JP2018146539A - Multi-wavelength fluorescence analyzer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a multi-wavelength fluorescence analyzer with which a measurement target component in a sample liquid can be obtained with high accuracy while eliminating influences of obstructive components such as turbidity, without requiring a separate turbidity meter or a light source.SOLUTION: Irradiation light E is guided to the liquid surface of a sample liquid by an irradiation optical system having a diffraction grating 33, and response light F emitted from the liquid surface of the sample liquid is guided to a detector 82 by a detection optical system having a diffraction grating 73 and a cut filter 85 that eliminates scattered light at the same wavelength as that of excitation light. Excitation light is guided to the liquid surface of the sample liquid by the irradiation optical system, and fluorescent light is guided to the detector 82 by the detection optical system to obtain a fluorescent intensity If. Correction irradiation light is guided to the liquid surface of the sample liquid by the irradiation optical system, and correction response light is guided to the detector 82 by the detection optical system to obtain an intensity Im of the correction response light. Then, the density of the measurement target component in the sample liquid is calculated based on the fluorescent intensity If. In the calculation, correction by using the intensity Im of the corrected response light is performed.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a fluorescence analyzer that obtains information about a measurement target component in a sample liquid by fluorescence emitted from the sample liquid. More specifically, the present invention relates to a multi-wavelength fluorescence analyzer that can accurately determine the fluorescence of a sample solution without the influence of interference components such as turbidity.

Presence / absence of the measurement target component by measuring fluorescence (including intensity and quenching time) emitted from the measurement target component in the sample liquid, a reaction product with the measurement target component, or a reagent that reacts with the measurement target component. Fluorescence analysis for measuring the concentration is widely used.
In the fluorescence analysis, if turbidity is present in the sample solution, a measurement error is given. Therefore, turbidity correction is performed by various methods.

For example, in Patent Document 1, when determining the algal concentration by the fluorescence method, the turbidity measured with a turbidimeter is used to eliminate an error caused by the excitation light scattered by the turbidity being added to the fluorescence intensity. It has been proposed to correct the fluorescence intensity based on the degree data.
Further, in Patent Document 2, when the chlorophyll a concentration is obtained by a fluorescence method, light having the same wavelength as that of fluorescence is transmitted in order to eliminate an error caused by the generated fluorescence reflected and scattered by turbidity and dimming. It has been proposed to correct the fluorescence intensity based on the transmitted light intensity.

JP 2001-83094 A JP 2001-33388 A

However, the method of Patent Document 1 does not consider the influence of fluorescence that is reflected and scattered by turbidity and dimmed. In addition to the fluorescence analyzer, a turbidimeter or turbidity measurement cell must be prepared separately.
In the method of Patent Document 2, the influence of the excitation light scattered by the turbid matter being measured on the fluorescence intensity is not considered. In addition, a light source for measuring fluorescence and a light source for measuring transmitted light intensity must be prepared.
In any of the methods of Patent Documents 1 and 2, the influence of disturbing components other than turbidity is not considered at all.
The present invention has been made in view of the above circumstances, and it is possible to accurately determine the component to be measured in the sample liquid by eliminating the influence of disturbing components such as turbidity. It is an object of the present invention to provide a multi-wavelength fluorescence analyzer that does not require a photometer or a light source.

In order to achieve the above object, the present invention employs the following configuration.
[1] A multi-wavelength fluorescence analyzer that obtains the concentration of a measurement target component in the sample solution based on the fluorescence intensity If obtained when the sample solution is irradiated with excitation light,
A light source;
An irradiation optical system for guiding the light source light emitted from the light source to the liquid surface of the sample liquid at an angle of 45 ± 15 ° with respect to the vertical direction;
A detector;
A detection optical system that receives response light emitted from the sample liquid in a vertical direction above the liquid surface of the sample liquid, and guides it to the detector;
A control apparatus for controlling the entire apparatus and performing a predetermined calculation by inputting the intensity of the response light obtained by the detector,
The irradiation optical system includes a diffraction grating, and includes at least one type of correction irradiation light for measuring excitation light for obtaining fluorescence by at least the measurement target component of the light source light and concentration of interference components. Including a plurality of light beams having different wavelengths, which are selectively guided to the liquid surface of the sample liquid,
The detection optical system has a diffraction grating and a cut filter that removes scattered light having the same wavelength as the excitation light, and measures at least the concentration of fluorescence and interference components due to the measurement target component in the response light. A plurality of lights having different wavelengths including one or more kinds of correction response lights are selectively guided to the detector;
A multi-wavelength fluorescence analyzer characterized by performing the following steps 1 to 3.
Step 1: Excitation light for obtaining fluorescence due to the measurement target component in the light source light is guided to the liquid surface of the sample solution by the irradiation optical system, and the response light includes the response light in the response light. Fluorescence due to the measurement target component is guided to the detector to obtain a fluorescence intensity If.
Step 2: Use the irradiation optical system to guide the correction irradiation light out of the light source light to the liquid surface of the sample liquid, and use the detection optical system to pass the correction response light out of the response light to the detector. Then, the intensity Im of the corrected response light is obtained.
Step 3: Calculate the concentration of the measurement target component in the sample solution based on the fluorescence intensity If. In this calculation, correction according to the concentration of the disturbing component obtained from the intensity Im of the correction response light is performed.
[2] The one or more types of corrected irradiation light includes turbidity correction irradiation light for measuring turbidity as a disturbing component,
The one or more types of corrected response light includes the turbidity corrected response light for measuring turbidity as a disturbing component, according to [1].
[3] The multiwavelength fluorescence according to [1] or [2], wherein the sensitivity of the detector is variable, and the arithmetic and control unit is capable of controlling the sensitivity of the detector according to the intensity of the response light. Analysis equipment.
[4] The multi-wavelength fluorescence analyzer according to any one of [1] to [3], wherein the calculation in the step 3 is performed based on the following formula (1).
C = a * If-k * g (Im) + b1 (1)
In Equation (1), C is the concentration of the component to be measured, g (Im) is the concentration of the disturbing component obtained from the intensity Im of the corrected response light, and a, b1, and k are coefficients.
[5] Furthermore, an upper end is formed as an opening, and an overflow cylinder into which the sample liquid flowing in from below is overflowed so that an overflow surface of the sample liquid is formed in the opening, and the liquid surface of the sample liquid is the sample The multiwavelength fluorescence analyzer according to any one of [1] to [4], which is a liquid overflow surface.

  According to the multi-wavelength fluorescence analyzer of the present invention, the component to be measured in the sample solution can be used to remove disturbing components such as turbidity in one measurement system without using a separate turbidimeter, light source, measurement cell, etc. The influence can be eliminated and it can be obtained with high accuracy.

1 is a schematic configuration diagram of a multiwavelength fluorescence analyzer according to an embodiment of the present invention.

  A multiwavelength fluorescence analyzer according to an embodiment of the present invention will be described. As shown in FIG. 1, the multi-wavelength fluorescence analyzer 1 of the present embodiment includes a light-shielding partition plate 2, a light-shielding detection unit case 3 installed above the partition plate 2, and a partition plate 2 below. A light-shielding sample case 4 to be installed and a light-shielding overflow cylinder 6 inserted obliquely from below the sample case 4 are provided.

A sample solution outlet 4 a is provided at the lower end of the sample case 4. The overflow cylinder 6 has a lower end as a sample liquid inlet 6a and an upper end as a substantially horizontal upper opening 6b.
The sample liquid S flows into the overflow cylinder 6 from the sample liquid inlet 6 a of the overflow cylinder 6, overflows from the substantially horizontal upper opening 6 b on the upper end side of the overflow cylinder 6, and forms an overflow surface 7. ing. The overflowed sample liquid S is guided to the sample liquid outlet 4a and discharged.
In the multi-wavelength fluorescence analyzer 1, the inflow and outflow of the sample liquid S are continuously continued in principle during continuous measurement, and the overflow surface 7 is always formed by the new sample liquid S.

The detection unit surrounded by the partition plate 2 and the detection unit case 3 includes a light source unit 10, an optical path unit 20, a light source side spectroscopic unit 30, an irradiation light unit 40, a light receiving unit 60, a detection side spectroscopic unit 70, and a detector unit. 80 is arranged.
In addition, the multi-wavelength fluorescence analyzer 1 includes an arithmetic control device 90, and the operation of the entire device is controlled by the arithmetic control device 90. In addition, a signal detected by the detector unit 80 is input to the arithmetic control device 90, and necessary calculations are performed based on the input value.
In addition, a display (not shown) is provided, and the measurement target component concentration, the interference component concentration, the response light intensity obtained by the detector, and the like calculated by the calculation control device 90 can be displayed as appropriate.

  In this embodiment, the light source unit 10 is a light source in the present invention, and the detector 82 in the detector unit 80 is a detector in the present invention. Further, the optical path unit 20, the light source side spectroscopic unit 30, and the irradiation light unit 40 constitute an irradiation optical system in the present invention. Further, the detector optical system other than the light receiving unit 60, the detection-side spectroscopic unit 70, and the detector 82 in the detector unit 80 constitutes the detection optical system in the present invention.

The light source unit 10, the optical path unit 20, the light source side spectroscopic unit 30, and the irradiation light unit 40, the light receiving unit 60, the detection side spectroscopic unit 70, and the detector unit 80 each have a lens, Two or more optical members such as a mask are fixed and unitized.
By dividing a complicated optical system into units, even if each part is downsized, it can be easily assembled with a minimum space and high accuracy. In addition, stray light is unlikely to enter the optical path even when the detector case is opened, and the optical path can be easily adjusted during maintenance work. In addition, by downsizing the optical system, it is possible to mount a light source, a detector, a drive system electrical unit such as a motor in the detection unit case 3, and the entire detection unit can be downsized. Further, by downsizing, it is possible to remove only the detection unit from the apparatus and use it as a portable device in combination with a PC, a display, or the like. Even when a commercial power source cannot be secured, the detection unit can perform measurement with power supplied from an external battery or a solar battery.

As the light source unit 10, Xe flash lamp (xenon discharge tube), D ₂ lamp (deuterium discharge tube) can be used.
The optical path unit 20 includes a mounting base 21, a mask 22 fixed to the mounting base 21, a lens 23, a mirror 24, a lens 25, and a mask 26. The optical path unit 20 is configured to guide the light beam from the light source unit 10 to the light source side spectroscopic unit 30 while adjusting the light beam from the light source unit 10 with the masks 22 and 26 and the lenses 23 and 25 while changing the direction with the mirror 24.

The light source side spectroscopic unit 30 includes a mounting base 31, a slit 32 fixed to the mounting base 31, a diffraction grating 33, and a slit 34. That is, the irradiation optical system has a diffraction grating 33.
The diffraction grating 33 separates the light source light incident from the optical path unit 20 through the slit 32. The diffraction grating 33 can be rotated by a motor (not shown). Depending on the rotation position, light having a specific wavelength out of the divided light source light passes through the slit 34 and enters the irradiation light unit 40. It is like that. That is, the wavelength of the irradiation light E irradiated on the overflow surface 7 via the irradiation light unit 40 can be selected depending on the rotational position of the diffraction grating 33.
The light having a specific wavelength that passes through the slit 34 includes at least a plurality of different wavelengths including excitation light (wavelength λe) for obtaining fluorescence by the measurement target component and correction irradiation light for measuring the concentration of the disturbing component. The light can be selected. The correction irradiation light to be selected may be one type (wavelength λx) or two or more types (wavelengths λx ₁ , λx ₂ ...).

The irradiation light unit 40 includes a mounting base 41, a mask 42, a lens 43, a mirror 45, a lens 46, a mask 47, a reference light detector 51, a mask 52, and a lens 53 fixed to the mounting base 41. Yes. The irradiation light E incident from the light source side spectroscopic unit 30 passes through the mirror 45 while being adjusted by the masks 42 and 52 and the lenses 43 and 46, and is irradiated to the overflow surface 7 due to the sample liquid S. It has become.
Further, part of the irradiation light E incident from the light source side spectroscopic unit 30 is reflected by the mirror 45, reaches the reference light detector 51 as the reference light R via the mask 52 and the lens 53, and the reference light R Is detected. The detection result of the light amount of the reference light R is sent to the arithmetic and control unit 90 so that the fluctuation of the light amount of the irradiation light E irradiated on the overflow surface 7 can be compensated.

  Irradiation light E is applied to the overflow surface 7 by the optical path unit 20, the light source side spectroscopic unit 30, and the irradiation light unit 40 from an angle θ with respect to the vertical direction of 45 ± 15 °.

  The angle θ is 45 ± 15 °, preferably 45 ± 10 °, preferably 45 ± 5 °, and particularly preferably 45 °. When the angle θ is larger than the preferable lower limit value, stray light such as scattered light and reflected light of the irradiation light E incident on the light receiving unit 60 can be reduced. Further, since the angle θ is smaller than the preferable upper limit value, the reflectance of the irradiation light E on the overflow surface 7 is prevented from increasing, and sufficient fluorescence is transmitted from the measurement target component in the sample liquid S as response light. F can be generated. Further, by making the angle θ smaller than the preferable upper limit value, it is easy to prevent the spot diameter of the irradiation light E on the overflow surface 7 from becoming too large.

When irradiating the overflow surface 7 with the irradiation light E, if the spot diameter is too large, it is difficult to stably detect the response light F due to the influence of fluctuation of the water surface. By adjusting the angle θ, the focal length of the lens of the irradiation optical system, and the like to sufficiently reduce the spot diameter, it is possible to reduce the influence of the fluctuation of the water surface on the response light F from the overflow surface 7.
On the other hand, increasing the spot diameter to some extent is advantageous in that it is easy to secure the amount of response light F. However, since the light quantity of the response light F can be increased by increasing the light quantity of the light source unit 10, the lower limit value of the spot diameter is not particularly limited.
The spot diameter is preferably 50 mm or less, more preferably 5 to 40 mm, and even more preferably 5 to 10 mm.

  The light receiving unit 60 includes a mounting base 61, a lens 62 fixed to the mounting base 61, and a lens 63. The light receiving unit 60 is disposed at a position where the response light F emitted from the overflow surface 7 by the irradiation light E can be captured vertically above the spot position where the irradiation light E is applied to the overflow surface 7. In other words, the response light F from the overflow surface 7 is arranged to be received vertically upward. The received response light F is adjusted in light flux by the lenses 62 and 63 and guided to the detection-side spectroscopic unit 70.

The detection-side spectroscopic unit 70 includes an attachment base 71, a slit 72 fixed to the attachment base 71, a diffraction grating 73, and a slit 74. That is, the detection optical system has a diffraction grating 73.
The diffraction grating 73 separates the response light F incident from the light receiving unit 60 through the slit 72. The diffraction grating 73 can be rotated by a motor (not shown). Depending on the rotation position, the light having a specific wavelength out of the response light F dispersed is incident on the detector unit 80 through the slit 74. It is supposed to be. That is, the wavelength of the response light F incident on the detector unit 80 can be selected according to the rotational position of the diffraction grating 73.
As the specific light passing through the slit 74, it is possible to select a plurality of lights having different wavelengths including at least the fluorescence (wavelength λf) by the measurement target component and the correction response light for measuring the concentration of the interference component. ing. Correction response light to be selected, kind (wavelength lambda] m) Any two or more (wavelength λm _1, λm _{2 ···,} however, the correction response light of wavelength lambda] m ₁ is obtained by correcting the irradiation light of wavelengths [lambda] x ₁ corrected response light , and the correction response light of wavelength lambda] m ₂ is a correction response light obtained by correcting the irradiation light having a wavelength of [lambda] x _2.) may be used.

The detector unit 80 includes a mounting base 81, a detector 82 fixed to the mounting base 81, a mask 83, a lens 84, and a cut filter 85. As the detector 82, a photomultiplier tube, a photodiode, a phototransistor, an avalanche photodiode, or the like can be used as appropriate.
The sensitivity of the detector 82 is variable. That is, an appropriate sensitivity can be selected under the control of the arithmetic and control unit 90 according to the amount of response light F (fluorescence or correction response light by the measurement target component) detected by the detector 82. For example, when the detector 82 is a photomultiplier tube, the arithmetic and control unit 90 can adjust the sensitivity by adjusting the applied voltage.

In the detector unit 80, after the spectral response light F guided from the detection-side spectroscopic unit 70 is adjusted by the mask 83 and the lens 84, and light having an unnecessary wavelength is removed by the cut filter 85. Are introduced into the detector 82. The cut filter 85 removes scattered light having the same wavelength as the excitation light. The result of detecting the response light F after the spectroscopy by the detector 82 is sent to the arithmetic and control unit 90, and the concentration and the like of the measurement target component are obtained.
The concentration of the measurement target component, the concentration of the interfering component, and the like are appropriately displayed on a display.

In the multi-wavelength fluorescence analyzer 1 of the present embodiment, the angle θ for irradiating the overflow surface 7 with the excitation light is 30 ° or more, and therefore the optical path of the excitation light and the optical path of the fluorescence can be separated. Therefore, unlike the epi-illumination method, a dichroic mirror and an interference filter are not required. Therefore, the wavelength of the light introduced into the detector 82 out of the wavelength of the irradiation light E irradiated on the overflow surface 7 and the response light F by spectroscopically arranging a diffraction grating in each optical path is used as a measurement step. It is possible to select them individually.
In addition, since the cell window is an overflow method that does not contact the sample solution, it is resistant to dirt and easy to maintain.

The multi-wavelength fluorescence analyzer 1 of the present embodiment performs the following steps 1 to 3 under the control of the arithmetic and control unit 90.
Step 1: Excitation light (wavelength λe) for obtaining fluorescence by the component to be measured among light source light is guided to the liquid surface of the sample liquid by the irradiation optical system, and emitted from the sample liquid by the detection optical system. Of the response light, the fluorescence (wavelength λf) due to the component to be measured is guided to the detector to obtain the fluorescence intensity If.
Step 2: Use the irradiation optical system to guide the correction irradiation light out of the light source light to the liquid surface of the sample liquid, and use the detection optical system to correct the correction response light (wavelength λx, in the case of a plurality of wavelengths). λx ₁ , λx ₂ ...) is guided to the detector, and the intensity Im of the corrected response light (intensity Im ₁ , intensity Im ₂ ... in a plurality of cases) is obtained.
However, the wavelength λm ₁ is the wavelength of the correction response light obtained by the correction irradiation light having the wavelength λx ₁ , and the intensity Im ₁ is the intensity of the correction response light at that time. Similarly, the wavelength λm ₂ is the wavelength of the corrected response light obtained by the corrected irradiation light having the wavelength λx ₂ , and the intensity Im ₂ is the intensity of the corrected response light at that time.
Step 3: Calculate the concentration of the measurement target component in the sample solution based on the fluorescence intensity If. In this calculation, correction according to the concentration of the disturbing component obtained from the intensity Im of the corrected response light (intensity Im ₁ , intensity Im ₂ ... In a plurality of cases) is performed.

Note that the multi-wavelength fluorescence analyzer 1 of the present embodiment detects the light amount of the reference light R. The fluorescence intensity If in step 1 and the correction response light intensity Im in step 2 (intensities Im ₁ , intensity Im ₂ ... In a plurality of cases) both vary in the amount of irradiation light E emitted from the light source unit 10. This is a value after correcting the influence by the amount of the reference light R.

The wavelength λe of the excitation light and the wavelength λf of the fluorescence in Step 1 can be appropriately selected according to the measurement target component.
For example, when the concentration of linear alkylbenzene sulfonates is determined, the wavelength λe of excitation light can be 230 nm, and the wavelength λf of fluorescence can be 290 nm. Further, the wavelength λe of excitation light when measuring BOD can be 320 nm, and the wavelength λf of fluorescence can be 440 nm. The wavelength λe of the excitation light when measuring the oil-in-water concentration is any one of 230 nm, 265 nm, 280 nm, and 365 nm, and the wavelength λf of the fluorescence is any of the wavelength λe of the excitation light being 230 nm, 265 nm, or 280 nm. In the case of 290 to 360 nm, the wavelength of excitation light can be 440 to 460 nm when the wavelength λe is 365 nm. In addition, when measuring the phenol concentration, the wavelength λe of the excitation light can be 250 nm, and the wavelength λf of the fluorescence can be 300 nm. In addition, the wavelength λe of excitation light when measuring the chlorophyll concentration can be 250 to 320 nm or 430 to 470 nm, and the wavelength λf of fluorescence can be 360 to 370 nm or 650 to 700 nm.

The corrected irradiation light in Step 2 includes turbidity correction irradiation light for measuring turbidity as a disturbing component, and the corrected response light includes turbidity correction response light for measuring turbidity as a disturbing component. preferable. The wavelengths of the turbidity correction irradiation light and the turbidity correction response light are preferably selected in the vicinity of 660 nm. Turbidity can be measured based on the intensity Im of the obtained corrected response light, and the concentration of the measurement target component can be corrected according to the measured turbidity.
In this embodiment, the sensitivity of the detector 82 is variable even if there is a large difference between the fluorescence intensity If in step 1 and the intensity Im of the corrected response light in step 2 (intensities I1, intensity Im ₂ ... In a plurality of cases). Therefore, each intensity can be accurately measured.

In step 3, the concentration C of the measurement target component in the sample solution S is calculated based on the fluorescence intensity If. In this calculation, correction is performed according to the concentration of the disturbing component obtained from the intensity Im of the corrected response light (intensity Im ₁ , intensity Im ₂ ... In a plurality of cases).
The following equation (1) is an example of an equation for obtaining the concentration C of the measurement target component while performing correction using the intensity Im of the corrected response light.
C = a * If-k * g (Im) + b1 (1)
In Equation (1), C is the concentration of the component to be measured, g (Im) is the concentration of the disturbing component obtained from the intensity Im of the corrected response light, and a, b1, and k are coefficients.
k × g (Im) corresponds to a measurement error corresponding to the concentration of the disturbing component.

  The coefficient a can be obtained, for example, from the relationship between the fluorescence intensity and the concentration of the measurement target component when a calibration solution containing the measurement target component having a known concentration is used. That is, it can be obtained from the fluorescence intensity If when the excitation light is irradiated to the liquid surface of the calibration liquid as the irradiation light E, and the measurement target component concentration of the calibration liquid. Specifically, it can be obtained by dividing the measurement target component concentration of the calibration solution by the fluorescence intensity If. Moreover, it can also obtain | require as the inclination a of a linear function (a * If + b) using the some calibration liquid containing the measurement object component of a different density | concentration.

  As the calibration solution, a standard solution containing no components other than the measurement target component can be used. Alternatively, an actual measurement system sample liquid in which the concentration of the measurement target component is obtained in advance by manual analysis or the like, or a sample liquid obtained by adding the measurement target component to the actual measurement system sample liquid may be used.

The function g (corrected calibration curve) for determining g (Im) is obtained in advance by using a calibration solution for the relational expression of the interference component concentration with respect to the intensity Im of the corrected response light. As the calibration solution, a standard solution that does not contain components other than the interfering components can be used. Further, it is possible to use an actual measurement system sample solution in which the concentration of the interfering component has been obtained in advance by manual analysis or the like, or a sample solution obtained by adding the interfering component to the actual measurement system sample solution.
In the case of turbidity correction, the function g (turbidity calibration curve) for obtaining g (Im) is obtained by uniformly expressing the relational expression of turbidity with respect to the intensity Im of the corrected response light in advance with the standard substances kaolin and formazine It is determined using a dispersed suspension turbidity standard solution.

The coefficient k corresponds to an error per unit concentration of the disturbing component. For example, it is obtained by the following equation (2) based on the measured value of the sample liquid of the actual measurement system including the interference component.
k = (Cx−Cs) / g (Im) (2)
However, Cx is a measurement target component concentration obtained only from If, Cs is a true concentration of the measurement target component, and g (Im) is an interference component concentration.
Cx may be obtained, for example, by multiplying the fluorescence intensity If by a coefficient a, or may be obtained by the formula a × If + b.

The coefficient b1 is obtained, for example, so that the concentration C of the measurement target component becomes zero when a sample liquid of an actual measurement system that does not include the measurement target component is measured. That is, the fluorescence intensity If and the intensity Im of the corrected response light are measured for the sample liquid of the actual measurement system that does not contain the measurement target component, and are obtained by the following equation (3).
b1 = −a × If + k × g (Im) (3)
By providing the term of the coefficient b1, it is possible to correct the zero point fluctuation due to various interference components in the actual measurement system.

  As described above, if the coefficients a, b1, k, and the function g are obtained in advance, the corrected measurement target component concentration is obtained by performing steps 1 to 3 for the sample liquid whose measurement target component concentration is unknown. Can do.

When correction is performed in consideration of the first disturbance component and the second disturbance component, the correction response light intensity Im ₁ corresponding to the first disturbance component and the correction response light intensity Im corresponding to the second disturbance component ₂ , for example, the corrected concentration C of the measurement target component can be obtained by the following equation (4).
_{C = a × If-k 1} × g 1 (Im 1) -k 2 × g 2 (Im 2) + b2 ··· (4)
However, a and If in equation (4) is the same as equation _(1), g 1 (Im ₁₎ the concentration of the first interference component obtained from the intensity Im ₁ correction response _light, g 2 (Im ₂ ) Is the concentration of the second disturbing component obtained from the intensity Im ₂ of the corrected response light, and b2, k ₁ and k ₂ are coefficients.

For example, b2 in the equation (4) is obtained such that the concentration C of the measurement target component becomes zero when the sample liquid of the actual measurement system that does not include the measurement target component is measured. That is, the fluorescence intensity If, the corrected response light intensity Im ₁ , and the corrected response light intensity Im ₂ are measured for the sample liquid of the actual measurement system that does not include the measurement target component, and are obtained by the following equation (5).
b2 = −a × If + k ₁ × g ₁ (Im ₁ ) + k ₂ × g ₂ (Im ₂ ) (5)
By providing the term of the coefficient b2, it is possible to correct the zero point fluctuation due to various interference components in the actual measurement system.

Furthermore, when correction is performed in consideration of the effects of the third and fourth interference components, a correction term is further added to Equation (5) to subtract a value obtained by multiplying the concentration of the third and fourth interference components by a coefficient. do it.
When performing correction in consideration of a plurality of disturbing components, one of the disturbing components is preferably turbidity.
Further, C obtained by the equation (1) may not be a component to be measured, but may be a concentration of a reagent or the like that reacts with the component to be measured. In this case, the concentration of the component to be measured is obtained indirectly from the value of C. It is done.

In the present invention, the influence of interference components such as turbidity can be corrected by the calculation in Step 3 by using a cut filter for removing scattered light having the same wavelength as the diffraction grating and the excitation light in the detection optical system, and detecting optical as stray light. This is because the influence of scattered light entering the system is eliminated. In the case of detecting fluorescence, if the scattered light of excitation light is strong, the influence (plus error) that enters the detection optical system as stray light and detects an intensity higher than the actual fluorescence intensity is very large.
In the present invention, the separation performance of scattered light is greatly improved by combining a diffraction filter and a cut filter that removes scattered light having the same wavelength as the excitation light. Therefore, it is possible to correct the influence other than the scattered light.

  As an influence other than the scattered light, first, a minus error due to turbidity can be mentioned. The minus error due to turbidity is caused by the fact that the turbidity interferes and excitation light does not reach the component to be measured and is not sufficiently excited, and fluorescence cannot be detected by the detection optical system. In addition, disturbing components other than turbidity also give negative and positive errors. Since these errors are overwhelmingly smaller than the plus error due to scattered light, the degree of the error can be detected and corrected for the first time only by sufficiently separating only the scattered light.

  In the present invention, a diffraction grating is also used for the irradiation optical system. Thereby, it is possible to irradiate a sample liquid with excitation light and 1 or more types of correction | amendment irradiation light, without using a several light source. Moreover, it is possible to irradiate the sample liquid with excitation light having a wavelength suitable for each of the plurality of components to be measured without using a plurality of light sources.

In the above embodiment, the liquid surface of the sample liquid is the overflow surface, but the liquid surface of the sample liquid may be the liquid surface of the liquid that flows while maintaining a constant water level. Further, it may be a stationary liquid surface of the sample liquid stored in the container.
In addition, a plurality of types of measurement target components may be used. In that case, the wavelength of the excitation light guided to the liquid surface of the sample liquid by the irradiation optical system may be selected for each measurement target component. Further, the wavelength of the fluorescence guided to the detector by the detection optical system may be selected for each measurement target component.
In addition, in the said embodiment, although it was set as the structure provided with a display device, a display device is not essential.

[Experimental Example 1]
The oil-in-water concentration was measured using the apparatus shown in FIG. However, the sample solution was not overflowed but was put in a sample solution container and measured in a stationary state. The wavelength λe of the excitation light in Step 1 was 230 nm. The wavelength λf of fluorescence was 350 nm. The wavelength of the turbidity correction light (turbidity correction irradiation light and turbidity correction response light) in Step 2 was 660 nm.
As the formula in step 3, the formula (1) was used. The coefficient and the like in equation (1) were obtained as follows.

The coefficient “a” was determined using a calibrating solution in which 1 mg of water-soluble oil as an oil-in-water component was dispersed in pure water to make 1 L, and pure water. The fluorescence intensity If of the wavelength λf when the liquid surface of the calibration liquid was irradiated with the excitation light of the wavelength λe was 480. The fluorescence intensity If of the long λf when the liquid surface of pure water was irradiated with the excitation light having the wavelength λe was 0 (zero).
Based on the above results, when the relationship between the water-soluble oil concentration and the intensity of response light was determined, a calibration curve of the following equation (6) was obtained, and a = 0.001.
Water-soluble oil concentration (mg / L) = 0.0021 × If (6)

Next, the function g in the formula (2) was obtained. The function g was obtained using a calibration solution having a turbidity of 100 degrees and 1 L obtained by dispersing 100 mg of kaolin in pure water and pure water. The intensity Im of the corrected response light having a wavelength of 660 nm when the correction irradiation light having a wavelength of 660 nm was irradiated on the liquid surface of the calibration liquid was 55.7. The intensity Im of the correction response light with a wavelength of 660 nm when the correction irradiation light with a wavelength of 660 nm was irradiated on the liquid surface of pure water was 0.06.
Based on the above results, the calibration curve (function g) of the following equation (7) was obtained when the relationship between turbidity and the intensity Im of turbidity correction response light was determined.
g (turbidity) = 1.773 × Im−0.1078 (7)

Next, river water that does not contain oil was sampled and used as sample solution A, and steps 1 and 2 were performed. As a result, the fluorescence intensity If was 70.7, and the corrected response light intensity Im was 7.1.
Separately from the sample solution A, a sample solution B was prepared by adding water-soluble oil and kaolin to a sample of river water containing no oil. The water-soluble oil was added so that the oil concentration of the sample solution B was 1 mg / L.
As a result of performing Step 1 and Step 2 for Sample Solution B, the fluorescence intensity If was 159.8 and the turbidity correction response light intensity Im was 64.

When the oil concentration of the sample solution A was determined by the above equation (6), it was 0.1485 mg / L (0.0021 × 70.7). Therefore, the above equation (6) was corrected so that the oil concentration of the sample liquid A was zero, and the following equation (8), which was a temporary oil concentration calibration curve not considering turbidity, was created.
Oil concentration (mg / L) = 0.0021 × If−0.1485 (8)

The oil concentration of the sample solution B (Cx in the above formula (2), that is, the concentration of the component to be measured obtained only from If) was obtained by the above formula (8), and 0.1871 mg / L (0.0021 × 159. 8-0.1485), and there was an error of minus 0.8129 mg / L with respect to 1 mg which is the added concentration of oil (Cs in the formula (2), that is, the true concentration of the component to be measured). Further, when the turbidity (g (Im) of the above formula (2), that is, the concentration of interference components) of the sample liquid B was determined by the above formula (7), 114.9 (1.7973 × 64−0.1078). )Met.
The coefficient k representing the error per turbidity (interfering component) per degree (per unit concentration) was obtained from the equation (2), and k = −0.8129 ÷ 114.98 = −0.00707.

Next, when the turbidity of the sample liquid A was calculated | required by said Formula (7), it was 12.65 (1.7973 * 7.1-0.1078).
Then, the coefficient b ₁ of the sample liquid A was obtained by the equation (3), and was b1 = −0.0021 × 70.7 + 0.00707 × 12.65 = −0.24.
Summarizing the above results, Equation (1) becomes Equation (9) below.
C = 0.0021 × If + 0.00707 × g (Im) −0.24 (9)

In order to evaluate the validity of the formula (9), a sample solution C was prepared by adding water-soluble oil to a sample of river water that does not contain oil separately from the sample solution A and sample B. In Sample Solution C, water-soluble oil was added so that the oil concentration was 0.1 mg / L.
As a result of performing Step 1 and Step 2 for Sample Solution C, the fluorescence intensity If was 98.7 and the turbidity correction response light intensity Im was 10.2.

  The turbidity of the sample liquid C was 18.22 (1.7973 × 10.2-0.1078) as determined by the above formula (7). Further, when the oil concentration of the sample solution C is obtained by the above formula (9), it is 0.096 mg / L (0.0021 × 98.7 + 0.00707 × 18.22−0.24), which is in good agreement with the added concentration. It was confirmed that

[Experiment 2]
The rhodamine B concentration was measured using the apparatus of FIG. However, the sample solution was not overflowed but was put in a sample solution container and measured in a stationary state. The wavelength λe of the excitation light in Step 1 was 335 nm. The fluorescence wavelength λf was 575 nm. The wavelength of the turbidity correction light (turbidity correction irradiation light and turbidity correction response light) in Step 2 was 660 nm.
As the formula in step 3, the formula (1) was used. The coefficient and the like in equation (1) were obtained as follows.

The coefficient a was determined by performing Step 1 on the sample solution D and pure water in which 2 mg of rhodamine B was dissolved in pure water to make 1 L. The fluorescence intensity If of the wavelength λf when the liquid surface of the sample liquid D was irradiated with the excitation light of the wavelength λe was 75.7. The fluorescence intensity If of wavelength λf when the liquid surface of pure water was irradiated with excitation light of wavelength λe was 0.01.
Based on the above results, the relationship between the rhodamine B concentration and the fluorescence intensity If was obtained, and a calibration curve of the following formula (10) was obtained, so a = 0.0264.
Rhodamine B concentration (mg / L) = 0.0264 × If−0.0003 (10)

Next, Steps 1 and 2 were performed on sample solution E in which 2 mg of rhodamine B and 100 mg of kaolin were dispersed in pure water to make 1 L. As a result, the fluorescence intensity If was 58.62, and the turbidity correction response light intensity Im was 62.62. It was 15.
Based on this fluorescence intensity If, the rhodamine B concentration determined by the formula (10) was 1.55 mg / L, and there was an error of minus 0.45 mg / L with respect to 2 mg which is the true value.
Moreover, the turbidity calculated | required by said Formula (7) based on intensity | strength Im of turbidity correction response light was 111.7 degree | times. Since the coefficient k in the equation (2) is an error per turbidity, the error of rhodamine B concentration in the calibration solution is divided by the turbidity of the calibration solution (−0.45 ÷ 111.7), and k = −. It was 0.004029.
From the above results, it was found that the concentration of rhodamine B corrected for turbidity can be obtained by the following equation (11).
C = 0.0264 × If + 0.004029 × g (Im) −0.0003 (11)

  The multi-wavelength fluorescence analyzer of the present invention can be used as a water quality analyzer for determining linear alkylbenzene sulfonate concentrations, BOD (biochemical oxygen demand), oil-in-water concentration, phenol concentration, and chlorophyll concentration in a sample solution. In addition, a substance having a property of emitting fluorescence, a reaction product with the substance, a fluorescent reagent or a probe (including intensity and quenching time) is measured, and the presence / absence and / or concentration of the measurement target component or the fluorescence It can be widely applied to any fluorescence analysis that measures the characteristics (fluorescence spectrum, excitation spectrum, etc.) itself.

DESCRIPTION OF SYMBOLS 1 ... Multiwavelength fluorescence analyzer, 2 ... Partition plate, 3 ... Detection part case, 4 ... Sample case,
6 ... overflow cylinder, 7 ... overflow surface,
DESCRIPTION OF SYMBOLS 10 ... Light source unit, 20 ... Optical path unit, 30 ... Light source side spectroscopic unit,
33 ... Diffraction grating, 40 ... Irradiating light unit, 60 ... Light receiving unit,
70 ... detection side spectroscopic unit, 73 ... diffraction grating, 80 ... detector unit,
85 ... Cut filter, 90 ... Calculation control device,
S: Sample liquid, E: Irradiation light, F: Response light, R: Reference light

Claims (5)

A multi-wavelength fluorescence analyzer that obtains the concentration of a component to be measured in the sample liquid based on the fluorescence intensity If obtained when the sample liquid is irradiated with excitation light,
A light source;
An irradiation optical system for guiding the light source light emitted from the light source to the liquid surface of the sample liquid at an angle of 45 ± 15 ° with respect to the vertical direction;
A detector;
A detection optical system that receives response light emitted from the sample liquid in a vertical direction above the liquid surface of the sample liquid, and guides it to the detector;
A control apparatus for controlling the entire apparatus and performing a predetermined calculation by inputting the intensity of the response light obtained by the detector,
The irradiation optical system includes a diffraction grating, and includes at least one type of correction irradiation light for measuring excitation light for obtaining fluorescence by at least the measurement target component of the light source light and concentration of interference components. Including a plurality of light beams having different wavelengths, which are selectively guided to the liquid surface of the sample liquid,
The detection optical system has a diffraction grating and a cut filter that removes scattered light having the same wavelength as the excitation light, and measures at least the concentration of fluorescence and interference components due to the measurement target component in the response light. A plurality of lights having different wavelengths including one or more kinds of correction response lights are selectively guided to the detector;
A multi-wavelength fluorescence analyzer characterized by performing the following steps 1 to 3.
Step 1: Excitation light for obtaining fluorescence due to the measurement target component in the light source light is guided to the liquid surface of the sample solution by the irradiation optical system, and the response light includes the response light in the response light. Fluorescence due to the measurement target component is guided to the detector to obtain a fluorescence intensity If.
Step 2: Use the irradiation optical system to guide the correction irradiation light out of the light source light to the liquid surface of the sample liquid, and use the detection optical system to pass the correction response light out of the response light to the detector. Then, the intensity Im of the corrected response light is obtained.
Step 3: Calculate the concentration of the measurement target component in the sample solution based on the fluorescence intensity If. In this calculation, correction according to the concentration of the disturbing component obtained from the intensity Im of the correction response light is performed. The one or more types of correction irradiation light includes turbidity correction irradiation light for measuring turbidity as a disturbing component,
The multi-wavelength fluorescence analyzer according to claim 1, wherein the one or more types of corrected response light include turbidity corrected response light for measuring turbidity as a disturbing component.   The multi-wavelength fluorescence analyzer according to claim 1 or 2, wherein the sensitivity of the detector is variable, and the arithmetic and control unit can control the sensitivity of the detector according to the intensity of the response light. The multiwavelength fluorescence analyzer according to any one of claims 1 to 3, wherein the calculation in the step 3 is performed based on the following formula (1).
C = a * If-k * g (Im) + b1 (1)
In Equation (1), C is the concentration of the component to be measured, g (Im) is the concentration of the disturbing component obtained from the intensity Im of the corrected response light, and a, b1, and k are coefficients.   Furthermore, an upper end is formed as an opening, and an overflow cylinder is provided to overflow the sample liquid flowing from below so that an overflow surface of the sample liquid is formed in the opening, and the liquid surface of the sample liquid overflows the sample liquid The multiwavelength fluorescence analyzer according to any one of claims 1 to 4, wherein the multiwavelength fluorescence analyzer is a surface.

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