JP4012169B2 - Instruments for measuring multiple types of ions - Google Patents

Description

  The present invention relates to an ion measuring instrument capable of measuring a plurality of types of ions in an aqueous sample.

  As one of water quality inspections for drinking water such as tap water and agricultural water such as river water and lake water, various ion concentrations are measured. A water quality test paper, a solution visual colorimetric kit, a simple photometer, and the like are used as means for easily and quickly measuring ions in sample water in the field where water as a sample exists.

  Water quality test paper is a paper in which an indicator (mainly a light-absorbing reagent) that responds to the target ions is applied to the paper. When the test paper is immersed in sample water or when sample water is dropped onto the test paper, the concentration of ions contained Depending on the color of the test paper changes. From this change, the target ions are quantified or detected visually.

  However, the water quality test paper has drawbacks such that the indicator that can be adsorbed and held on the paper is limited, only one type of ion can be measured with one test paper, the sensitivity is low, and the quantitative property is poor.

  A solution visual colorimetric kit or a simple photometer added thereto is used as an instrument for water quality inspection that solves the problems of these water quality test papers. In these instruments, a matrix material called an indicator mixed with an indicator is solidified on the inner wall of the container, a sample and necessary additives are added at the time of measurement, and the solidified indicator is eluted to react in the solution. . In this kind of known water quality inspection instrument, polyvinyl chloride is used as a film agent.

  However, these instruments can measure only one component at a time, and the analysis operation is different for each measurement component, which is complicated. Furthermore, the measurement sensitivity and accuracy cannot always be satisfied.

Ota Nobuhide, Bunseki, 661 (2001) Okauchi Kanji "Everyone can test the environment with a pack test" (2000), joint publication Toa DKK “Multi-item Water Quality Analyzer LASA1 Analysis Handbook”, 2002

  An object of the present invention is to provide a device for measuring a plurality of types of ions in a water-based sample, which can measure a plurality of types of ions easily and with high sensitivity to an acceptable level with a single device. It is.

As a result of intensive studies, the inventors of the present application have found that by using polyethylene glycol having a polymerization degree of 500 to 2000 as a film agent, ions in an aqueous sample can be accurately measured with high sensitivity to an acceptable level. heading further optode used in the measurement, and by immobilizing included in the polyethylene glycol the another component if measurements required other ingredients other than the optode, the plural kinds of ions It is possible to measure easily, and moreover, it is possible to measure multiple types of ions with a single instrument by using a substrate that can be provided with a plurality of reaction regions on a single plate such as a microplate. The present invention has been completed.

That is, in the present invention, the optode used for the measurement of ions, and when other components other than the optode are required for the measurement, the other components were mixed with polyethylene glycol having a polymerization degree of 500 to 2,000 . A plurality of species in an aqueous sample, each of which includes a substrate on which a plurality of regions solidified on a substrate are formed, and each of the plurality of regions has a different optode for measuring different ions immobilized on the substrate. An ion measuring instrument is provided.

  When the instrument of the present invention is used, a single instrument can measure a plurality of types of ions with high sensitivity and accuracy to a level that is simple and acceptable.

  The sample to which the instrument of the present invention is applied is not limited as long as it is a water-based sample containing water as a main component, drinking water such as tap water and well water, agricultural water such as river water and lake water, seawater Furthermore, various artificial drinks, foods, body fluids (blood, serum, plasma, lymph fluid, urine, saliva, tissue fluid, spinal fluid, wiping fluid, etc.), dilutions thereof, suspension water, aqueous solutions, etc. can be exemplified. It is not limited to these.

To be measured ions is not limited in any way, Ca ²⁺ is a major ions in river water, Mg ^2+, environmental standards have been established _{^{F -, BO 3 3-, Cd}} 2+, Pb 2 ^{+, Cr 6+, Hg 2+,} Zn 2+, Fe 3+, Cu 2+, Mn 2+, H +, As 5+, NO 3 -, NH 4 +, monitoring the content of agricultural water Required NO ₂ ⁻ , PO ₄ ³⁻ and K ⁺ , other H ⁺ , Cd ²⁺ , Pb ²⁺ , Cr ⁶⁺ , As ^5+, etc. can be mentioned, but are not limited to these is not. In the present invention, “ion measurement” means ion detection or ion concentration measurement (that is, quantification or semi-quantification of ions).

As described above, the plurality of types of ion measuring instruments of the present invention are prepared by mixing optodes and necessary additives used for measuring ions with polyethylene glycol having a polymerization degree of 500 to 2000 on a solid phase on a substrate. Including the converted area.

  Here, the optode is a substance that reacts with the ion to be measured and changes its photochemical properties (absorption, fluorescence, polarization, scattering, refraction, etc.), and a typical one is a so-called indicator. . The optode for measuring the target ions is known for each ion, and additives necessary for measuring ions using the optode are also known. In the present invention, known optodes and additives are used. be able to. A plurality of optodes may be used to measure a single ion. By combining a plurality of optodes having different measurable pH ranges, measurement in a wide pH range is possible. When multiple optodes are used to measure a single ion, the multiple optodes are usually solid-phased in different regions, but a calibration curve can be created even when solid-phased in the same region. In the case of a combination of various optodes, it can be solid-phased in the same region.

  Preferred examples of optodes and additives used for measuring each ion include those shown in Table 1 below. However, optodes and additives that can be used for measuring each ion are not limited to these.

  In the instrument of the present invention, when other components (so-called additives) other than the optode are necessary for the measurement, the other components (additives) are also solid-phased together with the optode. Examples of additives include, as shown in Table 1 above, buffers, masking agents that mask reaction with other ions, reducing agents and binders required for reaction with optode ions, and color development. Examples of the color former and the color stabilizer required for the present invention are not limited thereto. Additives other than optodes are preferably solid-phased on the substrate, so that the measurement can be performed simply by adding the aqueous sample to the substrate, which is convenient and preferable.

  The above-mentioned optode and necessary additives are mixed with polyethylene glycol, and the mixture is solid-phased on the substrate. Polyethylene glycol is used as a so-called film agent. The use of polyethylene glycol as a film agent is an important feature of the present invention. When optode and necessary additives are encapsulated in polyethylene glycol and solidified on a substrate, the optode and additives are stored well before use, and during use, polyethylene glycol dissolves and optodes and additives are dissolved. Elutes well, and the dissolved polyethylene glycol does not interfere with the reaction between the optode and ions, and further, since it is colorless and transparent, it does not interfere with the measurement. As a result, the measurement can be performed accurately with high sensitivity. .

Incidentally, NO ₃ ^- Measurements of the components shown in Table 1 immobilized, this sample may be performed by adding to it, NO ₂ is treated beforehand with metallic zinc sample ^- it was reduced to reduction treatment the already sample NO ₂ ^- can also be added to the region for measurement performing measurement based on the photochemical changes (see examples below). In this case, it is convenient and preferable to use granular metallic zinc as the metallic zinc, fill this into a micropipette tip, and add the sample using this tip (see the following examples).

The degree of polymerization of the polyethylene glycol used in the present invention (the number of polycondensed ethylene glycol units) is 500 to 2000 , preferably about 800 to 1200 , from the viewpoint of storage stability of reagents and solubility in water. It is.

  Solid-phase formation can be easily performed by dissolving optode, necessary additives, and polyethylene glycol in water and mixing them, and applying the resulting mixture onto a substrate and drying. The amount of optode and additive to be solid-phased is such that each reagent will have an appropriate concentration when dissolved in a predetermined amount of sample, depending on the type of optode and additive and the type of ion to be measured. It can be set as appropriate based on known information or through routine experiments. The amount of the polyethylene glycol to be solid-phased is not particularly limited, but the amount when the total amount is dissolved in a predetermined amount of sample is usually about 0.001 to 0.1% by weight, preferably about 0.005 to 0.2% by weight. is there.

  The film agent is preferably composed only of polyethylene glycol, but may contain a small amount of other components and impurities as long as the sensitivity and accuracy of measurement are not adversely affected. The total amount of such other components and impurities is not particularly limited as long as it does not adversely affect, but is usually 10% by weight or less, preferably 5% by weight or less based on polyethylene glycol. The polyethylene glycol is preferably a polycondensation of only ethylene glycol units, but may contain a small amount of other units as long as it does not adversely affect the sensitivity and accuracy of the measurement. The total amount of such other units is not particularly limited as long as it does not cause an adverse effect, but is usually 10 mol% or less, preferably 5 mol% or less in all units of polyethylene glycol.

  In water quality testing, since pH measurement is a widely performed item, it is possible to provide a single region where an optode capable of measuring pH is solidified in the same manner as the optode for ion measurement described above. It is convenient because it can be performed up to pH measurement with an instrument. For this purpose, a known pH indicator can be employed, and a wide range of pH can be measured by combining a plurality of pH indicators having different measurable pH ranges in combination. In addition, since the range of the pH of drinking water and agricultural water used for water quality inspection is relatively limited, a plurality of pH indicators with different pKa are included in the predicted range for water used for inspection. Use in combination is preferable because measurement accuracy within the range can be increased. Examples of preferred combinations used for such purposes include bromocresol purple (pKa 6.1), bromothymol blue (pKa7.2) and cresol red (pKa 8.3). According to this combination, the pH can be measured with high accuracy in the range of pH 6.0 to 8.5, and the pH of drinking water and agricultural water is almost always within this range. In addition, when using in combination with a pH indicator, it is preferable to solidify each pH indicator in a separate area | region, respectively. The solid phase amount of the pH indicator is not particularly limited and can be appropriately set according to the type of pH indicator to be used, but the concentration when the entire amount is dissolved in a predetermined amount of sample is usually about 0.01 to 0.1 mol%. Amount.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1 Preparation of Ion and pH Measurement Plate An aqueous solution containing polyethylene glycol (Aldrich) having a polymerization degree of 1000 as a reagent and a filming agent shown in Table 2 below was micrographed for each ion and pH measurement reagent. The plate was placed in a well of the plate (the pH indicator was put in a separate well), and the microplate containing the solution was dried under reduced pressure in a desiccator overnight. The prepared plate was sealed with a plate seal and stored in a desiccator until measurement. The amount of each reagent placed in the well was such that the concentration shown in Table 2 was obtained when the entire amount was dissolved with 200 μl of sample water. The amount of polyethylene glycol placed in the well was 0.01% by weight when the entire amount was dissolved with 200 μl of sample water. All reagents were commercially available.

Measurement of Standard Ion Aqueous Solution In order to confirm that ions can be measured using the ions prepared in Example 1 and the pH measurement plate, each of the aqueous solutions shown in Table 2 each containing one kind of measurement ions of various known concentrations. Prepared for ionic species. The concentration of each ion is described in FIGS. In the ion concentration display, for example, “5.0E-05” means 5.0 × 10 ⁻⁵ M. The salts used for the preparation of each aqueous solution containing each ionic species shown in Table 2 were calcium chloride, magnesium chloride, sodium fluoride, boric acid, sodium nitrite, potassium dihydrogen phosphate, and potassium chloride, respectively. It was.

  200 μl of the aqueous solution was taken with a micropipette and injected into the well of the microplate corresponding to each ion. At this time, a solution having the same concentration was injected into four wells. The plate was stirred for 1 minute by the stirring function of the microplate reader to dissolve the reagent, and then left for 5 minutes to complete the reaction. Thereafter, the solution was stirred again for 1 minute to make the solution uniform, and the absorption spectrum was measured with a microplate reader.

  The absorbance at the wavelength determined for each well was plotted against each ion concentration, and a calibration curve was drawn. Absorbance was the average of four wells, and error bars were created from the standard deviation values. The calibration curve was drawn from the theoretical formula on the assumption that the reagent and the measured ion have a one-to-one response. This formula is as follows.

Measured ion A responds to indicator I by the following reaction.
I + A ⇔ IA [I: Indicator, A: Measured ion]
The equilibrium constant K _a for this case,
K _a = [IA] / [I] [A]
The total amount of indicator and ions is as follows.
I _tot = [I] + [IA], A _tot = [A] + [IA]
On the other hand, if the constant α is defined as follows, the following equation holds.
α = [IA] / I _tot = (Abs Abs _free ) / (Abs _A Abs _free ) (1)
[Abs: Absorbance, Abs _free : Ion free, Abs _A : Saturation]
K _a = α / [(1 α) (A _tot α I _tot )] (2)
Each concentration at this time is
[I] = I _tot [IA], [A] = A _tot [IA]
(2) is substituted into equation complex formation constant K _a is
K _a = [IA] / {(I _tot [IA]) (A _tot [IA])} (3)
From equation (1)
A = [IA] (A _A A _free ) / I _tot + A _free ... (4)

According to the law of Lambert-Beer, measured absorbance Abs, Abs _free, and Abs _A more seeking alpha (1), to calculate the equilibrium constant K _a for the apparent (2). Based on these values, the theoretical value of absorbance was calculated from Eqs. (3) and (4), and a calibration curve was drawn.
The calibration range for each well was determined in consideration of the variation in measured values (error bar size), the slope of the calibration curve, and the accuracy of the machine.

  The results are shown in FIGS. In each figure, an absorption spectrum and a calibration curve are drawn. Of the calibration curve, the area where the plot is connected by a solid line is the calibration range described above. In each absorption spectrum, the absorbance at the peak wavelength is lowest at ion-free (ion concentration 0), and the higher the ion concentration, the greater the absorbance (in the case of adding magnesium ions, the ion concentration increases, Absorbance decreased). Therefore, it can be seen that each ion can be easily measured by the plate of Example 1.

Measurement of pH In order to confirm that it was possible to measure pH with the plate prepared in Example 1, a standard aqueous solution for a pH measurement test was prepared. The standard aqueous solution was as follows.
(1) For bromcresol purple
0.1 M TMACl + 0.1 M TMAOH + 0.01 M citric acid (pH 4.0-5.4)
0.1 M TMACl + 0.1 M TMAOH + 0.01 M MES (pH 5.8 to 7.8)
(2) For bromthymol blue
0.1 M TMACl + 0.1 M TMAOH + 0.01 M citric acid (pH 5.4)
0.1 M TMACl + 0.1 M TMAOH + 0.01 M MES (pH 5.8 to 7.8)
0.1 M TMACl + 0.1 M TMAOH + 0.01 M boric acid (pH 8.0-9.0)
(3) For cresol red
0.1 M TMACl + 0.1 M TMAOH + 0.01 M MES (pH 6.4 to 7.8)
0.1 M TMACl + 0.1 M TMAOH + 0.01 M boric acid (pH 8.0 to 10.0)
(TMACl: Tetramethylammonium chloride
TMAOH: Tetramethylammonium hydroxide
MES: 2-morpholinoethanesulfonic acid

  The same operation as in Example 2 was performed, and the absorption spectrum of each well was measured.

  The results are shown in FIGS. In each figure, an absorption spectrum and a calibration curve are drawn. Of the calibration curve, the region where the plot is connected by a solid line is the pH measurable region. 8 to 10, the absorbance of the peak increases in any case as the pH increases. Therefore, it can be seen that the pH can be easily measured by the plate of Example 1. In addition, since three kinds of pH indicators having different pKa are immobilized on the plate of Example 1, pH measurement is possible in the range of pH 5.0 to 9.0 as can be seen from FIGS. It is sufficient for water quality inspection of drinking water and agricultural water.

  Using the plate prepared in Example 1, the ion concentration and pH in actual river water (Tsurumi River and Yagami River) were measured by the method described in Example 2. For comparison, the ion concentration was also measured by a well-known ion chromatography method (IC) with high accuracy but complicated operation. The pH was also measured with a pH meter using a glass electrode. The results are shown in Tables 3 and 4 below. Table 3 shows the results for the Tsurumi River, and Table 4 shows the results for the Yagami River.

＊１： IC法の検出範囲外
＊２： 検出せず

* 1: Outside detection range of IC method * 2: Not detected

  From the above results, it can be seen that various ions and pH can be easily measured by the plate prepared in Example 1.

A plate was prepared in the same manner as in Example 1 except that the reagents shown in Table 5 below were used. However, NO ₃ ^- is metallic zinc used in the measurement of, rather than immobilized in the region of the plate, the granular metallic zinc is filled in a micropipette tip, the sample using the chip dispensing did. In this case, the sample was sucked into the chip and allowed to stand at room temperature for 3 minutes to perform a sufficient reduction reaction, and then added to the solid phase region.

  The standard samples used for the measurement were an aqueous ammonium chloride solution and an aqueous potassium nitrate solution, and the concentration of each aqueous solution was 50 mM to 1 μM. Measurement was performed in the same manner as in Example 2 using these standard samples. However, the standing time after stirring using the stirring function of the microplate reader was 15 minutes. In addition, absorbance was measured at intervals of 4 nm in the wavelength range of 350 to 700 nm.

In the measurement of ammonium ions, the relationship between the wavelength and the absorbance with respect to the standard sample of each concentration is shown in FIGS. 11-1 and 11-2. In addition, calibration curves showing the relationship between the logarithm of the ammonium ion concentration and the absorbance at wavelengths of 705 nm and 710 nm are shown in FIGS. 12-1 and 12-2. The maximum absorption wavelength shifts from [NH ₄ ⁺ ] = 1 × 10 ⁻⁴ M, and in the case of a concentration lower than this concentration, the absorbance decreases as the concentration decreases at the wavelength = 705 nm. In the case of a high concentration, the absorbance increases as the concentration decreases at a wavelength = 710 nm. And at [NH ₄ ⁺ ] = 1 × 10 ^-4 M, the maximum absorption is achieved. Therefore, a calibration curve for each of the two wavelengths was drawn (FIGS. 12-1 and 12-2). Compare the absorbance at wavelengths of 710 nm and 705 nm, and examine the concentration from the calibration curve indicating the greater absorbance. It can be seen that measurement is possible in a concentration range of approximately 5.0 × 10 ⁻² to 5.0 × 10 ⁻⁶ M.

In addition, in the measurement of nitrate ions, the relationship between the wavelength and the absorbance with respect to the standard sample of each concentration is shown in FIG. In addition, a calibration curve showing the relationship between the logarithm of nitrate ion concentration and absorbance at a wavelength of 512 nm is shown in FIG. As is clear from the calibration curve in FIG. 13-2, the calibration curve rises to the right in the range of about 2.5 × 10 ⁻² to 1.0 × 10 ⁻⁴ M, and the nitrate ion concentration can be measured in this range. It became clear.

It is a figure which shows the absorption spectrum and calibration curve at the time of measuring Ca2 ⁺ using the plate produced in Example 1. FIG. It is a figure which shows the absorption spectrum and calibration curve at the time of measuring Mg2 ⁺ using the plate produced in Example 1. FIG. FIG. 3 is a diagram showing an absorption spectrum and a calibration curve when F ⁻ is measured using the plate prepared in Example 1. FIG. 2 is a diagram showing an absorption spectrum and a calibration curve when BO ₃ ^3- is measured using the plate prepared in Example 1. ₂ is a diagram showing an absorption spectrum and a calibration curve when NO ₂ ⁻ is measured using the plate produced in Example 1. FIG. 2 is a diagram showing an absorption spectrum and a calibration curve when PO ₄ ³⁻ is measured using the plate prepared in Example 1. FIG. It is a figure which shows the absorption spectrum and calibration curve at the time of measuring K ⁺ using the plate produced in Example 1. FIG. It is a figure which shows the absorption spectrum and calibration curve at the time of measuring pH using the bromcresol purple solid-phase area | region in the plate produced in Example 1. FIG. It is a figure which shows the absorption spectrum and calibration curve at the time of measuring pH using the bromthymol blue solid-phase area | region in the plate produced in Example 1. FIG. It is a figure which shows the absorption spectrum and calibration curve at the time of measuring pH using the cresol red solid-phase area | region in the plate produced in Example 1. FIG. It is a figure which shows the absorption spectrum at the time of measuring ammonium ion using the plate produced in Example 5. FIG. It is a figure which shows the absorption spectrum at the time of measuring ammonium ion using the plate produced in Example 5. FIG. It is a figure which shows a calibration curve at the time of measuring ammonium ion using the plate produced in Example 5. FIG. It is a figure which shows a calibration curve at the time of measuring ammonium ion using the plate produced in Example 5. FIG. It is a figure which shows the absorption spectrum at the time of measuring nitrate ion using the plate produced in Example 5. FIG. It is a figure which shows a calibration curve at the time of measuring nitrate ion using the plate produced in Example 5. FIG.

Claims (8)

When an optode used for ion measurement and other components other than the optode are necessary for the measurement, a mixture of the other components with polyethylene glycol having a polymerization degree of 500 to 2000 is fixed on a substrate. An instrument for measuring a plurality of types of ions in an aqueous sample, comprising a substrate on which a plurality of phased regions are formed, wherein each of the plurality of regions has a different optode for measuring different ions immobilized thereon.   The instrument according to claim 1, further comprising a region in which an optode used for measuring pH is mixed with polyethylene glycol and solidified on the substrate, and the pH can also be measured. The other ingredients, buffers, masking agents, reducing agents, binders, instrument according to any one of claims 1 or 2 is at least one selected from the group consisting of color former and color stabilizers. A region for measuring at least two types of ions selected from the group consisting of Ca ²⁺ , Mg ²⁺ , F ⁻ , BO ₃ ³⁻ , NO ₂ ⁻ , PO ₄ ³⁻ , K ⁺ and NH ₄ ^+. The instrument according to any one of 1 to 3 . The instrument according to any one of claims 1 to 4 , wherein the substrate is a microplate, and each of the plurality of regions is formed in each well of the microplate. An aqueous system in which an aqueous sample is added to the plurality of regions on the instrument according to any one of claims 1 to 5 and a plurality of types of ions in the aqueous sample are measured based on a change in photochemical properties of the region. A method for measuring a plurality of types of ions in a sample. After reduction by treating the aqueous sample with zinc, the resulting reduced samples, NO ₂ ^- claim 6 comprising measuring the concentration ^- NO ₃ of the addition to the region for measurement aqueous sample the method of. Granular metal zinc filled into the micropipette tip, the aqueous sample NO ₂ with the chip ^- method of claim 7, comprising adding to the region for measurement.

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