JPH08145881A - Determination method for hydrogen peroxide by photo-absorption - Google Patents

Abstract

PURPOSE: To simply quantitative-analyze hydrogen peroxide in aqueous solution with the use of an optical analyzing means. CONSTITUTION: A measuring beam 10s containing the light in near infrared region is made incident on the sample solution provided in a light transmission type cell 3, and then, spectrum of the transmitted light is measured by a spectroscope 5 and a detector 6, and, based on the absorbance of one of absorption peaks in the range from 4300-4800cm<-1> to 5400-6600cm<-1> , hydrogen peroxide is determined.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to quality control of commercially available aqueous hydrogen peroxide solutions and other hydrogen peroxide-containing materials, and hydrogen peroxide in hydrogen peroxide generating or decomposing systems in chemical reactions such as enzyme reactions. It relates to a method for quantifying

[0002]

2. Description of the Related Art The following method is known as a method for quantifying hydrogen peroxide in an aqueous solution. (1) Method using hydrogen peroxide electrode: (2) Spectrophotometric method (leuco type, oxidative condensation type)
9-182361): As a typical method, hydrogen peroxide is reacted with 4-aminoantipyrine and phenol in the presence of peroxidase to develop color,
The absorption of the developed reaction solution at 505 nm is measured.

(3) Fluorescence method: Hydrogen peroxide is reacted with homovanillic acid to generate fluorescence, and the fluorescence is measured. (4) Chemiluminescence method: In the presence of a catalyst such as POD, a substrate (luminol, lucigenin, etc.) is excited using the oxidizing power of hydrogen peroxide, and light generated when the excited state returns to the ground state is detected. To do.

[0004]

There are problems in any of the above methods (1) to (4) for quantifying hydrogen peroxide in an aqueous solution sample. In the method (1), since the hydrogen peroxide electrode is covered with the hydrophilic film, it is affected by the reducing substance coexisting in the sample solution. Among the spectrophotometric methods of (2), the leuco-type measuring method tends to cause an error due to coloring of the reagent blank due to natural oxidation of the chromogen. In addition, in the oxidative condensation type measurement method, a negative error is likely to occur due to the reducing substance. further,
Two moles of hydrogen peroxide are required to produce one mole of dye, which is not suitable for quantifying trace components.

In the fluorescence method (3), the sensitivity greatly depends on the performance of the device. Therefore, the influence of temperature and mixed substances is large. In the chemiluminescence method of (4), a sufficient amount of luminescence cannot be obtained under alkaline conditions, and the reaction rate is slow,
It lacks reproducibility. Also, the coexistence of proteins reduces the luminescence intensity. An object of the present invention is to make it possible to easily and quantitatively analyze hydrogen peroxide in an aqueous solution by using an optical analysis means.

[0006]

In the present invention, measuring light including light in the near infrared region is made incident on a sample solution placed in a light transmission type cell, and 4300 to 4800 cm ^-1 and 5400 to 5400 to 1400 of the transmitted light are measured. Hydrogen peroxide is quantified based on the absorbance of any of the absorption peaks present at 6600 cm ^-1 .

The method of the present invention is used not only for measuring an aqueous solution sample that already contains hydrogen peroxide, but also for monitoring a reaction system such as an enzymatic reaction that produces or decomposes hydrogen peroxide. be able to. That is, there, the hydrogen peroxide produced by the specific reaction of various oxidases with biological components and metabolic components is measured by the method of the present invention. The following formula shows the case where the amount of the substance (S) or the product (P) is measured from the amount of hydrogen peroxide produced by the reaction with the enzyme (E).

Examples of such a combination of substance (S) and product (P) include glucose / glucose oxidase, cholesterol / cholesterol oxidase, urea / uricase, pyruvate / bilbate oxidase, hexose / pyranose oxidase and the like. Examples thereof include oxidases, but not limited thereto as long as they are enzymatic reactions that produce hydrogen peroxide.

Further, the reaction is carried out by using an enzyme which specifically reacts with hydrogen peroxide and decomposes, and the hydrogen peroxide is measured by the method of the present invention to measure the enzyme activity from the reduced amount of hydrogen peroxide. You can also The reaction formula in this case is shown in the following formula. Examples of the enzyme used here include dehydrogenases such as peroxidase and catalase. However, the present invention can be applied to reactions involving other enzymes as long as they are coupled reactions with hydrogen peroxide. .

Further, after labeling the compound to be measured with a compound having reactivity with hydrogen peroxide such as peroxidase or catalase, it is reacted with a certain amount of hydrogen peroxide, and the amount of the substance to be measured is calculated from the decrease amount of hydrogen peroxide. It can also be estimated. For example, in the case of measuring the amount of antibody, an anti-antibody labeled with peroxidase is reacted, and after B / F separation, it is reacted with hydrogen peroxide to calculate the decrease amount of hydrogen peroxide. Labeling is done by antigen / antibody or antibody / a
Either one of the nti-antibody combinations may be used. The antigen-antibody reaction is well known to those skilled in the art, and the method for carrying out the antigen-antibody reaction is not limited.

[0011]

1 is a schematic view showing an example of a measuring apparatus for carrying out the present invention. The light source 1 emits light including continuous wavelength light in the near infrared region, and a halogen lamp, a fluorescent lamp, a xenon lamp, a black body radiation source, or the like can be used. The optical path adjusting optical system 2 is an optical system that converts light from the light source 1 into parallel light, a beam splitter that splits the parallel light into measurement light 10s and reference light 10r, and the measurement light 1
It includes an optical system for making 0 s incident on the light transmissive cell 3. The light transmissive cell 3 is made of a material such as glass, quartz, or polyethylene terephthalate, and the cell solution is contained or circulated in the cell 3.

On the optical path of the measuring light 10s, the light transmission type cell 3
Optical path adjustment optical system 4 for adjusting the luminous flux of the measurement light transmitted through
And a spectroscopic device 5 such as an FTIR (Fourier Transform Infrared Spectrophotometer) that receives and disperses the measurement light adjusted by the optical system 4, and the spectroscopic measurement light is an infrared detector. 6 is detected. On the other hand, contrast light 10r
A spectroscopic optical system 9 that adjusts and disperses the luminous flux of the reference light 10r is arranged on the optical path of, and the reference light that has been separated is guided to the detector 6 and detected.

The detector 6 corrects the measurement light 10s transmitted through the light transmissive cell 4 and dispersed through the spectroscopic device 5 by the intensity of the spectral light of the reference light 10r representing the light source intensity to calculate the absorbance. Is configured to. Reference numeral 7 denotes a controller device, which controls the spectroscopic operation of the spectroscopic device 5 and the spectroscopic optical system 9 and, after the correction and amplification processing of the detection signal by the detector 6, performs the arithmetic processing and numerical analysis of the spectrum to perform the peroxide oxidation. The characteristic peak of hydrogen is identified and quantified. 8
Is an output device such as a recorder or a CRT that outputs a processing calculation result in the controller device 7.

A cell made of quartz crystal is used as the light transmission type cell 3, a halogen lamp is used as a light source, and an FTIR (PERKIN ELMER system 200) is used as a spectroscopic device.
An example of measurement using 0) will be described.

(Preparation of calibration curve) 30% (W / V) standard hydrogen peroxide reagent (Santoku Chemical Industry Lot 3018930428) was diluted with distilled water to obtain 30%, 20%, 10%, 2.5%, 1.
5%, 0.75%, 0.3%, 0.225%, 0.15%,
Hydrogen peroxide standard samples of 0.075% and 0.03% were prepared, and the absorption spectra of those standard samples and distilled water were measured. Some of the resulting spectra are divided into a high-concentration region and a low-concentration region, and are shown in FIGS. 2 and 3, respectively. The spectra of FIGS. 2 and 3 are obtained by subtracting the absorption spectrum of distilled water from the absorption spectrum of the standard sample, so that the absorption peak of hydrogen peroxide appears on the positive side of the absorbance. 4300 as the characteristic peak of hydrogen peroxide
~ 4800 cm ^-1 , 5500 cm ^-1, near 5860 cm
Absorption peaks are observed around ^{-1 and} around 6300 cm ^-1 .

In FIGS. 4 to 7, the absorbance at the peak position of each absorption wave number is plotted on the vertical axis and the concentration is plotted on the horizontal axis. This is a line.

(Sample Measurement) Next, an example of quantifying commercially available hydrogen peroxide solution using these calibration curves is shown below. (1) Measurement of a commercially available oxidol disinfectant: A commercially available oxidol disinfectant (a product of Fujimi Pharmaceutical Co., Ltd., labeled as 3 W / V%) was measured in the same manner as when the calibration curve was created, and the results are shown in FIG. The absorption spectrum shown was obtained. Near 4700cm ^-1 of the spectrum, 5500cm around ^-1, 58
Obtains absorbance in each absorption peak in the vicinity of 60cm ^-1 and around 6300cm ^-1, when estimating the concentration of hydrogen peroxide from 4 to absorbance at each absorption wave by applying the respective calibration curve of FIG. 7, respectively 3. 26%, 3.21
%, 3.31%, and 3.29%.

(2) Measurement of a commercially available contact lens cleaning liquid: A commercially available contact lens cleaning liquid (Concept F (trade name): import source; Burns Hind Co., Ltd.) (the indicated concentration) in the same manner as the measurement for preparing the calibration curve. From 2.9
8%) was obtained to obtain the absorption spectrum shown in FIG. 4300-4800 of that spectrum
cm ^-1, 5500Cm around ^-1, determine the absorbance in each absorption peak in the vicinity 5860Cm ^-1 and near 6300Cm ^-1, peroxide from 4 to absorbance at each absorption wave by applying the respective calibration curve of FIG. 7 Estimated hydrogen concentrations were 3.12%, 3.22%, 3.18%, and 3.08%, respectively.

Thus, the quantitative determination of hydrogen peroxide is carried out using the absorbance of any of the absorption peaks at 4300 to 4800 cm ^-1 and 5400 to 6600 cm ^-1 in the near infrared absorption spectrum of the sample solution containing hydrogen peroxide. Can be done.

[0020]

INDUSTRIAL APPLICABILITY In the present invention, the near-infrared absorption spectrum of a sample solution is measured, and its 4300-4800 cm ⁻¹
Since the hydrogen peroxide concentration can be quantified based on the absorbance of any absorption peak existing at 00 to 6600 cm ^-1 , the hydrogen peroxide can be directly quantified and the reduction of hydrogen peroxide as in the conventional method can be performed. Secondary operations such as reacting the luminescent substance with force or oxidizing power are not required, and the error associated with the reaction is reduced accordingly. Most of the enzyme reactions generate hydrogen peroxide. Conventionally, a color former is used to monitor the enzymatic reaction, but the method of the present invention allows the enzyme reaction to be directly monitored without using the color former.

[Brief description of drawings]

FIG. 1 is a block diagram showing an example of a measuring apparatus for carrying out the present invention.

FIG. 2 is a diagram showing a near-infrared absorption spectrum of a 30 to 10% hydrogen peroxide standard sample and distilled water.

FIG. 3 is a diagram showing a near-infrared absorption spectrum of a 2.5-0.3% hydrogen peroxide standard sample and distilled water.

FIG. 4 is an absorption wave number 4700 of the absorption spectra of FIGS.
It is a figure which shows the hydrogen peroxide calibration curve in cm ^<-1 >.

FIG. 5 is an absorption wave number 5550 of the absorption spectra of FIGS.
It is a figure which shows the hydrogen peroxide calibration curve in cm ^<-1 >.

FIG. 6 is an absorption wave number 5860 of the absorption spectra of FIGS.
It is a figure which shows the hydrogen peroxide calibration curve in cm ^<-1 >.

FIG. 7 is an absorption wave number 6300 of the absorption spectra of FIGS.
It is a figure which shows the hydrogen peroxide calibration curve in cm ^<-1 >.

FIG. 8 is a diagram showing an absorption spectrum of a commercially available oxide disinfectant solution.

FIG. 9 is a diagram showing an absorption spectrum of a commercially available contact lens cleaning liquid.

[Explanation of symbols]

 1 light source 3 light transmission type cell 5 spectroscopic device 6 detector 7 controller device

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Haruzo Uenoyama 57, Higashikujo Nishiamita-cho, Minami-ku, Kyoto-shi, Kyoto Prefecture Kyoto Daiichi Kagaku Co., Ltd.

Claims (1)

[Claims] 1. A measurement solution containing light in the near-infrared region is made incident on a sample solution contained in a light transmission type cell, and 4300 to 4800 cm ⁻¹ and 5400 to 6600 cm of the transmitted light.
Quantitative method for quantifying hydrogen peroxide based on the absorbance of any of the absorption peaks present in ^-1 .