JP6583159B2 - Method for measuring concentration by chromatograph and chromatograph - Google Patents

Description

  The present invention relates to an apparatus for measuring the concentration of a component contained in a sample using a chromatograph or a mass spectrometer, and more particularly to a technique for creating a calibration curve therefor.

  When measuring the concentration of various components contained in a sample, first, each component is temporally separated by a gas chromatograph or liquid chromatograph, and then the peak area and height appearing in the chromatograph for each separated component. There is a method of measuring the content (concentration) by, for example. The chromatographic peak is detected using a spectroscopic device, a mass spectrometer, etc., and its area and the like are measured. In order to determine the actual concentration value of the component in the sample, a known concentration is used. By analyzing a sample (standard sample), the relationship between the concentration value and the value of the peak area detected and measured by a spectroscope or a mass spectrometer must be obtained in advance. The relationship between the two is generally represented by a line called a “calibration curve” and stored. When a sample containing a component having an unknown concentration is analyzed under the same conditions, the concentration of the component can be determined by measuring the area of the peak appearing in the chromatograph and applying it to the calibration curve.

  The calibration curve differs for each apparatus such as a chromatograph, a spectroscopic apparatus, and a mass spectrometer, but generally does not form a straight line but presents a curve specific to these apparatuses. Therefore, it is not appropriate to measure a single standard sample and create a calibration curve.

  Therefore, in Patent Document 1, a mass spectrometer is used as a detector, a substance containing an element containing a plurality of stable isotopes is used as a standard sample, and two or more different concentrations (isotope abundance ratios) in one analysis. A method of obtaining a calibration curve by obtaining a measurement point of (concentration corresponding to 1) is disclosed.

JP 2000-065797

In the method described in Patent Document 1, two or more measurement points for creating a calibration curve can be obtained by one measurement of one type of standard sample, but in general, an element contained in a substance to be analyzed Even when a plurality of stable isotopes are present, the abundance ratio is largely biased. For example, hydrogen has a mass number of ¹ H of 99.984%, whereas mass 2 of ² H is 0.016%, carbon has ¹² C of 98.894%, whereas ¹³ C has 1.106%, and nitrogen has ¹⁴ %. N is ¹⁵ N is 0.366% whereas a 99.634%, oxygen ¹⁶ O is ¹⁷ O while a 99.762% is 0.038% ¹⁸ O becomes 0.200%. Therefore, when a standard sample is prepared so that the isotope with the higher abundance ratio is at an appropriate concentration for creating a calibration curve, the concentration of the isotope with the lower abundance ratio is extremely small, and the chromatogram A reasonable point cannot be constructed in the calibration curve because the peak is buried in noise. On the other hand, even if an attempt is made to prepare a standard sample based on an isotope having a smaller abundance ratio, the concentration of the isotope having a larger abundance ratio exceeds 100%, so that the production is impossible.

  Moreover, the method described in Patent Document 1 must be based on the use of a mass spectrometer as a detector, and cannot be applied to a general detector such as a spectrometer.

  The problem to be solved by the present invention is to provide a method and apparatus for creating a calibration curve that can determine the concentration of a component of an unknown sample as accurately as possible, reflecting the characteristics of the measurement apparatus as much as possible.

The concentration measurement method by the chromatograph according to the present invention made to solve the above problems is as follows.
a) preparing a plurality of samples having different concentrations of the target component;
b) passing the plurality of samples through chromatographic columns in different time zones;
c) measuring each sample passed through the column with a detector capable of measuring with a plurality of parameter values, obtaining a chromatogram for each sample and for each parameter;
d) preparing a calibration curve for the target component from the chromatogram obtained for each sample and each parameter by eliminating peaks with an intensity exceeding a predetermined standard or an intensity falling below another predetermined standard. Features.

  In the calibration curve creation step described above, peaks are detected from the chromatogram according to a predetermined standard, the intensity value of each peak is obtained, and a calibration curve is created by excluding excessive and excessive peaks from the peak intensity values. Here, the peak intensity refers to a parameter having a correlation with the concentration of the target component, such as a peak height or a peak area.

  In the concentration measurement method according to the present invention, since a plurality of samples having different concentrations are measured with a plurality of parameter values in the chromatograph, it is possible to obtain measurement values at many points of known concentrations for the target component. . If there is a point that is not appropriate for constructing a calibration curve because the intensity is too high or too low, remove the detector and create a calibration curve. It is possible to create a calibration curve that sufficiently reflects the characteristics of the chromatograph including it. By using this calibration curve, the concentration of the unknown sample containing the target component can be correctly determined over a wide range from a low concentration to a high concentration.

The chromatograph according to the present invention for carrying out the above method,
a) a column;
b) a detector capable of measuring the sample that has passed through the column with values of a plurality of parameters;
c) a chromatogram creation unit that creates a chromatogram for each parameter value based on the detection signal from the detector;
d) From a plurality of chromatograms obtained as a result of passing a plurality of samples having different concentrations of the target component through the column in different time zones, peaks having an intensity exceeding a predetermined standard or an intensity falling below another predetermined standard And a calibration curve creation unit that creates a calibration curve of the target component by eliminating.

Furthermore, the chromatographic program according to the present invention for carrying out the above method is a program used for a chromatograph including a detector capable of measuring with a plurality of parameter values,
a) a chromatogram creation unit that creates a chromatogram for each parameter value based on the detection signal from the detector;
b) Intensities exceeding a predetermined standard or lower than another predetermined standard from a plurality of chromatograms obtained by passing a plurality of samples having different concentrations of target components through the chromatographic column in different time zones And a calibration curve creation unit that creates a calibration curve of the target component by eliminating the intensity peak.

  In the concentration measurement method according to the present invention, since a plurality of samples having different concentrations are measured with a plurality of parameter values in the chromatograph, it is possible to obtain measurement values at many points of known concentrations for the target component. . If there is a point that is not appropriate for constructing a calibration curve because the intensity is too high or too low, remove the detector and create a calibration curve. It is possible to create a calibration curve that sufficiently reflects the characteristics of the chromatograph including it. By using this calibration curve, the concentration of the unknown sample containing the target component can be correctly determined over a wide range from a low concentration to a high concentration.

The schematic block diagram which shows one Embodiment of the chromatograph which concerns on this invention. Created from the chromatogram examples (a-1) and (a-2) and the peak intensities of the chromatograms that are created based on the data obtained by the chromatograph of the present embodiment and have different standard sample concentrations. The graph which shows the example (b) of a calibration curve. Examples of chromatograms (a-1) to (a-3) created based on data obtained by the chromatograph of the present embodiment and including peaks with different standard sample concentrations and peak intensities lower than a predetermined lower limit value ) And an example (b) of a calibration curve created from the peak intensities of the chromatograms. Examples of chromatograms (a-1) to (a-3) that are created based on data obtained by the chromatograph of the present embodiment and include peaks with different standard sample concentrations and peak intensities higher than a predetermined upper limit. ) And an example (b) of a calibration curve created from the peak intensities of the chromatograms. Examples of chromatograms (a-1) and (a-2) with different standard sample concentrations created when a mass spectrometer is used as a detector, and calibration curves created from the peak intensities of these chromatograms The graph which shows the example (b).

  An embodiment of a concentration measurement method, a chromatograph, and a chromatographic program according to the present invention will be described with reference to FIGS. In the present embodiment, a high-performance liquid chromatograph characterized by a configuration for concentration measurement, and a concentration measurement method and chromatograph program implemented in the chromatograph will be described as examples.

  FIG. 1 is a diagram showing a schematic configuration of a high performance liquid chromatograph 10 of the present embodiment. The high-speed liquid chromatograph 10 includes a mobile phase container 11, a liquid feed pump 12, a sample injection unit 13, a column 14, a detector 15, a data processing unit 16, and a control unit 17.

  The mobile phase container 11 is a container that stores a mobile phase, and the liquid feed pump 12 is a pump that sucks the mobile phase in the mobile phase container 11 and feeds it at a constant flow rate. The sample injection unit 13 selects one liquid sample from a plurality of liquid samples such as a standard sample and an unknown sample, and performs a pretreatment such as dilution or concentration on the sample if necessary, and then a liquid feed pump 12 is injected into the mobile phase fed from 12. The column 14 temporally separates the components contained in the sample while the sample injected into the mobile phase passes.

  The detector 15 is a detector using a spectroscopic measurement device, and includes a light source 151, a lens 152, a sample cell 153, a slit 154, a concave diffraction grating 155, and a photodiode array 156. The light source 151 generates light having a large number of wavelengths within a predetermined wavelength band. The lens 152 condenses the light from the light source 151 on the sample cell 153. In the sample cell 153, the sample introduced from the column 14 together with the mobile phase passes, and the light from the light source 151 is transmitted. In the sample cell 153, the sample is irradiated with light from the light source 151, and light having a wavelength determined by the components of the sample is absorbed. The light that has passed through the sample cell 153 is wavelength-dispersed by the concave diffraction grating 155 after passing through the slit 154. The photodiode array 156 includes a large number (for example, 1024) of detection elements arranged in a one-dimensional array. For each detection element, light having a specific wavelength out of the light wavelength-dispersed by the concave diffraction grating 155. Is detected. Thus, the detection intensity for each wavelength is obtained in the photodiode array 156.

  The data processing unit 16 is embodied by a central processing unit of a computer and a program (embodiment of the chromatographic program according to the present invention), and includes a chromatogram creation unit 161, a calibration curve creation unit 162, and an unknown A sample concentration determination unit 163 is included. The function of each part will be described later together with the description of the operation of the high performance liquid chromatograph 10.

  The control unit 17 controls operations such as suction and delivery of a mobile phase by the liquid feed pump 12, sample injection by the sample injection unit 13, and light generation in the light source 151 of the detector 15.

  Hereinafter, the operation of the high performance liquid chromatograph 10 and the embodiment of the concentration measuring method according to the present invention will be described. In this high-performance liquid chromatograph 10, a calibration curve is created as follows based on the measurement results of a plurality of standard samples having different concentrations (the concentrations are known), and then an unknown sample is measured and based on the calibration curve. Thus, the components of the unknown sample are obtained quantitatively. Here, the operation of measuring the unknown sample is the same as that of the conventional high-performance liquid chromatograph. Therefore, the following description will focus on the operation when creating a calibration curve based on the measurement results of a plurality of standard samples having different concentrations.

  First, while the mobile phase is sucked from the mobile phase container 11 by the liquid feed pump 12 and fed to the sample injection unit 13, the sample injection unit 13 has a known component content and a predetermined first concentration. A standard sample prepared as described above is injected into the mobile phase. Here, the standard sample may be prepared by an autosampler or an analyst. While the standard sample injected into the mobile phase passes through the column 14, the components contained in the sample are temporally separated. Each component reaches the sample cell 153 of the detector 15 with a time difference, and is detected at a different time (holding time) for each component as follows.

  In the detector 15, light having a large number of wavelengths within a predetermined wavelength band emitted from the light source 151 passes through the sample cell 153 and has a specific wavelength determined by the components of the standard sample existing in the sample cell 153 at that time. As described above, only light is absorbed and wavelength-dispersed by the concave diffraction grating 155, and the intensity is detected by the photodiode array 156 for each wavelength. The chromatogram creation unit 161 creates a chromatogram for each wavelength by obtaining data of the temporal change in detection intensity for each wavelength from the photodiode array 156.

  At the timing after all the components of the standard sample of the first concentration have passed through the sample cell 153 of the detector 15, the sample injection unit 13 is a standard sample prepared so as to have a predetermined second concentration different from the first concentration. Is injected into the mobile phase. The method for preparing the standard sample is the same as that for the first concentration. And the chromatogram for every wavelength is created about the standard sample of the 2nd concentration by the same method as the standard sample of the 1st concentration. Hereinafter, if necessary, the same operation is performed for one or more standard samples having a concentration different from the first concentration and the second concentration (three or more in combination with the standard samples of the first concentration and the second concentration).

  Fig. 2 (a-1) shows an example of a chromatogram created from the detected intensity obtained from the measurement of the standard sample of the first concentration. Fig. 2 (a-2) shows the measurement of the standard sample of the first concentration. An example of the chromatogram created from the detected intensity obtained in step 1 is shown. (a-1) and (a-2) show four chromatograms created for each wavelength based on the detected intensity of the photodiode array 156 obtained at four different wavelengths. . Of these four chromatograms, the two chromatograms labeled “CH1” and “CH2” in the figure show different amounts of light absorbed by the same component (referred to as “component A”) in the standard sample. It is the chromatogram created from the detection intensity of one wavelength. The peaks in the chromatograms of CH1 and CH2 have the same retention time, and their intensities (peak top height and peak integrated intensity (area)) are different. In addition, the two chromatograms labeled “CH3” and “CH4” in the figure show the detection intensities at two different wavelengths of light absorbed by the same component (component B) in a standard sample different from component A. It is the chromatogram created from each. The peaks of the chromatograms of CH3 and CH4 have the same retention time (different from the retention time of component A) and have different intensities.

  The calibration curve creation unit 162 determines the CH1 to CH4 for each of the CH1 to CH4 from the intensity of the CH1 to CH4 chromatograms obtained from the different (a-1) and (a-2) standard sample concentrations. As shown in FIG. 2 (b), a calibration curve showing the relationship between the concentration of the standard sample and the intensity of the obtained peak is prepared. In FIG. 2 (b), the horizontal axis is the concentration of the standard sample, and the points of the obtained peak intensity values are added to the values of the first concentration and the second concentration. However, the concentration of the standard sample is known. In addition, since the contents of components A and B in the standard sample are also known, the horizontal axis can be converted into the concentration of the components.

  When the unknown sample is quantified using the calibration curve thus obtained, the chromatogram creation unit 161 is obtained with the wavelengths (CH1 to CH4) detected by the photodiode array 156 by measuring the unknown sample. A chromatogram is created based on the detected intensity. Then, the unknown sample concentration determination unit 163 obtains the concentration of the component corresponding to the wavelength by reading the concentration at the peak intensity of the chromatogram obtained at the wavelength from the calibration curve at the wavelength. Here, one component can be measured with a plurality of wavelengths (CH1 and CH2 for component A, CH3 and CH4 for component B). Therefore, by averaging the concentration values obtained at the plurality of wavelengths, the accuracy can be made higher than when the concentration is obtained using only a calibration curve of one wavelength.

  In the examples shown in FIGS. 2 (a-1) and (a-2), none of the peaks in the chromatogram is so small that it is difficult to detect and is not so large that the peak is saturated. Therefore, all the peaks of the obtained chromatogram were used for preparing a calibration curve. On the other hand, if the peak intensity is too weak as in the latter of the chromatograms of CH1 and CH2 shown in FIG. 3 (a-1), the value of the peak intensity cannot be obtained accurately. Therefore, for each of CH1 and CH2, in addition to FIG. 3 (a-1), a chromatogram is prepared using a standard sample having a higher concentration as shown in (a-2) and (a-3), A calibration curve is created using the peak intensities obtained from these three chromatograms that are larger than a predetermined lower limit. An example of the created calibration curve is shown in FIG. In CH1, a calibration curve was created using all peak intensities obtained from the three chromatograms, whereas in CH2, the peak intensity lower than the lower limit (x mark in the figure) was excluded and the rest A calibration curve is created using two points. Data whose peak intensity is lower than the lower limit is low in accuracy, so it is out of the calibration curve created using the remaining two points, and including this point makes the calibration curve inaccurate. In this case, the accuracy of the calibration curve is prevented from being lowered by excluding this point.

  If the concentration of the component at the time of measurement is too high, a signal indicating the detection intensity of the wavelength corresponding to the component of the photodiode array 156 of the detector 15 is saturated. Then, as shown in FIG. 4 (a-3), the peak of the chromatogram is also saturated. Since the intensity of such a saturated peak is smaller than the true peak intensity, its accuracy is lowered when it is used to create a calibration curve. Therefore, in addition to Fig. 4 (a-3), a chromatogram was created using standard samples with lower concentrations as shown in (a-1) and (a-2), and these three chromatograms were obtained. A calibration curve is created using a peak intensity smaller than a predetermined upper limit value indicating that the peak is not saturated. An example of the created calibration curve is shown in FIG. In CH4, a calibration curve was created using all peak intensities obtained from the three chromatograms, whereas in CH3, peak intensity higher than the upper limit (marked with x in the figure) was excluded and the rest A calibration curve is created using two points. If this x-marked point is included, the calibration curve becomes inaccurate, but in this embodiment, the accuracy of the calibration curve is prevented from being lowered by excluding this point.

The present invention is not limited to the above embodiment.
For example, in the above embodiment, a detector including a photodiode array is used, but instead, a mass spectrometer may be used as a detector. In that case, using a plurality of different mass-to-charge ratios m / z as parameters, a chromatogram is created for each of the plurality of standard samples having different concentrations for each mass-to-charge ratio m / z. Here, in order to obtain a plurality of different mass-to-charge ratios m / z, the fact that the mass-to-charge ratios m / z are different depending on the presence of an isotope may be used (FIGS. 5A-1 and 5A). -2)), ions of the target component may be cleaved in a mass spectrometer. Based on the created chromatogram, a calibration curve of the target component can be created (FIG. 5 (b)).

  In the above-described embodiment, the case of the high performance liquid chromatograph has been described as an example. However, the same configuration for concentration measurement, concentration measurement method, and chromatograph program can be applied to the gas chromatograph.

DESCRIPTION OF SYMBOLS 10 ... High performance liquid chromatograph 11 ... Mobile phase container 12 ... Liquid feed pump 13 ... Sample injection part 14 ... Column 15 ... Detector 151 ... Light source 152 ... Lens 153 ... Sample cell 154 ... Slit 155 ... Concave diffraction grating 156 ... Photodiode Array 16 ... Data processing unit 161 ... Chromatogram creation unit 162 ... Calibration curve creation unit 163 ... Unknown sample concentration determination unit 17 ... Control unit

Claims (6)

a) preparing a plurality of samples having different concentrations of the target component;
b) passing the plurality of samples through chromatographic columns in different time zones;
c) measuring each sample passed through the column with a detector capable of measuring with a plurality of parameter values, obtaining a chromatogram for each sample and for each parameter;
d) preparing a calibration curve for the target component from the chromatogram obtained for each sample and each parameter by eliminating peaks with an intensity exceeding a predetermined standard or an intensity falling below another predetermined standard. Concentration measurement method using a characteristic chromatograph.   The concentration measuring method according to claim 1, wherein the parameter is a wavelength of light measured by a spectrometer. a) a column;
b) a detector capable of measuring the sample that has passed through the column with values of a plurality of parameters;
c) a chromatogram creation unit that creates a chromatogram for each parameter value based on the detection signal from the detector;
d) From a plurality of chromatograms obtained as a result of passing a plurality of samples having different concentrations of the target component through the column in different time zones, peaks having an intensity exceeding a predetermined standard or an intensity falling below another predetermined standard And a calibration curve creation unit that creates a calibration curve of the target component by eliminating the above.   The chromatograph according to claim 3, wherein the detector is a detector using a spectroscopic measurement device, and the parameter is a wavelength of light measured by the spectroscopic measurement device. A program used for a chromatograph equipped with a detector capable of measuring a plurality of parameter values,
a) a chromatogram creation unit that creates a chromatogram for each parameter value based on the detection signal from the detector;
b) Intensities exceeding a predetermined standard or lower than another predetermined standard from a plurality of chromatograms obtained by passing a plurality of samples having different concentrations of target components through the chromatographic column in different time zones A chromatograph program comprising: a calibration curve creation unit that creates a calibration curve of a target component by eliminating an intensity peak.   6. The chromatographic program according to claim 5, wherein the detector is a detector using a spectroscopic measurement device, and the parameter is a wavelength of light measured by the spectroscopic measurement device.

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