JPH0556467B2 - - Google Patents

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 using 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 most conventional liquid chromatographs, the eluent supply flow rate was 1 ml/min or more, so it was relatively easy to mix two eluents and perform gradient elution. That is, conventionally, a mixing chamber 3 as shown in FIG. 1 was provided in a mixer section of a gradient device. This mixing chamber 3 has a cylindrical shape, and the inside thereof is filled with glass balls 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, by making the flow pipe and separation column thinner,
When trying to perform gradient elution using a mixing chamber as shown in Figure 1 to perform so-called semi-microchromatography by setting the flow rate of the mixed eluent to a very small amount, for example, 0.1 ml/min, multiple liquids are Not mixed on the street. For example, change the flow rate to 0.1ml/
Even if you try to change the mixing ratio of liquids A and B step by step as in the case of Figure 7, the gradient curve will not match the set conditions as shown in Figure 8. do not have. This is thought to be due to the fact that both liquids are remixed in the glass ball type mixer.

[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 depending on the amount or nature of the eluent.

[Summary of the invention]

In the present invention, the flow path inlet and flow path outlet of the mixing section forming member that holds the mixing section can be fitted so that the mixing section can be connected in a chain between the confluence section of a plurality of eluents and the separation column. The above-mentioned
By providing a porous rigid body within the mixing section, the eluent can be rapidly mixed with the porous rigid body, and furthermore, remixing can be prevented.

[Embodiments of the invention]

According to a preferred embodiment of the present invention, the eluent is sufficiently mixed depending on the amount and properties of the eluent;
A stable mixed state is obtained.

FIG. 2 is a diagram illustrating the overall configuration of an embodiment of the present invention. Eluents 1A and 1B are pumped to pump 2A.
and 2B through the confluence 11 into the mixing chamber 3, respectively.
The liquid is sent to 1. 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. Then, it is detected by a photometer detector 6 equipped with a flow cell, and recorded by a recorder 7. The size of the separation column is 1 in diameter.
mm, length 20cm.

In this embodiment, the mixing section 31 is constructed as shown in FIG. That is, the mixing portion forming member 33a has a height of 17 mm and a porous element 10 with a diameter of 10 mmφ. 14 and 15 are Patsukin. This is an essential configuration in an embodiment described later in which the volume of the porous element 10 is configured to be cumulatively variable.

The porous rigid body is not limited to the shape described above, and various materials such as sintered alloys, porous glass filters, and ceramics can be used. The porous element may have a pore diameter of 1 to 10 μm, but is not limited to this size. When selecting various eluent flow rates, the flow path system, pump capacity, pore diameter and volume of the porous element, etc. are selected depending on the flow rate.

FIG. 4 shows an example of a gradient curve in which the mixing ratio is changed stepwise according to this embodiment. Very responsive to setting conditions, with a flow rate of 0.1ml/
It was found that no remixing occurred even at a flow rate of

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. FIG. 5 shows an example of three arrangement of the mixing section forming members 33. However, since only the mixing part forming member 34 disposed at the top has a flow path introduction part from the confluence point 11, its shape is different from the other mixing part forming members 33 as shown in the figure.
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 and 15 are Patsukin. body 3
5 is attached to a plate 18 with screws 19, and mixing members 34, 33a and 33b are attached to this body 35 with gaskets 16 to maintain airtightness. Incidentally, in the example shown in Fig. 5, the capacity of the mixing chamber is 150μ in the case of a single arrangement, but 450μ in the case of a triple arrangement. Increasing the flow rate of the eluent can be handled 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 has an inner diameter of 1.2 mmφ and a length of 150 mm.
Eluent A was 0.1% TFA, and eluent B was a mixture of isopropanol and CH ₃ CN.
%, TFA 0.1%, and water 19.9%. The detector is an ultraviolet photometer and the measurement wavelength is 220n.
It is m. The flow rate of the mixed eluent was 0.1 ml/min.
It is understood that gradient elution can be performed under a micro flow rate with sufficient measurement accuracy.

〔Effect of the invention〕

According to the present invention, it is possible to flexibly respond to changes in the amount of eluent and perform gradient solutions with high precision, making it possible to perform gradient operations in so-called semi-microchromatography and microchromatography. Enables short-time analysis.

[Brief explanation of the drawing]

Fig. 1 is a diagram for explaining the mixing section of a conventional gradient device, Fig. 2 is a schematic diagram of the overall configuration of an embodiment of the present invention, Fig. 3 is a diagram of a single arrangement of porous elements, and Fig. 4 5 is a diagram showing a gradient curve obtained based on the example shown in FIG. 2, FIG. 5 is a diagram showing a triple arrangement of porous elements, FIG. The figure shows a gradient curve for a relatively large volume using the conventional method, and FIG. 8 shows an example of a gradient curve when the conventional mixing chamber is applied to a minute flow rate. 1A, 1B...Eluent, 5...Column, 6...
Detector, 10... Porous element, 11... Merging point, 31, 32, 33a, 33b, 34... Mixing part forming member.

Claims (1)

[Claims] 1. Flow channels that send multiple eluents are merged at a confluence section, a mixing section is placed in the confluence channel led from the confluence section, and the mixed eluent from the mixing section is passed through a separation column to a detection section. In the liquid chromatograph, the mixing section includes a mixing section forming member having a porous element storage chamber containing a porous element, and a body section that fitably receives a downstream end of the mixing section forming member. A recess is formed in the upstream end of the mixing part forming member into which the downstream end of another mixing part forming member having the same shape as the mixing part forming member can be fitted in a liquid-tight manner, and The upstream end and the downstream end of the part forming member are communicated by a flow path passing through the porous element storage chamber, and the body part is provided with the liquid that has passed through the porous element storage chamber for separation. A microflow liquid chromatograph characterized in that a flow path is formed to allow flow toward a column.