JPH0214659B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a quantitative method and apparatus that can quickly and easily quantify extremely small amounts (1 ppm or less) of hydrogen peroxide (abbreviated as H ₂ O ₂ ). Traditionally, the iodine method has been commonly used to quantify H ₂ O ₂ , but its detection limit is 5 ppm.
(mg/Kg). A quantitative method using an oxygen electrode is also known, but its detection limit is 1 ppm. Additionally, an improved 4-aminoantipyrine colorimetric method (4-AA method) has recently been developed. However, although this method is highly sensitive, accurate, and has excellent reproducibility, it has drawbacks such as a long analysis process (about 3 hours) and the need for skill. An object of the present invention is to solve the drawbacks of the conventional methods described above and to provide a method and apparatus that can quickly and easily quantify trace amounts of H ₂ O ₂ of 1 ppm or less. In the present invention, a hydrogen peroxide-containing test solution treated with KBrO ₃ to suppress the decomposition of hydrogen peroxide is placed in a reaction container equipped with an oxygen detection element, and after the reaction container is sealed, the contents of the test solution and the test solution are By introducing an inert gas into the upper space, dissolved oxygen in the test solution and oxygen in the space are removed. Then, while introducing an inert gas only into the upper space, a hydrogen peroxide decomposer solution that does not contain dissolved oxygen is added. A method for quantifying hydrogen peroxide is provided, which comprises injecting hydrogen peroxide and measuring the amount of oxygen generated by decomposition of hydrogen peroxide based on a change in the output of the detection element. KBrO ₃ (oxidizing agent) is preferably used as a 0.05-0.5% aqueous solution. Furthermore, the H ₂ O ₂ decomposition suppressing treatment liquid contains 0.05 to 0.5% oxidizing agent and has a pH of about 7.
0.2 mol/phosphate buffer is preferred. Furthermore, it is preferable to use the above aqueous solution or phosphate buffer after previously removing oxygen with an inert gas such as N ₂ . Examples of the inert gas introduced into the reaction vessel include N ₂ and argon, but N ₂ is particularly preferred from a practical standpoint. In this method, after sealing the reaction container, an inert gas is introduced into the test solution and the space above it.
The test liquid and any oxygen in the headspace are removed by venting the gas through a suitable outlet in the headspace. Next, by introducing an inert gas only into the upper space, intrusion of oxygen from the outside is prevented. At this time, the reason why an inert gas is not introduced into the test liquid is because if it were introduced, the decomposed oxygen to be measured would be driven out, making accurate measurement impossible. H ₂ O ₂ decomposing catalase is usually used as the H ₂ O ₂ decomposing agent. The amount of catalase used is preferably 30 units or more per 2 ml of test solution. Note that one unit of catalase is the enzyme activity that decomposes 1 μM of H ₂ O ₂ in 1 minute. In the present invention, by adding an oxidizing agent to the H ₂ O _2- containing test solution in advance, the decomposition of H ₂ O ₂ in the test solution is extremely suppressed, although the reason is not yet clear. ₂ O ₂ can be quantitatively determined accurately. According to the present invention, the following effects can be obtained. (1) Calibration curve: Good linearity is obtained when the amount of H ₂ O ₂ is between 0.01 and 10 mg/. (2) Extraction liquid: When 1 ppm of H ₂ O ₂ is added to Shirasuboshi, water, 0.5% KBrO ₃ aqueous solution, 0.5% KBrO ₃
As a result of comparing four types of phosphate buffer solution containing phosphate buffer and phosphate buffer solution containing 0.5% KBrO ₃ with N ₂ gas replacement,
Recovery rates of 34.5, 65.9, 72.9 and 87.4% were obtained, respectively. (3) Recovery rates for several types of foods: Recovery rates when 1 ppm was added to raw noodles (udon, soba), hanpen, kamaboko, chikuwa, shirasuboshi, kazunoko, cheese, milk, and yogurt
It was over 80%. (4) Detection limit: solid food, 0.1ppm, liquid food,
0.01ppm (5) 4-Comparison with AA method: Measurements by both methods were in good agreement for raw noodles, hanpen, kamaboko, chikuwa, shirasuboshi, and kazunoko. (6) Time required: Measurement can be performed in approximately 10 minutes for liquid samples and approximately 20 minutes for solid samples. The present invention also provides a H ₂ O ₂ quantitative device including a gas inlet tube for introducing an inert gas into the bottom of the reaction container, a sealable reaction container equipped with an oxygen detection element and equipped with a stirrer, and a gas introduction pipe for introducing an inert gas into the bottom of the reaction container. a second gas introduction pipe for introducing an inert gas into the upper space of the reaction vessel; and an exhaust pipe for discharging the gas in the reaction vessel from the upper space of the reaction vessel; A switch is provided between the two gas introduction pipes and the gas supply source for simultaneously switching the gas introduction and the inert gas introduction through the second gas introduction pipe, and further, the exhaust pipe is discharged into the atmosphere. To provide a hydrogen peroxide metering device that is equipped with a gas backflow prevention means. According to this apparatus, the quantitative method of the present invention can be carried out suitably, and excellent effects can be obtained. A specific example of the method and apparatus of the present invention will be described below with reference to FIG. FIG. 1 is an example of an apparatus suitable for carrying out the invention. In Figure 1, 1
is an oxygen electrode as an oxygen detection element. 2 is 1
2' is a test liquid, and 2'' is an upper space. 2 contains a stirring bar 5 operated by a magnetic stirrer 6. 3 is a jacket for keeping the reaction vessel 2 at a constant temperature, 4 is a lid for sealing the reaction vessel 2, 7 is a four-way socket for switching N ₂ gas, and 8 is an N ₂ gas flow rate control tube. 9 is
A first gas introduction pipe is for introducing N ₂ gas into the test liquid, and 10 is a second gas introduction pipe for introducing N ₂ gas into the upper space. 11 is a needle valve;
12 is an exhaust pipe for discharging N ₂ gas from the upper space 2'' of the reaction vessel 2. 13 is a gas backflow prevention means, which is a trap in the embodiment.
This is a deoxidizing container that also serves as a storage tank for catalase solution for H ₂ O ₂ decomposition. Reference numeral 15 designates a syringe for injecting a hydrogen peroxide decomposition agent solution, which is equipped with an injection needle 15' capable of penetrating the sealing lid 4 of the reaction container 2, and is a syringe for injecting a catalase solution in the example. 16 is an amplifier for the output of the oxygen electrode, and 17 is a recorder. FIG. 2 is a chart showing the analytical trajectories. 1st
The operation of the present invention will now be described with reference to the figures and FIG. First, to describe the method for preparing or extracting a test solution from a solid sample, accurately take a certain weight of a finely chopped sample and place it into a homogenizer cup. to this
After purging with N ₂ gas, add about 8 times the weight of the treatment solution containing an oxidizing agent, and homogenize for about 2 to 3 minutes using a homogenizer. Wash the cup and blender with a small amount of processing solution, making the total volume 10 times that of the sample. Next No.5A
Filter through paper. Discard the first few ml of liquid,
Use the next filtrate as the test solution. A certain volume of this test solution is put into reaction container 2 and sealed with lid 4. Adjust the four-way _N2 gas switching knob 7 so that a solid line flow path is obtained. Open the needle valve 11 and operate the stirrer 5 at the same time.
As a result, N ₂ gas is introduced into the test liquid 2' of the reaction vessel 2 and its upper space 2'' via the tubes 9 and 10, and all the oxygen in the reaction vessel is exhausted via the tube 12. is captured by the oxygen electrode 1, amplified by the amplification device 16, and then recorded on the recorder 17 to obtain the descending curve A shown in Fig. 2.Next, the sensitivity of the amplification device is increased at point _B , and the dissolved When the amount reaches approximately _B2 point (when deoxidation is confirmed), switch the four-way knob 7 so that the flow path shown by the broken line is obtained.As a result, the N ₂ gas introduced into the test solution is The sum of the liquid pressure of the test liquid and the resistance of the _N2 gas flow control pipe 8 and the _N2 gas delivery pressure are balanced, and _N2 gas is not introduced into the test liquid but only into the upper space. Then, when the reading on the recorder reaches approximately point C (when the point has almost reached a steady state), zero point adjustment is performed (see point D).
Take a certain amount into syringe 15, and put the syringe into lid 4.
Inject the catalase solution into the test solution in the reaction container. Immediately, the following reaction occurs and dissolved oxygen is generated in the test solution, resulting in the rise curve E shown in FIG. H ₂ O ₂ Catalase――――――→ H ₂ O + 1/2 O ₂ From the height h of the peak of this rising curve E, the
The H ₂ O ₂ concentration is determined. In other words, the H ₂ O ₂ of the test solution is compared with the peak height h ₀ obtained by the same procedure for the H ₂ O 2 standard solution, and calculated using a calculation formula or calibration curve _.
Concentration is required. Next, the effects of the device of the present invention will be described. The apparatus of the present invention includes a first gas introduction pipe 9 for introducing an inert gas into the bottom of the reaction vessel 2, and a second gas introduction pipe 10 for introducing an inert gas into the upper space 2'' of the reaction vessel 2. and an upper space 2'' of the reaction vessel 2.
and an exhaust pipe 12 for discharging the gas in the reaction vessel, and a switch for simultaneously switching between inert gas introduction through the first gas introduction pipe 9 and inert gas introduction through the second gas introduction pipe 10. A hook 7 is provided between the two gas introduction pipes 9 and 10 and the gas supply source, and gas backflows to the atmosphere discharge part of the exhaust pipe 12 that communicates with the upper space 2'' in the reaction vessel 2. A prevention means 13 is provided, and the gas backflow prevention means 1
3 and is released into the atmosphere,
As a result of the fluid resistance at this point, the pressure within the exhaust pipe 12 and the upper space 2'' is slightly higher than the atmospheric pressure (external pressure), and no airflow (during measurements) into the upper space 2'' occurs. By constantly introducing inert gas through the two gas introduction pipes 10, the upper space 2''
The high pressure inside is maintained, and the infiltration of oxygen-containing air from outside is completely prevented.
This has a significant effect on reducing measurement errors. Test example (Preparation of H ₂ O ₂ standard solution) A 31% H ₂ O ₂ aqueous solution was diluted to 100 mg/dil with a treatment solution containing 0.5% KBrO ₃ (PH7.0, 0.2M phosphate buffer). It was further diluted to various concentrations and used. (Measurement conditions) Amount of H ₂ O ₂ standard solution collected: 2 ml Catalase solution: 0.1 ml (45 units) Reaction temperature: 30°C As a result of testing under the above conditions, the calibration curve shown in Figure 3 was obtained. Example 1 Add 40 ml of the treatment liquid shown in the table below to 5 g of whitebait, homogenize with a homogenizer, and then add the treatment liquid.
The volume was reduced to 50 ml, and after filtration, 2 ml of the filtrate was placed in a reaction vessel, and the H ₂ O ₂ concentration was detected. The results are shown in Table-1. 【table】

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing an example of the quantitative device of the present invention. FIG. 2 is a chart showing the analysis progress of the present invention. FIG. 3 is a graph showing the calibration curve obtained in the above test example. 1...Oxygen electrode, 2...Reaction container, 2'...Test liquid, 2''...Upper space, 4...Lid, 7...N ₂ gas switching four-way pot (switching pot), 8...N ₂ Gas flow rate control tube, 9...First gas introduction tube for introducing _N2 gas into the test liquid, 10...Second gas introduction tube for introducing _N2 gas into the upper space, 12... _N2 gas Exhaust pipe for discharging from the reaction container, 13...Gas backflow prevention means (trap), 14...Oxygen removal container that also serves as a storage tank, 15...Syringe for injecting hydrogen peroxide decomposer liquid, 15'...Lid 4. A needle that can be penetrated.

Claims (1)

[Claims] 1. A reaction vessel equipped with an oxygen detection element,
After adding the hydrogen peroxide-containing test solution treated with KBrO ₃ to suppress the decomposition of hydrogen peroxide and sealing the reaction vessel,
Dissolved oxygen in the test solution and oxygen in the upper space are removed by introducing an inert gas into the test solution and into the upper space of the test solution, and then, while introducing an inert gas only into the upper space, dissolved oxygen is removed. A method for quantifying hydrogen peroxide, which comprises injecting a hydrogen peroxide decomposer solution that does not contain hydrogen peroxide and measuring the amount of oxygen generated by decomposition of hydrogen peroxide based on a change in the output of the detection element. 2. A first gas introduction pipe for introducing an inert gas into the bottom of the reaction container and an inert gas into the upper space of the reaction container are introduced into a sealable stirrer-equipped reaction container equipped with an oxygen detection element. A second gas introduction pipe and an exhaust pipe for discharging the gas in the reaction vessel from the upper space of the reaction vessel are provided, and inert gas is introduced by the first gas introduction pipe and inert gas is introduced by the second gas introduction pipe. A switching device for simultaneously switching the active gas introduction and the active gas introduction is provided between the two gas introduction pipes and the gas supply source, and a gas backflow prevention means is provided at the discharge part of the exhaust pipe to the atmosphere, and further , a syringe for injecting a hydrogen peroxide decomposition agent solution, which is equipped with an injection needle capable of penetrating the sealing lid of the reaction container, as a means for introducing a hydrogen peroxide decomposition agent into the reaction container for hydrogen peroxide measurement; An apparatus for quantifying hydrogen peroxide, characterized in that it is provided separately from the reaction vessel and the like.