JPS6259774B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to an extremely sensitive method for quantifying hydrogen peroxide (hereinafter abbreviated as H ₂ O ₂ ), and more specifically,
A trace amount of 1 mgH ₂ O ₂ / or less (ppb order)
This provides a method suitable for quantifying H ₂ O ₂ .

Known methods for quantifying H ₂ O ₂ include redox titration methods such as the potassium permanganate method and iodine method, and colorimetric methods using titanium sulfate. The standard method could not quantify trace amounts of H ₂ O ₂ of less than 1 mg/kg.

The present inventors have already provided an electrochemical quantitative method for measuring the increase in dissolved oxygen concentration generated by the decomposition of H ₂ O ₂ using an oxygen electrode (Japanese Patent Publication No. 1973-
6094, Japanese Patent Publication No. 53-319), this method has advantages over the above-mentioned methods, such as ease of operation and no interference from coexisting components, so it is possible to reduce H ₂ O ₂ in food.
It has been used for simple determination of the remaining amount of
It was difficult to accurately quantify the following trace amounts.

The reason for this is that conventional methods use water saturated with atmospheric oxygen, so the amount of oxygen generated when low concentration hydrogen peroxide on the order of ppb is decomposed by catalase is extremely small compared to the saturated amount of dissolved oxygen. Therefore, small fluctuations in the amount of saturated oxygen can cause large errors, and such small changes can also be caused by fluctuations in the electrode indication, fluctuations in atmospheric pressure,
Detection was difficult due to the nozzle of the measurement system, the influence of temperature changes, etc.

A first object of the present invention is to provide a method for quantifying trace amounts of H ₂ O ₂ on the order of ppb that exist or remain in living organisms, foods, and the like.

A second object of the present invention is to provide a quantitative device required for carrying out the above method.

A third object of the present invention is to provide a method for quickly and accurately determining whether H ₂ O ₂ used in the food manufacturing process has been completely decomposed or removed.

A fourth object of the present invention is to streamline the concentration control of H ₂ O ₂ in the food manufacturing process using H ₂ O ₂ .

The present inventors have conducted research to establish a method for quantifying extremely small amounts of H ₂ O ₂ of 1 mg/or less, which has recently become strongly desired from the standpoint of achieving the above objectives.
When quantifying such extremely small amounts of H ₂ O ₂ ,
In the present inventors' H ₂ O ₂ decomposition oxygen detection method, the existing dissolved oxygen in the test solution is on the order of 10 mg/(10 ppm), and the slight relative fluctuation is on the order of ppb, making it difficult to quantify quantitatively. We discovered that the results were seriously impaired, and furthermore, we continued to blow inert gas into the test solution to keep the main part of the test solution free of dissolved oxygen (abbreviated as DO) while removing H ₂ O _2.
If a method is adopted in which a H ₂ O _{2 decomposition reaction occurs locally at the interface between the sensor and the test liquid, in which a decomposer is brought into close contact with the oxygen detection element, there will be no intrusion of atmospheric oxygen and the H 2 O 2} decomposition reaction will be reduced to 1 mg/
The present invention was achieved by discovering that the following trace amounts of H ₂ O ₂ can be determined quickly and accurately.

That is, in the present invention, a base solution is placed in a container equipped with an oxygen detection element with a fixed membrane of a hydrogen peroxide decomposer in close contact with the detection surface, dissolved oxygen is removed from this solution by blowing inert gas, and then dissolved oxygen is removed from this solution. This is a highly sensitive method for quantifying hydrogen peroxide concentration, which involves adding a test solution that does not contain dissolved oxygen while continuing to blow gas, and measuring the change in the output of the detection element. The present invention will be explained in detail below.

In the present invention, the H ₂ O ₂ decomposition agent refers to the enzyme catalase, living organisms and microbial cells containing it, as well as H ₂ O ₂ decomposition catalysts consisting of oxides such as iron, manganese, cobalt, and organic salts. collectively.

Furthermore, the membranes for immobilizing these decomposing agents can be prepared by appropriately applying conventional methods for preparing enzyme immobilization membranes.

In addition, as an oxygen detection element, a polarographic type or a galvanic cell type diaphragm-coated oxygen electrode,
An appropriate device such as a FET sensor can be used.

The immobilized membrane of the H ₂ O ₂ decomposer can also be used as the diaphragm of the enzyme detection electrode.

There are no particular limitations on the containers in which the above-mentioned detection elements are attached, but any number of containers made of glass or plastic can be used.
Small containers ranging from ml to 10ml can be used.

The detection element may be attached from above, or may be attached to the side wall or bottom of the container.

The container may or may not be stirred using a magnetic stirrer or the like.

The base liquid used in the present invention has a composition suitable for the progress of the H ₂ O ₂ decomposition reaction. For example, when a catalase-immobilized membrane is used, a buffer solution near neutrality is used. There is no particular limit to the amount of the base solution, but approximately the same amount to several times the amount of the test solution is used. If the amount is extremely small, the liquid amount will change due to gas blowing, which is undesirable. If the amount is too large, the amount of inert gas and gas blowing time required to remove dissolved oxygen will become large, making it uneconomical.

As the inert gas, any oxygen-free gas such as nitrogen, helium, hydrogen, etc. can be used.

Hydrogen generated from a small water electrolyzer may be used, or air from which oxygen has been removed using a molecular sieve agent with excellent oxygen absorption ability may also be used.

Any operation for removing dissolved oxygen using these inert gases can be used, but a thin tube may be inserted from above the container, or a side tube may be provided to blow in the oxygen. If electromagnetic stirring of the test solution is used in combination, the bubbles will become finer and the degassing time can be shortened. base liquid
In the case of 0.5 ml, dissolved oxygen can be sufficiently removed in about 2 to 3 minutes.

To deoxidize the test solution, methods such as passing an inert gas through it or suctioning it into a vacuum can be applied.

As a method for measuring changes in the output of the detection element, it is sufficient to use a digital or analog amplifier to measure changes over time and time differential values. Since the large output derived from existing DO is eliminated, this method can be used with higher sensitivity than the conventional method of the present inventors, so it is possible to magnify and capture minute changes in DO that were previously impossible to detect.

Note that standard calibration is prepared by appropriately diluting a standard solution whose concentration can be confirmed by the conventional method, but solutions with extremely low concentrations tend to be unstable, so they should be used immediately.

It goes without saying that the temperatures of the standard solution and the test solution should be as similar as possible, but the reaction temperature may be set to a temperature suitable for the action of the decomposer.

Normally, this can be done at room temperature. The test solution is used by adding an extraction solvent such as water or alcohol to food, etc., and homogenizing it, or by centrifuging and filtering it.

Example 1 Experimental materials and equipment: 1.1 Hydrogen peroxide immobilization membrane: Catalase immobilized membrane (thickness 0.07 mm) Membrane material: Polyacrylonitrile membrane 1.2 Oxygen detection element: Polarographic diaphragm oxygen electrode Cathode: Platinum disk (φ = 3mm) Anode: Silver pipe (% = 6mm, L = 40mm) Internal electrolyte: 1MkCl Diaphragm: FEP (fluorinated ethylene-propylene copolymer) thickness 0.0125mm (Applied voltage during use -0.6 to -0.8 V) 1.3 Reaction container: Made of acrylic resin φ = 10mm (depth 30mm) Total volume approximately 3ml 1.4 Base liquid composition: 0.1M phosphoric acid buffer PH6.8 Temperature: Room temperature (20℃) 1.5 Inert gas: _N2 gas, Blow rate: 0.6/min 1.6 Reaction liquid stirrer: magnetic stirrer 1.7 Output measuring device: amplifier (with differential circuit) and mV recorder An outline of the device of the present invention is shown in FIG.

2. Procedure: Attach a polarographic oxygen electrode with a catalase-immobilized membrane (approximately 1.3 times the cathode area) on the detection surface to the side wall of the reaction vessel. Base solution and phosphate buffer (0.1M, PH6.8)
Pour into a 0.6ml reaction vessel and add 600ml/nitrogen gas.
supply min. Dissolved oxygen in the base solution is removed in 2 to 4 minutes. Nitrogen gas is continuously supplied during measurement to prevent oxygen from redissolving.

Samples are also supplied with nitrogen gas in advance to remove enzymes, and during sampling, 0.5 ml is collected with a syringe while preventing oxygen from redissolving. Quickly inject the sample into the reaction vessel. Hydrogen peroxide in the sample was decomposed by the catalase-immobilized membrane, and the amount or rate of increase in the generated oxygen could be detected in 1 to 2 minutes using an oxygen electrode. (See Figures 2 and 3).

3 Experimental results: The recording using the differential circuit is as shown in Figure 2B, and the relationship between the peak height and concentration is as shown in Figure 2B.
It looks like Figure A.

The recording without using a differentiation circuit is as shown in Figure 3B, and the relationship between the increased current value and the H ₂ O ₂ concentration is as shown in Figure 3B.
It became a straight line as shown in figure A.

As in the above examples, in this method 1mg/
(1000ppb) or less, H ₂ O ₂
was found to be able to be quantified with considerable accuracy.

As is clear from Figures 2 and 3, the output measurement time is extremely short, about 30 seconds, which is significantly faster than the conventional electrode method, which takes 2 to 3 minutes. It turns out that it also has the unexpected advantage of being measurable.

In this method, while continuing to blow inert gas,
Since the operation involves detecting the extremely small amount of O ₂ generated by the decomposition of H ₂ O ₂ , the enzyme immediately dilutes in the liquid or escapes into the air along with the injected gas, making accurate quantification impossible. It is not clear why the quantification is successful in this way, but because the electrolytic reduction reaction of the generated oxygen by the oxygen electrode is extremely rapid, the oxygen generated by the action of the immobilized catalase membrane is not clear. The present inventors assume that this is because the ion is reduced and consumed at the electrode before it diffuses into the main part of the liquid.

The inert gas blowing operation in this method is essential to achieving the objective, and its effects can be summarized as follows.

(1) Removal of existing DO in the test solution (2) Prevention of dissolution of atmospheric oxygen (3) Instant mixing of the base solution and sample solution (4) Prevention of solid matter adhesion to the immobilization membrane In summary, this method extremely easy to implement;
A trace amount of ppb order, which was previously impossible.
Since H ₂ O ₂ can be measured in a short time of 1 minute or less, the above-mentioned objectives can be fully achieved.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram illustrating an example of a device suitable for carrying out the invention. 1... Oxygen electrode, 2... Catalase membrane, 3...
Reaction container, 4...dispenser, 5...drainage pump, 6
... _N2 gas, 7...amplifier, 8...differentiating circuit,
9,9'... Recorder, 10... Stirrer, 11
... Stirrer, 20 ... Base liquid, 30 ... Sample. FIG. 2B is a magnetic recording chart obtained from the experimental results of the example by differential method, and FIG. 2A is a calibration curve obtained from the chart. FIG. 3B is a magnetic recording chart obtained from the experimental results of the example by the incremental method, and FIG. 3A is a calibration curve obtained from the chart.

Claims (1)

[Claims] 1. Pour the base solution into a container equipped with an oxygen detection element with a fixed membrane of hydrogen peroxide decomposer in close contact with the detection surface, blow inert gas to remove dissolved oxygen from this liquid, and continue blowing this gas. A highly sensitive method for quantifying hydrogen peroxide concentration, which comprises adding a test solution that does not contain dissolved oxygen and measuring the change in the output of the detection element.