JP3829718B2 - High performance liquid chromatograph - Google Patents

Description

[0001]
[Field of the Invention]
The present invention relates to a high performance liquid chromatograph (hereinafter referred to as an HPLC apparatus) that performs a separation analysis of components contained in a liquid. More specifically, the present invention relates to a high performance liquid chromatograph that efficiently analyzes a specific target component contained in a sample having a complex matrix.
[0002]
[Prior art]
In the pharmaceutical and life science industries, for the purpose of disease prevention and treatment, search for compounds that act on specific diseases, study in vivo reactions to elucidate the mechanisms of diseases, and cause future diseases For the purpose of predicting the amount of target components contained in a sample with a complex matrix with high accuracy for various purposes such as identifying and quantifying genes, proteins, peptides, etc. present in each person's body There is. High performance liquid chromatography (hereinafter referred to as HPLC) is widely used as one of analytical methods corresponding to these purposes because it has very excellent characteristics.
[0003]
In HPLC, an HPLC apparatus including a mobile phase liquid feeding pump, a sample injection device, a separation column, and a detector as minimum requirements is used. A liquid chromatograph mass spectrometer (hereinafter referred to as LC / MS), which has been rapidly spreading in recent years, is an extremely effective analyzer for analyzing trace target components in a sample having a complex matrix containing many components. In many cases, even when performing LC / MS analysis, in order to ensure sufficient sensitivity and accuracy, cleanup, concentration, desalting, and substitution of target components It is necessary to perform in advance pre-processing such as multi-dimensional separation in which separation in a plurality of separation modes is continuously performed.
[0004]
In order to improve analysis throughput, ensure high accuracy, and save labor, it is essential to automate these processes. Usually, this process requires a complicated system using many switching valves. I was trying.
[0005]
[Problems to be solved by the invention]
An automated system using multiple flow path switching valves is 0.1 mL / min. In the case of using the above relatively high flow rate, it is considered that the system can be practically used although it has a drawback that the system becomes complicated and the price is high. Recently, however, microanalysis of the analysis size for the purpose of dramatically improving the sensitivity in LC / MS in addition to the reduction of the sample / mobile phase solvent is rapidly progressing, and 0.02 mL / min. Hereinafter, in some cases, several μL / min. To several hundred nL / min. It has been put to practical use at extremely low flow rates. In such a system, the use of a large number of flow path switching valves increases the dead volume of the flow path, significantly increases the separation peak of each component, and causes a decrease in separation performance. For this reason, the contents of preprocessing that can be performed online and the automation of multidimensional separation are limited.
[0006]
The present invention has been made to solve the above-described problems, and an object of the present invention is to provide an HPLC apparatus that enables advanced pretreatment and automation of multidimensional separation without causing a decrease in separation performance. .
[0007]
[Means for Solving the Problems]
In order to solve the above-described problem, the present invention has at least two combinations of inlets and outlets, and a stator to which at least four flow paths can be connected, and all the combinations of the inlets and outlets are sequentially connected. In the flow path switching valve comprising a rotor that is rotated and switched so as to form a flow path, the openings of the inlet and the outlet facing the rotor are arranged radially with respect to the rotation axis of the rotor, The rotor includes a pretreatment column, and the flow path switching valve is characterized in that at least one combination of the inlet and the outlet is connected through the pretreatment column.
[0008]
In addition, it has a combination of at least three inlets and outlets, and rotates and switches so as to form a stator to which at least six flow paths can be connected, and a flow path that is sequentially connected by all combinations of the inlets and outlets. The flow path switching valve is configured such that openings of the inlet and the outlet facing the rotor are arranged radially with respect to the rotation axis of the rotor, and the rotor is pretreated. A first separation column connected to the first set of inlets and outlets of the flow path switching valve, wherein at least one combination of the inlet and outlet is connected via the pretreatment column. A flow path connected to the flow path switching valve and the inlet of the flow path switching valve. And a high performance liquid chromatograph, characterized in that connecting the second set of channels that is connected to a second separation column to the outlet of the outlet.
[0009]
A set of inlet and outlet of a channel switching valve having a combination of at least two inlets and outlets includes a channel having a mobile phase liquid feeding pump and a sample injection device, and a channel connected to a separation column. The other set of inlets and outlets are connected to a flow path including a pretreatment solvent and a pump for feeding the solvent, and a flow path connected to the separation column. By making these two sets of inlets and outlets switchable, it is possible to automate pretreatment with only one flow path switching valve. Thereby, there is almost no increase in dead volume, and the separation performance does not deteriorate. In addition, one set of inlet and outlet of the flow path switching valve having at least three sets of inlets and outlets includes a flow path including a mobile phase liquid feeding pump, a sample injection device, and a first-dimensional separation column. The drain channel is connected, and another set of inlets and outlets are connected with a channel having a pretreatment solvent and a pump for feeding the solvent, and a channel connected to the drain, and the rest A set of inlets and outlets is connected to a flow path having a mobile phase liquid feeding pump and a flow path having a second-dimensional separation column. By making these three sets of inlets and outlets switchable, a two-dimensional liquid chromatograph in which pretreatment can be automated with only one flow path switching valve can be configured. Thereby, a plurality of separations can be performed in parallel, and throughput can be improved at the same time without degrading separation performance.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 is a schematic configuration diagram of an embodiment of a liquid chromatograph according to the present invention. Mobile phases 1, 2, 3, 4; pumps 5, 6, 7, 8 for feeding liquid; , A flow path switching valve 10, a trap column 11, a separation column 12 and a detector 13. The flow path switching valve 10 includes a rotatable rotor 21 (outer disk) and a non-rotatable stator 31 (inner disk). The rotor 21 has an inlet 23 and an outlet 26, and the inlet 23 and the outlet 26 are paired. The stator 31 has three inlets 33, 34, and 35 and three outlets 36, 37, and 38. The inlet 33 and the outlet 36, the inlet 34 and the outlet 37, and the inlet 35 and the outlet 38 are paired. . The trap column 11 is connected to the inlet 23 and the outlet 26. A mobile phase 1 for sample injection is connected to the inlet 33, a mobile phase 2 for purifying the target component for cleaning the trap column 11 is connected to the inlet 34, and mobile phases 3 and 4 for analysis are connected to the inlet 35. The separation column 12 is connected to the outlet 38.
[0011]
The flow path switching valve 10 has a rotatable rotor 21 and a non-rotatable stator 31, and the position can be switched by rotating the rotor 21. FIG. 2 shows the positions of the rotatable rotor 21 and the non-rotatable stator 31. In the position A of FIG. 2, the inlet 23 and the inlet 33 and the outlet 26 and the outlet 36 are connected. The sample injected from the autosampler 9 rides on the mobile phase 1 sent out by the pump 5 and is connected to the inlet 33, and is transported from the inlet 23 connected to the inlet 33 to the trap column 11. The sample is trapped in the trap column 11. Next, the rotor 21 is rotated to switch to the position B in FIG. At position B, the inlet 23 and the inlet 34 and the outlet 26 and the outlet 37 are connected. A mobile phase 2 and a pump 6 are connected to the inlet 34, and the mobile phase 2 for purifying the target component sent out by the pump 6 flows from the inlet 34 through the inlet 23 to the trap column 11. In the state in which the target component is held in the trap column 11 while being held in the trap column 11, contaminant components that are weaker than the target component are washed out and discharged from the outlet 37 through the outlet 26. Thereafter, the rotor 21 is rotated to switch to the position C in FIG. 2. At the position C, the inlet 23 and the inlet 35 and the outlet 26 and the outlet 38 are connected. The mobile phase 3, 4 and the pumps 7, 8 are connected to the inlet 35, and the mixed liquid of the mobile phase 3, 4 for analysis sent out by the pumps 7, 8 is supplied from the inlet 35 to the trap column 11. The target component in the trap column 11 is introduced into the separation column 12 and separated, and then detected by the detector 13. When the target component in the trap column 11 is quantitatively introduced into the separation column 12, the position of the flow path switching valve 10 returns to a position that is point-symmetric with respect to the position A in FIG. 23 and outlet 36 and outlet 26 and inlet 33 are connected, the trap column 11 is aged for a certain period of time by the mobile phase 1 for sample injection, and then the rotor 21 is rotated to return to position A, and the same operation is repeated thereafter. It is.
[0012]
FIG. 3 shows the structure of the flow path switching valve 10. The inlets 23 and outlets 26 of the rotor 21 and the inlets 33, 34, 35 and outlets 36, 37, 38 of the stator 31 are connected in a required combination by a pipe 39 by rotating the rotor 21.
[0013]
In the position A in FIG. 2, the dead volume is only the capacity of the pipe 39 connecting the inlet 23 and the inlet 33 and the capacity of the pipe 39 connecting the outlet 26 and the outlet 36, and is almost negligible compared to the column capacity. Thus, the separation performance does not deteriorate. Similarly, at the position B, the dead volume is only the capacity of the pipe 39 connecting the inlet 23 and the inlet 34 and the capacity of the pipe 39 connecting the outlet 26 and the outlet 37. At the position C, the dead volume is the inlet volume. The capacity of the pipe 39 connecting the outlet 23 and the inlet 35 and the capacity of the pipe 39 connecting the outlet 26 and the outlet 38 are both negligible.
[0014]
This makes it possible to easily and automatically perform pretreatment operations and connection to the separation column 12 by using only the flow path switching valve 10, and there is almost no increase in dead volume, and there is a decrease in separation performance. Does not happen.
[0015]
FIG. 4 is a schematic configuration diagram of a second embodiment of the liquid chromatograph of the present invention, which is applied to a two-dimensional HPLC apparatus. The parts composed of the mobile phases 2, 3, 4, the pumps 5, 6, 7, 8 for feeding liquid, the autosampler 9, the flow path switching valve 10, the trap column 11 and the detector 13 are shown in the figure. 1 is the same as the first embodiment. The structure of the flow path switching valve 10 is the same as that of the first embodiment in FIG. Mobile phases 41, 42, 43, and 44 having different compositions are connected to the mobile phase for sample injection, and is composed of a first-dimensional separation column 46 and a second-dimensional separation column 47.
[0016]
In the two-dimensional HPLC for separating proteins and peptides, when LC / MS is used for the detector 13, a cation exchange column is used for the first dimension separation column 46, and a reverse phase column is used for the second dimension separation column 47. In many cases, cation exchange chromatography usually switches the pH stepwise from acidic to basic in a high salt concentration mobile phase. For this purpose, mobile phases 41, 42, 43, and 44 having different compositions are connected to the mobile phase for sample injection. Since the protein and peptide in the sample are sequentially eluted from the separation column 46 in accordance with the charge at the pH, high resolution can be obtained by sequentially introducing each fraction into the second-dimensional separation column 47.
[0017]
The operation of the flow path switching valve 10 is the same as in the first embodiment, and the inlet 23 and the inlet 33 and the outlet 26 and the outlet 36 are connected at the position A in FIG. The sample injected from the autosampler 9 rides on the mobile phase 41 sent out by the pump 5, is separated by the separation column 46, is connected to the inlet 33, and is trapped from the inlet 23 connected to the inlet 33. 11 is conveyed. The sample is trapped in the trap column 11. Next, the rotor 21 is rotated to switch to the position B in FIG. At position B, the inlet 23 and the inlet 34 and the outlet 26 and the outlet 37 are connected. The mobile phase 2 and the pump 6 are connected to the inlet 34, and the mobile phase 2 for purifying the target component sent out by the pump 6 flows from the inlet 23 to the trap column 11 through the inlet 34, and is used for purifying the target component. In the state in which the target component is held in the trap column 11 while being held in the trap column 11, contaminant components that are weaker than the target component are washed out and discharged from the outlet 37 through the outlet 26. The main purpose of washing out in this case is to desalt the salt contained in the mobile phase 41. Thereafter, the rotor 21 is rotated to switch to the position C in FIG. 2. At the position C, the inlet 23 and the inlet 35 and the outlet 26 and the outlet 38 are connected. The mobile phase 3, 4 and the pumps 7, 8 are connected to the inlet 35, and the mixed liquid of the mobile phase 3, 4 for analysis sent out by the pumps 7, 8 passes through the inlet 35 from the inlet 23 to the trap column. 11, the target component in the trap column 11 is introduced into the second-dimensional separation column 47 and separated, and then detected by the detector 13. When the target component in the trap column 11 is quantitatively introduced into the separation column 47, the position returns to position A, the mobile phase 42 is connected to the pump 5, and the same operation is repeated thereafter.
[0018]
Also in the second embodiment, at the position A in FIG. 2, the dead volume is only the capacity of the pipe 39 connecting the inlet 23 and the inlet 33 and the capacity of the pipe 39 connecting the outlet 26 and the outlet 36. In B, the dead volume is only the capacity of the pipe 39 connecting the inlet 23 and the inlet 34 and the capacity of the pipe 39 connecting the outlet 26 and the outlet 37. In the position C, the dead volume is the inlet 23 and the inlet 35. The capacity of the pipe 39 connecting the outlet 26 and the capacity of the pipe 39 connecting the outlet 26 and the outlet 38 are both negligible.
[0019]
FIG. 5 is a schematic configuration diagram of a third embodiment of the liquid chromatograph of the present invention, which is applied to a two-dimensional HPLC apparatus. Mobile phases 41, 42, 43, 44, 2, 3, 4, pumps 5, 6, 7, 8 for liquid feeding, autosampler 9, flow path switching valve 10, and first-dimensional separation column 46 The portion constituted by the second-dimensional separation column 47 and the detector 13 is the same as that of the first embodiment in FIG. The rotor 21 capable of rotating the flow path switching valve 10 has four inlets 51, 52, 53, 54 and four outlets 55, 56, 57, 58, the inlet 51 and outlet 55, the inlet 52 and outlet 56, and the inlet 53. And outlet 57, and inlet 54 and outlet 58 are in pairs. The stator 31 has the same structure as in the first and second embodiments. In the present embodiment, four trap columns 61, 62, 63, 64 are connected to the inlet 51 and outlet 55, the inlet 52 and outlet 56, the inlet 53 and outlet 57, and the inlet 54 and outlet 58, respectively. A mobile phase 41, 42, 43, 44 for sample injection is connected to the inlet 33, and a mobile phase 2 for purification of a target component for cleaning the trap columns 61, 62, 63, 64 is connected to the inlet 34. The mobile phase for analysis 3 and 4 is connected to 35, and the analytical column 12 is connected to the outlet 38.
[0020]
Next, the operation will be described. First, the inlet 51 and the inlet 33 and the outlet 55 and the outlet 36 are connected. A separation column 46 is connected to the inlet 33, and the sample injected from the autosampler 9 rides on the mobile phase 41 sent out by the pump 5, is separated by the separation column 46, and then passes through the inlet 33. 51 to the trap column 61. The sample is trapped in the trap column 61. The mobile phase sent out by the pump 5 is switched to mobile phases 41, 42, 43, and 44 at regular intervals. Further, the position of the flow path switching valve 10 is switched at regular intervals, the rotor 21 is rotated, and the inlet 51 and the inlet 34 and the outlet 55 and the outlet 37 are connected. The mobile phase 2 and the pump 6 are connected to the inlet 34, and the mobile phase 2 for purifying the target component sent out by the pump 6 flows to the trap column 61 and is washed by the mobile phase 2 for purifying the target component, While the target component is held in the trap column 61, a contaminant component that is weaker than the target component is washed out and discharged from the outlet 55 through the outlet 37. The main purpose of the washing out in this case is to desalt the salt contained in the mobile phase 41. At this time, the inlet 52 and the inlet 33 and the outlet 56 and the outlet 36 are connected at the same time. A separation column 46 is connected to the inlet 33, and the sample injected from the autosampler 9 rides on the mobile phase 41, 42, 43 or 44 sent out by the pump 5 and is separated from the inlet 33 through the inlet 52. After reaching the column 46 and separated by the separation column 42, it is conveyed to the trap column 62. The sample is trapped in the trap column 62.
[0021]
Thereafter, the rotor 21 is rotated, and the inlet 51 and the inlet 35 and the outlet 55 and the outlet 38 are connected. The mobile phase 3, 4 and the pumps 7, 8 are connected to the inlet 35, and the mixed liquid of the mobile phase 3, 4 for analysis sent out by the pumps 7, 8 passes through the inlet 35 from the inlet 51 to the trap column. The target component in the trap column 61 is introduced into the second dimension separation column 47 by the mixed liquid of the mobile phases 3 and 4 and separated, and then detected by the detector 13. At this time, the inlet 52 and the inlet 34 and the outlet 56 and the outlet 37 are connected simultaneously. The mobile phase 2 and the pump 6 are connected to the inlet 34, and the mobile phase 2 for purifying the target component sent out by the pump 6 flows from the inlet 51 through the inlet 34 to the trap column 62. In the state where the target component is retained in the trap column 62, the contaminated component having a weaker retention than the target component is washed out and discharged from the outlet 56 through the outlet 37. At the same time, the inlet 53 and the inlet 33 and the outlet 57 and the outlet 36 are connected. A separation column 46 is connected to the inlet 33, and the sample injected from the autosampler 9 rides on the mobile phase 41, 42, 43 or 44 sent out by the pump 5 and is separated by the separation column 46. It is conveyed from the inlet 53 to the trap column 63 through the inlet 33. The sample is trapped in the trap column 63. Thereafter, the inlets 33, 34, 35 and the inlets 51 to 54 and the outlets 36, 37, 38 and the outlets 55 to 58 are sequentially connected, and the fraction components eluted from the first-dimensional separation column 46 are trap columns 61 to 64, the mobile phase 2 is used for washing and desalting, and the mobile phases 3 and 4 are used for separation and analysis by the second-dimensional separation column 47.
[0022]
Here, trapping of elution components from the first-dimensional separation column 46, purification / desalting, and separation / analysis by the second-dimensional separation column 47 can be performed in parallel. The dead volume is a negligible capacity as in the first and second embodiments, and a plurality of separations can be performed in parallel without a decrease in separation performance, and throughput can be improved at the same time.
[0023]
As mentioned above, although the Example of this invention was described, this invention is not limited to the said Example, A various change can be made within the range of the summary of this invention described in the claim. . For example, in the second and third embodiments, mobile phases 41, 42, 43, and 44 having different compositions are connected to the mobile phase for sample injection, but this number is not limited. Secondly, in the embodiment, in order to more reliably hold the target component by the trap column 11, another trap column is added by adding another flow path, and the trap operation by one trap column and the other trap column are added. The aging operation may be performed at the same time. In the third embodiment, four trap columns 61, 62, 63, 64 are used, but this number is not limited.
[0024]
【The invention's effect】
According to the present invention, since it is possible to easily and automatically perform connection switching of a large number of flow paths such as a pretreatment column and a separation column with only one switching valve, without causing a decrease in separation performance, A liquid chromatograph capable of automating advanced pretreatment and multidimensional separation can be constructed, and at the same time, throughput can be improved.
[Brief description of the drawings]
FIG. 1 is a schematic configuration diagram of an embodiment of a liquid chromatograph of the present invention.
FIG. 2 is a diagram showing a positional relationship between an inlet and an outlet in a flow path switching valve.
FIG. 3 is a view showing a structure of a switching valve.
FIG. 4 is a schematic configuration diagram of a second embodiment of the liquid chromatograph of the present invention.
FIG. 5 is a schematic configuration diagram of a third embodiment of the liquid chromatograph of the present invention.
[Explanation of symbols]
1, 2, 3, 4, 41, 42, 43, 44 --- mobile phase 5, 6, 7, 8 --- pump 9 --- autosampler 10 --- flow path switching valve 11 --- trap Column 12, 46, 47 --- Separation column 13 --- Detector 21 --- Rotor 31 --- Stator 39 --- Piping

Claims (2)

A stator having at least two sets of inlets and outlets and capable of connecting at least four flow paths;
In the flow path switching valve composed of a rotor that is rotated and switched so as to form a flow path that is sequentially connected by all combinations of the inlet and the outlet,
The openings of the inlet and the outlet facing the rotor are arranged radially with respect to the rotation axis of the rotor,
The rotor includes a pretreatment column,
A flow path switching valve, wherein at least one combination of the inlet and outlet is connected via the pretreatment column. A stator having at least three combinations of inlets and outlets and capable of connecting at least six flow paths;
A flow path switching valve comprising a rotor that is rotated and switched so as to form a flow path that is sequentially connected in all combinations of the inlet and the outlet,
In the flow path switching valve, the openings of the inlet and the outlet facing the rotor are arranged radially with respect to the rotation axis of the rotor,
The rotor includes a pretreatment column,
At least one set of the inlet and outlet combination is connected via the pretreatment column;
Connecting a flow path connected to a first separation column to a first set of inlets of the inlet and outlet of the flow path switching valve;
A high-performance liquid chromatograph characterized in that a flow path connected to a second separation column is connected to a second set of outlets of the inlet and outlet of the flow path switching valve.

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