JPS6259775B2 - - 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 less than 1 mgH ₂ O ₂ (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 noise in the measurement system, effects 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 necessary 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. After clarifying the cause of the significantly impaired results and removing dissolved oxygen (hereinafter also referred to as DO) from the test solution by blowing inert gas into the test solution, gas ventilation was stopped and the airtight container was removed. By adopting a method of injecting a H ₂ O ₂ decomposer that removes dissolved oxygen in the same way as the test solution while maintaining zero DO, it is possible to quickly and accurately remove extremely small amounts of H ₂ O ₂ of less than 1 mg/dl. It was discovered that it could be quantified, and the present invention was obtained.

That is, in the present invention, a test liquid is placed in a container equipped with an oxygen detection element, dissolved oxygen is removed from the liquid by blowing inert gas, and then the dissolved oxygen is removed under conditions where no oxygen is dissolved from the outside. This is a highly sensitive method for quantifying hydrogen peroxide concentration, which consists of adding a H ₂ O ₂ decomposer solution that does not contain H 2 O 2 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.

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 container in which the detection element is mounted is not particularly limited, but a small container of several ml to about 10 ml and made of glass or plastic with low oxygen permeability and of any shape 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.

If the amount of sample is very small, the liquid volume will change due to gas blowing, which is undesirable, and if it is too large, the amount of inert gas required to remove dissolved oxygen and the gas blowing time 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 also used, the bubbles will become finer and the degassing time can be shortened. Test solution 1.0
In the case of ml, dissolved oxygen can be sufficiently removed in about 4 to 5 minutes.

To deoxidize the H ₂ O ₂ decomposer, 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 the known DO is eliminated, it 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 could not be detected conventionally.

If the substance to be quantified is a solid such as food, add an extraction solvent such as water or alcohol and use it as it is homogenized or as a liquid by centrifugation and filtration.

1 Experimental materials and equipment: 1.1 Hydrogen peroxide decomposer: Catalase solution (activity: 400 units/ml) 1.2 Oxygen detection element: Polarographic diaphragm oxygen electrode Cathode: Platinum disk (φ = 3 mm) Anode: Silver pipe (% = 6 mm, L = 40 mm) Internal electrolyte: 1M kCl Diaphragm: FEP (Fluorized ethylene-propylene copolymer) Thickness 0.0125 mm (Applied voltage -0.6 to -0.8 V during use) 1.3 Reaction container: Made of acrylic resin φ = 10 mm (depth 30 mm) Total capacity approximately 3 ml 1.4 Inert gas: N ₂ gas, blowing rate 30 ml/min 1.5 Reaction liquid stirrer: magnetic stirrer 1.6 Output measuring device: amplifier (with differential circuit) and mV Recorder An outline of the apparatus of the present invention is shown in FIG.

2 Operation Attach the polarographic oxygen electrode to the side wall of the reaction vessel. Pour 1 ml of the test solution into the reaction container, seal it with a septum, and then supply nitrogen gas at 30 ml/min. Dissolved oxygen in the test solution is removed in 4 to 5 minutes. After confirming that it has been removed, stop supplying nitrogen gas. The reaction vessel is hermetically sealed, ensuring zero dissolved oxygen for detection.

The catalase solution is also supplied with nitrogen gas in advance to remove oxygen and prevent oxygen from redissolving, while collecting 20μ (8U) with a syringe. Quickly inject through the septum into the reaction vessel. Hydrogen peroxide in the sample is decomposed by catalase, and the increase in the amount of oxygen generated is 1 to 1 at the oxygen electrode.
Detection was possible in 2 minutes (see Figures 1 and 2).

3 Experimental results: The relationship between the increased current value and the H ₂ O ₂ concentration is a straight line as shown in Figure 2, and in the method of the present invention, the relationship between the increased current value and the H 2 O 2 concentration is a straight line.
(1.000ppb) or less in the extremely low concentration region H ₂ O ₂
could be quantified with considerable accuracy.

As described above, the present invention can fully achieve the above-mentioned objects because it is possible to measure trace amounts of H ₂ O ₂ on the order of ppb in a short time of one minute or less, which was previously impossible.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing an example of a device suitable for carrying out the invention. FIG. 2 is a calibration curve showing the relationship between hydrogen peroxide concentration and detected current value obtained by the method of the present invention. 1...Oxygen electrode, 2...Detection, 3...Reaction container, 4...Septum, 5...Stirrer bar, 6...Magnetic stirrer, 7...Stop valve, 8
......Amplifier, 9...Recorder, 10...Inert gas, 11...Discharge, 20... _{H2O2 decomposer} solution.

Claims (1)

[Claims] 1. Pour the test solution into a container equipped with an oxygen detection element, blow inert gas to remove dissolved oxygen from the solution, and then remove the dissolved oxygen-free filtrate under conditions that no oxygen will be dissolved from the outside. A highly sensitive method for quantifying hydrogen peroxide concentration, which comprises adding a hydrogen oxide decomposer solution and measuring a change in the output of the detection element.