JPH0827271B2 - Analytical method using liquid chromatography - Google Patents

Description

TECHNICAL FIELD The present invention relates to an analysis method using liquid chromatography, and more particularly to an analysis method suitable for measuring a trace amount of a test substance in a sample.

[Conventional technology]

It is generally said that a solution containing no impurities should be used as an eluent used when separating and analyzing a test substance (which may be referred to as a test component) in a sample using liquid chromatography. ing. The reason is that the presence of impurities is believed to adversely affect the analytical results.

On the other hand, "Baked Liquid Chromatography, No. 8.1 ~
8.20, Varian Airgraph (1971) ”(Basic
Liquid Chromatography, pp 8.1 ~ 8.20, Varian Aerograp
h (1971)) teaches that impurities in the eluent do not interfere with or make an error in the measurement. In the first prior art, even if the eluent contains the same substance as the test substance in the sample as an impurity, the eluent continuously flowed is once supplied to the packing material in the separation column. , When it reaches an equilibrium state, it becomes a constant background,
It is explained that the test substance in the sample appears on the background.

However, “Analytical Chemistry, Vol. 38, pp. 94-97 (19
89) ”, when analyzing alkali metal ions and ammonium ions by ion chromatography, if the test sample is a strongly acidic sample such as a hydrochloric acid extract,
It has been reported that the retention force of the column is weakened by the influence of hydrogen ions in the sample and the analysis accuracy is reduced. In this second prior art, nothing is discussed about the relationship between impurities in the eluent and matrix components in the sample.

[Problems to be Solved by the Invention]

The present inventors, in the examination of liquid chromatography analysis method, when the sample is injected, the equilibrium state achieved between the eluent and the packing material in the separation column is temporarily collapsed, It was found that it affects the analysis value. It is considered that the cause of such an analysis error is that the eluent contains a substance of the same type as the test substance in the sample. Therefore, if impurities in the eluent can be removed, such analysis error may be reduced.
However, in all cases, it is difficult to completely remove impurities in the eluent.

It is an object of the present invention that even if the eluent contains impurities composed of the same substance as the test substance in the sample,
An object of the present invention is to provide a liquid chromatography analysis method capable of measuring a test substance with high accuracy.

[Means for solving the problem]

According to the present inventors, the measurement value of a test substance in a sample by liquid chromatography causes an error when the eluent contains the same impurity type as the test substance. Was confirmed. As a result of further experiments conducted by the present inventors, the present inventors found that such a measurement error is related to the concentration of the matrix component contained in the liquid sample. The present invention has been made as a result of examination of such findings.

The matrix component in this case is a general term for other components excluding the test substance and the solvent in the liquid sample.

The feature of the present invention is that an analysis using liquid chromatography in which a sample is introduced into a separation column in which an eluent is flowed to separate the components contained in the sample and a test substance in the sample is detected by a detector. In the method, equilibrating the inside of the separation column with an eluent, introducing a plurality of sample solutions derived from the same original sample and having different concentrations of matrix components into the separation column at different timings, respectively. Obtaining a first measured value based on the test substance eluted from the separation column with the chromatographic separation of one sample solution, and eluting from the separation column with the chromatographic separation of the other sample solution Obtaining a second measurement value based on a test substance of the same type as the test substance; if the first measurement value and the second measurement value are not substantially the same, a substance of the same type as the test substance It determines that the information includes the eluent,
Compensating the effect of the matrix component based on the substantially proportional relationship between the concentration of the matrix component and the negative error of the measured value, using the first measurement value and the second measurement value. Is characterized by.

[Action]

When the liquid sample containing the matrix component is introduced into the separation column after the eluent containing impurities reaches an equilibrium state with the packing material in the separation column, the following behavior is considered.

When a liquid sample containing a high concentration of matrix components is introduced into the separation column, the equilibrium state in the separation column temporarily changes. Before introducing the liquid sample, the impurities in the eluent were kept in an equilibrium state while being adsorbed by the packing material in the separation column. Are released with a large ratio and are carried out of the separation column within a short period of time along with the flow of the eluent. Assuming that the impurities are the same as the test substance in the sample, the amount of adsorption to the filler will be extremely small. Next, the test substance in the liquid sample is adsorbed on the filler, and the test substance must also compensate for the insufficient adsorption of impurities. When the band of the liquid sample passes through the separation column, the equilibrium state in the separation column returns to its original state, and the test substance is moved in the separation column. Is much less than the amount introduced as a sample. That is, the measured value of the test substance causes a negative error. The magnitude of this error corresponds to the amount that compensates for the insufficient adsorption.

On the other hand, when a liquid sample containing a low concentration of matrix component is introduced into the separation column, the degree of influence is smaller than that in the case of high concentration, but the measured value of the test substance is the same for the same reason. , Will result in a negative error.

The present inventors have found that there is a substantially proportional relationship between the concentration of the matrix component in the sample and the negative error that the measured value of the test substance undergoes. As the measured value, it is preferable to adopt the peak area based on the test substance, but in some cases, the peak height may be used. if,
If the matrix component is not present in the liquid sample, the measured value of the test substance has no error. Therefore, in the present invention, by using a value obtained by measuring a plurality of sample solutions having different contents of the matrix component, by predicting a state in which only the test substance is contained in the solvent of the sample, An accurate analysis result of the amount of the test substance was obtained.

The number of sample solutions having different matrix component concentrations prepared from the same original sample is not limited to two. Rather, the greater the number of three or more, the higher the reliability of the analysis result.

〔Example〕

Prior to describing the embodiment based on the present invention, the results obtained in the preliminary experiment will be described with reference to FIGS. 4 to 6.

In the first and second preliminary experiments, a column packed with a cation exchange resin was used as the separation column, a conductivity monitor was used as the detector, and 0.5 mM was used as the eluent.
An aqueous solution containing 0.5 mM tartaric acid and 0.5 mM ethylenediamine was used.

The first preliminary experiment will be described with reference to FIGS. 4 and 5. A commercially available tartaric acid and ethylenediamine were dissolved in pure water to prepare an eluent. On the other hand, five kinds of sample solutions were prepared. Tartaric acid and ethylenediamine were added as matrix components to the sample solution. However, the matrix component concentration was 0.5 mM for the first sample, 1.0 mM for the second sample, 2.0 mM for the third sample, 5.0 mM for the fourth sample, and 5 for the fifth sample. The sample is 20.0 mM. The solvent of each sample solution is pure water. All sample solutions are Mg ⁺⁺ , Ca
⁺⁺ and Sr ⁺⁺ were prepared so as to contain 1 ppm each. The injection volumes of the sample solutions into the separation columns were all the same. Here, the introduction volume of the sample solution is 20 μl.

After equilibrating the separation column, the first to fifth sample solutions were introduced into the separation column one by one, and the corresponding chromatograms were output on the chart paper of the recorder. The measurement results thus obtained are shown in FIG.

In FIG. 4, the chromatograms (1) to (5) are based on the first to fifth sample solutions. Focusing on the Ca ⁺⁺ peak in each chromatogram, the matrix component concentration was 5.0 mM (chromatogram (4)) and 20.0 mM.
In the case of (Chromatogram (5)), Ca ⁺⁺ shows a negative peak. This is because Ca ⁺⁺ is contained as an impurity in the eluent. By the way, as a result of measuring this eluent by atomic absorption method, a total of 10 ppb Ca
⁺⁺ was detected.

Next, the test substances Ca ⁺⁺ , Mg ⁺⁺ , and Sr in FIG.
The relationship between the ⁺⁺ peak area and the matrix component concentrations of the first to fifth sample solutions is shown in FIG.

In FIG. 5, the peak area value of Ca ⁺⁺ changes in inverse proportion to the matrix concentration. Matrice concentration 3m
The area is zero at M, which is considered to be due to the balance of the adsorption amount of Ca ⁺⁺ which is an impurity in the eluent used. Similarly, Mg ⁺⁺ also shows a tendency that the peak area tends to decrease with increasing matrix concentration, but the content of Mg ⁺⁺ as an impurity in the eluent is extremely small compared to Ca ^++. I understand. Sr ⁺⁺ is the matrix density River connexion also, since no change is the peak area, is in the eluent Sr ⁺⁺ is estimated not included as an impurity.

Next, the second preliminary experiment will be described with reference to FIG.
The eluent compositions, tartaric acid and ethylenediamine, were sufficiently purified to be free of impurities. And
Instead of impurities, Mg ⁺⁺ , Ca ⁺⁺ , Sr ⁺⁺ , Ba ⁺⁺ in the eluent
Was contained in an amount of 1 ppm.

On the other hand, five kinds of sample solutions were prepared. In these sample solutions, the solvent is pure water, the matrix components are tartaric acid and ethylenediamine, and the test substances are Mg ⁺⁺ , Ca ⁺⁺ , Sr.
⁺⁺ and Ba ⁺⁺ . Tartaric acid and ethylenediamine were prepared at the same molar concentration. The matrix component concentration is zero mM for the first sample solution and 0.5 mM for the second sample solution,
The third sample solution is 1.0 mM and the fourth sample solution is 2.0 mM
And the fifth sample solution is 5.0 mM. That is, the second
The matrix concentration of the sample solution is the same as that of the eluent, and the matrix concentration of the third to fifth sample solutions is higher than that of the eluent. In all of the first to fifth sample solutions,
Mg ⁺⁺ , Ca ⁺⁺ , Sr ⁺⁺ , and Ba ⁺⁺ were added at 1 ppm each. These concentrations are the same as the concentrations of the same type of substance in the eluent. The introduction volume of each sample solution is 20 μl.

These sample solutions were measured as in the first preliminary experiment. The analytical results obtained are shown in FIG. As shown in FIG. 6, the measured values (peak areas) obtained for the first to fifth sample solutions are closely related to the matrix concentration in each sample solution. When the matrix concentration and the test substance concentration match the eluent as in the second sample, the peak of the test substance cannot be obtained. As in the case of the third to fifth sample solutions, even if the concentration of the test substance in the sample and the concentration of the same kind of impurities in the eluent are the same, the higher the matrix concentration is, the more negative error the test substance has growing.

Here, an important finding is that the peak area values of each test substance when the matrix concentration obtained based on the first sample solution is zero were obtained based on the second to fifth sample solutions. By connecting the peak area values with a straight line, the values are exactly the same as the values obtained by extrapolating to the point where the matrix concentration is zero.

This means that even if the concentration of the matrix component cannot be made zero in a practical analytical work, the amount of the test substance in such an infinite dilution state should be adjusted from the same original sample. It teaches the possibility that it can be estimated from the measured values of a plurality of sample solutions having different matrix concentrations.

The correct amount of each test substance obtained from the results of FIG. 6 is obtained by plotting the area value of each test substance using the matrix concentration of the second to fifth sample solutions as a parameter, and calculating the linear values of these areas. It can be obtained by using the method of extrapolation to the point where the matrix concentration is zero by tying with, so if you devise the dilution method of the original sample and the method of data processing, Such a method can be applied.

Next, an embodiment of the present invention will be described with reference to FIGS. 1 to 3B.

In the analyzer shown in FIG. 1, the eluent in the eluent tank 1 is supplied to the separation column 7 by the liquid feed pump 2 through the sample diluting device 3, and further through the flow cell 8 to the outlet.
Spilled from 15. The liquid feed pump 2 is a pump capable of controlling the liquid feed flow rate up to the order of μl / min, and is a reciprocating type pump having a double piston. A pump of this kind is shown, for example, in U.S. Pat. No. 4,810,168. In the apparatus shown in FIG. 1, the eluent was sent by setting the flow rate of the solution sending pump 2 to 1 ml / min.

The separation column 7 and the flow cell 8 are housed in the column oven 6 and maintained at a temperature of 40 ° C. The separation column 7 has a stainless steel cylinder having an inner diameter of 4 mm and a length of 50 mm, and a cation exchange resin having a low exchange capacity is closely packed in the cylinder. This type of separation column is commercially available, for example, as Hitachi packed column # 2710IC. In the flow cell 8, a detection electrode associated with the conductivity monitor 10 is arranged. Conductivity monitor 10
It incorporates an amplifier capable of changing the amplification factor of the detection signal according to an instruction from the microcomputer 5 functioning as a control device. Flow cell 8 and conductivity monitor 10
The analog signal obtained by the detector including the above is taken into the arithmetic and control unit 5 as a digital signal through the analog / digital converter 11 and subjected to necessary data processing. On the other hand, the analog signal from the detector is
Recorded as a chromatogram on 14 chart papers.

In the analyzer shown in FIG. 1, as a detector, Hitachi L
-3700 conductivity monitor and flow cell are used. As the detector, a spectrophotometer, a fluorometer, an electrochemical detector, or the like can be adopted depending on the type of sample to be analyzed.

The automatic diluting device 3 may have a function of diluting the original sample in the channel so as to prepare a sample solution having a plurality of dilution ratios, or alternatively, the original sample may be stored in a container arranged outside the channel. After diluting, the diluted sample solution may be automatically introduced into the channel. Here, an example of diluting in the flow channel is shown in FIGS. 2A to 3B. The operation of this diluter will be described later.

After deciding how many sample solutions with different dilution ratios are to be used for the original sample to be measured, the operator inputs the data of the dilution ratios on the operation panel 4. The data input from the operation panel 4 is taken into the arithmetic / control unit 5 and stored in the built-in storage unit. Based on this data, the arithmetic / control device 5 operates the automatic diluting device 3 to introduce the sample solution into the separation column 7 one by one at a predetermined timing. When all of the instructed number of sample solutions have been introduced into the separation column 7, the automatic diluter 3 finishes the sampling operation for one original sample. When it is desired to measure other original samples, the information is input in advance from the operation panel 4.

When a certain amount of the first sample solution diluted to a predetermined dilution ratio is introduced into the separation column 7 from the diluting device 3 in a state where the separation column is equilibrated, the sample is separated in the separation column 7. The components are separated and eluted from the separation column 7. Along with this, the peaks of the respective components separated by the detector are detected, and the peak area value of the test substance is stored in the storage unit of the micro computer 5.

Meanwhile, the diluting device 3 mixes the original sample and the pure solvent,
A second sample solution having a dilution ratio that matches the input second dilution ratio is prepared. After the measurement operation of the first sample solution is completed, the diluting device 3 is configured to equilibrate the separation column 7 to the second sample solution having the same volume as the introduction volume of the first sample solution. Is introduced into the separation column 7. The dilution ratio of the second sample solution is
Since the dilution ratio of the first sample solution is different, the matrix concentration in these sample solutions is different. Also for this second sample solution, the peak area values of the same kind of test substance as in the first case are measured, and those values are stored in the storage unit.

In order to improve the analysis accuracy, it is desirable to measure three or more sample solutions, but here, in order to avoid the complexity of the explanation, the corrected amount of the test substance is obtained from the measured values of the two sample solutions. An example will be described.

When the scheduled measurement of all the diluted samples is completed, the arithmetic and control unit 5 gives the measured values (peak area value or peak height value) based on each sample solution obtained for the original sample, Based on the instruction input from the operation panel 4. It can be output to the printer 12 and / or the CRT 13.

After the measurement of a plurality of sample solutions derived from the same original sample is completed, the arithmetic / control device 5 sets the peak area value or the peak height value obtained for each sample solution to the corresponding input dilution factor. By multiplying, the converted value for each measured value is obtained. These converted values will correspond to the amount of test substance, assuming that the original sample was introduced into the separation column 7.

Then, the arithmetic and control unit 5 uses all the converted values in a state where it is assumed that the matrix component is not contained in the original sample, that is, only the test substance is contained in the pure solvent. The peak area value or peak height value of each test substance in the state assumed to exist is calculated. Such corrected values are obtained in the same number as the number of test substances. This modified value can also be output to the printer 12 and / or the CRT 13 if necessary. Such calculation is performed by extrapolating the relationship straight line when the dilution ratio is infinite by obtaining the slope of the relationship straight line using the matrix concentration as a parameter using the converted values having different dilution ratios. To be achieved.

Further, the calculation / control device 5 calculates the concentration of each test substance by associating the calibration curve stored in advance with the corrected value obtained by extrapolation, and the result is calculated. Is output to the printer 12 and / or the CRT 13.

Next, an example of the diluting device 3 used in the analyzer of FIG. 1 will be described with reference to FIGS. 2A to 3B. Second
FIGS. A and 2B show the operation when the dilution ratio for the original sample is high, and FIGS. 3A and 3B show the operation when the dilution ratio for the original sample is low. Regardless of the dilution ratio, a plurality of sample solutions derived from the same original sample have the same volume introduced into the separation column. Further, in the case of performing an analysis operation on a plurality of sample solutions derived from the same original sample, the same eluent is supplied to the separation column.

When obtaining a sample solution with a high dilution ratio from the original sample,
Sampling valve 21 according to instructions from control / calculation unit 5
The hexagonal valve 26 is first set to the state as shown in FIG. 2A, and the diluting device 3 is supplied with a diluting liquid made of a pure solvent. In this state, the eluent fed by the liquid feed pump 2 is circulated in the separation column 7, and the equilibrium between the eluent and the packing is achieved in the separation column 7.

A predetermined volume of the original sample from the sample injector 27 and the diluent (pure solvent) from the diluent tank 30 are introduced into the flow path of FIG. 2A. The diluting liquid is sent by the liquid sending pump 29 at a predetermined flow rate. When a predetermined amount of the original sample is injected into the sample injection unit 28 from the sample injector 27, the sample is sandwiched by the diluent and transferred. The band of the original sample spreads in the diluent while flowing through the large capacity coil 25 and the dilution coil 23. The longer these coils, the greater the dilution factor. When the center of the diffused sample band reaches the sampling coil 22 of the sampling valve 21, the sampling valve 21 is switched by a command from the arithmetic / control device 5.

When the sampling valve 21 is switched, the flow path shown in FIG. 2B is formed. The sample solution of a predetermined magnification cut off by the sampling coil 22 of a predetermined volume is introduced into the separation column 7 by the flow of the eluent sent from the pump 2.

After the measurement of the previous sample solution is completed, control is performed to introduce the same original sample as that previously introduced. Third
As shown in FIG. A, in order to prepare a sample solution having a low dilution ratio, the hexagonal switching valve 26 is switched so that the small capacity coil 24 is in communication with the sample injection part 28 and the dilution coil 23. When a certain amount of original sample is injected from the sample injector 27,
While the sample band is diffused by the diluent, it passes through the small-capacity coil 24 and the dilution coil 23, and then the sampling coil 22.
Be led to. The degree of dilution of the sample solution filling the sampling coil 22 is lower than the dilution rate of the previous sample solution. This is because the length of the small capacity coil 24 is shorter than the length of the large capacity coil 25.

The center of the spread of the sample band is the sampling coil 22.
3B, the valve 21 is switched as shown in FIG. 3B, and the sample solution cut out to a constant volume by the sampling coil 22 is introduced into the separation column by the eluent and separated and analyzed. The lengths of the coils 23, 24, 25 and the flow rate of the diluent supplied by the pump 29 are set in advance so that a desired dilution ratio can be obtained in the sampling coil 22. The relationship between the sample injection timing by the sample injector and the switching timing of the sampling valve 21 is also input to the arithmetic / control device 5 in advance to set conditions.

Next, a measurement example using the analyzer of FIG. 1 will be described.

Measurement Example 1 The eluent was prepared by dissolving sufficiently purified tartaric acid and ethylenediamine in pure water so that each contained 0.5 mM. In this eluent, instead of impurities, Mg ⁺⁺ ,
Ca ⁺⁺ , Sr ⁺⁺ , and Ba ⁺⁺ were added at 1 ppm each. The original samples supplied to the diluter 3 are Mg ⁺⁺ , Ca ⁺⁺ , Sr ⁺⁺ , Ba ^++.
A liquid containing 25 ppm of each was prepared. The solvent of this original sample was pure water, and the matrix components were 2 mM tartaric acid and 2 mM.
It is ethylenediamine. The diluent for diluting the original sample is pure water.

Four kinds of sample solutions having different matrix concentrations derived from one original sample were prepared. The first sample solution has a dilution ratio of 1
The original sample itself was injected into the separation column 7.
The matrix concentration ratio of this first sample solution is 1.0. The second sample solution had a dilution factor of 2 and was prepared such that the original sample was diluted with pure water by a factor of 2. This second
The matrix concentration ratio of the sample solution of is 0.5. The sample solution in FIG. 3 had a dilution ratio of 4 times, and was prepared so that the original sample was diluted 4 times with pure water. The matrix concentration ratio of this third sample solution is 0.25. The dilution ratio of the fourth sample solution was 10 times, and the original sample was diluted 10 times with pure water. The matrix concentration ratio of this fourth sample solution is 0.10
Is.

20 μl of each of the first to fourth sample solutions was sequentially introduced into the separation column 7, and the test substances Mg ⁺⁺ , Ca ⁺⁺ , Sr ⁺⁺ ,
Ba ⁺⁺ was detected. For each test substance, the product of the peak area value and the dilution rate was calculated by the calculation / control device 5, and the result was output to the printer 12.

The relationship between the value of this product and the matrix concentration is shown in FIG. For Mg ⁺⁺ , Ca ⁺⁺ , Sr ⁺⁺ , Ba ⁺⁺
The slope of the line is obtained from the product seed based on the fourth sample solution,
Find the value at the point where the matrix concentration ratio is zero. The concentration of the test substance in the original sample was determined from this corrected value, which was not affected by the matrix component, and was in agreement with the added amount of the prepared original sample.

Measurement Example 2 The eluent was prepared as a 1.6 mM nitric acid aqueous solution by mixing commercially available concentrated nitric acid with pure water. The flow rate of the eluent by the pump 2 is 0.7 ml / min. This measurement example was performed to examine the impurity concentration in the eluent. Four kinds of sample solutions were prepared. In order to know the contents of Na ₊ and NH _{4+ in} the eluent, 2 ppm each of Na ₊ and NH _{4 +} was contained in all sample solutions. Nitric acid with high purity was added as a common matrix component of each sample solution. The content of the matrix component is
The first sample solution is 2 mM, the second sample solution is 3 mM, the third sample solution is 6.5 mM, and the fourth sample solution is 8 mM. The solvent of each sample solution is pure water.

20 μl of each of these sample solutions was sequentially introduced into the separation column 7, and the component peak heights of the respective sample solutions were measured. The results are shown in Fig. 8. The measured values decreased as the concentrations of Na ₊ and the matrix component in the sample increased, and the values extrapolated from the slope of the graph to the point where the matrix concentration was zero corresponded to the Na ₊ concentration of 2 ppm. From these results, it was estimated that about 30 ppb Na ₊ was contained as an impurity in the eluent. On the other hand, since the NH ₄₊ it is has failed change in the measured value even if the change in the concentration of the matrix components, NH ₄₊ was divide is not included as impurities in the eluent.

In the analyzer of FIG. 1, based on the information of the dilution ratio input from the operation panel 4, the detector is operated so as to correspond to those dilution ratios by the command from the control arithmetic unit 5.
You can also increase the output sensitivity of 10. In this case, there is no need to calculate the product of the measured value and the dilution ratio, and the same data as in FIG. 7 is obtained in the detector 10, so that it is output to the recorder 14 and / or the printer 12.

Next, another embodiment of the present invention will be described. The overall structure of this analyzer is mostly the same as that of FIG. 1, but the structure of the diluter 3 is different from that of FIG. 2A.
That is, for example, an autosampler having a diluting function, which is commercially available as Hitachi AS-4000 type, is used. In this autosampler, the pipetting nozzle moves on the plate in the X, Y, and vertical directions by placing the sample and the diluent on the plate,
Add the sample and diluent into the container. The sample solution diluted to a predetermined ratio is injected into the sample injection port of the liquid chromatograph by a pipetting nozzle in a predetermined volume.

Next, an example of removing the matrix component from the sample will be described. If the matrix component can be removed when the sample is introduced into the channel, the measured value of the test substance can be obtained with a low error. In this case, it is preferable to arrange a column capable of removing the matrix component between the separation column and the sample injection part.

Sample solution comprises sodium hydroxide as the matrix component, Cl ^- and SO ₄ ^- in the case of measuring a test substance in, the use of a column filled with H-type cation-exchange resin as the matrix component removal column You can As a matrix component removal column, the inner diameter is 6 mm and the length is 10 m
A column in which a Hitachi custom ion exchange resin # 2613 was packed in a cylindrical tube of m was used. An aqueous solution containing 0.2 N NaOH was used as a sample. The sample was processed through 2ml to the removal column, Cl treated sample was liquid chromatography separation ^-
And SO ₄ ^- were measured. As a result, the test substance consisting of anions could be measured with high sensitivity.

Further, when analyzing a trace amount of metal ions in an acidic hydrochloric acid solution, it is preferable to use a column packed with an OH type strongly basic anion exchange resin. As a result, the anions in the sample can be removed, so that the metal ions that are the test components can be analyzed with high accuracy.

To check whether or not the matrix component has been removed from the sample, at least two samples with different dilution ratios are prepared by diluting the same sample with a diluent (pure solvent), and analyze in the same manner as above. It can be easily determined by comparing the product of the peak area value at the time of tanning and the dilution rate. If there is a difference between the two values, the matrix is further removed and used.

When it is desired to remove impurities in the eluent, a column for removing impurities in the eluent may be provided between the pump 3 and the diluting device 3 shown in FIG.

It is clear from the results of the above-described examples that if the same substance as the test substance is not contained as an impurity in the eluent, the analysis can be performed without being affected by the sample matrix. To remove impurities in such eluent, Hitachi Custom Ion Exchange Resin # 2650L is used as an impurity removal column.
The obtained product was washed with acid to form H, and used in a column having an inner diameter of 4 mm and a length of 40 mm. This can remove impurities such as alkali metals, ammonium, alkaline earth metals, and heavy metals, and analysis can be performed without being affected by the matrix concentration.

〔The invention's effect〕

According to the present invention, even if the sample contains a matrix component other than the test substance, it is possible to eliminate the influence of the matrix component on the test substance measurement value,
Accurate analysis results are obtained.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the overall configuration of an embodiment of an analyzer to which the present invention is applied, and FIGS. 2A and 2B are for explaining the operation of the diluter in the embodiment of FIG. Illustration of
3A and 3B are diagrams for explaining the operation of the same diluting device at a low dilution rate, and FIG.
The figure which shows the example of the chromatogram obtained in the preliminary experiment, FIG. 5 is the figure which represented the result of FIG. 4 in the graph, FIG. 6 is the figure which shows another experimental result, FIG. First
Diagram for explaining the measurement results obtained with the analyzer of the figure,
FIG. 8 is a diagram for explaining another measurement result obtained by the analyzer of FIG. 1 ... Eluent tank, 3 ... Diluter, 4 ... Operation panel,
5 ... Arithmetic / control device, 7 ... Separation column, 10 ... Conductivity monitor, 12 ... Printer, 14 ... Recorder, 21 ... Sampling valve, 24 ... Small capacity coil, 25 ... Large capacity Coil, 27 …… Sample injector, 29 …… Diluent delivery pump.

Front page continuation (72) Inventor Iwabuchi, et al. 882 Ige, Katsuta City, Ibaraki Hitachi Measurement Engineering Co., Ltd. (56) References JP-A 64-73251 (JP, A) JP-A 62-231132 ( JP, A)

Claims (1)

[Claims] 1. An analysis using liquid chromatography in which a sample is introduced into a separation column in which an eluent is flowed to separate the components contained in the sample, and a test substance in the sample is detected by a detector. In the method, equilibrating the inside of the separation column with the eluent, and introducing a plurality of sample solutions derived from the same original sample and having different concentrations of matrix components into the separation column at different timings. To obtain a first measurement value based on the test substance eluted from the separation column with the chromatographic separation of one sample solution, and to perform the above separation with the chromatographic separation of the other sample solution. Obtaining a second measurement value based on the same type of test substance as the test substance eluted from the column, wherein the first measurement value and the second measurement value are not substantially the same. If so, it is determined that the same kind of substance as the test substance is contained in the eluent, and the concentration of the matrix component and the measurement are performed using the first measurement value and the second measurement value. A method for analyzing using liquid chromatography, which comprises correcting the influence of the matrix component based on a substantially proportional relationship with a negative error that the value receives.