JP2001318087A - Column for liquid chromatography and its using method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To elute a constituent having large affinity with performance equal to that of an HPLC column in gravitating type chromatography. SOLUTION: A column 1 is used which is equipped with a pair of inner tubes 3 one end side of each of which is slidingly inserted into an outer tube 2 from either end of the outer tube 2 and which can be fixed to the outer tube at arbitrary insertion positions. A filler 11 is put in the outer tube 2 and end parts of the inner tubes 3 are brought into close contact with the both ends of the filler 11. Then, the separation and recovery of a mixture are conducted by the gravitating type chromatography. Thereafter, the column 1 is reversed upside down to elute the constituent having large affinity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a column for liquid chromatography capable of efficiently separating and purifying a plurality of substances by gravity flow type liquid chromatography using a column, and a method for using the same.

[0002]

2. Description of the Related Art In chromatography, a sample mixture placed at one end of a stationary phase (packing layer) is moved by an appropriate mobile phase (for example, a solvent), and a moving speed is determined based on a difference in adsorptivity and a distribution coefficient of each component. This is a technique that uses the difference to separate components from each other. Chromatography is widely used as a means for separating and purifying substances. In liquid chromatography, a column packed with a packing material is used, the addition of solvent is continued, the adsorption band of each component is lowered, and development is performed to better separate the components. Separately outflow from the weaker components. In this case, if the affinity of the substance intended for separation and recovery with the column packing material is too large, a large amount of elution solvent (developing solvent) will be used for elution of the component.
In some cases, making it difficult to recover the target substance. In such a case, it was necessary to judge that the filler could not be applied.

[0003] This will be described with reference to FIGS. FIG. 3 is a side view of a conventional column for liquid chromatography. The column 20 is a straight tube filled with the packing material 11 and has a cock 8 at the lower end. The sample loading and the elution solvent (developing solvent) 13 are added from above. In the open column 20 shown in FIG.
m of filler are used. 4 (a) to 4 (e) are explanatory diagrams showing a method of using the same, which will be described as follows.

(A) At the bottom of a column 20, a porous glass 2
1 and the column 20 is filled with the filler 11 together with the elution solvent.

(B) The sample 12 is loaded, and the elution solvent 13 is added from above.

(C) The components in the sample are sequentially captured by the filler 11 like the components 12a, 12b, and 12c in accordance with the affinity for the filler.

(D) The elution solvent 13 is added from above and allowed to flow out from the lower end of the column. The elution solvent 13 elutes components trapped in the filler 11 and passes through the filler 11, and the passed component 12 c is collected in the pan 14.

(E) When the operation is further continued, component 12b
Is also collected in the pan 14. The component 12a having a high affinity for the filler is hardly eluted, and it is difficult to recover the eluted component remaining in the filler 11.

As shown in the "Manual for Standard Measurement and Analysis of Dioxins in Waste Disposal" (Environmental Improvement Division, Water Environment Department, Health and Welfare Bureau, Ministry of Health and Welfare, March 1997), cleanup for dioxin analysis is always carried out. In the method, after performing a sulfuric acid treatment, silica gel chromatography is performed, and then alumina chromatography is performed. If cleanup is not enough in these series of processes,
Further, it is usual to perform a cleanup using a carbon-based column. As the method, the manual describes a treatment method by HPLC (high performance liquid chromatography) using a graphite carbon packing column. The HPLC column is commercially available as a closed type packed with a packing material having a particle size of several μm in a very dense state.
It has a pressure-resistant structure made of stainless steel or the like that elutes at a high pressure of about MPa. In the HPLC method, the flow direction of the elution solvent is reversed during the column operation to recover dioxins, but this is for eluting highly chlorinated dioxins having high affinity for the packing material. HPLC
The method using the method requires a dedicated apparatus for that purpose, and the column is expensive.

[0010]

In the case where there is the above problem, since the component having a high affinity for the packing material is retained at the upper end of the column, the elution solvent is reversed (from bottom to top). It is thought that the components at the top of the column can be eluted and recovered without using a large amount of the elution solvent by flowing. But,
If this is done with a normal gravity flow column, air bubbles are mixed into the column packing material and the packing material is disturbed, so that column operation is practically impossible.

According to the present invention, in a gravity-flow chromatography, the chromatography is carried out by reversing the flow direction of the eluting solvent without causing air bubbles to enter the column packing and disturbing the packing as described above. And a method for using the same.

[0012]

In order to solve the above problems, it is necessary to create a mechanism that allows the elution solvent to flow in the reverse direction while maintaining the form of chromatography in which the elution solvent flows in the direction of gravity. is there. At this time, it is necessary to prevent bubbles from being mixed or disturbance of the column packing. Also, the column must be easy to clean and refill the column packing material. As a result of intensive studies, the inventor
A column capable of performing the above operations has been developed, and the present invention has been completed.

[0013] The present invention is a column used for gravity flow type liquid chromatography, comprising an outer cylinder, and an inner cylinder having one end inserted into the outer cylinder from both sides of the outer cylinder. It is a column for liquid chromatography.
It is preferable that one or both of the inner cylinders is provided with a fixing means that can be fixed at an arbitrary insertion position so that the method can be applied when the size of the filler is not constant.

In the method of the present invention, the column for liquid chromatography of the present invention is filled with a chromatography filler in an outer cylinder, and the ends of the inner cylinder are brought into close contact with both ends of the filler. A method for using a column for liquid chromatography, wherein separation and recovery of a mixture are carried out by gravity flow type chromatography using, and elution is performed by inverting the column upside down.

[0015]

Embodiments of the present invention will be described below with reference to the drawings. The column 1 of the example is shown in FIG.
The column 1 of the embodiment includes an outer cylinder 2 and a pair of inner cylinders 3. The outer cylinder 2 and the inner cylinder 3 are made of glass, and the inner surface of the outer cylinder 2 and the outer surface of the inner cylinder 3 are machined into an injection cylinder to form the outer cylinder 2.
To allow the inner cylinder 3 to slide on the inner surface. The inner tube 3 is provided with a thin tube 6 in the center longitudinal direction. Screw threads are provided outside the upper and lower ends of the outer cylinder 2 so that the inner cylinder 3 can be fixed at an arbitrary position by the screw lid 4 and the seal ring 5.

The seal ring 5 has a trapezoidal cross section as shown in the figure, and fixes the inner cylinder 3 to the outer cylinder 1 by tightening the screw cap 4. The seal ring 5 is preferably made of polyfluorinated ethylene or silicone rubber, but may be made of any other material as long as there is no deterioration or elution due to the solvent used. The screw cap 4 is preferably made of a hard resin, for example, polyfluoroethylene, polyethylene, or the like. The outer end of the inner cylinder 3 has a common fitting or transparent fitting structure so that it can be connected to the cock 8 or the solvent reservoir 9. There are no particular restrictions on the dimensions of these column components, and they may be arbitrarily determined depending on the amount of the filler used, the purpose of chromatographic separation, and the like.

The actual use of the column 1 according to the invention is as follows. First, preparation for column assembly is performed. The inner cylinder 3 is fixed to an appropriate position below the outer cylinder 2 with a seal ring 5 and a screw lid 4. A liquid-permeable stopper is provided on the distal end portion 7 of the central thin tube of the inner cylinder 3 with glass wool, absorbent cotton or the like. This is to prevent the chromatography packing 11 from leaking. Alternatively, the tip 7 may be made of porous glass. The two-way cock 8 is connected to the lower part of the inner cylinder 3, but the inner cylinder 3 and the cock 8 should be fixed with a clamp or the like so that the inner cylinder 3 and the cock 8 do not come off during operation. Is preferred. The upper part of the outer cylinder 2 is left open without attaching the inner cylinder 3.

Next, the filling of the filler 11 will be described. After an appropriate amount of the solvent is previously filled in the outer cylinder 2, the chromatography filler slurry is filled from the upper part of the outer cylinder 2. At this time, the lower part and the upper part of the filler 11 may be filled with sea sand, sodium sulfate, or the like.
After filling a predetermined amount of the filler 11, it is allowed to stand, and when the filler 11 has sufficiently settled, the upper inner cylinder 3 is left in a state where the solvent is left at the top.
Is inserted until its tip touches the upper end of the filler 11,
The upper inner cylinder 3 is fixed with a screw lid 4 and a seal ring 5.
A liquid permeable stopper made of glass wool, absorbent cotton, or the like is provided at the tip 7 of the inner cylinder 3. Alternatively, the tip 7 may be made of porous glass.

Next, sample addition and elution operations are performed.
The column chromatography operation in the forward direction can be performed in the same manner as ordinary gravity-flow chromatography. That is, a sample solution is added from the upper part of the inner cylinder 3 and washed with a small amount of a solvent, and then an elution solvent is flowed to separate and separate each component. At this time, a solvent reservoir 9 shown in FIG. 2 can be connected to and used above the upper inner cylinder 3. In order to perform elution in the reverse direction, the lower cock 8 is closed after completion of the elution in the forward direction, and if the solvent reservoir 9 is used, it is removed. The same thing as the cock 8 is connected to the fitting part on the upper part of the cylinder 3.

With the upper and lower cocks closed, the entire column is turned upside down, the cock 8 which is to be newly positioned at the top is removed, and a solvent reservoir 9 is connected and eluted instead. Elution can be performed.

FIGS. 5 (a) to 5 (d) are process diagrams schematically showing the method of using the column for liquid chromatography according to the present invention. (A) Using the column 1 of the present invention, the elution solvent is allowed to flow down from above as indicated by the arrow 15. (B) The component 12b with a low affinity flows out of the system as indicated by an arrow 16, but the component 12a with a high affinity is
1 remains on top. (C) Turn the column 1 upside down and let the elution solvent flow down.
At this time, the component 12 a having a high affinity is located at the lower end of the filler 11. (D) The component 12a having a high affinity can be easily eluted. (Example 1) As an example, application of dioxin in soil to pretreatment for analysis will be described. Weigh 10g of soil precisely,
After performing extraction, sulfuric acid treatment and silica gel chromatography treatment according to a conventional method, the silica gel chromatography eluate was concentrated to several ml to obtain a concentrate (A). On the other hand, the column 1 for liquid chromatography of the example of the present invention shown in FIG.
mm), and 1.0 g of graphite powder was wet-filled with hexane. Above and below the graphite layer, anhydrous sodium sulfate was filled to a height of about 1 cm. The concentrated solution (A) was loaded on the column 1 and washed with a small amount of hexane. Here, alumina chromatography was not performed after silica gel chromatography, and the process was shifted to graphite column processing.

First, elution was carried out with 50 ml of hexane.
Fractions were obtained. Next, the top and bottom of the column were inverted as described above, and eluted with 150 ml of toluene to obtain a second fraction. The second fraction contained dioxins, and this fraction was concentrated, and dioxins were separated and quantified by gas chromatography-mass spectrometry. Dioxin concentration (pg-T
The analysis result of (EQ / g) was equivalent to the conventional method as follows.
Here, the ordinary method is based on the analysis method performed in accordance with the above-mentioned "Manual for Standard Measurement and Analysis of Dioxins in Waste Disposal" (Environmental Improvement Section, Water Supply Environment Department, Ministry of Health and Welfare, March 1997).

Example Measured value: 33 (pg-TEQ / g) Standard method Measured value: 30 (pg-TEQ / g) As described above, conventionally, as an effective cleanup method for dioxins analysis, HPLC (high performance liquid chromatography) using a graphite carbon packing column has been used. In the HPLC method, in order to elute highly chlorinated dioxins having high affinity for the packing material, the flow direction of the elution solvent was reversed during the column operation. To use the HPLC method, a dedicated apparatus is required, and the HPLC column is expensive.
By using the column of the present invention, it has become possible to carry out the same operation as the above-mentioned HPLC method by gravity-flow chromatography with low cost and simple operation without using a special apparatus.

In the embodiment, for both of the inner cylinders,
A liquid chromatography column with a structure that can be fixed at any insertion position has been described. However, a liquid chromatography column with a structure where one inner tube has a fixed insertion position and the other inner tube can be fixed at any insertion position. Has the same effect. Further, if the size of the filler is constant, the same effect can be obtained by using a liquid chromatography column having a structure in which the insertion positions of both inner cylinders are fixed.

[0025]

According to the present invention, it is possible to easily reverse the direction in which an elution solvent flows in gravity-flow chromatography, and a substance having a strong affinity for chromatography packing, which has been difficult in the past. Can be collected. In addition, unlike HPLC, which requires a dedicated apparatus and a column is expensive, the use of the column of the present invention allows the packing material to be disposable, so that the reliability of analysis can be improved. INDUSTRIAL APPLICABILITY The present invention can be used, for example, in a pretreatment process for analyzing dioxins contained in exhaust gas, soil, combustion ash, and the like.

[Brief description of the drawings]

FIG. 1 is a sectional side view of a column according to an embodiment.

FIG. 2 is a side view of a solvent reservoir.

FIG. 3 is a side view of a conventional column.

FIG. 4 is an explanatory view showing a method of using a conventional column.

FIG. 5 is a process chart showing a method of using a column of an example.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Column 2 Outer cylinder 3 Inner cylinder 4 Screw cap 5 Seal ring 6 Capillary tube 7 Tip 8 Cock 9 Solvent reservoir 11 Filler 12 Samples 12a, 12b, 12c Components 13 Elution solvent (developing solvent) 14 Pan 15 Arrow 16 Arrow 20 Column 21 Porous glass

Claims (3)

[Claims] 1. A column for use in gravity-flow liquid chromatography, comprising: an outer cylinder; and an inner cylinder having one end inserted into the outer cylinder from both sides of the outer cylinder. Chromatography column. 2. The column for liquid chromatography according to claim 1, wherein one or both of the inner cylinders is provided with fixing means that can be fixed to the outer cylinder at an arbitrary insertion position. 3. An outer cylinder is filled with a chromatography filler using the column for liquid chromatography according to claim 1 or 2, and the ends of the inner cylinder are brought into close contact with both ends of the filler. A method for using a column for liquid chromatography, comprising separating and recovering a mixture by gravity flow chromatography using a column, and performing elution by inverting the column upside down.