KR100384795B1 - Residual agrichemical analysis of kinetic type using spectrophotometer - Google Patents

Abstract

The present invention relates to a method for analyzing residual pesticides using a spectrophotometer (property analysis method), wherein a change in absorbance during chemical reaction of a sample without adding a reaction stopper by introducing a time concept (Kinetic Scan) into a conventional property analysis method By calculating the inhibition rate using the method, a kinetic type attribute analysis method capable of accurately detecting residual pesticides in a sample is provided.

Description

Kinetic type residual pesticide analysis method using spectrophotometer {RESIDUAL AGRICHEMICAL ANALYSIS OF KINETIC TYPE USING SPECTROPHOTOMETER}

The present invention relates to a method for quantitatively analyzing residual pesticides of plants using a spectrophotometer, and more particularly, a kinetic using a spectrophotometer that can accurately detect residual pesticides in a sample by correcting errors occurring during preparation of an analytical sample. Type Residual Pesticide Analysis Method.

<Principle and Operation of Spectrophotometer>

First, the principle and operation of the spectrophotometer essential for the attribution analysis method of the present invention will be briefly described.

A spectrophotometer is a device for measuring light transmittance and absorbance (absorbance) by spectroscopy light having a specific wavelength and passing it through a sample.

The transmittance represents the amount of light passing through the sample, and the absorbance is expressed as a logarithmic function of transmittance, which is obtained according to the following equation.

Here, I ₀ is the intensity of light incident on the sample and I is the intensity of light passing through the sample.

A typical spectrophotometer first remembers 100% of the intensity of the light that passes through a reference sample called blank (zero adjustment) and then measures the intensity of the light that has passed through the sample sample. It is represented by T (= t × 100) value.

Next, the operation of the spectrophotometer will be briefly described with reference to FIG. 1 schematically shows a model of a spectrophotometer in conjunction with a computer.

The user 1 drives the spectrophotometer 2 using the keypad 21, and can also be driven by the keyboard 41 of the computer when the interface of the spectrophotometer and the computer 4 is possible. The measured data (dotted line in FIG. 1 is the flow of data) is processed in the spectrophotometer and the CPUs 22 and 42 embedded in the computer and transmitted 150 to the user via the LCD 25 and the computer monitor 45, respectively. .

Actually performing the measurement is the part of the spectrophotometer that contains the spectrometer (monochromator: 23), which is when the user puts the sample in a part of the spectrophotometer (the Cell Holder) and orders it ( 100), the CPU operates the monochromator, the sensor, the cell holder and the like to obtain the necessary data 130.

The data 130 is converted into a form of absorbance and transmittance 140 by the CPU 22 and 42, and the processed data is output to the LCD 25 and the computer monitor 45 so that the user can easily determine the characteristics of the sample. To judge.

In addition to the simple absorbance / transmittance measurement described above, a typical spectrophotometer system offers a versatile mode that is convenient for sample analysis, including a Survey Scan that outputs absorbance / transmittance values at multiple wavelengths at one time. Kinetic scan that continuously measures samples at intervals and outputs the type of change. Standard curve that calculates the concentration of an unknown sample using absorbance by measuring multiple samples at different wavelengths. Etc.

<Residual pesticide analysis method (property analysis method)>

In pesticide analysis, property analysis is one of the simple analysis methods using organic enzymes to analyze organophosphates and carbamates pesticides that inhibit neurotransmission. These pesticides cause anxiety, paralysis, convulsions, exhaustion, and death by disrupting the function of the enzyme called acetylcholinestrase, which is responsible for the breakdown of acetylcholine in the insect nervous system. .

In the property analysis, acetylcholinesterase is used as an enzyme to add a color reaction so that the reaction of the enzyme can be easily understood. As a substrate, acetylthiocholine is used. Acetyl thiocholine is degraded into acetate and thiocholine by enzymes, and the produced thiocholine reacts with a colorant to increase color development.

Therefore, in the case of the aforementioned pesticides, these pesticides interfere with the production of thiocholine and inhibit the color development. Without the pesticide, the activity of the enzyme is maintained to increase the color development (absorbance). In the analytical method, the absorbance is finally used to calculate the inhibition rate. The more pesticides, the higher the inhibition rate.

Attribute analysis generally requires two reference samples, a blank sample and a control sample. The blank sample is a solvent in which distilled water or solute is removed and serves to set the spectrophotometer to zero. The control sample is a reagent that is developed in the absence of pesticides and has the highest absorbance. The absorbance of the sample containing pesticide is higher than that of the blank sample and smaller than that of the control sample.

By using the absorbance of the sample sample and the absorbance of the control sample, the inhibition (Inhibition), which is a measure of the residual pesticide, is obtained according to the following equation.

As can be seen from Equation 3, the higher the absorbance of the sample sample, the lower the inhibition rate. Therefore, since the residual amount of pesticide is inversely proportional to the absorbance of the sample, the higher the inhibition rate, the higher the residual amount of pesticide.

Figure 2 is a graph for explaining the occurrence of errors in the absorbance of the sample sample according to the conventional attribution analysis method.

The conventional attribution analysis described in FIG. 2 is an ending type attribution analysis, in which the absorbance at 412 nm of a reagent developed for a predetermined time t in the aforementioned attribution analysis is measured using a spectrophotometer. It is a method to determine the residual amount of pesticide by measuring the reaction degree of enzyme by measuring at.

This ending type attribute analysis uses a reaction stopper to ensure that each sample reacts for the same time.

However, during the process of making the sample, pigments and suspended solids extracted from the leaves of the plant are added to the sample sample, which causes an error in absorbance measurement.

In FIG. 2, reference numerals 20 and 30 denote absorbance change curves according to reaction times of the control sample and the blank sample, respectively. As described above, the control sample is a pesticide-free sample, the absorbance changes as the reaction proceeds to the maximum, and the blank sample serves as a reference point for measuring the absorbance of the control sample and the sample samples, and the change in absorbance even when the reaction proceeds. There is almost no. Reference numeral 40 denotes a curve of absorbance change according to the reaction time of the sample sample when the absorbance value is obtained by actual measurement, and reference numeral 40 'denotes the reaction of the sample sample in an ideal state (no color or suspended solids added). Absorbance change curve with time.

FIG. 2 shows that the absorbance of the sample sample should generally vary in the form of reference numeral 40 ', but is measured in the form of reference numeral 40 including an error 200 due to plant pigments and suspensions inevitably added in the sample preparation process. Shows.

In the case of using the conventional property analysis method to detect residual pesticides in the sample, such error cannot be corrected. Therefore, the inhibition rate, which is finally measured as a measure of residual pesticides, is determined by absorbance. Will include errors.

Moreover, because the spectrophotometer and the sample used for measurement are affected by the environment, especially time, the kinetic scan of only one sample at a time and the next measurement of different samples like the conventional spectrophotometer, each sample under the same conditions Difficult to measure

Therefore, there is a need for a novel method of attribute analysis that can accurately detect the amount of pesticide residue in a sample by correcting the error shown in the conventional attribute analysis to calculate the inhibition rate of the sample.

Accordingly, the present invention has been made in accordance with such a demand, and an object of the present invention is to provide an attribution analysis method capable of more precisely calculating the amount of residual pesticide by correcting errors occurring in the conventional attribution analysis method.

In order to achieve this object, according to the present invention, a property analysis method for analyzing the residual amount of pesticide in a sample sample using a spectrophotometer, comprising: a first step of preparing a control sample and a sample sample; Attaching the prepared control sample and the sample sample to the spectrophotometer and setting a measurement time; And the residual amount of pesticide in the sample sample by measuring a plurality of absorbances for each of the control sample and the sample sample by a spectrophotometer for a set time and using the time-dependent change of the plurality of absorbances calculated for each of the control sample and the sample sample. Provided is a property analysis method comprising a third step of analyzing the.

Through this attribution analysis method of the present invention, the spectrophotometer and its software program perform hourly adjustments necessary for absorbance measurement, and kinetic scans of control samples and a plurality of sample samples to correct errors occurring in conventional attribution analysis methods. Accurate inhibition rates can be calculated, increasing the accuracy of the measurement and effectively reducing the measurement time.

1 is a block diagram schematically illustrating an attribution analysis system including a typical spectrophotometer system in which attribution analysis is performed;

Figure 2 is a graph for explaining the occurrence of errors in absorbance of the sample sample according to the conventional attribution analysis method.

3 schematically shows a spectrophotometer system in which an attribution analysis according to the invention is carried out.

Figure 4 is a graph for explaining the absorbance error correction of the sample sample according to the kinetic type attribute analysis method of the present invention.

5 is a flowchart for explaining an attribute analysis method of a kinetic type of the present invention.

<Description of the symbols for the main parts of the drawings>

1: user

10: user input and commands

11: Spectrophotometer Control

12: user sample preparation and sample loading

13: output of the analyzed data

22: CPU

22 ': Property analysis software

300: preparation of sample

340 light source

341: Monochromator

342: Cell Holder

343: sample

344: Sensor

345: light that is emitted by spectroscopy

Hereinafter, with reference to the accompanying drawings will be described in detail the property analysis method of the present invention.

3 shows a spectrophotometer system to which an attribution analysis according to the present invention is applied.

The diagram shows how the kinetic type attribute analysis method is applied, along with the method of measuring absorbance and transmittance in the spectrophotometer.

In response to the user's command 10, the CPU 22 applies a predetermined control signal 11 to the drive of the spectrophotometer, whereby the monochromator 341 generates light rays 345 of a particular wavelength and The sample 343 held in the holder 342 is radiated. This light beam passes through the sample to reach the sensor 344, and the light amount of the light beam reached here is converted into an electrical signal and transmitted to the CPU 22.

The data transmitted to the CPU 22 is basically processed in the form of transmittance and absorbance by the attribute analysis software 22 'built in the CPU 22. The blank sample which is a reference sample is used for calculation of a transmittance | permeability.

First, the spectrophotometer measures the amount of light that has passed through the blank sample and stores it as 100%. The next step is to measure the sample you want to measure in the same way and calculate what percentage of the light passes through the blank sample, which is the transmittance. Therefore, as the measured sample passes more light, the transmittance becomes higher.

The absorbance is calculated from the logarithm of the decimal log by dividing the transmittance obtained above by 100. Since absorbance has a characteristic proportional to concentration in a certain section, it is common to use absorbance rather than transmittance in concentration-related experiments. As the sample passes more light, the absorbance becomes lower.

If several samples can be mounted on the cell holder of the spectrophotometer and the spectrophotometer and its software provide an automation function for controlling the cell holder and the sensor, the measurement of the transmittance and the absorbance can be very simple. .

Next, a method of correcting the absorbance error 200 of the sample sample described in FIG. 2 in the kinetic type attribute analysis method according to the present invention will be described.

4 is a graph for explaining the absorbance error correction of the sample sample according to the kinetic type attribute analysis method of the present invention.

As shown in Figure 4, the kinetic type attribute analysis method according to the present invention by continuously measuring the samples without the addition of the reaction stopper for a certain time (T1) to store all the changes in the absorbance (color development degree) Next, select the section in which the sample reacts with a relatively constant rate of change. In this case, the absolute value of absorbance of each sample (control sample and sample samples) is not directly applied to the inhibition rate calculation, but the change rate or amount of change of the sample in the selected time period is applied to the inhibition rate calculation.

Therefore, in the property analysis method of the present invention, the inhibition rate is calculated according to the following equation.

The error in absorbance generated by the conventional ending type attribution analysis was that the absorbance of the measured sample was affected by the impurities added at the beginning of sample preparation. However, the kinetic type attribute analysis method according to the present invention does not use the absorbance value itself to calculate the inhibition rate, but selectively uses the absorbance change rate (change amount) in a specific section where the change in absorbance is constant, thereby canceling the influence of impurities. Absorbance error, that is, the error of the inhibition rate generated in the ending type attribute analysis method, can be automatically corrected to obtain an accurate inhibition rate.

The whole process of the property analysis method of the present invention by the spectrophotometer system shown in FIG. 3 is divided into the following.

Step 1: preparing a control sample and a sample sample before operating the spectrophotometer,

Step 2: mounting the prepared control sample and the sample sample on the spectrophotometer and setting the measurement time,

Step 3: Measure a plurality of absorbances for each of the control sample and the sample sample by the spectrophotometer for a set time and use the time-varying variation of the plurality of absorbances calculated for each of the control sample and the sample sample to The process of analyzing pesticide residues.

As described above, in the first step, the user starts the measurement with the spectrophotometer immediately after the reaction of the sample is initiated, if possible by slightly changing the sample preparation process in the conventional attribute analysis method before operating the spectrophotometer. . At this time, since the absorbance of the sample in the reaction must be continuously measured, the addition of the reaction stopper, which was performed in the conventional attribution analysis method, should not be included in the sample preparation process 300.

In addition, two reference samples (blank sample and control sample) and a plurality of sample samples for residual pesticide measurement are mounted in the cell holder of the spectrophotometer to complete the measurement preparation.

Steps 2 to 3 except for step 1 of the above 3 steps are further simplified by a series of methods as shown in FIG. 5 provided by the spectrophotometer and its software program.

5 shows a process in which a spectrophotometer and a software program thereof perform attribute analysis in the manner of the present invention to calculate the inhibition rate, and summarizes each process provided by the spectrophotometer step by step.

Step a: Start the measurement based on the data regarding the sample and time you entered

Step b: emit light in the wavelength band you want to measure and adjust the zero point

Step c: continuously measure the reaction state (absorbance) of each sample (blank sample, control sample, and sample sample)

Step d: Analyze the trend of changes in the absorbance resulting from the measurement

Step e: calculate inhibition rate by trend analysis of step d

Step f: feedback from the user to the interval and sample to be applied to the calculation

step g: outputting each process in real time to the user-spectrophotometer interface in steps c, d, e, and f

It consists of seven steps.

At the command of the user, the spectrophotometer starts the measurement by inputting the number and location of the mounted sample and the time T1 to continue the measurement. (410)

When the measurement is started, the monochromator generates light in the wavelength band where the property analysis is performed, emits it to the sample part, and stores the zero point of the light amount by measuring the amount of light in a state where no light is input. (420)

In the next step, a kind of loop 430 is constructed which continuously measures the absorbance of the sample for the time T1.

In the absorbance measurement 431 of the blank sample, the zero point of the absorbance is adjusted, and the absorbance of the control sample is measured (432). The cell holder of the spectrophotometer is equipped with a plurality of sample samples, and the spectrophotometer measures 443 and 434 the absorbances of the plurality of sample samples received in step 410 while driving the cell holder. Absorbance measurement operation of the samples is performed until the measurement time (T1) (435).

Since the driving of the cell holder is precisely controlled for the above multi-cell measurement, each sample is continuously measured at regular time intervals during the measurement time T1 by the CPU internal clock. The measurement can be completed without any action.

The trend of step 450 is a process of converting the change in absorbance of each sample during the measurement time T1 into a standardized multi-order curve form to make more reliable data. In general, the data on the absorbance change of the measured sample is a form that increases slightly less regularly, so it is converted into the data required by the user, that is, the form of the multi-order curve closest to the actual absorbance change curve. Through this process, it is possible to find out which trend the absorbance of each sample changes, and express the trend in a constant equation so that it can be applied to the calculation.

The data on the change in absorbance after the above trend process is applied to the inhibition rate calculation formula of Equation 4 to calculate the inhibition rate (460) and output the same. Thereafter, the user selects 470 only necessary sections among data of all sections measured as necessary and feeds back to the above trend step 450. This feedback repeats the process after the trend or terminates measurement and analysis. The graph of a typical chemical reaction is in the form of an S curve (see Figure 4), which is also possible by this measurement, in which case the user can select only those sections where the reaction is constant.

Each measurement process calculates data in real time and outputs the data to the user 480 to transmit the progress of the experiment and the change in absorbance to the user. The user can get help by restarting the experiment or selecting only the necessary part of the data if there is a problem during the experiment.

The measurement ends when the user selects the required data and the spectrophotometer outputs the final data.

The present invention implements a series of methods in the spectrophotometer system to introduce a time concept (Kinetic Scan) to the existing method to continuously measure the absorbance value and correct the error while the sample is reacted without adding the reaction stopper. In addition, it is possible to solve the error occurring in the existing property analysis method.

By using this method, it is possible to obtain an accurate inhibition rate in which errors generated during sample preparation are corrected.

In addition, multiple samples can be mounted in the cell holder of the spectrophotometer, and the spectrophotometer and its software program provide automated functions to control the cell holder and the sensor. It allows for small errors and very compact measurements.

Claims (4)

In the property analysis method for analyzing the pesticide residual amount of the sample sample using a spectrophotometer, A first step of preparing a control sample and a sample sample; A second step of mounting the prepared control sample and the sample sample on the spectrophotometer and setting a measurement time; And Measure the plurality of absorbances for each of the control sample and the sample sample by the spectrophotometer for the set time and use the time-varying amount of the plurality of absorbances calculated for each of the control sample and the sample sample Third step to analyze pesticide residue in sample Attribute analysis method comprising a. The method of claim 1, wherein the third step, Calculating the inhibition rate of the sample sample based on the calculated amount of change in absorbance over time; Property analysis method further comprising. The method of claim 2, wherein the inhibition rate is the following equation, % Inhibition = {(the amount of change in absorbance of the control sample minus the absorbance of the sample sample Amount of change) / amount of change in absorbance of the control sample} × 100 Attribute analysis method, characterized in that calculated by. The method of claim 3, wherein the set measurement time includes a section in which absorbances of the control sample and the sample sample change linearly with time.