JPH0715465B2 - Multichannel detection data processor for liquid chromatograph - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention measures the absorbance values at a plurality of wavelengths simultaneously in an ultraviolet / visible absorptiometer detector in a liquid chromatograph, analyzes the absorbance values, and then The present invention relates to a multi-channel detection data processing device that outputs a processing result.

(B) Conventional technology The purpose of liquid chromatography is to separate each component in a sample from the beginning, but not all components contained under the same analysis conditions can be separated. Some components may be insufficiently separated due to the similar behavior, or may be eluted without being separated at all.

When a conventional detector with a single wavelength is used, it is often impossible to determine whether or not the peaks are unseparated peaks, and it is difficult to quantify the components of these unseparated peaks. When such a problem occurs, change the analysis conditions such as the column and mobile phase, and study the conditions under which these unseparated peaks are separated,
Alternatively, the sample itself needs to be pretreated. However, it takes a lot of time to examine the analysis conditions, and it is often difficult to obtain the optimum conditions. In addition, when the pretreatment is performed, the operation is often complicated or causes a large error on the quantitative value.

(C) Purpose The present invention has been made in view of the above circumstances, and an object thereof is to provide a multichannel detection data processing device for a liquid chromatograph capable of quantifying components of unseparated peaks without changing analysis conditions. Is.

(D) Configuration And the configuration of the present invention, a plurality of detection means for simultaneously detecting the absorbance information at a plurality of wavelengths of the light irradiated to the column outflow fluid of the liquid chromatograph, and the absorbance information obtained by the detection means Sampling means for sampling at arbitrary time intervals, storage means for storing the absorbance information extracted by the sampling means for each wavelength, and a chromatogram for outputting the absorbance information as a chromatogram showing the temporal change of the absorbance for each wavelength. The output means and two predetermined wavelengths having the same absorbance information for any one component in the outflow fluid are selected, and the difference between the absorbance information of the outflow fluid at the two wavelengths is calculated on the chromatogram. Calculation means for separating and quantifying the peak value of the absorbance information of each component contained in the outflow fluid And a multi-channel detection data processing device for a liquid chromatograph.

(E) Embodiments Embodiments of the present invention will be described in detail below with reference to the drawings, but the present invention is not limited to the following embodiments.

The configuration of the embodiment of the present invention will be described with reference to FIG.

(1) is a sampling means which samples a plurality of detection output signals obtained by a plurality of types of detection means (2) of a liquid chromatograph at arbitrary time intervals.
The detecting means (2) uses, for example, a photodiode array, and each channel of the detecting means (2) corresponds to each element of the photodiode. The absorption spectrum at a wavelength of 200 to 699 mm is constantly measured every 1 nm. Then, the detection output signal is output to the sampling means (1). A storage means (3) stores the sample signal sampled by the sampling means (1), and a required sample signal is output from the storage means and transmitted to the chromatogram output means (4). (5) is a calculation means for calculating the difference between two different sample signals output from the storage means (3) as a chromatogram and the peak area of the obtained chromatogram. (6) is a display means for displaying and outputting a plurality of sample signals at arbitrary times from the storage means (3) as spectra.

The operation of the embodiment of the present invention will be described below with reference to FIGS.

As shown in FIG. 2, it was found that a certain peak on the chromatogram was obtained by overlapping and eluting the component A and the component B without separating them, and the components A and B respectively. Differences in the absorption spectrum characteristics of the above shall be recognized. At this time, the elution times of the components A and B are often slightly different. (The dotted line in FIG. 2 shows the elution peak of component A, the one-dot chain line shows the elution peak of component B, and the solid line shows the components A and B.
Is the peak when the two overlap. ) Furthermore, the absorption spectra of the components A and B are as shown in FIG. In FIG. ₃ , it can be seen that the component A has the same absorbance at the wavelengths λ ₁ and λ ₂ , and the component B has the same absorbance at the wavelengths λ ₃ and λ ₄ . Now, when the absorbance at the time t at the wavelength λ is represented by Aλ (t), the wavelength λ
₂ absorbance A λ ₂ (t) and wavelength λ ₁ absorbance A λ
_When the difference from ₁ (t) is calculated, A ₁ (t) = Aλ ₂ (t) −Aλ ₁ (t) ... (1-1). In this chromatogram, the component A is removed and a peak is obtained only from the component B. Similarly, the wavelength lambda ₃ of the absorbance A? ₃ (t) and the determining a difference between the absorbance A? ₄ wavelength _{λ 4 (t), A 2} (t) = Aλ 3 (t) -Aλ 4 (t) ... ( 1-2), and in this chromatogram, the component B is removed and the peak due to the component A alone is obtained. Then, for each of the component A, the respective peak areas are calculated by the chromatogram A ₂ (t) shown by the equation (1-2) and also by the chromatogram A ₁ (t) shown by the equation (1-1) for the component B. By doing so, it becomes possible to quantify.

Next, as a sample, a mixture of hippuric acid, O-methylhippuric acid, m-methylhippuric acid, and P-methylhippuric acid at a ratio of 1 mg / ml, and m-methylhippuric acid and P-methylhippuric acid were prepared. The operation when each of them is used individually will be described with reference to FIGS.

First, only the m-methylhippuric acid is injected, and after 5 minutes, the data obtained by injecting P-methylhippuric acid is stored in the storage means (3). The spectra of m-methylhippuric acid and P-methylhippuric acid are output from the stored data. The output spectrum is shown in FIG. 4, where the solid line is m-methylhippuric acid and the dotted line is P-methylhippuric acid. In λ ₁ and λ ₂ of FIG. 4, the absorbances of m-methylhippuric acid are equal,
Since the absorbances of P-methylhippuric acid are the same at λ ₃ and λ ₄ , the difference between the absorbances of λ ₁ and λ ₂ is output as a chromatogram, the peak of m-methylhippuric acid disappears, and λ ₃ and λ _When the difference in absorbance at ₄ is output as a chromatogram, the peak of P-methylhippuric acid is eliminated. Chromatograms output in FIGS. 5A and 5B are shown.
In FIG. 5A, the dotted line is the chromatogram at λ ₄ , the broken line is the chromatogram at λ ₃ , and the solid line is the chromatogram obtained by subtracting the absorbance at λ ₄ from the absorbance at λ ₃ at the same time. At, the peak of P-methylhippuric acid is eliminated. (The peak after 11.1 minutes shows m-methylhippuric acid and the peak after 16.2 minutes shows P-methylhippuric acid.) Similarly, in FIG. 5, the dotted line is the chromatogram at λ ₁ , and the wavy line is λ ₁ . chromatogram of _2, the solid line obtained by outputting the chromatogram minus the absorbance at lambda ₁ at the same time from the absorbance at lambda _2, the peak of m- methyl hippuric acid has been erased. That is, in order to quantify P-methylhippuric acid, the formula (1-2) is used, and a chromatogram of Ap (t) = Aλ ₂ (t) −Aλ ₁ (t) ... (1-3) is used. , To quantify m-methylhippuric acid, use the formula (-1) and use the chromatogram Am (t) = Aλ ₃ (t) -Aλ ₄ (t) ... (1-4). It can be seen that both isomers can be individually quantified even if they are eluted without being separated.

Next, hippuric acid, O-methyl hippuric acid, m-methyl hippuric acid,
P-Methylhippuric acid, 1 mg / ml, was mixed and measured as a sample.

The chromatogram obtained as a result is shown in FIG. The wavelengths recorded are λ _{1 to} λ ₄ , as above. From this chromatogram, hippuric acid (marked as HA in the figure) and O-methyl hippuric acid (marked as O-MHA in the figure) were separated well, but m-
It can be seen that methyl hippuric acid (indicated by m-MHA in the figure) and P-methyl hippuric acid (indicated by P-MHA in the figure) are not separated but are overlapped and eluted. Since there is a time difference between the peaks of the peak in the chromatogram at wavelength λ ₃ and the peak at wavelength λ ₁ , it can be seen that this peak contains a plurality of insufficiently separated components. From this, it is expected that the insufficiently separated components are m-methylhippuric acid and P-methylhippuric acid. Further, FIG. 7 is a diagram in which the data obtained by the measurement of the mixed solution is output as a three-dimensional chromatogram with time-absorbance-wavelength as the three axes. Figure 8 shows the same data for the absorbance step 0.12Ab.
s, contour lines are displayed. In FIG. 8, the elution peak of C seems to be tilted slightly obliquely, which shows that the time at the peak apex is shifted depending on the wavelength, and it is expected to be an unseparated peak.

Now, from the same data obtained and stored by the measurement of the mixed solution, from the equations (1-3) and (1-4),
Calculate and output two chromatograms. The result is 9th
As shown in the figure, the dotted line in FIG. 9A is the chromatogram at λ ₃ , the broken line is the chromatogram at λ ₄ ,
The solid line is the chromatogram called Am (t). And the ninth
In Figure B, the dotted line is the chromatogram at λ ₂ ,
The broken line is the chromatogram at λ ₁ , and the solid line is the chromatogram at Ap (t). As a result of calculating the area of each peak by this, in the chromatogram Am (t), (a) in FIG. 9A is hippuric acid, (b) is O-methylhippuric acid, and (c) is m-methylhorse. It is the peak of uric acid. Similarly, as a result of performing peak area calculation for the chromatogram Ap (t), (d) in FIG. 9B was the peak of P-methylhippuric acid.

The peak of interest from the quantitative value obtained next is m
It is necessary to confirm that it is an unseparated peak consisting of -methyl hippuric acid and p-methyl hippuric acid alone and does not contain the third and fourth components.

First, from the data of m-methyl hippuric acid and P-methyl hippuric acid injected at 5-minute intervals, Am (t) = Aλ ₃ (t) -Aλ ₄ (t) ... (1-5) Gram is calculated, and the peak height Hm of the peak of m-methylhippuric acid and the time tm of the peak apex are obtained. Times of Day
Absorption spectrum in tm-absorption spectrum of methylhippuric acid) represents Sm.

Furthermore, a chromatogram of Ap (t) = Aλ ₂ (t) -Aλ ₁ (t) ... (1-6) is calculated, and the peak height Hp of the peak of P-methylhippuric acid and the time tp at the peak time are calculated. Ask. The absorption spectrum at time tp (the absorption spectrum of P-methylhippuric acid) is represented by Sp.

Next, hippuric acid, O-methyl hippuric acid, m-methyl hippuric acid,
From the data of the mixed sample with P-methylhippuric acid, hm = Aλ ₃ (to) −Aλ ₄ (to) at a certain time to when both m-methylhippuric acid and P-methylhippuric acid were eluted ( 1-7) hp = Aλ ₂ (to) −Aλ ₁ (to) ... (1-8) At this time, the absorption spectrum at time to
Represented as Sto.

If the unseparated peak does not include anything other than m-methylhippuric acid and P-methylhippuric acid, the absorption spectrum Sto
And the absorption spectrum Sx shown in the following equation should agree with each other.

FIG. 10 shows the output of the two spectra thus obtained in an overlapping manner. The solid line is the absorption spectrum for Sx, and the dotted line is the absorption spectrum for Sto. It was confirmed that this non-separated peak did not contain components other than m-methylhippuric acid and P-methylhippuric acid.

(F) Effect According to the present invention, since the absorbances at a plurality of wavelengths can be measured at the same time, a multi-channel detection data processing device for liquid chromatograph capable of quantifying components of unseparated peaks without changing analysis conditions. can get. Furthermore, since simultaneous detection is carried out at a plurality of wavelengths, it is possible to separate and quantify unseparated peaks from the data obtained in only one analysis, which enables quick and simple processing.

[Brief description of drawings]

FIG. 1 is a block diagram of an embodiment of the present invention, FIG. 2 is a chromatogram showing unseparated peaks, FIG. 3 is a spectrum diagram of components A and B of the unseparated peaks of FIG. 2, and FIG. A spectrum diagram of methyl hippuric acid and P-methyl hippuric acid, FIGS. 5A and 5B are chromatograms of m-methyl hippuric acid and P-methyl hippuric acid, respectively, and FIG. 6 is hippuric acid, O-methyl hippuric acid, Chromatogram of a mixed sample of P-methylhippuric acid, m-methylhippuric acid and P-methylhippuric acid, FIG. 7 is a three-dimensional chromatogram of FIG. 6, and FIG. 8 is the data of FIG. 9A and 9B are chromatograms for each wavelength of unseparated peaks from the data of FIG. 6, and FIG. 10 is a spectrum diagram of absorption spectra Sto and Sx. (1) ... Sampling means, (2) ... Detection means, (3) ... Storage means, (4) ... Chromatogram output means, (5) ... Calculation means, (6) ... Display means.

Claims (1)

[Claims] 1. A plurality of detecting means for simultaneously detecting absorbance information at a plurality of wavelengths of light irradiated to a column outflow fluid of a liquid chromatograph, and the absorbance information obtained by the detecting means is sampled at arbitrary time intervals. Sampling means, storage means for storing the absorbance information extracted by the sampling means for each wavelength, chromatogram output means for outputting the absorbance information as a chromatogram showing the temporal change of the absorbance for each wavelength, and the outflow By selecting two predetermined wavelengths at which the absorbance information for any one component in the fluid is equal, calculating the difference between the absorbance information of the outflow fluid at the two wavelengths on the chromatogram, and calculating the difference in the outflow fluid. And a calculation means for separating and quantifying the peak value of the absorbance information of each contained component. Multi-channel detection data processor for liquid chromatograph.