JP2005017226A - Fraction collector for liquid chromatograph - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fraction collector for efficiently fractionating the minute quantity of a sample. <P>SOLUTION: A metallic capillary 22 is provided in the vicinity of the exit of a capillary 2, connected to a power supply 10 for supplying a high voltage of 2-5 kV via a switch 12, and conducts by turning on the switch 12. A counter electrode 14 is connected to a connector of the power supply 10 opposite to the metal capillary 22, and the electrode 14 is disposed under the metal capillary 22. When a sample component to be fractionated comes to the metallic capillary 22, it becomes a drop ions, is emitted in the direction of the counter electrode 14 by an electrospray phenomenon due to application of the high voltage between the metallic capillary 22 and the counter electrode 14, and is collected on a sample plate 6. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a fraction collector for dropping and fractionating only an eluate sent from a liquid chromatograph from a capillary without mixing with another solution.
[0002]
[Prior art]
Conventionally, for protein structure analysis, proteins separated by liquid chromatography are fractionated, each component is decomposed into peptides by oxygen digestion reaction, separated again by another liquid chromatograph, and then analyzed by mass spectrometry. Post-translational modification analysis and sequence analysis using a peptide sequencer are performed. In order to analyze the physiological activity of a protein, the protein separated by liquid chromatography is fractionated, and the activity of each component is measured, a protein / low molecular interaction experiment, and a protein / protein interaction experiment.
[0003]
When the sample is a biopolymer (protein, nucleic acid, sugar, etc.), the amount of flow is limited because the amount of the sample is often very small for structural analysis and bioactivity analysis of these biopolymers. It is preferable to use a micro flow rate high performance liquid chromatograph that is μL / min or less, preferably a nano flow rate high performance liquid chromatograph that is a flow rate range of several nL / min.
[0004]
In the case of fractionation in the flow rate region in a normal high performance liquid chromatograph, since the flow rate is large, a fraction collector using a solenoid valve or the like is usually used. However, a solenoid valve cannot be used in the case of a micro flow rate high-performance liquid chromatograph or a nano flow rate high-performance liquid chromatograph in a minute flow rate range. Therefore, when fractionating and collecting the eluate, place the probe that will be the final outlet above the target spot on the sample plate, bring the sample plate close to the probe, and the eluate contacts the sample plate. And waiting until the eluate adheres to the target spot of the sample plate or waiting for the eluate to drop (see Non-Patent Document 1).
[Non-Patent Document 1]
"Interfacing Capillary or Nano LC With MALDI MS", [online], [November 15, 2002 search], Internet <URL: http: // www. lcpackings. com / products / Instruments / Probot01. html>
[0005]
[Problems to be solved by the invention]
When sorting is performed using the method described above, if the receiving member such as a microplate is made of a material that does not soak in liquid, the sample component may become a needle at the elution outlet depending on the surface tension of the mobile phase (eluent). May not move to the receiving member. For example, in the case of a sample that frequently uses an aqueous mobile phase such as protein, it is difficult for a droplet to move due to surface tension, and it is particularly difficult to sort in a minute volume of nL order.
[0006]
Therefore, an object of the present invention is to provide a fraction collector capable of separating a minute amount of eluate droplets from a liquid chromatograph into a sample plate such as a microplate regardless of its components.
[0007]
[Means for Solving the Problems]
One aspect of the present invention is a fraction collector for dropping and fractionating only an eluate sent from a liquid chromatograph from a capillary without mixing with another solution, and gas in the tip direction of the capillary. And a probe for detaching the eluate from the liquid chromatograph from the tip of the capillary with the gas ejected from the ejection port.
Since the droplets of the eluate are forcibly dropped by the gas ejection, the separation can be performed without being affected by the surface tension of the eluate components and the receiving material such as the sample plate.
Furthermore, this fraction collector drops only the eluate from the liquid chromatograph from its tip, thereby further decomposing the sample components separated by the liquid chromatograph by adding digestive fluid etc., and again using the liquid chromatograph etc. It can be set as the pre-processing apparatus for isolate | separating.
[0008]
Another aspect of the present invention includes a conductive capillary installed at an outlet tip of a flow path of an eluate from a high performance liquid chromatograph, a counter electrode disposed below the conductive capillary, and the conductive capillary. A power source for applying a voltage for discharging a droplet containing ions from the conductive capillary by an electrospray phenomenon between the counter electrode and a counter electrode disposed on the plate disposed between the conductive capillary and the counter electrode. It will be sorted out.
[0009]
If the eluate is moved to a microplate well or a test tube by the electrospray method, even a very small amount of liquid in the nL region can be moved with high efficiency.
In addition, since electrospray is a gentle ionization method, biopolymer denaturation is unlikely to occur.
[0010]
In the electrospray method, as shown in FIG. 3, when a high voltage is applied to the opposing electrode 14 by applying a high voltage to the outlet tip of the conductive capillary, the components in the solution accumulated at the tip of the capillary 22 are removed. The ionized droplets are attracted to the opposing electrode 14 and jump out to the electrode 14 side.
[0011]
【Example】
(Example 1)
Embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a schematic view showing an embodiment of one aspect together with a micro flow rate high performance liquid chromatograph.
A capillary 2 having an inner diameter of 0.1 mm is connected to send an eluate from a high-speed liquid chromatograph column 44 via a detector 42, and a probe 4 is attached to the tip of the capillary 2. A tube 18 for supplying air is connected to the probe 4 via an open / close valve 9 by a three-way joint 16. At the outlet of the probe 4, an air outlet is disposed around the tip of the capillary 2, and air from the air outlet is ejected toward the tip of the capillary 2. The sample plate 6 is installed below the outlet of the probe 4 and is moved in a horizontal plane (XY direction).
[0012]
Further, the capillary 2 branches before the probe 4 and can be operated by selecting whether the fluid is sent to the probe 2 or discharged by the micro volume electromagnetic valve 5.
[0013]
Channel switching and channel opening / closing operations of the micro volume solenoid valve 5 and the opening / closing valve 9 are controlled by the controller 20. The controller 20 can read the signal transmitted from the detector 42 and operate the micro volume solenoid valve 5 and the opening / closing valve 9 independently.
[0014]
The distances from the detector 42 to the micro volume solenoid valve 5 and from the detector 42 to the exit tip of the probe 4 are known, and the flow rate of the eluate sent from the high-performance liquid chromatograph is constant at 1 to 5 μL / min. Therefore, it is possible to calculate the time required for the sample component detected by the detector 42 to reach a predetermined position. Therefore, when the sample is detected by the detector 42, the controller 20 calculates the time for the sample component to reach the position of the microvolume solenoid valve 5 and the tip of the outlet of the probe 4 based on the signal from the detector 42. .
[0015]
Based on the calculated result, the controller 20 controls the micro volume electromagnetic valve 5 to flow only the liquid containing the sample component detected to the probe 4 side. Furthermore, when the liquid containing the sample component reaches the outlet tip of the probe 4 and accumulates, the controller 20 opens the open / close valve 9 to send air into the probe 4 and drop it onto the sample plate.
[0016]
The operation will be described below.
The sample is injected from the injector 46 and sent to the column 44. The sample separated by the column 44 is dissolved in the eluent sent by the pump 48 to become an eluent, detected by the detector 42 and sent by the capillary 2, but the eluent containing no sample component is also flowing. Therefore, by switching the micro volume solenoid valve 5 controlled by the controller 20, only the eluate containing the sample component to be collected is sent to the probe 4. The eluate sent to the probe 4 accumulates as a droplet at the outlet tip of the probe 4 due to surface tension, and the valve 9 opens when the target sample component accumulates at the tip, and the liquid is detached from the tip by the pressure of the inflowing air. And dripped.
[0017]
(Example 2)
Next, an example of another aspect is shown below.
A highly polar compound having a charge in a solvent can be electrostatically moved in space as a sample-derived ion generated by an electrospray phenomenon. A conductive capillary is used at the outlet of the eluate after the column separation, and a voltage of 2 to 5 kV is applied thereto for electrospraying. As a result, the target compound such as a biopolymer is sprayed and dispensed into a receiving part (such as a microplate) placed in front of the paired electrodes.
[0018]
FIG. 2 is a schematic view showing the fraction collector of the same example together with the nano flow rate high performance liquid chromatograph. The configuration of the liquid chromatograph is basically the same as that of FIG. 1, but since the flow rate is smaller than that, the inner diameter of the column 44a is smaller than that of the column 44 of FIG.
The vicinity of the outlet of the capillary 2a having an inner diameter of 75 μm is a metal capillary 22, which is connected to a power source 10 that supplies a high voltage of 2 to 5 kV via a switch 12, and a high voltage is applied by turning on the switch 12. Is done. A counter electrode 14 is connected to the connector on the opposite side of the metal capillary 22 of the power supply 10, and the counter electrode 14 is disposed below the metal capillary 22. The sample plate 6 is installed between the metal capillary 22 and the counter electrode 14 and is moved in a horizontal plane (XY direction).
[0019]
Further, the capillary 2a branches before the metal capillary 22, and the microvolume solenoid valve 5 can select whether the sample solution is sent to or discharged from the metal capillary 22 side.
The micro volume solenoid valve 5 and the switch 12 are connected to the controller 20 and controlled in operation. The controller 20 can read the signal transmitted from the detector 42 and operate the micro volume solenoid valve 5 and the switch 12 independently.
[0020]
The distances from the detector 42 to the micro volume solenoid valve 5 and from the detector 42 to the outlet tip of the capillary 2a are known, and the flow rate of the eluate sent from the high performance liquid chromatograph is constant at 50 to 100 nL / min. Therefore, it is possible to calculate the time required for the sample component detected by the detector to reach a predetermined position. Therefore, when the sample is detected by the detector 42, the controller 20 calculates the time for the sample component to reach the position of the micro volume solenoid valve 5 and the outlet tip of the capillary 2a based on the signal from the detector 42. .
[0021]
Based on the calculated result, the controller 20 controls the micro volume solenoid valve 5 to flow only the liquid containing the detected sample component in the direction of the dropping outlet. When the liquid containing the sample component reaches the tip of the outlet of the capillary 2a, the switch 12 is closed at the time of dropping, and a high electric field is generated between the metal capillary 22 and the counter electrode 14 positioned below the switch 12, and the eluate is removed. The sample plate 6 is moved by an electrospray phenomenon.
[0022]
The operation will be described below.
A sample plate 6 for collecting a sample is installed between the tip of the metal capillary 22 and the counter electrode 14.
The sample is injected from the injector 46 and sent to the column 44a. The sample separated by the column 44a is dissolved in the eluent sent by the pump 48, becomes an eluent, is detected by the detector 42, and is sent through the capillary 2a, but the eluent containing no sample component also flows. Therefore, the controller 20 controls the micro volume solenoid valve 5 so that only the eluate containing the sample component to be collected is sent in the direction of the dropping outlet.
[0023]
The eluate sent in the direction of the dropping outlet collects as droplets at the outlet tip of the capillary 2a due to surface tension. When the target sample component accumulates at the tip, the controller 20 closes the switch 12 and generates a high electric field between the metal capillary 22 and the counter electrode 14. The solution containing the sample component jumps out toward the electrode 14 by the electrospray phenomenon. At this time, since the sample plate is provided between the tip of the metal capillary 22 and the counter electrode 14, the sample component that has jumped out is collected on the sample plate 6. When the target sample component moves onto the sample plate 6, the switch 12 is opened to disconnect the power supply 10. When the collection of one fraction is completed, the sample plate 6 is moved in the horizontal plane to prepare for the next sorting at a new position on the sample plate 6. This operation is repeated to collect the sample components.
[0024]
【The invention's effect】
According to one aspect of the present invention, when a liquid containing a sample component is forcibly dropped by gas ejection, the sample is unlikely to remain at the tip of the outlet of the capillary, so that cross-contamination can be prevented.
Moreover, if the amount of gas ejection is adjusted, the effect of preventing the elution fraction from drying can be expected, and even a sample such as protein that may be denatured by drying can be fractionated without being deactivated.
[0025]
According to another aspect of the present invention, if a sample is moved by electrospray, it is a gentle ionization method, so that biopolymer denaturation hardly occurs and a small amount of sample solution can be collected with high efficiency. It is.
[Brief description of the drawings]
FIG. 1 is a schematic configuration diagram showing an example of one aspect together with a liquid chromatograph.
FIG. 2 is a schematic configuration diagram showing an example of another aspect together with a liquid chromatograph.
FIG. 3 is an explanatory diagram of electrospray ionization.
[Explanation of symbols]
2,2a Capillary 4 Probe 5 Micro volume solenoid valve 6 Sample plate 9 Open / close valve 10 Power supply 12 Switch 14 Electrode 16 Three-way joint 18 Air supply pipe 20 Controller 22 Metal capillary 42 Detector 44 Column 46 Injector 48 Pump

Claims (2)

In a fraction collector that drops and fractionates only the eluate sent from the liquid chromatograph by dropping from the capillary without mixing with other solutions,
A fraction collector, comprising: a probe at a tip of the capillary for ejecting gas in a tip direction, and a probe for detaching an eluate from a liquid chromatograph from the tip of the capillary by the gas ejected from the nozzle. A conductive capillary installed at the outlet tip of the flow path of the eluate from the high performance liquid chromatograph;
A counter electrode disposed below the conductive capillary;
A power source for applying a voltage for discharging a droplet containing ions from the conductive capillary by an electrospray phenomenon between the conductive capillary and the counter electrode;
A fraction collector for sorting on a plate disposed between the conductive capillary and the counter electrode.

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