JP6904059B2 - Automatic method for determining the peak detection sensitivity of the chromatogram - Google Patents

Description

The present invention relates to a method for automatically determining an appropriate peak detection sensitivity with respect to a chromatogram of a measurement sample.

Chromatography is a method of separating and quantifying a sample containing a plurality of components with a column. In general, qualification is performed from the elution time of each component peak, and quantification is performed based on the output degree of the detector. When performing a quantitative analysis of an unknown sample, it is necessary to prepare a calibration curve of the target peak using a plurality of standard samples having different dilution ratios. In order to realize accurate qualitative and quantitative analysis, it is important to accurately detect peaks in both standard samples and unknown samples and calculate the area or height of the peaks.

The peak is generally detected based on the amount of change in the output of the detector per hour, that is, the change in the differential value of the chromatogram. The peak start point and peak end point are determined using the peak detection sensitivity for which the amount of change is predetermined as a threshold value, and the peak height, area, and the like are calculated.

The peak detection sensitivity is manually changed from the peak detection sensitivity specified as the default threshold value to the optimum peak detection sensitivity for each chromatogram so that all peaks of measurement samples with different concentrations can be detected, and for each chromatogram. It is necessary to check the peak detection result and manually change the peak detection sensitivity again if the peak cannot be detected normally.

Figures 1 and 2 show the results of attempting peak detection using standard samples with different concentrations using a single peak detection sensitivity. When the peak detection sensitivity was set to 2.000 μS / min at the peak start and -2,000 μS / min at the peak end, the first derivative value of the chromatogram shown in FIG. 1a is FIG. 1b, and the peak start point and the peak end point are one point. It is detected one by one, and it can be seen that the peak is detected correctly. On the other hand, the first derivative value of the chromatogram shown in FIG. 2a is shown in FIG. 2b, and neither the peak start point nor the peak end point is detected, indicating that no peak is detected.

For chromatograms in which the target peak is not automatically detected, a method is adopted in which the operator manually specifies the peak detection sensitivity for each chromatogram. However, the operator-dependent peak detection method impairs the quantitativeness of chromatography and loses its reliability as a measurement method because the analysis result changes depending on the operator's subjective judgment and work proficiency. In addition, when trying to automatically detect all target peaks contained in the chromatograms of multiple standard samples with different concentrations with a single detection sensitivity, the peak detection sensitivity is specified so that the peak has the smallest differential value or less. Must. In this case, there is a problem that unnecessary peaks due to noise and impurities contained in the chromatogram are detected.

The present invention provides a method that automatically adjusts the peak detection sensitivity when measuring a sample and enables appropriate peak detection.

As a result of diligent studies to solve the above problems, the present inventors have focused on the fact that the first derivative value of the peak of the chromatogram of the sample changes depending on the height of the chromatogram, and completed the present invention. I arrived.

That is, the present invention
It is a method of analyzing a sample by chromatography.
When the stable point is any time when there is no peak in the chromatogram,
For standard samples with known concentrations of specific components
The reference range is the range of elution time in which the peak related to the specific component can be detected in the chromatogram.
The difference between the maximum output value within the reference range and the output value at the stable point is the standard maximum peak value.
The peak detection sensitivity during measurement is used as the standard peak detection sensitivity.
When analyzing a sample containing the specific component,
The maximum peak value, which is the difference between the maximum output value within the same reference range as the standard sample and the output value at the same or different stable point as the standard sample in the chromatogram of the sample, is calculated.
A value obtained by multiplying the standard peak detection sensitivity by the ratio of the maximum peak value to the standard maximum peak value is used as the peak detection sensitivity.

Hereinafter, the present invention will be described in detail.

The stable point in the present invention means an arbitrary time in which a peak does not exist in the chromatogram. You can select any time in the chromatogram that is on the baseline that is not interfered with by the peaks derived from the measurement sample or impurities, but it is preferable to select the first or last part of the chromatogram. , It is preferable that the determination is automatically made by the device in order to eliminate the subjective judgment of the operator.

The reference range in the present invention refers to the range of elution time at which a peak related to a specific component can be detected in the chromatogram. As the specific component, it is necessary to select a component that is always included, such as an internal standard component and a known component when creating a calibration curve. When preparing a calibration curve, it is preferable to select a component that is contained only in a small amount in the sample. Further, in the case of a sample having only one type of main component or a molecular weight distribution measurement, there is no problem even if the time is such that the entire chromatogram is included in the reference range.

In the present invention, first, a standard sample having a known concentration of a specific component described above is subjected to chromatographic measurement, and the difference between the maximum output value within the reference range and the output value at the stable point of the obtained chromatogram is the standard maximum peak. Calculate as a value. The peak detection sensitivity set at the time of chromatographic measurement of the standard sample is the standard peak detection sensitivity. If the value specified in advance such as the default value is set to a value that detects unnecessary peaks due to noise or impurities, it is preferable to lower the peak detection sensitivity and measure the standard sample. When the value specified in advance such as the default value is set to a value that cannot correctly detect the target peak, it is preferable to increase the peak detection sensitivity for measurement. However, there is no problem if the peak detection sensitivity used when actually measuring the standard sample is set as the standard peak detection sensitivity.

Next, the sample containing the above-mentioned specific component is analyzed. At this time, the maximum output value within the same reference range as the standard sample in the chromatogram of the measurement sample is the same as or different from the standard sample. The maximum peak value, which is the difference from the output value at the stable point, is calculated, and the value obtained by multiplying the standard peak detection sensitivity by the ratio of the maximum peak value to the standard maximum peak value is used as the peak detection sensitivity. For example, when the standard maximum peak value is 2 μS, the standard peak detection sensitivity is 2.000 μS / min at the peak start, and -2000 μS / min at the peak end, if the maximum peak value of the measurement sample is 1 μS, the standard maximum peak Since the ratio of the maximum peak value (1 μS) to the value (2 μS) is 0.5 (1 μS / 2 μS), the standard peak detection sensitivity (2.000 μS / min) is multiplied by 0.5, and the peak start is 1. A peak detection sensitivity of 000 μS / min and -1,000 μS / min at the peak end may be used.

Since it is necessary to compare the specific component in the standard sample with the same component, the reference range needs to be the same as that of the standard sample, but the stability point is not necessarily the same as that of the standard sample for the measurement sample. Since it is not always the stable point, it may be the same as the standard sample or it may be different. When the reference range is set to a time that includes the entire chromatogram, there must be no component contained in the measurement sample in a larger amount than the specific component. In this case, it is necessary to reset the reference range.

The present invention can also be suitably used for producing a calibration curve. Select a sample obtained by diluting the standard sample as the measurement sample, calculate the maximum peak value in the same reference range and stable point as the standard sample, and as described above, set the ratio of the maximum peak value to the standard maximum peak value as the standard peak. The value obtained by multiplying the detection sensitivity may be used as the peak detection sensitivity.

The method of the present invention may be carried out by manually changing the peak detection sensitivity automatically calculated by the measurer himself, or there is no problem even if the apparatus automatically changes the peak detection sensitivity by a program. ..

In the present invention, after the chromatogram is acquired, the quantitative calculation can be appropriately and automatically performed by the appropriate peak detection sensitivity, and the burden on the operator can be reduced and the reliability of the quantitative calculation can be improved. It was.

It is a figure which showed the peak detection result with respect to the measurement sample which a general dilution ratio is low. It is a figure which showed the peak detection result with respect to the measurement sample with a general high dilution ratio. It is a figure which showed the system configuration used in Example 1. FIG. It is a figure which showed the calibration curve which made using the general peak detection result. It is a figure which showed the peak detection result with respect to the measurement sample with a high dilution ratio in this invention. It is a figure which showed the calibration curve prepared using the peak detection result of this invention. It is a figure which showed the system configuration used in Example 2. It is a figure which showed the chromatogram obtained in Example 2. The peak detection result by the conventional method and the peak detection result (# 7 to # 4) in the present invention are shown. The peak detection result by the conventional method and the peak detection result (# 3 to # 1) in the present invention are shown. This is a comparison of the area of the peak detected by the conventional method and the area detected by the method of the present invention. This is a comparison between the integrated molecular weight distribution curve of the peak detected by the conventional method and the integrated molecular weight distribution curve of the peak detected by the present invention method.

Hereinafter, the present invention will be described with reference to examples, but these are embodiments of the present invention and do not limit the present invention.

(Example 1)
The method of the present invention was verified to produce a calibration curve by measuring a plurality of standard substances using the ion chromatography system shown in FIG. The calibration curve was prepared for chloride ions (reference elution time: 1.8 to 2.05 minutes).

The system consists of a solvent degassing device (2), a liquid feed pump (3), a sample injection valve (4), a column oven (6), a suppressor (7), and an electrical conductivity detector (8). The data processing apparatus (10) used the data processing program IC-2010 Workstation manufactured by Tosoh Corporation.
As the analysis column (5), TSKgel SuperIC-Anion HS manufactured by Tosoh Corporation was used.

The measurement samples are standard anion samples (fluoride ion 1 mg / L, chloride ion 1 mg / L, nitrite ion 5 mg / L, bromide ion 5 mg / L, nitrate ion 5 mg / L, phosphate ion 10 mg / L, sulfate ion. 5 mg / L) 20-fold dilution (concentration 1), 40-fold dilution (concentration 2), 80-fold dilution (concentration 3), 160-fold dilution (concentration 4), 320-fold dilution (concentration 5), 640-fold dilution (concentration 5) 6) Dilution was used.
Other conditions are as follows.
Injection volume: 30 μL, column oven temperature: 40 ° C., eluent flow rate: 1.5 mL / min, eluent: 3.8 mM sodium hydrogen carbonate + 3.0 mM sodium carbonate, suppressor gel: TSKgel supress IC-A.

First, according to the conventional method, the sample was measured by setting the peak start detection sensitivity, which is a single detection sensitivity, to 2.000 μS / min and the peak end detection sensitivity to -2,000 μS / min. As a result, in the sample diluted 640 times (concentration 6), the peak could not be detected and could not be plotted as a calibration curve (see FIG. 4).

Next, a calibration curve was prepared by the method of the present invention. The elution time at which chloride ions appear is set in the reference range of 1.8 to 2.05 minutes, a stable point (7.0 minutes, which is the last part of the chromatogram) is selected from the baseline, and a sample with a concentration of 1 is standardized. The standard maximum peak value as a sample and the maximum peak value of a sample having a concentration of 2 to 6 were calculated, respectively. The peak start detection sensitivity of 2.000 μS / min and the peak end detection sensitivity of -2000 μS / min are set as the standard peak detection sensitivity, and Table 1 shows the peak detection sensitivity of each sample calculated using this method.

When measuring a sample with a concentration of 6, the standard peak detection sensitivity is multiplied by the ratio of the maximum peak value of the sample with a concentration of 6 to the standard maximum peak value (0.023), and the peak start detection sensitivity is 0.046 μS / min. The measurement was performed with the end detection sensitivity set to −0.046 μS / min. As a result, a peak could be detected even in a sample having a concentration of 6 which could not be detected by the conventional method (see FIG. 5), and a calibration curve plotting the points of all the measurement samples could be created (see FIG. 6).

(Example 2)
In this example, the chromatography system shown in FIG. 7 was used and verified by a system for measuring the molecular weight of a polymer component by molecular exclusion chromatography (GPC).

The system includes a solvent degassing device (2), a sample side liquid feed pump (11), a reference side liquid feed pump (12), a sample injection valve (4), a column oven (6), and a differential refractometer detector (13). , Consists of a data processing device (10). As the data processing device (10), a multi-station GPC-8020II manufactured by Tosoh Corporation was used.
As the analysis column (5), two TSKgel G-Oligo-PWs (7.8 mm ID × 30 cm) manufactured by Tosoh Corporation were connected in series and used.

The measurement sample is polyethylene glycol: average molecular weight 1000 (manufactured by Wako Pure Chemical Industries, Ltd.) 250 mg / mL (# 7), diluted 2-fold (# 6), 4-fold diluted (# 5), 8-fold diluted. (# 4), 16-fold dilution (# 3), 32-fold dilution (# 2), and 64-fold dilution (# 1) were used (see Table 2).
Other conditions are as follows.
Injection volume: 50 μL, column oven temperature: 40 ° C., eluent flow rate: 1.0 mL / min (sample side), 0.5 mL / min (reference side), eluent: 200 mM sodium nitrate aqueous solution

FIG. 8 is a diagram showing the obtained chromatogram. The legend in the figure shows measurement #.

First, the sample of # 7 was used as a standard, and the peak detection conditions including the peak detection sensitivity were set.
The peak detection conditions of the data processing apparatus used in this embodiment are as follows.
[Detection sensitivity]: 2.000 mV / min (chromatogram first derivative threshold for peak detection)
[Base judgment value]: 0.000 mV / min (Judgment value of whether the detected peak is a "base peak" or a "valley peak")
[Excluded area value]: 0.000 mV x sec (peak area threshold not judged to be a peak)
[Exclusion height]: 0.020 mV
(Peak height threshold that is not judged to be a peak)
[Exclusion half width]: 0.000 seconds (peak half width threshold not judged to be a peak)
The reference range is chromatogram 0.0 to 25.0 min, the maximum peak value of # 7 is the standard maximum peak value, and the peak start detection sensitivity is 2.000 mV / min and the peak end detection sensitivity is -2000 mV / min. Sensitivity. Table 3 shows the peak detection sensitivity of each sample calculated using this method.

For example, in the # 4 chromatogram, the maximum output with time zero as the stable point is 11.834 mV, and the ratio of the maximum peak value (11.834 mV) of the sample to the standard maximum peak value (58.334 mV) is 0. It becomes 203 (11.834 / 58.334), and the peak detection sensitivity is calculated as 0.406 mV / min (2.000 mV / min × 0.203).

The peak detection sensitivities calculated as described above are applied to each chromatogram, and the results obtained by performing peak detection are shown in FIGS. 9b and 10b.

For comparison with the conventional method, the peak detection sensitivity is set to 2.000 mV / min, applied to all chromatograms, and the results of peak detection are shown in FIGS. 9a and 10a. The peak start point and peak end point are indicated by black circles in the figure.

As is clear from FIGS. 9 and 10, when the peak detection sensitivity is set to a constant value, the peak start point is delayed from the normal time or the peak end point is delayed from the normal time as the peak intensity (concentration) decreases. The phenomenon of accelerating becomes remarkable. When the peak detection sensitivity according to the peak intensity is used by the method of the present invention, the peak start point and the peak end point are normally detected even if the peak intensity (concentration) becomes small.

The purpose of the GPC shown in this example is to obtain the molecular weight distribution of an unknown sample. Since the start side and the end side of the peak represent the vicinity of the maximum value and the vicinity of the minimum value in the molecular weight distribution of the unknown sample, respectively, it is necessary to accurately specify the start position and the end position of the broad polymer component peak. Therefore, after the peak detection is automatically executed, if the peak start position and the end position are inappropriate, trial and error is performed so that the peak detection condition can be changed again to perform accurate peak detection. If an appropriate peak cannot be detected by any trial and error, it is finally necessary to manually identify the peak start position and the end position at the operator's discretion. In particular, when the peak height changes or the elution position changes, accurate peak detection is often not performed.

FIG. 11 is a diagram showing the difference between the peak start point obtained by the above operation and the area value obtained from the peak end point. The area% obtained by the conventional method (fixed peak detection sensitivity) was calculated and plotted, assuming that the area obtained by the detection method (variable peak detection sensitivity) of the present invention was 100. The horizontal axis is the logarithmic value of the concentration, the black triangle indicates the conventional method (fixed peak detection sensitivity), and the white circle indicates this method (variable peak detection sensitivity). As is clear from this figure, it can be seen that even for samples of all concentrations, when the peak detection method is performed by the conventional method (fixed peak detection sensitivity), the size is reduced by up to 25%.

FIG. 12 is an integrated molecular weight distribution curve created based on the results obtained in FIGS. 9 and 10. The horizontal axis is the molecular weight and the vertical axis is the ratio of the integral distribution. The broken line in the legend is the integrated molecular weight distribution curve when the peak is detected with the peak detection sensitivity fixed by the conventional method, and the solid line in the legend is the integrated molecular weight distribution curve when the peak is detected by making the peak detection sensitivity variable according to the present invention. .. As can be seen from this, it can be seen that the present invention can more accurately obtain the vicinity of the maximum value and the vicinity of the minimum value in the molecular weight distribution of the peak as compared with the conventional method.

1. 1. Eluent 2. Degassing device 3. Liquid feed pump 4. Sample injection valve 5. Analytical column 6. Column constant temperature bath 7. Suppressor 8. Electrical conductivity detector 9. Waste liquid 10. System control and data processing equipment 11. Liquid feed pump (sample side)
12. Liquid feed pump (reference side)
13. Differential refractometer

Claims (4)

It is a method of analyzing a sample by chromatography.
When the stable point is any time when there is no peak in the chromatogram,
For standard samples with known concentrations of specific components
The reference range is the range of elution time in which the peak related to the specific component can be detected in the chromatogram.
The difference between the maximum output value within the reference range and the output value at the stable point is the standard maximum peak value.
The peak detection sensitivity during measurement is used as the standard peak detection sensitivity.
When analyzing a sample containing the specific component,
The maximum peak value, which is the difference between the maximum output value within the same reference range as the standard sample and the output value at the same or different stable point as the standard sample in the chromatogram of the sample, is calculated.
An analysis method characterized in that a value obtained by multiplying the standard peak detection sensitivity by the ratio of the maximum peak value to the standard maximum peak value is used as the peak detection sensitivity. The analysis method according to claim 1, wherein the stable point is automatically determined as the first part or the last part of the chromatogram. The analysis method according to claim 1 or 2, wherein the reference range is the entire chromatogram. The sample containing the specific component is a sample obtained by diluting the standard sample.
The maximum peak value, which is the difference between the maximum output value within the same reference range as the standard sample and the output value at the same stable point as the standard sample in the chromatogram of the sample, is calculated.
A method for creating a calibration curve, which comprises performing the analysis method according to any one of claims 1 to 3.

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