a liquid specimen which is carried out by employing the composition of this invention is hardly affected by the pH of the specimen. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a perspective view of a test piece embodying this invention; Fig. 2 is a graph showing the relation between the reflexivity and the wavelength of a color analyzer as measured of urine specimens by employing the test pieces of this invention; Fig. 3 is a graph showing the K/S function as measured of urine specimens by employing the test pieces of this invention in relation to their specific gravity; and Fig. 4 is a graph showing the relation between the absorbance and the wavelength of a spectrophotometer as measured of urine specimens by employing the composition of this invention. BEST MODE OF CARRYING OUT THE INVENTION The invention will be described in further detail with reference to examples. EXAMPLE 1 to 3: Preparation of Compositions of this invention The compositions of this invention as shown below were prepared by dissolving the respective components in purified water. % means weight/volume %, otherwise specified. EXAMPLE 1: Sodium heparinate - 1 % Methylene Blue - 1 mM EXAMPLE 2: Sodium dextran sulfate (M w : 8000) - 1 % Safranine 0 - 1 mM EXAMPLE 3: Sodium chondroitin sulfate - 1 % Nile Blue - 1mM EXAMPLES 4 to 6: Preparation of Test Pieces of this Invention EXAMPLE 4: Filter paper (product of Toppan Shoji, 51A) was impregnated with the composition of EXAMPLE 1, dried, and cut to a size of 5x5 mm to prepare a piece of test paper 1. The test paper 1 was stuck to a plastic piece 2 having a size of 5x40mm, whereby a test piece embodying this invention was prepared, as shown in Fig. 1. Example 5: A test piece embodying this invention was prepared by repeating EXAMPLE 4, except for the use of the composition of EXAMPLE 2. EXAMPLE 6: A test piece embodying this invention was prepared by repeating EXAMPLE 4, except for the use of the composition of EXAMPLE 3. EXAMPLES 7 to 9: Measurements with the Test Pieces of this invention. EXAMPLE 7: Measurement 1 of Specific Gravity of Urine Specimen The test pieces according to EXAMPLE 4 were dipped for one or two seconds in a number of urine specimens differing form one another in specific gravity, respectively, and the reflectivity thereof was measured by color analyzer TC-1800M. The results are shown in Fig. 2. As is obvious from Fig. 2, the difference in specific gravity gave a difference in color from room one specimen of urine to another. EXAMPLE 8: Measurement 2 of Specific Gravity of Urine Specimens The reflectivity of each of urine specimens having different specific gravities was measured by repeating EXAMPLE 7, except for the use of test pieces according to EXAMPLE 6, and was employed for calculating the K/S function thereof at 680 pm by equation I: K/S = (1-R) 2 /2R (where K = absorption coefficient, S = scattering coefficient, and R = reflectivity ). The K/S function was theoretically derived from R to determine the reflectivity of a coated surface, or a woven fabric. The K/S function as calculated was proportional to the specific gravity of urine, as shown in Fig. 3. EXAMPLE 9: Measurement 3 of Specific Gravity of Urine Specimen Specimens (Having Different pH Values) Test pieces according to EXAMPLE 6 were dipped for one or two seconds in urine specimens (1) to (3) as described below, respectively, and the reflectivity thereof was measured by a color analyzer, TC-1800M. The K/S function at 680nm, was determined from Fig. 3. The specific gravity of each of urine specimens (1) to (3) was also measured by a refractometer. Urine specimen (1): Having a pH 5, and a specific gravity of 1.020 as measured by the refractometer; Urine specimen (2): Having a pH 6, and a specific gravity of 1.020 as measured by the refractometer; Urine specimen (3): Having a pH 7, and a specific gravity of 1.020 as measured by refractometer. All of urine specimens (1) to (3) showed a specific gravity of 1.020 as measured by employing the test pieces of this invention, and their difference in pH did not give any difference in specific gravity. EXAMPLE 10: Measurement of specific Gravity of Urine Specimens by the Composition of this Invention 250 µ l of each of a number of urine specimens having different specific gravities was added to, and mixed thoroughly in an aqueous solution of a composition embodying this invention which consisted of 3 ml of purified water, 50 µℓ of 1 % aqueous solution of sodium dextran sulfate and 50 µℓ of 1nM aqueous solution of Methylene Blue, and absorption curves were obtained by employing a Hitachi 200-20 spectrophotometer. The results are shown in Fig.4. It is obvious from Fig. 4 that the difference in specific gravity gives different forms of absorbance can be utilized for measuring the specific gravity of such a specimen. INDUSTRIAL UTILITY The composition of this invention which comprises an anionic reagent of the inonically associable type and a cationic reagent of the inonically associable type, and further contains a pH buffer agent, if required, develops a color by the association of ions, and can be used to measure the ionic strength, or specific gravity of a liquid specimen characteristics of the specimen, such as its pH and temperature. As there are a wide variety of such anionic and cationic reagents, and as they are commercially available, it is possible to obtain a wide variety of combinations of reagents easily choose the optimum composition for a particular purpose. The test piece of this invention is easy to prepare in a variety of forms, is simple in construction, small in size and light in weight, and is, therefore, very easy to use, carry and store, while is can be prepared at a low cost.

Claims: A composition for measuring the ionic strength, and/or the specific gravity of a liquid specimen, said composition comprising: (i) an ion-associable anion, (ii) an ion-associable basic dye cation, and (iii) optionally a pH buffer agent, wherein the ion-associable anion associates with the basic dye cation to give a colour, by metachromasy, colour which changes when salts are present in said liquid specimen. A composition according to Claim 1, wherein the ion-associable anion is a polymer, or chain compound having a sulfonate, sulfate or phosphate ester group. A composition according to Claim 1, wherein the basic dye is selected from thiazine, oxazine, azine, triphenylmethane, diphenylmethane, azo, indoaniline and aminoindophenol dyes. A composition according to Claim 1, wherein the pH buffer agent maintains the composition at a pH of 5-10. A test piece for measuring the ionic strength, and/or the specific gravity of a liquid specimen, said test piece comprising an absorbent carrier impregnated with a composition as defined in Claim 1. A method for measuring the ionic strength and/or the specific gravity of a liquid specimen, which method uses a specific relationship between ionic strength and/or specific gravity and the colour resulting by metachromasy from the association of an ion-associable anion and a ion-associable basic dye cation, a colour that changes when salts are present in said liquid specimen, the method comprising the steps of: (a) preparing a solution comprising (i) an ion-associable anion, (ii) a basic dye cation, and (iii) optionally a pH buffer agent, (b) mixing the solution with the liquid specimen to be measured, to effect a colour change by metachromasy in the solution, (c) measuring the absorbance of the solution, (d) calculating the ionic strength and/or the specific gravity of the specimen on the basis of the colour change, using said specific relationship. A method for measuring the ionic strength and/or the specific gravity of a liquid specimen, which method uses a specific relationship between ionic strength and/or specific gravity and the colour resulting by metachromasy from the association of an ion-associable anion and an ion-associable basic dye cation, a colour that changes when salts are present in said liquid specimen, the method comprising the steps of: (a) preparing a test piece by impregnating it with a solution comprising (i) an ion-associable anion, (ii) an ion-associable basic dye cation, and (iii) optionally a pH buffer agent, (b) contacting said test piece with the liquid specimen to be measured, to effect a colour change by metachromasy in the test piece, (c) calculating the ionic strength and/or the specific gravity of the specimen on the basis of the colour change, using said specific relationship. A method according to Claim 6 or Claim 7, wherein the ion-associable anion is a polymer or chain compound having a sulfonate, sulfate or phosphate ester group.

Inventors: HARAKO MIEKO, HARAKO, MIEKO
Applicants: EIKEN CHEMICAL, EIKEN KAGAKU KABUSHIKI KAISHA
Application year: 1992
Main classification: G01N31
IPC codes: G01N31, G01N9, G01N33