JP6926628B2 - Automatic method for determining the peak detection sensitivity of the chromatogram - Google Patents

Description

The present invention relates to a method for automatically determining the peak detection sensitivity for each measurement sample for chromatograms of a plurality of measurement samples having different dilution ratios.

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 quantitative analysis, it is necessary to create a calibration curve for the target peak using multiple standard samples with different dilution ratios, and it is important to accurately detect the peak and calculate the area or height of that peak. Become.

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 dilution ratios 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 dilution ratios with a single detection sensitivity, the peak detection sensitivity is set so that the peak detection sensitivity is equal to or less than the peak with the smallest differential value. Must be specified. 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 enables appropriate peak detection by automatically adjusting the peak detection sensitivity for each sample when measuring a plurality of samples having different dilution ratios at the time of preparing a calibration curve.

As a result of diligent studies to solve the above problems, the present inventors have focused on the fact that the first-order differential value of the chromatographic peak changes depending on the dilution ratio of the measurement sample, and the target peak is correctly detected. By correcting the peak detection sensitivity of the reference chromatogram using the dilution ratio of the measurement sample, we have found a method for automatically determining the peak detection sensitivity that does not require the judgment of the operator.

That is, the present invention
A method for preparing a calibration curve by measuring samples with different dilution ratios by chromatography.
The standard dilution factor is the dilution ratio of the sample for which a chromatogram in which a specific peak can be detected is obtained.
The peak detection sensitivity when measuring the sample at the standard dilution ratio is defined as the standard peak detection sensitivity.
When measuring a sample other than the sample having the standard dilution ratio, a value obtained by multiplying the standard peak detection sensitivity by the ratio of the standard dilution ratio to the dilution ratio of the sample is used as the peak detection sensitivity.

The method for creating a calibration curve of the present invention will be described in detail below.

In the calibration curve preparation method of the present invention, as in the general preparation method, a plurality of samples obtained by diluting known samples at different dilution ratios are prepared, chromatographic measurement is performed under the same conditions, and the vertical axis is the concentration and the horizontal axis for each peak. Plot the axis as the peak area. The number of samples to be prepared is at least two, but it is preferably two or more because it affects the accuracy of the calibration curve. The dilution ratio depends on the content of the components contained in the known sample, but there is no problem if it is selected from the range of 1 to 1000 times.

First, the dilution ratio of a sample from which a chromatogram in which a specific peak can be detected is obtained is defined as a standard dilution ratio. The "specific peak" mentioned here can be any one, but it is a component that contains only a small amount in the sample (only a small peak can be obtained on the chromatogram) among the peaks for the purpose of preparing a calibration curve. It is preferable to do so. The sample to be used as the standard dilution ratio is not particularly limited as long as a specific peak can be detected, but it is preferable to select the sample having the lowest dilution ratio among the samples prepared for preparing the calibration curve.

In the present invention, the peak detection sensitivity set when measuring a sample having a standard dilution ratio 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 sample at the standard dilution ratio. Further, 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 and measure the sample at the standard dilution ratio. However, there is no problem if the peak detection sensitivity used when actually measuring the sample at the standard dilution ratio is set as the standard peak detection sensitivity.

After the standard dilution ratio and the standard peak detection sensitivity are determined, the samples other than the sample with the standard dilution ratio are measured. At this time, the value obtained by multiplying the standard peak detection sensitivity by the ratio of the standard dilution ratio to the dilution ratio of the sample to be measured is used as the peak detection sensitivity. For example, when the standard dilution ratio is 10 times, the standard peak detection sensitivity is 2.000 μS / min at the peak start, and -2000 μS / min at the peak end, if the dilution ratio of the sample to be measured is 20 times, the measurement is performed. Since the ratio of the standard dilution ratio (10 times) to the dilution ratio (20 times) of the sample is 0.5 (10 times / 20 times), the standard peak detection sensitivity 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.

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 acquiring the chromatogram, it is possible to appropriately and automatically perform the quantitative calculation by the appropriate peak detection sensitivity, and it is possible to reduce the burden on the operator and improve the reliability of the quantitative calculation. rice field.

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 general measurement sample with a 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 prepared 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.

The effects of the present invention have been verified using an ion chromatography apparatus, but these are embodiments of the present invention and do not limit the present invention.

In this example, the ion chromatography system shown in FIG. 3 was used.

The system includes a solvent degassing device (2), a liquid feed pump (3), a sample injection valve (4), a column oven (6), a suppressor (7), an electrical conductivity detector (8), and a data processing device (10). ).

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 (relative concentration 1.000), 40-fold dilution (relative concentration 0.500), 80-fold dilution (relative concentration 0.250), 160-fold dilution (relative concentration 0.1250), 320 Those diluted 2-fold (relative concentration 0.063) and 640-fold diluted (relative concentration 0.031) were 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
(Example 1)
The method of the present invention was verified using the measurement sample and the ion chromatography system. The calibration curve was prepared for chloride ions (reference elution time: 1.8 to 2.05 minutes).

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 (relative concentration 0.031), 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 sample diluted 20 times was used as the standard dilution ratio, the peak start detection sensitivity was set to 2.000 μS / min, and the peak end detection sensitivity was set to -2000 μS / min standard peak detection sensitivity. Table 1 shows the peak detection sensitivity for each dilution ratio.

６４０倍希釈した試料の測定の際は、標準ピーク検出感度を希釈倍率６４０倍に対する標準希釈倍率２０倍の比０．０３１で乗算し、ピークスタート検出感度を０．０６３μＳ／ｍｉｎ、ピークエンド検出感度を−０．０６３μＳ／ｍｉｎに設定して、測定を行った。その結果、希釈倍率６４０倍の試料でもピークを検出することができ（図５参照）、全ての測定試料の点をプロットした検量線が作成できた（図６参照）。

When measuring a sample diluted 640 times, the standard peak detection sensitivity is multiplied by 0.031, which is a ratio of 20 times the standard dilution ratio to 640 times the dilution ratio, and the peak start detection sensitivity is 0.063 μS / min, and the peak end detection sensitivity. Was set to −0.063 μS / min, and the measurement was performed. As a result, the peak could be detected even in a sample having a dilution ratio of 640 times (see FIG. 5), and a calibration curve plotting the points of all the measurement samples could be created (see FIG. 6).

1. 1. Eluent 2. Degassing device 3. Liquid transfer 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

Claims (1)

A method for preparing a calibration curve by measuring samples with different dilution ratios by chromatography.
The standard dilution factor is the dilution ratio of the sample for which a chromatogram in which a specific peak can be detected is obtained.
It is a threshold value for peak detection, and the threshold value in the differential value of the chromatogram is used as the peak detection sensitivity.
The peak detection sensitivity at the time of peak detection of the sample at the standard dilution ratio is defined as the standard peak detection sensitivity.
When measuring a sample other than the sample having the standard dilution ratio, the value obtained by multiplying the standard peak detection sensitivity by the ratio of the standard dilution ratio to the dilution ratio of the sample is used as the peak detection sensitivity. Method.

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