JP2000338094A - Liquid chromatograph apparatus and method for fractional refining of sample using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To realize a liquid chromatograph apparatus capable of confirming a residue of an eluent from the outside of a container and adding the eluent while supplying the eluent to a column irrespective of small condensation and composition change of the eluent and a method for fractionally refining a sample using the apparatus. SOLUTION: The liquid chromatograph apparatus comprises an eluent container 1 for preserving an eluent 2, a separate column 6 for fractionally refining a sample, an eluent supply means 4 for supplying the eluent 2 from the container 1 to the column 6 while controlling its liquid feeding conditions, and a sample pouring means 5 for supplying the sample to the column 6. In this case, the eluent in the container 1 can communicate with the atmosphere, and when communicating, a sectional area of the communicating portion becomes 4 mm2 or below.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liquid chromatograph and a method for separating and purifying a sample using the same.

[0002]

2. Description of the Related Art Liquid chromatography is known as a technique for separating and purifying an object to be separated and contaminant components contained in a sample, subjecting the separated object to qualitative and quantitative analysis, and concentrating and purifying the separated object. ing.

[0003] In this technique, a sample dissolved in an eluent is supplied to a separation column, whereby the sample is sequentially eluted from those having a small interaction with the separation column, and the separated material is separated. The purpose is achieved by appropriately selecting the eluent and the separation column according to the conditions.

When a sample is subjected to qualitative or quantitative analysis using the above eluent, recently, a dedicated eluent corresponding to the sample is used in order to make the analysis routine, and the eluent is made of glass or plastic. It is housed in an eluent container (hereinafter, simply referred to as “container”) having good storage stability, with a lid attached, sealed and commercially available.

To use such a commercially available eluent,
After removing the lid, as shown in FIG.
Is set in a predetermined position, and this container 10a is connected to the pipe 4a on the suction side of the liquid sending pump P, thereby sucking out the eluent 2a from the container 10a, and passing the column through the injector 5a. 6a.

However, according to this method, the eluent 2a is actually handled while being stored in the container 10a. As the eluent 2a decreases, air flows into the container 10a, and the air enters the container 10a. There is a problem that the volatile component of the eluent 2a is full, the eluent 2a condenses, and the composition changes.

Therefore, a method has been proposed in which the above-mentioned container is a flexible container having airtightness, and the eluent is sealed in a container in a vacuum-packed state (Japanese Patent Application Laid-Open No. Hei 10-82773).

[0008]

However, according to the above-mentioned method, it is necessary to use a film laminated with an aluminum foil or the like in order to ensure the gas barrier property and the water vapor gas barrier property of the container. As a result, there is a problem that the remaining amount of the eluent inside the container cannot be confirmed from the outside. In such a bag type container,
There is also a problem that it becomes difficult to add the eluent to the column during the analysis.

The present invention solves the above-mentioned problems, and it is possible to check the remaining amount of the eluent from the outside of the container, despite the fact that the eluent is less condensed and the composition does not change. It is an object of the present invention to provide a liquid chromatograph device that can be added and a method for separating and purifying a sample using the same.

[0010]

The liquid chromatographic apparatus according to claim 1 (hereinafter referred to as "the apparatus of the present invention")
A container for storing the eluent, a separation column for separating and purifying the sample, an eluent supply means for supplying the eluent from the container to the separation column while controlling the conditions for sending the eluent, and a sample supply for supplying the sample to the separation column Means, the eluent in the eluent container and the atmosphere can be communicated with each other, and the cross-sectional area of the communicating portion when the eluent is communicated is 4 mm ² or less.

In a third aspect of the present invention, there is provided a method for separating and purifying a sample using a liquid chromatograph apparatus (hereinafter referred to as the "separation and purification method of the present invention"). When the eluent is supplied to the separation column, it is communicated, and when it is not supplied, it is separated.

The eluent and the separation column used in the apparatus and the separation and purification method of the present invention are those used in ordinary liquid chromatography, and the optimum eluent and the separation column are selected according to the components to be separated. Is done.

Next, preferred eluents and separation columns are shown for each component to be separated.

(1) A phosphate buffer or the like is used as a protein eluent, and GP is used as a separation column.
A column packed with a packing material for C or a column packed with an ion exchange type packing material in which the packing surface is modified with various amino groups, carboxyl groups or the like is preferably used.

(2) In the case of sugar A mixed solvent such as acetonitrile / aqueous buffer is preferably used as an eluent, and a column packed with a filler whose surface is modified with an amino group is preferably used as a separation column.

(3) In the case of peptide Acetonitrile / water-based buffer or the like is preferably used as an eluent, and an ordinary ODS column is suitably used as a separation column.

(4) In the case of amino acids A citrate buffer or a phosphate buffer is preferably used as an eluent, and a column packed with a packing material whose surface is modified with a sulfonyl group is preferably used as a separation column.

(5) In the case of nucleic acids A mixed solvent such as acetonitrile / aqueous buffer is preferably used as an eluent, and a column packed with a filler whose surface is modified with an amino group is preferably used as a separation column.

As the eluent, a single component may be used, or two or more types may be used. When the measurement is performed using two or more types, gradient methods such as a step gradient and a linear gradient are applied. In particular, when a mixture is separated for each component by liquid chromatography, a gradient method represented by the above two methods is suitable for obtaining a chromatogram with high resolution in a short time.

The material of the eluent container used in the apparatus and the separation and purification method of the present invention includes, for example, resin materials such as polyethylene, polypropylene, polyvinyl chloride, and polyfluoroethylene; and glass. It can be manufactured by blow molding.

The eluent supply means used in the apparatus and the separation and purification method of the present invention is not particularly limited as long as the eluent can be supplied from the eluent container to the separation column while controlling the conditions for sending the eluent. Instead, for example, a liquid sending pump is preferably used.

The sample supply means used in the apparatus and the separation and purification method of the present invention is not particularly limited as long as it can supply a sample to a separation column, and a usual injector is used.

In the apparatus and the separation and purification method of the present invention,
The eluent in the eluent container and the atmosphere can communicate with each other. When the eluent and the atmosphere communicate with each other, the cross-sectional area of the communicating portion is too large, the eluent evaporates into the atmosphere, condenses and condenses, and the composition changes.
m ² are not limited to, there is a possibility that too small eluent clogged when crystallized and dried, 0.0
1 mm ² or more is preferable.

If the communication section is provided with a valve for opening and closing the communication section, the communication section is opened only when the eluent is supplied to the separation column in conjunction with the eluent supply means. can do.

The valve used is not particularly limited, and for example, an electromagnetic valve (including an electromagnetic pinch valve) and the like are preferably used.

In the apparatus and the separation and purification method of the present invention,
When concentrating and purifying the separated material, the separated material is
If the conditions to be eluted from the separation column are determined, a means for detecting an object to be separated is not necessarily required, but, of course, a detection means is required for qualitative and quantitative analysis.

The means for detecting the object to be separated is not particularly limited, but usually a spectrophotometer (either ultraviolet or visible light) is used.

(Operation) The apparatus of the present invention comprises an eluent container for storing an eluent, a separation column for separating and purifying a sample, and an eluent supplied from the eluent container to the separation column while controlling the conditions for sending the eluent. And an eluent supply means for supplying the sample to the separation column. The eluent in the eluent container and the atmosphere can be communicated with each other. ^Since it is set to ² or less, the amount of the eluent evaporating into the atmosphere is reduced, the concentration of the eluent is suppressed, and the remaining amount of the eluent can be confirmed from the outside of the container.

The separation and purification method of the present invention uses the apparatus of the present invention, and the eluent and the atmosphere are communicated when the eluent is supplied to the separation column, and are separated when the eluent is not supplied. When is not supplied to the separation column, there is no component that evaporates into the atmosphere, so that the concentration of the eluent can be further suppressed, and the remaining amount of the eluent can be confirmed from the outside of the container.

[0030]

Next, embodiments of the present invention will be described in detail with reference to the drawings.

FIG. 1 is a schematic view showing an example of the main part of the apparatus of the present invention. FIG. 1 (a) shows a state in which a commercially available eluent is contained in a container, and FIG. 1 (b) shows the container of FIG. (C) shows another example of the apparatus of the present invention utilizing the above.

In the commercially available eluent, as shown in FIG. 1A, an eluent 2 is contained in a container 1, and the container 1 has a lid.
11 is attached and sealed.

As shown in FIG. 1 (b), the lid 11 of the container 1 is provided with an eluent suction nozzle 12 and a communication hole 13 having a cross-sectional area of 4 mm ² or less, whereby the apparatus of the present invention (claim Item 1) is completed.

On the other hand, as shown in FIG. 1C, an eluent suction nozzle 12 is provided on one lid 11, a communication hole is provided on the other lid 14, and a piping member 15 having the electromagnetic valve 3 is provided. Thereby, the device of the present invention (claim 2) is completed. Although not shown, one lid may be provided with a piping member having an eluent suction nozzle and an electromagnetic valve.

FIG. 2 is a flowchart showing still another example of the apparatus of the present invention, showing a case where a plurality of eluents are used.

As shown in FIG. 2, the apparatus of the present invention comprises containers 1a, 1b, and 1c each containing a different eluent, valves 3a, 3b, and 3c for allowing the eluent to communicate with the atmosphere. It comprises an eluent supply means 4, a sample injection means 5, a separation column 6, and a detection means 7. In this liquid chromatograph, the containers 1a, 1b, 1c can be switched sequentially, and the valves 3a, 3b, 3c are switched in response to the switching of the containers 1a, 1b, 1c. Instead of switching the containers 1a, 1b, 1c, an eluent supply means (for example, a liquid feed pump) 4
May be turned on and off.

For example, when the eluent contained in the container 1a is used, the eluent in the container 1a is
At the same time, the valve 3a is opened. Then, the eluent contained in the container 1a is supplied to the separation column 6 from the valve 3a to the container 1a, which communicates with the atmosphere, the pressure in this system is hardly reduced. on the other hand,
From the sample injection means (for example, an injector) 5, a sample to be analyzed is dissolved or dispersed in an eluent and supplied to the separation column 6. At this time, of the components in the sample, those having a smaller interaction with the separation column 6 are sequentially eluted and subjected to qualitative and quantitative analysis by the detector 7.

Next, instead of supplying the eluent from the container 1a to the separation column 6, the container 1b is transferred from the container 1b to the separation column 6.
Supply the eluent inside. Here, the switching of the eluent is performed by the step gradient method, but each of the containers 1a, 1b, 1c is connected to a dedicated liquid sending pump, and the linear gradient method of adjusting the liquid sending amount (speed) is used. It may be changed continuously with a change in the liquid sending speed of the liquid sending pump.

[0039]

EXAMPLES (Examples 1 and 2, Comparative Examples 1 and 2) Automatic Glycohemoglobin Analyzer (manufactured by Kyoto Daiichi Kagaku Co., Ltd .; Model "HA")
-8131 ") and 500 mL of the dedicated eluent (31
A) The container (having a capacity of about 3 L) in which a hole having a diameter shown in Table 1 was opened in a lid and left at room temperature. Pipe (Sekisui Chemical Co., Ltd .; Model "SP-0502E
M "), glycohemoglobin was analyzed using whole blood collected from healthy and the change in retention time was measured. In addition,
During the measurement, water condensed (condensed) on the inner wall of the container was shaken off to the container. The above results are summarized in Table 1.

[0040]

[Table 1]

[0041]

Since the liquid chromatograph of the present invention is as described above, the remaining amount of the eluent in the container can be confirmed from the outside, so that the eluent can be added during the measurement. In addition, since the composition change (concentration) due to vaporization (evaporation) of the eluent is reduced, the retention time does not fluctuate, so the peak area and height do not fluctuate. The amount of liquid that cannot be used due to a change in composition can be reduced.

Since the method for fractional purification of the sample of the present invention is as described above, in addition to the above effects, the concentration of the eluate can be further suppressed.

[Brief description of the drawings]

FIG. 1 is a schematic view showing an example of a main part of an apparatus of the present invention, in which (a) shows a state in which a commercially available eluent is contained in a container, and (b) uses the container of (a). One example of the apparatus of the present invention, (c) shows another example.

FIG. 2 is a flow chart showing still another example of the apparatus of the present invention, showing a case where a plurality of eluents are used.

FIG. 3 is a flowchart illustrating an example of a conventional liquid chromatograph apparatus.

[Explanation of symbols]

 1, 1a, 1b, 1c, 10a Eluent container 11,14 Lid 2a, 2a Eluent 3, 3a, 3b, 3c Valve (electromagnetic valve) 4 Eluent supply means 5 Sample injection means 6 Separation column

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] August 18, 1999 (1999.8.1)
8)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction contents]

[0008]

However, according to the above-mentioned method, it is necessary to use a film laminated with an aluminum foil or the like in order to ensure the gas barrier property and the water vapor gas barrier property of the container. As a result, there is a problem that the remaining amount of the eluent inside the container cannot be confirmed from the outside. In such a bag type container,
There is also a problem that it becomes difficult to add the eluent during the analysis.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0039

[Correction method] Change

[Correction contents]

[0039]

EXAMPLES (Examples 1 and 2, Comparative Examples 1 and 2) Automatic glycohemoglobin measuring device (manufactured by Kyoto Daiichi Kagaku Co., Ltd .; Model "HA")
-8131 ") and 500 mL of the dedicated eluent (31
The lid of the container A) is accommodated (volume about 3L), opened diameter of the hole as shown in Table 1, while left at room temperature, as a sample, a vacuum vote of EDTA-2Na is accommodated as an anticoagulant Pipe (Sekisui Chemical Co., Ltd .; Model "SP-0502E
Using a whole blood blood adopted from a healthy person at M "), carried out the analysis of glycated hemoglobin, and measuring the change in the retention time. At the time of measurement, water condensed (condensed) on the inner wall of the container was shaken off to the container. The above results are summarized in Table 1.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] Explanation of sign

[Correction method] Change

[Correction contents]

[Description of Signs] 1, 1a, 1b, 1c, 10a Eluent container 11, 14 Lid 2 , 2a Eluent 3, 3a, 3b, 3c Valve (electromagnetic valve) 4 Eluent supply means 5 Sample injection means 6 Separation column

[Procedure amendment 4]

[Document name to be amended] Drawing

[Correction target item name] Fig. 2

[Correction method] Change

[Correction contents]

FIG. 2

Claims (3)

[Claims] An eluent container for storing an eluent, a separation column for separating and purifying a sample, an eluent supply means for supplying the eluent from the eluent container to the separation column while controlling the conditions for sending the eluent, A sample supply means for supplying the sample to the separation column, wherein the eluent in the eluent container and the atmosphere can communicate with each other;
A liquid chromatograph apparatus, wherein a cross-sectional area of the communication portion when communicating is 4 mm ² or less. 2. The liquid chromatograph device according to claim 1, wherein a valve for opening and sealing the communication part is provided in the communication part. 3. A liquid using the liquid chromatograph device according to claim 1 or 2, wherein the eluent and the atmosphere are communicated when the eluent is supplied to the separation column, and are separated when the eluent is not supplied. A method for separating and purifying a sample using a chromatographic apparatus.