JP2010139448A - Liquid chromatograph, liquid feed pump for liquid chromatograph, and cleaning method of liquid chromatograph - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liquid chromatograph capable of cleaning a holding column without affecting an elution time of a sample from the holding column. <P>SOLUTION: The liquid chromatograph includes the holding column 9 for holding the sample, a first pump 1 for feeding the sample to the holding column 9, using a mobile phase solvent 2, a second pump 10 for eluting the sample held in the holding column 9, using a sample separating solvent 11, a separating column 12 for separating the sample eluted from the holding column 9, and an auto sampler 4, and further includes a control section for switching a liquid to be fed from the first pump 1 from the mobile phase solvent 2 to a cleaning solvent 3 after separating the sample at the separating column 12, and controlling cleaning of the holding column 9. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a liquid chromatograph for separating and analyzing mixed samples such as proteins, peptides, and nucleic acids, a liquid chromatograph feed pump, and a liquid chromatograph cleaning method.

  Separation and analysis of proteins, peptides, nucleic acids, and the like by liquid chromatography is performed by using the difference in sample retention time by using an appropriate separation column for each sample.

  In recent years, in the field of proteomics research, an analysis system in which a liquid chromatograph (LC) having a flow rate of microliter to nanoliter per minute and a mass spectrometer (MS) are frequently used.

  When analyzing a sample as described above, in order to perform detection with higher sensitivity, the sample is temporarily held in a concentration holding column (trap column) placed in the previous stage, and then the flow path is switched. In some cases, a method of sending a sample to an analysis column at a later stage is employed.

  When the flow path switching valve to which the trap column is connected switches the flow path and the side that uses the flow path from the pump (gradient pump) that sends the solvent for separation and analysis of the sample is connected, the gradient liquid feeds from the trap column. Gradually elutes the sample. The eluted sample is sent to a separation column at the subsequent stage, further detected by a detector placed at the subsequent stage, and subjected to separation analysis.

  When performing a liquid chromatograph analysis using a holding column, the sample may remain in the holding column to which the sample is sent, and contamination such as the next analysis or adsorption between samples will occur accurately. There was a problem that made it difficult to do.

  Regarding the cleaning function in the liquid chromatograph apparatus, in WO 2004-102182 (Patent Document 1), a cleaning liquid is sucked from a sample needle in the same manner as the sample and is discharged as a mobile phase, thereby cleaning the needle. However, in a liquid chromatograph having a holding column, it cannot be applied because the site to be washed is different.

  In JP 2001-194355 A (Patent Document 2), a cleaning liquid having a specific composition is used for cleaning a separation column.

In JP 2003-194791 A (Patent Document 3), the detector cell of the sample separated from the liquid chromatograph is washed.
In analysis using a liquid chromatograph, the sample to be analyzed is held in a temporary holding column, or a fixed amount of sample is introduced into a sample loop, etc., and after switching the flow path, the sample is sent to the subsequent analysis column. In many cases, however, the sample remains in the holding column, causing contamination in subsequent analyzes, which makes accurate analysis impossible.

  If the sample remains as described above, a solution that does not contain the sample (for example, pure water) is measured once or multiple times by the same method and conditions as the analysis sample (before and after measurement) ( Blank measurement), it is necessary to elute the remaining sample.

  Alternatively, it is necessary to change the solvent for each analysis to a washing solvent and manually wash the holding column by setting the flow rate.

  The object of the present invention is to wash the holding column, and the liquid chromatograph in which the detector is not placed downstream of the holding column is not suitable because the configuration is different.

WO 2004-102182 JP 2001-194355 A JP 2003-194791 A

  However, if the holding column is manually washed, there is a problem that the analysis time varies, and the time during which the sample elutes from the holding column varies every measurement. Therefore, accurate data evaluation may be difficult.

  The present invention has been made to solve the above-mentioned problems, and its purpose is to wash a holding column so as not to affect the elution time of the sample from the holding column, and to wash the liquid chromatograph. A method is provided.

In order to solve the above problems, the liquid chromatograph of the present invention includes a holding column for holding a sample,
A first pump for feeding a sample to the holding column using a first solvent;
A second pump for eluting the sample held in the holding column using the second solvent;
A separation column for separating the sample eluted from the holding column;
A liquid chromatograph comprising a sample injection part,
After the sample is separated by the separation column, a control unit is provided that controls to wash the holding column by switching the liquid fed from the first pump from the first solvent to the washing solvent. .

  According to the above configuration, since the control unit is configured to send the cleaning solvent to the holding column using the first pump that has sent the sample to the holding column, the cleaning solvent is sent to the holding column. Can be automated. By automating the feeding of the washing solvent to the holding column in this way, it is possible to eliminate variations in the washing time of the holding column every time. Therefore, accurate data evaluation can be performed.

  According to the present invention, variations in the washing time of the holding column each time can be eliminated.

  FIG. 1 is a schematic configuration diagram of a liquid chromatograph of the present invention.

[Configuration of liquid chromatograph]
As shown in the figure, the liquid chromatograph includes a holding column (trap column) 9 for holding a sample, a flow path switching six-way valve 8 to which the holding column 9 is connected, and a sample to the holding column 9. A first liquid feed pump (first pump) 1 for feeding liquid, an autosampler 4, and a second liquid feed pump (second pump) for feeding a sample separation solvent 11 for eluting the sample held in the holding column 9 10, a separation column 12 that separates the sample eluted from the holding column, and a detection unit 13 that detects the sample separated by the separation column 12. In addition, as shown in FIG. 1, the waste liquid bottle 14 which stores a waste liquid can be used with a liquid chromatograph.

  The first pump 1 can switch between two solvents, ie, the mobile phase solvent 2 and the washing solvent 3 to send the solution. The mobile phase solvent 2 moves the sample to the holding column 9, and the washing solvent 3 is a solvent for washing the holding column 9. In addition, what added these solvents 2 and 3 may be called a liquid chromatograph.

  The autosampler 4 is located between the first pump 1 and the 6-way valve 8 and includes a needle 5, a sample injection port 6, and a washing unit 7. The needle 5 sucks the sample and is connected to the sample injection port 6. The needle 5 is used to inject the sucked sample into the flow path, and can be cleaned by the cleaning unit 7. That is, the needle 5 is configured to be movable between the sample injection port 6 and the cleaning unit 7.

The 6-way valve 8 switches the following two flow paths. The first flow path is a flow path formed when the position of the 6-way valve 8 is the valve position A (FIG. 1), and the second flow path is the position of the 6-way valve 8 at the valve position B (FIG. 2). ).
(First flow path) First pump 1 → autosampler 4 → holding column 9 → waste bottle 14
Second pump 10 → separation column 12 → detector 13
(Second flow path) First pump → autosampler 4 → waste liquid bottle 14
Second pump 10 → holding column 9 → separation column 12 → detector 13
In addition, although not shown in figure, the control part for controlling each said member as shown below is provided. The washing solvent 3 is not particularly limited as long as it is a solution that does not significantly deteriorate the performance of the holding column 9.

  Next, an example of the operation of the liquid chromatograph will be described.

[Operation of liquid chromatograph]
(Holding step)
First, when the position of the 6-way valve 8 is the valve position A, the mobile phase solvent 2 is sent from the first pump 1 to the holding column 9 via the autosampler 4. Here, for example, as the mobile phase solvent 2, a 0.1% aqueous solution of formic acid can be used, and the mobile phase solvent 2 can be fed at 10 μL / min. The needle 5 connected to the sample injection port 6 collects a sample, and the collected sample is sent to the holding column 9 by the mobile phase solvent 2. When the sample is sent to the holding column 9 in this way, substances such as salts that are not held in the holding column 9 are sent to the waste liquid bottle 14 together with the mobile phase solvent 2.

  The sample separation solvent 11 is sent from the second pump 10 to the separation column 12 through a flow path that does not pass through the holding column 9. Here, for example, as the sample separation solvent 11, a 50% acetonitrile aqueous solution containing 0.1% formic acid can be used.

(Analysis step)
When the position of the six-way valve 8 is switched from the valve position A to the valve position B, the sample separation solvent 11 is sent from the second pump 10 to the holding column 9 and further to the subsequent separation column 12. Thus, the sample held in the holding column 9 is eluted by the second pump 10, separated by the separation column 12, and detected by the detection unit 13. The first pump 1 continues liquid feeding even when the position of the six-way valve 8 is switched from the valve position A to the valve position B, and the mobile phase solvent 2 to be fed flows to the waste bottle 14. After injection of the sample in the holding step, the needle 5 is disconnected from the flow path and returns to the idle position (not shown).

(Holding column washing step)
After the detection by the detector 13 is completed, the position of the six-way valve 8 is switched from the valve position B to the valve position A. Thereafter, the solvent fed by the first pump 1 is changed from the mobile phase solvent 2 to the washing solvent 3, and the needle 5 of the autosampler 4 is moved from the sample injection port 6 to the washing unit 7. After the movement, the flow rate of the first pump 1 is changed from 10 μL / min to 1 mL / min, and the solution in the flow path from the first pump 1 to the needle 5 is replaced with a high flow rate. Replacing the solution in the flow path is called purging. The purge time can be, for example, 5 minutes. After completion of the purge, the flow rate of the first pump 1 is returned to 10 μL / min, and the needle 5 is moved to the sample injection port 6. Thereby, the cleaning solvent 3 is sent from the first pump 1 to the holding column 9 via the autosampler 4, and the holding column 9 can be cleaned. The cleaning solvent 3 after cleaning the holding column 9 is stored in the waste liquid bottle 14.

  For example, the cleaning solvent 3 is fed by the first pump 1 for 10 minutes to wash the holding column 9.

(Replacement step in holding column)
Thereafter, the solvent sent from the first pump 1 is changed from the cleaning solvent 3 to the mobile phase solvent 2. Further, the needle 5 of the autosampler 4 is moved from the sample injection port 6 to the cleaning unit 7. After the needle 5 is moved, the flow rate of the first pump 1 is changed from 10 μL / min to 1 mL / min, and the solution in the flow path from the first pump 1 to the needle 5 is transferred from the washing solvent 3 to the mobile phase solvent 2 at a high flow rate. (Purge) The purge time can be 5 minutes, for example. After completion of the purge, the flow rate of the first pump 1 is returned from 1 mL / min to 10 μL / min, and the needle 5 is moved from the cleaning unit 7 to the sample injection port 6. The mobile phase solvent 2 is sent from the first pump 1 to the holding column 9 via the autosampler 4. In this state, for example, by carrying out liquid feeding for 20 minutes, the solvent in the holding column 9 is replaced with the mobile phase solvent from the washing solvent 3 and equilibrated, so that an initial state in which the next analysis can be performed.

  Note that the 6-way valve 8 may be a 10-way valve as long as the flow path can be switched instead. Alternatively, as shown in FIG. 3 to be described later, the sample separation solvent 11 is a 2% acetonitrile aqueous solution containing 0.1% formic acid, and, for example, a second pump using a 98% acetonitrile aqueous solution containing 0.1% formic acid. Gradient analysis may be performed using 10 as a pump capable of supplying gradient liquid. Furthermore, although the autosampler 4 is used in the above liquid chromatograph, the sample may be injected with a manual injector or the like.

  In the above configuration, a single type of washing liquid is added. However, since the liquid feeding pump used can feed up to four kinds of solvents as standard, two or more kinds of washing liquid can be efficiently washed. Solutions may be added. In addition, the liquid feed pump for sample introduction is changed to a pump that mixes and feeds a plurality of solutions with a determined composition, and the liquid is fed from the control unit with a solution composition that gradually increases the cleaning efficiency. Control may be performed to wash the holding column more efficiently.

  Further, the liquid chromatograph of the present embodiment is not limited to the one described above, and may be a nano-flow rate liquid chromatograph having a cleaning function for the holding column. The nano flow rate liquid chromatograph will be described with reference to FIG.

  In FIG. 3, only the configuration different from that in FIG. 1 will be described, and the description of the same configuration will be omitted. The nano-flow rate liquid chromatograph uses a 10-way valve 15 instead of the 6-way valve 8 of the liquid chromatograph shown in FIG. A pump 20, a nano flow rate pump (nanoflow pump) 17, and a 10-way valve 19 connected to the third pump 20 and the nanoflow pump 17 for creating a gradient solution are provided. Further, the third pump 20 sends sample separation solvents (sample elution solvents) 11 and 16 for eluting the sample from the holding column 9 at a predetermined mixing ratio. The nanoflow pump 17 sends pure water 18.

  Next, the operation will be described. First, the 10-way valve 15 holds the sample on the holding column 9 at the valve position C shown in FIG. Next, the flow path of the 10-way valve 15 is switched to a valve position D shown in FIG.

  Thereafter, the sample separation solvents 11 and 16 mixed at a mixing ratio determined from the third pump 20 are sent. The valve position of the 10-way valve 19 changes every certain time (for example, 1 minute). The 10-way valve 19 is provided with two loops, and solutions having different mixing ratios of the sample separation solvents 11 and 16 flowing into the respective loops at regular intervals are stored. The solution stored in the loop is pushed out into the flow path toward the 10-way valve 15 by the amount of nanoflowing water flowing from the nanoflow pump 17. The nano flow rate solvent created by the third pump 20, the nanoflow pump 17, and the 10-way valve 19 is sent to the holding column 9, and the sample is eluted from the holding column 9.

  The eluted sample is gradually separated by the separation column 12 and detected by the detection unit 13. After detection, the valve position of the 10-way valve 15 is returned from the valve position D to the valve position C, the solvent sent from the first pump 1 is switched from the mobile phase solvent 2 to the cleaning solvent 3, and the same cleaning as described above is performed. The action is done.

  Further, two six-way valves 21 and 22 as shown in FIG. 5 may be added to the nano-flow rate liquid chromatograph shown in FIG.

FIG. 6 shows a time chart of operations of the first pump 1 (upper stage) and the autosampler 4 (lower stage) in the holding column cleaning step of the present invention. In the washing step
(A) The needle 5 of the autosampler 4 is moved to the cleaning portion 7 purge position.
(B) The mobile phase solvent 2 of the first pump 1 is switched to the cleaning solvent 3, and the flow rate is changed from 10 μL / min to 1 mL / min. For example, after 5 minutes have elapsed, liquid feeding is temporarily stopped.
(C) The needle 5 of the autosampler 4 returns to the position of the sample injection port 6.
(D) The holding column 9 is washed as the washing solvent 3 flows in the order of the first pump 1 → the autosampler 4 → the holding column 9 → the waste liquid bottle 14. The flow rate is 10 μL / min. For example, after 20 passes, the liquid feeding is temporarily stopped.
(E) The needle 5 of the autosampler 4 moves to the cleaning unit 7 purge position.
(F) The cleaning solvent 3 of the first pump 1 is switched to the mobile phase solvent 2, and the flow rate is changed from 10 μL / min to 1 mL / min. For example, after 5 minutes have elapsed, liquid feeding is temporarily stopped.
(G) The needle 5 of the autosampler 4 returns to the position of the sample injection port 6.
(H) The mobile phase solvent 2 flows through the first pump 1 → the autosampler 4 → the holding column 9 → the waste liquid bottle 14, whereby the solvent in the holding column 9 is replaced with the mobile phase solvent 2. The flow rate is 10 μL / min.
(I) For example, by carrying out liquid feeding for 20 minutes, solvent replacement in the holding column 9 and equilibration are completed.

  In the present invention, the washing solvent 3 is a 98% acetonitrile solution containing 0.1% formic acid. In addition, a solution having a composition that sufficiently elutes the residue of the sample from the holding column 9 such as a 100% acetonitrile solution or a 50% methanol solution may be used. Depending on the properties of the holding column 9, if pure water or a 0.1% formic acid aqueous solution functions as a strong solvent, such a solution may be used.

  According to the present invention, the holding column for holding the sample, the first liquid feeding pump for feeding the sample to the holding column, the second liquid feeding pump for eluting the sample held in the holding column, and the holding column In a liquid chromatograph comprising a separation column for separating a sample eluted from the liquid and a washing solvent sent from the first pump, the holding column can be automatically washed after the analysis. A liquid chromatograph that can be used is provided.

  The holding column can be washed, and by adding one or more washing solvents to the pump for feeding the sample to the holding column, it is possible to wash the holding column powerfully and efficiently, As a result, it is not necessary to perform blank measurement between analyses, so that the total analysis time can be shortened. Furthermore, by shortening the total analysis time, it is possible to improve data accuracy, reduce sample deterioration, and reduce the amount of reagent used.

  In addition, if a valve and a flow path capable of supplying a cleaning solvent are added to the pump for supplying the sample, cleaning operation by purging can be performed, but the cost increases significantly. Is a technology that can be used in

  In this example, a type of washing liquid was added. However, the liquid feed pump used in the example can send up to four kinds of solvents as standard, so the holding column can be washed efficiently. Two or more kinds of solutions may be added.

  In addition, the liquid feed pump for sample introduction is changed to a pump that mixes and feeds a plurality of solutions with a predetermined composition, and the liquid is fed from the control unit with a solution composition that gradually increases the cleaning efficiency. Control may be performed to wash the holding column more efficiently.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows this invention and shows schematic structure of the liquid chromatograph of one Embodiment. It is a figure which shows the state which switched the valve position of the 6-way valve of the liquid chromatograph shown in FIG. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates a schematic configuration of a nano flow rate liquid chromatograph according to an embodiment of the present invention. It is a figure which shows the state which switched the valve position of the 10-way valve of the liquid chromatograph shown in FIG. It is a figure which shows schematic structure which added two 6-way valves to the liquid chromatograph shown in FIG. 3 is a time chart showing the operation of the first pump and the autosampler in the holding column cleaning step of the present invention.

Explanation of symbols

1 First Pump 2 Mobile Phase Solvent (First Solvent)
3 Solvent for cleaning 4 Autosampler (sample injection part)
8 6-way valve (switching valve)
9 Holding column 10 Second pump 11, 16 Solvent for sample separation (second solvent)
12 Separation column 15 10-way valve (switching valve)

Claims (9)

A holding column for holding the sample;
A first pump for feeding a sample to the holding column using a first solvent;
A second pump for eluting the sample held in the holding column using the second solvent;
A separation column for separating the sample eluted from the holding column;
A liquid chromatograph comprising a sample injection part,
A control unit is provided for controlling the liquid to be fed from the first pump after the sample is separated in the separation column from the first solvent to the washing solvent to wash the holding column. Liquid chromatograph. In claim 1,
The control unit changes the liquid sent from the first pump from the first solvent to the cleaning solvent, and then temporarily shorts the flow path between the first pump and the holding column at the sample injection unit. The liquid chromatograph is characterized in that the flow rate per unit time from one pump is increased, and then the flow path and the flow rate are returned to the original state before the cleaning solvent is fed to the holding column. . In claim 1,
The control unit returns the liquid sent from the first pump from the cleaning solvent to the first solvent, and then temporarily shorts the flow path between the first pump and the holding column at the sample injection unit. A liquid chromatograph characterized in that the liquid flow rate per unit time from one pump is increased, and then the flow path and the liquid flow rate are returned to the original state before the first solvent is fed to the holding column. . In any one of Claims 1-3,
A switching valve is provided for switching between a flow path for sending the first solvent from the first pump or the cleaning solvent to the holding column and a flow path for sending the second solvent from the second pump to the holding column. A liquid chromatograph characterized by that. A holding column for holding the sample;
A first pump for feeding a sample to the holding column using a first solvent;
A second pump for eluting the sample held in the holding column using the second solvent;
A separation column for separating the sample eluted from the holding column;
A liquid chromatograph comprising a sample injection part,
The liquid chromatograph, wherein the first pump is configured to wash the holding column using a washing solvent. A liquid feeding pump for liquid chromatography that uses a solvent to feed a sample to a holding column,
A liquid feeding pump, wherein the holding column is washed using a washing solvent different from the solvent. A holding column for holding the sample;
A first pump for feeding a sample to the holding column using a first solvent;
A second pump for eluting the sample held in the holding column using the second solvent;
A liquid chromatograph washing method comprising a separation column for separating a sample eluted from a holding column,
A method for washing a liquid chromatograph, comprising: separating a sample with a separation column, and switching a liquid sent from a first pump from a first solvent to a washing solvent to wash the holding column. In claim 7,
The control unit changes the liquid sent from the first pump from the first solvent to the cleaning solvent, and then temporarily shorts the flow path between the first pump and the holding column at the sample injection unit. A method for washing a liquid chromatograph, in which the flow rate per unit time from one pump is increased, and then the flow path and the flow rate are returned to the original state before the washing solvent is fed to the holding column. In claim 7,
After the liquid sent from the first pump is returned from the cleaning solvent to the first solvent, the flow path between the first pump and the holding column is temporarily short-circuited at the sample injection section to remove the liquid from the first pump. A method for washing a liquid chromatograph, in which the liquid flow rate per unit time is increased, and then the flow path and the liquid flow rate are returned to the original state, and then the first solvent is fed to the holding column.

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