JPS60183554A - Microflow liquid chromatograph - Google Patents

Abstract

PURPOSE:To effect gradient elution with good responsiveness even if the flow rate of eluates are as slight as <=1ml/min by joining the plural eluates then passing the joined eluate to a mixing chamber having a porous rigid body and detecting the same with a liquid chromatograph. CONSTITUTION:For example, two kinds of eluates 1A, 1B are joined 11 by pumps 2A, 2B and are fed to a mixing chamber 31. The eluate is then fed to a sample 4 and a sample is introduced therein. The sample is fed by the eluate to a sepn. column 5 and is subjected to a gradient elution. The separated sample is detected 6 and the liquid chromatogram is measured. An empty chamber 40 is provided between the inlet member 35 and outlet member 37 of the chamber 31 and, for example, a stainless steel porous element 10 is put therein. The pore size of the element is made about 5mum. Since the porous rigid body is provided in the mixing chamber, the accuracy of the gradient elution is increased even if the flow rate of the eluate is as slight as <=1ml/min. The measurement in short time is thus made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a liquid chromatograph, and particularly to a liquid chromatograph equipped with a gradient device suitable for minute flow rates.

[Background of the invention]

When separating and analyzing biological samples and the like by liquid chromatography, it is often beneficial to use a gradient technique. A gradient device changes the mixing ratio of eluents with different compositions over time and supplies them to a separation column, and is used to quickly separate mixed components that are difficult to separate using the same eluent. .

In conventional liquid chromatographs, the supply flow rate of the solution 1f solution was 1rnt, and most of them were over 7 minutes.
It was relatively easy to perform gradient elution by mixing two eluents. In other words, conventionally, a mixing chamber 3 as shown in FIG. 1 was provided in the mixer section of a gradient device. This mixing chamber has three cylindrical shapes, and the inside thereof is filled with glass poles 8. When such a mixing chamber 3 is provided and the liquid is fed at a flow rate of 2 ml/min and a gradient curve is obtained, a result that follows the set conditions as shown in FIG. 8 is obtained.

However, if the flow tube and column are made thinner,
When trying to perform gradient elution using a mixing chamber as shown in Figure 1 to perform so-called semi-micro chromatography by setting the flowmeter of the mixed eluent to a micrometer, for example, 0.1 me for 7 minutes, multiple The liquids are not mixed as per the settings. For example, even if you set the flow rate to 0.1 ml for 7 minutes and try to change the mixing ratio of liquids A and B step by step as in the case of Figure 8, the gradient curve will not change as shown in Figure 9. I can't get what I want. This is considered to be because in the glass pole type mixer, both liquids are remixed.

[Purpose of the invention]

An object of the present invention is to provide a microflow liquid chromatograph that can perform gradient elution with good responsiveness to set conditions even when the eluent flow rate is 1 rneZ minute or less.

[Summary of the invention]

In the present invention, a mixing section is provided between a confluence section of a plurality of eluents and a separation column, and a porous rigid body is provided within this mixing section, so that rapid mixing of eluents can be achieved using this porous rigid body. According to a preferred embodiment of the present invention, the two liquids are sufficiently mixed even at a minute flow rate of about 0.1-min, and a stable mixing state can be obtained. . Furthermore, since the dead volume inside the mixing section is small, the residence time or delay time for the liquid entering the mixing section to exit the mixing section can be minimized. Figure 2 is a diagram illustrating the overall configuration of one embodiment of the present invention. be. Eluents IA and IB are sent to mixing chamber 31 via confluence 11 by pumps 2 and 2B, respectively. The eluent mixed in the mixing chamber 31 is sent to the separation column 5. Now, when the sample is introduced into the channel by the sampler 4, it is sent to the column 5 using a gradient mixed eluent and separated into each component. It is then detected by a photometer detector 6 with 70 cells and recorded by a recorder 7. The size of the separation column is 1 rrvn in diameter.

The length is 20cm.

In this embodiment 2, the mixing section 31 is constructed as shown in FIG. That is, the member 3 having the inlet passage 38
5 and a member 37 having an exit passage 39, forming a chamber;
In this part, a cylindrical element 10 made of a porous rigid body is housed indoors.

This element 10 has a diameter of Low and a thickness of 3 mm, is formed by sintering stainless steel, and has a pore diameter of about 5 μm. This element 10 can be effectively used for electrical distribution with an eluent flow rate of at least 0.01 rne/min or more.

The porous rigid body is not limited to the shapes described above,
- MAZOKO materials include various sintered alloys, porous glass filters,
Ceramics etc. can be used. The porous element may have a pore diameter of 1 to 1-0 μm, but is not limited to this size. When selecting various eluent flow rates, the flow path system, pump capacity,
The pore size and volume of the porous element are selected.

FIG. 4 shows an example of a gradient curve in which the mixing ratio is changed stepwise according to this embodiment. It was found that the response to the set conditions was very good, and no remixing occurred even at a flow rate of 0.1 m67 min.

When changing the flow rate of the eluent, a plurality of porous elements may be arranged in series in the same flow path to change the volume within the mixing section. Figure 5 shows such an example, (4)
is a single shape, and [F]) is an example of a double arrangement. In FIG. 5, the mixing member 33 has a height of 17 mm and a porous element 10 with a diameter of 10 mmφ. 14.15 is Patsukin. The body 35 is attached to the plate 18 with screws 19, and the mixing members 34, 33a and 33b are attached to the body 35 with gaskets 16 so as to maintain airtightness. Incidentally, in the example shown in FIG. 5, the capacity of the mixing chamber is 150 .mu.t in the case of a single arrangement, but 450 .mu.t in the case of a three-speed arrangement. Increasing the flow rate of the eluent can be achieved simply by increasing the number of porous elements.

FIG. 6 is a diagram showing an example of a chromatogram when a protein sample is subjected to gradient elution using the present invention. The separation column used was one with an inner diameter of 1.2 mm and a length of 150 mm.
Eluent A & 0.1% TFAX Eluent B contained a mixture of 80% Improper Tool and CH3CN, 0.1% TFA, and 19.9% water. The detector is an ultraviolet photometer and the measurement wavelength is 220 nm. The flow rate of the mixed eluent was 0.1me1 minute. It is understood that gradient elution can be performed at a minute flow rate with sufficient measurement accuracy.

FIG. 7 is an explanatory diagram of a mixing section used in another embodiment of the present invention, in which a mixing chamber 32 formed by a member 71 and a member 72 is wedge-shaped and gradually narrows at the inlet and outlet sides. shows. The porous element 9 is made according to the shape of this room. In such an example, dead volume within the mixing chamber can be further eliminated, and even a trace amount of eluent can be mixed sufficiently.

〔Effect of the invention〕

According to the present invention, even if the eluent has a minute flow rate, it is possible to perform a gradient solution with high precision, making it possible to perform gradient operations in so-called semi-micro chromatography and micro chromatography, making it possible to perform gradient operations even more quickly. Allows time analysis.

[Brief explanation of the drawing]

FIG. 1 is a diagram for explaining the mixing section of a conventional gradient apparatus, and FIG. 2 is a schematic diagram of the overall configuration of an embodiment of the present invention.
Figure 3 is an explanatory diagram of the mixing section in the embodiment shown in Figure 2;
The figure shows an example of a gradient curve obtained based on the implementation sequence of Fig. 2, and Fig. 5 shows an example of the arrangement of porous elements.
(E) shows a single shape, [Yes]) shows a double series, FIG. 6 shows an example of a chromatogram to which the present invention is applied, and FIG. 7 shows another embodiment of the present invention. An explanatory diagram of the mixing section in the example, Figure 8 is a diagram showing an example of a gradient curve in the case of a relatively large volume when using the conventional method, and Figure 9 is a diagram showing an example of the gradient curve when the conventional mixing chamber is applied to a minute flow rate. It is a figure showing an example of a gradient curve. IA, IB... Eluent, 5... Column, 6... Detector, lO... Porous element, 11... Confluence point, 31° force 1 Figure 2 Figure 3 Figure 1 4th - 5th Snake (A) (β) 5th Snake Figure 7

Claims (1)

[Scope of Claims] 1. In a liquid chromatograph in which a plurality of eluents are fed, combined at a confluence section, and distributed to a detection section via a separation column, the amount of liquid sent after the above-mentioned confluence is 1 m7! 1. A microflow liquid chromatograph having a microflow rate of less than /min, characterized in that a mixing section is provided between the merging section and the separation column, and a porous rigid body is provided within the mixing section. 2. The liquid chromatograph according to claim 1, wherein the mixing section includes a plurality of porous rigid bodies arranged in series.