JP3299362B2 - Method and apparatus for gradient analysis of liquid chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for liquid chromatographic gradient analysis which enable rapid and high-precision analysis in liquid chromatography, and more particularly to a method and a liquid chromatograph which can perform highly sensitive analysis with a small amount of sample. The present invention relates to a method and an apparatus for gradient analysis of liquid chromatography, which are optimal for liquid chromatography analysis using a small-diameter separation column and a suppressor.

[0002]

2. Description of the Related Art When analyzing a sample containing an unnecessary component (hereinafter, referred to as a matrix component) having a longer elution time (retention time) than a target component by using liquid chromatography, high-precision analysis is performed. In order to prevent interference of the detection of the target component by the matrix component, it is necessary to wait for the next measurement until the matrix component elutes from the separation column.

For example, when a sulfate ion in a sample containing perchlorate ion as a matrix component is measured using a widely used AS4A anion separation column manufactured by Dionex, the sulfate ion elutes in about 10 minutes. However, it takes about 1 hour from the start of measurement to start elution of perchlorate ions, and at least about 1 hour and 20 minutes from the start of measurement to complete the elution of perchlorate ions. is necessary. Here, in order to increase the measurement efficiency, when the samples are successively injected and measured in a state where the perchlorate ion is not eluted, as shown in FIGS. 8A to 8C after the measurement of 5 to 6 samples. In addition, the peak of the perchlorate ion often overlaps with the peak signal of the sulfate ion, which is the measurement target component, and interferes with the measurement of the sulfate ion.

Further, when new samples are continuously injected while perchlorate ions remain in the separation column, perchlorate ions and the like remain in the separation column and affect the performance of the separation column. Fluctuations in the sulfate ion elution time and changes in the shape of the detected peak may occur, leading to undesirable qualitative and quantitative conditions. For this reason, various studies have been made on a method for rapidly eluting the matrix component after eluting the target component, thereby enabling the next measurement to be performed quickly.

[0005]

As a method for eluting a matrix component quickly without affecting the measurement sensitivity and reproducibility of a target component, a method of increasing the flow rate of an eluent after elution of a target component, and a method of eluting a target component after elution There are known methods for increasing the concentration of eluent. However, when the flow rate of the eluent is increased, the pressure of the liquid chromatograph increases, so that the flow rate of the eluent can be increased only up to the usable pressure of the apparatus. For this reason, the flow rate of the eluent can be increased only up to about 2 to 3 times the normal amount, so that the effect is weak.

On the other hand, during the measurement, a dilute eluent is used to increase the elution time difference between the components to sharpen the peak of the chromatogram. After the target component has been eluted, the concentration of the eluent is increased. Gradient analysis, which promotes elution of matrix components, is more effective than the above-described method of increasing the amount of eluent, and is known as an effective means. However, the concentration of the eluent can be improved only to the limit of the apparatus, and an eluent having a too high concentration may adversely affect a suppressor, a detector, and the like. In particular, when a liquid chromatograph apparatus using a separation column and a suppressor, and a gradient analysis method is used in a device using a small-diameter separation column and a suppressor that has recently attracted attention,
It has been discovered that eluent components remain in the suppressor, causing a decrease in measurement sensitivity and measurement accuracy.

A liquid chromatograph apparatus using a small-diameter separation column and a suppressor (hereinafter, referred to as a small-diameter chromatograph apparatus) is a small-diameter separation column and a suppressor having a smaller diameter than a normal one. The sensitivity can be analyzed. The analysis can be performed with a small amount of eluent (if the caliber is half, the eluent can be reduced to 1/4), and the measurement sensitivity is good (if the caliber is half). The sample concentration with respect to the eluent is quadrupled, and the sensitivity is approximately quadrupled).

However, according to the study of the present inventors, in the case of a small-diameter type chromatographic apparatus, when the eluent concentration is increased to a level that the apparatus (separation column and suppressor) can withstand, the high-performance chromatographic apparatus cannot be used. After the matrix component is eluted with a high-concentration eluent, even if the eluent concentration is reduced to the normal measurement concentration, the high-concentration eluent component remains in the suppressor, causing a decrease in the measurement sensitivity and measurement accuracy of the target component. I found that.

For example, when anions are analyzed, a sodium salt solution is usually used as an eluent, but when a gradient analysis method is used in a small-diameter type chromatograph, the eluent concentration is increased. In the suppressor, sodium ion and hydrogen ion are not sufficiently ion-exchanged,
Sodium ions stay on the surface and inside the ion exchange membrane of the suppressor. Therefore, it is presumed that sodium ions remain on the ion exchange membrane surface of the suppressor and inside the membrane even when the eluent concentration is reduced to the normal use concentration.

When the next sample is injected before the sodium ion concentration on the surface of the ion exchange membrane and inside the membrane has decreased to the same level as the concentration of the eluent at the time of measurement, the sodium salt of the target component is converted into an acid in the suppressor. Since the change due to ion exchange to the above-mentioned form is performed non-quantitatively and incompletely compared to the normal state, a non-quantitative decrease in the sensitivity of the measurement target component occurs. To prevent this, washing may be performed with an eluent having a concentration of about water to the concentration at the time of measurement, and the inside of the flow of the apparatus may be returned to a normal state. Yes,
This leads to an increase in measurement time.

On the other hand, in order to solve the problems caused by the matrix components, as shown in FIG. 9, upstream of the separation column 62 for measurement, a guard column or the like having a shorter elution time for the separation components than the separation column 62 is used. A configuration is also conceivable in which the matrix separation column 60 is attached, and a three-way switching valve 64 for switching the path is provided between the matrix separation column 60 and the separation column 62. In this chromatographic apparatus, the sample a flows into the matrix separation column 60, and then flows into the separation column 62. The target component passing through the separation column 62 flows into the suppressor and is detected by the electric conductivity detector. Is done.

On the other hand, when it is confirmed that the target component in the sample has passed through the matrix separation column 60, the three-way switching valve 64 is switched so that the matrix component flows into the waste liquid bottle 66 without being sent to the separation column 62. here,
As described above, since the elution time of the matrix component is short in the matrix separation column 60, the matrix component quickly passes through the matrix separation column 60, and the next measurement can be quickly performed.

However, since the elution time of the target component from the matrix separation column 60 changes depending on the concentration of the matrix component, and the elution time difference between the matrix component and the target component for measurement is short, the switching timing of the three-way switching valve 64 is set. It is difficult and practical. If the matrix separation column 60 has a large difference in the elution time between the target component and the matrix component to facilitate the setting of the switching timing of the three-way switching valve 64, the matrix separation column 60 for the target component and the separation column 6
The elution time from Step 2 becomes longer, leading to an increase in analysis time.

On the other hand, in Japanese Patent Application Laid-Open No. 3-242552, a switching valve for selectively switching between a sample flow path and a cleaning liquid supply source is provided downstream of the separation column, and the cleaning liquid is supplied to the separation column from the three-way switching valve. A liquid chromatograph device (metal component analyzer) that supplies the separated column so that it can be washed is disclosed.

[0015] However, the apparatus disclosed in this publication has a separation (concentration) whose capacity has been reduced by many uses.
The purpose is to restore the separation ability by washing the column, and it is impossible to wash the separation column only after the analysis operation by the apparatus is completely stopped. Therefore, in the apparatus disclosed in this publication, rapid continuous analysis using a gradient analysis method is impossible in liquid chromatography. It is not possible to perform a rapid continuous analysis by solving the above-mentioned problems inherent in a small-diameter type chromatograph apparatus using the method.

An object of the present invention is to solve the above-mentioned problems of the prior art, and in liquid chromatography using a gradient method, a matrix component is rapidly eluted from a separation column, and measurement caused by the matrix component is performed. High-precision continuous measurement without loss of sensitivity and accuracy can be performed quickly. Especially, in liquid chromatography (ion chromatography) using a separation column and a suppressor, a small-diameter separation column and It is an object of the present invention to provide a gradient analysis method for liquid chromatography, which is suitably used for liquid chromatography using a suppressor, and an apparatus using the method.

[0017]

In order to achieve the above-mentioned object, a gradient analysis method for liquid chromatography according to the present invention is directed to a liquid chromatography using a separation column and a suppressor. A gradient analysis method in which, after flowing out of the separation column, a removal eluent having a higher elution capacity than the measurement eluent flows into the separation column to cause unnecessary components to flow out of the separation column. Flowing into the separation column and switching the flow path out of the separation column to a path different from the suppressor, and at the same time, flowing an auxiliary eluent having a lower elution capacity than the eluent for removal into the suppressor. A gradient analysis method for liquid chromatography is provided.

The diameter of the separation column and the suppressor is preferably 3 mm or less.

Furthermore, the gradient analyzer for liquid chromatography of the present invention utilizing the above-mentioned analysis method,
A liquid chromatography gradient analyzer for performing a gradient analysis in which, after the target component flows out of the separation column, a removal eluent having a higher elution capacity than the measurement eluent flows into the separation column and unnecessary components flow out of the separation column. A separation column, a suppressor disposed downstream of the separation column, a detection means for detecting a target component flowing into the suppressor, a sample, and a measurement eluent for eluting components in the separation column. Means for supplying an eluent for removal having a higher elution capacity than the eluent for measurement to the separation column, and an auxiliary for supplying an auxiliary eluent having a lower elution capacity than the eluent for removal to the suppressor. An eluent supply means, disposed between the separation column and the suppressor, for supplying the separation column or the auxiliary solution; And a multi-way switching valve and the flow path to freely connect the the stepped suppressor or waste path, after the detection of the target component has been completed by the detection means,
Supplying the eluent for removal to the separation column,
A gradient analyzer for liquid chromatography, characterized in that the multidirectional switching valve is switched, an outflow path from the separation column is connected to a waste liquid path, and an auxiliary solution supply means and the suppressor are connected.

Further, it is preferable to use the suppressor according to claim 3, wherein the diameter of the separation column and the suppressor is 3 mm or less.

Hereinafter, the method and apparatus for gradient analysis of liquid chromatography of the present invention will be described in detail based on preferred embodiments shown in the accompanying drawings. FIG. 1 shows a conceptual diagram of a liquid chromatography gradient analysis method (hereinafter, referred to as an analyzer) of the present invention for performing the liquid chromatography gradient analysis method (hereinafter, referred to as an analysis method) of the present invention.

The analyzer shown in the drawing is an apparatus for performing so-called ion chromatography (IC) using a separation column 24 for separating each component, a suppressor 32, an electric conductivity detection cell 34 and an electric conductivity detector 36. hand,
In addition to the above parts, an autosampler 10, a first eluent tank 12, a second eluent tank 14, a concentration control pump 2
0, sample / eluent switching valve 16, multi-directional switching valve 26, third
Various parts such as the eluent tank 28 and the first waste liquid bottle 40 are appropriately combined and connected. Such an analyzer according to the present invention employs a so-called gradient analysis method in which, after a target component is eluted from the separation column 24, an eluent for removal having a high elution force flows into the separation column 24 to rapidly elute the matrix component. Is what you do.

The autosampler 10 collects a fixed amount of the adjusted sample and supplies it to the sample loop 22. In the illustrated apparatus, a sample is injected from the autosampler 10 into a sample loop 22 connected to the sample / eluent switching valve 16, and flows into the separation column 24 from the sample loop 22. In the present invention, various known autosamplers can all be used. In the analyzer of the present invention, the autosampler 10 does not always need to be used.

On the other hand, the first eluent tank 12 is filled with a dilute eluent such as water, and the second eluent tank 14 is about 5 to 100 times, preferably 10 times, the eluent for measurement used for measurement. Fill the eluent with a concentration approximately twice as high.

A deaerator 18 and a concentration control pump 20 are connected to both eluent tanks. The concentration control pump 20 pumps up the eluent filled in both eluent tanks, adjusts the mixing and concentration, and elutes the target component from the separation column 24 during measurement. The eluent for removal is also adjusted and discharged with a high concentration and a high elution ability, and is supplied to the sample / eluent switching valve 16.

In the present invention, the use of the concentration control pump 20 is not limited, and the eluent for measurement and the eluent for removal are adjusted in advance, and supplied to the separation column 24 by different pumps. For example, an eluent for measurement or an eluent for removal may be selectively used in the separation column 24.
All known methods that can be supplied to are available.

The autosampler 10 and the concentration control pump 20 are both connected to the sample / eluent switching valve 16. The sample / eluent switching valve 16 is composed of two four-way switching valves connected directly or via a sample loop 22, and connects the autosampler 10 and the concentration control pump 20 with or without the sample loop 22.
It is connected to the separation column 24 and the first waste liquid bottle 40.

In the present invention, the sample, the eluent for measurement and the eluent for removal can be used for each of the sample, the eluent for measurement and the eluent for removal without using the sample / eluent switching valve 16 in which two such four-way switching valves are combined. Any configuration that can independently supply the sample, the eluent for measurement and the eluent for removal to the sample loop 22, the separation column 24, and the like, such as a configuration in which an independent pump and a path switching unit are provided, can be used.

A separation column 24 is arranged downstream of the sample / eluent switching valve 16. The separation column 24 separates each component in the sample by temporarily retaining (retaining) each component contained in the sample and then eluting it. Each component in the sample depends on the flow rate and elution capacity of the eluent. According to the corresponding elution time (retention time) for each component, the components are sequentially eluted from the one with the shorter elution time and separated.

In the analyzer (method) of the present invention, the separation column 24 is not particularly limited, and all known separation columns can be used according to the target component to be measured. Here, the present invention performs a gradient analysis, which will be described in detail later. Since the eluent for removal does not flow into the suppressor 32, the measurement sensitivity and accuracy due to the retention of the eluent components in the suppressor 32 are described. There is no decline. This advantage is particularly important for small diameter suppressors. Therefore, a small-diameter type separation column having a smaller diameter than normal, preferably having a diameter of 3 mm or less, which can perform analysis with a small amount of sample and a small amount of eluent and can improve the measurement sensitivity, is particularly suitably used. It is possible.

Downstream of the separation column 24, a multi-way switching valve 26, which is a four-way switching valve, is arranged. Multi-directional switching valve 2
No. 6 has a lower elution capacity than the eluent for removal, preferably has substantially the same elution power as the eluent for measurement, that is, the same elution power as the eluent for measurement (or has the same composition as the eluent for measurement)
A third eluent tank 28 for filling and supplying the auxiliary eluent is connected. Further, the remaining port of the multi-way switching valve 26 is provided with a suppressor 3 for changing the eluent component to a form having a low electric conductivity to lower the background level of the electric conductivity measurement.
2 and the first waste liquid bottle 40.

That is, in the analyzer shown in the drawing,
The separation column 24 and the third eluent tank 28 are connected to the suppressor 32 and the first waste liquid bottle 40 by the multi-directional switching valve 26.
Can be connected freely.

The analyzer (method) of the present invention having such a configuration allows the removal eluent for rapidly eluting the matrix components from the separation column 24 to flow into the suppressor 32 in the gradient analysis method. Without flowing, the liquid can flow into the first waste liquid bottle 40.

As is well known, the gradient analysis method means that a dilute eluent for measurement (eluent for measurement) is used for measurement.
The peak of the chromatogram is sharpened by using, and after the target component is eluted from the separation column 24, the elution of the late-eluting component from the separation column 24 is performed using a high-concentration eluent (removal eluent). This is a method that enables rapid and continuous analysis by a liquid chromatograph device by promoting it. It should be noted that originally, it means that the density is gradually changed (a density gradient is applied), but there is no problem even if the density is changed from a low density to a high density at once. According to a new finding of the present inventors, the diameter of the separation column and the suppressor is 3 mm.
Although the following small-diameter type chromatographic apparatus was particularly prominently confirmed, when a high-concentration eluent was used, even after the matrix component was eluted from the separation column 24, the eluent was changed to a dilute one, the high-concentration eluent was changed. As described above, the eluent component stays in the suppressor, causing a decrease in measurement sensitivity and a decrease in measurement accuracy of the target component.

On the other hand, according to the analyzer (method) of the present invention, when the eluent for removal is supplied to the separation column 24, the separation column 24 and the first waste liquid bottle 40 are connected by the multidirectional switching valve 26. And remove the eluent for removal with the suppressor 32.
The matrix components remaining in the separation column 24 can be quickly eluted without flowing into the separation column 24. In addition, at this time, the third eluent tank 28 is connected to the suppressor 32, and an auxiliary eluent equivalent to the eluent for measurement is supplied, so that the suppressor 32 is stably started. After the elution of the matrix component from the separation column 24, the next measurement can be performed immediately. Therefore, according to the present invention, in the liquid chromatography utilizing the gradient method, the matrix component is quickly eluted from the separation column without giving the adverse effect of the eluent for removal to the suppressor or the detector, and the matrix component is converted to the matrix component. High-accuracy continuous measurement can be rapidly performed without a decrease in measurement sensitivity and accuracy caused by the measurement, and it can be suitably used particularly for liquid chromatography using a small-diameter separation column and a suppressor.

The suppressor 32 that can be used in the present invention is not particularly limited, and any known separation columns can be used according to the target component to be measured. It may be. Here, as described above, in the present invention, since the eluent for removal does not flow into the suppressor 32 and the measurement sensitivity and accuracy do not decrease due to the retention of the eluent components in the suppressor 32, a small amount of sample and a small amount of In particular, a small-diameter type suppressor capable of performing analysis with an eluent and improving measurement sensitivity can be suitably used.

In the present invention, it is not limited to providing the third eluent tank 28 as shown in the illustrated example and supplying the auxiliary eluent to the suppressor, but providing a supply means to fill the first eluent tank. It may be configured to supply a diluted eluent.

The suppressor 32 is connected to a regenerating liquid supply means 30 for regenerating the capability of the suppressor 32. The used regenerating liquid is used in the second waste liquid bottle 42.
Is discharged to

Downstream of the suppressor 32, an electric conductivity detection cell 34 is arranged.
Is connected to a third waste liquid bottle 44. An electric conductivity detector 36 for measuring the electric conductivity of the eluent and detecting the target component is (electrically) connected to the electric conductivity detection cell 34. The result is recorded in the recording device 38.

Hereinafter, the operation of the analyzer (method) of the present invention will be described. First, the sample / eluent switching valve 16 and the multi-directional switching valve 26 are switched to the state shown in FIG. 1, and the autosampler 10 and the sample loop 22, the concentration control pump 20 and the separation column 24, and the separation column 24 and the suppressor 32 And are respectively connected.
In this state, a sample is injected from the autosampler 10 into the sample loop 22. In addition, the first eluent tank 12 and the second
The dilute and high-concentration eluents filled in the eluent tank 14 are:
Concentration control pump 2 while being deaerated by deaerator 18
It is pumped up by 0, mixed / concentrated and adjusted to be an eluent for measurement, supplied to the sample / eluent switching valve 16, and flows through the main route from the separation column 24 to the suppressor 32 to the third waste liquid bottle 44. It is desirable that the degassing device 18 be installed, but in the case of the present invention, the concentration changes suddenly. Therefore, even if degassing is not performed, the temporary fluctuation due to gas bubbles is not so fatal, is not.

When the injection of the sample into the sample loop 22 is completed, the sample / eluent switching valve 16 is switched to the state shown in FIG. 2, and the concentration control pump 20, the sample loop 22, and the separation column 24 are connected. The measurement starts. That is, the eluent for measurement is supplied to the sample loop 22, and the sample flows into the separation column 24 together with the eluent for measurement. When the sample (eluent for measurement) flows into the separation column 24 and the eluent for measurement is further supplied, the components in the sample having the shorter elution time are separated by the separation column 2.
4, passes through the multi-way switching valve 26, the background of the eluent component is reduced by the suppressor 32, and then flows into the electric conductivity detection cell 34, where the electric conductivity is detected by the electric conductivity detector 36. The measured component is detected and recorded in the recording device 38. The eluent for measurement (the target component) that has passed through the electric conductivity detection cell 34
Is discharged to the third waste liquid bottle 44.

The electric conductivity detector 36 or the recording device 3
8, it is confirmed that all the target components have flowed out of the separation column 24, the multi-directional switching valve 26 is switched to the state shown in FIG. 3, and the separation column 24 and the first waste liquid bottle 40 The tank 28 and the suppressor 32
Connected respectively. At the same time, the auxiliary eluent is supplied from the third eluent tank 28 to the suppressor 32, while the concentration control pump 20 adjusts the mixing amount of each eluent from the first eluent tank 12 and the second eluent tank 14. The eluent is then supplied to the separation column 24 to quickly elute the remaining matrix components. At this time, if necessary, the flow rate of the eluent for removal may be increased. The eluent for removal and the matrix component that have passed through the separation column 24 are discharged to the first waste liquid bottle 40 without flowing into the suppressor 32.

When the elution of the matrix component from the separation column 24 is completed, the concentration control pump 20 adjusts the concentration of the eluent again and supplies the eluent for measurement for several minutes, thereby supplying the eluent for removal. When the eluent flow rate is adjusted, the flow is returned to the measured flow rate.

When the replacement with the eluent for measurement is completed, the multi-directional switching valve 26 is switched to connect the separation column 24 and the suppressor 32 as shown in FIG. (3) Stabilize the system by flowing through the entire main route to the waste liquid bottle 44, and after a predetermined time elapses,
The eluent switching valve 16 is switched to the state shown in FIG. 1 to wait for the next measurement.

Although the method and apparatus for gradient analysis of liquid chromatography of the present invention have been described in detail above, the present invention is not limited to the above-described examples, and various changes and modifications can be made without departing from the spirit of the present invention. Of course, improvements may be made.

[0046]

EXAMPLES Hereinafter, specific examples of the method and apparatus for gradient analysis of liquid chromatography of the present invention will be described.
The present invention will be described in more detail. Note that the present invention is not limited to the following embodiments.

Example 1 Sulfate ions having a concentration of 100 μg / l to several mg / l in a solution of 100 mg / l perchloric acid were measured using the analyzer of the present invention shown in FIGS. The analysis was performed according to the analysis method of the present invention. In addition, each eluent, measurement conditions, etc. are as follows.

First eluent tank 12 ion-exchanged water Second eluent tank 14 18 mM Na ₂ CO ₃ , 17 m
M NaHCO ₃ third eluent tank 28 1.8 mM Na ₂ CO ₃ , 1.
7 mM NaHCO ₃ eluent flow rate 0.5 e.
ml / min or 0.8 ml / min Third eluent 0.5 ml / min Regenerating solution 25 mM H ₂ SO ₄ Regenerating solution flow rate 5 ml / min Sample amount 10 μl Separation column 24 Dionex IONPAC
AS4A-SC2mm Suppressor 32 Dionex AMMS (2m
m)

The sample was analyzed under the above conditions. First,
In the state shown in FIG. 1, 10 μl of a sample is injected from the autosampler 10 into the sample loop 22. During this time, 1.8 mM Na ₂ was supplied from the concentration control pump 20.
The eluent for measurement adjusted to CO ₃ and 1.7 mM NaHCO ₃ is supplied to the sample / eluent switching valve 16 at a flow rate of 0.5 ml / min.

Next, the sample / eluent switching valve 16 is switched as shown in FIG.
0, connecting the sample loop 22 and the separation column 24,
The measurement was started.

Under the above conditions, it was confirmed that the detection of the target component (sulfate ion) was completed after 10 minutes.
6 is switched to the state shown in FIG. 3, and the suppressor 32 is filled with the 1.8 mM Na
An auxiliary eluent of ₂ CO ₃ and 1.7 mM NaHCO ₃ was supplied, and at the same time, the concentration of the eluent was adjusted by the concentration control pump 20 to obtain 18 mM Na ₂ CO ₃ and 17 mM.
The eluent for removing NaHCO ₃ was supplied to the sample loop 22 and the separation column 24. At the time of switching to the eluent for removal, the flow rate was increased to 0.8 ml / min.
The eluent for removal passes through the multi-way switching valve 26 to the first eluent.
It is discharged to a waste liquid bottle 40.

At the lapse of 18 minutes from the start of the measurement, the concentration of the eluent was adjusted by the concentration control pump 20 and the eluent supplied to the separation column 24 was 1.8 mM N 2.
The eluent for measurement was changed to a ₂ CO ₃ and 1.7 mM NaHCO ₃ , and the flow rate was again set to 0.5 ml / min.

After a lapse of 23 minutes, elution of the matrix components
It is considered that the replacement of the eluent for measurement in the system is almost completed, and the multi-directional switching valve 26 is switched to the state shown in FIG. 2 to place the eluent for measurement on the main path from the separation column 24 to the third waste bottle 44. After 30 minutes, the sample / eluent switching valve 16 was switched to the next measurement standby state shown in FIG. 1, one measurement routine was completed, and the next measurement was started immediately. That is, in this example (invention example), one measurement operation was completed in 30 minutes. The replacement with the eluent for measurement may be confirmed by a method of performing a preliminary measurement by flowing the eluent through the suppressor 32, or a method of mixing with a coloring agent and measuring by post-column absorption spectroscopy.

FIG. 4 shows the result of repeating the above measurement routine. As shown in FIG. 4, according to the present invention, even if the next sample was measured immediately after the measurement, the sensitivity did not decrease and good reproducibility was obtained.

<Example 2> In Example 1, the supply of the eluent for removal was terminated without switching the multi-directional switching valve 26 after 10 minutes had elapsed from the start of measurement (when 22 minutes had elapsed in Example 1). )), The same measurement was performed by changing the supply time of the eluent for measurement in various ways, sulfate ions were measured, and the fluctuation of the detection sensitivity was examined. FIG. 5 shows the result of performing the same measurement three times.
Shown in For the purpose of comparison, as a method of the present invention, the same experiment as described above was performed by switching the multi-directional switching valve 26 after the lapse of 10 minutes from the start of the measurement, and the result of measuring the change in sensitivity is shown in FIG. .

As shown in FIG. 6, the multi-way switching valve 26
In the present invention, the sensitivity is not changed in the present invention, in which the eluent for removal is switched and the removal is performed, but as shown in FIG. 5, when the present invention is not used, the sensitivity fluctuates, and the recovery is stable for about 2 hours. Then, it is necessary to replace the eluent concentration in the flow of the apparatus.

Example 3 A change in sensitivity was measured in the same manner as in Example 2 except that the eluent was changed to boric acid and sodium hydroxide. The concentration of the eluent for measurement was 4 for both the boric acid eluent and the sodium hydroxide eluent.
0 mM and the concentration of the eluent for removal were 400 mM. FIG. 7 shows the results. In FIG. 7, △ shows the results with the sodium hydroxide eluent, and □ shows the results with the boric acid eluent. In addition, the results in Example 2 (in the case where the present invention is used and in the case where the present invention is not used) are also shown as ○.

As shown in FIG. 7, in the conventional method, even if the type of the eluent is changed, the sensitivity is restored for about 2 hours to recover the sensitivity similarly to the case of the mixed eluent of sodium carbonate and sodium hydrogen carbonate. It is necessary to replace the eluent concentration in the flow. Also, when measuring at the usual constant eluent concentration,
The analysis time requires at least 75 minutes per sample.

In the above embodiment, the suppressor 32
This is an example using an ion exchange membrane type suppressor (AMMS (2 mm) manufactured by Dionex), but almost the same results were obtained when an electrodialysis type suppressor was used.

From the above results, the effect of the present invention is clear.

[0061]

As described above in detail, according to the liquid chromatography method and apparatus using the gradient method of the present invention, the eluent for removal having a high elution power does not flow into the suppressor, but flows from the separation column. The matrix components can be rapidly eluted, and high-precision continuous measurement can be quickly performed without a decrease in measurement sensitivity and accuracy due to the matrix components. Since the eluent for removal does not flow into the suppressor, it is particularly suitable for liquid chromatography using a small-diameter separation column and a suppressor in which sensitivity and accuracy are likely to be degraded by the eluent components to the suppressor. .

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing an example of a gradient analyzer for liquid chromatography of the present invention and its operation.

FIG. 2 is a conceptual diagram showing the operation of the gradient analyzer for liquid chromatography shown in FIG.

FIG. 3 is a conceptual diagram showing the operation of the gradient analyzer for liquid chromatography shown in FIG.

FIG. 4 is a graph showing the results of routine analysis by the liquid chromatography gradient analysis method of the present invention.

FIG. 5 is a graph showing the relationship between the stabilization time and the measurement sensitivity according to a conventional liquid chromatography gradient analysis method.

FIG. 6 is a graph showing the relationship between the stabilization time and the measurement sensitivity according to the gradient analysis method for liquid chromatography of the present invention.

FIG. 7 is a graph showing the relationship between the stabilization time and the measurement sensitivity according to another example of the gradient analysis method of the present invention and the conventional liquid chromatography.

FIGS. 8 (a), (b) and (c) are chromatograms obtained by performing routine analysis in conventional liquid chromatography.

FIG. 9 is a view conceptually showing a part of an apparatus for performing a conventional liquid chromatography gradient analysis method.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 10 Autosampler 12 1st eluent tank 14 2nd eluent tank 16 Sample / eluent switching valve 18 Deaerator 20 Concentration control pump 22 Sample loop 24 Separation column 26 Multidirectional switching valve 28 3rd eluent tank 30 Regeneration liquid Tank 32 Suppressor 34 Electric Conductivity Cell 36 Electric Conductivity Detector 38 Recording Device 40 Waste Liquid Bottle 1 42 Waste Liquid Bottle 2 44 Waste Liquid Bottle 3

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 30/34 G01N 30/02

Claims (4)

(57) [Claims] In liquid chromatography using a separation column and a suppressor, after a target component flows out of the separation column by an eluent for measurement, the eluent for removal having a higher elution capacity than the eluent for measurement is used as the eluent for removal. A gradient analysis method in which an unnecessary component flows into a separation column and flows out of the separation column, wherein the eluent for removal flows into the separation column, and an outflow route from the separation column is switched to a route different from a suppressor. A gradient analysis method for liquid chromatography, wherein an auxiliary eluent having a lower elution capacity than the eluent for removal is simultaneously flowed into the suppressor. 2. The gradient analysis method for liquid chromatography according to claim 1, wherein the diameter of the separation column and the suppressor is 3 mm or less. 3. After the target component flows out of the separation column,
A gradient analyzer for liquid chromatography for performing a gradient analysis in which a removing eluent having a higher eluting ability than a measuring eluent flows into the separation column and unnecessary components are caused to flow out of the separation column, comprising: a separation column; A suppressor arranged downstream of the column, a detecting means for detecting a target component flowing into the suppressor, a sample, an eluent for measurement for eluting the components in the separation column, and a more eluent than the eluent for measurement Means for supplying a high-performance removal eluent to the separation column, auxiliary eluant supply means for supplying an auxiliary eluent having a lower elution capacity than the removal eluent to the suppressor, the separation column and Disposed between the suppressor, the separation column or auxiliary solution supply means and the suppressor or waste liquid path, After the detection of the target component by the detection means is completed, the removal eluent is supplied to the separation column, and the multidirectional switch valve is connected to the separation column. A gradient analyzer for liquid chromatography, wherein the switching is performed, an outflow path from the separation column is connected to a waste liquid path, and an auxiliary solution supply means and the suppressor are connected. 4. The gradient analyzer for liquid chromatography according to claim 3, wherein the diameters of the separation column and the suppressor are 3 mm or less.