JPS6250659A - Introducing method for sample into liquid chromatography - Google Patents

Abstract

PURPOSE:To reduce the loss at the time of sample injection and to inject the sample into a column by sandwiching the specific amount of sample between air bubbles when sucking the sample into a sample intake passage and filling only the sample in a sample loop, and then injecting the sample into the column. CONSTITUTION:The flow passage connecting with a cleaning pump 22 from the tip of a sample suction stylus 30 through a six-port valve 10 and a three-way valve 12 is filled with cleaning liquid for cleaning before the sample is sucked in. The sample stylus 30 is inserted into a sample bottle 44 and the sample a little bit more than the amount V0 of sample injected into the column is sucked. The stylus 30 is extracted from the bottle 44 and air 54 is sucked through a syringe 18 for measurement until the tip of the sample 50 reaches the X point of the port 1 of the valve 10. The valve 10 is rotated counterclockwise to link ports 2, 3 and 4, and 5 and 6 mutually, and the sample is sucked into the sample loop 32 by the amount V0 through the syringe 18. The valve is rotated clockwise to link ports 1 and 2, 3 and 4, and 5 and 6 and the sample in the loop 32 is injected into the column 34 through a return pump 40.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for introducing a sample into a liquid chromatograph using a sample introduction device.

(Prior Art) FIG. 1 shows an example of a sample introduction device for a liquid chromatograph.

10 is a six-port valve as a switching valve. A flow path 14 or 16 is connected to port 1 of the six-port valve IO via a three-way valve 12. The flow path 14 is connected to a metering syringe 18, and
It is connected to a cleaning pump 22 via a solenoid valve 20.

24 is a cleaning liquid.

The other flow path 16 connected to the three-way valve 12 is the check valve 2.
6 and is led to the drain.

A sample suction channel 28 is connected to port 2 of the six-port valve 10, and a sample suction needle 30 is provided at the tip of the sample suction channel 28. Port 3 of six boat valves 10
A sample loop 32 is connected between and port 6. In addition, column 3 is connected to port 4 of six-port valve 10.
4 is connected, and a detector 36 is provided at the outlet of the column 34. A liquid feeding pump 40 supplies the mobile phase in the mobile phase reservoir 38 to port 5 of the six-port valve IO.
is connected. A resistance pipe 42 is provided between ports 4 and 5 to alleviate pressure changes when switching the six-boat valve. 44 is a sample bottle, and 46 is a washing port.

To introduce a sample into the column 34 using this sample introduction device, first, the six-boat valve 10 and the three-way valve 12 are set to the state shown in the figure, and the sample suction needle 30 is inserted into the cleaning port 46.
The electromagnetic valve 20 is opened and the cleaning pump 22 is operated to clean the flow path.

Next, after the electromagnetic valve 20 is closed, the sample suction needle 30 is inserted into the sample bottle 44, and the sample is aspirated by the measuring syringe 18. At this time, the unexpanded portion of the tip of the aspirated sample reaches port 1 of the six-port valve 10.

After the six-port valve 10 is rotated counterclockwise and ports 2 and 3, ports 4 and 5, and ports 6 and 1 are electrically connected,
A sample is aspirated into the sample loop 32 by the metering syringe 18 in an amount to be introduced into the column. When the sample is drawn into the sample loop 32, the six-port valve 1
0 is rotated clockwise to connect ports L and 2 as shown.
, ports 3 and 4, and ports 5 and 6 are switched to conduction, and the sample in the sample loop 32 is swept away by the mobile phase from the liquid pump 40 and introduced into the column 34.

The flow path 16 is used, for example, when the set sample suction amount is greater than the suction amount of one stroke of the metering syringe 18. In that case, the three-way valve 12 is temporarily connected to port 8.
After switching to the position where ports 7 and 9 are electrically connected, the liquid in the measuring syringe 18 is discharged to the drain, and the three-way valve 12 is again switched to the position where ports 7 and 8 are electrically conducting, and the sample is aspirated by the measuring syringe 18. will be carried out.

(Problems to be Solved by the Invention) When the sample introduction device shown in FIG. 1 is operated as described above,
The sample sucked into the sample suction flow path from the tip of the sample suction needle 30 to port 1 of the six-boat valve 10 is discharged from the cleaning port 46 to the drain before the next sample introduction, resulting in sample loss.

By the way, in recent years in the field of liquid chromatography, there has been a strong demand to introduce the entire amount of a precious sample, such as a biological sample, into a column because the detection sensitivity is insufficient. ing. However, as described above, conventional sample introduction methods involve a large amount of loss during sample injection, and cannot meet these requirements.

Also, when introducing the sample into the column, it is necessary to prevent bubbles (air) from entering the column along with the sample.
When an ultraviolet spectrophotometer is used as a detector for a liquid chromatograph and the detection wavelength is set to a short wavelength (for example, 210 nm), an air peak appears on the chromatogram. In the chromatogram shown in Figure 2, peak P+ is benzene.

p= is naphthalene, P3 is diphenyl, P4 is solvent, P
a is air. The numbers in the figure are retention times. If air bubbles enter the column in this way, it becomes impossible to identify or quantify sample components whose retention times in the chromatogram are close to those of air.

An object of the present invention is to provide a sample introduction method for introducing a sample into a column while minimizing loss during injection into a liquid chromatograph.

(Means for Solving the Problems) The present invention provides a sample suction channel (
28) and the measuring syringe (18), the connection of the sample suction channel (28), the sample loop (32) and the measuring syringe (18), and the mobile phase channel (37) and the sample loop (32). and a sample introduction method using a sample introduction device in which the connection of the column (34) is switched by a switching valve (10) and the sample is introduced into the column (34), the sample (50) being introduced into the sample suction channel (28). ), air bubbles (52°54) are placed before and after a predetermined amount of sample (50).
In this method, only the sample (50) is filled into the sample loop (32), and then the sample (50) is introduced into the column (34).

(Example) An example will be explained in order of steps using the sample introduction device shown in FIG.

The channel is cleaned before the sample is aspirated. Therefore, the six-port valve IO and the three-way valve 12 are in the state shown in the figure, and the flow path leading from the tip of the sample suction needle 30 to the cleaning pump 22 is filled with cleaning liquid.

(1) Before the sample suction needle 30 is inserted into the sample bottle 44,
Air 52 (volume 2 to 3 μQ) with measuring syringe 18
is attracted.

(2) The sample suction needle 30 is inserted into the sample bottle 44, and an amount slightly larger than the sample amount VoμQ introduced into the column (Vo+
2α) A sample of μQ is aspirated.

(3) The sample suction needle 30 is pulled out from the sample bottle 44, the air 54 is aspirated by the measuring syringe 18, and the tip of the sample 50 is inserted into the measuring syringe 1 of port 1 of the six-port valve 10.
It is sucked until it reaches the X point of the 8th tsuba. The internal volume of the flow path from the exit point Y of port 1 of the six-port valve 10 to the point X is αμQ.

(4) In this state, the six-port valve 10 is rotated counterclockwise, and ports 2 and 3. Ports 4 and 5, Port 6
and 1 are brought into conduction, and the metering syringe 18 aspirates into the sample loop 32 an amount vOμΩ of sample to be introduced into the column.

(5) When the six-port valve 10 is rotated clockwise and ports 1 and 2, ports 3 and 4, and ports 5 and 6 are brought into conduction, the sample in the sample loop 32 is transferred to the liquid sending pump 4.
It is forced out by the mobile phase from zero and introduced into the column 34.

(6) The sample suction needle 30 is inserted into the cleaning boat 46, and the sample 2αμQ and air at the front and rear ends of the sample are washed away by the cleaning liquid supplied by the cleaning pump 20 by opening the solenoid valve 20. It will be done.

The above operations can be performed automatically under control from the controller by measuring the volume from the tip of the sample suction needle 30 to the outlet Y point of the port 1 of the six-boat valve 10 in advance.

In the above embodiments, a six-port valve is used as the switching valve, but the invention is not limited to this.

(Effects of the Invention) According to the conventional sample introduction method, the flow path volume from the tip of the sample suction needle to the switching valve results in sample loss. However, according to the sample introduction method of the present invention, regardless of the size of the flow path volume from the tip of the sample suction needle to the switching valve,
Sample loss (2α) during sample injection can be reduced to 1 to 2 μQ. Therefore, it is very effective for introducing valuable biological samples into columns that are often used in the field of liquid chromatography.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing an example of a sample introduction device for a liquid chromatograph in which the method of the present invention is carried out, and FIG. 2 is a diagram showing a liquid chromatogram in which air is also injected. 10...Six-port valve as a switching valve, 18...Measuring syringe, 28...Sample suction channel, 32...Sample loop, 34 ... Column, 37 ... Mobile phase channel, 50 ... Sample. 52.54...Bubble.

Claims (1)

[Claims] (1) The connection between the sample intake channel and the metering syringe, the connection between the sample intake channel, sample loop, and the metering syringe, and the connection between the mobile phase channel, sample loop, and column are switched by the switching valve to the column. When sucking the sample into the sample suction channel using a sample introduction device into which the sample is introduced, a predetermined amount of the sample is sandwiched between the front and back sides of air bubbles, and only the sample is filled into the sample loop, and then the sample is transferred to the column. A method for introducing a sample into a liquid chromatograph, characterized by introducing.