JP4211651B2 - Liquid fractionator - Google Patents

Description

  The present invention relates to an analyzer such as a high performance liquid chromatograph, and more particularly to an apparatus for dropping a liquid such as a sample or a mobile phase used in these analyzers onto a sample plate.

Conventionally, an eluate of sample components separated by a separation column of a liquid chromatograph is automatically dropped onto a sample plate for MALDI-TOF-MS (Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry) or the like for separation. When collecting fractions, the eluate from the separation column is sent to the tip of the pipe outlet (referred to as the probe), waits for a certain amount of time until the amount of liquid droplets defined by the probe tip is formed, and then the sample is applied to the sample plate. Move the droplet.
As a means to move the droplet to the sample plate, a stage with the sample plate fixed, or a method of moving the droplet to the sample plate by moving the probe itself up and down to move the droplet to the sample plate, or air to the tip of the probe There is a method in which droplets are ejected from a portion and sprayed onto a sample plate.

Usually, when protein analysis is performed using a high performance liquid chromatograph, a solution of water and acetonitrile is fed as a mobile phase while changing the composition by a gradient type liquid feeding mechanism. In this gradient analysis, the composition of the mobile phase transitions from a water-rich state containing a lot of water components to an acetonitrile-rich state containing a lot of acetonitrile.
In a device that forms a droplet at the tip of the probe and moves the droplet by moving the probe or sample plate up and down to bring the droplet into contact with the sample plate when the droplet reaches a certain size, The components in the droplets are different immediately after the start of fractionation and immediately before the end of fractionation, with a lot of water components immediately after the start of fractionation and a lot of acetonitrile immediately before the end of fractionation. As a result, the uniform droplet size is not formed.

One of the causes is a change in the surface tension accompanying a change in the composition of the droplet.
In addition, when adding a matrix solution to the eluate from the separation column at the tip of the probe and fractionating from the tip, as a material for the matrix addition tube of the probe, a droplet should not crawl up the matrix addition tube. A hydrophobic substance such as Teflon (registered trademark) is used, and immediately after the start of fractionation, the droplet contains a lot of water components, so a droplet is formed on the inner diameter side of the matrix addition tube. Since the previous droplet contains a lot of acetonitrile, the droplet may be formed to the outer diameter side of the matrix addition tube. If the volume of the droplet is constant, the outer shape of the droplet formed on the probe tip is different immediately after the start of fractionation and immediately before the end of fractionation. As a result, the height of the droplets formed at the probe tip in the direction of the sample plate is different, so even with a sensor that keeps the distance between the probe tip and the sample plate constant, the droplet volume is particularly small. In this case, the droplet does not come into contact with the sample plate, and the droplet may not be moved to the sample plate.

  Accordingly, the present invention provides a liquid fractionation device that can move a droplet to the sample plate reliably while keeping the height of the droplet formed at the probe tip in the direction of the sample plate constant. Objective.

  The present invention is a liquid fractionation apparatus comprising a liquid feeding mechanism for feeding a mobile phase while changing its composition, and a probe for dropping an eluate from a separation column from the tip, the probe being a liquid feeding device A flow path for sending a second mobile phase different from the mobile phase fed by the mechanism to the tip, and the second mobile phase has a composition of the mobile phase sent by the liquid feed mechanism at the tip of the probe. It has a composition close to a certain composition.

The probe can have a multi-tube structure in which two or more flow paths are formed.
In that case, the probe has a triple tube structure, the eluate from the separation column flows on the innermost side, and the outer side of the probe is a matrix compound for sample preparation for analysis with a mass spectrometer based on matrix-assisted laser desorption / ionization The matrix liquid, which is a solution of the above, flows, and the second mobile phase flows on the outermost side, or the eluate from the separation column flows on the innermost side, and the second mobile phase flows on the outermost side. It is preferable that the matrix liquid flows on the outside.

  Since the probe is provided with a flow path for sending the second mobile phase to the probe tip, which makes the mobile phase composition in the droplet formed at the tip of the probe constant, the probe tip even if the composition of the mobile phase changes In this case, since the composition of the droplet is kept constant, a change in the outer shape of the droplet can be suppressed, and a stable fractionation operation can be performed.

When adding a matrix solution, the matrix solution is usually a saturated solution in which water: acetonitrile = 1: 1 is used as a solvent and the matrix compound is dissolved therein. When the matrix solution is added to the eluate from the separation column at the tip of the probe and fractionation is performed from the tip, the mobile phase composition immediately after the start of fractionation contains a large amount of water components. As a result, the matrix in the saturated matrix solution is deposited on the probe tip, which inhibits the stable fractionation operation, and there is a problem of carry-over due to the penetration of the component to be measured into the matrix.
However, according to the present invention, since the mobile phase composition in the droplet formed at the probe tip is kept constant, there is also an effect of preventing matrix precipitation at the probe tip.

One embodiment will be described below. FIG. 1 is a diagram schematically showing a configuration of a fractionation apparatus to which the present invention is applied together with a high performance liquid chromatograph.
As a liquid feed mechanism for supplying a mobile phase to the analysis flow path, a flow path switching valve 51, a liquid feed pump 48, a mixer 52, and an opening / closing valve 53 are provided. In this liquid feeding mechanism, the liquid A and the B liquid are sucked by the liquid feeding pump 48 and mixed by being sent to the mixer 52 while switching the flow path by the flow path switching valve 51, and these solutions are used as a mobile phase for the analysis flow path. This is a gradient-type liquid feeding mechanism to be supplied to the vehicle. The A liquid and the B liquid are, for example, water and acetonitrile, and the composition of the solution mixed by the mixer 52 is sent to the analysis channel while changing with time.

The capillary 2 is used as the analysis flow path, and the capillary 2 is provided with an injection port 46, a separation column 44, and a detector 42 along the flow of the mobile phase. The capillary 2 is connected to the tip of the probe 1.
When the sample is injected into the injection port 46, it is separated and eluted by the separation column 44. After the eluate is detected by the detector 42, it flows through the capillary 2 to the tip of the probe 1 and drops onto the sample plate. Is done.

  In this embodiment, the probe 1 can move in the vertical direction and the horizontal direction. Although not shown, the probe 1 includes a sensor for detecting the distance between the sample plate and the tip of the probe 1 on the side. When fractionating the liquid on the sample plate, when the droplet formed at the tip of the probe 1 reaches a predetermined size, the probe 1 moves to approach a predetermined position on the sample plate, and the droplet and the sample. The plate is brought into contact to move the droplet onto the sample plate.

  J1 is a three-way joint that joins the pipe 16 for feeding the matrix liquid and the probe 1 together. The pipe 16 includes a pump 49 and supplies the matrix liquid to the probe 1.

  J2 is a three-way joint that joins the pipe 17 for feeding the second mobile phase and the probe 1 together. The pipe 17 includes a flow path switching valve 54, a liquid feed pump 50, a mixer 56, and an open / close valve 57 as a liquid feed mechanism for supplying the second mobile phase. In this liquid feeding mechanism, the A liquid and the B liquid are sucked by the liquid feed pump 50 while being switched by the flow path switching valve 54 and sent to the mixer 56 to be mixed, and these solutions are used as the mobile phase for the analysis flow path. This is a gradient-type liquid feeding mechanism that is supplied to the vehicle. The mixing ratio of the solution A and the solution B mixed by the mixer 56 is adjusted to be opposite to the mixing ratio of the solution A and the solution B mixed by the mixer 52.

  Although not shown in the drawing, the tip of the probe 1 has a triple tube structure, and the eluate from the separation column 44, the matrix liquid merged at the three-way joint J1, and the second merged at the three-way joint J2. These mobile phases are mixed at the tip of the probe 1 to form droplets and dropped onto the sample plate.

FIG. 2 shows temporal changes in the composition of the mobile phase (first mobile phase) and the second mobile phase fed by the pump 48. (A) is a figure which shows the time change of the composition of a 1st mobile phase, (B) is a figure which shows the time change of the composition of a 2nd mobile phase.
As shown in FIG. 2 (A), the first mobile phase occupies most of the liquid A (water) in the solution immediately after the start of fractionation, but the ratio of liquid B increases linearly with time. I will do it. On the other hand, as shown in (B), the proportion of the second mobile phase in the solution B (acetonitrile) is almost immediately after the start of fractionation, but the proportion of solution A is linear with time. It will increase.
The composition of the second mobile phase is adjusted so that the composition of the mixed liquid of the first mobile phase and the second mobile phase is always A liquid: B liquid = 1: 1.
For example, assuming that the composition of the first mobile phase is 90% of liquid A and 10% of liquid B after a certain time has elapsed after the start of fractionation, the composition of the second mobile phase is 10% of liquid A and liquid B. It is adjusted to be 90%. Therefore, the mixing ratio of the eluate mixed at the tip of the probe 1 and the water and acetonitrile in the second mobile phase is water: acetonitrile = 1: 1.

Hereinafter, the probe 1 of this embodiment will be described in more detail with reference to FIG. FIG. 3 is a sectional view showing in detail the structure of the probe portion of this embodiment.
The thinnest capillary 2 to which the eluate from the high-performance liquid chromatograph passes through two of the non-orthogonal joints a and b of the first T-shaped three-way joint J1 on the upstream side. The joint a on the upstream side is sealed using a piping part 10a such as a mail nut, and at that time, a sleeve 12 or the like is used as necessary.

  A pipe 16 through which the matrix liquid is fed is connected to the orthogonal joint c of the T-shaped three-way joint J1, and is sealed with a pipe part 10c such as a mail nut. The joint b from which the thinnest capillary 2 comes out is covered with a capillary 4 and sealed with a piping component 10b such as a mail nut. In this case, a sleeve 22 or the like is used as necessary.

  The downstream side T-shaped three-way joint J2 is inserted with capillaries 2 and 4 from the upstream side joint a, and is sealed using a piping part 20a such as a mail nut. Use. A pipe 17 that supplies the second mobile phase is connected to the joint c orthogonal to the capillaries 2 and 4, and is sealed with a pipe component 20 c such as a mail nut. The most downstream joint b is covered with a pipe 8 and sealed with a pipe part 20b such as a mail nut.

  The tip of the probe 1 has a triple tube structure with capillaries 2, 4 and a pipe 8, the eluate from the liquid chromatograph flows through the innermost flow path, and the matrix liquid flows through the outer flow path, The second mobile phase flows through the outermost flow path. These liquids are mixed at the tip of the probe 1 to form droplets, and are dropped onto the sample plate when a predetermined droplet amount is reached.

4 and 5 are diagrams showing the state of liquid fractionation at the tip of the probe 1, FIG. 4 shows a conventional case in which the second mobile phase is not added, and FIG. 5 shows the second mobile phase. The case where is added is shown. Further, (A) shows immediately after the start of fractionation, and (B) shows just before the end of fractionation.
As shown in FIG. 4, in the case where the second mobile phase that adjusts the composition of the mobile phase component in the eluate is not added, (A) at the start of fractionation and (B) at the end of fractionation, the probe 1 Since the composition of the droplet formed at the tip changes and the outer shape of the droplet changes, the height in the direction of the sample plate (vertical direction) changes, and the droplet moves reliably to the sample plate. It may not be possible to In addition, when the matrix liquid is a saturated solution of water: acetonitrile = 1: 1, the mobile phase composition immediately after the start of fractionation contains a large amount of water components. The matrix inside is deposited on the probe tip, which hinders stable fractionation.

  As shown in FIG. 5, when the second mobile phase that adjusts the composition of the mobile phase component in the eluate is added, the composition of the mobile phase component in the droplet formed at the tip of the probe 1 is always constant. Therefore, the outer shape of the droplet formed on the tip of the probe 1 is the same at the start of (A) fractionation and at the end of (B) fractionation, and a stable fractionation operation can be performed. In addition, since the composition of the mobile phase component in the droplet is always water: acetonitrile = 1: 1, the matrix does not precipitate even if the eluate is mixed with the matrix liquid at the tip of the probe 1.

In the MALDI-TOF-MS analysis, since it is premised that the amount of droplets to be fractionated is small and constant, as described above, when the composition of the mobile phase in the droplet changes, it is formed at the probe tip. The shape of the droplet to be changed will change. The distance between the probe tip and the droplet tip immediately after the start of fractionation increases, and the distance decreases immediately before the end of fractionation. Becomes prominent, and at a predetermined distance between the probe and the plate, the droplet may not be able to move to the plate.
In the present invention, since the mobile phase composition in the droplet formed at the probe tip is made constant, the size (height) of the droplet formed at the probe tip can be made constant and stable. Fractionation operation can be performed.

In the embodiment, the mobile phase liquid feeding of the low pressure gradient method is taken as an example, but the high pressure gradient method may be used. The fractionation apparatus of the embodiment is of a type in which the probe 1 moves in the vertical direction or the horizontal direction and performs a fractionation operation. However, the fractionation device in which the stage for fixing the sample plate moves and performs the fractionation operation is used. It may be a thing.
The present invention can be applied to a probe having a structure of a double tube or a quadruple tube or more. For example, the probe may have a flow path through which a cleaning liquid such as acetone flows. In this embodiment, the eluate from the liquid chromatograph flows through the innermost flow path, the matrix liquid flows through the outer flow path, and the second mobile phase flows through the outermost flow path. The eluate from the separation column may flow on the innermost side, the second mobile phase may flow on the outer side, and the matrix liquid may flow on the outermost side.

It is a figure which shows roughly the structure of the fractionation apparatus to which this invention is applied with a high performance liquid chromatograph. The time change of the composition of a 1st mobile phase and a 2nd mobile phase is shown, (A) is a figure which shows the time change of the composition of a 1st mobile phase, (B) is a 2nd mobile phase. It is a figure which shows the time change of a composition. It is sectional drawing which shows the structure of the probe part of the Example in detail. It is a figure which shows the mode of the probe front-end | tip part at the time of the liquid fraction when not liquid-feeding a 2nd mobile phase, (A) is a figure which shows a state just before the end of fractionation, (B) immediately after the start of fractionation, respectively. It is. It is a figure which shows the mode of the probe front-end | tip part at the time of the liquid fraction at the time of liquid-feeding a 2nd mobile phase, (A) is a figure which shows a state just before the end of fractionation, (B) immediately after the start of fractionation, respectively. It is.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Probe 2,4 Capillary 8 Piping 10a, 10b, 10c, 20a, 20b, 20c, 30a, 30b, 30c Piping component 12, 22, 32 Sleeve 16 Matrix supply pipe 18 Second mobile phase supply pipe 42 Detector 44 Column 46 Injection port 48, 49, 50 Pump 51, 54 Flow path switching valve 52, 56 Mixer 53, 57 Stop valve S Sample plate J1, J2 T-shaped three-way joint

Claims (4)

In a liquid fractionation apparatus comprising a liquid feeding mechanism for feeding a mobile phase while changing its composition, and a probe for dropping an eluate from a separation column from the tip.
The probe includes a flow path for sending a second mobile phase different from the mobile phase fed by the liquid feeding mechanism to the tip,
The liquid fractionation apparatus, wherein the second mobile phase has a composition that brings the composition of the mobile phase fed by the liquid feeding mechanism close to a constant composition at the tip of the probe.   The liquid fractionation apparatus according to claim 1, wherein the probe has a multi-tube structure in which two or more flow paths are formed.   The probe has a triple tube structure, and the eluate from the separation column flows on the innermost side, and the outer side of the probe is a solution of a matrix compound for sample preparation for analysis by a mass spectrometer based on matrix-assisted laser desorption / ionization. The liquid fractionation device according to claim 1 or 2, wherein the matrix liquid flows and the second mobile phase flows on the outermost side.   The probe has a triple tube structure. The eluate from the separation column flows on the innermost side, the second mobile phase flows on the outer side, and the outermost side is analyzed by a mass spectrometer based on matrix-assisted laser desorption / ionization. The liquid fractionation device according to claim 1 or 2, wherein a matrix liquid, which is a solution of a matrix compound for sample preparation for flowing, flows.

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