JPS61117451A - Analyzing method of sample component by high-speed liquid chromatography using absorbance detector - Google Patents

Abstract

PURPOSE:To identify easily a sample by detecting the absorbance with the light of a wavelength at which an eluate indicates the absorbance of a specified level as a detection wavelength and obtaining a chromatogram indicating the difference of the absorbance between the eluate and sample component. CONSTITUTION:An eluate tank 1, a pipe 2, a liquid feed pump 3, a sample injector 4, a column 5, an absorbance detector 6, a waste liquid tank 7 and a recorder 8 are provided. The eluate lifted from the tank 1 by the pump 3 is discharged through the pipe 2 to the tank 7. The sample is injected from the injector 4 into the eluate during the course of said discharge and is subjected to sepn. and elution in the elution time intrinsic to components by the interaction in the stage of passing through the column 5 and passes through the detector 6. The detector 6 detects the change of the absorbance of the eluate with time and applies the same to the recorder 8 so that the change is recorded by the recorder 8. The simultaneous analysis of the sample is thus made possible.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for analyzing sample components by high-performance liquid chromatography using an absorbance detector, and in particular, a method for analyzing sample components by high-performance liquid chromatography using an absorbance detector. The present invention relates to a method for analyzing sample components by high-performance liquid chromatography using an absorbance detector designed to obtain a chromatogram that shows the difference in chromatograms.

[Conventional technology]

High performance liquid chromatography using an absorbance detector is currently indispensable as a method for quantitatively and qualitatively determining components contained in a sample.゛As is well known, in high-performance liquid chromatography using an absorbance detector, when the sample is injected and the eluent is passed through the column, each component elutes at its own elution time due to differences in interactions within the column. When the eluate from this column is fed to an absorbance detector, a chromatogram is obtained with time as the horizontal axis and the absorbance of the eluate as a variable.This chromatogram can be used to quantify and qualitatively analyze the components contained in the sample. It can be used as a material for carrying out.

In high-speed liquid atomography using a conventional absorbance detector, the detection wavelength is the wavelength at which a component expected to be contained in the sample exhibits absorption, and elution that does not exhibit absorption at this detection wavelength is used. Obtain a chromatogram as a rise in baseline, or
A wavelength at which the expected component in the sample does not exhibit absorption is used as the detection wavelength, and an eluent that exhibits absorption at this detection wavelength is used to obtain a chromatogram as a decrease in the baseline.

[Problems to be solved by the invention]

For this reason, in high-performance liquid chromatography using the conventional absorbance detector described above, it has been pointed out that it is difficult to simultaneously analyze samples containing components that exhibit absorption and components that do not exhibit absorption at the detection wavelength, and it is also difficult to analyze qualitatively. It has also been pointed out that since the primary measure for this is only the expected elution time of the component, it is difficult to identify it.

[Means for Solving the Problems and Their Effects] The present invention was made to solve these problems, and consists of components that exhibit absorption at the detection wavelength and components that do not exhibit absorption or have small absorption. In addition to the expected elution time of each component, by providing the difference between the absorbance of the i1ilI solution and the absorbance of the sample component at the detection wavelength of each component as a qualitative measure. The purpose is to facilitate identification.

In the method for analyzing sample components by high-performance liquid chromatography using the absorbance detector according to the present invention, when a wavelength that exhibits an appropriate absorbance with respect to the eluent is used as the detection wavelength, a sample having a higher absorbance than the eluent and the eluent are detected. This was done based on the fact that samples with low absorbance yield peaks with different polarities, and the detection wavelength is selected as the detection wavelength, which exhibits a constant absorption for the eluent that satisfies the separation conditions. Obtain a chromatogram showing the difference between the absorbance of the eluent and the absorbance of the sample components with respect to the light of
The components are qualitatively and quantitatively determined based on the peak time, peak polarity, and peak amount of the obtained chromatogram.

For example, in FIG. 1, the horizontal axis is the detection wavelength and the vertical axis is the absorbance. The absorption spectral characteristics of the second sample are shown by the dashed-dotted line S2, and the absorption spectral characteristics of the third sample among the samples are shown by the dashed-double line S3.
In the case shown by , in the conventional analysis method, absorbance detection is performed using the wavelength λ1, which has no absorption (low absorption) for the eluent, as the detection wavelength.

Therefore, if the elution time of the first sample is tl, the elution time of the second sample is t2, and the elution time of the third sample is t3, a romatodalum as shown in FIG. 2 is obtained, for example.

When the detection wavelength is λ1, the first sample and the second sample exhibit absorption, so peaks appear on the chromatogram at times t1 and t2, but the third sample does not exhibit absorption, so at time t3 Although the third sample has been eluted, only noise peaks appear on the chromatogram, and the detection of the third sample is difficult.

In order to detect the third sample that does not exhibit absorption at the detection wavelength λ1 in this manner, it is necessary to change the detection wavelength to a wavelength at which the third sample exhibits absorption. In this case, it is necessary to select an eluent that does not exhibit absorption at the changed detection wavelength, and the eluent must also satisfy the separation conditions in relation to the column and sample components. Since absolute conditions are imposed, the selection of eluent and column becomes very difficult.

Also, regarding the first and second samples, considering the presence of unknown components that may be contained in the sample, it is not uncommon to identify only the elution time tl - t2 as a criterion for qualitative judgment. It's dangerous.

However, in the present invention, as described above, a wavelength that exhibits a certain absorption is selected as the detection wavelength, and analysis is performed based on the peak time and peak polarity of the obtained chromatogram. It is possible to simultaneously detect components that exhibit absorption and components that do not exhibit absorption, and to increase the certainty of identification.

For example, when analyzing a sample exhibiting absorption spectroscopic characteristics as shown in FIG. 1, the present invention uses wavelengths λ2 and λ3 that exhibit absorption in the eluent as detection wavelengths.

The detection wavelength is /+2, and the elution time of the first sample is
- If the elution time of the second sample is t2 and the elution time of the third sample is L3, the eluent exhibits a constant absorption at the detection wavelength λ2, so for example, as shown in Figure 3,
A chromatogram is obtained in which the baseline has a constant level and the peak polarity is determined according to the difference in absorbance between the sample and the eluent at the elution time of each sample.

Also, the detection wavelength is λ3, and the elution time of the first sample is t.
Even if the elution time of the second sample is t2 and the elution time of the third sample is t3, the eluent exhibits a certain absorption at the detection wavelength λ3, so for example, as shown in FIG. , a chromatogram is obtained in which the baseline has a constant level and the polarity of the peak is determined according to the difference in absorbance between the sample and the eluent at the elution time of each sample.

Therefore, even samples containing a mixture of components that absorb at the detection wavelength and components that do not absorb at the detection wavelength can usually be detected reliably, and in addition to the elution time, the polarity of the peak can also be determined for qualitative analysis. This improves the certainty of identification, especially when the detection wavelength is set to 2.
If the above methods are used and analyzed, the certainty of identification will be extremely high.

In summary, the method for analyzing sample components by high performance liquid chromatography using the absorbance detector according to the present invention satisfies the separation conditions for components expected to be contained, and also uses an eluent whose absorbance spectroscopic characteristics are known in advance. A sample is injected into the solution and passed through the column, the eluate eluted from the column is given to an absorbance detector, and the absorbance detector detects light at a wavelength at which the eluent exhibits a certain level of absorption. The absorbance is detected as a wavelength to obtain a chromatogram showing the difference between the absorbance of the eluent and the absorbance of the sample component.

[Effect]

That is, according to the present invention, the detection wavelength is the wavelength at which the eluent exhibits a certain absorption, so components whose absorbance at the detection wavelength is different from the absorbance of the eluent are detected as positive or negative peaks, regardless of the presence or absence of absorption. Furthermore, if the detection wavelength is set to a wavelength at which the absorbance of the sample component is equal to the absorbance of the eluent, only the peak of the sample component can be removed. Further, qualitative determination can be performed based on the appearance time and polarity of this peak, and quantitative determination can be performed more accurately based on the peak amount.

〔Example〕

Hereinafter, one embodiment of the present invention will be described in detail with reference to the drawings.

First, Figure 5 shows the principle diagram of a chromatograph.
1 is an eluent tank, 2 is a pipe, 3 is a liquid pump, 4 is a sample injector, 5 is a column, 6 is an absorbance detector, 7 is a waste liquid tank, and 8 is a recorder. The eluent pumped up from the eluent tank 1 by the pipe 2f
is and is discharged to the waste liquid tank 7. During this process, a sample is injected into the eluent from the sample injector 4, and due to interaction as it passes through the column 5, it is separated and eluted at a component-specific elution time, and then passes through the absorbance detector 6. . The absorbance detector 6 detects changes in the absorbance of the eluate over time and supplies the detected changes to the recorder 8, which records the change.

In this example, a commercially available wavelength variable ultraviolet absorbance detector was used as the absorbance detector 6 in order to use a wavelength that is moderately absorbed by the eluent as the detection wavelength.

In addition, column 5 was filled with Zipax SAX, and i
Fist? (Disodium phthalate was mainly used as l, and the flow rate was 1 to 2 ml per minute. Also, the temperature inside the column 5 was adjusted to 20" to 40" Celsius, and a commercially available sodium salt was used as the sample solution. 1o to 100 μm was implanted.

First, 5X10-4M di-phthalate as an eluent,
In addition to using sodium nitrate (sample ion is N03-), sodium sulfate (sample ion is 5Oa2-), sodium iodide (sample ion is shows an experimental example using I-).

Figure 6 (A), Figure 6 (B), and Figure 6 (C) show the absorbance of the separation column eluate when using the above eluent and sample solution, and the absorbance of the eluate from the separation column when exposed to ultraviolet rays at wavelengths of 239 nm, 245 nm, and 254 nm. The graphs show chromatograms obtained by using and tracking over time.

Peak I, peak ■, and peak ■ that appear in the chromatograms of Figure 6 (A), Figure 6 (B), and Figure 6 (C) are the same elution time in each figure, regardless of the detection wavelength. It appears that the same component was eluted in peaks ■, peak ■, and peak ■ in each figure.

Judging from the elution time previously investigated for pure products of nitrate ion (NO3-), sulfate ion (SO42-), and iodide ion (I-), the appearance time of peak ■ is the elution time of nitrate ion (NO3-). coincides with the peak H
The appearance time of sulfate ion (S.

42-), and the appearance time of peak ■ corresponds to the elution time of iodide ion (I-), so peak ■
indicates nitrate ion (NO3-), and peak ■ indicates sulfate ion (
It is estimated that the peak ① indicates the iodide ion (I-).

Figure 7 shows the eluent disodium phthalate (eluent ion is pnthalate2-) and the sample solution sodium nitrate (sample ion is N03-), sodium sulfate (sample ion is 5042-), and iodide. sodium(
The sample ion shows the absorption spectroscopic characteristics of I-). The curve A shows the eluent ion (ph th a la
t e2-5 Ion (S 042-5 XIO-5
The absorption spectroscopic characteristics of M) are expressed by the curve iii of iodide ion (
1-I XIO-4M) are shown.

First, in the chromatogram shown in FIG. 6(A) when the detection wavelength is 239 nm, the peaks of I and H are negative, and the peak of ■ is positive. Therefore, when the detection wavelength is 239 nm, the I peak component, which is presumed to be nitrate ion, and the n peak component, which is presumed to be sulfate ion, have lower absorbance than the eluent, and are presumed to be iodide ions. It is understood that the component of the peak (2) has higher absorbance than the eluent.

Therefore, looking at the absorption spectral characteristics in Figure 7, the wavelength is 239n.
The absorbance of the eluent and each component for ultraviolet light of m is arranged in descending order of iodide ion (curve iii) - solution (curve A), - nitrate ion (curve i), and sulfate ion is not absorbed at all. does not indicate. This is consistent with the chromatogram shown in Figure 6 (A) obtained with the detection wavelength of 239 nm, and the peak ■ is nitrate ion (
It is consistent with the estimation that the peak ① indicates sulfate ion (3042-), and the peak ▪ indicates iodine ion (I-).

do.

Next, in the chromatogram shown in FIG. 6(B) when the detection wavelength is 245 nm, the peaks of I and ■ are negative, and the peak of ■ does not appear. -And from this, the detection wavelength is set to 2
4. ．． When the wavelength is 5 nm, I is assumed to be a nitrate ion.
It is understood that the peak component (2) and the H peak component (assumed to be sulfate ions) have a lower absorbance than the eluent (and the component (2), which is identified as iodide ion, has an absorbance equal to that of the soluble liquid).

Therefore, looking at the absorption spectral characteristics in Figure 7, we see that the wavelength is 245n.
Looking at the absorbance of the eluent and each component against ultraviolet rays of m, the iodide ion (curve iii) and the eluent (curve A)
The absorbance of is equal, followed by nitrate ion (curve i), and sulfate ion shows no absorption at all. This is consistent with the chromatogram obtained with the detection wavelength of 245r+m shown in Figure 6(B), where the peak ■ represents nitrate ions (NO3-) and the peak ■ represents sulfate ions (5042-).
, the peak ■ [in Fig. 6 (B), the peak ■ does not appear in reality. ] is consistent with the assumption that it represents an iodide ion (1-).

Furthermore, in the chromatogram shown in FIG. 6(C) when the detection wavelength is 254 nm, the peaks of ■, ■, and ■I are all negative. From this, when the detection wavelength is set to 254 nm, the components of the I peak, which is estimated to be nitrate ions, the H peak component, which is estimated to be sulfate ions, and the component of the ■ peak, which is estimated to be iodide ions, are all eluted. It is understood that the absorbance is lower than that of the liquid.

Therefore, looking at the absorption spectral characteristics in Figure 7, we see that the wavelength is 254n.
Looking at the absorbance of the eluent and each component against ultraviolet rays of m, only the eluent (curve A) shows light absorption, and each sample component does not show light absorption. This is consistent with the chromatogram obtained with the detection wavelength of 254 nm shown in FIG. is consistent with the assumption that it represents an iodide ion (1-).

As a result of comparing the chromatograms obtained at three different wavelengths with the absorption spectroscopic characteristics, we can confirm the above assumption, and the peak ■ represents nitrate ion (NOx-), and the peak ■ represents sulfate ion. (504'-)
When qualitatively determining that the peaks 1 and 2 represent iodide ions (I-), the accuracy is much more reliable than the qualitative determination based only on the elution time.

Next, looking at quantitative performance under the same conditions as above, the area of the elution peak is proportional to the product of the difference in absorbance between sample ions and eluent ions and the injection amount of sample ions, regardless of whether the polarity is positive or negative. Therefore, under conditions where the elution peak width is constant, the calibration curve will be a straight line regardless of whether the peak height method or the area method is used.

Furthermore, erasing the peak seen in peak (i) (iodine ion) is also useful for erasing component peaks that impede qualitative and quantitative determination, thereby facilitating qualitative and quantitative determination of target sample components.

In addition, Fig. 8 shows that the detection wavelength was changed to 2 while keeping the elution conditions constant.
The calibration curve obtained by changing the sample concentration at 39 nm is shown, and it was possible to obtain linear characteristics for nitrate ion, sulfate ion, and iodine ion.

Although the above example shows an example in which the present invention is applied to ion exchange chromatography, it goes without saying that the present invention can be applied to chromatography other than ion exchange chromatography. When using an eluent that does not exhibit absorption in all wavelength ranges used in the instrument, this method can be used by mixing a small amount of a reagent that exhibits absorption at the detection wavelength within a range that does not disturb the separation conditions. It can be passed.

C effect] As explained above, according to the present invention, the chromatogram showing the difference in absorbance between /g syneresis and the components in the sample is expressed as (q). For example, it is possible to simultaneously analyze a sample containing a mixture of components that exhibit absorption at the detection wavelength and components that exhibit no absorption or have low absorption.

In addition, in the case of the present invention, when selecting an eluent, there is no need to select an eluent that does not exhibit absorption at the detection wavelength after determining the absorbance of each sample component against the detection 6JLU as in the conventional method. As for the eluent, it is only necessary to consider and select the elution conditions, so selection of the eluent becomes extremely easy.

Furthermore, since the positive/negative peak polarity is newly added in addition to the elution time as a criterion for the qualitative determination of the components, the qualitative determination becomes easier.

In addition, interfering peaks can be easily eliminated, making qualitative and quantitative determination easier.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the absorption spectroscopic characteristics of a hypothetical sample and eluent, Figure 2 is a chromatogram exploded using the conventional method for a sample having the absorption spectroscopic characteristics shown in Figure 1, and Figures 3 and 4. The figure shows a chromatogram prepared by the method of the present invention for a sample having the absorption spectroscopic characteristics shown in Figure 1, Figure 5 is a schematic diagram of the chromatography equipment system, and Figure 6 (A
＞ ・Figure 6 (B) ・Figure 6 (C) shows the chromatogram obtained in the experimental example of the present invention, and Figure 7 shows the absorption spectroscopic characteristics of the eluent and sample used as the experimental example of the present invention. Figure 8 shows calibration curves for iodide ions, nitrate ions, and sulfate ions. ■... Eluent tank 2... Pipe 3... Liquid pump 4... Sample injector 5... Column ゛ 6... Absorbance detector 7... Waste liquid tank
8...Recorder patent applicant Representative of Applied Spectroscopic Instruments Co., Ltd. Patent attorney Figure 1 for Hikaru Murakami

Claims (1)

[Claims] In addition to satisfying the separation conditions for components expected to be included,
A sample is injected into an eluent whose absorbance spectroscopic characteristics are known in advance and allowed to pass through the column, and the eluate eluted from the column is applied to an absorbance detector. A high-speed absorbance detector using an absorbance detector, characterized in that the absorbance is detected using light at a wavelength that indicates a level of absorption as the detection wavelength, and a chromatogram showing the difference between the absorbance of the eluent and the absorbance of the sample component is obtained. A method for analyzing sample components using liquid chromatography.