JPH08304367A - Method and device for introducing sample into liquid chromatograph - Google Patents

Abstract

(57) [Summary] [Purpose] To reduce the amount of wasted sample when introducing a sample into a liquid chromatograph using a sampling valve. [Structure] The following steps are performed in order. (A) The sample supply flow path 6 is directly connected to the waste liquid flow path 8, and the eluent supply flow path 1
In the first channel 0 connected to the column channel 12 via the sample loop 4, the sample (thick solid line) liquid feed and the eluent (thick broken line) liquid feed are started, and the inlet sensor 14 When is detected, the sample transfer is stopped. (B) The sample supply channel 6 is connected to the waste liquid channel 8 via the sample loop 4, and the eluent supply channel 10 is switched to the second channel directly connected to the column channel 12 to transfer the sample. Restart the fluid.
(C) After the sample is sent to the sample loop 4 and the outlet sensor 16 detects the liquid, if the liquid detection is interrupted, the sample feeding is stopped. (D) Switching to the first flow path again, the sample in the sample loop 4 is introduced into the separation column by the eluent.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention In the fields of biochemistry, pharmacology, medicine, etc., the present invention collects a sample in a sample loop of a sampling valve, switches the sampling valve, and collects the sample in the sample loop in a column of a liquid chromatograph. Regarding the sample introduction method and the apparatus to be introduced, for example, in order to determine the amino acid sequence of a protein or peptide, a solution obtained by cutting out one amino acid from a peptide sequencer is introduced into a liquid chromatograph as a sample, and the amount of the sample is limited. The present invention relates to a sample introduction method that is effective in some cases and a sample introduction device that is convenient in such cases.

[0002]

2. Description of the Related Art Proteins or peptides (both of which are amino acids bound by amide bonds,
Peptide sequencers are used to determine the amino acid sequence of both (hereinafter simply referred to as proteins) because they are distinguished only by the size of their molecular weight. In a peptide sequencer, phenylisothiocyanate was used to cut out only one amino acid from the N-terminal (amino group terminal) of a sample protein as a 3-phenyl-2-thiohydantoin derivative (PTH amino acid).
Dissolved in an aqueous solution of acetonitrile, and identified by high performance liquid chromatography. This process is one cycle,
The same process is repeated again for the remaining part of the sample protein from which one amino acid has been cut off. This process is n
By repeating the cycle, the sequence of n amino acids counting from the N-terminus of the original sample protein can be determined.

Upon introduction into high performance liquid chromatography, the solution is introduced into the sample loop of the sampling valve of the sample introduction device of the high performance liquid chromatograph, and then the flow path of the sampling valve is switched to introduce into the column by the eluent. As shown in FIG. 1 (A), the sample introduction device includes a sampling valve 2 composed of a 6-port valve, a sample loop 4 having a constant volume, a sample supply channel 6,
The waste liquid flow path 8 and the eluent supply flow path 10 are connected to each other, an inlet sensor 14 for detecting the liquid near the sampling valve of the sample supply flow path 6, and an outlet for detecting the liquid near the sampling valve of the waste liquid flow path 8. The sensor 16 is provided. Reference numeral 3 denotes a slit that connects ports in the sampling valve 2, the sample supply flow path 6 is directly connected to the waste liquid flow path 8, and the eluent supply flow path 10 is connected via the sample loop 4 by switching the sampling valve 2. First flow path connected to the column flow path 12 and the sample supply flow path 6 are connected to the waste liquid flow path 8 via the sample loop 4, and the eluent supply flow path 10 is directly connected to the column flow path 12. The flow path is switched between the second flow path and the second flow path. The eluent is supplied to the eluent supply channel 10 by the pump 22, and the column channel 12 is connected to the separation column 26. 24 is a column oven, 2
8 is a detector. The pump 22, the column oven 24, and the detector 28 constitute a high performance liquid chromatograph (HPLC).

When this sample introduction device is applied to a device for determining the amino acid sequence of a protein, the sample supply channel 6
Supplies the solution of the amino acid derivative cleaved from the peptide sequencer 20, which performs the Edman reaction to cleave N-terminal amino acids one by one.

As shown in FIG. 1B, the inlet sensor 14 is provided in the sample supply flow path 6 near the sampling valve 2, and the sample supply flow path is connected to the valve 2 by the nut 22 via the ferrule 20. It is connected. The outlet sensor 16 provided in the waste liquid flow path 8 is also attached in the same manner as the inlet sensor 14 and is arranged at the same distance from the sampling valve 2. The distance from the sampling valve 2 to the sensors 14 and 16 is, for example, 25 mm, and the inner diameters of the flow paths 6 and 8 are, for example, 0.5 mm.

FIG. 2 shows a method of introducing a sample into a column by using such a sample introduction device. (A) Set the sampling valve 2 to the second position as shown in FIG.
And a sample (thick solid line) is supplied from the sample supply flow path 6 to flow through the sample loop 4 to the waste liquid flow path 8.
The eluent (thick broken line) is caused to flow from the eluent supply channel 10 directly to the column channel 12. When the outlet sensor 16 detects the liquid, the liquid feeding of the sample is stopped. At this time, the entrance sensor 14
It is determined that the sample loop 4 is filled with the sample by confirming that the sample loop 4 is detecting the liquid. (B) The sampling valve 2 is switched clockwise as shown in FIG. As a result, the sample collected in the sample loop 4 is introduced into the separation column by the eluent.

[0007]

The volume of the sample loop 4 is, for example, 20 μl, and the volume of one of the slits 3 is 0.3.
μl, the capacity from the slit 3 to the sensors 14 and 16 is 4.9 μl, and when the liquid feeding is stopped, the remaining pressure in the flow passage is 10 mm from the time when the sensor 16 detects the liquid until the liquid feeding is stopped. Since the liquid progresses to some extent, 2 μl of waste sample exists on the downstream side of the outlet sensor 16. Also entrance sensor 1
On the upstream side of 4, it is difficult to control the movement of the liquid due to the residual pressure after the liquid supply is stopped. Therefore, it is necessary to allow for a safe amount of about 20 mm, so about 4 μl is wasted. Therefore, the amount of sample required by introducing the sample once by the method of FIG. 2 is 20 + 0.3 × 2 + 4.9 × 2 + 2 + 4 = 36.4 (μl). Of these, what is actually introduced into the column is (20
+0.3) μl, so 5 of the samples used
Only 6% would have been effectively analyzed.

A sample in which the PTH amino acid cut out as an amino acid derivative by the peptide sequencer 20 is a solution is
By pressurizing the flow path with gas, the sample is supplied from the sample supply flow path 6 to the sampling valve 2, and whether or not the sample loop 4 is filled with the sample is determined by the outputs of the inlet sensor 14 and the outlet sensor 16. However, since it is difficult to control the flow velocity with only the gas pressure for liquid delivery, in order to reliably detect the liquid with the outlet sensor 16, the liquid delivery is stopped after the liquid has passed the outlet sensor 16 to some extent. There is a need to. Therefore, an excessive amount of sample is needed with respect to the volume of the sample loop 4, and in the case of a peptide sequencer, for example, the amount of PTH amino acid solution required is large, so the amount of dissolution is large and the concentration is low. Therefore, a first object of the present invention is to reduce the amount of wasted sample when introducing a sample into a liquid chromatograph using a sampling valve. A second object of the present invention is to accurately grasp the amount of introduced liquid when introducing a sample into a liquid chromatograph by using a sampling valve, and perform liquid feeding control.

[0009]

The sample introduction method of the present invention introduces a fixed amount of sample into a liquid chromatograph by switching a sampling valve using a sample introduction device as shown in FIG. 1 (A). This is a method, and the following steps (A) to (C) are sequentially performed. (A) In the first flow path in which the sample supply flow path is directly connected to the waste liquid flow path and the eluent supply flow path is connected to the column flow path via the sample loop, the sample transfer is started and the inlet When the sensor detects the sample, the sample feeding is stopped. (B) The sample supply flow path is connected to the waste liquid flow path through the sample loop, and the eluent supply flow path is switched to the second flow path which is directly connected to the column flow path to restart the sample transfer. After the sample is sent to the sample loop, the outlet sensor detects the eluent, and when the eluent detection is interrupted, the sample transfer is stopped. (C) Switching to the first flow path again, the sample in the sample loop is introduced into the separation column by the eluent.

The sample introducing device of the present invention is the same as the sample introducing device shown in FIG. 1 (A), in which a branch channel is provided in the waste liquid channel through a three-way switching valve, and the branch channel is formed by a motor. The syringe to be driven is connected, and the liquid sending speed when the sample is sent to the sample loop in the second channel is controlled by the syringe.

[0011]

[Function] If the amount of wasteful sample in the sampling valve is reduced, the amount of sample to be supplied to the sampling valve can be reduced. Therefore, for example, in a peptide sequencer, the amount of solvent dissolving the sample is reduced and the sample is concentrated in a concentrated state. It can be introduced into a liquid chromatograph, and the sensitivity is improved accordingly.

[0012]

EXAMPLE FIG. 3 shows a first example in which a sample is introduced into a liquid chromatograph by using the sample introduction device shown in FIG. 1 (A). (A) In the first channel in which the sample supply channel 6 is directly connected to the waste fluid channel 8 and the eluent supply channel 10 is connected to the column channel 12 via the sample loop 4, the sample (thick solid line ) And the eluent (thick broken line) are started, and when the inlet sensor 14 detects the sample, the sample transfer is stopped.

(B) The sample supply channel 6 is the sample loop 4
And the eluent supply channel 10 is switched to the second channel directly connected to the column channel 12 to restart the sample feeding. (C) After the sample is sent to the sample loop 4 and the outlet sensor 16 detects the eluent, if the eluent detection is interrupted, the sample sending is stopped. (D) Switching to the first flow path again, the sample in the sample loop 4 is introduced into the separation column by the eluent.

In the embodiment of FIG. 3, as shown in the flow path of (C) or (D), the sample amount required for one sample introduction is
The volume of the sample loop 4 is 20 μl, the volume of one slit is 0.3 μl, the volume from the slit to the inlet sensor 14 is 4.9 μl, the volume of the liquid advancing when the liquid feeding is stopped is 2 μl, and the upstream side of the inlet sensor 14 is safe. A total of 31.2 μl in a volume of 4 μl. Since 20.3 μl of them are introduced into the separation column, the effective rate of the sample is 65%.
It is improved by 16% as compared with the conventional method.

FIG. 4 shows a second embodiment.
In this embodiment, as shown in (A), a second inlet sensor 14a is provided upstream of the inlet sensor 14, and the volume of the flow path between the two inlet sensors 14 and 14a is set to the volume of the sample loop 4. , And the capacity for one slit and + α.

The operation of the embodiment shown in FIG. 4 will be described. (A) In the first channel, the liquid feeding of the sample is started, and when the inlet sensor 14 detects the sample, the liquid feeding of the sample is stopped.
At this time, it is confirmed that the inlet sensor 14a is also detecting the sample. (B) Switching to the second flow path, restarting the sample feeding, feeding the sample into the sample loop 4, detecting the eluent by the outlet sensor 16, and then stopping the eluent detection, the sample feeding is stopped. Stop. (C) Switching to the first flow path again, the sample in the sample loop 4 is introduced into the separation column by the eluent.

In the case of the embodiment of FIG. 4, the amount of sample required for introducing the sample once is the volume of the flow path 26 between the sensors 14 and 14a of FIG. 4 (A), and specifically, the sample loop. 4 volume 20 μl, volume for one slit 0.3 μl, amount 2 μl of liquid advancing when liquid feeding is stopped, and amount 4 μl left for safety on the upstream side of sensor 14a, total 26.3 μl, of which 20 Since 0.3 μl is introduced into the separation column, the effective rate of the sample is 77%, which is a 37% improvement over the conventional direction of FIG.

FIG. 5 shows an embodiment of the invention in which a syringe is provided in the waste liquid flow path to control the flow rate when the sample is supplied to the sample loop 4. Reference numeral 30 is a reaction vessel of the peptide sequencer, and a solvent 32 is supplied to the reaction vessel 30 in order to dissolve the amino acid derivative cut out. The solvent 32 is supplied via the opening / closing valve 38 by the pressurized gas supplied from the gas supply unit 34 via the opening / closing valve 36. The sample solution in the reaction container 30 is supplied to the sample supply channel 6
The pressurized gas from the gas supply unit 34 is supplied to the reaction container 30 via the on-off valve 40 to be sent to the reaction container 3
The sample solution of 0 is supplied to the sampling valve 2 via the opening / closing valve 42. A branch channel is connected to the waste fluid channel 8 by a three-way valve 44, and a syringe 46 is connected to the branch channel. The syringe 46 is driven by the pulse motor 48.

The operation of the embodiment shown in FIG. 5 will be described. (A) The on-off valve 42 is opened, the reaction container 30 is pressurized with gas, the sample solution is supplied to the sample loop 4 of the sampling valve 2, the three-way valve 44 is set on the syringe 46 side, and the syringe 46 sucks it. The sample solution is introduced into the sample loop 4. At this time, the number of pulses of the pulse motor 48 is counted while the sample solution is passing through the inlet sensor 14. (B) On condition that the amount of liquid calculated from the count number of the pulse motor 48 exceeds a predetermined amount, the sampling valve 2 is switched when the outlet sensor 16 detects the liquid, and the sample measured in the sample loop 4 is sampled. Is introduced into the separation column.

If the amount of liquid passing through the inlet sensor 14 does not reach the predetermined amount when the sample passes through the inlet sensor 14, it means that the sample amount necessary for the sample loop 4 has not been collected. The syringe 46 is pushed back without switching the sampling valve 2, and the sample solution is returned to the reaction container 30. Then, the reaction container 30
Is dried once to volatilize the solvent, and then the solvent 3
2 is supplied to the reaction container 30 to dissolve again, and the sampling operation is repeated again.

In this embodiment, since the amount of liquid is measured by the syringe 46, the flow velocity can be accurately controlled, and the distance that the liquid travels after the outlet sensor 16 detects the liquid is not provided with the syringe 46. In the case of 10 mm, it can be reduced to about 3 mm (0.6 μl) in FIG. 5, and the amount of wasted sample can be reduced accordingly. Here, the amount of sample required for one sample introduction is 20 μl for the sample loop 4 and 0.6 μl for the slit 2 minutes (=
0.3 μl × 2), the capacity from the slit to the outlet sensor 16 is 4.9 μl, and the safety amount of 0.6 μl (= 0.3 μl × 2) on the upstream side and the downstream side is 26.7 μl in total. Since 20.3 μl of which is introduced into the separation column, the effective rate of the sample is improved to 76% or more.

Further, the motor 4 for driving the syringe 46
In order to reduce the predetermined amount of liquid by counting the number of pulses of 8 by the amount of 4.9 μl from the slit to the outlet sensor 16, after detecting the liquid with the outlet sensor 16, return the syringe by the number of pulses of 4.9 μl. Good. In this case, the amount of sample required for introducing the sample once is 20 μl for the sample loop 4 and 0.6 μl for the slit 2 minutes (=
0.3 μl x 2), and a total amount of 21.8 μl, which is a safety-friendly amount of 0.6 μl (= 0.3 μl x 2) on the upstream side and the downstream side.
Is. Since 20.3 μl of which is introduced into the separation column, the effective rate of the sample is improved to 90% or more. Further, in the embodiment of FIG. 5, since the liquid amount can be measured by the pulse number of the motor 48, it is not necessary to confirm the liquid by the inlet sensor 14, and the accurate volume setting from the sampling valve 2 to the inlet sensor 14 is set. Becomes unnecessary.

The use of the embodiment of FIG. 5 simplifies the structure for introducing a standard sample for confirmation of retention time on a liquid chromatograph. FIG. 6A shows a conventional method. In order to introduce the standard sample, a manual sampling valve 50 that is manually introduced into the eluent supply channel 10 is provided, the standard solution is collected in a sample loop 52 provided in the sampling valve 50, and the sampling valve 50 is switched. The standard sample is injected into the separation column via the sampling valve 2.

However, according to the embodiment of FIG.
The manual sampling valve 50 as shown in FIG. 6A becomes unnecessary, and it is only necessary to connect the standard sample injection line 56 to the sample supply channel 6 via the three-way valve 54 as shown in FIG. 6B. . Reference numeral 58 is a standard sample. This is because the syringe 46 is provided in the waste liquid flow path, and the sampling valve 2 is attached to the sampling valve 2 in FIG.
As shown in (B), switching to the second flow path, the three-way valve 54
The standard sample 58 is introduced into the sample loop 4 of the sampling valve 2 by suctioning with the syringe 46 in a state where the standard sample 58 is switched to the standard sample 58 side.

As described above, since the standard sample is injected infrequently, it is not necessary to attach a new sampling valve, and the structure is simple and the cost is low.
Further, it is possible to eliminate the relative error that occurs when the actual sample and the standard sample are quantified by different sample loops.

Further, according to the embodiment shown in FIG. 6 (B), the amount of the sample injected into the separation column can be easily measured and automation becomes possible. Next, a method of measuring the inner volume of the sample loop 4 will be described. (1) A non-volatile liquid (for example, water but other liquid may be used) is placed in a container (standard sample container) in which the standard sample 58 is placed. The three-way valve 54 is placed on the standard sample container side. (2) The non-volatile liquid contained in the standard sample container is sucked by the syringe 46. (3) When the inlet sensor 14 detects the liquid, the syringe 46
Count the number of pulses of the motor driving the. (4) The liquid fills the sample loop 4 and the outlet sensor 16
When it reaches, the counting is finished. The count number at this time is N1. (5) After returning the liquid into the standard sample container with the syringe 46, turn the sampling valve 2 clockwise by 60 °.
Rotate to switch channels. (6) The above operations (2) to (4) are repeated. However, at this time, the liquid reaches the outlet sensor 16 without passing through the sample loop 4. The count number at this time is N2. When N = N1-N2 is obtained, the amount N is the total of the volume of the sample loop 4 and the volume for one slit, and the sample volume actually injected into the separation column has been calculated.

The weighing accuracy of the embodiment shown in FIG. 6B depends on the linearity between the number of pulses of the motor and the movement of the syringe, but when a ball screw with a pitch of 1 mm is used to drive the syringe, The amount of liquid sent per pulse is 0.02 μl, and the error is small. In the above example of measuring the amount of sample injected into the separation column, the liquid contained in the standard sample container was used.However, if the washing liquid installed in the device is used, the amount of sample injected into the separation column The measurement is automated. However, in this case, the liquid that has filled the sample loop 4 in the above step (5) cannot be restored, and therefore the liquid is discharged to the waste liquid bottle by the gas.

[0028]

Industrial Applicability According to the present invention, it is possible to reduce a waste amount of a sample when introducing the sample into the liquid chromatograph.
If a syringe is provided in the waste liquid flow path to control the flow rate at the time of supplying the sample to the sample loop, it is possible to reduce the waste amount of the sample when introducing the sample. In this way, it is possible to reduce the wasted amount of the sample, so that it becomes possible to analyze the sample by concentrating it even more when a limited sample such as a peptide sequencer can be obtained, thus improving the sensitivity. To do. Also, with a peptide sequencer, if the amount of the solution becomes less than the specified amount due to bubbling when dissolving the amino acid derivative in the solvent, etc. When the amount becomes insufficient to the capacity of the sample loop, there is no means for returning the sample once supplied to the sampling valve to the original reaction container, and the measurement at that time is treated as an error. However, by providing the syringe in the waste liquid flow path, the sample solution supplied to the sample loop can be returned to the reaction container again by the syringe, so that the sample solution can be redissolved and introduced again into the sampling valve. Therefore, it is possible to effectively use the operation that has been conventionally processed as an error. Further, when introducing the standard sample, it is not necessary to separately provide a manual sampling valve having a sample loop, and the configuration is simplified.

[Brief description of drawings]

FIG. 1 is a diagram showing a sample introduction device to which the present invention is applied, (A) is a schematic flow path diagram, and (B) is a sectional view showing a liquid sensor portion.

FIG. 2 is a flow chart showing a conventional sample introduction method in the order of steps.

FIG. 3 is a flow chart showing the sample introduction method of the first embodiment in process order.

FIG. 4 is a flow chart showing the sample introduction method of the second embodiment in the order of steps.

FIG. 5 is a flow chart showing a third embodiment.

6A and 6B are flow charts showing a case where a standard sample is introduced. FIG. 6A is a conventional case, and FIG. 6B is a case of the embodiment of FIG.

[Explanation of symbols]

 2 Sampling valve 4 Sample loop 6 Sample supply flow path 8 Waste liquid flow path 10 Eluent supply flow path 12 Column flow path 14 Inlet sensor 16 Outlet sensor 20 Peptide sequencer 22 Eluent feed pump 24 High-performance liquid chromatograph 46 Syringe 48 Pulse motor

Claims (2)

[Claims] 1. A sampling valve is connected to a sample loop of a fixed volume, a sample supply flow path, a waste liquid flow path, an eluent supply flow path of a liquid chromatograph and a column flow path leading to a separation column, and sampling of the sample supply flow path. Use a sample introduction device that has an inlet sensor that detects liquid near the valve and an outlet sensor that detects liquid near the sampling valve in the waste liquid flow path. A first flow path that is directly connected to the flow path and an eluent supply flow path is connected to a column flow path via a sample loop, and a sample supply flow path is connected to a waste liquid flow path via a sample loop, In a method for introducing a fixed amount of sample into a liquid chromatograph by switching the flow path between the supply flow path and a second flow path directly connected to the column flow path. Sample introduction method characterized by performing the following steps (A) to (C) in this order. (A) The liquid feeding of the sample is started in the first channel, and when the inlet sensor detects the sample, the liquid feeding of the sample is stopped. (B) Switching to the second flow path to restart the liquid feeding of the sample to feed the sample into the sample loop, and after the outlet sensor detects the eluent, when the eluent detection is interrupted, the liquid feeding of the sample is stopped. . (C) Switching to the first flow path again, the sample in the sample loop is introduced into the column by the eluent. 2. The sampling valve is connected to a sample loop having a constant volume, a sample supply flow path, a waste liquid flow path, an eluent supply flow path of a liquid chromatograph, and a column flow path leading to a separation column, and by switching the sampling valve. A first flow path in which the sample supply flow path is directly connected to the waste liquid flow path and the eluent supply flow path is connected to the column flow path through the sample loop, and the sample supply flow path is the waste liquid through the sample loop In a sample introduction device for introducing a fixed amount of sample into a liquid chromatograph by switching the flow path between a second flow path connected to the flow path and an eluent supply flow path directly connected to the column flow path A branch channel is provided in the waste fluid channel through a three-way switching valve, and a syringe driven by a motor is connected to the branch channel, and the sample is sampled in the second channel. Sample introduction device, characterized in that controlled by the syringe liquid transfer rate of the time of feeding the Rurupu.