JP5446515B2 - Sample liquid dilution method and liquid chromatograph apparatus using the same - Google Patents

Description

  The present invention relates to a method for diluting a sample solution in an analysis using a liquid chromatograph, and a liquid chromatograph apparatus using the method.

As shown in FIG. 1, the liquid chromatograph is mainly composed of a container (100) containing an eluent, a liquid feed pump (200), a sample injection device (300), an analysis column (400), and a detector (500). Configured. Of these, the sample injection device (300) is at least
A sample container (301) containing a sample solution to be analyzed;
A sample loop (302) for containing the sample liquid;
A sample injection valve (303) to which the sample loop (302) is connected, and a means for introducing a sample into part or all of the sample loop (302);
A means (313) for switching the sample injection valve (303).

  As the sample injection valve (303), the hexagonal valve shown in FIG. 1 is usually used, and the sample liquid stored in the sample container (301) is introduced into the sample loop (302) by rotating the valve by 60 degrees. A flow path (loading position, shown by a dotted line in FIG. 1), and a flow path (introduction position, shown by a solid line in FIG. 1) for introducing the sample liquid introduced into the sample loop (302) into the analysis column (400). It is possible to switch the flow path.

  When a sample is introduced into the analysis column (400) using the sample injection device (300) shown in FIG. 1, first, the sample accommodated in the sample container (301) with the sample injection valve (303) set to the load position. The liquid is introduced into the sample loop (302) by suction from the needle (305) using a syringe pump (304, 314). At this point, the sample liquid is introduced into part or all of the sample loop (302). In addition, since the flow path from the needle (305) to the sample injection valve (303) becomes a dead volume, it is preferable to reduce the capacity of the flow path. If the capacity of the channel is not negligible, the needle (305) is raised from the sample liquid level of the sample container (301) by the moving mechanism (308), and then air is sucked halfway through the channel. Also good. After completing the introduction of the sample liquid into the sample loop (302), the sample injection valve (303) is switched to the introduction position, and the sample liquid introduced into the sample loop (302) is fed by the liquid feed pump (200). The eluent is introduced into the analysis column (400), and the eluate from the column is detected by the detector (500).

  However, since there is usually an upper limit on the load capacity of the analysis column (400) and the concentration detectable by the detector (500), if the concentration of the analyte component contained in the sample solution is high, the column and detection It is necessary to dilute the sample solution before introducing it into the analytical column (400) so as to be within the upper limit of the vessel.

As a method for diluting the sample solution,
[1] A method of diluting using a whole pipette and a volumetric flask,
[2] After the sample solution and the diluted solution are weighed and discharged to the dilution container by the suction / discharge means, suction / discharge by the means, rotation by a spatula or stirrer, vibration by a spatula, ultrasonic wave or vortex mixer, air flow The method of stirring and diluting by discharge etc. can be illustrated.

  Among the dilution methods of [2] above, the method of using a pipetter equipped with a disposable tip as a suction / discharge means and stirring and diluting by suction / discharge by the pipetter is usually used in an automatic analyzer (Patent Document 1). In general, after aspiration at a position near the bottom of the dilution container, the pipetter is lifted upward and discharged to stir and dilute the sample solution. However, when the method is applied to a liquid chromatograph apparatus, the diluted sample solution sucked by the pipetter cannot be directly introduced into the sample injection valve. This necessitates a needle and a syringe pump for separately sucking a diluted sample solution and introducing the diluted sample solution into the analysis column, which complicates the apparatus. Further, when a diluted sample solution obtained by stirring by suction / discharge is analyzed by a liquid chromatograph apparatus, there is a problem that reproducibility when the same sample is analyzed is not sufficient.

  Patent Document 2 discloses a liquid chromatograph apparatus in which a needle for sucking a sample liquid is immersed in a sample liquid housed in a sample container, and the needle is vibrated to stir and then suck the sample liquid with the needle. Yes. However, the stirring method according to Patent Document 2 has a problem that fine droplets are generated, and there is a problem that the apparatus configuration is complicated.

  In Patent Document 3, after the flow path from the needle to the syringe pump is filled with the hydraulic fluid in advance, [A] a small amount of air, [B] a predetermined amount of diluent, [C] a small amount of air, [D A dilution apparatus for a liquid chromatograph apparatus is disclosed in which a predetermined amount of sample solution is sucked in order and the sample solution is diluted by discharging the [B] to [D] into a dilution container with the pump. However, the apparatus of Patent Document 3 does not disclose a mechanism for introducing the diluted sample solution into the analysis column, and requires a needle and a syringe pump for sucking and introducing the diluted sample solution into the column separately.

Japanese Patent Laid-Open No. 10-62432 Japanese Patent Application Laid-Open No. 11-304779 Japanese Patent Publication No.8-10214

  An object of the present invention is to provide a sample liquid dilution method having high reproducibility in a liquid chromatograph analysis and a liquid chromatograph apparatus using the method.

The present invention made in view of the above problems includes the following inventions:
The first invention is
A sample container for containing a sample solution,
A hydraulic fluid container for containing the hydraulic fluid,
A diluent container containing the diluent,
Syringe pumps and needles for aspirating / discharging these liquids or air,
And using a sample diluting device equipped with a dilution container containing a mixed solution of the sample liquid and the diluting liquid,
[1] Fill the flow path from the needle to the syringe pump with the working fluid using the syringe pump,
[2] Using the syringe pump, (A) a small amount of air, (B) a predetermined amount of diluent, (C) a small amount of air, and (D) a predetermined amount of sample liquid are sucked from the needle in this order.
[3] Discharging (D) to (D) into the dilution container,
[4] In the method of diluting the sample liquid by sucking / discharging the whole or a part of the mixed liquid of the sample liquid and the diluted liquid discharged in [3],
The sample liquid dilution method is characterized in that the liquid level when the liquid mixture discharged in [3] is stored in the dilution container is not more than 3 times the inner diameter of the dilution container.

The second invention is
A sample container for containing a sample solution,
A diluent container containing the diluent,
Syringe pumps and needles for aspirating / discharging these liquids or air,
And using a sample diluting device equipped with a dilution container containing a mixed solution of the sample liquid and the diluting liquid,
[1] Fill the flow path from the needle to the syringe pump with the diluent using the syringe pump,
[2] Using the syringe pump, (A) a small amount of air and (B) a predetermined amount of sample liquid are sucked from the needle in this order,
[3] (A), (B) and a part of the diluent are discharged into the dilution container,
[4] In the method of diluting the sample liquid by sucking / discharging the whole or a part of the mixed liquid of the sample liquid and the diluted liquid discharged in [3],
The sample liquid dilution method is characterized in that the liquid level when the liquid mixture discharged in [3] is stored in the dilution container is not more than 3 times the inner diameter of the dilution container.

The third invention is
In a liquid chromatograph apparatus provided with a container containing an eluent, a pump for feeding the eluent, a sample injection apparatus for diluting and introducing a sample liquid to be analyzed, an analytical column, and a detector, the sample injection apparatus comprises:
A sample container containing a sample liquid, a working liquid container containing a working liquid, a diluting liquid container containing a diluting liquid, a syringe pump and a needle for sucking / discharging these liquids or air, and a sample liquid and diluting liquid A sample diluting device including a diluting container for containing a mixed solution of
A sample liquid diluted by the apparatus is introduced into a sample loop, and a sample injection valve capable of introducing the sample liquid introduced into the sample loop into the analysis column is provided.
The sample diluting apparatus dilutes the sample liquid by the method described in the first invention.

The fourth invention is
In a liquid chromatograph apparatus provided with a container containing an eluent, a pump for feeding the eluent, a sample injection apparatus for diluting and introducing a sample liquid to be analyzed, an analytical column, and a detector, the sample injection apparatus comprises:
A sample container containing a sample liquid, a dilution container containing a diluent, a syringe pump and a needle for sucking / discharging these liquids or air, and a dilution container containing a mixed liquid of the sample liquid and the diluent Sample dilution device,
A sample liquid diluted by the apparatus is introduced into a sample loop, and a sample injection valve capable of introducing the sample liquid introduced into the sample loop into the analysis column is provided.
The sample diluting apparatus dilutes the sample liquid by the method described in the second invention.

  Hereinafter, the present invention will be described in detail.

The sample solution dilution method of the present invention comprises:
[1] A syringe pump is used to fill the flow path from the needle to the syringe pump with a working fluid,
[2] Using the syringe pump, (A) a small amount of air, (B) a predetermined amount of diluent, (C) a small amount of air, and (D) a predetermined amount of sample liquid are sucked from the needle in this order.
[3] (B) to (D) are discharged into a dilution container,
[4] In the method of diluting the sample liquid by sucking / discharging the whole or a part of the mixed liquid of the sample liquid and the diluted liquid discharged in [3],
The liquid level when the liquid mixture discharged in [3] is stored in the dilution container is not more than three times the inner diameter of the dilution container. Since the dilution method can dilute the sample solution with high accuracy, the peak reproducibility of the same diluted sample solution in the analysis using the liquid chromatograph can be improved.

  The shape of the dilution container used in the dilution method of the present invention may be cylindrical or prismatic, and may be further tapered. When a prismatic container is used, the liquid level when the liquid mixture discharged in [3] is stored should be three times or less the length of the short side of the quadrangle that forms the bottom surface. In the case of using a container with a taper, the liquid level when the mixed liquid discharged in [3] is accommodated should be three times or less the inner diameter of the bottom surface.

  The size of the dilution container used in the dilution method of the present invention may be appropriately selected within the range satisfying the above-described conditions based on the amount of the liquid mixture discharged in [3]. If the liquid level when the mixed liquid discharged in [3] is stored in the sample container is not more than 3 times the inner diameter of the sample container, the same container is used for the sample container and the dilution container. can do.

  A preferred embodiment of the dilution container used in the dilution method of the present invention is a container in which the liquid level when the mixed liquid discharged in [3] is stored in the dilution container is not more than twice the inner diameter of the dilution container. It is. By diluting the sample liquid using the preferred dilution container, the sample liquid can be diluted with higher accuracy, so that the peak reproducibility of the same diluted sample liquid in the analysis using the liquid chromatograph is further improved. Can do.

  The material of the dilution container used in the dilution method of the present invention may be a commonly used resin such as polypropylene or glass, but a material having good wettability to the sample solution and the dilution liquid (for example, glass) is used as the material of the dilution container. When used, it is preferable because the peak reproducibility when the same sample diluent is analyzed by liquid chromatography is further improved.

  In the dilution method of the present invention, the suction amount of the air sucked in the above [2] may be any amount that does not mix the working fluid and the diluent, and the diluent and the sample solution, and the pipe from the needle and the needle to the syringe pump. May be determined in consideration of the inner diameter.

  In the dilution method of the present invention, when the discharge operation of [4] is performed away from the liquid surface in the dilution container and the droplet is dropped, stirring by the suction / discharge operation of [4] is efficiently performed. It is preferable in that it can be performed.

Examples of the diluent used in the dilution method of the present invention include pure water, a buffer solution, and an organic solvent that are usually used in a liquid chromatograph apparatus. Note that it is preferable to use a liquid having the same composition as the eluent used in the liquid chromatographic analysis described later as a diluent because the fluctuation of the baseline of the chromatogram can be suppressed. The working fluid used in the dilution method of the present invention may be any fluid that can transmit the volume change of the syringe pump to the needle, and examples thereof include pure water, buffer solution, and organic solvent. In addition, it is preferable to use a liquid having the same composition as the washing liquid as the working liquid in that the needle can be washed by discharging the working liquid remaining in the flow channel after introducing the diluted sample liquid into the analytical column described later. In addition, when a fluid having the same composition as the diluent is used as the working fluid, the working fluid container also has a role as a diluent container, so there is no need to install a diluent container separately, and the device configuration can be simplified / miniaturized. This is preferable. If the hydraulic fluid and diluent have the same composition,
[1] Using the syringe pump, fill the flow path from the needle to the syringe pump with a working fluid (diluent),
[2] Using the syringe pump, (A) a small amount of air and (B) a predetermined amount of sample liquid are sucked in this order,
[3] (A), (B) and a part of the hydraulic fluid (diluent) are discharged into a dilution container,
[4] The sample liquid may be diluted by a method of sucking / discharging the whole or a part of the mixed liquid of the sample liquid and the diluted liquid discharged in [3].

  As another aspect of the method for diluting the sample liquid with high accuracy, the suction / discharge operation of [4] is performed a plurality of times, and the suction / discharge amount of [4] is decreased as the number of times of suction / discharge is repeated. The dilution method used can be raised. The dilution method improves the peak reproducibility of the same diluted sample solution in the analysis using the liquid chromatograph as compared with the method of diluting with the suction / discharge amount always constant in [4]. Note that if the suction / discharge amount of [4] is less than 30% of the mixed liquid amount discharged in [3], sufficient agitation is not performed, so 30% to 100% of the mixed liquid amount discharged in [3]. % Must be done. In addition, it is preferable that the suction / discharge amount of [4] is 50% to 100% of the mixed liquid amount discharged in [3] because peak reproducibility is further improved.

  In the dilution method, the number of suction / discharge of [4] may be a plurality of times, that is, two or more times. What is necessary is just to determine the frequency | count. If the amount of suction / discharge amount in the suction / discharge operation in [4] is small relative to the previous amount, liquid chromatographic analysis of the same diluted sample liquid as in the dilution method in which suction / discharge is always performed at a constant amount. Since the peak reproducibility is reduced, the reduction width of the suction / discharge amount in [4] is preferably 15% or more. Furthermore, it is more preferable that the reduction width is 20% or more in terms of improving peak reproducibility.

  An embodiment of a liquid chromatograph apparatus using the sample liquid dilution method of the present invention is shown in FIG. In the sample injection device (300) shown in FIG. 2, the sample injection valve (303) is a valve that can introduce the diluted sample liquid introduced into the sample loop (302) into the analysis column (400) by switching the flow path. In addition to the rotary six-way valve shown in FIG. 2, two rotary four-way valves or two three-way solenoid valves can be used. As the switching valve (307) for connecting the flow path on the syringe pump (304) side and the flow path on the needle (305) side or the hydraulic fluid container (306) side, a three-way electromagnetic valve can be exemplified. In order to prevent the diluted sample liquid from entering the syringe pump (304), a holding loop (309) for holding the diluted solution in the flow path from the sample injection valve (303) to the switching valve (307). Is provided. The material from the needle (305) to the sample injection valve (303), the sample loop (302), and the holding loop (309) may be resistant to the mobile phase used in the liquid chromatograph. Tetrafluoroethylene (PTFE), polyether ether ketone (PEEK), fluorinated ethylene propylene (FEP) and the like are preferably used. The sample container (301) for storing the sample liquid may be a container having a lid for preventing evaporation. The diluent container (310) containing the diluent may be installed in a range where the needle (305) can be moved by the moving mechanism (308). When a plurality of diluents are used or when a large amount of diluent is used, a plurality of diluent containers may be installed.

An example of a method for diluting a sample solution and introducing it into an analytical column using the sample injection device (300) shown in FIG. 2 will be described below.
[1] The switching valve (307) is switched to the hydraulic fluid container (306) side, and the hydraulic fluid is sucked using the syringe pumps (304, 314). The sample injection valve (303) is set to the introduction position (shown by a solid line in FIG. 2).
[2] The switching valve (307) is switched to the needle (305) side, and the tip of the needle (305) is filled with the working fluid using the syringe pumps (304, 314).
[3] A small amount of air is sucked using a syringe pump (304, 314).
[4] After the needle (305) is moved to a position where it comes into contact with the diluent contained in the diluent container (310) by the moving mechanism (308), the diluent is sucked using the syringe pump (304, 314). .
[5] After raising the needle (305) by the moving mechanism (308), a small amount of air is sucked using the syringe pump (304, 314).
[6] After the needle (305) is moved to a position where it comes into contact with the sample solution stored in the sample container (301) by the moving mechanism (308), the sample solution is sucked using the syringe pumps (304, 314).
[7] After moving the needle (305) to the position of the dilution container (311) by the moving mechanism (308), the diluted solution sucked by [4] using the syringe pump (304, 314) and sucked by [5] The sample liquid sucked with air and [6] is discharged to the dilution container (311).
[8] A small amount of air is sucked using the syringe pump (304, 314).
[9] After moving the needle (305) to the position in contact with the discharge liquid of [7] housed in the dilution container (311) by the moving mechanism (308), the discharge liquid is used using a syringe pump (304, 314). Aspirate the entire amount or a part of it.
[10] After the needle (305) is raised by the moving mechanism (308), the liquid aspirated in [9] is discharged by using the syringe pumps (304, 314) and stirred.
[11] Repeat the operations [8] to [10] one or more times as necessary. However, when repeating, it is preferable that the suction / discharge amount is smaller than the previous suction / discharge amount.
[12] After raising the needle (305) by the moving mechanism (308), a small amount of air is sucked using the syringe pump (304, 314).
[13] The moving mechanism (308) moves the needle (305) to a position where it comes into contact with the diluted sample liquid stored in the dilution container (311), and the switching device (313) moves the sample injection valve (303) to the loading position ( After switching to the dotted line in FIG. 2, the diluted sample solution is sucked up to the sample loop (302) using the syringe pump (304, 314).
[14] The sample injection valve (303) is switched to the introduction position (shown by a solid line in FIG. 2) by the switching device (313), and the diluted sample held in the sample loop (302) is transferred by the liquid feed pump (200). The introduced eluent is introduced into the analysis column (400), and the components in the diluted sample liquid are detected by the detector (500).

  Another embodiment of the liquid chromatograph apparatus using the sample liquid dilution method of the present invention is shown in FIG. The difference from the apparatus of FIG. 2 is that there is no installation of the diluent container (310) and the working fluid also serves as the diluent.

An example of a method for diluting a sample solution and introducing it into an analytical column using the sample injection device (300) shown in FIG. 3 will be described below.
[1] The switching valve (307) is switched to the hydraulic fluid (diluent) container (306) side, and the hydraulic fluid (diluent) is sucked using the syringe pumps (304, 314). The sample injection valve (303) is set to the introduction position (shown by a solid line in FIG. 3).
[2] The switching valve (307) is switched to the needle (305) side, and the tip of the needle (305) is filled with the working fluid (diluent) using the syringe pumps (304, 314).
[3] A small amount of air is sucked using a syringe pump (304, 314).
[4] After the needle (305) is moved to a position where it comes into contact with the sample liquid stored in the sample container (301) by the moving mechanism (308), the sample liquid is sucked using the syringe pumps (304, 314).
[5] After the needle (305) is moved to the position of the dilution container (311) by the moving mechanism (308), a necessary amount of hydraulic fluid (diluent) is sucked using the syringe pump (304, 314), and suction is performed with [3]. The air sucked and the sample sucked in [4] are discharged to the dilution container (311).
[6] A small amount of air is sucked using the syringe pump (304, 314).
[7] After moving the needle (305) to the position in contact with the discharge liquid of [5] housed in the dilution container (311) by the moving mechanism (308), the discharge is performed using a syringe pump (304, 314). Aspirate all or part of the fluid.
[8] After the needle (305) is raised by the moving mechanism (308), the liquid sucked in [7] is discharged using the syringe pump (304, 314), and the mixture is stirred.
[9] If necessary, repeat the operations [6] to [8] one or more times. However, when repeating, it is preferable that the suction / discharge amount is smaller than the previous suction / discharge amount.
[10] After raising the needle (305) by the moving mechanism (308), a small amount of air is sucked using the syringe pump (304, 314).
[11] The moving mechanism (308) moves the needle (305) to a position where it comes into contact with the diluted sample liquid stored in the dilution container (311), and the switching device (313) moves the sample injection valve (303) to the loading position ( After switching to the dotted line in FIG. 3, the diluted sample solution is aspirated to the sample loop (302) using the syringe pump (304, 314).
[12] The sample injection valve (303) is switched to the introduction position (illustrated by a solid line in FIG. 3) by the switching device (313), and the diluted sample liquid held in the sample loop (302) is fed to the liquid feed pump (200). Is introduced into the analytical column (400) by the eluent sent in step, and the components in the diluted sample liquid are detected by the detector (500).

  2 and 3, when the inner diameter, suction speed, and discharge speed of the needle (305) and the holding loop (309) are inappropriate, the working fluid (in the case of the device of FIG. This is not preferable because the interface between a small amount of air and the liquid inserted between the diluted sample liquid (mixed liquid of the sample liquid and the diluted liquid) is disturbed and the diluted sample liquid is diluted with the working liquid or the diluted liquid. Therefore, the pipe from the needle (305) to the sample injection valve (303), the pipe in the sample injection valve (303), and the holding loop (309) is changed depending on the wettability between the diluted sample solution and the material constituting the pipe. In any case, the inner diameter is preferably 0.4 mm or more and 1 mm or less. The suction / discharge speed is preferably 1 mL / min or more and 10 mL / min or less.

  In the apparatuses 2 and 3, the position of the needle (305) when the mixed liquid of the sample liquid and the diluted liquid stored in the dilution container (311) is sucked and when the mixed liquid is discharged is constant. Or may be changed during each operation. As an example of the change, when the mixed liquid of the sample liquid and the diluent stored in the dilution container (311) is sucked, the tip of the needle (305) is always the height from the bottom of the container (311) to the liquid level. A method of sucking the liquid while lowering the needle (305) so that the position becomes 1/2 of the above is mentioned.

The present invention
A sample container for containing a sample solution,
A hydraulic fluid container for containing the hydraulic fluid,
A diluent container containing the diluent,
Syringe pumps and needles for aspirating / discharging these liquids or air,
And using a sample diluting device equipped with a dilution container containing a mixed solution of the sample liquid and the diluting liquid,
[1] Fill the flow path from the needle to the syringe pump with the working fluid using the syringe pump,
[2] Using the syringe pump, (A) a small amount of air, (B) a predetermined amount of diluent, (C) a small amount of air, and (D) a predetermined amount of sample liquid are sucked from the needle in this order.
[3] Discharging (D) to (D) into the dilution container,
[4] In the method of diluting the sample liquid by sucking / discharging the whole or a part of the mixed liquid of the sample liquid and the diluted liquid discharged in [3],
The liquid level when the liquid mixture discharged in [3] is stored in the dilution container is not more than three times the inner diameter of the dilution container. By the dilution method, the sample solution can be diluted with high accuracy. Moreover, the peak reproducibility of the same diluted sample liquid in the liquid chromatographic analysis is improved by applying the dilution method to the liquid chromatograph apparatus.

In the dilution method, the working fluid and the diluent may have the same composition. In this case, the operations of [2] and [3] are performed using [2] the syringe pump (A) a small amount of air, (B) A predetermined amount of sample liquid is sucked from the needle in this order,
[3] The same effects as in the dilution method can be obtained even when the operations (A) and (B) and a part of the hydraulic fluid (diluent) are discharged to the dilution container.

The figure which shows the liquid chromatograph apparatus provided with the sample injection apparatus which does not dilute a sample liquid. The figure which shows the liquid chromatograph apparatus provided with the sample injection apparatus which can dilute a sample liquid. The figure which shows the liquid chromatograph apparatus provided with the sample injection apparatus which can dilute a sample liquid. The figure which shows the analysis result of the sample liquid diluted by the method of the comparative example 1. The figure which shows the analysis result of the sample liquid diluted by the method of Example 1. FIG. The figure which shows the analysis result of the sample liquid diluted by the method of Example 2. FIG. The figure which shows the analysis result of the sample liquid diluted with the method of Example 3. FIG. The figure which shows the analysis result of the sample liquid diluted by the method of Example 4. FIG. The figure which shows the analysis result of the sample liquid diluted by the method of the comparative example 2. FIG. The figure which shows the analysis result of the sample liquid diluted by the method of the comparative example 3. FIG.

  Examples will be shown below to describe the present invention in more detail. However, these examples are only examples of the present invention, and the present invention is not limited to the examples.

Comparative Example 1
Using the apparatus shown in FIG. 3, the peak reproducibility of the same diluted sample solution was confirmed.

  As the analytical column (400), a column filled with an ion exchange resin was used. The working fluid container (306) contained pure water as a working fluid (also serving as a diluent). The sample container (301) contained a 10 mM KCl aqueous solution as a sample solution. A polypropylene container having an outer diameter of φ11.3 mm × 32 mm (height) and an inner diameter of φ6.0 mm × 31 mm (height) was used for both the sample container (301) and the dilution container (311). When 700 μL of pure water is put into the container, the height of the liquid becomes about 24.8 mm. The needle (305) was a hard resin tube having an outer diameter of 2.0 mm and an inner diameter of 0.5 mm. A PTFE tube was used for piping from the needle (305) to the sample injection valve (303). A PEEK tube having an inner diameter of 0.75 mm and a length of 2 m was used as the holding loop (309). The suction / discharge speed of the syringe pump (304, 314) was 3.0 mL / min. An electric conductivity meter was used for the detector (500).

The sample solution was diluted and introduced into the analytical column as follows.
[1] With the sample injection valve (303) at the introduction position, 3 μL of air is sucked from the tip of the needle (305) from the state filled with the diluent (pure water) from the working fluid container (306) to the tip of the needle (305). Thereafter, 70 μL of the sample solution (10 mM KCl) was aspirated.
[2] 70 μL of the sample solution, 3 μL of the air, and 630 μL of pure water (total 703 μL) were discharged into a dilution container (311).
[3] After sucking 20 μL of air from the tip of the needle (305), 560 μL of pure water and sample liquid mixed with pure water and sample at a position of about 3 mm from the bottom of the dilution container (311) 80% of the liquid) was aspirated.
[4] The needle (305) was raised from the bottom of the dilution container (311) to a position of about 30 mm by the moving mechanism (308), and 560 μL was discharged.
[5] The operations of [3] and [4] were repeated twice to prepare a diluted sample solution.
[6] After 2 μL of air is sucked from the tip of the needle (305), the switching device (313) switches the sample injection valve (303) to the loading position, and the syringe pump (304, 314) is used to sample the diluted sample solution. Aspirated to (302).
[7] The sample injection valve (303) is switched to the introduction position, the diluted sample liquid introduced into the sample loop (302) is introduced into the analysis column (400), and the peak of the sample is confirmed with the detector (500). .
[8] The operations [6] and [7] were performed again.
[9] The operations [1] to [8] were performed again.

  The chromatograph of the analysis result is shown in FIG. In the following drawings, A1 and A2 represent the analysis results of the diluted sample solution prepared at the first time, and B1 and B2 represent the analysis results of the diluted sample solution prepared at the second time. A1 and B1 represent the first analysis result of each diluted sample solution, and A2 and B2 represent the second analysis result of each diluted sample solution, respectively. Although the peak area was almost constant and showed good reproducibility, when the chromatogram was enlarged and examined, there was a difference in measurement between the peak heights (A1 and A2, B1 and B2).

Example 1
In Comparative Example 1, the dilution container (311) is a polypropylene container having an outer diameter of φ11.8 mm × 32 mm (height) and an inner diameter of φ10.1 mm × 30.6 mm (height). (When water is added, the height of the liquid becomes approximately 8.74 mm), and the suction / discharge operation of [3] and [4] is changed to 500 μL at one time. Implemented.

  The chromatograph of the analysis result is shown in FIG. The variation in peak height between measurements (A1 and A2, B1 and B2) was smaller than in Comparative Example 1. As a result, the liquid level when the pure water and the sample solution of [2] are discharged to the dilution container (311) is changed to the dilution container (311) which is not more than three times the inner diameter of the container. It can be seen that the peak reproducibility is improved.

Example 2
In Comparative Example 1, the dilution container (311) has an outer diameter of φ10 (bottom) to 12 mm (top) × 21 mm (height), and an inner diameter of φ8.0 (bottom) to 10 (top) mm × 20 mm (height). The number of suction / discharge cycles of [3] and [4] above is placed in a polypropylene container having a narrow bottom and a wide upper part (when 700 μL of pure water is added to the container, the height of the liquid is about 12 mm). Was carried out in the same manner as Comparative Example 1 except that the number of times was changed from 3 times to 2 times.

  The chromatograph of the analysis result is shown in FIG. The variation in peak height between measurements (A1 and A2, B1 and B2) was smaller than in Comparative Example 1. Accordingly, if the pure water and the sample liquid of [2] are discharged into the dilution container (311), the liquid surface height is 3 times or less of the inner diameter of the container. It can be seen that the peak reproducibility is improved even in the case where the container is installed as in the case of the dilution container used in Example 1.

Example 3
In Comparative Example 1, the dilution container (311) is a glass container having an outer diameter of φ11.3 mm × 32 mm (height) and an inner diameter of φ9.0 mm × 31 mm (height) (700 μL of pure water is added to the container). In this case, the height of the liquid becomes about 11.0 mm, and the number of suction / discharge of [3] and [4] is changed from 3 times to 2 times. Carried out.

  The chromatograph of the analysis result is shown in FIG. The variation in peak height between measurements (A1 and A2, B1 and B2) was smaller than in Comparative Example 1. Thus, the peak reproducibility is improved by changing the material of the dilution container (311) from a material (polypropylene) having low wettability to pure water and a sample solution (10 mM KCl aqueous solution) to a material (glass). I understand that.

Example 4
In Comparative Example 1, the suction / discharge amounts of [3] and [4] are set to 700 μL for the first time (100% of pure water and sample liquid discharged in [2]), and 560 μL for the second time ([2] above). 80% amount of pure water and sample solution discharged in step 3), and the same procedure as in Comparative Example 1 except that the third time was 350 μL (50% amount of pure water and sample solution discharged in [2] above).

  The chromatograph of the analysis result is shown in FIG. The variation in peak height between measurements (A1 and A2, B1 and B2) was smaller than in Comparative Example 1. From this, it can be seen that the peak reproducibility is improved by decreasing the suction / discharge amount at the time of dilution as the number of times is increased.

Comparative Example 2
In Comparative Example 1, the suction / discharge amount of [3] and [4] is 350 μL for the first time (50% of the pure water and sample liquid discharged in [2]), and the second time is 560 μL (the above [2] 70% amount of pure water and sample solution discharged in step 3), and the same procedure as in Comparative Example 1 except that the third time was 700 μL (100% amount of pure water and sample solution discharged in [2] above).

  The chromatograph of the analysis result is shown in FIG. The variation in peak height between measurements (A1 and A2, B1 and B2) was greater than that in Comparative Example 1. As a result, when the dilution container (311) has a liquid surface height that exceeds three times the inner diameter of the container when the pure water and the sample solution of [2] are discharged to the dilution container (311), dilution is performed. It can be seen that the peak reproducibility is not improved if the suction / discharge amount is increased as the number of times is increased.

Comparative Example 3
In Comparative Example 1, the suction / discharge amounts of [3] and [4] are 700 μL for the first time (100% of pure water and sample liquid discharged in [2]), and 630 μL for the second time ([2] above). 90% amount of pure water and sample liquid discharged in step 2), and the same procedure as in comparative example 1 except that the third time was 200 μL (28.6% amount of pure water and sample liquid discharged in [2] above). .

  The chromatograph of the analysis result is shown in FIG. The variation in peak height between measurements (A1 and A2, B1 and B2) was greater than that in Comparative Example 1. As a result, when the dilution container (311) has a liquid surface height that exceeds three times the inner diameter of the container when the pure water and the sample solution of [2] are discharged to the dilution container (311), dilution is performed. Even if the suction / discharge amount is decreased as the number of times is increased, peak reproducibility may not be improved if suction / discharge is performed with less than 30% of the pure water and sample liquid discharged in [2]. Recognize.

  The sample liquid dilution method of the present invention and the liquid chromatograph apparatus using the method can dilute the sample liquid with high accuracy when analyzing a high concentration sample liquid with a liquid chromatograph. The components contained in can be analyzed with higher accuracy.

100: Eluent container 200: Liquid feed pump 300: Sample injection device 301: Sample container 302: Sample loop 303: Sample injection valve 304: Syringe 305: Needle 306: Working liquid container 307: Switching valve 308: Needle moving mechanism 309: Retention loop 310: Dilution liquid container 311: Dilution container 312: Washing port 313: Sample injection valve switching device 314: Suction / discharge pump 315: Control device 400: Analytical column 500: Detector 600: Waste liquid bottle

Claims (4)

A sample container for containing a sample solution,
A hydraulic fluid container for containing the hydraulic fluid,
A diluent container containing the diluent,
Syringe pumps and needles for aspirating / discharging these liquids or air,
And using a sample diluting device equipped with a dilution container containing a mixed solution of the sample liquid and the diluting liquid,
[1] Fill the flow path from the needle to the syringe pump with the working fluid using the syringe pump,
[2] Using the syringe pump, (A) a small amount of air, (B) a predetermined amount of diluent, (C) a small amount of air, and (D) a predetermined amount of sample liquid are sucked from the needle in this order.
[3] Discharging (D) to (D) into the dilution container,
[4] In the method of diluting the sample liquid by aspirating / discharging the whole or a part of the mixed liquid of the sample liquid and the diluted liquid discharged in [3], a plurality of times ,
Liquid level when containing a liquid mixture was discharged in the [3] in the dilution container state, and are 3 times or less of the inner diameter of the dilution container,
The method for diluting a sample liquid according to [4], wherein the suction / discharge amount is 30% or more of the liquid mixture discharged in [3] and decreases as the number of times increases . A sample container for containing a sample solution,
A diluent container containing the diluent,
Syringe pumps and needles for aspirating / discharging these liquids or air,
And using a sample diluting device equipped with a dilution container containing a mixed solution of the sample liquid and the diluting liquid,
[1] Fill the flow path from the needle to the syringe pump with the diluent using the syringe pump,
[2] Using the syringe pump, (A) a small amount of air and (B) a predetermined amount of sample liquid are sucked from the needle in this order,
[3] (A), (B) and a part of the diluent are discharged into the dilution container,
[4] In the method of diluting the sample liquid by aspirating / discharging the whole or a part of the mixed liquid of the sample liquid and the diluted liquid discharged in [3], a plurality of times ,
Liquid level when containing a liquid mixture was discharged in the [3] in the dilution container state, and are 3 times or less of the inner diameter of the dilution container,
The method for diluting a sample liquid according to [4], wherein the suction / discharge amount is 30% or more of the liquid mixture discharged in [3] and decreases as the number of times increases . In a liquid chromatograph apparatus provided with a container containing an eluent, a pump for feeding the eluent, a sample injection apparatus for diluting and introducing a sample liquid to be analyzed, an analytical column, and a detector, the sample injection apparatus comprises:
Sample container containing sample liquid, working liquid container containing working liquid, dilution container containing diluting liquid, syringe pump and needle for aspirating / discharging these liquids or air, and sample liquid and diluting liquid A sample diluting device having a dilution container for containing the mixed solution;
A sample liquid diluted by the apparatus is introduced into a sample loop, and a sample injection valve capable of introducing the sample liquid introduced into the sample loop into the analysis column is provided.
A liquid chromatograph apparatus, wherein the sample diluting apparatus dilutes the sample liquid by the method according to claim 1. In a liquid chromatograph apparatus provided with a container containing an eluent, a pump for feeding the eluent, a sample injection apparatus for diluting and introducing a sample liquid to be analyzed, an analytical column, and a detector, the sample injection apparatus comprises:
A sample container containing a sample liquid, a dilution container containing a diluent, a syringe pump and a needle for sucking / discharging these liquids or air, and a dilution container containing a mixed liquid of the sample liquid and the diluent Sample dilution device,
A sample liquid diluted by the apparatus is introduced into a sample loop, and a sample injection valve capable of introducing the sample liquid introduced into the sample loop into the analysis column is provided.
A liquid chromatograph apparatus, wherein the sample diluting apparatus dilutes the sample liquid by the method according to claim 2.

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