JP2001033388A - Method and device for measuring concentration of chlorophyll a - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately measure the concentration of chlorophyll (a) in inspection water. SOLUTION: A turbidity meter 10 measures the turbidity of inspection water by transmission high with wavelength close to 660 nm and supplies the measured value to a chlorophyll (a) concentration computing element 12. A fluorescence photometer 11 measures the fluorescence lighting intensity of inspection water near an excitation light wavelength of 436 nm and a fluorescence reception light intensity wavelength of 670 nm, and supplies the measured value to the chlorophyll (a) concentration computing element 12. The fluorescence light intensity and turbidity of inspection water are simultaneously measured, and the measured values are supplied to the chlorophyll (a) concentration computing element 12. The chlorophyll (a) concentration computing element 12 stores the measured values being supplied from the turbidity meter 10 and the fluorescence photometer 11 and then uses an operation expression indicating the relationship between the concentration of chlorophyll (a), fluorescence light intensity, and turbidity, and calculates the concentration of the chlorophyll (a).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for measuring chlorophyll a concentration in water.

[0002]

2. Description of the Related Art At present, methods for measuring the concentration of chlorophyll a in water include single-wavelength absorptiometry, three-wavelength absorptiometry, high-performance liquid chromatography, and fluorometry. Among them, the fluorometer method is also used as a chlorophyll a concentration in-situ measurement method.

[0003] In the case of in-situ measurement, unlike laboratory analysis,
There is an advantage that the chlorophyll a concentration can be known on the spot at the site. In addition, if continuous measurement can be performed on site, there is an advantage that the chlorophyll a concentration can be continuously known and the state of eutrophication in the water area can be continuously monitored.

[0004] The principle and method of measuring chlorophyll a concentration by the fluorescence method will be briefly described. (Principle) When chlorophyll a is irradiated with ultraviolet rays, it emits red fluorescence, and its intensity is proportional to the intensity of the excitation light. Therefore, a highly sensitive measurement can be performed by using a strong light source. (Measurement equipment) A fluorometer or a fluorescence spectrophotometer is used. (Measurement operation) 1) Set the excitation wavelength of the fluorometer or the fluorescence spectrophotometer to 436n.
m, adjust the wavelength of the fluorescent part to 670 nm.

2) A certain amount of a test solution is accurately taken and put into a cell.

3) The cell is set in a fluorimeter and fluorescence is measured at an excitation wavelength of 436 nm and a reception wavelength of 670 nm.

4) The chlorophyll a concentration is calculated from the measured fluorescence reading. At this time, 1 μg of chlorophyll a /
The intrinsic fluorescence per 1 (calibration curve of fluorescence intensity and chlorophyll a concentration) is determined in advance.

In the case of a filter-type fluorometer in which the wavelengths of excitation light and light reception are selected by a filter, an interference filter of 436 nm or a color glass filter having a local maximum near it is used as a primary filter (excitation light source). Used. In addition, as a secondary filter (light receiving unit side), 650n
A red glass filter that transmits only light of m or more is used.

Here, the principle of the above-described quantification method by fluorescence analysis will be described.

[0010] Fluorescence is generated by light absorption. Now, when a solution having a concentration c of a fluorescent substance is put into a cell having a thickness b and irradiated with an excitation light source, if the intensity of incident light is I ₀ and the intensity of transmitted light is I, light absorption by the fluorescent substance is possible. About Lamb
The law of ert-Beer is established, and the following equation (1) is obtained.

I = I ₀ · exp (−a · b · c) (1) where a is an extinction coefficient.

Therefore, the intensity of the absorbed light is given by the following equation (2).

I ₀ −I = I ₀ · (1−exp (−a · b · c)) (2) If the intensity of the fluorescence is measured by placing the light receiver at right angles to the light irradiation direction, Assuming that the fluorescence intensity F itself is not absorbed by the solution and is proportional to the absorption of the irradiation light, the following expression (3) is obtained.

F = K ′ · (I ₀ -I) = K · I ₀ · (1-exp (−a · b · c)) · φ (3) where K is the irradiation area of the solution, A constant depending on the device such as the size of the light receiver and the response, and φ is the fluorescence yield, that is, the ratio of the total fluorescence amount to the absorbed excitation light amount. Therefore, the following equations (4) and (5) are obtained.

F = K · I ₀ · φ · a · b · c, (a · b · c <0.05) (4) F = K · I ₀ · φ · a · b · c · (1− (abc / 2), (0.05 <abc <0.25) (5) Since the equation (4) holds for a diluted solution, if the various conditions of the measuring device are fixed, the fluorescence intensity F is proportional to the fluorescent substance concentration c, and an unknown concentration can be obtained by obtaining a relational expression between F and c in advance.

Next, a method for measuring chlorophyll a in the field will be described.

There are two types of on-site measuring devices: a type in which the sensor is directly submerged in the water at the site, and a type in which the sensor is installed on land at the site and water is passed through the device. The former is referred to as "in situ fluorometer" and the latter is referred to as "normal fluorometer".

The in-situ fluorimeter is characterized in that after the sensor is sunk to the point and depth to be measured, the luminous intensity at that point is measured, and the measured value is converted into chlorophyll a concentration. The concentration conversion formula needs to be set in advance by examining the relationship between the fluorescence intensity and the chlorophyll a concentration by a preliminary test. In addition, in order to realize continuous measurement, some detection units are provided with a wiper for removing dirt that may interfere with measurement.

An ordinary fluorometer is characterized in that water at a point and a depth to be measured is pumped up, the water is passed through a flow cell, the fluorescence is measured, and the measured value is converted into chlorophyll a concentration. I have. As in the case of the in-situ fluorometer, it is necessary to investigate the relationship between the fluorescence intensity and the chlorophyll a concentration by a preliminary test and to set a concentration conversion formula in advance. Hereinafter, the measurement method using the present fluorometer is referred to as “in-situ measurement using an in-situ fluorometer”.

The marine environment survey method, edited by the Oceanographic Society of Japan (Koseisha Koseikaku) includes chlorophyll a using an in-situ fluorometer.
The following statement is made regarding the measurement. By utilizing the property that chlorophyll a contained in a living body of phytoplankton emits fluorescence, chlorophyll a can be measured at a marine site. The on-site fluorometer is extremely convenient because it does not require operations such as water sampling, filtration, extraction, etc., and it is possible to continuously record the amount of chlorophyll a by changing the depth or towing the detector directly into water. The accuracy is inferior to that of the extraction method. The most important thing in using an in-situ fluorometer is that a reliable calibration curve must be created in advance for each measuring device. Care must be taken even when the turbidity due to fine particles or dissolved substances other than phytoplankton is remarkable because the measurement error may increase.

[0021]

When measuring water at a site with a fluorometer, as described above, the measurement error can be large even when turbidity due to fine particles other than phytoplankton or dissolved matter is remarkable. As a result, there is a great possibility that the measured luminous intensity includes a measurement error due to the constantly changing sample water quality. Thus, in the field measurement,
The turbidity and coexisting substances in the test water are particularly problematic. This is common to in-situ fluorimeters and ordinary fluorimeters.

Further, when there are a lot of turbid substances and coexisting substances, there are the following problems. The fluorescence emitted by the fluorescent substance is reduced by reflection, scattering, and absorption by the turbidity and coexisting substances in the test water, and the fluorescence received by the fluorescence receiving unit becomes weaker than the fluorescence actually emitted. At present, the above-mentioned in-situ fluorometer only measures the fluorescence intensity by immersing the detection unit in water and does not address the above-mentioned problems relating to the turbidity and dissolved substances of the sample.

Accordingly, the present invention has been made in view of the above circumstances, and even if pollutants coexist in the test water, it is possible to measure the chlorophyll a concentration in the test water with high accuracy. It is an object to provide a method and an apparatus therefor.

[0024]

According to the present invention, in order to achieve the above-mentioned object, a first invention is to measure the fluorescence and turbidity of a test water, and calculate from the measured values of the fluorescence and turbidity. The chlorophyll a concentration of the test water is calculated by the following method.

According to a second aspect of the present invention, the turbidity of the sample is calculated by the following equation.

T = a · L·log (I ₀ / I) T: turbidity a: constant L: measuring cell length I ₀ : intensity of incident light on test water I: transmitted light intensity of test water The calculation for calculating the chlorophyll a concentration is based on the following equation.

C = K ′ · 10 ^{T / K} · FC C: Chlorophyll a concentration in the test water T: Turbidity of the test water F: Fluorescence intensity of the test water K, K ′: Constant The supplied measuring cell is irradiated with light of a certain wavelength from a light source to measure the fluorescence intensity of the sample water, and the measuring cell supplied with the sample is irradiated with light of a certain wavelength from the light source. Turbidity measuring means for measuring the turbidity of the test water, storing the measured values supplied from the fluorescence luminosity measuring means and the turbidity measuring means, and calculating the measured water turbidity in the measuring cell from these measured values. And an arithmetic processing means for calculating the chlorophyll a concentration.

According to a fifth aspect of the present invention, the turbidity measuring means measures the intensity of light emitted from the light source to the measuring cell and the intensity of light transmitted from the measuring cell, and then measures the intensity from the measured values. It is characterized in that the turbidity of the water sample in the cell is calculated.

According to a sixth aspect of the present invention, the measuring cell is a shared measuring cell of the fluorescence luminosity measuring means and the turbidity measuring means, and blocks irradiation light for turbidity measurement at the time of measuring the fluorescence and measures the fluorescence at the time of measuring the turbidity. It is characterized in that the measurement illuminance is switched at regular intervals by shutting off the measurement irradiation light.

According to a seventh aspect of the present invention, there is provided a fluorescent light measuring means for irradiating a light having a predetermined wavelength from a light source to a measuring cell to which a test water is supplied, and measuring a fluorescent intensity of the test water, and a measuring cell to which the test water is supplied. Incident light / transmitted light intensity measuring means for measuring the intensity of light from the light source irradiated to the light source and the intensity of light transmitted from the measuring cell; and supplied from the fluorescent light intensity measuring means and the incident light / transmitted light intensity measuring means. The measured values are stored, and the chlorophyll a of the test water in the measuring cell is calculated from these measured values.
And a calculation processing means for calculating the density.

According to an eighth aspect of the present invention, the measuring cell is a shared measuring cell of the fluorescence luminosity measuring means and the incident light / transmitted light intensity measuring means. It is characterized in that the measurement is switched between the fluorescence intensity and the incident light / transmitted light intensity at regular intervals by shutting off and interrupting the irradiation light for fluorescence intensity measurement when measuring the incident light intensity / transmitted light intensity.

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION The fluorometer used in the method for measuring chlorophyll a according to the present invention is based on the principle of measuring the concentration of chlorophyll a by the fluorometry (for example, when the excitation wavelength is around 436 nm and the light receiving side wavelength is 650 nm or more). Fluorescent intensity can be measured). The fluorescence intensity according to the present invention,
This is the fluorescence intensity around the excitation wavelength of 436 nm and the light receiving side wavelength of around 670 nm, which is the fluorescence intensity for measuring the concentration of chlorophyll a.

In describing the embodiment of the present invention, first, improvement of the measurement accuracy of chlorophyll a concentration in the presence of a turbid substance will be considered.

The conventional method of measuring chlorophyll-a concentration using a fluorometer is to measure the fluorescence intensity of a test sample using a fluorometer, and to calculate the fluorescence intensity using a relational expression between the fluorescence intensity and the chlorophyll-a concentration prepared in advance. This is a method of calculating the chlorophyll a concentration in the test water from the luminous intensity and the relational expression. The relational expression here is represented by the following expression (6).

(Concentration of chlorophyll a) = A × (fluorescence intensity) + B (6) Equation (6) is obtained by conducting a preliminary test in advance.
Equation (6) is obtained from a relationship between the chlorophyll a concentration and the fluorescence intensity after preparing a concentration sequence of chlorophyll a samples of known concentrations and measuring their fluorescence intensity. The concentration sequence of a chlorophyll-a sample whose concentration is known is prepared by diluting actual test water containing chlorophyll-a (the concentration is measured separately) with pure water or the like. In this case, the former concentration column has no coexisting substances such as turbidity, while the latter concentration column has coexisting substances such as turbidity in the sample water at the actual measurement site depending on the dilution ratio. As described above, the chlorophyll a concentration conversion formula (6) is created under certain test conditions in the preliminary test.

However, the actual water sample at the site does not always show the same water test state as that of the preliminary test, and the conditions are rather different in many cases. Water quality always fluctuates on site.
The fluctuation of the water quality and the difference of the water quality appear as an error when converting the chlorophyll a concentration in the equation (6), and the measurement accuracy is deteriorated.

It is considered that the turbid matter has the greatest influence on the chlorophyll a concentration measurement accuracy among the fluctuating sample water qualities. In the fluorescence measurement of the test water, chlorophyll a absorbs the excitation light in proportion to its concentration and emits fluorescence in proportion to the absorption (for the quantification method of chlorophyll a by the fluorescence analysis described in the related art). It is assumed that it is a diluted solution in principle). The emitted fluorescence is reflected and scattered by the turbidity in the test water, and as a result,
The fluorescence which is attenuated and received by the fluorescence receiving unit is measured to be smaller than the actually emitted fluorescence. Further, the degree of the extinction of the fluorescence also varies depending on the sample turbidity. In an extreme example, if the fluorescence emitted by the trace amount of chlorophyll a is not detected by the fluorescence detection unit, but is reflected, scattered, or dimmed by the turbidity, chlorophyll a = 0 may result in the test water. .

As described above, since the conventional method of converting the chlorophyll a concentration from the fluorescence intensity uses the equation (6), if the turbidity of the test water changes, the conversion accuracy will decrease.

Here, how much the turbidity of the test water affects the fluorescence intensity will be considered. The turbidity measured by a turbidimeter is used as an index of the turbidity in the test water. The turbidimeter uses a transmitted light measurement method. The transmitted light of the turbidimeter uses a wavelength around 660 nm.

The fluorometer is used at an excitation light wavelength of around 436 nm and a fluorescence reception wavelength of around 670 nm.

The calibration curve used in the transmitted light turbidimeter is represented by the following equation (7). That is, as shown in the following equation, the relationship between the turbidity and the absorbance is set in a range where the relationship is established by a linear equation and in a water area.

T = a · L · (E 660) (7) Turbidity: T (degrees) L: Cell length (cm) E 660: Absorbance (abs / cm) near wavelength 660 nm a: Constant (a> 0) ) Above 600nm wavelength, there is almost no absorption of chromaticity components in the test water, so the absorbance at 660nm (E660) and 670n wavelength
The absorbance of m (hereinafter referred to as E670 (abs / cm)) is considered to be substantially equal.

[0043] Thus, assuming that the E660 = E670, following (8) from the definition of the way becomes T = a · L · (E670 ) ...... (8) absorbance of the _{equation, E670 = log (I 0 /} I) ... ... (9) I _0: incident light intensity of the wavelength 670nm I: substituting the transmitted light intensity of the wavelength 670nm (8) equation (9), the equation (10).

T = a · L · (Log (I ₀ / I)) ∴I ₀ / I = 10 ^{T / a} · ^L (10) Let C be the chlorophyll a concentration of the test sample. Suppose now that chlorophyll a absorbs excitation light in proportion to its concentration and emits fluorescence in proportion to its absorption. That is, it is assumed that the chlorophyll a concentration in the diluted solution and the test water in the above-described conventional technique is low. If the fluorescence intensity at this time is F ₀ , the following equation (11) is obtained from the principle of fluorescence.

F ₀ = k · L ′ · C (11) F ₀ : wavelength 670 nm of fluorescence emitted from chlorophyll a in the sample water
L ': Cell length of the fluorometer (cm) C: Chlorophyll a concentration in the test water (μg / l) k: Constant (k> 0) Also, the light receiving wavelength 670 nm detected by the fluorometer at this time
Is the fluorescence intensity of F. The fluorescence is reflected and scattered by the turbidity of the sample in the cell, and is dimmed, so that F becomes smaller than F ₀ .

Since the fluorescence detection is performed at 670 nm, I in equation (10) can be replaced with F, and the following equation (12) is obtained.

F ₀ / F = 10 ^{T / a} · ^L (12) However, since the fluorescence does not actually pass through the cell having the length L ′, the fluorescence is emitted from the water sample in the cell, The degree to which the fluorescence is dimmed by the turbidity is smaller than when transmitted through a cell of length L '. Here, assuming that the fluorescence of the chlorophyll a in the sample passes through the apparent cell length L ″ and the apparent cell length L ″ = b · L ′ (b is a constant, 0 ≦ b ≦ 1), (12) The equation becomes the following equation (13).

F ₀ / F = 10 ^{T / a} · ^{L ”} (13) If the cell length L ′ of the fluorometer is not changed, only the turbidity T is the variable on the right side of the equation (13). , A · L ″ = a ·
If b · L ′ = K (K is a constant, K> 0), the equation (13) becomes the following equations (14) and (15).

F ₀ / F = 10 ^{T / K} (14) F / F ₀ = 1 / (10 ^{T / K} ) (15) Further, F ₀ is deleted from the equations (11) and (15). Then, when summarized by C, the following equation (16) is obtained.

C = (1 / (k · L ′)) · F ₀ = (1 / (k · L ′)) · 10 ^{T / K} · F (16) Since k is a constant and L ′ is constant, , 1 / (k · L ′) = K ′
If (K ′ is a constant, K ′> 0), the equation (16) becomes the following equation (17).

C = K ′ · 10 ^{T / K} · F (17) From equation (15), the relationship between T and F / F ₀ is a characteristic diagram shown in FIG.

As the turbidity of the sample increases, the F / F
₀ value decreases. That is, as the turbidity of the test water increases, the fluorimeter of the fluorimeter detected by the fluorimeter becomes smaller than the luminous intensity actually emitted by the chlorophyll a in the test water.

From the equation (17), the relationship between F and C is a characteristic diagram shown in FIG. From FIG. 2, it can be seen that when the turbidity of the test water is 0 when the turbidity of the test water is 0, the chlorophyll a concentration becomes C ″, whereas the turbidity of the test water is T ′.
Then, the chlorophyll a concentration becomes C ′, and it can be seen that the chlorophyll a concentration differs depending on the turbidity of the test sample even when the fluorescence intensity is equal. Similarly, it can be seen that, even when the turbidity is different even in the water sample having a chlorophyll a concentration of C ″, the detected fluorescence intensity is different.

In FIG. 2, a measurement error in the conventional method is considered. It is assumed that a calibration curve at the time of turbidity = 0 in FIG. 2 is obtained by a preliminary test using a concentration column of water turbidity 0 by a conventional method using the relationship between the fluorescence intensity and the chlorophyll a concentration as in the equation (6). Next, the actual test water was measured at the site, and turbidity = 0 and fluorescence luminosity F ′ were obtained as the first result, and the turbidity was obtained as the second result.
Assuming that T ′ and fluorescence intensity = F ′ are obtained, the chlorophyll a concentration is measured to be lower than the chlorophyll a concentration of both samples by C ″ −C ′ from the calibration curve. This is a measurement error of chlorophyll a due to turbidity fluctuation. is there.

Equation (17) is obtained in advance in a preliminary test. In the preliminary test, it was confirmed that the absorbance at the measurement wavelength of the turbidimeter used (E660 in the above case) and the absorbance at the fluorescence reception wavelength of the fluorometer (E670 in the above case) were almost equal, and the difference was negligible. Using a water sample with a turbidity of 0, K ′ of the expression (17) was calculated using a water sample with a known turbidity of (1).
7) Calculate K in the formula and create (17).

Using the chlorophyll a concentration conversion formula (17), the chlorophyll a concentration can be measured accurately even if the turbidity of the test sample varies. Further, even if the turbidity of the test water differs from the turbidity of the actual test water at the time of the preliminary test, the chlorophyll a concentration can be accurately measured.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. (First Embodiment) FIGS. 3A and 3B are schematic diagrams showing a method for measuring chlorophyll a concentration according to the present invention. FIG. 3A shows a case where a sample is passed through a measuring device, and FIG. Part) is described in the case of immersion in a sample.

The chlorophyll a concentration measuring apparatus according to the present embodiment comprises a turbidimeter 10, a fluorimeter 11, and a chlorophyll a concentration calculator 12.

The turbidity meter 10 measures turbidity with transmitted light having a wavelength near 660 nm, and uses the measured value as a chlorophyll a concentration calculator.
Supply to 12.

The fluorimeter 11 measures the fluorescence intensity around the excitation light wavelength 436 nm and the fluorescence intensity wavelength 670 nm, and supplies the measured value to the chlorophyll a concentration calculator 12. Also,
The fluorometer 11 has a stain removing function, and has a function of accurately measuring the fluorescence intensity even in a long-time continuous measurement.

Incidentally, the fluorescence intensity and turbidity of the sample are measured at the same time, and these measured values are supplied to the chlorophyll a concentration calculator 12.

The chlorophyll a concentration calculator 12 has a turbidimeter 10
And after storing the measurement value supplied from the fluorometer 11,
The following chlorophyll a expressed as a function of fluorescence intensity and turbidity
The chlorophyll a concentration is calculated using the concentration conversion formula.

C = K ′ · 10 ^{T / K} · F (18) C: chlorophyll a concentration in the test water T: turbidity of the test water F: fluorescence intensity of the test water K, K ′: constants (K, K ′> 0) When calculating the chlorophyll a concentration, it is necessary to obtain the constants K and K ′ in equation (18) in advance. That is, turbidity meter
After confirming that the absorbance at the measurement wavelength of 10 and the absorbance at the fluorescence reception wavelength of the fluorometer 11 are almost equal and the difference can be ignored, the K ′ value of the equation (18) is calculated using a water sample with a turbidity of 0. The K value is determined using a water sample with a known turbidity.

The chlorophyll a measuring apparatus according to the present embodiment
The absorbance at the measurement wavelength of the turbidimeter to be used is almost equal to the absorbance at the fluorescence receiving wavelength of the fluorometer, and the present invention can be applied to an aqueous system in which the difference is negligible.

The chlorophyll a measuring device according to the present embodiment
It can be used either on-site installation (method using a normal fluorometer) or direct immersion in water sample (in the case of using an on-site fluorometer). In the former case, it is necessary to pass the test water to each measuring instrument by a pump or the like. At the site, water at a place (position, water depth) to be measured may be taken out with a pump or the like. In the latter case, this is not necessary, and the measuring instrument or the detecting section of the measuring instrument may be immersed in the water sample to be measured.

The apparatus for measuring chlorophyll a according to the present embodiment is supposed to be used on site where the turbidity of the sample varies, but can be applied not only on site but also in a laboratory. In this case, water is sampled from the site, and measurement is performed in a laboratory in a batch system. (Second embodiment) In the first embodiment, a method for correcting turbidity of water sample in measurement of chlorophyll-a concentration in water using a fluorometer was created. In the first embodiment, two types of fluorometer and transmitted light turbidimeter are used.
Different types of water quality measuring instruments were used. In this case, there is an advantage that it can be configured using existing water quality equipment.

However, when performing on-site continuous measurement with a water quality meter, contamination of the equipment becomes a problem. Equipment stains include biofilms,
If the detection unit becomes contaminated due to adhesion of iron and manganese components, it is highly likely that normal measurement cannot be performed. For this reason, the measuring instruments for continuous measurement often take measures to remove dirt (for example, cleaning wipers). When two water quality meters are used as in the first embodiment, the structure of the meters,
There is a possibility that there is a difference in the degree of dirt due to a difference in the details of the dirt removal measures. The dirt on the instrument itself may hinder the measurement of the single instrument, but the difference in the dirt between the instruments results in an error in the chlorophyll a concentration calculated using the measured value of each instrument. Therefore, it is better that there is no difference in dirt between the measuring instruments.

Therefore, in the present embodiment, the inventor measured the fluorescence intensity and turbidity of the test sample alternately using two light sources in the same measurement cell in order to cancel the difference in cell contamination between the instruments. Then, a chlorophyll a concentration measuring device for calculating chlorophyll a concentration was proposed.

The apparatus for measuring chlorophyll a comprises: a luminous intensity measuring means for irradiating a light of a predetermined wavelength from a light source to a measuring cell supplied with the water to measure the luminous intensity of the water; Turbidity measuring means for irradiating light of a certain wavelength to measure the turbidity of the test water, and storing the measured values supplied from the fluorescence luminosity measuring means and the turbidity measuring means, and calculating from these measured values And arithmetic processing means for calculating the chlorophyll a concentration of the test water in the measurement cell.

Here, the turbidity measuring means includes an intensity of light emitted from the light source to the measuring cell (hereinafter referred to as transmitted light reference intensity) and an intensity of light transmitted from the measuring cell (hereinafter referred to as transmitted light). (Hereinafter referred to as intensity), and has a function of calculating the turbidity of the test water in the measurement cell by calculation from these measured values.

Further, in order to switch the measurement of the fluorescence and turbidity of the same sample at regular intervals, the measurement cell is a shared measurement cell of the fluorescence luminosity measurement means and the turbidity measurement means. It is characterized in that irradiation light for turbidity measurement is blocked during luminosity measurement and irradiation light for fluorescence luminosity measurement is blocked during turbidity measurement.

FIG. 4 is an explanatory view of a chlorophyll a concentration measuring device and a fluorescence photometric measurement according to the second embodiment. In FIG. 4, the components related to the fluorescence photometric measurement means are excitation light sources 20 and 43 for fluorescence measurement, which are light sources capable of outputting excitation light near 436 nm.
Optical filter 21 for extracting light around 6 nm, shutter 22 for blocking irradiation light for transmitted light measurement during fluorescence photometry, filter 23 for blocking reflection and scattering of excitation light and extracting light around 670 nm, and water sampling is continuous And a light receiver 24 that receives the fluorescence / transmitted light from the flow cell 40 and supplies the measured value to the arithmetic processing unit 50.

The component relating to the turbidity measuring means outputs light of about 670 nm for the signal for turbidity correction (it is not necessary to have the wavelength of about 660 nm used in the turbidity measurement by the transmission method). Turbidity correction signal light source 30, optical filter 31 for extracting light near 670 nm (31 and 23 use filters with the same optical specifications), excitation light shutter 32 for blocking excitation light when measuring transmitted light, transmission A half mirror 33 for extracting the transmitted light reference intensity from the light source and the transmitted light reference intensity are measured, and a transmitted light reference intensity light receiver 34 for supplying the measured value to the arithmetic processing unit 50 for turbidity measurement, It comprises the above-described flow cell 40 for measuring the intensity of transmitted light, the optical filter 23, and the light receiver 24.

The flow cell 40 has the same optical path length in the excitation light irradiation direction and the same light path length in the transmitted light irradiation direction.
Since the flow cell 40 is a common component for the measurement of the fluorescence intensity and the measurement of the transmitted light intensity, the degree of contamination on the excitation light incidence surface / transmission surface and the transmission light incidence surface / transmission surface is equal.

The arithmetic processing unit 50 has a function of storing the measured values supplied from the light receivers 24 and 34, calculating the turbidity of the test water and the chlorophyll a concentration by calculation, and displaying the calculated values. are doing.

Next, measurement of chlorophyll a concentration by the apparatus according to the present embodiment will be described with reference to FIG.

In the fluorescence intensity measuring step, the transmitted light shutter 22 is inserted between the turbidity correction signal light source 30 and the flow cell 40. This is for blocking the transmitted light measurement light. When the excitation light shutter 32 is opened, excitation light is emitted from the fluorescence measurement excitation light source 20 to the flow cell 40 storing the water sample. Here, light of a certain wavelength (for example, 436 nm) is extracted from the excitation light by the optical filter 21 provided between the light source 20 and the flow cell 40. The water sample in the flow cell that has received the excitation light of a certain wavelength emits light of a long wavelength due to the fluorescent substance in the water sample. Again, by the optical filter 23 installed between the flow cell 40 and the light receiving unit 24,
A certain wavelength (eg, 670nm) from the light emitted from the sample
Is taken out. The light receiver 24 detects the light of the above-mentioned fixed wavelength and measures it as a fluorescence intensity. This measured value is supplied to the arithmetic processing unit for calculating the chlorophyll a concentration of the test water. After measuring the fluorescence intensity, the process proceeds to the turbidity measurement step.

In the turbidity measurement step, as shown in FIG. 5 for explaining the transmitted light measurement for turbidity correction, an excitation light shutter is used to cut off the excitation light from the excitation light source 20 for fluorescence measurement.
When 32 is inserted between the fluorescence measurement excitation light source 30 and the flow cell 40, the transmitted light shutter 22 is opened, and the transmitted light measurement light is transmitted from the turbidity correction signal light source 30 to the flow cell 40 storing the water sample. Is irradiated. Here, a certain wavelength (for example, 670 nm) is extracted from the transmitted light measurement light by the optical filter 31 provided between the light source 30 and the half mirror 33. The half mirror 33 extracts the reference light from the extracted light and supplies it to the transmitted light reference intensity light receiver 34, and simultaneously irradiates the flow cell 40 with the transmitted light measurement light. The light receiver 34 detects the reference light taken out by the half mirror 33 and measures it as a transmitted light reference light intensity I ₀ ,
This measured value is supplied to the arithmetic processing unit 50 for calculating turbidity and the like. Even in the irradiation light transmitted through the flow cell 40, light of a certain wavelength is extracted by the optical filter 23 as in the case of measuring the fluorescence intensity. The extracted light is measured as the transmitted light intensity I in the light receiver 24. The measured transmitted light intensity I is supplied to the arithmetic processing unit 50 for calculating the chlorophyll a concentration of the test water.

The arithmetic processing unit 50 calculates the turbidity from the transmitted light reference intensity supplied from the light receiving device 34 and the transmitted light intensity supplied from the light receiving device 24 by calculation using equations (8) and (9). The chlorophyll a concentration is calculated from the calculated value of the turbidity and the measured value of the fluorescent luminous intensity supplied from the light receiver 24 by the calculation using the equation (18).

The fluorescence luminosity measurement step and the turbidity measurement step are switched by taking in and out the excitation light shutter and the transmitted light shutter. The excitation light shutter and the transmitted light shutter are automatically switched at regular time intervals, and each process is continued for the same time.

By using the chlorophyll a measuring apparatus according to the present embodiment, the fluorescence intensity and the turbidity are measured with the same flow cell, so that the influence of the contamination of the flow cell is cancelled.

[0082]

According to the method and apparatus for measuring chlorophyll-a according to the present invention, the turbidity is measured together with the fluorescence of the sample, and the chlorophyll-a concentration is determined from the measured value of the turbidity of the sample and the fluorescence of the sample. Since the conversion to へ is corrected, the measurement error of the chlorophyll a concentration due to the turbidity of the test water is reduced, and the chlorophyll a concentration can be accurately measured with respect to the test water in which the turbidity tends to fluctuate.

In the chlorophyll-a measuring apparatus according to the present invention, the fluorescence intensity and turbidity of the sample are alternately measured in the same measuring cell, thereby canceling the chlorophyll-a concentration measurement error caused by the contamination of the measuring cell. effective.

[Brief description of the drawings]

FIG. 1 is a characteristic diagram showing a relationship (image) between turbidity T and fluorescence luminous intensity F / F _{0 in} a water sample.

FIG. 2 is a characteristic diagram showing a relationship (image) between the measured value F of the fluorometer and the concentration of chlorophyll a.

FIG. 3 is a schematic diagram of a chlorophyll a concentration measuring system according to the present invention.

FIG. 4 is an explanatory view of a chlorophyll a concentration measuring device and a fluorescence photometric measurement according to a second embodiment.

FIG. 5 is an explanatory diagram of turbidity correction transmitted light measurement according to a second embodiment.

[Explanation of symbols]

 10 turbidimeter 11 fluorimeter 12 chlorophyll-a concentration calculator 20 excitation light source for fluorescence measurement 21 optical filter (for 436 nm) 22 transmitted light shutter 23, 31 optical filter (for 670 nm) 24 light reception 30 ... Light source for turbidity correction signal 32 ... Excitation light shutter 33 ... Half mirror 34 ... Light receiver for transmitted light reference intensity 40 ... Flow cell 50 ... Arithmetic processing unit

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G043 AA01 BA14 CA03 EA01 EA13 GA02 GB01 GB19 GB21 HA09 HA11 JA03 KA02 KA05 LA01 NA01 NA11 2G054 AA03 AB01 CA20 EA03 EA04 EA09 EB01 FA09 FA19 FA22 FA33 FB01 GA03 GB01 GB02 JA01 BB05 CC12 EE01 EE07 HH02 HH06 JJ03 JJ23 KK01 MM01 MM12

Claims (8)

[Claims] 1. A method for measuring the chlorophyll a concentration of chlorophyll a, wherein the chlorophyll a concentration of the sample is measured by measuring the fluorescence and turbidity of the sample and calculating the chlorophyll a from the measured values of the fluorescence and turbidity. 2. The chlorophyll a concentration measuring method according to claim 1, wherein the turbidity of the sample is calculated by the following equation. T = a · L·log (I ₀ / I) T: turbidity a: constant L: measuring cell length I ₀ : intensity of incident light to test water I: intensity of transmitted light to test water 3. The calculation for calculating the chlorophyll a concentration according to the following equation.
The method for measuring chlorophyll a concentration described in the above. C = K '· 10 ^{T / K} · F C: Chlorophyll a concentration in the test water T: Turbidity of the test water F: Fluorescence intensity of the test water K, K': Constant 4. A fluorescent light measuring means for irradiating a light of a predetermined wavelength from a light source to a measuring cell to which the test water has been supplied to measure the fluorescent light of the test water, and a light source for the measuring cell to which the test water has been supplied. Turbidity measuring means for irradiating light of a certain wavelength to measure the turbidity of the test water, and storing the measured values supplied from the fluorescence luminosity measuring means and the turbidity measuring means, and calculating from these measured values A chlorophyll-a concentration measuring device, comprising: arithmetic processing means for calculating the chlorophyll-a concentration of the test water in the measurement cell. 5. The turbidity measuring means measures the intensity of light emitted from the light source to the measuring cell and the intensity of light transmitted from the measuring cell, and calculates the intensity of the light in the measuring cell from the measured values. The chlorophyll a concentration measuring device according to claim 4, wherein the turbidity of the sample is calculated. 6. The measurement cell is a shared measurement cell of the fluorescence luminosity measurement means and the turbidity measurement means, and blocks irradiation light for turbidity measurement during fluorescence measurement and irradiation for fluorescence measurement during turbidity measurement. 6. The chlorophyll-a concentration measuring apparatus according to claim 4, wherein switching of measurement of fluorescence intensity and turbidity is performed at regular intervals by blocking light. 7. A fluorescent light measuring means for irradiating a light having a predetermined wavelength from a light source to a measuring cell to which the test water has been supplied, and measuring a fluorescent intensity of the test water, and irradiating the measuring cell to which the test water has been supplied. Incident light / transmitted light intensity measuring means for measuring the intensity of light from the light source and the intensity of light transmitted from the measuring cell; and measurement supplied from the fluorescent light intensity measuring means and the incident light / transmitted light intensity measuring means. A chlorophyll-a concentration measuring device, comprising: a processor for storing values and calculating the chlorophyll-a concentration of the test water in the measuring cell from the measured values by calculation. 8. The measuring cell is a shared measuring cell of the fluorescence luminosity measuring means and the incident light / transmitted light intensity measuring means,
By blocking the irradiation light for measuring the incident light intensity and transmitted light intensity when measuring the fluorescence intensity, and blocking the irradiation light for measuring the fluorescence intensity when measuring the incident light intensity and transmitted light intensity, the fluorescence intensity and the incident light The clofil a concentration measuring apparatus according to claim 7, wherein measurement of transmitted light intensity is switched.