JPH08233740A - Method and apparatus for determining hydrogen peroxide through raman scattering - Google Patents

Abstract

PURPOSE: To quantitatively analyze hydrogen peroxide in aqueous solution easily using an optical analytic means. CONSTITUTION: An exciting light emitted from a light source 1 is split through an optical system 2 for adjusting the optical path including a beam splitter into a measuring light 10s and a reference light 10r. A sample in a cell 3 is irradiated with the measuring light 10s. Raman scattering light from the sample passes through an optical system 4 for adjusting the optical path of scattering light and a spectrometric unit 5 before being detected by a spectroscopic detector 6 including a spectrometer. Hydrogen peroxide is measured quantitatively using the peak of Raman scattering present in the spectroscopic spectrum, detected through the spectroscopic detector 6, having wavelength being shifted by 800-920cm<-1> from that of the exciting light.

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. And a device used therefor.

[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.

On the other hand, one of the optical analysis methods is a method called Raman scattering analysis method. In Raman scattering analysis, when a particular molecule is irradiated with radiant energy in the form of an electromagnetic wave, a small part of the molecules that retain photons do not return to the original vibrational level even after releasing the retained photons, We use the phenomenon that the ground states of electrons fall to different vibrational levels.
Therefore, the energy emitted from these molecules is unique to the molecule, and a specific molecule can be identified by detecting the emitted energy as an electromagnetic wave.

Energetic light emitted by Raman scattering is in an energy state lower than the absorbed energy (Stokes Raman scattering) and in a state higher than the absorbed energy (Anti-Stokes Raman scattering). , But the number of electrons in the excited state is much smaller than the number of electrons in the ground state.
-Stokes Raman scattering intensity is extremely weak, and Stokes Raman scattering is usually used as a method for identifying a specific molecule.

However, there has been no report of qualitative or quantitative analysis of hydrogen peroxide in an aqueous solution using Raman scattering. There is a report of an example of measuring Raman scattering of hydrogen peroxide in the gas phase (Journal of Raman Spectroscopy 2
(1974) 125-132), where three peaks of 3607 cm ^-1 , 1393.5 cm ^-1 and 863.1 cm ^-1 are observed as Raman spectrum peaks.
This is to detect -OO- moiety in hydrogen peroxide,
It is used for structural analysis and cannot quantify the concentration of hydrogen peroxide.

[0007]

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 change in current obtained when hydrogen peroxide is electrically oxidized is measured, 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. Furthermore, 2 moles of hydrogen peroxide are required for the production of 1 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 coexisting 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.

The Raman measurement example of hydrogen peroxide in the gas phase is intended for structural analysis, and the concentration of hydrogen peroxide cannot be quantified. Even if the detection of -O-O- moiety in hydrogen peroxide is attempted in the water-liquid phase, the hydrogen peroxide characteristics will be different in the aqueous solution due to the effect of hydrogen bond of water.
Difficult to detect. Therefore, the quantification of hydrogen peroxide is performed by the above methods (1) to (4). 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.

[0010]

According to the present invention, a sample solution contained in a cell is irradiated with excitation light of a single wavelength, and scattered light from the sample solution is dispersed. Wavelength shift from wavelength is 800 ~ 920cm ^-1
Quantitative measurement is carried out using the Raman scattering peaks present in.

[0011] When coherent light is incident on hydrogen peroxide, classically speaking, the inside of hydrogen peroxide is polarized by vibrating to cause a specific angular vibration, and a spectrum due to the vibration is obtained. This means that when coherent light is applied to hydrogen peroxide molecules in quantum theory, it does not return to the original level even after releasing the photons held by a small part of the specific molecules holding the photons. The vibrational energy peculiar to hydrogen peroxide is released by dropping to different vibrational levels in the ground state. That is, by vibrating excited state is changed by energy vibration of photons, or by vibrating excited state by dispersing a specific spectrum of hydrogen peroxide generated by changing the vibrating state of the adjacent hydrogen peroxide molecule, Perform qualitative and quantitative determination of hydrogen peroxide.

This method is characterized by irradiating molecules of hydrogen peroxide with light energy by radiant energy light in the form of all kinds of electromagnetic waves (especially coherent light such as various laser lights), and dispersing the scattered light. The peak of the wavelength is quantified and the hydrogen peroxide is quantified. It was found that the spectral spectrum of hydrogen peroxide by this method exists at 800 to 920 cm ⁻¹ in terms of wavelength shift (Raman scattering shift wave number). In the method of the present invention, the peak is used to quantify hydrogen peroxide.

A novel and useful point of the method of the present invention is that hydrogen peroxide molecules can be directly quantified, and a luminescent substance is reacted using the reducing power or oxidizing power of hydrogen peroxide as in the conventional method. No secondary operation is added, so there is no reaction error.

The measuring apparatus for carrying out this method is an integrating spherical cell holder having an inner surface serving as a reflecting surface, a spherical cell in which a portion containing a sample solution is fitted in the cell holder, and a cell holder in the cell holder. The light source section that irradiates the sample solution in the cell with excitation light of a single wavelength and the scattered light of the excitation light by the sample solution in the cell inside the cell holder are received and dispersed to detect the Raman scattered light intensity of hydrogen peroxide. It has a spectroscopic and detection unit. By making the cell spherical and fitting the cell into an integrating spherical cell holder whose inner surface is a reflecting surface, the excitation light undergoes multiple reflection in the cell, and Raman scattering can be enhanced.

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 combinations of such substances (S) and enzymes (E) include glucose / glucose oxidase, cholesterol / cholesterol oxidase, urea / uricase, pyruvate / bilbate oxidase, hexose / pyranose oxidase and other oxidases. Examples thereof include, but are not limited to, as long as they are enzymatic reactions that produce hydrogen peroxide.

Further, the enzyme activity is measured from the reduced amount of hydrogen peroxide by reacting with an enzyme which specifically reacts with hydrogen peroxide and decomposes, and measuring hydrogen peroxide by the method of the present invention. 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 measurement substance 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 measurement substance is determined 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.

[0019]

FIG. 1 shows an embodiment of a measuring device for quantifying hydrogen peroxide according to the present invention. Reference numeral 1 denotes an excitation light source for measuring Raman scattered light, which uses a laser device. As the laser device, a continuous wave Ar ion laser, a Kr ion laser, a He-Ne laser, a He-Cd laser, or a pulsed laser such as an Nd: YAG laser can be used. It is possible to select and use from a laser having a wide wavelength range over the infrared region. When using only the oscillation line of the laser device as excitation light,
A laser device may be used in combination with an interference filter or a spectroscope to shield spontaneous emission lines. However, the spontaneous emission ray may be irradiated at the same time to calibrate the wavelength of the spectrum.

The excitation light generated from the light source 1 is separated into the measurement light 10s and the reference light 10r by the optical path adjusting optical system 2 including the beam splitter, and the measurement light 10s is applied to the sample in the cell 3. Raman scattered light generated from the sample is detected by a spectroscopic detector 6 including a spectroscope after passing through a scattered light optical path adjusting optical system 4 for adjusting a light flux and a spectroscopic device 5 such as a filter for removing an excitation light component from the scattered light. To be done.

On the other hand, in order to correct the fluctuation of the excitation light intensity, the reference light 10r is a spectroscopic optical system including a spectroscopic device such as a filter having the same characteristics as the spectroscopic device 5 on the measurement light side and an optical system for adjusting the optical path. It is detected by the spectroscopic detector 6 via 9.
The controller device 7 controls the operation of the spectroscope of the spectroscopic detector 6, corrects the Raman scattered light detection value by the spectroscopic detector 6 with the reference light detection value indicating the light source intensity, obtains a Raman spectrum, and quantifies hydrogen peroxide. Do. 8 is a printer or CRT
Is an output device such as.

2A and 2B show examples of the cell 3 portion, respectively. In (A), the cell 3 is a round-bottomed flask-shaped cell made of a transparent material such as glass, quartz, or polyethylene terephthalate, in which the sample solution is placed. The cell 3 is fitted in a cell holder 10a having an integrating spherical shape. The cell holder 10a has a reflecting surface on the inner surface thereof, and is provided with a window through which the excitation light from the excitation light source is incident and the scattered light is extracted in a direction 180 ° from the incident direction. The excitation light 12 is bent by the mirror 14 and enters the cell 3 through the window of the cell holder 10a.
Reference numeral 16 is a condenser lens for condensing scattered light emitted from the window of the cell holder 10a. The excitation light applied to the sample solution in the cell 3 is repeatedly reflected on the inner surface of the cell holder 10a, is extracted from the window of the cell holder 10a with Raman scattering, and is guided toward the spectroscopic detector.

(B) is an example in which a window is opened in the cell holder 10b so as to extract scattered light in a direction of 90 ° with respect to the incident direction of the excitation light. The cell 3 is fitted into an integrating spherical cell holder 10b having an inner surface serving as a reflecting surface, and the cell holder 10b is provided with a window for allowing the excitation light 12 to enter from the y direction and a scattered light 18 in the x direction forming 90 ° with the incident direction. The window to take out is open.

Returning to FIG. 1 and continuing the description, the scattered light extracted from the cell 3 is condensed by the optical system 4, and the spectroscopic device 5
The excitation light component is removed by and the Raman scattered light is converted into the spectroscopic detector 6
Is detected and dispersed. In the spectroscopic detector 6, the signal is corrected by the intensity of the reference light, and after amplification processing is performed, the signal is taken into the controller device 7 and arithmetic processing for peak detection of hydrogen peroxide is performed, and processing such as numerical calculation is performed. Is performed to identify and quantify the characteristic peaks of hydrogen peroxide.

The measuring apparatus of FIG. 1 is equipped with an argon laser having an output of 100 mW as the excitation light source 1, and its output is 514.5n.
m oscillation line is used, and the cell of FIG.
An example of measurement using an ultraviolet / visible spectrophotometer equipped with a Pelze-type CCD detection element as the spectral detector 6 will be described.

(Preparation of calibration curve) 30% standard hydrogen peroxide reagent (Santoku Chemical Industry Lot 3018930428) was diluted with distilled water to obtain 15%, 10%, 5%, 3%, 1%, 0.5%. Hydrogen peroxide standard samples were prepared, and Raman scattered light of these standard samples and water was measured. The Raman spectrum of the result is shown in FIG. The Raman spectrum of FIG. 3 is obtained by subtracting the spectrum of distilled water from the spectrum of each standard sample as the background. A peak is observed at a position shifted by 878.8 cm ^-1 from the position of the excitation light wavelength. FIG. 4 shows the Raman shift 87 shown in FIG.
The vertical axis represents the peak intensity at 8.8 cm ⁻¹ , and the horizontal axis represents the concentration.

(Sample Measurement) Next, an example of quantifying commercially available hydrogen peroxide solution using this calibration curve is shown below. (1) Measurement of commercially available contact lens cleaning liquid: Commercial contact lens cleaning liquid (Concept F (trade name): Import source; Burns Hind Co., Ltd.) (from the indicated concentration to 2. (Calculated as 98%) was obtained to obtain the Raman spectrum of FIG. This Raman spectrum is shown by subtracting the spectrum of distilled water as the background from the spectrum of the sample. Raman shift wavenumber of this spectrum 878.8
FIG. 6 shows the peak intensity of cm ⁻¹ fitted to the calibration curve of FIG. 4, and the hydrogen peroxide concentration was estimated to be 3.13% based on the results.

(2) 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 the measurement for preparing the calibration curve The Raman spectrum of FIG. 7 was obtained.
This Raman spectrum is also shown by subtracting the spectrum of distilled water from the spectrum of the sample as the background. Raman shift of this spectrum 87
FIG. 8 shows the peak intensity at 8.8 cm ⁻¹ applied to the calibration curve of FIG. 4, and the hydrogen peroxide concentration was estimated to be 3.04% based on the results. In this way, the Raman shift wave number of the sample solution containing hydrogen peroxide is from 800 to 9
The peak at 20 cm ^-1 can be used to quantify hydrogen peroxide.

[0029]

INDUSTRIAL APPLICABILITY In the present invention, the sample solution is irradiated with excitation light and the Raman scattered light is detected to detect Raman shift wavenumber of 80.
Since the peak of 0 to 920 cm ^-1 is detected and the concentration of hydrogen peroxide can be quantified based on the peak intensity, hydrogen peroxide can be directly quantified and the reducing power of hydrogen peroxide can be reduced as in the conventional case. The secondary operation such as reacting the luminescent substance with the use of oxidative power or oxidizing becomes unnecessary, and the error due to 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 schematically showing a hydrogen peroxide quantification device according to the present invention.

FIG. 2 is a cross-sectional view showing a cell portion of the device,
(A) is an example of extracting 180 ° enhanced scattering, (B) is 90
° This is an example of extracting enhanced scattering.

FIG. 3 is a diagram showing a Raman spectrum measured using a standard sample in which the hydrogen peroxide concentration was changed from 0 to 30%.

FIG. 4 is a diagram showing a hydrogen peroxide calibration curve prepared using the peaks in FIG.

FIG. 5 is a diagram showing a Raman spectrum of a commercially available contact lens cleaning liquid.

6 is a diagram showing an example in which the results of FIG. 5 are fitted to the calibration curve of FIG.

FIG. 7 is a diagram showing a Raman spectrum of a commercially available oxide disinfectant solution.

8 is a diagram showing an example in which the results of FIG. 7 are applied to the calibration curve of FIG.

[Explanation of symbols]

 1 Excitation Light Source 3 Cell 6 Spectral Detector 7 Controller Device 10a, 10b Cell Holder 12 Excitation Light 18 Raman Scattered Light

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Akira Aki 57 57 Higashikujo Nishi-Ameracho, Minami-ku, Kyoto-shi, Kyoto Prefecture Kyoto Daiichi Kagaku Co., Ltd.

Claims (2)

[Claims] 1. A sample solution contained in a cell is irradiated with excitation light of a single wavelength, and scattered light from the sample solution is dispersed,
A method for quantifying hydrogen peroxide, which comprises performing a quantitative measurement by using a Raman scattering peak existing in a wavelength shift of 800 to 920 cm ⁻¹ from the wavelength of excitation light in the spectrum. 2. An integral spherical cell holder having an inner surface as a reflecting surface, a spherical cell in which a portion containing a sample solution is fitted in the cell holder, and a single sample solution in the cell in the cell holder. A light source section for irradiating excitation light of a wavelength, and a spectroscopic and detection section for detecting the Raman scattered light intensity of hydrogen peroxide by receiving and dispersing scattered light of the excitation light by the sample solution in the cell in the cell holder, An apparatus for quantifying hydrogen peroxide, which is characterized in that it is provided.