JP5028109B2 - Liquid chromatograph - Google Patents

Description

  The present invention relates to a liquid chromatography technique.

  In recent years, the importance of the field of proteome research that comprehensively observes proteins and performs profiling has been widely recognized. The research results in this field are expected to be widely applied in diagnosis and treatment. When introducing proteomic analysis to the clinical field, the protein is enzymatically digested in the form of a mixture, and the resulting peptides are separated by high-resolution liquid chromatography, and the mass spectrometer provides high resolution, high sensitivity, and high sensitivity. It is necessary to make functional measurements. For clinical applications, the target components are blood, serum and other blood-derived components, urine, ascites, saliva, tears, cerebral bone marrow fluid, pleural effusion, and protein extracts from cellular tissues. Serum is said to contain more than 22,000 proteins and must be separated by liquid chromatography as much as possible before being introduced into the mass spectrometer. In addition, in order to improve the resolution, a multi-dimensional liquid chromatography technique in which a plurality of columns having different separation modes are connected is also widespread. In multidimensional liquid chromatography, an ion exchange column and a reverse phase column are connected by a column switching valve, and the peptide fraction separated using the difference in charge is once concentrated in a trap column, and then further hydrophobic and hydrophilic. By separating two-dimensionally using the difference, it is an apparatus that can also separate complex samples.

  Japanese Examined Patent Publication No. 4-62024 discloses that in a one-dimensional liquid chromatography apparatus, the pressure applied to the column is measured and abnormality of the column is automatically detected.

Japanese Examined Patent Publication No. 4-62024

  However, in the two-dimensional liquid chromatography apparatus, the apparatus configuration includes a first dimension separation column, at least two trap columns, and a second dimension separation column. The flow path is also significantly more complicated than a general one-dimensional chromatographic apparatus. Problems such as clogging of pipes or columns in the flow path due to impurities derived from the sample and solvent may occur.Each time, remove the pipe or column from the downstream flow path of the mass spectrometer, and perform reconnection or exchange adjustment. Yes. The inner diameters of the pipes and columns constituting the apparatus are 20 μm to 300 μm, and reconnection may cause the construction of dead volume and contamination of impurities, which may deteriorate the analysis accuracy.

  An object of the present invention relates to facilitating detection of a failure in a separation column in a liquid chromatography apparatus.

  The present invention includes a separation column, a pump capable of feeding liquid to the separation column, a sensor capable of detecting pressure of the liquid fed by the pump, an upstream side switching valve provided on the upstream side of the separation column and capable of communicating with a waste liquid container. The present invention relates to a liquid chromatography apparatus comprising a downstream switching valve provided on the downstream flow side of a separation column and capable of communicating with a waste liquid container. By switching the upstream and downstream switching valves of the separation column so as to communicate with the waste liquid container, the liquid is fed by the pump, and the pressure of the liquid fed by the pump is detected, so that the malfunction of the separation column can be detected.

  Preferably, an upstream switching valve, a downstream switching valve, a pump, and a control mechanism for controlling the sensor are provided.

  Preferably, the pump can send the cleaning liquid, and the cleaning liquid is sent to the separation column where the abnormality is detected.

  According to the present invention, the abnormality of the separation column can be easily detected.

  The above and other novel features and effects of the present invention will be described below. It should be noted that the drawings are for explanation only and do not reduce the scope of rights.

  In this embodiment, a flow path is constructed before and after the two separation columns to branch to the waste liquid bottle through the clogging detection valve on the waste liquid bottle side. When a problem occurs, the valve is automatically switched to the waste bottle side, and the problem location is grasped from the fluctuation value of the pressure sensor in the pump. In addition, maintenance is performed by trying to eliminate clogging by performing a cleaning process.

  FIG. 1 shows a schematic diagram of two-dimensional liquid chromatography as an example of a multidimensional liquid chromatograph mass spectrometer with a simple maintenance function.

  The apparatus includes a first separation column, a first pump capable of feeding liquid to the first separation column, a first sensor capable of detecting pressure of liquid fed by the first pump, and a first separation column. A first upstream switching valve provided on the upstream side and capable of communicating with the waste liquid container; and a first downstream switching valve provided on the downstream flow side of the first separation column and capable of communicating with the waste liquid container; , A second separation column, a first pump that can send liquid to the second separation column, a first sensor that can detect the pressure of the liquid sent by the second pump, and an upstream side of the second separation column A second upstream switching valve provided and capable of communicating with the waste liquid container; and a second downstream switching valve provided on the downstream flow side of the second separation column and capable of communicating with the waste liquid container. .

More specifically, the apparatus configuration includes a front separation column part A, a trap column part B, and a rear separation column part C. The pre-stage separation column part A is arranged at the rear stage of the pump 12 having a pressure sensor function for feeding the eluent 13, the eluent 14, and the eluents 13 and 14, and the sample introduction apparatus for introducing the sample into the separation column 10. 11 and a separation column 10 for separating a sample. The separation column 10 is usually a strong cation ion column (strong cation ion).
chromatography column) is used. The eluent 13 is appropriately selected depending on the type of column used for the separation column 10. In this example, 0.1% HCOOH (pH 3.0) was used, and the solution was fed at a flow rate of 10 μL / min. The eluent 14 is a CH ₃ CN solution and is used for pipe cleaning. The sample introduction device 11 is located at the rear stage of the pump 12 and the front stage of the separation column 10 and can introduce the sample into the separation column 10 together with the eluate from the pump 12 by introducing the sample into the flow path with a syringe. The separation column 10 is separated according to the ionic strength. Eluents having different ionic strengths are installed at the port of the sample introduction device, and the sample is adsorbed on the separation column 10 and then introduced sequentially. Exchange separation was performed. In this example, human serum (SIGMA, H1388) was removed from the serum as a sample using Multiple Affinity Removal Column System (Agilent Technologies, 5185-5984), and then reduced alkyl was used. 1 μL was prepared using a 5 mg / mL serum sample subjected to trypsin digestion. Eluents with different ionic strengths are 0 mM HCOONH ₄ , 50 M HCOONH ₄ , 100 mM HCOONH ₄ , 1000 containing 0.1% HCOOH.
mM HCOONH ₄ was placed in the sample port of the sample introduction apparatus 11 and introduced in 20 μL aliquots. In addition, an eluent containing a non-volatile salt such as CH ₃ COONH ₄ can be used as the eluent having different ionic strength.

The trap column section B includes an eluent 7, an eluent 8, a pump 9 having a pressure sensor function for feeding the eluents 7 and 8, a trap column 4 and a trap column 5 for concentrating the sample separated by the separation column 10, and a trap. The column 3 and the valve 3 that enables switching between the flow path from the pump 9 and the flow path from the pump 12 are configured. Clogging detection valves 17 and 18 are installed before and after the separation column 10 to enable switching of the flow path when clogging occurs. The valve 3, the clogging detection valve 17 and the clogging detection valve 18 are controlled by the control device 21, and the timing for switching the valve 3 is created in advance in the control device 21, and the valve 3 is switched at regular time intervals. Thus, the flow path can be switched from the separation column 10 ⇒ trap column 4 ⇒ waste liquid bottle 6 to the separation column 10 ⇒ trap column 5 ⇒ waste liquid bottle 6. By switching the valve 3, the sample component eluted from the separation column 10 can be concentrated in the trap column 4 and the trap column 5 at regular time intervals. The clogging detection valve 18 is switched by switching the clogging detection valve 18 under the control of the control device 21 when clogging occurs in the piping or column in the apparatus, and the separation column 10 ⇒ clogging detection valve 18 ⇒ valve 3 to separation column. 10⇒Clogging detection valve 18⇒Change the flow path to the waste liquid bottle 15. Whether or not clogging has occurred is determined by the value of the pressure sensor of the pump 12, and a pressure value when clogging has occurred is set in advance in the control device 21, and it is determined that clogging has occurred if the pressure value exceeds that value. . This value is appropriately determined depending on the back pressure values of the separation column 10 and the trap columns 4 and 5 used for the measurement. The SCX column (manufactured by Poly LC) and the trap column (manufactured by Kyoto Monotech) used here have a back pressure value of about 9 MPa at a flow rate of 10 μL / min. The pressure value was 10 MPa or more in this example. When the pressure value of the pump 12 indicates 10 MPa or more, it is determined that clogging has occurred in the flow path or column in the apparatus, the valve 18 is switched, and the fluctuation range of the pressure value is observed. When there is no change in the pressure value of the pump 12, the upstream side of the clogging detection valve 18 is provided, and when the pressure value changes to a value obtained by subtracting the back pressure of the trap column from the back pressure of the separation column 2, the downstream side of the clogging detection valve. It can be seen that there is a clogged portion. If there is no fluctuation in the pressure value, the clogging detection valve 17 is then automatically switched under the control of the control device 21, and the sample introduction device 11⇒clogging detection valve 17⇒the separation column 10 to the sample introduction device 11⇒clogging. The flow path is changed from the detection valve 17 to the waste liquid bottle 15. When there is no change in the pressure value of the pump 12, there is a clogging point before the clogging detection valve 17, and when the pressure value fluctuates to a value of 0 to 0.5 Mpa, there is a clogging point after the clogging detection valve 17. You can see that it exists. FIG. 2 shows a flowchart up to grasping a clogged portion by the control mechanism of the clogging detection valves 17 and 18.

The latter-stage separation column section C includes clogging detection valves 19 and 20 that enable switching of the flow path when clogging occurs before and after the mass spectrometer 1, the separation column 2 that separates the sample, and the separation column 2. Composed. As the separation column 2, a reverse phase column or a monolith column is used, and the separation is performed by hydrophobic interaction. The composition of the eluent 7 and the eluent 8 is appropriately selected depending on the column type. In this embodiment, the eluent 7 is a 2% CH ₃ CN aqueous solution containing 0.1% HCOOH. Using a 98% CH ₃ CN aqueous solution containing 0.1% HCOOH, the eluent 7 and the eluent 8 were mixed in accordance with a program preset in the controller 21 and fed at a flow rate of 200 nL / min. The valve 3, the clogging detection valve 19 and the clogging detection valve 20 are controlled by the control device 21, and the timing for switching the valve 3 is created in advance in the control device 21, and the valve 3 is switched at regular time intervals. Thus, the flow path can be switched from pump 9 ⇒ trap column 4 ⇒ separation column 2 to pump 9 ⇒ trap column 5 ⇒ separation column 2. The mixing of the eluent 7 and the eluent 8 is started in synchronization with the switching of the valve 3, and the sample component concentrated in the trap column is eluted by the liquid sent from the pump 9 and separated by the separation column 2. Is possible. Switching of the clogging detection valve 20 is performed by switching the clogging detection valve 20 under the control of the control device 21 when clogging occurs in the piping or the column in the apparatus, and from the separation column 2 ⇒ clogging detection valve 20 ⇒ mass spectrometer 1. The flow path is changed from separation column 2 to clogging detection valve 20 to waste liquid bottle 16. Whether or not clogging has occurred is determined by the value of the pressure sensor of the pump 9, and a pressure value when clogging has occurred is set in advance in the control device 21, and it is determined that clogging has occurred when the value exceeds that value. . This value is appropriately determined depending on the back pressure values of the separation column 2 and the trap columns 4 and 5 used for the measurement. Monolith column (GL Science Co., Ltd.) and trap column (Kyoto Monotech Co., Ltd.) used at this time, flow rate 200 nL /
Since the back pressure value at min is about 3 MPa in total, the pressure value for judging whether clogging has occurred or not is 5 Mpa or more in this embodiment. When the pressure value of the pump 9 indicates 5 Mpa or more, it is determined that clogging has occurred in the flow path or column in the apparatus, the valve 20 is switched, and the fluctuation range of the pressure value is observed. When there is no change in the pressure value of the pump 9, it can be seen that there is a clogging occurrence position after the clogging detection valve 20 when the front stage from the clogging detection valve 20 fluctuates to 5 Mpa or less. If there is no fluctuation in the pressure value, the clogging detection valve 19 is then automatically switched under the control of the control device 21. Valve 3⇒clogging detection valve 19⇒separation column 2 to valve 3⇒clogging detection valve 19⇒ The flow path is changed to the waste liquid bottle 16. When there is no fluctuation in the pressure value of the pump 12, there is a clogging location before the clogging detection valve 19, and when the pressure value fluctuates to the back pressure value of the trap column, there is a clogging occurrence position after the clogging detection valve 19. I understand that. FIG. 3 shows a flowchart up to grasping a clogged portion by the control mechanism of the clogging detection valves 19 and 20.

After the location where the clogging has occurred is grasped in this way, cleaning is performed under the control of the control device 21 to remove the clogging. Switching between the clogging detection valves 17 and 18 causes clogging in either (1) the front stage from the clogging detection valve 17, (2) between the clogging detection valve 17 and the clogging detection valve 18, or (3) any stage after the clogging detection valve 18. It is confirmed that the location exists. After the location of the clogging is grasped, the valve 3 and the clogging detection valves 17, 18, 19 are automatically switched by the control of the control device 21, and the cleaning liquid suitable for the clogging location is sent from the pump 12 or the pump 9. And cleaning is performed. In the case of (1), the clogging detection valve 17 is switched to the flow path of the sample introduction device 11 ⇒ clogging detection valve 17 ⇒ waste liquid bottle 15, and the eluent 14 is sent at a composition of 100% by the pump 12 for a certain time. Cleaning is performed. In the case of (2), the clogging detection valve 18 is switched to the flow path of the separation column 10 ⇒ clogging detection valve 18 ⇒ waste liquid bottle 15, and 1000 mM HCOONH ₄ installed in the sample port of the sample introduction apparatus 11 is eluted by the pump 12. 13 is introduced into the flow path through which the liquid is fed, and the separation column 10 is washed for a certain period of time. In the case of (3), the clogging detection valve 19 is switched to the flow path of valve 3⇒clogging detection valve 19⇒waste liquid bottle 16, and the valve 3 is also switched to switch the pump 12⇒trap column 4⇒waste liquid bottle 6 to pump 9⇒trap The flow path is switched from column 4 to waste liquid bottle 16 or pump 12 to trap column 5 to waste liquid bottle 6 to pump 9 to trap column 5 to waste liquid bottle 16 and the eluent 8 is fed by pump 9 with a composition of 100%. Washing is performed for a certain time. Cleaning is performed for a certain period of time, and maintenance is completed when the pressure value of the pump 12 returns to 10 Mpa or less. If the effect is not found by washing, replace the piping and column and perform maintenance.

  Similarly, by switching the clogging detection valves 19 and 20, either (4) the clogging detection valve 19 to the front stage, (5) the clogging detection valve 19 to the clogging detection valve 20, or (6) any of the following stages from the clogging detection valve 20 It is confirmed that there are places where clogging occurs. After the location of the clogging is grasped, the valve 3 and the clogging detection valves 19 and 20 are automatically switched by the control of the control device 21, and the cleaning liquid suitable for the clogging location is sent from the pump 9 to perform cleaning. . In the case of (4), the clogging detection valve 19 is switched to the flow path of valve 3⇒clogging detection valve 19⇒waste liquid bottle 16, and the eluent 8 is fed at a composition of 100% by the pump 9, and the trap column 4 or The trap column 5 is washed. In the case of (5), the clogging detection valve 20 is switched to the flow path of the separation column 2⇒clogging detection valve 20⇒waste liquid bottle 16, and the eluent 8 is sent at a composition of 100% by the pump 9, and the separation column 2 Cleaning is performed. In the case of (6), if cleaning is performed, impurities are introduced into the mass spectrometer, which may lead to deterioration in measurement accuracy. Therefore, cleaning is not performed, and the clogging detection valve 20 and the mass spectrometer 1 are not cleaned. Replace the piping. Cleaning is performed for a certain period of time, and the maintenance is completed when the pump 9 returns to 5 MPa or less. If the effect is not found by washing, replace the piping and column manually and perform maintenance.

  When piping or clogging occurs, usually the front stage of the device, in Fig. 1 the piping and columns are removed and replaced sequentially from the side close to the mass spectrometer, and the clogging location is identified from the fluctuation range of the pump pressure value. The device was being restored. With this method, maintenance is troublesome, and at the same time, there is a possibility that dead volume may be formed or impurities may be mixed when the pipe or column is detached. Therefore, the measurement accuracy, throughput, or reproducibility is greatly affected. However, by using this example, it is possible to easily find the exact clogging location, and the removal and replacement of columns and pipes can be minimized by performing the washing process and trying to eliminate the clogging. Therefore, it is possible to minimize the formation of dead volume and the mixing of impurities. Therefore, the maintenance of the apparatus is completed in a short time, and stable data can be acquired while ensuring measurement accuracy and reproducibility.

  According to this embodiment, it is possible to automatically grasp the clogged portion and remove the clogging in the cleaning process. Even if the clogging cannot be removed in the washing process, the clogged portion can be grasped, so that troublesome work of piping and column exchange and reconnection by the measurer is reduced as much as possible, and simple equipment maintenance is enabled. In addition, it is possible to prevent the deterioration of analysis accuracy due to the construction of dead volume and contamination of impurities, and to obtain highly reliable data.

  Example 2 relates to a liquid chromatography mass spectrometer that separates a sample by a single column without using a plurality of columns as in Example 1. The main use of this example is used for biomarker search research such as disease-related markers and metabolism-related markers in the disease proteome field, and blood-derived components such as blood and serum, urine, ascites, saliva as samples Protein extracts from tear fluid, cerebral bone marrow fluid, pleural effusion, and cellular tissue are used.

Hereinafter, the present embodiment will be described with reference to FIG. This apparatus includes an eluent 405 and an eluent 406, a pump 404 having a pressure sensor function for feeding the eluent 405 and the eluent 406, a sample introduction device 403 for introducing a sample by the liquid feed of the pump 404, a separation column 402, It consists of a mass spectrometer 401. Further, before and after the separation column 402, a clogging detection valve 407 and a clogging detection valve 408, which can change the flow path under the control of the control device 410, are arranged, and the flow path is changed to the waste liquid bottle 409. Can do. In this example, separation is performed by hydrophobic interaction, and a column that can be used under a high back pressure of 10 MPa or more is used. The eluent 405 is a 2% CH ₃ CN aqueous solution containing 0.1% HCOOH, and the eluent 6 is a 98% CH ₃ CN aqueous solution containing 0.1% HCOOH. Liquid 405 and eluent 406 were mixed and fed at a flow rate of 5 μL / min. Whether or not clogging has occurred is determined based on the value of the pressure sensor of the pump 404, and a pressure value when clogging has occurred is set in advance in the control device 410, and when the value exceeds that value, it is determined that clogging has occurred. . This value is appropriately determined depending on the back pressure value of the separation column 402, but since a reverse phase column (manufactured by Shiseido) is used as the separation column 402 this time, the back pressure value at a flow rate of 5 μL / min is about 3 MPa in total. In this embodiment, the pressure value for determining whether clogging has occurred is 5 Mpa or more. When the pressure value of the pump 404 indicates 5 Mpa or more, it is determined that clogging has occurred in the flow path or column in the apparatus, the valve 408 is switched, and the separation column 402 ⇒ clogging detection valve 408 ⇒ mass spectrometer 401. To the separation column 402 → the clogging detection valve 408 → the waste bottle 409, and the fluctuation range of the pressure value is observed. When there is no change in the pressure value of the pump 404, it can be seen that there is a clogging location at a stage after the clogging detection valve 408 when the stage before the clogging detection valve 408 fluctuates to 5 Mpa or less. If there is no change in the pressure value, then the clogging detection valve 407 is automatically switched under the control of the control device 410, and the sample introduction device 403⇒clogging detection valve 407⇒from the separation column 402 to the sample introduction device 403⇒ The flow path is changed from clogging detection valve 407 to waste liquid bottle 409. When there is no change in the pressure value of the pump 404, 0 to 0 before the clogging detection valve 407.
When the back pressure value changes to 0.5 MPa, it can be seen that there is a clogging occurrence position downstream of the clogging detection valve 407.

  After the location where the clogging has occurred is ascertained in this way, cleaning is performed under the control of the control device 410 to remove the clogging. Switching between the clogging detection valves 407 and 408 causes clogging at either (1) the clogging detection valve 407 before, (2) between the clogging detection valve 7 and the clogging detection valve 408, or (3) either after the clogging detection valve 408. It is confirmed that the location exists. After the clogging location is grasped, clogging detection valves 407 and 408 are automatically switched under the control of the control device 410, and a cleaning liquid suitable for the clogging location is sent from the pump 404 to perform cleaning. In the case of (1), the clogging detection valve 7 is switched to the flow path of the sample introduction device 403⇒clogging detection valve 407⇒waste liquid bottle 409, and the eluent 406 is fed at a composition of 100% by the pump 404 for a certain period of time. Cleaning is performed. In the case of (2), the clogging detection valve 8 is switched to the flow path of the separation column 2⇒clogging detection valve 408⇒waste liquid bottle 409, and the eluent 406 is sent at a composition of 100% by the pump 404 and washed for a certain period of time. Is done. In the case of (3), the clogging detection valve 407 is switched between the sample introduction device 403 ⇒ clogging detection valve 407 ⇒ waste liquid bottle 409 and the flow path, the eluent 406 is fed by the pump 404 with a composition of 100%, and the washing is constant. Done for hours. Cleaning is performed for a certain period of time, and the maintenance is completed when the pressure value of the pump 404 returns to 5 Mpa or less. If the effect is not found by washing, replace the piping and column manually and perform maintenance.

1 is a schematic diagram of a multidimensional liquid chromatograph apparatus according to Embodiment 1. FIG. Flowchart up to grasping the clogging location by the control mechanism of the detection valves 17 and 18. Flowchart from the detection mechanism of the detection valves 19 and 20 until the clogging point is grasped. FIG. 3 is a schematic diagram of a liquid liquid chromatograph apparatus according to a second embodiment.

Explanation of symbols

1,401 Mass spectrometer 2,10,402 Separation column 3 Valve 4,5 Trap column 6,15,16,409 Waste liquid bottle 7,8,13,14,405,406 Eluent 9,12,404 Pump 11, 403 Sample introduction device 17, 18, 19, 20, 407, 408 Clogging detection valve 21, 410 Control device

Claims (4)

A first separation column;
A first pump capable of feeding liquid to the first separation column;
A first sensor capable of detecting the pressure of liquid fed by the first pump;
A first switching valve provided upstream of the first separation column and capable of communicating with a waste liquid container;
A second switching valve provided downstream of the first separation column and capable of communicating with a waste liquid container;
A control device for controlling the second switching valve and the first switching Rikawa example valves,
Wherein the control device, based on the pressure fluctuations of the first sensor when switching the second switching Rikawa example valves and / or the first switching valve, an upstream side of the first switch valve, Between the first switching valve and the second switching valve, a clogging point located downstream of the second switching valve is specified, and the control device includes the clogging point from the first pump. A liquid chromatograph apparatus , wherein a cleaning liquid is sent to a flow path by the first pump . The liquid chromatograph apparatus according to claim 1,
A third switching valve is provided downstream of the second switching valve;
The third switching valve
A flow path connected to the second separation column;
A flow path connected to a second pump capable of sending liquid to the second separation column;
A liquid chromatograph characterized by switching a flow path connected to a third separation column via a fourth switching valve capable of communicating with a waste liquid container . A liquid chromatograph apparatus according to claim 2 ,
A liquid chromatograph apparatus comprising a fifth switching valve provided downstream of the third separation column and capable of communicating with a waste liquid container . A liquid chromatograph apparatus according to claim 3,
A second sensor capable of detecting the pressure of the liquid sent by the second pump;
The control device is configured to detect the fourth switching valve upstream and the fourth switching valve based on a pressure fluctuation of the second sensor when the fourth switching valve and / or the fifth switching valve is switched. Between the switching valve and the fifth switching valve, any one of the clogging locations downstream of the fifth switching valve is specified, and the control device changes the flow path including the clogging location from the second pump. On the other hand , a liquid chromatograph apparatus in which a cleaning liquid is fed by the second pump .

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