JP2004325168A - Separation isolation device and separation isolation method by liquid chromatography - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a separation isolation device by a liquid chromatography suitable for high-throughput analysis. <P>SOLUTION: This separation isolation device by the liquid chromatography is equipped with a separation means equipped with a supply means 10 of a moving phase and two or more separation columns 6 corresponding to one moving phase supply means, and a means 30 for isolating eluate flowing out of the separation columns 6. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to component separation by column chromatography and fractionation of separated components (particularly, a technique of fractionating by spot application (blotting)), and particularly to a separation and fractionation technique suitable for high-throughput analysis.
[0002]
[Prior art]
Functional analysis of proteins is the first step in drug discovery research. In recent years, its importance has increased, and high-speed processing of a large number of samples has been required. Further, in analyzing the function of a protein, knowing its primary structure and higher-order structure is an important element for obtaining knowledge about the relationship between post-translational modification and function. For such structural analysis, an analysis system such as MALDI-TOF / MS (matrix assisted laser desorption ionization time-of-flight mass spectrometer) is particularly useful.
[0003]
Here, heretofore, a technique in which chromatography and a fractionation system are connected has been provided (Patent Document 1). However, even with such a technique, only one sample can be simultaneously separated and separated by one chromatography system, and it has been difficult to meet the recent demand for high-throughput analysis. It is also important that the eluate is in a form suitable for identification analysis after the fractionation, in order to improve the efficiency of the treatment. There is a demand for sorting (blotting) in the form of application (blotting).
[0004]
[Patent Document 1]
Japanese Patent No. 2603434
[0005]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a chromatography and fractionation system that can supply many samples in an appropriate state to an analysis system after component separation, that is, is suitable for high-throughput analysis. .
[0006]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors branched a flow path of one liquid sending pump for column chromatography to form a double track in parallel, and set a separation column for each branched flow path. Thus, the present inventors have found that a multi-column system and a preparative (blotting) system for collectively collecting eluates eluted from each column can be constructed, thereby completing the present invention.
That is, according to the present invention, the following means are provided.
[0007]
(1) An apparatus for separation and separation by liquid chromatography,
Separation means comprising a mobile phase supply means, and at least two separation columns corresponding to one mobile phase supply means,
Means for separating the eluate flowing out of the separation column,
An apparatus comprising:
(2) The apparatus according to (1), wherein a column having an inner diameter of 50 μm or more and 100 μm or less and a length of 5 cm or more and 20 cm or less is used as the separation column.
(3) The mobile phase supply means has a liquid sending capacity within a flow rate range of 2 μL / min to 10 μL / min and supplies the mobile phase to the separation column at a flow rate of 1000 nL / min or less. Or the apparatus according to (2).
(4) The apparatus according to any one of (1) to (3), wherein a precolumn is provided at a stage preceding each of the separation columns.
(5) The separation means has a target for accommodating the eluate from the separation column corresponding to the number of the separation columns, and moving these targets to elute the eluate from the separation column. The device according to any one of (1) to (4), wherein the device is formed so as to be able to sequentially separate the components.
(6) The separation means has a support connected to the separation column and supporting at least two ends of an outflow pipe of the eluate directed to the target, and the holder is provided below the holder. It has a cylindrical targeting guide provided in a protruding shape, and in the guide, each of the outflow pipes is held so as to be movable in the insertion direction in a state where the maximum protruding length from the guide tip opening is regulated, The device according to (5).
(7) The apparatus according to (6), wherein the plate-shaped holder has targeting guides corresponding to the total number of the separation columns.
(8) The apparatus according to any one of (1) to (7), which is for preparing a sample for protein mass spectrometry.
(9) The apparatus according to (8), which is for matrix-assisted laser desorption type mass spectrometry, and further includes a matrix supply means capable of supplying the matrix for mass spectrometry to the eluate in the outlet tube.
(10) A method for separation and separation by liquid chromatography,
A step of introducing a test sample into each of a plurality of separation columns provided in parallel by branching the mobile phase flow path to the mobile phase supply means, and separating them,
A fractionation step of fractionating the eluate eluted from each of the separation columns,
A method comprising:
(11) The method according to (10), wherein the separation step and the fractionation step are performed simultaneously on a plurality of test samples.
(12) performing an adsorption step of adsorbing a test sample on a precolumn connected to a stage preceding the separation column for each separation column, and after completing the adsorption step corresponding to all separation columns, performing the separation step for all separation columns Starting, the method of (11).
(13) A method for preparing a protein mass spectrometry sample, comprising the separation step and the fractionation step according to any one of (10) to (12).
(14) A method for preparing a sample for matrix-assisted laser desorption type mass spectrometry, comprising the separation step and the fractionation step according to any one of (10) to (12).
(15) The method according to (14), further comprising a step of supplying a matrix for mass spectrometry to the eluate before the fractionation step.
[0008]
According to the present invention, a large number of test samples can be efficiently processed by branching the flow path from one mobile phase supply unit and providing a plurality of separation columns in parallel. In particular, by applying the present invention to a nanocolumn having an inner diameter of 50 μm or more and 100 μm or less and a length of about 5 cm or more and 20 cm or less, efficient analysis of a small amount of a test sample becomes possible. Further, by providing the separation column with a mobile phase supply means for supplying a mobile phase at a mobile phase speed of 1000 nL / min or less, the eluate from the separation column can be blotted at the nanomicroliter level. Further, by providing a pre-column before the separation column, the test sample can be retained or concentrated in the pre-column, and the supply timing of the test sample to a plurality of separation columns can be synchronized. As a result, the number of the blotting means can be reduced to one or less. In addition, when the plate-shaped holder is provided in the blotting means, it becomes easy to synchronously blot the eluate from a plurality of separation columns by one blotting means.
[0009]
Further, according to the fractionation method of the present invention, a large number of test samples can be efficiently separated and fractionated for analysis at a later stage. Particularly, by performing the separation step and the blotting step synchronously, more efficient processing can be performed. Further, an adsorption step of adsorbing the test sample on the pre-column is performed, and after performing the adsorption step on all the separation columns, the separation step is performed on all the separation columns at once, so that the separation step and the fractionation step can be efficiently performed. Can be implemented.
Further, a preferred embodiment of the apparatus and method of the present invention is a form applied to an apparatus or method for preparing a sample for protein mass spectrometry, and particularly, an apparatus and a method for preparing a matrix assisted laser desorption type mass spectrometry sample. It is a preferred embodiment to apply to the method. In particular, when the present invention is applied to an apparatus and a method for preparing a matrix-assisted laser desorption-type mass spectrometry sample, a means or a step of supplying a matrix to an eluate prior to fractionation is provided, so that component separation can be performed. And the preparation of a sample for analysis can be realized at the same time.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
The fractionation method of the present invention is a method for fractionating eluted components in liquid chromatography, wherein each of a plurality of separation columns provided in parallel by branching a mobile phase flow path with respect to a mobile phase supply means. The method is characterized by comprising a step of loading a test sample onto a column to separate each column, and a fractionation step of blotting the eluate eluted from each of the separation columns.
Further, as an apparatus suitable for carrying out the method, the present invention is a separation / fractionation apparatus using liquid chromatography, which comprises a mobile phase supply unit and a mobile phase supply unit corresponding to one mobile phase supply unit. And at least two separation columns, and means for blotting an eluate flowing out of the separation column.
Hereinafter, the apparatus and method of the present invention will be described in detail with reference to the entire apparatus 2 of the present invention illustrated in FIG.
[0011]
(Separation means by liquid chromatography)
The liquid chromatography as the separation means 4 has separation columns 6a to 6f (hereinafter, when these are not particularly distinguished, the figure number is described as "6"). (Mobile phase) is provided.
As the separation column 6, a known column and a packed carrier used for liquid chromatography can be used. That is, a packing carrier and a column used in various known liquid chromatography such as ion exchange chromatography, affinity chromatography, and hydrophobic chromatography can be used. For example, a separation column 6 using a silicon compound having ODS (octadecylsilyl group) as a separation carrier can be used.
The column size is not particularly limited, but a capillary column having an inner diameter of 50 μm to 100 μm and a length of 5 cm to 20 cm can be used. The column is particularly preferable as a column for nanoliquid chromatography.
The number of the separation columns 6 is not particularly limited, but is preferably 3 or more, more preferably 4 or more, and still more preferably 5 or more. Although the upper limit is not particularly limited, when the mobile phase supply means 10 has a liquid sending capacity within a flow rate range of 2 μL / min to 10 μL / min, an appropriate flow rate range in the separation column 6 is used. It is preferable to use 10 or less separation columns 6 from the viewpoint of ensuring the following.
[0012]
Further, the device 2 and the method of the present invention can be applied to micro liquid chromatography with a flow rate of 1000 μL / min or less and nano liquid chromatography with a flow rate of 1000 nL / min or less. Preferably, it applies to nano-liquid chromatography. This is because the application to the nano LC enables blotting with a very small amount of sample and a very small amount. In particular, nanoLC can supply an appropriate sample amount as a sample for mass spectrometry for the purpose of protein function analysis and the like.
[0013]
The mobile phase supply means 10 is not particularly limited, and a conventionally known pump for various liquid chromatography can be used. In particular, when applied to nano LC, it is preferable to use a pump for a capillary column. As for the liquid sending capacity, when applied to the nano LC, when the flow path is branched into a plurality of channels and a plurality of separation columns 6 are provided in parallel, the liquid sending capacity is 1000 nL / min or less. Preferably, it has the ability. Usually, as the mobile phase supply means 10 which achieves such a flow rate, there is a mobile phase supply means 10 having a liquid sending capacity (preferably also having a concentration gradient forming ability) in a range of 2 μL / min to 10 μL / min. be able to.
[0014]
The present invention is characterized in that the flow path of the mobile phase from the mobile phase supply means 10 is branched into a plurality of rows, and a plurality of columns can be connected in parallel.
In order to branch the flow path from the mobile phase supply means 10 into a plurality of flow paths, for example, an appropriate flow divider is used. One or more flow dividers can be used to correspond to the finally required number of flow paths. For example, as shown in FIG. 1, when it is desired to finally branch into six flow paths, a flow divider 14a having two branches is used. On the other hand, using a flow divider 14b having a three-way branch, branching in two stages can be branched.
It is preferable to use a tube having an inner diameter suitable for the flow rate between the column 6 and the flow divider 14 and between the flow divider 14 and the flow divider 14. In addition, it is preferable to use a fused silica tube for the nano LC. In addition, the connection between the various tubes and the flow divider 14 and the valve 12 described later is performed by inserting the tube into a union that covers a connection portion between the tube and the tube or a connector that forms a connection portion between the tube and the flow divider 14, and Can be connected by minimizing or minimizing dead volume by fixing the ferrules and bolts that match the shape in the union or connector.
[0015]
The flow paths branched into a desired number of branches are provided via flow path switching valves (flow selector valves) 12a to 12f (hereinafter, simply described as "12" when these valves are not particularly distinguished). Each of the separation columns 6a to 6f can be communicated. By using a valve having a plurality of ports, that is, a multi-port, for example, as shown in FIG. 1, a flow path for sample loading from the autosampler 18 can be connected to the separation column 6. In addition, pre-columns 16a to 16f for adsorbing and holding the test sample in the preceding stage of the columns 6a to 6f (hereinafter, simply referred to as "16" when these pre-columns are not distinguished) are provided along with the valve 12. It is possible to do. Thus, by switching the valve 12, it is possible to switch between a flow path system that reaches the separation column 6 without passing the mobile phase through the pre-column 16 and a flow path system that passes through and reaches the separation column 6. In FIG. 1, a flow path system in which the mobile phase passes through the pre-column 16 and reaches the separation column 6 is formed by the valve 12. When the pre-column passage channel system is formed after the test sample is adsorbed and held on the pre-column 16, the test sample adsorbed on the pre-column 16 is eluted by the mobile phase and reaches the separation column 6.
[0016]
As described above, the present apparatus 2 can be provided with the pre-column 16. The pre-column 16 is attached to the front stage of the separation column 6, and by pre-adsorbing and holding the test sample on the pre-column 16, for example, The sample can be concentrated, or components having an adverse effect on the separation column 6 can be retained in the precolumn 16 for purification. When a plurality of separation columns 6 are used as in the present invention, a test sample is sequentially supplied to a precolumn 16 after shutting off a flow path system to the separation column 6 by a valve 12, and finally, a test sample is After the test sample is adsorbed on the supplied precolumn 6, the valve 12 is simultaneously operated to open the flow path system from the precolumn 16 to the separation column 6, thereby simultaneously supplying each test sample to the plurality of separation columns 6. be able to. In addition, in order to temporarily hold the test sample, a sample loop that can temporarily store the test sample can be used instead of the precolumn 16.
[0017]
The device 2 can also be equipped with an autosampler (automatic sample supply device) 18 as described above. The autosampler 18 can be used without any particular limitation as long as it is a conventionally known various autosampler 18 that can be used in an LC system. For example, as shown in FIG. 1, the autosampler 18 can include a sample loading pump 20, a valve 22, and a sample loop 24.
The autosampler 18 and the separation means having the separation column 6 are connected via a flow selector valve 26. The valve 26 is configured to form an appropriate channel system so as to distribute the test sample transported together with the mobile phase by the sample loading pump 20 from the sample loop 24 of the autosampler 18 to the corresponding separation column 6. I have.
[0018]
In the autosampler 18, in the state of waiting for the introduction of the test sample, a predetermined mobile phase is conveyed by the sample loading pump 20 so as to avoid the sample loop 24, and goes out of the separation system via the valves 26 and 12. It is being discharged.
On the other hand, in the introduction period of the test sample in the autosampler 18, a predetermined amount of the test sample is sucked into the sample vial under the mobile phase transport state on the autosampler 18 side and held in the sample loop 24 ( Thereafter, the valve 22 is switched and the mobile phase conveyed by the sample loading pump 20 is caused to flow out to the sample loop 24, so that the test sample held in the sample loop 24 is separated from the separation column 6 (see FIG. 1). It is conveyed to the side.
[0019]
(Sampling means)
An outflow pipe 28 is connected to the elution end of the separation column 6, and a separation means 30 is provided at the tip of the outflow pipe 28. As described above, typically, a tube made of fused silica can be used as the outflow tube 28. In the case of the nano LC, the inner diameter can be about several tens of μm (typically, about 20 to 30 μm in inner diameter).
The sorting means 30 includes a target 32 having a large number of eluate storage sections, and a table 42 on which the target 32 is placed and which can be controlled to move in the x, y, and z directions. The targets 32 can be individually provided for the respective separation columns 6, and each target 32 usually has a large number of storage sections capable of storing a predetermined amount of eluate. When LC is used to separate a nano LC or a sample for MS, a fractionation form is a point-like coating, that is, a so-called blotting form. In the case of blotting, the target 32 has a plate shape as a whole, and the form of the eluate container is not particularly limited, but may be a circular recess having a diameter of about 1 mm. On the target 32, about several tens to several hundreds of such concave eluate storage sections are formed. Note that the target 32 may be simply planar.
[0020]
The table 42 is moved in three axial directions with the target 32 placed thereon, and the eluate eluted from the separation column 6 is sorted and sorted in advance in the eluate container arranged on the target 32. (Blot). Although this table 42 can be provided individually for all the separation columns 6, it is preferable that one table 42 corresponds to two or more separation columns 6, that is, two or more targets 32. Preferably, a single table 42 corresponds to all separation columns 6, ie, all targets 32. Such a correspondence form is particularly effective when the separation is started by loading the sample in all the separation columns 6 at the same time (synchronously). In addition, since a single table 42 is used for a plurality of targets 32, it is possible to save space and efficiently perform sorting.
[0021]
In order to accurately blot the eluate eluted from the separation column 6, it is necessary to accurately contact the tip of the outflow tube 28 with the solution storage section of the target 32. Therefore, in the case of simultaneous analysis, collective blotting becomes difficult unless the tips of a plurality of outflow pipes 28 are simultaneously brought into contact with the target 32 accurately. In the present invention, for accurate blotting in the simultaneous analysis, the holding means 52 for holding the distal ends of the plurality of outflow pipes 28 can be used. The holding means 52 is configured such that the tips of the plurality of outflow pipes 28 are arranged at appropriate intervals corresponding to the plurality of targets 32, and can always be brought into contact with the target when blotting the eluate.
[0022]
The holding means 52 typically has a plate-shaped support 54, and has a through hole 58 with an inner diameter that allows the elution tube to be freely inserted into a predetermined position of the support 54 in a substantially straight state. And a targeting guide 56 in the form of a sleeve. The tip of the targeting guide 56 projects below the lower surface of the support 54, that is, a predetermined distance toward the target 32. This protruding length can be, for example, about several cm. The outflow pipe 28 is inserted into the through hole 58 of the targeting guide 56, and the tip of the outflow pipe 28 is projected downward so that the tip does not project more than a predetermined length (a maximum projection length, for example, about 1 to 2 mm) from the tip of the targeting guide 56. A regulating member 62 that regulates but does not regulate upward movement is provided. The restricting member 62 can be, for example, a retaining material having an outer diameter larger than the through hole 58 provided at any part of the outflow pipe 28 projecting upward from the through hole 58. By arranging such a retaining material, the outflow pipe 28 does not protrude beyond the predetermined length below the holding means 52, and the tip of the protruding outflow pipe 28 abuts on the target 32, for example. In this case, the elution tube is moved upward, and protrudes from the targeting guide 56 with a protruding length shorter than the predetermined length. The targeting guide 56 may be formed on the support 54 in any form, and may be, for example, a nut having a male screw portion that is screwed into a female screw portion formed on the support 56. It is preferable that the targeting guide 56 be integrated by screwing because the length of the targeting guide 56 projecting from the lower surface of the support 54 can be adjusted. The targeting guide 56 does not hinder the three-dimensional movement of the table 42 when the portion projecting from the lower surface of the support 54 is tapered at the tip, and the targeting guide 56 also This is convenient because the eluate flowing out hardly adheres to the tip of the sleeve 56.
[0023]
According to the holding means 52, as shown in FIG. 2, when the table 42 moves and the tip of the outflow pipe 28 abuts on the eluate storage part of the target 32, the outflow pipe 28 becomes a through hole 58 of the targeting guide 56. The inside is pushed up together with the regulating member 62. At this time, by causing the outflow pipe 28 to protrude from the targeting guide 56 and abut against the bottom of the eluate storage unit, contamination of the eluate is effectively prevented, and each eluate is collected into each eluate storage unit. Alternatively, it can be blotted. Assuming the length of the outflow pipe 28 projecting from the tip of the targeting guide 56, assuming a range in which the setting of the table 42 or the support 54 can deviate from horizontal, the state in which the tips of all the elution tubes project from the tip of the sleeve 54 By setting the length to be in contact with the bottom of the eluate container, it is possible for all the outflow pipes 28 to have a state in which the tip is in contact with the bottom of the eluate container, The eluate from all of the plurality of outflow pipes 28 can be blotted to each of the storage portions in an accurate drop amount and while preventing contamination.
[0024]
In addition, the present apparatus 2 may be provided with means for mixing the eluate eluted from the separation column 6 with another liquid. The other liquid can be supplied to the eluate storage section of the target 32, but preferably, the other liquid is supplied to the elution tube at any part of the outflow pipe 28 from the end of the separation column 6 to the target 32. It is preferable to supply them and mix them. Such in-line mixing is particularly preferable when analysis of the eluate requires another liquid (matrix) in addition to the eluate, such as when preparing a sample for mass spectrometry. Specifically, it is preferably applied to sample preparation for matrix aid laser desorption type mass spectrometry.
[0025]
For such in-line mixing, for example, a flow divider 72 is provided in the middle of an outflow pipe from the separation column 6 to the target 32, and another liquid supply means can be connected to one port of the flow divider 72. . As a result, the eluate and the other liquid are conveyed to the target 32 in a state where they are merged in the flow divider 72. For example, in the embodiment shown in FIG. 1, the flow path from the matrix supply unit 74 is divided according to the number of the separation columns 6 via the flow dividers 76, and the flow dividers 72 are connected to the outlet pipes 28 from the respective separation columns 6. Are joined via the.
[0026]
The target 32 is preferably in a form that can be applied as it is to the following analysis means, for example, a sampling means of an identification means such as a mass spectrum or a nuclear magnetic resonance spectrum, or a preprocessing means of the identification means.
[0027]
Next, the method of the present invention will be described. Note that the following steps will be described using the case where the apparatus 2 shown in FIG. 1 is used, but these steps are not limited to the case where the apparatus 2 shown in FIG. 1 is applied.
(Separation process)
First, it can be stabilized by flowing a predetermined mobile phase through the LC separation system. The mobile phase is not particularly limited, but a fixed solvent can be used throughout the separation step, or a gradient can be applied. Moreover, it is possible to combine both. The flow rate in the mobile phase supply means 10 can be 2 to 10 μL / min. For example, when the flow is divided into six as shown in FIG. It can be at the LC level.
[0028]
After sufficiently stabilizing the separation system (including the separation column 6, the flow path, and the precolumn 16) with the initial mobile phase, the test sample is introduced into the separation system. The test sample set in the autosampler 18 is sequentially sucked through the valve 22 and trapped in the sample loop 24, and at the same time, the valve 22 is switched to be pushed out to the separation system side by the sample loading pump 20. It is pushed out to a predetermined pre-column 16 by the sequential outlet opening / closing control of the path switching valve 26. The separation component in the test sample is adsorbed and held on the pre-column 16. In introducing the test sample into the pre-column 16, the valve 12 is at a control position different from the control position of the valve 12 shown in FIG. 1, and the flow path from the valve 26 and the pre-column 16 are connected in series. Will be contacted. After such test samples are sequentially introduced and all the test samples are adsorbed to the precolumn 16, the test samples are introduced into all the separation columns 6 at the same timing.
[0029]
The introduction of the test sample into the separation column 6 is achieved by switching the valve 12. FIG. 1 illustrates a state in which the separation column 6 and the precolumn 16 are connected in series by switching the valve 12. When the valve 12 is switched to this state, the test component adsorbed and held on the precolumn 16 is introduced into the separation column 16. According to the method of the present invention, a single mobile phase supply means 10 is used to branch the flow path, thereby enabling simultaneous analysis using a plurality of separation columns 6 at a time. For this reason, high-speed processing of separation and blotting of components in a test sample is possible. Conventionally, a plurality of separation columns 6 have not been connected to one mobile phase supply means 10 in parallel. In the case where a gradient is applied to the mobile phase, a liquid sending state that forms a gradient at an appropriate time is formed. A mobile phase is sent to each separation column 6 at a flow rate of a nanoliter per minute, a test component is separated, and each component is sequentially eluted from the separation column 6.
[0030]
(Preparation process)
The eluate eluted from each separation column 6 passes through the outflow pipe 28 and is sequentially fractionated (blotted) into the eluate storage recess of the target 32 on the table 42 of the fractionating means 30. The blotting is performed by moving the table 42 at predetermined time intervals and positioning the next eluate storage recess at the tip of the outflow pipe 28. At a flow rate of 500 nL / min, approximately 250 nL will be blotted by positioning one eluate containing recess for 30 seconds.
[0031]
When the tip of each outflow pipe 28 is held by the holding means 52, the eluate from all the separation columns 6 can be blotted by one movable table 42. Even when the horizontality of the table 42 and the target 32 is not completely ensured, since the outflow pipe 28 is guided by the targeting guide 56, the tip thereof does not bend and the tip of the eluate storage recess is formed. Reached to the bottom or its vicinity, exactly the specified amount is blotted into the receiving recess, and contamination is also prevented, resulting in reliable blotting of the eluate and sample collection suitable for subsequent analysis It becomes.
[0032]
In particular, sample preparation for matrix-assisted laser desorption mass spectrometry can be realized simultaneously with the blotting step. In this case, it is preferable that the matrix is mixed with the eluate before the eluate flowing out of the separation column 6 reaches the target 32. For this purpose, a matrix is supplied by the matrix supply means 74 shown in FIG. In addition to the matrix, reagents or other liquids necessary for identification analysis in the subsequent stage can be mixed with the eluate in the same manner as the matrix.
[0033]
As described above, the separation step and the fractionation step in the present invention can be performed. According to the present invention, it is possible to carry out a plurality of separation steps with a plurality of separation columns 6 using one mobile phase supply means 10, and particularly, to introduce a test sample into a plurality of separation columns 6 at the same time. As a result, simultaneous analysis is possible. Since one mobile phase supply means 10 is used, it is convenient for simultaneous analysis, for example, when a gradient is applied to the mobile phase. Further, according to the present invention, the blotting step can be performed on the eluates from the plurality of separation columns 6 by a single blotting means. In particular, when a test sample is introduced into a plurality of separation columns 6 at the same time, the elution components from each separation column 6 can be simultaneously blotted, so that a large number of elutions from each separation column 6 can be achieved. The fractions can be set under the same elution conditions (retention time, mobile phase gradient, etc.) as those of a large number of eluate fractions from the other separation columns 6. This facilitates identification and structural analysis of the eluted components in the identification analysis at a later stage for each eluted fraction.
[0034]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the separation and separation apparatus and method by liquid chromatography suitable for high-throughput analysis can be provided.
[Brief description of the drawings]
FIG. 1 is a diagram schematically illustrating the entire separation and fractionation apparatus of the present invention.
FIG. 2 is a diagram showing a holding means for holding an outflow tube from a separation column.
[Explanation of symbols]
2 Equipment
6 separation column
10 Mobile phase supply means
16 pre-column
18 Autosampler
28 Outflow pipe
30 Preparative means

Claims (15)

A separation / fractionation apparatus using liquid chromatography,
Separation means comprising a mobile phase supply means, and at least two separation columns corresponding to one mobile phase supply means,
Means for separating the eluate flowing out of the separation column,
An apparatus comprising: The apparatus according to claim 1, wherein a column having an inner diameter of 50 μm to 100 μm and a length of 5 cm to 20 cm is used as the separation column. 3. The mobile phase supply unit according to claim 1, wherein the mobile phase supply unit has a liquid sending capacity within a flow rate range of 2 μL / min or more and 10 μL / min or less, and supplies the mobile phase to the separation column at a flow rate of 1000 nL / min or less. 4. The described device. The apparatus according to any one of claims 1 to 3, wherein a precolumn is provided at a stage preceding each of the separation columns. The separation means has a target for accommodating the eluate from the separation column corresponding to the number of the separation columns, and by moving these targets, the eluate from the separation column is sequentially separated. Apparatus according to any of the preceding claims, wherein the apparatus is removably formed. The separation means has a holder connected to the separation column and holding two or more ends of the outflow pipe of the eluate directed to the target, and the holder is protruded below the holder. 6. A tubular targeting guide provided, wherein each of the outflow pipes is held in the guide so as to be movable in the insertion direction in a state where the maximum protruding length from the guide tip opening is regulated. The described device. The apparatus according to claim 6, wherein the plate-shaped holder has a targeting guide corresponding to the total number of the separation columns. The device according to any one of claims 1 to 7, which is for preparing a sample for protein mass spectrometry. 9. The apparatus according to claim 8, further comprising a matrix supply means for a matrix-assisted laser desorption type mass spectrometry, wherein said matrix supply means is capable of supplying said mass analysis matrix to said eluate in said outlet tube. A method for separation and separation by liquid chromatography,
A step of introducing a test sample into each of a plurality of separation columns provided in parallel by branching the mobile phase flow path to the mobile phase supply means, and separating them,
A fractionation step of fractionating the eluate eluted from each of the separation columns,
A method comprising: The method according to claim 10, wherein the separation step and the fractionation step are performed simultaneously on a plurality of test samples. An adsorption step of adsorbing a test sample on a pre-column connected to the preceding stage of the separation column is performed for each separation column, and after the completion of the adsorption step corresponding to all separation columns, the separation step for all separation columns is started. The method of claim 11. A method for preparing a sample for protein mass spectrometry, comprising the separation step and the fractionation step according to claim 10. A method for preparing a sample for matrix assisted laser desorption mass spectrometry, comprising the separation step and the fractionation step according to claim 10. The method according to claim 14, further comprising a step of supplying a mass spectrometric matrix to the eluate before the fractionation step.

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