JPH10288571A - Pipet chip and material extracting method with it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To extract and separate only an analysis object in a liquid sample at high purity and high concentration with a simple structure and a simple action as an analysis sample. SOLUTION: Methanol and water are sucked into an air phase 6 through a silica gel fixed phase 5 from a suction port 4 in sequence then discharged from the suction port 4. The methanol and water are held in the fixed phase 5. Blood serum is likewise sucked and discharged from the suction port 4. The analysis object in the blood serum is held in the fixed phase 5. Water is likewise sucked as a cleaning liquid then discharged from the suction port 4. The eluant of the analysis object is likewise sucked then discharged from the suction port 4, and the analysis object is extracted.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention can provide a target substance for analysis by extracting, purifying and separating a target substance for analysis from a liquid sample when analyzing a liquid sample such as blood, urine, culture solution or the like by various analyzers. For pipette tips. In addition, the present invention relates to a method for extracting a liquid sample that can extract and separate only a target substance for analysis from a liquid sample by a simple operation using the pipette tip.

[0002]

2. Description of the Related Art When analyzing a specific component in a liquid sample such as blood or urine, it is necessary to extract and separate only the specific component or wash and remove unnecessary substances before injecting the sample into an analyzer. Pre-processing such as is required.

Conventionally, when performing such pretreatment for analysis, a stationary phase made of silica gel or the like is formed at a predetermined portion inside a long tube or a column-shaped instrument in order to facilitate the treatment. Structured instruments are used.
Such a device is used in a vertically suspended state, and the organic solvent, the liquid sample, the washing solution, and the eluent are sequentially flowed downward from above, so that in the stationary phase,
It carries out each step of holding, washing and eluting and extracting the target substance in the liquid sample.

[0004]

The pretreatment method for a liquid sample using the above-mentioned conventional instrument has the following problems. First, the operation of flowing an organic solvent or the like from above is complicated, and a complicated and precise mechanism such as a supply switching device for each liquid flow and its control circuit is required. In addition, when the apparatus is an open system, the vaporized organic solvent may have an adverse effect on humans, and the volatile organic solvent or the like may be caused to fall by gravity or by pressurization from above or suction from below. In order for the method to pass and discharge a sufficient amount for the extraction operation in the stationary phase, it is necessary to flow a relatively large amount of an organic solvent or the like. Furthermore, when blood, urine, or other body fluids with a limited collection amount are used as a sample, the number of extractions is restricted by the amount that remains in the flow path such as a tube, or the amount retained in the stationary phase is reduced. Analysis accuracy may be affected. Further, in the case of using a mechanism such as pressurization or suction, there is a problem that a large difference occurs in the analysis result value after extraction depending on the quality of the apparatus.

An object of the present invention is to solve the above-mentioned problems, and it is possible to extract and separate only an object of analysis from a liquid sample with high purity and high concentration.
It is another object of the present invention to provide a pipette tip that can be operated simply and quickly, and that can extract and separate only an analysis target substance with high extraction efficiency and quick operation, and a method for extracting a liquid sample using the same.

Another object of the present invention is to provide a pipette tip capable of achieving the above object by simply attaching and detaching it to the tip of each suction and discharge portion of a conventional syringe-type manual suction and discharge device or automatic extraction device, and a pipette tip using the same. To provide an extraction method.

Another object of the present invention is to simplify the structure.
An object of the present invention is to provide a pipette tip which is small in size, safe and easy to handle, has a low manufacturing cost, and can achieve the above object.

[0008]

In order to achieve the above object, the present invention is characterized by comprising, in order from the suction port side of a pipette tip, a stationary phase containing a porous material, an air phase, and a filter. To provide a pipette tip.

In order to achieve the above object, the present invention provides an extraction method using the pipette tip, wherein an organic solvent and water are sucked through a stationary phase from a suction port to an air phase, and then flowed back. Discharging from the suction port
A process in which the liquid sample is passed through the stationary phase from the suction port to the air phase and then backflowed and discharged from the suction port, and a step in which the cleaning liquid is passed through the stationary phase from the suction port to the air phase and then backflowed and suctioned A step of discharging from a mouth, and a step of discharging a liquid for dissolving a component serving as an analyte in a liquid sample from a suction port to an air phase by passing through a stationary phase from a suction port and then flowing back and discharging from a suction port, And a method for extracting a substance characterized by having the following.

[0010]

Next, an example of a pipette tip according to the present invention will be described with reference to FIG.

A pipette 1 has a plunger 2 in a cylinder.
a comprises a syringe-type main body 2 slidably disposed and a tip cone-type pipette tip 3 connected thereto. The pipette tip 3 is formed so as to be able to be closely attached to and detached from the main body 2 by means such as screwing or fitting by pressing force.

In the interior of the hollow pipette tip 3, a stationary phase 5 containing a porous material and an air phase 6 are arranged in this order from the suction port 4 side.
And a filter 7. Pipette tip 3
The shape, size, length, and the like of are determined as appropriate so that the stationary phase 5, air phase 6, and filter 7 having the required volumes can be formed according to the purpose.

The stationary phase 5 is formed by inserting and fixing a material molded to conform to the internal shape of the pipette tip 3 to be attached thereto, for example, a substantially disk-shaped or substantially frustoconical porous material. Is formed. This attachment site is preferably near the suction port 4 in order to secure the required volume of the air phase 6. As the porous material used in the stationary phase 5, silica gel, porous glass,
In addition to inorganic materials such as zeolite, organic materials represented by porous polymers such as ion exchange resins can be used. The type and amount of these porous materials are appropriately selected according to the type and amount of the liquid sample to be analyzed.

The air phase 6 comprises a space provided between the stationary phase 5 and the filter 7. The capacity of the air phase 6 is set in consideration of the amount of the organic solvent, liquid sample, and the like to be sucked in practical use.

The filter 7 is formed of a material that is air-permeable and that prevents or makes it difficult to pass a vaporized organic solvent. Such a filter 7
For example, a carbon filter made of carbon fiber or the like can be used.

Next, a method of extracting a liquid sample using the pipette 1 will be described for each operation.

First, the organic solvent or water passes through the stationary phase 5 from the suction port 4 and is sucked up to the air phase 6, and then flows backward, passes through the stationary phase 5 again, and is discharged from the suction port 4. This operation can be repeated two or more times with the same solvent or with a different solvent.
, And can be discharged at a time. Further, in this operation, a part of the organic solvent sucked into the air phase 6 is vaporized, and the pressure balances the inside of the air phase 6 to stably and reliably perform dispensing operations such as suction and discharge. I do. At this time, due to the action of the filter 7, the organic solvent or water passes through the filter 7 and
Is prevented from flowing to the suction side.

In this treatment operation, when the organic solvent or water passes through the stationary phase 5 twice in the forward and reverse directions, the organic solvent or water is sufficiently and evenly contained in the pores of the porous material constituting the stationary phase 5. It is adsorbed and held. At this time, the stationary phase 5 contains a mixture of pores where the organic solvent or water is adsorbed and held and non-porous portions where the organic solvent or water is not held. Of these, the pores where the organic solvent is adsorbed and held are solid. Functions as a shared bed. That is, the solid phase bed becomes an active part in which the adsorption and retention of the analysis target substance, washing and purification, and extraction are performed in the subsequent steps.

The type and amount of the organic solvent used in the step of forming the solid phase bed are appropriately selected according to the type and amount of the liquid sample. If necessary, two or more organic solvents can be combined, or a mixture of an organic solvent and water can be used.

Next, the liquid sample passes through the stationary phase 5 from the suction port 4 and is sucked up to the air phase 6, and then flows backward by the plunger 2. The liquid sample passes through the stationary phase 5 again and passes through the suction port 4. A discharging step is performed. By the reciprocating suction and discharge operation of the plunger 2, the liquid sample passes through the stationary phase twice, and the analyte in the liquid sample is adsorbed and held at a high concentration on the solid phase bed inside the stationary phase 5.

After that, the washing liquid is passed through the stationary phase 5 from the suction port 4 to be sucked up to the air phase 6, and then the stationary phase 5 is again passed backward and discharged from the suction port 4.
Also in this case, since the washing solution passes through the stationary phase twice in the forward and reverse directions, unnecessary substances other than the analysis object are sufficiently washed and discharged, and the analysis object is highly purified with a small amount of washing solution.
The washing liquid used in this step is appropriately determined in consideration of the type of the object to be analyzed. For example, usually, water or an organic solvent such as methanol can be used.

Next, a liquid (hereinafter referred to as “eluent”) for eluting a component to be analyzed from the liquid sample is
After passing through the stationary phase 5 from the suction port 4 and sucking up to the air phase 6, a step of passing the stationary phase 5 backflow again and discharging from the suction port 4 is performed. This step is performed by reciprocating the plunger 2 as described above. As a result of this operation, the eluent passes through the stationary phase 5 twice in reverse order, so that the analyte to be analyzed which has been adsorbed and held on the solid phase bed is sufficiently dissolved in the eluent, and is extracted and separated without waste. The discharged eluent containing the analysis target is stored in an appropriate container and then provided as a liquid to be analyzed. In this case, the suction port 4 may be connected to a sample injection port of a desired analyzer (not shown) so that the discharged eluent can be directly injected into the analyzer.

The pipette tip according to the present invention can be prepared by appropriately selecting and combining an appropriate stationary phase, an organic solvent, a washing solution, an eluent, etc., according to the liquid sample to be analyzed, and thus, such as blood and lymph. In addition to body fluids and urine,
It can be applied as a pretreatment instrument for analyzing liquid samples in various fields such as drinking water, river water, eluents / extracts of solids, eluents / extracts of synthetic substances, and the like.

In the figure, a syringe-type manual device is shown as the suction main body 2, but instead, a suction / discharge mechanism using the power of an electric motor or the like, and an electronic circuit / program software for controlling the same are provided. One or more pipette tips 3 may be attached to the automatic extraction device and used for automatic continuous operation.

[0025]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 As a stationary phase, silica gel (approximately 25
mg, about 60 to 100 angstroms and a specific surface area of about 50 to 450 m ² / g for adsorption chromatography), and a carbon filter (about 5 mm in thickness) as a filter. Pipette tips were obtained. The volume of the air phase was about 10 cc.

Example 2 Using the pipette tip obtained in Example 1, human serum was extracted by the following steps.

First, about 0.3 ml of methanol is passed through the stationary phase from the suction port to be sucked into the air phase, and then about 0.3 ml of water is similarly passed through the stationary phase from the suction port to form the air phase. Was sucked up. Thereafter, methanol and water were caused to flow backward from the air phase side, passed through the stationary phase again, and then discharged from the suction port. This step resulted in an average formation of a solid bed of sufficient bed thickness in the stationary phase.

Next, after about 0.3 ml of human serum was passed through the stationary phase through the suction port and aspirated to the air phase, the stationary phase was again passed back through the stationary phase and discharged through the suction port. Human serum is composed mostly of water, contains plasma proteins and inorganic salts as other components, and further contains non-protein nitrogen compounds, carbohydrates, organic acids, lipids, bilirubin, etc. as the remaining components. As a result, a sufficient amount of the analyte such as metabolites was adsorbed and held on the solid phase bed at a high concentration.

Thereafter, as a washing step, about 0.3 ml of water was passed through the stationary phase from the suction port to be sucked into the air phase, and then the stationary phase was again passed backward and discharged from the suction port. By this step, impurities remaining in the stationary phase were almost washed away, and only the adsorbed analytes were retained at a high purity.

Next, about 0.3 ml of a mixed solvent of methanol / acetonitrile and water was eluted as an eluent from the suction port to elute the metabolites, which are the analytes, adsorbed and held on the solid phase bed. , And then suctioned to the air phase. Then, the stationary phase was again passed backflow, discharged from the suction port into the beaker, and collected. Also at this time, the eluent passes through the stationary phase twice in the forward and reverse directions, so that the adsorbed material is eluted without any excess, and a lean extraction amount can be secured.

The time required for this series of steps was about 50 seconds. The sample thus extracted and separated was directly provided to an analyzer.

[0033]

By using the pipette tip of the present invention, complicated operation by a complicated and large-sized extraction device is not required, and the pretreatment of the liquid sample to be provided to the analyzer becomes easy. In addition, safety is high because the extraction operation is performed in a closed system. Furthermore, compared to the conventional technology, the liquid movement distance in each process is extremely short and there are two movement passes from the forward and reverse directions, so that the amount of liquid sample, amount of solvent, washing solution and eluent used is greatly increased. And a high-purity / high-concentration analyte can be obtained. In addition, analysis costs can be reduced. Further, since the pipette tip of the present invention can be easily attached and detached and its attachment / exchange can be carried out quickly, a large number of different specimens can be quickly extracted without cross-contamination. Further, since the pipette tip of the present invention has a simple structure and is small and lightweight, it can be mass-produced at low cost and can be easily handled for storage and transportation. Further, application to not only a manual extraction device but also an automatic extraction device is easy.

[Brief description of the drawings]

FIG. 1 is a schematic sectional view of a pipette tip.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Pipette 2 Main part 3 Pipette tip 4 Suction port 5 Stationary phase 6 Air phase 7 Filter

Claims (4)

[Claims] 1. A pipette tip comprising, in order from the suction port side of the pipette tip, a stationary phase containing a porous material, an air phase, and a filter. 2. The pipette tip according to claim 1, wherein the stationary phase has an inorganic or organic porous material layer. 3. The pipette tip according to claim 1, wherein the stationary phase has an inorganic or organic non-porous material layer. 4. A method for extracting a substance using a pipette tip according to claim 1, wherein the organic solvent and water are passed through a stationary phase from a suction port to an air phase, and then flow backward and discharged from the suction port. And passing the liquid sample through the stationary phase from the suction port to the air phase and then backflowing and discharging the liquid sample from the suction port, and after drawing the washing liquid through the stationary phase from the suction port to the air phase. Backflow and discharge from the suction port, and liquid for dissolving the component to be analyzed in the liquid sample passed through the stationary phase from the suction port to the air phase and then backflow and discharged from the suction port And extracting the substance.