JPS61213668A - Multi-wavelength absorption detection for liquid chromatograph - Google Patents

Abstract

PURPOSE:To determine the true spectrum of an elution component by determining the base line spectra over the entire wavelength at the peak point by computation and subtracting the same from the spectrum of the peak to be observed. CONSTITUTION:Light from a lamp 8 is converted by a concave mirror 10 and is made incident on a flow cell 6. The transmitted light from the flow cell 6 is reflected by a concave mirror 12 and after the light is divided by a grating 14, the light is reflected by a concave mirror 16 by which the reflected light is made incident on a photodiode array 18. The UV and visible spectra of 500 wavelengths are subjected to simultaneous multi-wavelength measurement in the array 18. 500 pieces of the data I (i) (i=1-500) on the light intensity are fed to a computer system 20. The computer differentiates the detected output per wavelength and determines the differentiated value as the base line when said value is within the specified fluctuation value; otherwise, the value is determined as the peak time. The true spectrum of the elution component is obtd. by subtracting the base line spectrum from the spectrum of the next peak.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a method for processing measurement data in a liquid chromatograph that measures the absorption spectrum of an eluate in an on-flow state without stopping liquid feeding. It is something.

(Prior art) When performing gradient analysis in liquid chromatography, the baseline drifts.

Even if a spectrum is obtained in an on-flow state, the detected spectrum does not necessarily represent the true spectrum of the eluted components.

Further, the same applies to the spectrum of a peak appearing on the tailing of a large peak, and the detected spectrum cannot necessarily be said to represent the true spectrum of the eluted component.

In such cases, conventionally the baseline spectrum closest to the peak of interest is subtracted from the peak spectrum.

(Problems to be Solved by the Invention) However, the baseline drifts even during peak elution, and a true spectrum cannot be obtained even with the above method.

The object of the present invention is to obtain the true spectrum of the eluted component by calculating the baseline spectrum over all wavelengths at the peak point and subtracting it from the spectrum of the observed peak.

(Means for Solving the Problems) The multi-wavelength absorption detection method of the present invention is comprised of the following steps.

(b) Sampling data related to multiple detection outputs obtained by multiple wavelength detection means for liquid chromatograph column elution fluid at arbitrary time intervals, (b) Differentiating the detection output for each wavelength, (h) ) Determine whether the differential value is within a certain range of fluctuation, and when it is within the range of fluctuation, it is the baseline time, and when it is outside the range of fluctuation, it is the peak time. (d) Slope of the rise of the peak. The baseline time point closest to t! , the spectrum S2 at the baseline time point t2 closest to the peak falling slope and the spectrum So at an arbitrary time to at the peak time are stored; (e) the two stored baseline spectra Sl;
52, the baseline spectrum SO at an arbitrary time to at the peak time is calculated from the following equation.

5b=(Ss(t2-t0)+S2(to-t1)}/
(t2-t+) (1) (to) Subtract the obtained baseline spectrum from the already stored spectrum at time to.

(Operation) Assume that chromatogram 2 shown in FIG. 1(A) is obtained for a certain wavelength. When this chromatogram 2 is differentiated, a differential chromatogram 4 as shown in FIG. 1 (CB) is obtained. Therefore, we set a constant fluctuation range Δ in the differential chromatogram, and the spectrum whose differential value is within this constant fluctuation range Δ is called the baseline spectrum, and its fluctuation range Δ
Let the spectrum outside the peak be the peak spectrum. The baseline point t1 closest to the rising slope of the peak is determined as the point at which the differential chromatogram 4 and the upper limit of the variation width Δ intersect, and the baseline point t2 closest to the falling slope of the peak is determined as the point in time when the differential chromatogram 4 intersects with the upper limit of the variation width Δ. It is determined as the point at which the lower limit of the width Δ intersects. Baseline spectrum s at any point in time of the peak, e.g. to
b can be calculated using the above equation (1). Then, if the spectrum (go-5b) is obtained, this becomes the true peak spectrum.

(Example) FIG. 2 shows an example of a liquid chromatograph detection device to which the present invention is applied.

Reference numeral 6 denotes a flow cell through which the eluate from the liquid chromatograph column flows, and light from a deuterium (D2) lamp 8 as a light source is focused by a concave mirror 10 and incident on the flow cell 6. The transmitted light from the flow cell 6 passes through the concave mirror 12
After being reflected by the grating 14 and separated by the grating 14, the light is reflected by the concave mirror 16 and enters a 500-channel photodiode array 18 as a detection means. The photodiode array 18 simultaneously measures multiple wavelengths of ultraviolet and visible spectra of 500 wavelengths. 500 pieces of light intensity data I
(i) (t=1 to 500) is sent to the computer system 20 at, for example, a one-second period.

Next, the procedure of one embodiment will be explained with reference to the flowchart of FIG.

Immediately from the data I (i) input to the computer, 1og I (i) is calculated and converted to absorbance (step 81.82), and the 500 converted data are once stored in a buffer ( Step S3), the buffer always stores some spectra in ascending order of time. In addition, spectrum data log I
The data corresponding to the preset wavelength i of (i) is combined into a chromatogram and its peak is detected (step S4), and the peak detection is performed as shown in Fig. 1 (A) and (B).
As a result, as shown in FIG. 1(A), the baseline time t1. and the baseline time t2 closest to the falling slope of the peak, and calculate the respective spectra SL, S
2 as the baseline time spectrum (step S5), and any time t between the baseline time 1+ and t2.

(to is not limited to the time corresponding to the top of the peak.

(including other sampled peak times) is recognized as the peak time, and the spectrum at that time is stored as the peak time spectrum So (step S6).

After that, the peak time t is determined by the above equation (1).

Calculate the baseline spectrum sb at (step S7). This calculation of the baseline spectrum sb is performed for each of the 500 pieces of data.

Next, (So-Sb) is calculated for each of the 500 pieces of data, and each is used as a spectrum of the eluted component at the peak time to.

In addition, although the method of detecting an eluted component by ultraviolet/visible absorption spectrum is illustrated in the example, the eluted component may be detected by other means, for example, by infrared absorption.

(Effects of the Invention) According to the present invention, since the baseline at the peak point is correctly determined, it is possible to achieve the first-order effect.

(1) Accurate spectra of eluted components can be obtained even in gradient analysis.

(2) Accurate spectra can also be obtained for tailing peak components.

[Brief explanation of drawings]

Figure 1 (A) is a diagram showing a chromatogram, Figure 1 (CB)
is a diagram representing the differential chromatogram of the same chromatogram,
FIG. 2 is a schematic diagram showing an example of a detection device for a liquid chromatograph, and FIG. 3 is a flowchart showing one embodiment.

Claims (1)

[Claims] (1) A multi-wavelength absorption detection method for a liquid chromatograph, which is characterized in that a spectrum of an eluted component is determined by the following steps. (b) Sampling data regarding multiple detection outputs obtained by detection means of multiple wavelengths for liquid chromatograph column elution fluid at arbitrary time intervals, (b) Differentiating the detection outputs for each wavelength, (c) ) Determine whether the differential value is within a certain fluctuation range,
Spectrum S_1 at the baseline time t_1 closest to the rising slope of the peak, the peak falling slope is defined as the baseline time when it is within a certain fluctuation range, and the peak time when it is outside the fixed fluctuation range. Storing the spectrum S_2 at the nearest baseline time t_2 and the spectrum S_0 at an arbitrary time t_0 at the peak time; (e) the two stored baseline spectra S_1;
, calculate the baseline spectrum S_0 at an arbitrary time t_0 at the peak from S_2 from the following formula, S_b=
{S_1(t_2-t_0)+S_2(t_0-t_1
)}/(t_2-t_1) (f) Subtract the obtained baseline spectrum from the already stored spectrum at time t_0.