JP2000342982A - Fractional refiner for repeating type dispenser - Google Patents

Abstract

PROBLEM TO BE SOLVED: To effect the fractional refining of objective substances and impurities easily and efficiently even if the amount of a sample is very small by mounting a fractional refiner, to which a solid-phase carrier is secured, releasably to a repeating type dispenser. SOLUTION: The fractional refiner 2 being a chip is releasably mounted to an opening 3 of a repeating type dispenser 1. A solid-phase carrier 10 for a liquid chromatograph in the refiner 2 is fixed by a pair of upper and lower plugs 8, 9. A button 4 in association with a filling and discharging piston of the dispenser 1 is operated to depress the piston, whereby a liquid sample of minute amount in the dispenser 1 (several microliters to several milliliters) is poured into the chip 2, and further, if necessary, by repeatedly operating the piston of the dispenser 1, the sample is filled and discharged repeatedly, following which the sample is discharged from a chip filling, discharging port 7. Thus, objective substances or impurities can be fractionally refined from the sample easily and reliably.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a preparative purifier capable of efficiently purifying and purifying a target substance from a sample or a specimen.

[0002]

2. Description of the Related Art Conventionally, when separating and purifying a target substance or impurity, a dedicated separation column for purification is connected to an expensive liquid chromatograph, and a liquid sample and a buffer for separation and purification are sent. It is done by that. However, even in the case of a small amount of sample, it is easier and more efficient to carry out fractionation and purification, especially in the fields of biochemistry, agricultural chemistry, molecular biology, medicine, clinical medicine and clinical medical examination, etc. Are also required at the laboratory level.

[0003] As a relatively simple purification device, there is a preparative purification column having the same shape as an injection cylinder (FIGS. 4 and 5). This means that a liquid sample is injected from the sample inlet (21) of FIG. 5, and then a buffer for preparative purification is injected, and pressure is applied from the sample inlet (21) or the outlet (23).
The pressure is reduced using a vacuum pump or the like, so that the liquid sample in the column body (22) and the buffer for preparative purification pass through the solid support for liquid chromatography (26), and The target biopolymer and impurities are separated and purified.

Further, a centrifugal tube as shown in FIG. 6 is also used. In this method, a part of a plastic cup (29) on which a solid support for liquid chromatography is fixed is placed in a centrifuge tube body (28), a liquid sample is injected into the plastic cup (29), and the centrifuge tube is placed in the tube. The liquid sample is mounted on a centrifuge and operated to allow the liquid sample to pass through the solid phase carrier for liquid chromatography (32) by using the generated centrifugal force, so that the target living body height in the liquid sample is increased. In this method, molecules and impurities are separated and purified and collected at the bottom of the centrifuge tube (28). Further, as shown in FIG. 7, a column body (33) is filled with a solid support (39) for liquid chromatography, and a liquid sample and a buffer for preparative purification (35) are filled with a column (34) using a peristaltic pump (34). An apparatus for fractionating and purifying a target biopolymer or impurities by sending a liquid at a low flow rate into 33) is also used.

That is, in the prior art, a liquid sample and a preparative buffer are separated by passing through a solid phase carrier by pressurizing an inlet, depressurizing an outlet, or using centrifugal force. Although purification is performed, in such a method, the liquid sample and the preparative buffer can only move in a certain direction in the solid support, and there is an opportunity for the liquid sample to come into contact with the solid support. And the efficiency of preparative purification cannot be increased. Further, in order to perform this operation, a complicated and large-sized apparatus is still required, and it is difficult to carry out simple and inexpensive separation and purification of a target substance or an impurity from a very small amount of liquid sample.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an instrument for easily and efficiently separating and purifying a target substance or impurity even in a small amount of a liquid sample.

[0007]

Means for Solving the Problems The present inventor has conducted intensive studies to solve the above-mentioned problems, and as a result, the objective preparative purification can be carried out simply and efficiently, and the structure is extremely simple. As a result, the present invention concerning a preparative purifier that can be provided at low cost has been completed.

[0008] That is, the present invention is a preparative purifier detachable from a repetitive type dispenser, wherein the solid phase carrier is fixed in the preparative purifier. It relates to a purifier. The preparative purifier of the present invention is configured such that the liquid is brought into and out of contact with the solid-phase carrier by repeatedly reciprocating the liquid with a repetitive dispenser and repeatedly passing through an internal carrier such as a solid-phase carrier for liquid chromatography. Since the probability of performing this process can be easily increased, even a small amount of liquid sample can be efficiently fractionated and purified. The repetitive dispenser to which the preparative purifier of the present invention is attached may be a normal repetitive dispenser used for titration or the like, and typically, the repetitive dispenser has a liquid inside. Is a pipette having a cylindrical passage through which a liquid flows, an opening provided at one end of the cylindrical passage through which liquid is injected and discharged, and a liquid injection / discharge piston provided at the other end. .

The fractionating / purifying apparatus of the present invention can be detachably attached to an arbitrary position in a liquid flow path in a repetitive dispenser. It is composed of a tip that is attached to the tip of the opening of the injector. Further, the preparative purifier of the present invention may be shaped so as to be mounted inside a chip, and may be a preparative purifier that can be used by appropriately loading a commercially available chip. In the preparative purifier of the present invention, the solid phase carrier may be fixed by any means, but is typically fixed by a pair of upper and lower stoppers through which gas and liquid can pass. As the stopper, a disc-shaped glass fiber filter, a disc-shaped plastic, or the like is desirable, and a product obtained by rolling cotton or fiber can also be used.

The solid phase carrier may be a conventional solid phase carrier for liquid chromatography such as a silica-based carrier, a synthetic polymer-based carrier, and a cellulose-based carrier. These may be an amino group, a carboxyl group, In addition, it should have chemical properties such as functional groups, anion exchange groups, cation exchange groups, hydrophobicity, reverse phase, etc. for specifically binding various antibodies to the target biopolymer or impurities. it can.

Accordingly, the following can be exemplified as embodiments of the present invention. A cylindrical passage through which liquid passes, an opening provided at one end of the cylindrical passage through which liquid is injected and discharged, and a liquid injection / discharge piston provided at the other end. A separation / purification device that can be attached to and detached from a repetitive dispenser. -The preparative purifier described above, which is detachable from an opening of the repetitive type dispenser. The dispenser / purifier described above, wherein the dispenser is mounted inside a chip detachable from an opening of the repetitive dispenser. -The preparative purifier as described above, wherein the solid phase carrier is fixed by a pair of upper and lower stoppers through which gas and liquid can pass. -The preparative purifier as described above, wherein the solid phase carrier comprises at least one selected from a silica carrier, a synthetic polymer carrier and a cellulosic carrier. -The preparative purifier as described above, wherein the solid support has at least one selected from an amino group, a carboxyl group, an anion exchange group and a cation exchange group. The preparative purifier as described above, wherein the solid support has hydrophobic or reversed-phase chemistry.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, in order to explain the present invention,
Embodiments will be described with reference to the drawings, but the present invention is not limited thereto. FIGS. 1A, 1B and 2 show one embodiment of the present invention. here,
The tip (2) itself that can be attached to and detached from the opening (3) of the repetitive dispenser (1) is the separation / purification device of the present invention. The solid support (10) for liquid chromatography in the preparative purifier (2) is fixed by a pair of upper and lower stoppers (8, 9).

By operating the button (4) linked to the injection / discharge piston of the repetitive dispenser (1), the piston is depressed, and a small amount of liquid sample in the repetitive dispenser (about several microliters to several milliliters) Into the chip (2),
If necessary, the injection into and out of the tip (2) is repeated by the repetitive operation of the piston of the repetitive dispenser. Thereafter, the liquid sample is discharged from the discharge port of the chip. By this operation, the target substance or impurity can be easily separated and purified from the liquid sample.

According to this aspect of the present invention, preparation and purification can be performed by a simple operation of attaching the purification / purification unit to the opening (3) of the repetitive dispenser (1). By repeatedly operating the button (4), a small amount of liquid sample (several microliters to several milliliters) can be repeatedly injected and discharged into the preparative separation / purification device. The separation and purification of substances and impurities can be easily performed. Moreover, since the chip is inexpensive and easily detachable, a disposable preparative purifier can be obtained. FIG. 3 shows another embodiment of the preparative purifier of the present invention.
The preparative purifier here is a cylindrical tube which is closed at the top and bottom with a pair of stoppers and molded so that a solid phase carrier for liquid chromatography can be introduced and held therein. The pipe body (11) is detachable from the chip body (2 '). The separation and purification of the liquid sample can be performed by the same operation as that described with reference to FIGS. 1 (a), 1 (b) and 2 described above.

As described above, according to the present invention, the injection / discharge button (4) of the repetitive dispenser (1) is provided in any of the embodiments.
The disposable preparative purifier can be provided, which can increase the probability that the liquid sample repeatedly moves in the solid phase carrier and the target substance or impurity comes into contact with the solid phase carrier by operating .

[0016]

EXAMPLE As a solid support for liquid chromatography, using a preparative purifier of the present invention in which a strongly basic anion exchange solid support was fixed, 100 microliters of 1 mg of bovine serum albumin was used. The following experiment for fractionating and purifying a liquid was performed. 1) Attach a preparative purifier to the opening of the repetitive dispenser. 2) operating the repetitive dispenser, injecting the liquid sample into the preparative purifier, and repeatedly moving the liquid sample in the preparative purifier to make more contact with the strongly basic anion exchange solid phase carrier; Drain all liquid. This discharged liquid is referred to as a. 3) A washing solution is injected into the chip to remove unnecessary substances remaining on the strongly basic anion exchange solid phase carrier,
Wash and drain all the washing liquid. This discharged liquid is referred to as B. 4) A buffer for eluting bovine serum albumin retained on a strongly basic anion exchange solid phase carrier is injected into the chip, and the buffer is repeatedly moved within the chip to enhance the elution effect. Drain the liquid. This liquid is designated as C. As a result, as shown in the table below, 1 milligram of bovine serum albumin in 100 microliters was finally separated and purified by 98% or more.

[0017]

[Table 1] Table 1: Bovine serum albumin content (%) in each liquid

[Brief description of the drawings]

FIG. 1A is an overall view showing an example of a repetitive dispenser to which a general chip is connected. (B) is a perspective view of a dispenser (tip).

FIG. 2 is a cross-sectional view showing the internal structure of a preparative purifier (chip) according to the present invention, in which a solid support is fixed by a pair of upper and lower stoppers.

FIG. 3 is a cross-sectional view of a chip loaded with a cylindrical tubular body which is a preparative purifier of the present invention.

FIG. 4 is a perspective view of a conventional purification device having a cylindrical shape.

FIG. 5 is a cross-sectional view of a conventional purification device having a cylindrical shape.

FIG. 6A is a perspective view of a conventional liquid chromatograph using a centrifuge tube. (B) is a sectional view of the centrifuge tube.

FIG. 7A is a perspective view of a conventional liquid chromatograph apparatus for forcibly sending a preparative buffer. (B) is a cross-sectional view of a glass column.

[Explanation of symbols]

 1: Repetitive preparative purifier 2: Preparative purifier (chip) 2 ': Chip body 3: Opening 4: Injection / ejection button 5: Mounting part 6: Chip 7: Injection / emission port 8,8': Top Plug 9,9 ': Lower plug 10,10', 26,32,39: Solid support 11: Cylindrical tube 21,27: Sample inlet 22,33: Column body 23: Outlet 24,30 , 37: upper stopper 25, 31, 38: lower stopper 28: centrifuge tube body 29: plastic cup with solid phase carrier fixed 34: peristaltic pump 35: buffer for preparative purification 40: outlet

Claims (8)

[Claims] 1. A preparative purifier detachable from a repetitive dispenser, wherein a solid phase carrier is fixed in the preparative purifier. 2. An iterative dispenser comprising: a tubular passage through which a liquid passes; an opening provided at one end of the tubular passage through which a liquid is injected and discharged; and an other end. The liquid purifying piston according to claim 1, further comprising a liquid injection / discharge piston provided in the liquid purifier. 3. The fractionating / purifying device according to claim 1, wherein the fractionating / purifying device is detachable from an opening of the repetitive type dispenser. 4. The preparative purifier according to claim 3, wherein the preparative purifier is mounted inside a chip detachable from an opening of the repetitive type dispenser. 5. The preparative purifier according to claim 1, wherein the solid support is fixed by a pair of upper and lower stoppers through which gas and liquid can pass. . 6. The fractionation method according to claim 1, wherein the solid phase carrier comprises at least one selected from a silica-based carrier, a synthetic polymer-based carrier, and a cellulosic-based carrier. Purifier. 7. The solid support according to claim 1, wherein the solid support has at least one selected from an amino group, a carboxyl group, an anion exchange group and a cation exchange group. Preparative purifier. 8. The preparative purifier according to claim 1, wherein the solid phase carrier has hydrophobic or reversed-phase chemical properties.