JP2018185158A - Automatic determination method of peak detection sensitivity of chromatogram - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for automatically determining peak detection sensitivity with respect to chromatogram of multiple measurement samples different in dilution magnification and concentration.SOLUTION: An analytical curve generation method is the method for measuring samples different in dilution magnification by chromatography. In the method, dilution magnification of a sample for obtaining chromatogram that can detect a specified peak is defined as standard dilution magnification, and peak detection sensitivity in measuring a sample of the standard dilution magnification is defined as standard peak detection sensitivity. Then, when a sample other than the sample of the standard dilution magnification is measured, a value obtained by multiplying the standard peak detection sensitivity by a ratio of the standard dilution magnification to dilution magnification of the sample is used as peak detection sensitivity.SELECTED DRAWING: Figure 5

Description

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

  Chromatography is a method for separating and quantifying a sample containing multiple components on a column. Generally, qualitative analysis is performed based on the elution time of each component peak, and quantification is performed based on the output level 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. It is important to accurately detect the peak and calculate the area or height of the peak. Become.

  The detection of the peak is generally performed based on the amount of change in the output of the detector per time, that is, the change in the differential value of the chromatogram. A peak start point and a peak end point are determined using a peak detection sensitivity determined in advance as the threshold value, and a peak height, an 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. When the peak detection result is confirmed and the peak cannot be detected normally, it is necessary to manually change the peak detection sensitivity again.

  FIGS. 1 and 2 show the results of attempting peak detection using a single peak detection sensitivity for standard samples having different dilution ratios. When the peak detection sensitivity is 2.000 μS / min at the peak start and −2,000 μS / min at the peak end, the first derivative 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 that each peak is detected correctly. On the other hand, the first derivative value of the chromatogram shown in FIG. 2a is as shown in FIG. 2b, indicating that neither the peak start point nor the peak end point is detected, and no peak is detected.

  For a chromatogram in which the target peak is not automatically detected, a method is adopted in which the operator manually designates the peak detection sensitivity for each chromatogram. However, the peak detection method depending on the operator changes the analysis result depending on the operator's subjective judgment and work proficiency, so that the quantitativeness of chromatography is impaired and the reliability as a measurement method is lost. 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 should be set so that the differential value is below the smallest peak. Must be specified. In this case, there is a problem that an unnecessary peak due to noise or impurities included in the chromatogram is detected.

  The present invention provides a method capable of automatically adjusting peak detection sensitivity for each sample to enable appropriate peak detection when measuring a plurality of samples having different dilution ratios when preparing a calibration curve.

  As a result of intensive studies to solve the above problems, the present inventors have focused on the fact that the primary differential value of the chromatogram peak varies depending on the dilution factor of the measurement sample, and the target peak is correctly detected. The present inventors have found an automatic determination method of peak detection sensitivity that eliminates the need for operator judgment by correcting the peak detection sensitivity of the reference chromatogram using the dilution factor of the measurement sample.

That is, the present invention
A calibration curve creation method by measuring samples with different dilution ratios by chromatography,
The standard dilution factor is the dilution factor of the sample from which a chromatogram capable of detecting a specific peak is obtained.
The peak detection sensitivity when measuring the sample at the standard dilution factor is the standard peak detection sensitivity,
When measuring a sample other than the sample at the standard dilution factor, a value obtained by multiplying the standard peak detection sensitivity by the ratio of the standard dilution factor to the dilution factor of the sample is used as the peak detection sensitivity.

  Hereinafter, the calibration curve creation method of the present invention will be described in detail.

  In the calibration curve preparation method of the present invention, as in the general preparation method, a plurality of samples prepared by diluting a known sample at different dilution ratios are prepared, and chromatographic measurement is performed under the same conditions. Plot the axis as the peak area. The number of samples to be prepared is at least two. However, since it also affects the accuracy of the calibration curve, it is preferably 2 or more. Further, although the dilution factor depends on the content of components contained in the known sample, there is no problem if it is selected from a range of 1 to 1000 times.

  First, the dilution ratio of a sample from which a chromatogram capable of detecting a specific peak is obtained is determined as the standard dilution ratio. The “specific peak” mentioned here is arbitrary, and there is no problem, but among the peaks for the purpose of creating a calibration curve, only a small amount is contained in the sample (only a small peak on the chromatogram can be obtained) It is preferable to do. The sample used as the standard dilution factor is not particularly limited as long as a specific peak can be detected, but it is preferable to select a sample having the lowest dilution factor among samples prepared for preparing a calibration curve.

  In the present invention, the peak detection sensitivity set when measuring a sample with a standard dilution factor is the standard peak detection sensitivity. If the value specified in advance, such as the default value, is set to a value that will detect unwanted peaks due to noise or impurities, it is preferable to lower the peak detection sensitivity and measure the sample at the standard dilution factor. In addition, when a value specified in advance as a default value or the like is set so that the target peak cannot be detected correctly, it is preferable to increase the peak detection sensitivity and measure the sample at the standard dilution factor. However, there is no problem if the peak detection sensitivity used when actually measuring the sample at the standard dilution factor is the standard peak detection sensitivity.

  Once the standard dilution factor and standard peak detection sensitivity are determined, samples other than the standard dilution factor are then measured. At this time, 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 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 -2,000 μS / min at the peak end, the measurement is performed if the dilution ratio of the sample to be measured is 20 times. Since the ratio of the standard dilution factor (10 times) to the sample dilution factor (20 times) is 0.5 (10 times / 20 times), the standard peak detection sensitivity is multiplied by 0.5, and 1. A peak detection sensitivity of -1.000 μS / min at the peak end may be used.

  The method of the present invention may be implemented by manually changing the peak detection sensitivity calculated automatically by the measurer, and 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 perform quantitative calculation appropriately and automatically by appropriate peak detection sensitivity, and it is possible to reduce the burden on the operator and increase the reliability of the quantitative calculation. It was.

It is the figure which showed the peak detection result with respect to the measurement sample with a general low dilution rate. It is the figure which showed the peak detection result with respect to the measurement sample with a general high dilution rate. It is the figure which showed the system configuration used in Example 1. FIG. It is the figure which showed the calibration curve created using the general peak detection result. It is the figure which showed the peak detection result with respect to the measurement sample with a high dilution rate in this invention. It is the figure which showed the analytical curve created using the peak detection result of this invention.

  Although the effect of the present invention was verified using an ion chromatography apparatus, these are one embodiment 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 analytical column (5), TSKgel SuperIC-Anion HS manufactured by Tosoh Corporation was used.

  Standard anion samples (fluoride ions 1 mg / L, chloride ions 1 mg / L, nitrite ions 5 mg / L, bromide ions 5 mg / L, nitrate ions 5 mg / L, phosphate ions 10 mg / L, sulfate ions 5 mg / L) diluted 20 times (relative concentration 1.000), 40 times diluted (relative concentration 0.500), 80 times diluted (relative concentration 0.250), 160 times diluted (relative concentration 0.1250), 320 Two-fold diluted (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 bicarbonate + 3.0 mM sodium carbonate, suppressor gel: TSKgel suppression 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 using chloride ions (standard 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 20-fold diluted sample was used as the standard dilution factor, the peak start detection sensitivity was 2.000 μS / min, and the peak end detection sensitivity was −2,000 μS / min standard peak detection sensitivity. Table 1 shows the peak detection sensitivity for each dilution factor.

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 a ratio 0.031 of the standard dilution factor 20 times to the dilution factor 640 times, the peak start detection sensitivity is 0.063 μS / min, and the peak end detection sensitivity. Was set to −0.063 μS / min. As a result, it was possible to detect a peak even in a sample with a dilution factor of 640 (see FIG. 5), and to create a calibration curve in which the points of all measurement samples were plotted (see FIG. 6).

1. 1. Eluent 2. Deaeration device 3. Liquid feed pump 4. Sample injection valve Analysis column 6. 6. Column constant temperature bath Suppressor8. Electrical conductivity detector 9. Waste liquid10. System control and data processing equipment

Claims (1)

A calibration curve creation method by measuring samples with different dilution ratios by chromatography,
The standard dilution factor is the dilution factor of the sample from which a chromatogram capable of detecting a specific peak is obtained.
The peak detection sensitivity when measuring the sample at the standard dilution factor is the standard peak detection sensitivity,
When measuring a sample other than the sample at the standard dilution factor, a value obtained by multiplying the standard peak detection sensitivity by a ratio of the standard dilution factor to the dilution factor of the sample is used as the peak detection sensitivity. Method.

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