JP6832782B2 - Spectral analysis method, spectroscopic analyzer - Google Patents

Description

The present invention relates to a technique for analyzing a sample using light.

Spectroscopy quantifies the concentration of chemicals present in a gas / liquid / solid by measuring the spectrum of light as the substance absorbs or emits light, the turbidity of a particle-dispersed solution. It is widely used in applications such as measuring. Among the spectroscopic analysis methods, the absorption spectroscopic analysis method is most commonly used because of its high quantitativeness.

In absorption spectroscopy, the concentration of a specific molecule in a substance is quantified by measuring the spectrum of the light absorbed by the substance in the irradiated light. However, for example, when the turbidity of a substance is high, it is difficult to use absorption spectroscopy because the amount of transmitted light is small. In such cases, diffuse reflection spectroscopy may be used in place of absorption spectroscopy. In the diffuse reflection spectroscopy, a substance is irradiated with light, and the irradiated light is repeatedly reflected and refracted inside the substance to detect the reflected light emitted from the inside again. Diffuse spectroscopic analysis is used for quantitative analysis of, for example, chemical substances in soil, chemical substances in tablets, chemical substances in cell suspensions, and pigment components in milk.

In absorption spectroscopic analysis, it is common to put the substance to be measured in a spectroscopic analysis cell and analyze it. If a spectroscopic analysis cell cannot be prepared (for example, when measuring chemical substances in a chemical plant on the spot), a light source and a light receiving part are built in a probe that can be inserted into the plant, and the optical system is used. And carry out the measurement.

When absorption spectroscopic analysis is performed using such an optical system, the optical path length is defined by the length of the spectroscopic analysis cell and the distance between the light source and the light receiving portion in the probe. Therefore, the concentration of substances contained in the analysis target can be quantified by the Bale-Lambert law.

In diffuse reflection spectroscopic analysis, the light radiated to the analysis target reaches the detector outside the substance while repeating reflection and refraction inside the substance. Light is absorbed inside the substance before it reaches the detector. Information on the inside of the substance is obtained from the amount of absorbed light. The optical path length until light is repeatedly reflected and refracted inside a substance is affected by various parameters of the substance. For example, if the light absorption by a substance is large, the number of times of repeating reflection and refraction before being absorbed by the substance is reduced. On the other hand, when the light absorption by a substance is small, reflection / refraction occurs a relatively large number of times before the absorption occurs. That is, there is a tendency that the optical path length becomes shorter when the absorption of the substance is large, and the optical path length becomes longer when the absorption of the substance is small. As a result, the magnitude of absorption for diffusely reflected light does not correspond to the magnitude of absorption actually inherent in the substance. Therefore, the linear relationship between the absorbance and the substance concentration is broken, and it becomes difficult to quantify the concentration based on the Bale-Lambert law.

In spectroscopic analysis, mathematical techniques such as multivariate analysis may be used to quantify concentrations in systems containing many types of substances. Partial least squares regression (PLS) is widely known as a technique for multivariate analysis, and is used for concentration quantification of various systems. In multivariate analysis techniques such as PLS, a linear relationship is established between the explanatory variable (in the case of spectroscopic analysis, the absorbance obtained from the obtained spectral spectrum) and the objective variable (for example, the concentration of a specific substance). May be a prerequisite. Therefore, in diffuse reflection spectroscopic analysis, it may be difficult to quantify the concentration using a multivariate analysis method such as PLS.

The following Non-Patent Document 1 discloses a method for solving such a problem of diffuse reflection spectroscopic analysis. In the same document, among the diffuse reflected light components, the intensity of the component in which polarized light is conserved and the intensity of the total reflected light are obtained based on the measurement results, and based on these, the representative layer theory (RLT; Representative Layer Theory) ) Is used to analyze the concentration of a substance.

Analytical Chmica Acta, 853, pp.486-494 (2015)

As a result of diligent studies by the present inventors, the method described in Non-Patent Document 1 may reduce the linearity between the concentration and the absorbance, especially in a measurement region where the concentration of the measurement target is high. Found. It is considered that this is because the substance has anisotropy with respect to the reflected light, and the measurement result is affected by the anisotropy.

The present invention has been made to solve the above problems, and an object of the present invention is to improve the linearity of the correspondence between concentration and absorbance in a spectroscopic analysis method for analyzing a sample by diffuse reflection spectroscopy. To do.

In the spectroscopic analysis method according to the present invention, a parameter that is not affected by the anisotropy of the reflected light of the sample is calculated from the intensity of the light reflected from the surface of the sample, and the average absorption rate of the sample is used using the parameter. Ask for.

According to the present invention, in diffuse reflection spectroscopy, it is possible to obtain measurement results with good linearity in a wide dynamic range from low concentration to high concentration. As a result, the accuracy of quantifying the concentration of the substance can be improved.

It is a figure which shows the structure of the spectroscopic analyzer 100 which concerns on Embodiment 1. FIG. It is a graph which shows the result of having measured the _{absorbance A surf} with the dye rhodamine B dissolved in milk as the substance to be measured 15. It is a schematic diagram which shows the state of single scattering. It is a graph which shows the result of having measured the reflectance of the surface reflected light for each when the concentration of the dye rhodamine B is 100 μg / mL and 0 μg / mL. It is a graph which shows the absorbance A _surf. It is a flowchart which summarized the procedure for determining _{the absorbance Asurf} of a representative layer in Embodiment 1. It is a figure which shows the structural example at the time of estimating the scattering parameter g using a spectroscopic analyzer 100. It is a graph which shows the result of having measured the reflectance of a standard sample and the reflectance of the substance to be measured 15 at a plurality of light wavelengths, respectively.

<Embodiment 1: Device Configuration>
FIG. 1 is a diagram showing a configuration of a spectroscopic analyzer 100 according to a first embodiment of the present invention. The light emitted from the light source 11 passes through the collimating lens 13 via the optical fiber 12. In the light collimated by the collimating lens 13, only a part of the polarizing components is extracted by the polarizing element 14, and the light is irradiated to the substance to be measured 15. When performing spectroscopic analysis, it is conceivable to use a white light source, for example, a halogen lamp as the light source 11.

The light irradiated to the measurement target substance 15 is diffused by repeating reflection and refraction inside the measurement target substance 15. Some light is radiated to the outside of the measurement target substance 15 without being absorbed by the measurement target substance 15. The light radiated to the outside enters the optical fiber 18 through the polarizing element 16 and the collimating lens 17. The optical fiber 18 is connected to the light receiving unit 19. The light receiving unit 19 measures the light intensity at each wavelength by using, for example, a spectroscope. The calculation unit 20 calculates a parameter such as the concentration of the substance to be measured 15 by using the light intensity measured by the light receiving unit 19.

An angle Φ21 is provided between the emitted light and the reflected light. The angle Φ21 is preferably set to, for example, 45 ° so that the light reflected from the substance to be measured 15 does not include the specularly reflected light.

<Embodiment 1: Measurement using representative layer theory>
In the following, in order to clarify the object of the present invention, the measurement procedure and its problems in the conventional diffuse reflection spectroscopic analysis will be described first. After that, the spectroscopic analysis method according to the present invention will be described.

First, the intensity of the reflected light is measured in a state where the polarization direction of the polarizer 14 and the polarization direction of the polarizer 16 are aligned (parallel). The calculation unit 20 stores the _{light intensity I ||} measured by the light receiving unit 19.

Next, the polarizer 14 (or the polarizer 16) is rotated. The angle of rotation can be set arbitrarily, but for example, it is conceivable to rotate the polarization direction by 90 ° in order to irradiate light having a polarization state orthogonal to the emitted light at the time of the previous measurement. After rotating the polarizer 14, the intensity of the reflected light is measured in the same manner. The calculation unit 20 stores the _{light intensity I ⊥} measured by the light receiving unit 19.

Next, the intensity of the incident light incident from the light source 11 is measured. The intensity of the incident light is also measured before and after rotating the polarizer 14. The calculation unit 20 stores the incident light intensities I _{0 ||} (before rotation) and I _{0 ⊥} (after rotation) acquired by the light receiving unit 19.

Since the light scattered many times inside the substance 15 to be measured no longer holds the polarization direction of the incident light, I _|| = I _⊥ . On the other hand, the light reflected from the surface maintains the polarization direction of the incident light. Similarly, it is considered that the polarization direction of the incident light is maintained for the light diffusely reflected near the surface. Hereinafter, the light reflected from the surface and the light diffusely reflected from the vicinity of the surface are collectively referred to as surface reflected light. For surface reflected light, I _⊥ = 0. Using this, the calculation unit 20 calculates the intensity ΔI of the surface reflected light of the substance to be measured 15 according to the following equation 1.

ΔI = I _|| −I _⊥ (1)

_{The calculation unit 20 obtains the intensity Itot} of the total reflected light including the light diffusely reflected from the inside of the measurement target substance 15 and the light reflected from the surface according to the following equation 2.

I _tot = I _|| + I _⊥ (2)

Reflectance is the ratio of the intensity of incident light to the intensity of reflected light. The calculation unit 20 calculates the reflectance r of the surface reflected light and the reflectance R _tot of the total reflected light according to the following equations 3 and 4, respectively.

r = ΔI / I ₀ (3)
R _tot = I _tot / I ₀ (4)

In the representative layer theory (RLT; Representative Layer Theory), it is shown that the following equation 5 has a relationship under the condition that the transmitted light intensity of the light emitted from the light source 11 to the substance to be measured 15 can be ignored. There is. a _REP is the light absorption rate in the representative layer, and r _REP is the reflectance of surface reflected light in the representative layer theory.

(1-R _tot ² ) / R _tot = (a _REP / r _REP ) (2-r _REP -2a _REP ) (5)

In the conventional method, the light absorption rate a _REP in the representative layer theory is calculated by _{assuming that r REP and r are approximately equal in Equation 5. The light absorption rate a REP} in the representative layer theory is the light absorption rate in the representative layer having a thickness that is not affected by multiple scattering. Therefore, the absorbance A _surf of the representative layer can be calculated from the following formula 6. Absorbance A _surf is not affected by multiple scattering and therefore follows the Veil-Lambert rule. Therefore, the concentration of the substance can be estimated from the absorbance A _surf.

A _surf = -log (1-a _REP ) (6)

FIG. 2 is a graph showing the results of measuring the _{absorbance A surf} using the dye rhodamine B dissolved in milk as the substance to be measured 15. Here it was measured _{A surf} by changing the concentration of the dye rhodamine B from 1.6 [mu] g / mL to 100 [mu] g / mL is. For comparison, it is also shown the absorbance A _ref measured from the total reflectance. To make it easier to compare, _{the vertical axis of Surf} is multiplied by 20. The black square in FIG. 2 _{indicates A ref} , and the white square indicates A _surf .

A _surf and A _ref have different detection depths. A _surf is the calculation of the absorbance of the representative layer, while A _ref is the calculation of the absorbance at the total depth reached by the light. A _ref is the absorbance without correction for the difference due to the optical path length.

According to FIG. 2, A _ref is seen that significant tilt is decreased in accordance with the concentration of the dye rhodamine B becomes a high concentration. On the other hand, _{it can be seen that A surf} improves the linearity of the correspondence between the concentration and the absorbance even in the high concentration region. However, there is still a tendency for the slope to decrease in the high concentration region. The present inventors considered this factor as follows.

_{The reflectance r REP} used in the representative layer theory is an average value of the reflectance of the surface reflected light of the substance to be measured 15. However, an actual optical system for measuring reflected light usually measures reflected light only at a specific light receiving angle. The same applies to the optical system shown in FIG. Since it is considered that the light diffusely reflected many times inside the measurement target substance 15 loses most of the anisotropy, even if the reflected light measured at a specific light receiving angle is measured, the measurement target substance 15 It is considered that it is not so affected by the anisotropy of the reflected light. That is, it is considered that the influence of the light receiving angle on _{R tot is small.} On the other hand, in the representative layer theory, the density is estimated using the surface reflected light. In the above method, it is assumed that the reflectance r of the surface reflected light having sufficient anisotropy is _{equal to r REP.} However, in reality, r _REP in the representative layer theory is the average reflectance of surface reflected light at each light receiving angle. Therefore, since the concentration is estimated without considering the influence of the anisotropy of r, it is presumed that the phenomenon of reduced linearity as shown in FIG. 2 occurs.

Therefore, we considered to calculate _{r REP} by the following method. First, the relationship between r and r _{REP was considered.}

FIG. 3 is a schematic view showing the state of single scattering. It is assumed that the scattering of the substance to be measured 15 from the surface is one-time scattering. In this case, the reflectance of the component in which polarized light is conserved is considered to be surface reflected light in which single scattering contributes as a main component. The incident light intensity I ₀ and the surface reflected light intensity ΔI are expressed by the following equation 7. s is the probability that the incident light is scattered once, p is the phase function that expresses the anisotropy of scattering (dependence on the light receiving angle), σ _a is the light absorption coefficient of the medium 27, and d is the thickness of the representative layer. Is. The light receiving angle is 45 °.

ΔI = I ₀ × s × p (θ = 45 °) × exp (−σ _a (d + d / cos (45 °))) (7)

Dividing this by I ₀ gives r _{45 °} .

r _{45 °} = s × p (θ = 45 °) × exp (−σ _a (d + d / cos (45 °))) (8)

r _REP is the average value of the reflectance at each light receiving angle from each direction, and is expressed by the following formula.

r _REP = ∫s × p (θ) × sinθ × exp (−σ _a (d + d / cosθ)) dθ) / ∫sinθdθ (9)

Further, a _REP is an absorption rate in a representative layer having a thickness d, and is represented by the following formula.

_{a REP = exp (-σ a d} ) (10)

As a phase function representing the anisotropy of one-time scattering, for example, the Henyey Greenstein function represented by the following equation is known. g is a scattering parameter representing the anisotropy of scattering, and has a value from -1 to 1 depending on the anisotropy of scattering. For example, if g = 0, the phase function corresponds to isotropic scattering that does not depend on the scattering direction. When the value is positive, the forward scattering is dominant, and when the value is negative, the backscatter is dominant.

p (g, θ) = ( 1-g 2) / (4π (1 + g 2 -2gcosθ) 3/2) (11)

In the following, we propose a method to mitigate the influence of the anisotropy of reflected light when estimating the concentration of the substance to be measured 15 using the representative layer theory.

FIG. 4 is a graph showing the results of measuring the reflectance of surface reflected light when the concentrations of the dye rhodamine B are 100 μg / mL and 0 μg / mL, respectively. Reference numeral 28 indicates a measurement result of 100 μg / mL, and reference numeral 29 indicates a measurement result of 0 μg / mL. When the dye rhodamine B is dissolved at 100 μg / mL, light is absorbed by this, and it can be seen that the reflectance is significantly reduced as compared with the case where the dye rhodamine B is not dissolved. As described above, when the concentration of the substance that absorbs light is high, the effect of absorption appears on the reflectance of the surface reflected light.

_{In the single scattering as illustrated in FIG. 3, at the reflectance r 45 °} (100 μg / mL) at the light receiving angle θ when the concentration of the dye rhodamine B is 100 μg / mL, and at the light receiving angle θ when the dye Rhodamine B concentration is 0 μg / mL. The ratio of reflectance r _{45 °} (0 μg / mL) can be expressed by the following equation 12. σa is the absorption coefficient and d is the thickness of the representative layer. When the concentration of the dye rhodamine B is 0 μg / mL, σa = 0. When the concentration of the dye rhodamine B is 100 μg / mL, it is expressed as _{σa = σ a, 100 μg / mL.} Since Equation 12 holds at all wavelengths, it is preferable to substitute the measurement result at the wavelength at which the difference 30 appears most prominently into Equation 12.

_{r 45 ° (100μg / mL)} / r 45 ° (0μg / mL) = exp (-σ a, 100μg / mL (d + d / cos (45 °))) (12)

Since r _{45 °} (100μg / mL) and _{r 45 ° (0μg} / mL) are those measured at both the same reflection angle 45 °, by obtaining these ratios according to Equation 12, the anisotropic reflective light It is considered that the influence of sex is canceled and the obtained ratio is not affected by anisotropy.

From Equation 12, σ _{a and 100 μg / mL} d can be obtained. By using the obtained σ _{a, 100 μg / mL} d and the formula 10, a _REP (100 μg / mL) can be obtained.

By substituting the obtained a _REP (100 μg / mL) into Equation 5 of the representative layer theory, the r _REP (100 μg / mL) can be obtained.

The _{scattering parameter g can be determined using r REP} (100 μg / mL), r _{45 °} (100 μg / mL), equations 8, 9, and 11. Therefore, the Hennyy Greenstein function p (g, θ) can be obtained.

The obtained g is determined only by the properties of the scatterer, regardless of the concentration of the medium 27. Therefore, by using this obtained g, r _{45 °} measured under arbitrary concentration conditions can be converted into r _REP. In the following, as a specific example, the procedure for converting _{r 45 °} (0 μg / mL) _{already measured in the above procedure into r REP will be described.}

When the concentration of the dye rhodamine B is 0 μg / mL, it is considered that light is not absorbed, so σ _a = 0. Substituting this into Equations 8 and 9, the following Equation 13 is obtained.

r _REP / r _{45 °} (0 μg / mL)
= (∫s × p (θ) × sinθdθ / ∫sinθdθ) / s × p (θ = 45 °)
= (∫p (θ) × sinθdθ / ∫sinθdθ) / p (θ = 45 °) (13)

Using the value of g and Equation 11, ∫p (θ) × sinθdθ / ∫sinθdθ can be calculated numerically. p (θ = 45 °) can be obtained from the value of g and Equation 11. r _{45 °} (0 μg / mL) has been measured. By substituting these values into Equation 13, r _REP can be obtained.

By substituting r _REP into Equation 5, a _REP can be obtained. By substituting a _REP into Equation 6, the absorbance A _surf of the representative layer can be obtained.

_{In the above description, the procedure for obtaining the} g value and the r REP using the g value using the measurement result under the concentration condition of 100 μg / mL and the measurement result under the concentration condition of 0 μg / mL has been described. _{The r REP} can be obtained by the same procedure under other concentration conditions.

FIG. 5 is a graph showing the _{absorbance A surf} corrected by the above procedure. The white square is the same as the white square shown in FIG. Black circles indicate the corrected absorbance A _surf . As shown in FIG. 5, it can be seen that the method according to the first embodiment further improves the linearity of the correspondence between the concentration and the absorbance even in the high concentration region.

FIG. 6 is a flowchart summarizing the procedure for _obtaining the absorbance Asurf of the representative layer in the first embodiment. Each step of FIG. 6 will be described below.

(FIG. 6: Step S601)
_{The operator measures r 45 °} (100 μg / mL) and r _{45 °} (0 μg / mL) using the spectroscopic analyzer 100. This step corresponds to obtaining the measurement results illustrated in FIG. The calculation unit 20 holds the measurement result.

(FIG. 6: Steps S602 to S604)
_{The calculation unit 20 obtains σ a and 100 μg / mL} d using the measurement result in step S601 and the equation 12 (S602). The calculation unit 20 obtains _{a REP} (100 μg / mL) _{by using σ a, 100 μg / mL} d and Equation 10 (S603). The calculation unit 20 obtains _{r REP} (100 μg / mL) _{by using a REP} (100 μg / mL) and Equation 5 (S604).

(FIG. 6: Step S605)
The calculation unit 20 _{obtains the scattering parameter g using r REP} (100 μg / mL), r _{45 °} (100 μg / mL), equation 8, equation 9, and equation 11.

(FIG. 6: Steps S606 to S608)
The calculation unit 20 _{obtains r REP according to Equation 13 using g and r 45 °} (0 μg / mL) (S606). The calculation unit 20 _{obtains a REP using r REP} and Equation 5 (S607). The calculation unit 20 _{obtains A surf using a REP} and Equation 6 (S608).

(FIG. 6: Steps S601 to S608: Supplement)
If for other density conditions (tentatively referred to as xμg / mL) Request _{A surf,} its density conditions (xμg / mL), to get the same measurement results _{(a) r 45 ° (0μg} / mL), ( b) Before using Equation 5, R _tot is calculated according to Equations 3 and 4. Other procedures are the same as above.

<Embodiment 1: Summary>
The spectroscopic analyzer 100 according to the first embodiment measures the reflectance of the sample in which the medium 27 is dissolved and the reflectance of the sample in which the medium 27 is not dissolved, and by combining these with the representative layer theory, the anisotropic parameter g of scattering is g. Can be calculated. As a result, _{the linearity of the correspondence between the absorbance A surf} and the concentration of the substance to be measured 15 can be improved, and the concentration can be obtained more accurately.

<Embodiment 2>
In the first embodiment, it has been explained that the linearity of the correspondence between the absorbance and the concentration is improved by obtaining the parameters that are not affected by the anisotropy of the surface reflected light of the substance to be measured 15. On the other hand, the anisotropy of the surface reflected light of the substance to be measured 15 is measured in advance, or if this is known, the effect of the anisotropy is known in advance, so this is used. Absorbance and concentration can also be used. In the second embodiment of the present invention, a specific method thereof will be described.

FIG. 7 is a diagram showing a configuration example in the case of estimating the scattering parameter g of the Henyey Greenstein function represented by the formula 11 using the spectroscopic analyzer 100. The configuration of the spectroscopic analyzer 100 is the same as that of the first embodiment, but the measurement is performed at a plurality of light receiving angles (light receiving angles 111 and 112 in FIG. 7). After the measurement is performed at the light receiving angle 111, the polarizer 16 / collimating lens 17 / optical fiber 18 / light receiving unit 19 is moved to a position corresponding to the light receiving angle 112 to perform the measurement.

The phase function of Equation 11 is a function that draws an elliptical orbit with reference to the position of the substance to be measured 15, and g can be specified if two or more values of p (g, θ) are known. Therefore, the function of g and Equation 11 can be obtained by acquiring two or more values of p (g, θ) using the measurement results at two or more light receiving angles and Equation 7. Although the measurement at two light receiving angles 111 and 112 is illustrated in FIG. 7, the measurement may be performed at three or more light receiving angles, or may be measured using two or more light receiving optical systems. When measuring the substance to be measured 15 having a known value of g, the measurement process as shown in FIG. 7 can be omitted.

If g and the phase function of Equation 11 can be specified, the subsequent procedure is the same as that of the first embodiment. However, _{if the same measurement result as r 45 °} (0 μg / mL) has not yet been obtained for the concentration condition (xμg / mL) for which _{A surf} is to be obtained, it is necessary to obtain it again.

Depending on the characteristics of the substance to be measured 15, it may be appropriate to use a phase function other than Equation 11. For example, when the size of the substance to be measured 15 is sufficiently small with respect to the wavelength scale, scattering called Rayleigh scattering becomes dominant. On the other hand, when the size of the substance to be measured 15 is about the same as the wavelength scale, scattering called Mie scattering becomes dominant.

The following equation 14 can be used as the phase function in the case of Rayleigh scattering. Furthermore, since Rayleigh scattering has the property of eliminating polarized light, the following equation 15 can be used in consideration of its influence. Δ is the degree of depolarization.

p (θ) = 3 (1 + cos ² θ) (14)
p (θ) = (3/4) (2 / (2 + Δ)) (1 + Δ) + (1-Δ) cos ² θ (15)

In the case of diffusive scattering in which the size of the substance 15 to be measured is sufficiently larger than the wavelength scale, scattering depending on the shape of the substance surface occurs. In this case, information on the shape of the surface of the substance (for example, the refractive index, unevenness, surface roughness, etc. of the surface) is input to the spectroscopic analyzer 100, and the calculation unit 20 uses the information to correspond to the phase function. Functions can be defined.

Each piece of information for identifying the phase function (eg, the distinction between equations 11, 14, and 15, the degree of depolarization Δ, the shape of the substance to be measured 15, etc.) is provided to the spectroscopic analyzer 100 by the operator via an appropriate interface. Can be given.

<Embodiment 2: Summary>
In the spectroscopic analyzer 100 according to the second embodiment, the calculation unit 20 obtains the scattering parameter g by measuring or giving it by the operator, and uses the scattering parameter g to obtain the average reflectance over the entire light receiving angle. it can. As a result, even when the substance 15 to be measured has anisotropy of reflected light and the measurement is performed at a specific light receiving angle, the influence of the anisotropy is removed and the concentration of the substance 15 to be measured is increased. It can be calculated accurately.

<Embodiment 3>
Depending on the measurement conditions such as the characteristics of the substance to be measured 15, the intensity of the surface reflected light may be weak and the measurement may be difficult. In Embodiment 3 of the present invention, a method of estimating the reflectance of the surface reflected light of the substance 15 to be measured will be described using the reflectance of the surface reflected light of the standard sample.

FIG. 8 is a graph showing the results of measuring the reflectance of the standard sample and the reflectance of the substance to be measured 15 at a plurality of light wavelengths. First, the reflectance 113 of the surface reflected light and the reflectance 114 of the total reflected light are calculated for the standard sample in the same manner as in the first embodiment. As the standard sample, it is desirable to use a substance that is the same as the substance to be measured 15 and has a high concentration at which the influence of absorption is recognized at the reflectance 113.

Next, the reflectance 115 of the total reflected light is calculated for the substance 15 to be measured having a concentration different from that of the standard sample, as in the first embodiment. Regarding the substance to be measured 15, it does not matter whether or not the surface reflected light is affected by absorption. It is assumed that the surface reflected light of the substance to be measured 15 has a weak intensity. The reflectance in this case is shown in the dotted line curve of FIG. 8, and it is difficult to accurately obtain the reflectance.

The relationship between the reflectance 114 of the total reflected light and the reflectance 113 of the surface reflected light in the standard sample is similar to the relationship between the reflectance 115 of the total reflected light and the reflectance shown by the dotted line in the material 15 to be measured. It is presumed that it is. In the third embodiment, the surface reflectance of the substance to be measured 15 is obtained by utilizing this. The specific procedure will be described below. In general, the reflectance changes according to the light wavelength, but in the following procedure, it is assumed that the change is slight and the influence can be ignored when determining the reflectance.

The calculation unit 20 identifies _{the light wavelength λ peak} when the reflectance 115 takes an extreme value based on the measurement result of the reflectance 115. The calculation unit 20 specifies _{the light wavelength λ ref} when the reflectance 114 has the same value as the reflectance 115 at the _{light wavelength λ peak (R ref in FIG. 8).} If the candidate of the optical wavelength lambda _ref there are _multiple, | λ _peak -λ _ref | it is desirable to select a light wavelength lambda _ref to best smaller.

Calculation unit 20, the reflectance r _ref surface reflection light of the standard sample in the optical wavelength lambda _ref, is specified as the reflectance of surface reflection light of the measurement target material 15 in the optical wavelength lambda _ref. This is because the relationship between the reflectances 114 and 115 is presumed to be similar to the relationship between the reflectance 113 and the reflectance of the surface reflected light of the substance 15 to be measured.

_{The reflectance r 0} of the surface reflected light of the standard sample when the light is not absorbed can be expressed by the following equation 16 using the reflectance r _ref , the absorption coefficient σa, and θ and d in FIG. _{The light absorption rate a REP} in the representative layer can be obtained from the formulas 16 and 9 _{, and the absorbance A surf} can be further obtained by the formula 6.

r _ref / r ₀ = exp (−σa (d + d / cosθ)) (16)

Since the absorbance A _surf is not affected by multiple scattering, it is considered to follow the Veil-Lambert law. If _{the assumption that the dependence on the wavelength of r 0} is small is established, the concentration of the substance to be measured 15 can be calculated by the above procedure.

<Embodiment 4>
In the first to third embodiments, the intensity _Itot of the total reflected light is determined by Equation 2. However, depending on the measurement target, the optical properties may change depending on the depth direction. For example, the skin is composed of epidermis / dermis / subcutaneous fat in order from the surface, and each layer has a different composition. In such a substance to be measured 15, if the _{total reflectance is determined by using Itot} as the total reflected light, the total reflection may include characteristics other than the layer to be measured. In particular, in the third embodiment, since it is premised that the reflectance does not change (or the influence of the change can be ignored) depending on the light wavelength, it is not desirable to be affected by a layer other than the measurement target.

Therefore, for such a substance to be measured 15, I _|| can be used _{instead of Itot as the total reflected light.} As a result, total reflectance can be obtained in a portion shallower than _Itot. Using this, the concentration can be determined by the method of the third embodiment. Depending on the characteristics of the substance to be measured 15, I _⊥ may be used as the total reflected light, or the polarization angle may be set to an arbitrary value from 0 ° to 90 °.

In general, it is considered that I _|| gives the reflectance in the shallow part, and I _⊥ gives the total reflectance including all layers up to the deepest part. Therefore, when the substance 15 to be measured has different characteristics for each layer, it is considered desirable to use _{I ||} as the total reflected light instead of _Itot.

<About a modified example of the present invention>
The present invention is not limited to the above-described examples, and includes various modifications. For example, the above-described embodiment has been described in detail in order to explain the present invention in an easy-to-understand manner, and is not necessarily limited to those having all the described configurations. Further, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Further, it is possible to add / delete / replace a part of the configuration of each embodiment with another configuration.

In the first embodiment, the absorbance is calculated using the ratio of the two surface reflectances r (100 μg / mL) and r (0 μg / mL). When these surface reflectance values are small, for example, the third embodiment can be used in combination.

In the first embodiment, the light source 11 may be turned off and the background may be measured in each rotation state of the polarizer 14. _{The calculation unit 20 stores the background light intensity I b ||} when the polarized light states are parallel and the background light intensity I _{b ⊥} when the polarized light states are vertical, and sets the reflected light intensity. These can be deducted when asking.

When measuring the incident light intensity I ₀ , the incident light may be directly guided to the light receiving unit 19 through a mirror or the like. If that is difficult, the incident light intensity I ₀ may be measured using a diffuse reflection standard or the like.

In the above embodiments, milk in which the dye rhodamine B is dissolved is taken as an example of the substance to be measured 15, but the method of the present invention more generally means the concentration of components in a solution or powder containing a scatterer. Can be applied when quantifying by diffuse reflection spectroscopy.

In the third embodiment, since the light intensity signal when measuring the reflectance 113 is often weak, it is desirable to smooth the obtained reflectance 113. As the smoothing method, a method based on a moving average, a Savitzky-Goray method, a method based on the Fourier transform, a method based on the Wavelet transform, and the like can be considered. Further, since the portion affected by absorption is considered to be a constant multiple of the absorption spectrum of the pure substance, the constant may be obtained in advance.

In the above embodiments, the light reflected from the vicinity of the surface is also referred to as surface reflected light. Specifically, in view of various conditions such as measurement accuracy, the reflected light in the range where Equation 1 can be considered to hold can be treated as the surface reflected light.

In the above embodiment, the arithmetic unit 20 can be configured by hardware such as a circuit device that implements these functions, or software that implements these functions is provided by an arithmetic unit such as a CPU (Central Processing Unit). It can also be configured by executing.

11: Light source 12: Optical fiber 13: Collimating lens 14: Polarizer 15: Substance to be measured 16: Polarizer 17: Collimating lens 18: Optical fiber 19: Light receiving unit 20: Calculation unit 21: Light receiving angle 27: Medium 111: Light receiving angle 112 : Light receiving angle

Claims (12)

A spectroscopic analysis method that analyzes a sample using light.
A step of receiving the surface reflected light reflected from the surface of the sample and measuring the intensity of the surface reflected light.
A step of using the intensity of the surface reflected light to calculate a parameter represented by the light absorption coefficient of the sample and not affected by the anisotropy of the reflected light of the sample.
Using the parameters, the step of calculating the average absorption rate corresponding to the average of the light absorption rates of the sample over the entire light receiving angle.
A spectroscopic analysis method characterized by having. The spectroscopic analysis method further
After aligning the polarization state of the polarizer of the measuring instrument that measures the intensity of the light reflected from the sample with the polarization state of the emitted light, the first intensity of the light reflected from the sample is determined by using the measuring instrument. Steps to measure,
A step of measuring the second intensity of the light reflected from the sample by using the measuring instrument after rotating the polarization state of the polarizer.
Have,
The first aspect of claim 1, wherein in the step of measuring the intensity of the surface reflected light, the intensity of the surface reflected light is measured by using the difference between the first intensity and the second intensity. Spectral analysis method. The spectroscopic analysis method according to claim 2, wherein in the step of measuring the second intensity, the polarization state of the polarizer is rotated by 90 degrees. The spectroscopic analysis method further
A step of measuring the first reflectance of the surface reflected light by irradiating a liquid containing the sample at a first concentration with light.
A step of measuring the second reflectance of the surface reflected light by irradiating a liquid containing the sample at a second concentration with light at the same light receiving angle and polarization state as when measuring the first reflectance. ,
A step of calculating a scattering parameter representing the anisotropy of the reflected light of the sample by using the ratio of the first reflectance and the second reflectance.
The spectroscopic analysis method according to claim 1, wherein the method is characterized by the above. In the step of calculating the average absorption rate,
A function representing the anisotropy of the reflected light of the sample, defined using the scattering parameters, is integrated over the total light receiving angle.
The spectroscopic analysis method according to claim 4 , wherein the average absorptivity is calculated by using the result of the integration and the second reflectance. The spectroscopic analysis method further
A step of specifying a scattering parameter representing the anisotropy of the reflected light of the sample.
A step of measuring the reflectance of the surface reflected light by irradiating a liquid containing the sample with light.
Have,
In the step of calculating the average absorption rate,
A function representing the anisotropy of the reflected light of the sample, defined using the scattering parameters, is integrated over the total light receiving angle.
The spectroscopic analysis method according to claim 1 , wherein the average absorption rate is calculated by using the result of the integration and the reflectance. The claim is characterized in that the function is either a Henyey Greenstein function, a function representing the anisotropy of light reflection in Rayleigh scattering, or a function representing the anisotropy of light reflection and the degree of depolarization in Rayleigh scattering. Item 6. The spectral analysis method according to Item 6. The spectroscopic analysis method further
A step of measuring the reflectance of the first surface reflected light reflected from the surface of a reference sample over a predetermined range of light frequencies.
A step of measuring the reflectance of the first total reflected light including the diffusely reflected light diffusely reflected inside the reference sample and the first surface reflected light over an optical frequency in the predetermined range.
The step of specifying the first light frequency at which the reflectance of the first total reflected light takes an extreme value,
A step of measuring the reflectance of the second total reflected light including the diffusely reflected light diffusely reflected inside the sample and the second surface reflected light reflected from the surface of the sample over an optical frequency within the predetermined range.
A step of specifying a second light frequency when the reflectance of the first total reflected light is the same as the extreme value of the reflectance of the second total reflected light.
A step of specifying the light reflectance of the first surface reflected light measured at the second light frequency as the light reflectance of the second surface reflected light at the first light frequency.
The spectroscopic analysis method according to claim 1, wherein the method is characterized by the above. The spectroscopic analysis method further
After aligning the polarization state of the polarizer of the measuring instrument for measuring the intensity of the light reflected from the reference sample with the polarization state of the emitted light, the third of the light reflected from the reference sample using the measuring instrument. Steps to measure strength,
A step of measuring the fourth intensity of the light reflected from the reference sample using the measuring instrument after rotating the polarization state of the polarizer.
A step of measuring the fifth intensity of the light reflected from the sample by using the measuring instrument after aligning the polarization state of the polarizer with the polarization state of the emitted light.
A step of measuring the sixth intensity of light reflected from the sample using the measuring instrument after rotating the polarization state of the polarizer.
Have,
In the step of measuring the reflectance of the first total reflected light, the intensity of the first total reflected light is measured by using the sum of the third intensity and the fourth intensity.
8. The eighth aspect of claim 8, wherein in the step of measuring the reflectance of the second total reflected light, the intensity of the second total reflected light is measured by using the sum of the fifth intensity and the sixth intensity. Spectral analysis method. The spectroscopic analysis method according to claim 1, further comprising the step of calculating the absorbance of the sample using the average absorptivity. The spectroscopic analysis method further
A step of specifying the concentration of the sample using the absorbance of the sample according to the correspondence between the absorbance of the sample and the concentration of the sample based on the Bale-Lambert law.
10. The spectroscopic analysis method according to claim 10. A spectroscopic analyzer that analyzes a sample using light.
A measuring instrument that measures the intensity of surface reflected light reflected from the surface of the sample.
An arithmetic unit that calculates the light absorption rate of the sample,
With
Using the intensity of the surface reflected light, the calculation unit calculates a parameter represented by the light absorption coefficient of the sample and not affected by the anisotropy of the reflected light of the sample.
The calculation unit is a spectroscopic analyzer characterized in that the calculation unit calculates an average absorption rate corresponding to an average of the light absorption rates of the sample over all light receiving angles using the parameters.