JPH02254343A - Spectroscopic measurement apparatus - Google Patents

Abstract

PURPOSE:To enable accurate assay by providing a correction function to determine a correction absorbance from actually measured absorbance by a function containing a coefficient not depending on a concentration of a sample, indicating a band width of a spectroscope and an inclination of a spectrum of a sample to be measured as parameter. CONSTITUTION:Sample cells C1, C2... are set on an automatic sample exchanger T and moved to a measuring position sequentially being controlled by a computer K. Then, the computer K takes in outputs of one or more of photo detectors Di, Dj... specified previously in the order of the cells C1, C2... on the exchanger T according to a given program and absorbance corrected to a desired substance in a sample is calculated according to a specified formula. A concentration of the desired substance is determined from a data of a calibration curve given about the substance to be recorded on a recorder together with a sample number.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to an absorbance-corrected spectrophotometer.

(Prior art) When determining the concentration of a target component in a sample by measuring absorbance fl, the absorbance at the peak center wavelength of the absorption spectrum of the target component is usually measured. There are cases where it is necessary to set measurement wavelengths on the descending slopes and perform quantitative determination. For example, because the wavelength is not continuously variable in the spectrophotometer of an automatic analyzer, it is not possible to set the measurement wavelength at the peak wavelength, or the peak absorbance is too large and accurate measurement cannot be performed. This is the case, for example, when setting the wavelength at the hem. In such a case, the calibration curve becomes curved and the quantitative accuracy decreases. How measurements at the peak wavelength differ from measurements taken off-peak are shown very clearly in Annex a and b. This means that a spectrum with a half-width of 50 nm (a Gaussian shape is assumed for ease of calculation) as shown in Figure 3 has a spectrometer bandwidth of 2,5.5 *
Numerical measurement n was performed assuming that measurements were taken in four cases of 10r and 15nm. The ratio of the expected measured value to the correct measured value is calculated for the case of the peak wavelength (a) and for the third wavelength 25 nm (1/2 half width) away from the peak.
The arrow B in the figure shows the calculated value. This ratio is 1000
Close to■? means the value is correct. Naturally, the smaller the bandwidth, the more accurate the value, but (a>
The most notable difference between cases (b) and (b) is whether or not this "ratio" remains constant when the absorbance of the sample increases.

For example, in the case of a band width of 10 nm, if the peak wavelength (a>), the absorbance ranges from 0.2 to 4.
For samples up to 0, the value of the above "ratio" changes only slightly from 0.9818 to 0.9797 (0,
9797÷0.9818=0.9978). In other words, the change is only 0.2%.

On the other hand, in case (b), which is 25 nmfi from the peak, the same sample has half the absorbance, so the corresponding absorbance changes from 0.1 to 2.0, but the value of the above ratio is 0.
．． From 9955 to 0.8281, i.e. (0,8281
Since ÷0.9955=0°8318), it changes by 17%. As the absorbance increases, the ratio value decreases, so
This indicates that the so-called calibration curve is significantly curved.

Therefore, in the case of (a>), the calibration curve remains a straight line and the slope becomes only slightly smaller, so it can be easily corrected by simply multiplying by a coefficient slightly larger than 1, whereas (
In the case of 1], the degree of absorbance is high and the absorbance decreases greatly, so Ffff cannot be positive by simple calculation of 81 coefficients.

If the bandwidth is infinitesimal, the calibration curve will be a straight line whether measured at the peak wavelength or at a point deviating from the peak wavelength, but if the band width is wide to some extent, the calibration curve will be straight at a wavelength deviating from the peak wavelength. In particular, spectrophotometers used in automatic analyzers have a relatively wide band width of about 10 nm, and the wavelength can only be set in steps. Since there are many cases where selection is not possible, the above-mentioned problem becomes a problem.

However, until now, there was no known method to predict and correct the curvature of the calibration curve, so in such cases, you should either use the uncorrected quantitative value as is, or prepare various standard solutions with different concentrations. Either method was used to create a curved inspection ffi line. The cause of this curve in the calibration curve is roughly as follows. The wavelength of transmitted light from a spectrometer has a wide wavelength range (bandwidth) centered around the set wavelength λ0, so in Figure 2, A(λ) is the absorbance curve of the slope of the sample peak, and S( If λ) is the wavelength transmission characteristic of the spectrometer, then
The wavelength distribution of the light that has passed through the sample and spectrometer is not symmetrical about the spectrometer's set wavelength λ0, but is asymmetrical, with the center wavelength of the transmitted light shifting toward shorter wavelengths. , the absorbance is substantially measured at a wavelength shorter than the set wavelength λ0 of the spectrometer, and the actually measured absorbance appears smaller than the absorbance Ao at the wavelength λ0. This tendency increases as the slope of the absorbance curve increases, so as the absorbance increases, that is, the sample concentration increases, the apparent absorbance decreases, and the relationship between concentration and absorbance becomes distorted.

(Problems to be Solved by the Invention) The present invention provides a spectrophotometer that can correct the curvature of the calibration curve and measure the correct concentration even when the measurement wavelength cannot be set to the center wavelength of the absorption peak in quantitative analysis using absorbance measurement. This is what we are trying to provide.

(Means for solving the problem) The bandwidth B of the spectrometer and the coefficient k corresponding to the slope at the measurement wavelength of the absorption spectrum as a characteristic value of the sample to be measured
(K is defined in detail as in Equation 4 and is a quantity independent of the concentration of the sample), where α constant number Xk2B2 The sample concentration is found. The above formula (1) has 1-n″A in the denominator and numeral.
Based on the formula o=A constant number Xk2B2Δ2-(2), Δ
The spectrophotometer was equipped with arithmetic means for determining 0.

(Function) In the above equations (1) and (2), the bandwidth B of the spectrometer is known in advance by a constant unique to the spectrometer, and k, which is a characteristic value of the sample, is determined by the appropriate standard solution of the sample to be measured. It can be determined in advance by actual measurements using a standard spectrophotometer with a sufficiently narrow band width and continuously variable wavelength. By inputting the values of B and B in advance into the calculation means and measuring the absorbance A of the sample to be measured, the corrected absorbance Ao can be determined from A. Since this absorbance Ao has been corrected for the curvature of the calibration curve, it can be rewritten as correct using a linear calibration curve created by measuring standard solutions of appropriate concentrations.

The establishment of formula (1) will be explained below.

In FIG. 2, Δ(λ) represents the absorbance characteristic of a liquid having the concentration of the sample to be measured. (λ0) is the designed center wavelength of the spectrometer, and Ao is the absorbance of the sample solution at the wavelength (λ0). In Fig. 2, the difference between the arbitrary wavelength and (λ0) is rewritten as λ, and the slope characteristic A(λ) of the above sample solution is expressed by the formula: A(λ) = Ao + k'λ... (3) Here, ゛ is positive when A increases with λ. Figure 2 (B) shows the transmission characteristics of the spectrometer, and the wavelength scale is shown in Figure 2 (Δ).
It is set the same as . If this transmission characteristic is S(λ), then B is the above-mentioned bandwidth and the half-width of the above-mentioned transmission characteristic. The value of B is about 10 nm. The above S(λ) is given as the wavelength distribution of the intensity of the light transmitted by the spectrometer when light from a light source whose intensity is constant within the wavelength range of the light transmitted by the spectrometer is incident on the spectrometer. Here, the measured value T of the transmittance of the sample solution using the spectrometer assuming the light source is as follows, where T(λ) is the transmittance wavelength characteristic of the sample solution. The numerator of formula (5) can be rearranged as (enlo) ・k
'・B=x and is expressed by the exponent of equation (8). (
5) To specifically calculate the formula, absorbance characteristic A(λ)
must be converted into a transmittance characteristic T(λ). That is -Ao -Bon72 T(λ) = 10-””’) = 10 X 1
0・・・・・・・・・・・・(6) Since the denominator of equation (5) is oB 10・・・・・・ in the triangular interview shown in Figure 2 B, we can change the above T to an absorption oddball. After conversion, we obtain equation (1) as follows: (Two quadratic equations＼
where A is the absorbance of the sample solution actually measured with a spectrometer. ° used in the above calculation is the slope of the absorbance of the sample solution with a concentration of It exists as a characteristic value. That is, k is an amount that does not depend on the concentration of the sample. Specifically, in a standard solution of known concentration, the wavelength λ0
By actually measuring the slope of absorbance at k =
k is determined by k'/Ao. In the above equation (9), '' is rewritten as k. In equation (11), A is the measured absorbance and Ao is a constant that is close to the corrected value, so it is common to set α=constant x r2B2 (12). It can also be written that equation (11) can be solved using the following approximate method. The second term on the right side of the equation (11)' is an equation that gives the difference (Ao - A) from Ao, and is a small value when compared to Δ itself. Therefore, (1
1) Solution for the value of Ao in the equation (For the reason, it is set as a constant.

If we compare equations (1) and (2), we can see that equation (1) involves a square root operation, so the calculation program is quite long, which can be a bit troublesome, but equation 0 is just a quadratic equation. Because of the formula, it is easy to incorporate into the device.

(Example) FIG. 1 shows an example of the present invention. L is the light source, C1, C
2, C3... is the sample cell, S is the entrance slit of the spectrometer, G is the diffraction grating, DI; D2...Dn is the diffraction grating G
A photodetector is placed on the spectral image plane formed by λ1°, λ2, . ...It is designed to detect light with a wavelength of λn. K is a computer that controls the entire device, and also includes photodetectors DI, D2...
The concentration of each sample is calculated by importing the output and performing arithmetic processing. The sample cells CI, C2, etc. are set in the automatic sample exchanger T, and are sequentially moved to the measurement position under the control of the computer K. According to the program given to the computer, the sample cells on the automatic sample exchanger are - or a plurality of photodetectors Di, Dj, specified in advance according to the
Calculate the corrected absorbance of the target substance in the sample according to formula (1) or C) by taking in the output of ..., and determine the concentration of the target substance from the calibration curve data given for that substance. Record it on the recording device along with the sample number.

A spectrophotometer as shown in FIG. 1 is usually used in a multi-item biochemical automatic analyzer. In this type of device, the measurement items are fixed (for example, 30f111), so the value of k as a characteristic of the sample to be measured for each item is fixed.

Further, the band width B is, for example, about 10 nm, and although it is somewhat dependent on the wavelength, it is a known value once the wavelength is determined.

Normally, k, measurement wavelength, sample collection amount, reagent dispensing amount, etc. are set for each item as so-called "measurement parameters," so the values of k and B introduced this time are set as parameters for each item. By additionally setting the values, it is possible to automatically perform correction calculations using appropriate values of k and B according to each item.

The calculation operation of the corrected absorbance Ao according to the above formula (1) or (2) may be actually performed by the calculation program according to the formula (1) or (2), or the calculation operation between A and Ao A relational table may be created and the corrected absorbance Ao may be derived from the actually measured absorbance value A.

In the explanation so far, the case of one wavelength has been described. However, it is also effective to apply the method to a two-wavelength method or a multi-wavelength method that is often used in automatic analyzers. In the example of the three-wavelength method, two wavelengths λ1 and λ2 are set on the absorption peak k as shown in FIG. 4, but either λ1 or λ2 is often the part of the slope. When the bandwidth is wide, [A(λ
1) - A(λ2)] will curve the calibration curve, so after making corrections to Ao(λ1) and Ao(λ2) at each wavelength, [Ao(λ1)-Ao(λ2)] The correct value can be found by calculating .

Attachment A Calculation results for four bandwidths when measuring at the peak wavelength shown in Figure 3) Attachment B (Effects of the invention) According to the present invention, the measurement wavelength cannot be set to exactly the center wavelength of the peak of the sample to be measured. Even in such a case, the correct concentration can be determined by correcting the curvature of the calibration curve due to the influence of the spectrometer's bandwidth, allowing highly accurate quantification.

[Brief explanation of drawings]

FIG. 1 is a block diagram of an apparatus according to an embodiment of the present invention, FIG. 2 is an explanatory diagram of the operation of the present invention, and FIG. 3 is a diagram showing measurement at a peak wavelength.
FIG. 4 is an explanatory diagram of a specific example showing the difference when measuring at a wavelength far from the peak, and FIG. 4 is an explanatory diagram when the present invention is applied to a two-wavelength method. L...Light sources C1, C2...Sample cell, T...Automatic sample exchanger, S...Entrance slit, G...Diffraction grating, Dl, D2...Photodetector, K... Computer. Agent Hiroshi Agata, Patent Attorney Figure 3: Calculation results for four bandwidths when measuring at a point (wavelength 25nrn away from the peak)

Claims (2)

[Claims] (1) Calculate the corrected absorbance Ao from the measured absorbance A using the following function that includes as parameters the band width B of the spectrometer and the coefficient k defined below, which represents the slope of the spectrum of the sample to be measured and does not depend on the concentration of the sample. A spectrometer with the desired correction function. However, Ao = F (B, k, A) where k is the ratio of the correct absorbance As of the standard product at an appropriate concentration of the sample to be measured to the slope ks of the spectrum of the standard product at the wavelength to be measured. It is assumed that k=ks/As, and f is a function of B, k, and A. (2) The spectrometer according to claim 1, wherein the function is Ao=2A/[1+√(1-4Aα)] where α=(ln10/12)k^2B^2 or an approximate expression thereof.