JPS63285459A - Measurement of concentration of organic substance and apparatus used therefor - Google Patents

Abstract

PURPOSE:To expand the range of measurable concn., by making a fixed amount of a sample injected react with an enzyme electrode to determine an integratal value of a response current. CONSTITUTION:A DEAE cellulose film is impregnated with an alcohol oxidase solution, washed by a phosphoric acid buffer and covered with a polycarbonate film to form an immobilized enzyme film, which is combined with an H2O2 electrode to build an enzyme electrode 3. A 0.1M phosphoric acid buffer flows to a flowcell 2 at a flow velocity of 5.2ml/min through a conduit 5 from a buffer tank 4. Prior to the measurement of a sample, a reference ethanol solution with a known concn. is injected from a sample injecting section 8 to determine a reference current integrated value. The integrated current value of the subsequent sample is obtained by translation according to proportional calculation from the reference value to calculate the concentration of alcohol of the sample.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention provides a method for rapid determination of organic substances such as alcohol using an enzyme electrode equipped with an immobilized membrane of an enzyme that specifically reacts with the organic substance, and The present invention relates to an apparatus used in the method.

[Conventional technology]

Conventionally, the concentration of organic substances such as alcohol was measured using an enzyme electrode.
As a method and apparatus for determining, for example, a sample is diluted as a pretreatment for measuring the alcohol concentration of an alcoholic beverage, and then a steady-state current value (i.e., a steady-state rate of an enzyme reaction) for a fixed concentration of alcohol, for example, is measured. A method of calculating the alcohol concentration from the speed or acceleration until the steady current value is reached after diluting the sample has been proposed or used (Am,
J, Enol, Vit ic, Vo134, No, 3
(198B), 173-175, Analytica
Chimica Acta75 (1975) 16
9-180, Japan Brewing Association Magazine Vol. 80 No. 3 (1985
) 206-207, App 1. Mic rob
io IBiotechnol 19 (1984
) 181-185, Biotechnology a
ndBioengineering Vo125(1
983) 1049-1055, AnnalsNew
York Academy of Science
Vol. 434 (1984) 515-519).

Furthermore, there is a flow injection method in which an enzyme electrode is installed in a flow cell, a buffer solution is constantly allowed to flow at a constant flow rate, the sample is diluted, and a minute amount of sample is injected into the line to measure the steady current value (Ana). lyst, Vo 1.
106 (1981) 1309-1317), and
There is a flow injection device (Glucose Analyzer manufactured by Toa Denki) that does not require dilution.

[Problem that the invention seeks to solve]

In the conventional method of measuring steady-state current values for organic substances,
The measurable range is narrow, within several hundred ppm, and normal organic substance concentration measurements require dilution of the sample. Further, even the flow injection method described above, which does not require dilution, has problems such as requiring a small-capacity precision cell and a minute starchy to stir the liquid within the cell.

This invention was made based on the above background,
The purpose is to provide a measurement method that does not require pretreatment such as sample dilution, has a wide measurable concentration range, and can perform rapid measurements, and an apparatus used in the method. .

[Means for solving problems]

As a result of various tests and research on methods for quantitative analysis of organic concentration, the inventors of the present invention discovered that by constantly flowing a buffer solution through the enzyme electrode at a constant flow rate and injecting a small amount of sample into the line, the amount of the injected sample was measured. It would be effective to achieve the purpose of the invention if a certain amount of the organic matter, rather than the entire amount, was reacted with the enzyme electrode, and the total amount of organic matter involved in the enzyme reaction was measured from the obtained response current settlement value to determine the concentration of the organic matter in the sample. I discovered something,
This invention was completed.

That is, the method for measuring the concentration of an organic substance such as alcohol according to the present invention is characterized in that it includes the following steps. What is the method of measuring substance concentration?

(a) A process of injecting a certain amount of organic substance-containing sample into a buffer solution flow at a constant flow rate before applying it to the surface of the immobilized enzyme membrane and supplying the solution to the enzyme electrode. (b) A process of injecting the buffer solution flow to the enzyme electrode. A process of reacting a part of the organic matter in the injected sample with an enzyme electrode by immediately draining the liquid, and measuring the organic concentration by measuring the integral value of the response current generated from the enzyme electrode.This invention The device used in the method for measuring the concentration of organic substances such as alcohol has an inlet for a buffer flow at the bottom;
A cylindrical vertical flow cell with a drain port at the top;
An enzyme electrode is inserted into the flow cell so that the immobilized enzyme membrane surface provided at the lower end is located above the inlet, and a buffer is supplied to the inlet of the flow cell at a constant flow rate via a conduit. A liquid feeding device that injects a sample into a certain amount of organic material provided in a conduit between the flow cell and the liquid feeding device, and a sample injector that injects a certain amount of sample into the conduit between the flow cell and the liquid feeding device and inside the flow cell. a temperature control device that adjusts the temperature of the liquid to a constant temperature; a part of the substance in the injected sample is reacted with an enzyme electrode, and a response current generated from the enzyme electrode is detected;
This is an apparatus for measuring the concentration of organic matter, which is composed of a measuring section that measures the integral value.

This invention will be explained in more detail.

Organic Substance Concentration Measuring Method The object to be measured in this invention is a sample containing organic substances, and examples of such organic substances include alcohol, glucose, galactose, and lactic acid. Examples of organic substance-containing samples that can be used in the present invention include regular beer, other specialty beers, various alcoholic beverages such as Japanese sake, and beverages containing sugar and lactic acid.

The enzyme electrode used in this invention has an immobilized enzyme that reacts specifically and selectively with the organic substance to be measured. For example, when the organic substance is alcohol, the enzyme alcohol oxidase is transferred to a DEAE cellulose membrane. The detection part of the enzyme electrode was created by fixing the probe to a platinum electrode, attaching it to a platinum electrode, and covering it with a polycarbonate membrane. In this example, the concentration of hydrogen peroxide, a product of the enzymatic reaction, can be converted into a current value. The present invention is not limited to examples relating to alcohol; for example, when the target is glucose, glucose oxidase is used as the enzyme, and when the target is galactose, galactose oxidase is used as the enzyme.
It can be applied to the measurement of various organic substances.

One of the characteristics of the measurement method of this invention is that only a predetermined portion of the organic substance in the sample injected into the buffer solution flow at a constant flow rate reacts at the enzyme electrode, and the response current generated from the enzyme electrode due to the reaction is An integral value is measured, and an organic substance concentration is calculated based on the integral value. Next, a measuring device that can exhibit such characteristics will be explained.

q Substance Concentration Measuring Apparatus The measuring apparatus of the present invention is shown in the first part schematically illustrating an example of the apparatus.
This will be explained with reference to the figures.

This apparatus 1 includes a cylindrical vertical flow cell 2, an enzyme electrode 3 inserted into the flow cell 2, and a buffer solution tank 4 that supplies a buffer solution to a bottom inlet 6 of the enzyme electrode 3 via a conduit 5. a liquid feeding device (pump) 7 for injecting a certain amount of organic material into the buffer solution stream; It includes a control device (thermal bath) 9 for adjustment, and a measuring section 10 for detecting the response current generated from the enzyme electrode 3 due to the enzyme reaction and measuring its integral value.

FIG. 2 shows a partially enlarged sectional view of the flow cell and enzyme electrode of this device example. As shown in FIG. 2, this flow cell has an inlet 6 for the buffer flow 11 at the bottom;
The enzyme electrode 3 has a drain port 12 at its upper part, and an immobilized enzyme membrane 13 at its lower end. inserted inside.

The flow cell of the device of this invention is preferably a vertical type,
It is of small capacity. Although it varies depending on the flow rate of the buffer solution, the amount of sample injection, etc., for example, when the flow rate of the buffer solution is 5 mN/min and the sample injection amount is 5 μg, the outer diameter of the enzyme electrode is 14.4 n + mφ,
If the inflow port diameter is 1 mmφ, the flow cell inner diameter is 14.6~
15.4+n+sφ, distance from inlet to electrode 1 to 4
It can be a city.

[For production]

This invention is a system in which a buffer solution flow at a constant flow rate is applied to the surface of an immobilized enzyme membrane to supply the solution to an enzyme electrode, and the solution is immediately drained,
When an organic-containing sample is injected into the buffer flow before this liquid supply,
The organic matter is uniformly mixed with the buffer solution and hits the surface of the immobilized enzyme membrane together with the buffer solution.There is an enzyme that reacts specifically and selectively with the organic matter on this surface of the immobilized enzyme membrane, so the flowing organic matter is If the resulting product is, for example, an oxidizing substance, it is detected as a response current at the redox electrode. In this invention, a buffer stream is applied to the enzyme electrode and immediately drained, so that only a certain percentage of the injected organic material reacts with the enzyme, rather than the entire amount. The detected response current corresponds to the reaction rate of the enzyme reaction, and the concentration was conventionally measured from the peak value of this current value. Determine the total amount of , and then calculate the concentration of organic matter in the injected sample. This is because the integral value has a linear relationship with the concentration of organic matter in the sample.

The operation and effect of the present invention will be explained using the apparatus of the present invention that uses this method.

A buffer solution is supplied to the inlet of the flow cell through the conduit by the liquid supply device at a constant flow rate, and is caused to flow into the flow cell.

The buffer solution hits the surface of the immobilized enzyme membrane of the enzyme electrode above the inlet and is immediately drained. A certain amount of the organic substance-containing sample is injected into the buffer flow from the sample injection part provided in the conduit between the flow cell and the liquid feeding device, and the organic substance is mixed with the buffer solution and reaches the surface of the immobilized enzyme membrane. As explained in the above method, only a part of the injected organic substance reacts with the enzyme, and the concentration of the organic substance in the injected sample is calculated.

〔Effect of the invention〕

The concentration measuring method and device of the present invention can provide the following effects.

(a) Since a small amount of sample is directly injected into the buffer flow, the alcohol concentration range that can be determined is greatly expanded, for example, from 0 to 25%, and there is no need to dilute the sample. It can be used as a sensor.

(b) Since the integral value of the response current has a good linear relationship with the concentration of organic matter in the sample, organic matter can be measured accurately and quickly.

〔Example〕 This invention will be specifically explained by the following examples.

Example I DEAE cellulose membrane (Whatman thickness 120 μm) was impregnated with alcohol oxidase solution and washed with phosphate buffer, and the membrane was covered with a polycarbonate membrane (Nomura Microscience Nueleporemca+brane, pore size 0.03.czm, thickness 5 μm). Immobilized enzyme membrane and H2O2 electrode (manufactured by Toa Denpa Kogyo, PS-115G)
were assembled as shown in FIG. 2, and the apparatus was assembled as shown in FIG.

A 0.1M phosphate buffer solution was flowed from the buffer tank to the flow cell via a conduit at a flow rate of 5.2ml/1min, and before sample measurement, a standard ethanol solution of known concentration was injected from the sample injection part (injector) to inject the standard. Find the current integral value. From this standard value, the subsequent current integral values of the sample are converted by proportional calculation to calculate the alcohol concentration of the sample.

Using regular beer (Light Beer, manufactured by Kirin Brewery) as a sample, the alcohol concentration of the beer was measured under the following measurement conditions. In addition, the alcohol concentration of the sample was measured in parallel using the distillation method, which has been established as an accurate method for measuring alcohol concentration.

The results are shown in Table 1, with the values obtained by the method according to the present invention as measured values and the values obtained by the distillation method as true values.

Measurement conditions Flow rate 5.2 m, Q/min Temperature 20.0°C pH 8.00 (0.1M phosphate buffer) Injection amount
5 μg Examples 2 to 11 Measurements were made in the same manner as in Example 1, except that the samples shown in Table 1 were used. The results are shown in Table 1.

Table 1 Example No. Sample Measured value (X) True value (X) Difference (%)
)I A 2.789 2.87 +
0.1192.662' -
0,0082B 2.798 2.81
-0.0122.825 〃+0.015 3 C5,0825,12-0,0385,058〃
-0.064 4 D 5.050 5.13 -0.
0805.084 〃-0,04B 5 E 4JQ3 4.43 -Q, 1
274.347 〃-0,083 6 F 4.329 4.39
-0.0814JO6〃 -0,084 7G 6.822 B, 71
−0.0886.647 〃−0,0[i
38 H6,8591i, 78 +0.0
796.795 〃 +0.01
59 1 12.476 12.38
+0.09612.419 〃
0.03910 J 12.8
09 12.74 +0.08912.7
5 〃 +0.01011
K 11.863 11.82
+0.04311.83 〃
+0.010*Note) Sample A Nilight Beer (manufactured by Kirin Brewery) Sample B: Regular beer (manufactured by Kirin Brewery, different lot from light beer sample A) Sample C: Regular beer (manufactured by Kirin Brewery, Minebroi Beer) Sample D : Regular beer (manufactured by Kirin Brewery, separate loft from Mainbroi beer sample C) Sample E: Dark beer (manufactured by Kirin Brewery, dark beer) Sample F: Dark beer (manufactured by Kirin Brewery, separate loft from dark beer sample E) Sample G : Stout beer (manufactured by Kirin Brewery, stout beer) Sample H: Stout beer (manufactured by Kirin Brewery, different lot from stout beer sample G) Sample I: Sake (manufactured by Nishinomiya Shuzo, Nihonmori)
(manufactured by Jido Shuzo Co., Ltd., 0 Tsuru) Sample: Japanese sake (manufactured by Kizakura Sake Brewery Co., Ltd., Kizakura Co., Ltd.) From the above results, the measurement error in each case was ±0.1% in terms of alcohol concentration.

Also, the response time was within 5-6 minutes.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing an outline of an example of a measuring device that can be used in the method of the present invention, and FIG. 2 is a partial sectional view showing the flow cell and enzyme electrode of FIG. 1. DESCRIPTION OF SYMBOLS 1... Measuring device, 2... Flow cell, 3... Enzyme electrode, 4... Buffer tank, 5... Conduit, 6...
Inflow port, 7... Pump, 8... Sample injection section (injector), 9... Constant temperature chamber, 10... Measurement section, 11
... Buffer flow, 12 ... Drain port, 13 ... Immobilized enzyme membrane Applicant's agent Mr. Sato - Figure 2

Claims (1)

[Scope of Claims] 1. A method for measuring the concentration of an organic substance in a sample using an enzyme electrode having an immobilized enzyme membrane that specifically reacts with the organic substance, which comprises the following steps. (a) A process of injecting a certain amount of organic substance-containing sample into a buffer solution flow at a constant flow rate before applying it to the surface of the immobilized enzyme membrane and supplying the solution to the enzyme electrode. (b) A process of injecting the buffer solution flow to the enzyme electrode. step 2, in which a portion of the organic matter in the injected sample is reacted with the enzyme electrode by immediately draining the liquid, and the concentration of the organic matter is measured by measuring the integral value of the response current generated from the enzyme electrode; The measuring method according to claim 1, wherein the organic substance is alcohol. 3. A cylindrical vertical flow cell having an inlet for buffer flow at the bottom and a drain port at the top, and a cylindrical vertical flow cell with an immobilized enzyme membrane surface provided at the lower end positioned above the inlet. An enzyme electrode inserted into the flow cell, a liquid feeding device that supplies a buffer solution to the inlet of the flow cell at a constant flow rate via a conduit, and a constant flow rate provided in the conduit between the flow cell and the liquid feeding device. a sample injection unit for injecting a quantity of an organic substance-containing sample;
A temperature control device that adjusts the liquid temperature in the conduit between the flow cell and the liquid feeding device and in the flow cell to a constant temperature, and a part of the organic substance in the injected sample is reacted with an enzyme electrode and transferred from the enzyme electrode. An organic substance concentration measuring device consisting of a measuring section that detects the generated response current and measures its integral value.