JP7135689B2 - Peak detection method that is immune to negative peaks - Google Patents

Description

The present invention relates to a method for detecting a target positive peak when negative peaks appear before and after a chromatogram.

Chromatography is a method of separating and detecting a sample containing multiple components with an analytical column to quantify and qualify the components. In general, qualitative determination is performed from the elution time of each component peak, and quantitative determination is performed based on the degree of detector output. For quantitative analysis, it is necessary to determine the peak baseline and calculate the peak area or height from the chromatogram. The baseline can be determined manually by the operator, or by referring to a preset threshold as a peak detection condition based on the amount of change in chromatogram output over time. , or a method of automatically determining the peak end point (see FIG. 1(a)). For example, when the amount of output change monotonously increases and exceeds the peak detection condition A (0<A), it is determined as a peak start, and when the amount of output change monotonously increases and exceeds the peak detection condition B (B<0) There are methods such as determining peak end.

However, when a negative peak appears before or after a positive peak for the purpose of quantification, the above method may not be able to detect the peak correctly (see FIG. 1(b)). In this case, since the same baseline determination condition as in FIG. It was necessary for the operator to set special peak detection conditions such as manually re-specifying the baseline, prohibiting peak detection in a specified time period, and specifying a specified time as the peak start point.

The present invention provides a method for detecting a target positive peak without the influence of the negative peak in a chromatogram in which a negative peak exists in the vicinity of a positive peak that is the target of detection. It is.

In the chromatogram peak processing method according to the present invention,
A negative peak appearing before the target peak prevents the peak start of the target peak from being smaller than the output value of the peak end by more than an arbitrary threshold. The true peak start time is the time at which the amount of change in output is the minimum between the time when the time is multiplied by a factor exceeding 1.0 and the time when the peak top time of the target peak is multiplied by a factor of less than 1.0. and let the peak start time of the target peak be from the true peak start time.
Further, in one aspect of the chromatogram peak processing method according to the present invention, the output value of the peak end of the target peak is arbitrarily higher than the output value of the peak start due to the negative peak appearing behind the target peak. For the target peak smaller than the threshold, from the time obtained by multiplying the peak top time of the target peak by a factor exceeding 1.0 to the time obtained by multiplying the peak end time of the target peak by a factor of less than 1.0 The time at which the amount of change in output is maximized is defined as the true peak end time, and the peak end time of the target peak is defined as the true peak end time.

According to the present invention, in a chromatogram in which a negative-going peak exists in the vicinity of a positive-going peak to be detected, the baseline of the target positive-going peak is determined without the influence of the negative-going peak. became possible.

(a) Results of peak detection using a conventional method in a chromatogram with no negative peaks. (b) Results of peak detection using a conventional method in a chromatogram in which negative peaks are present. FIG. 10 is a diagram showing a difference in output difference between peak start and peak end depending on the presence or absence of a peak in the negative direction; (a) there is no negative peak, (b) there is a negative peak in the front, and (c) there is a negative peak in the rear. It is an example of implementing the present invention on a chromatogram with a peak start anomaly. It is an example of implementing the present invention on a chromatogram with peak end anomalies. 1 is a configuration diagram of an ion chromatograph system used in Example 1. FIG. Fig. 10 shows the implementation of the present invention on Sample 1 to correct the peak start anomaly; FIG. 10 illustrates the implementation of the present invention on sample 2 to correct the peak start anomaly; 1 is a configuration diagram of a size exclusion chromatography system used in Example 2. FIG. FIG. 10 shows the implementation of the present invention on sample 3 to correct the peak start anomaly;

The "target peak" in the present invention means obtaining the amount of output change per time for the chromatogram (hereinafter sometimes simply referred to as "output change amount"), and calculating the output change amount and the predetermined threshold value. Referred to as a peak detection condition, it indicates a specific peak when peak detection is performed by determining the peak start point and peak end point. The amount of change in chromatogram output can be obtained by time differentiation of the chromatogram in the case of continuous data, and can be obtained by time difference in the chromatogram in the case of discrete data. Also, an index of the amount of change in a certain time may be obtained based on the outputs of several points before and after, and the method of obtaining the amount of output change is not particularly limited. If the amount of change in output fluctuates greatly due to noise in the chromatogram, use a means to reduce the noise component, such as applying a filter such as a low-pass filter to the amount of change in output, or obtaining a moving average. can be

The term "negative peak" as used in the present invention refers to a concave peak, which is fluctuation in the chromatogram caused by a decrease in the output signal of the detector due to the elution component.

In the case of positive-going peaks without the influence of negative-going peaks, the output difference between peak start and peak end points is as small as the signal difference caused by baseline drift (Fig. 2a). On the other hand, the positive-going peak ahead of the negative-going peak becomes smaller when the output value at the peak start is larger than the output value at the peak end, and the signal difference caused by the drift of the baseline is larger (Fig. 2b). . In addition, the positive peak behind the negative peak becomes smaller when the output value at the peak end is larger than the output value at the peak start and the signal difference caused by the drift of the baseline (Fig. 2c). . Therefore, the “arbitrary threshold value” in the present invention should be set to a value that can ignore the influence of baseline drift, and is desirably changeable by the operator.

1.0 at the peak start time of the target peak when the peak start output value of the target peak is smaller than the peak end output value by more than an arbitrary threshold due to a negative peak appearing before the target peak to the peak top time of the target peak multiplied by a factor less than 1.0. As an example, in a chromatogram with a negative peak appearing at 3.200 minutes and a positive peak (target peak) appearing at 5.000 minutes, the peak start time is multiplied by 1.100, and the peak FIG. 3A shows the case where the top time is multiplied by 0.950. In this case, the search range is from 3.520 minutes to 4.750 minutes. It can be seen from FIG. 3(b) that the time at which the amount of change in output is the minimum within this search range is 4.450 minutes. Therefore, the "true peak start time" of the target peak is 4.450 minutes. It is clear from FIG. 3(c) that the baseline to be taken for the target peak is from the true peak start time to the peak end time. If there are multiple times when the amount of change in output is minimum, it is preferable to set the latest time as the true peak start time.

In addition, when the output value of the peak end of the target peak is smaller than the output value of the peak start by exceeding an arbitrary threshold due to a negative peak appearing behind the target peak, the peak top time of the target peak is 1 A search is made for the time at which the amount of change in output is maximized, from the time when the peak end time of the target peak is multiplied by a factor of less than 1.0 to the time when the peak end time of the target peak is multiplied by a factor of less than 1.0. As an example, in a chromatogram with a positive peak (target peak) appearing at 5.000 minutes and a negative peak appearing at 6.500 minutes, multiply the peak top time by 1.050 to obtain the peak FIG. 4A shows the case where the end time is multiplied by 0.950. In this case, the search range is from 5.250 minutes to 6.175 minutes. It can be seen from FIG. 4(b) that the time at which the amount of change in output is maximum within this search range is 5.800 minutes. Therefore, the "true peak end time" of the target peak is 5.800 minutes. It is clear from FIG. 4(c) that the baseline to be taken for the target peak is from the peak start time to the true peak end time. If there are multiple times when the amount of change in output is maximum, it is preferable to set the earliest time as the true peak end time.

The coefficient is not limited as long as it exceeds 1.0 or less than 1.0, but it is determined by the separation mode, the shape of the peak to be detected, etc., and it is desirable that the operator can change it. Basically, it is preferable to narrow the allowable range when the peak shape is sharp, and widen the allowable range when the peak shape is broad.

Furthermore, the present invention calculates the difference between the peak start output value and the peak end output value for each peak on the chromatogram, and the peak start output value exceeds the peak end output value by an arbitrary threshold. A small peak and a peak whose output value at the peak end is smaller than the output value at the peak start by exceeding an arbitrary threshold value are detected, and these peaks are processed by the method described above. .

EXAMPLES The embodiments of the present invention will be described in more detail below with reference to Examples. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible for the details. Furthermore, the present invention is not limited to the above-described embodiments, and can be modified in various ways within the scope of the claims. It is included in the technical scope of the invention. In addition, all documents described in this specification are incorporated by reference.

(Example 1)
The method of the present invention was performed using the ion chromatographic system shown in FIG. The system includes a mobile phase and a mobile phase bottle 1, a mobile phase degassing device 2, a mobile phase pump 3, a sample injection part 4, a column oven 5, a suppressor 6, an electrical conductivity detector 7, a waste liquid bottle 8, and a controller. It is composed of a data processing program 9 and the like. TSKgel SuperIC-Anion HS was used as the analytical column 10, TSKgel guardcolumn SuperIC-A HS was used as the guard column 11, TSKgel suppress IC-A was used as the suppressor gel, and IC-2010 was used as the control data processing program 9. Workstation was used (both manufactured by Tosoh Corporation).
In ion chromatography, a solution containing ions is usually used as the mobile phase, but since the water component in the sample is not retained in the separation column, the negative direction of the water component originating from the water component occurs before the ion component appears. A peak appears. In this example, in the chromatogram obtained by analyzing the following sample with this system, the fluoride ion that causes a peak start abnormality that appears immediately after the negative peak derived from the water component was analyzed. Using the method, we verified whether peak detection without the influence of negative peaks is possible.
Sample 1: Anion mixed standard solution 1 manufactured by Fujifilm Wako Pure Chemical Co., Ltd. diluted 10 times with ultrapure water Sample 2: Fujifilm Wako Purechemical Co., Ltd. Anion mixed standard solution 1 diluted with ultrapure water at 1280 2-fold dilution Sample injection volume: 30 μL
Column oven temperature: 40°C
Mobile phase flow rate: 1.5 mL/min
Mobile phase composition: 7.5 mmol/L sodium hydrogen carbonate + 0.8 mmol/L sodium carbonate Sampling pitch: 50 ms
6(a) and 7(a) show the results of detecting the peak of fluoride by the conventional method and designating the baseline. Peak detection was performed under the conditions shown in Table 1.

First, the threshold value was set to 0.050 μS for the chromatogram for which peak detection was completed, and it was confirmed whether there was a peak affected by the peak in the negative direction. The sample 1 has a difference of 1.344 μS, and the sample 2 has a difference of 1.525 μS, indicating that the peak start is abnormal.
Next, the peak start time was multiplied by 1.030 and the peak top time by 0.970 to search for the time at which the amount of change in output was minimized. In sample 1, the peak start time was 0.948 minutes and the peak top time was 1.752 minutes. found to be the smallest. In sample 2, the peak start time was 0.950 minutes and the peak top time was 1.750 minutes. It was found that
Next, the peak start abnormality was corrected by setting the peak start time to the time when the amount of change in the output became the minimum within the search range. By implementing the present invention, the peak start can be corrected correctly for the fluoride ion peak whose baseline has become abnormal due to the previous negative peak, and the correct baseline can be determined without the operator's judgment. It was confirmed that it is possible to obtain Further, it was found that the present invention is capable of detecting peaks that are not affected by peaks in the negative direction with a common set value even for samples that differ in concentration by a factor of 100 or more.

(Example 2)
The method of the present invention was performed using the size exclusion chromatographic apparatus shown in FIG. The chromatographic apparatus comprises a mobile phase and a mobile phase bottle 1, a mobile phase degassing device 2, a mobile phase pump 3, a sample injection part 4, a column oven 5, a differential refractive index detector 12 and the like. GPC-8020 model II was used as the control data processing program 9, and two TSKgel SuperMultipore PW-N (both manufactured by Tosoh Corporation) connected in series were used as the analysis column 10.
In the chromatogram obtained by size exclusion chromatography, after the molecular weight distribution of polymeric materials, additives in the sample, the solvent used to dissolve the sample, and gases unintentionally dissolved in the sample originating from air. A peak in the negative direction may appear due to, for example, In this example, in the chromatogram obtained by analyzing the following samples with this chromatographic apparatus, the peak of polyoxyethylene lauryl ether appearing immediately before the peak in the negative direction was negatively detected using the present invention. We verified whether it is possible to detect peaks that are not affected by directional peaks.
Sample 3: 4.0 g of polyoxyethylene lauryl ether dissolved in 100 mL of a solution of 200 mmol/L sodium nitrate + 20% acetonitrile Sample injection volume: 50 μL
Column oven temperature: 40°C
Mobile phase flow rate: 0.6 mL/min
Mobile phase composition: 200 mmol/L sodium nitrate + 30% acetonitrile Sampling pitch: 50 ms
Fig. 9(a) shows the result of peak detection in the chromatogram obtained by measuring the sample 3 by the conventional method and designating the baseline. Peak detection was performed under the conditions shown in Table 2.

First, the threshold value was set to 0.100 mV for the chromatogram for which peak detection was completed, and it was confirmed whether there was a peak affected by the peak in the negative direction. It was found that there was a difference of 54.848 mV and that the peak end was abnormal.
Next, the peak top time was multiplied by 1.030 and the peak end time by 0.970, and the time at which the amount of change in output was maximized was searched for. In this chromatogram, the peak top time was 7.805 minutes and the peak end time was 9.770 minutes. found to be the maximum.
Next, the peak end abnormality was corrected by setting the peak end time to the time when the amount of output change was maximum. Regarding the polyoxyethylene lauryl ether peak whose baseline was abnormal due to the negative peak immediately after, by carrying out the present invention, the peak end can be corrected correctly, and the correct baseline can be determined by the operator. It has been confirmed that it is possible to obtain it without needing it.

1. Mobile phase and mobile phase bottle2. Mobile phase deaerator3. 4. mobile phase liquid delivery pump; Sample injection section 5 . column oven6. suppressor7. electrical conductivity detector8. waste liquid tank 9 . data processing program 10. analytical column 11 . guard column 12 . Differential refractive index detector

Claims (4)

A chromatogram peak processing method comprising:
For the target peak whose output value at the peak start of the target peak is smaller than the output value at the peak end by more than an arbitrary threshold due to a negative peak appearing before the target peak,
The time at which the amount of change in output is minimized between the time when the peak start time of the target peak is multiplied by a factor exceeding 1.0 and the time when the peak top time of the target peak is multiplied by a factor of less than 1.0. Let be the true peak start time, and
The method, wherein the peak start time of the target peak is taken from the true peak start time. A chromatogram peak processing method comprising:
For the target peak whose output value at the peak end of the target peak is smaller than the output value at the peak start by more than an arbitrary threshold due to a negative peak appearing behind the target peak,
The time at which the amount of change in output is maximized between the time when the peak top time of the target peak is multiplied by a factor exceeding 1.0 and the time when the peak end time of the target peak is multiplied by a factor of less than 1.0. be the true peak-end time, and
The method, wherein the peak end time of the target peak is set to the true peak end time. For each peak on the chromatogram, calculate the difference between the peak start output value and the peak end output value,
The method according to claim 1 is performed for peaks in which the output value at the peak start is smaller than the output value at the peak end by an arbitrary threshold, and the output value at the peak end is less than the output value at the peak start by an arbitrary threshold. 3. A method of processing a chromatogram, wherein the method of claim 2 is performed for peaks smaller than a threshold of . A method according to any one of claims 1 to 3, characterized in that said threshold and/or said coefficient are specified by an operator.

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