JP2007038130A - Apparatus for liquid-liquid extraction or solid- liquid extraction, and its extraction method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus matching with various liquid-liquid extraction or solid-liquid extraction and capable of conducting the repeated extraction for attempting the efficiency of the liquid-liquid extraction or solid-liquid extraction, and to provide a kit and its using method. <P>SOLUTION: In the apparatus for the liquid-liquid exchange or solid-liquid exchange dispense extraction by combination of high-density liquid and low-density liquid forming two layers which are not mixed with each other, it has a column having a space to conduct the exchange dispense reaction of the solute in the liquid-liquid interface, an opening in the upper part for injecting both liquids into the space and an opening which can be closed in order to discharge one of liquids and being equipped with a porous material which has a strong affinity with the liquid to be discharged from the lower part of the apparatus and has weak affinity with the liquid to be retained in the space of the column between the opening in the lower part and the space to conduct the exchange dispense reaction of the solute. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an apparatus for liquid-liquid extraction or solid-liquid extraction, an extraction method using the apparatus, a method for detecting and measuring a substance, and a kit used therefor.

The liquid-liquid extraction process using two liquids that are not mixed with each other and the solid-liquid extraction process using a liquid that does not completely dissolve the solid and the solid are soluble and distributable in each liquid. It is a technology that has been widely used in processes such as extraction of food components, synthesis and purification of chemical substances such as drugs and pigments, and analysis of environment-related substances.
So far, continuous extraction technology in the liquid phase has been developed for the purpose of increasing the scale and efficiency of the extraction / purification process. For example, Nippon Dionex Co., Ltd. sells the product “ASE-200”, and METTLER TOLEDO Co., Ltd. sells a liquid-liquid extraction device “ALLEXis”. Even now, a separatory funnel for liquid-liquid extraction is routinely used in many organic synthesis processes, extraction component separation processes, and the like.

On the other hand, in the screening process of high value-added compounds such as drug seed compounds, it is necessary to process a small amount and a large number of samples quickly, and improvement of the extraction processing speed has become an important factor.
Correspondingly, a solid-phase extraction method, which can be called a modified method of column chromatography technology, has been developed and used in various situations. However, for solid-phase extraction methods, a method development method for each target compound to be extracted, an appropriate amount of sample to be added to the column adsorption capacity, the possibility of diluting the recovered liquid, and the organic matter from the column It is necessary to consider the effects of elution. For this reason, there is still a growing need for efficiency improvement such as an improvement in the processing speed of the extraction technique in various screening situations.

  The essence of liquid-liquid or solid-liquid extraction is the migration phenomenon at the two-phase interface that does not mix with each other. When a substance to be analyzed or a substance that hinders analysis is dissolved or dispersed in the liquid or solid that is the first phase, the substance moves to the second phase separated by the interface by the boundary layer diffusion at the interface. Is possible. Mass transfer at this time is bidirectional, and when mass transfer from the first phase to the second phase and mass transfer from the second phase to the first phase antagonize, the mass distribution to each phase reaches equilibrium. . Also, mechanical operations such as agitation are often performed in order to quickly reach equilibrium, which increases the interfacial area where diffusion occurs and thins the two-phase boundary layer by constantly updating the interface. It is a thing aimed at. The larger the distribution ratio of the extracted components in the two phases, the more efficiently the extraction is performed. However, in some cases, the extraction is repeated several times by exchanging the second phase. In any case, if the extraction operation is to be performed continuously and efficiently with the previous and subsequent operations, the key is how efficiently the two phases can be separated.

When the two phases are both liquids, separatory funnels have long been known as a means to efficiently separate them and are still often used today in analytical experiments. A Soxhlet extractor is well known for extracting a liquid from a solid. However, both the separatory funnel and the Soxhlet extractor are tools premised on reuse, and the operation is largely dependent on human hands, making it difficult to reduce the cost and time required for the analysis operation.
As a means to replace the separatory funnel, two layers separated into the upper and lower layers in the test tube are often collected by manual pipetting, but the reproducibility of the analysis varies greatly depending on the experimenter's technique. In addition, it is difficult to handle a large number of samples in parallel.

In order to efficiently separate the two phases after liquid-liquid distribution, a hydrophobic filter paper having a property of selectively allowing only an organic solvent to permeate without passing water, and a column on which the filter is fixed are known. . However, in such a filter paper, a part of special silicon used for imparting water repellency to cellulose, which is a hydrophilic substrate, is eluted at the time of use, so that there is a limitation in use. In addition, the column is possible when the nonpolar liquid is in the lower layer during liquid-liquid extraction, but in the reverse case, the nonpolar liquid cannot contact the hydrophobic filter paper on the bottom surface and does not function sufficiently.
Examples of the solvent having a higher specific gravity than water include chloroform and dichloromethane, and examples of the solvent having a lower specific gravity than water include hexane and ethyl acetate. In the case of liquid-liquid extraction, it is necessary to pay attention to whether the liquid to be recovered is a polar liquid or a nonpolar liquid, and whether the liquid to be recovered is in the upper layer or the lower layer.
Furthermore, in the liquid-liquid separation step, in addition to the problem of separation between liquids, it is necessary to consider a problem related to a method of efficiently performing an interfacial exchange reaction between the two and a problem related to a method of performing repeated extraction. The same applies to solid-liquid extraction. Existing liquid-liquid separators such as filter paper and columns do not accurately deal with these problems. To date, basic technologies and solutions for solving these problems and achieving high throughput have been proposed. No column or extraction system has been developed.

As a field where there is a high need for the development of liquid-liquid extraction technology, for example, there is a field of high-sensitivity detection and quantitative analysis technology by derivatization of substances. This is a technique in which a derivatizing reagent is allowed to act on a substance to convert the substance into a labeled compound labeled with a partial chemical structure suitable for high-sensitivity detection / quantitative analysis for detection and quantification. For this purpose, labeling is often performed with a partial chemical structure having a pi-electron system, which is highly sensitive to light absorption and fluorescence characteristics in the visible and ultraviolet wavelength regions, as well as increased ionization efficiency under mass spectrometry conditions. It is aimed at detection and quantitative analysis.
Many derivatization reagents for the purpose of high-sensitivity detection and quantitative analysis use a specific functional group originally possessed by the substance to be analyzed as an action point. So far, many derivatization reagents have been developed and marketed focusing on carbonyl groups, carboxyl groups, amino groups, alcohol hydroxyl groups, thiol groups, etc. as functional groups serving as action points. For example, a reagent for labeling a carbonyl group includes “Alexa Fluor 350 Hydrazide, Sodium Salt”, “Fluorescein-5-Thiosemicarbazide”, and other reagents for labeling a carboxyl group include “9-Anthryldiazanthane”, “5- (amino) Reagents for labeling amino groups such as “Fluorescein” are “AMCA-NHS”, “Fluorescamine”, etc., and reagents for labeling alcohol hydroxyl groups are “9-Anthrylnitrile”, “Fluorescein-5-Carbonyl Azide, Diacetate”, etc., and Reagents for labeling thiol groups include “Alexa Fluoro 350 C5 Maleimid” "," Such as IC3-OSu ", are many types exist.

However, in this highly sensitive measurement technique, in many cases, a large excess of unreacted derivatization reagent and impurities derived therefrom remain in the reaction solution after derivatization. In such a case, even if the absorbance or fluorescence intensity of the reaction solution is directly measured, the background noise is too large to detect and measure the presence of the labeled substance contained in the reaction solution.
Therefore, in many cases, detection and quantification of a derivatized substance must wait for an analysis result combined with separation analysis such as high performance liquid chromatography or electrophoresis. This is a problem that hinders efficiency in performing screening for detecting a target reaction from hundreds, thousands, or more samples.

  Specific examples of the field of high-sensitivity detection / measurement technology by derivatization of substances include detection / measurement technology for reducing sugars. In general, carbohydrates are known as compounds that do not have clear absorption in the ultraviolet / visible wavelength region, and have low ionization efficiency during mass spectrometry, and are difficult to perform highly sensitive measurement and structure determination. Therefore, a method of detecting and measuring a sugar molecule with high sensitivity by modifying a carbonyl group at the reducing end of a sugar with a labeling compound such as a fluorescent substance (see Patent Document 1) has been developed.

Until now, several types of devices and kits have been commercially available for detection and measurement of reducing sugars. For example, a reaction apparatus (such as Takara Shuzo “PALSTATION”, “GlycoTAG”), 4-aminobenzoic acid ethyl ester (ABEE), 4- Reagent kits modified with aminobenzoic acid octyl ester (ABOE) and separated and identified by high performance liquid chromatography (Seikagaku Corporation "J-oil ABEE sugar composition analysis kit", "J-oil ABOE") A method for detecting and quantifying bands by electrophoresis after fluorescently labeling a sugar chain with a water-soluble fluorescent compound 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), etc. Toyobo Co., Ltd. “FACE Carbohydrate Analysis Kits” and “F ACE SE1000 Workstation ") and the like are commercially available.
However, since there is no method for purifying a labeled compound derived from a saccharide to be analyzed from a large excess of fluorescently labeled derivatization reagent mixed at that time, it cannot sufficiently cope with the movement of high throughput. is the current situation.

Research on carbohydrates is highly important not only in basic research but also in a wide range of research fields such as medical / pharmaceutical research, food utilization research, and biomass utilization research. In the medical / pharmaceutical research field, attention has been paid to the maintenance of complex life activity through the synthesis of sugar chains and the control of metabolism, and the development of diseases due to its breakdown.
As a result of research on sugar chain biology, it is known that, for example, the structure of the displayed sugar chain and the activity change of sugar chain-related enzymes are important disease markers. So far, methods have been developed in which a sugar chain is cut out from a glycoprotein, glycolipid, and the like and mapped. In addition, glycan-related enzymes have been vigorously screened as a basic technology that supports such research.

  In food utilization research, development of food materials such as low-calorie sweeteners and oligosaccharides having prebiotic activity, and screening of useful enzymes have been energetically performed. As a result of these screenings, mass production techniques for new food materials such as cyclodextrin and erythritol have been established through the establishment of enzyme conversion methods and fermentation production methods. In addition, enzyme preparations aimed at improving the physical properties of processed foods have been developed, and screening of amylase, cellulase, pectinase, etc. from microbial resources has been performed.

In biomass utilization research, screening of microorganisms and enzymes for efficiently degrading carbohydrates such as cellulose, chitin, hemicellulose, starch and the like has been performed. Research aimed at commercialization, such as production of more powerful cellulases by genetic recombination technology and screening of raw starch degrading enzymes, is ongoing. Furthermore, in ethanol fermentation, since yeast cannot metabolize pentose derived from hemicellulose, research is being conducted to increase the efficiency of ethanol production by imparting the ability of pentose to metabolize using metabolic engineering techniques. Yes. In addition, research is underway on conversion to useful materials such as fungal polysaccharides, seaweed polysaccharides, and animal polysaccharides.
Thus, it is considered that there is an extremely great need for the efficiency improvement of carbohydrate screening technology that supports carbohydrate research.

Japanese Patent Laid-Open No. 10-267931

As described above, development of liquid-liquid extraction or solid-liquid extraction technology for increasing the throughput of various screenings is desired.
The problem to be solved by the present invention is mainly the development of an apparatus and kit for improving the efficiency of liquid-liquid extraction or solid-liquid extraction, and is compatible with various liquid-liquid extraction or solid-liquid extraction systems. The present invention relates to the development of an apparatus or kit that can repeatedly perform extraction. In addition, the screening method after derivatizing a substance with a labeled compound, including a liquid-liquid extraction or solid-liquid extraction process, aims to greatly increase the efficiency.

The present inventors paid attention to the behavior of substances at the solid-liquid and liquid-liquid interfaces, whether the two-phase liquid separation operation can be performed more simply and efficiently. As a result, when one of the two liquids after liquid-liquid extraction shows a remarkable capillary action with a rough surface or porous material having higher affinity with the liquid, the other liquid does not mix with the liquid. It was found that the transportation of can be controlled.
Consider the behavior in the air of a porous material that has two liquids that do not mix with each other and a surface wetted by one of the liquids. First, when the first liquid that can wet the surface of the material comes into contact with the surface of the porous material, it immediately penetrates and fills the pores. The air that has filled the pores is expelled at that time. Even when the second liquid is brought into contact with the porous material impregnated with the first liquid in this way, it is not easy for the second liquid to penetrate and penetrate the first liquid. The reason is that the second liquid enters the area where the pore internal surface of the porous material is wetted and filled by the first liquid, and that the interface between the first liquid and the second liquid has a complicated shape. This is because it is necessary to enter while increasing the area by changing, and a large amount of energy is required to overcome the strong interfacial tension acting on the interface between these two liquids.
Even if the second liquid is placed on the porous material already impregnated with the first liquid and the interface tension is overcome by gravity, the first liquid is replaced if the porous material has sufficiently small pores. And cannot enter the pores.
On the other hand, even in the same situation, if the first liquid comes into contact with the porous material that has already been impregnated with the first liquid, the interface does not form and the solution is integrated so that it penetrates easily and in one direction. In a situation where gravity works, the first liquid that has permeated flows in the porous material according to gravity and drops.
That is, the porous material having a surface that gets wet with the first liquid can be used for the purpose of selectively transmitting only the first liquid without transmitting the second liquid.

  As a result of intensive studies to solve this problem by paying attention to such a phenomenon, the present inventors have found that one of the two liquids that do not mix with each other and the porous material having a high affinity with the other liquid has a low affinity. Screening using equipment and kits that can selectively discharge the target liquid after promoting the interfacial exchange reaction while appropriately preventing the liquid from being discharged from the lower end of the column and scattered from the upper end. As a result, the inventors have found that liquid-liquid extraction and solid-liquid extraction in various scenes such as the above are greatly improved in efficiency, and have completed the present invention.

  That is, the first invention for solving the above-mentioned problem is an apparatus for liquid-liquid exchange or solid-liquid exchange distributed extraction by a combination of a high-density liquid and a low-density liquid that form two layers without mixing with each other. A column having a space for exchanging and distributing solutes at the liquid-liquid interface, an upper opening for injecting both liquids into the space, and a lower pluggable opening for discharging one liquid. A porous material with a strong affinity for the liquid to be discharged from the lower part of the apparatus and a weak affinity for the liquid to be retained in the column space is installed between the opening of the lower part and the space for performing the exchange / distribution reaction of the solute. It is related with the apparatus characterized by this.

  A second invention for solving the above-mentioned problems is the device according to the first invention, characterized in that it has a function of closing the opening at the top of the device.

  A third invention for solving the above-mentioned problems is characterized in that the porous material is continuously attached from the front of the lower part of the apparatus to the side of the column space where the solute exchange partition reaction is performed. It is an apparatus as described in 1 invention.

  A fourth invention for solving the above-mentioned problems is the apparatus according to the third invention, characterized in that it has a function of closing the opening at the top of the apparatus.

  According to a fifth aspect of the present invention for solving the above problems, when performing liquid-liquid exchange distribution extraction using the apparatus described in the first aspect, the liquid to be discharged from the lower part of the apparatus is less than the liquid to be retained in the apparatus. If the density is high, the porous material installed in the device is impregnated with only the high-density liquid used for liquid-liquid extraction, and then liquid-liquid partition extraction is performed from the low-density liquid using the high-density liquid. This is a liquid-liquid exchange partition extraction method.

  The sixth invention for solving the above-mentioned problem prevents the outflow of liquid from the upper part of the apparatus during the liquid-liquid distributed extraction operation by closing the opening at the upper part of the apparatus during the liquid-liquid distributed extraction operation. The method according to the fifth invention, characterized in that:

  According to a seventh aspect of the present invention for solving the above problems, when performing liquid-liquid exchange distribution extraction using the apparatus described in the third aspect of the invention, the liquid to be discharged from the lower part of the apparatus is less than the liquid to be retained in the apparatus. If the density is low, the porous material mounted on the device is impregnated with the low-density liquid used for liquid-liquid extraction, and then liquid-liquid partition extraction is performed from the high-density liquid with the low-density liquid. This is a liquid-liquid exchange partition extraction method.

  The eighth invention for solving the above problem prevents the outflow of liquid from the upper part of the apparatus during the liquid-liquid distributed extraction operation by closing the opening at the upper part of the apparatus during the liquid-liquid distributed extraction operation. A method according to the seventh invention, characterized in that:

  According to a ninth aspect of the present invention for solving the above problems, a porous material is clogged with a liquid to be retained in the apparatus space when performing solid-liquid exchange distribution extraction using the apparatus according to the first aspect. The solid-liquid exchange partition extraction method is characterized in that solid-liquid extraction is performed by replacing a solid sample that is not used.

  According to a tenth aspect of the present invention for solving the above-mentioned problems, liquid is prevented from flowing out from the upper part of the apparatus during the solid-liquid distribution extraction operation by closing the opening at the upper part of the apparatus during the solid-liquid exchange distribution extraction. The method according to the ninth aspect, characterized in that:

  An eleventh invention for solving the above-mentioned problems is a liquid-liquid exchange distribution extraction device characterized in that a plurality of the devices described in the first or second invention are connected in parallel.

  A twelfth invention for solving the above-mentioned problems is a liquid-liquid exchange distribution extraction device characterized in that a plurality of devices according to the third or fourth invention are connected in parallel.

  According to a thirteenth invention for solving the above-mentioned problem, a measurement interfering substance containing an unreacted derivatization reagent present in a reaction mixture is obtained after reacting a substance with a derivatization reagent to convert it into a labeled compound. A method for purifying a labeled compound, characterized in that an operation for removal by liquid-liquid extraction is performed using the apparatus according to any one of the first to fourth inventions and the 11th to 12th inventions.

  A fourteenth invention for solving the above-mentioned problems is a method for derivatizing a substance according to the method described in the thirteenth invention, wherein a reaction for converting the substance into a labeled compound is carried out inside the apparatus.

  According to a fifteenth aspect of the invention for solving the above-mentioned problems, in the method according to the thirteenth or fourteenth aspect of the invention, the analysis is performed by the instrument analyzer while the liquid containing the labeled compound after liquid-liquid extraction is kept inside the apparatus. How to do it.

  A sixteenth invention for solving the above-mentioned problems is characterized in that a derivatizing reagent characterized by imparting ultraviolet / visible absorption characteristics is used, and a labeled compound is detected / quantified by a spectrophotometer. The present invention relates to the detection / quantification method described in the fifteenth invention.

  A seventeenth invention for solving the above-mentioned problems is characterized in that a derivatization reagent characterized by imparting a fluorescence emission property is used, and a labeled compound is detected and quantified by a fluorescence spectrophotometer. The present invention relates to a detection / quantification method described in 15 inventions.

  According to an eighteenth invention for solving the above-mentioned problems, a labeled compound is detected and quantified using a derivatization reagent characterized by imparting a strong ionization characteristic and / or a characteristic giving a characteristic product ion. The present invention relates to the detection / quantification method described in the fifteenth aspect of the present invention, wherein

  A nineteenth invention for solving the above-mentioned problems relates to a method for purifying a labeled compound as described in the thirteenth invention, wherein the substance is a reducing sugar.

  A twentieth invention for solving the above-mentioned problems relates to a method for derivatizing a substance described in the fourteenth aspect, wherein the substance is a reducing sugar.

  A twenty-first invention for solving the above problem relates to the analytical method described in the fifteenth invention, wherein the substance is a reducing sugar.

  A twenty-second invention for solving the above problem relates to the detection / quantification method described in the sixteenth invention, wherein the substance is a reducing sugar.

  A twenty-third invention for solving the above-described problems relates to the detection / quantification method described in the seventeenth invention, wherein the substance is a reducing sugar.

  A twenty-fourth invention for solving the above-mentioned problems relates to the detection / quantification method described in the eighteenth invention, wherein the substance is a reducing sugar.

  A twenty-fifth invention for solving the above-mentioned problems relates to the method according to any of the nineteenth to twenty-fourth inventions, wherein the derivatization reagent is a reductive amination reaction reagent.

  A twenty-sixth aspect of the present invention for solving the above problems relates to the method described in the twenty-fifth aspect of the present invention, wherein 4-aminobenzoic acid ethyl ester and a reducing agent are used as a derivatizing reagent.

  A twenty-seventh aspect of the present invention for solving the above problems comprises the column according to any one of the first to fourth aspects of the present invention, a stopper having a function of closing the opening at the lower end of the column, and a plurality of these. A high-throughput kit for liquid-liquid exchange or solid-liquid exchange distribution extraction, characterized by having instruments for connection and assembly.

By using the extraction apparatus and the extraction kit according to the present invention, the liquid-liquid extraction or solid-liquid extraction process becomes efficient, and it becomes possible to easily remove complicated impurities and purify the target substance. High throughput of screening becomes possible. In addition, by using this extraction device or extraction kit, a series of steps to convert a substance into a labeled compound with a derivatization reagent and detect and measure the substance with high sensitivity has been developed, making it easy to screen the substance. You can do it.
Therefore, the present invention is not only used for basic research seeking new scientific knowledge and analysis of environmental pollutants, but also in the pharmaceutical industry, agrochemical / animal medicine industry, food industry, enzyme industry that conducts daily screening. It is expected to greatly improve the screening work efficiency in the material industry.

Hereinafter, the present invention will be described in detail.
The first half of the present invention, that is, the first invention to the twelfth invention relates to an apparatus, a kit and a method for using the same for improving the efficiency of liquid-liquid extraction or solid-liquid extraction. In this specification, the kit means a device that is disposable in part or in whole and can be efficiently separated, detected, analyzed, etc. by operating according to manuals or conventional methods. .

  Liquid-liquid extraction is performed by combining two liquids of a high-density liquid and a low-density liquid that form two layers without mixing with each other. The definition of a high density liquid and a low density liquid here is relative, and a liquid having a large specific gravity corresponds to a high density liquid and a liquid having a small specific gravity corresponds to a low density liquid. In general, different liquids have different specific gravity, so it is important that the two liquid combinations in liquid-liquid extraction do not mix with each other. As an example suitable for liquid-liquid extraction, a combination of an aqueous system and an organic system, which is a combination of a high polarity liquid and a low polarity liquid, can be given. The term “aqueous” as used herein refers to water or a liquid containing a solute in water, and includes those containing a buffer component in addition to acid and alkali. An organic system means an organic solvent with low miscibility with water, or an organic solvent containing a solute. Examples of organic solvents include chloroform, dichloromethane, carbon tetrachloride, hexane, ethyl acetate, and benzene. , Toluene and the like.

After liquid-liquid extraction, it is necessary to separate the two liquids. For this reason, this device has a strong affinity for the liquid to be discharged from the device and a weak affinity for the liquid to be left in the device. Is installed. The strength of affinity here is relative. The affinity between the liquid and the porous material is affected by two factors: wettability and ease of penetration.
On the liquid side, dissolved solute molecules influence in addition to the functional groups and chemical structures of the molecules that make up the liquid. In addition, in porous materials, the physical shape of the material surface, such as the size and shape of the pores, is affected in addition to the properties at the molecular level such as the electron affinity exposed to the material surface and the ease of inducing effects. Giving. Whether the two types of liquid have higher affinity with the porous material is determined regardless of the surface shape of the porous material, so the contact angle between the smooth material surface without pores and the liquid is examined. This can be easily determined. That is, a liquid with a smaller contact angle has a higher affinity with the material.

If liquid-liquid extraction using a combination of aqueous and organic materials is performed as a porous material with a material that is clearly stated to be hydrophilic and lipophilic, the configuration of the apparatus according to the purpose of extraction is easy. In addition, although there is a part that has not been theoretically handled for the wetting phenomenon, the basic knowledge of measurement and evaluation methods has been accumulated and published, so the affinity of porous materials for any liquid It is possible to control sex.
Solid-liquid extraction, on the other hand, uses the apparatus of the present invention that can be used for liquid-liquid extraction to extract a target substance from a solid or semi-solid sample containing the target substance into a liquid phase, and then perform solid-liquid separation / The step of removing is performed.

  Next, the latter half of the present invention, that is, the thirteenth to twenty-sixth aspects of the present invention relates to a method for screening a substance using the invention of the first half and a kit for the same.

Features of the apparatus according to the first invention are shown in FIGS. 1A and 1B. It is desirable that the material of the device should be such that components such as plasticizers do not dissolve, or the function of the structure does not deteriorate due to liquid leakage, cracks, etc. Examples thereof include polycarbonate, polyethylene, polystyrene, polypropylene, polyethylene terephthalate, polytetrafluoroethylene (PTFE), and glass. However, these materials have different resistance and strength characteristics against various solvents, acids and bases, and the materials should be selected according to the extraction system to be used. In addition, materials having different characteristics such as stability to liquid and mechanical strength may be used in appropriate combination.
Furthermore, although there is no restriction on the size of the apparatus, it is desirable to reduce the size to a size that can accommodate the size of a 96-well microplate or a 384-well microplate when performing high-throughput screening.

  Since a solid-phase extraction column having a size corresponding to a 96-well microplate size is commercially available, it is possible to newly manufacture the device according to the first invention by improving the manufacturing process. Become. In that case, it is expected that hundreds of microliters of solution can be recovered by extracting with a device having a column of about 1 milliliter or less, and a sufficient amount of liquid can be detected and measured by a spectrophotometer. It is considered possible.

For materials having pores to be fixed to the bottom of the column described in the first invention, cellulose, glass fiber, polycarbonate, polyethylene, polypropylene, PTFE, polyvinylidene fluoride (PVDF), nylon, 1PS (Whatman Japan Co., Ltd.) Company). However, these materials have different resistances to various solvents, acids and bases, and should be selected according to the extraction system to be used.
The appropriate pore diameter is determined by relative influence factors such as the layer thickness, surface water repellency, oil repellency, liquid surface tension, liquid volume and external force. Because the interfacial tension works greatly, the device functions reliably.
However, as long as the function capable of blocking the passage of the liquid desired to remain inside the apparatus is maintained, the larger the pore diameter as much as possible, the higher affinity liquid that can penetrate into the pore is discharged from the system. It is expected that the process will proceed smoothly and lead to an improvement in the processing capacity of the entire apparatus. A material having pores with appropriate diameters can be produced by a known method, and some of them are commercially available. For example, Varian's polyethylene frit has a pore size of 20 micrometers, and Whatman Japan's PTFE resin has a pore diameter of 1.0 micrometers. Both exhibit the expected functions.

With respect to the plug to be provided at the lower end of the apparatus described in the first invention, a lid that covers the lower end, a rotary cock structure, a plug for preventing a drop by air pressure, and the like are conceivable. Obviously, the stopper works in the same way whether it is integrated with the column or attached to the bottom of the device.
For example, Biorad's two-way polycarbonate stopcock (polycarbonate / polypropylene) or female luer plug (polypropylene), or Waters' Sep-PAK cartridge accessory / vacuum stopcock. (Made of polyethylene) can be used.

In the apparatus described in the first invention, when a target substance is extracted or removed from a solid sample using a liquid having a high affinity with the porous material at the lower part of the apparatus, or when the target substance has an affinity with the porous material at the lower part of the apparatus. When extracting or removing objects using low-density liquid with high affinity and high-density liquid with high affinity, liquid with gentle pipetting and shaking with extremely small volume compared to the size of the device, stirring- Used when conducting a liquid interface exchange reaction.
When water is used as the low affinity liquid, examples of the high density liquid having a higher specific gravity include chloroform, dichloromethane and carbon tetrachloride.

  The device described in the second invention is provided with a stopper for closing the opening at the upper end of the device described in the first invention. The apparatus of the second invention is used to prevent liquid from spilling from the upper end of the apparatus when the interface exchange reaction is promoted by a mixing operation such as turning the entire apparatus upside down, shaking, or stirring. is there.

  The features of the apparatus described in the third invention are shown in FIGS. In the apparatus described in the third invention, the porous material continuous from the front of the lower part of the apparatus has a lower density than the liquid to be discharged from the lower part of the apparatus when liquid-liquid extraction is performed. Therefore, even if it is located in the upper layer, it is in direct contact. The liquid to be discharged from the lower part of the apparatus comes into contact with a porous material having high affinity, penetrates through the material, is guided below the column, and is discharged from the lower end.

  Moreover, although it is a form of the porous material which continues from before the opening of the apparatus lower part, for example, it is a cross section as shown to FIG. 3 (1), (2), (4) besides the form shown to FIG. The continuous form shown in the figure is easily conceived. Further, as shown in FIG. 3 (5), when the apparatus described in the first invention is inclined and an appropriate amount of a combination of polar liquid and nonpolar liquid is added, it is described in the third invention. It is obvious that the same function as that of the apparatus is exhibited (FIG. 3 (6)). FIG. 3 (3) is the first invention and is not the third invention, but has an equivalent function intended by the third invention.

The apparatus described in the third invention is a case where a high-density liquid having a low affinity and a low-density liquid having a high affinity with a porous material at the bottom of the apparatus are used for extraction or removal of an object. It is used when conducting a liquid-liquid interface exchange reaction by shaking and stirring with a very small amount of liquid compared to the size of the apparatus. When water is used as the low affinity liquid, examples of the low density liquid having a smaller specific gravity include hexane, ethyl acetate, benzene, and toluene.
The device described in the fourth invention is a device in which a stopper is provided on the upper end of the device described in the third. As in the second invention, the apparatus of the fourth invention prevents liquid from spilling from the upper end of the apparatus when promoting the interfacial exchange reaction by a mixing operation such as turning the entire apparatus upside down, shaking, or stirring. It is used to make it.

  Using the apparatus described in the first invention, liquid-liquid extraction and high-density liquid discharge operation can be performed by the method described in the fifth invention. Prior to extraction, the bottom of the device is opened as necessary, and the porous material attached to the bottom of the device is impregnated with a high-density liquid with high affinity to fill the pores of the porous material. A liquid surface of high density liquid is formed on the surface of the material.

  After closing the stopper at the lower end so that the liquid in the apparatus is not discharged from the lower end, a combination of low density and high density liquid to be subjected to the interface exchange reaction is introduced from the upper end of the apparatus. Next, the interface exchange reaction is promoted by a mixing operation such as shaking, stirring, and pipetting. Thereafter, when the stopper at the lower end is opened, the high-density liquid is discharged from the lower end by gravity or appropriately performing suction, pressurization, and centrifugation. Since the low density liquid cannot penetrate into the pores due to the interfacial tension with the high density liquid, the penetration into the porous material is blocked during the discharging operation of the high density liquid and remains in the apparatus.

If only a high-density liquid is discharged, a new high-density liquid is added as necessary, and the mixing operation and the discharge are repeated, so that liquid-liquid extraction can be performed repeatedly.
When the target substance to be recovered remains in the low-density liquid inside the apparatus, the target substance can be recovered as a low-density liquid solution from the upper part or the lower part of the apparatus after sufficient extraction is performed in this manner. To recover from the upper part, for example, it may be collected by sucking up with a pipette, a syringe needle, etc., and to recover from the lower part, to press and extrude from the top of the device, to make negative pressure under the device, centrifugal force What is necessary is just to discharge | emit from a lower end by the method of utilizing. When discharging from the lower end, there is a possibility that a small amount of nonpolar liquid held in the porous material at the bottom is also discharged together.

  Using the apparatus described in the second invention, liquid-liquid extraction and high-density liquid discharge operation can be performed by the method described in the sixth invention. In the operation of the sixth invention, in addition to the operation of the fifth invention, the opening at the upper part of the apparatus is opened when the liquid is supplied from the upper part of the apparatus, and the opening is closed during the mixing operation for promoting the interface exchange reaction. It is characterized by doing. Thereby, it is possible to prevent the liquid from flowing out from the upper part of the apparatus and perform a mixing operation by an operation such as upside down.

  Using the apparatus described in the third invention, the liquid-liquid extraction and the discharge operation of the low density liquid can be performed by the method described in the seventh invention. Prior to extraction, after opening the opening at the bottom of the device as necessary, a high-density liquid having high affinity with this porous material is transferred from the bottom opening to the porous material that continues to the side of the space where the exchange distribution reaction is performed. Through the impregnation step, the pores of the porous material are filled with the low density liquid, and a liquid film of the low density liquid is formed on the surface of the porous material.

  After closing the stopper at the lower end so that the liquid in the apparatus is not discharged from the lower end, a combination of a low density liquid and a high density liquid to be subjected to the interface exchange reaction is introduced from the upper end of the apparatus. Next, the interface exchange reaction is promoted by a mixing operation such as shaking, stirring, and pipetting. Thereafter, the low-density liquid is discharged from the lower end by opening the stopper at the lower end and performing gravity or appropriate suction, pressurization and centrifugation. Since the high-density liquid cannot penetrate into the pores due to the interfacial tension with the low-density liquid, the penetration into the porous material is blocked during the discharging operation of the low-density liquid and remains in the apparatus.

If only a low-density liquid is discharged, a new low-density liquid is added as necessary, and the mixing operation and the discharge are repeated, so that liquid-liquid extraction can be performed repeatedly.
In addition, when the target substance to be recovered remains in the high-density liquid inside the apparatus, the target substance can be recovered as a low-density liquid solution from the upper part or the lower part of the apparatus after sufficient extraction is performed in this manner. To recover from the upper part, for example, it may be collected by sucking up with a pipette, a syringe needle, etc., and to recover from the lower part, to press and extrude from the top of the device, to make negative pressure under the device, centrifugal force What is necessary is just to discharge | emit from a lower end by the method of utilizing. When discharging from the lower end, there is a possibility that a small amount of nonpolar liquid held in the porous material at the bottom is also discharged together.

  Using the apparatus described in the fourth invention, the liquid-liquid extraction and the discharge operation of the low density liquid can be performed by the method described in the eighth invention. In the operation of the eighth invention, in addition to the operation of the seventh invention, the opening at the upper part of the apparatus is opened when the liquid is supplied from the upper part of the apparatus, and the opening is closed during the mixing operation for promoting the interface exchange reaction. It is characterized by doing. As a result, the liquid can be prevented from flowing out from the upper part of the apparatus, and a mixing operation such as upside down can be performed.

Using the apparatus described in the first invention, solid-liquid extraction and liquid discharge operation can be performed by the method described in the ninth invention. The operation of the ninth invention is characterized in that the liquid to be retained in the apparatus space is used by being replaced with a solid sample in which the porous material is not clogged. The solid sample may be a plant slice, a plastic piece, food, or the like, and is not particularly limited as long as it does not hinder the discharge of the liquid after the extraction operation by pulverization and clogging. Moreover, the liquid used for extraction will not be specifically limited if it has affinity with the pore of a porous material.
After closing the stopper at the lower end so that the liquid in the apparatus is not discharged from the lower end, a combination of solid and liquid to be subjected to the interface exchange reaction is introduced from the upper end of the apparatus. Next, the interface exchange reaction is promoted by a mixing operation such as shaking, stirring, and pipetting. Thereafter, the plug at the lower end is opened, and the high-density liquid is discharged from the lower end by gravity or appropriately performing suction, pressurization, and centrifugation. Since the solid cannot penetrate into the pores, the penetration into the porous material is blocked during the liquid discharging operation and remains in the apparatus.
If only a liquid is discharged, a new liquid is input as necessary, and the mixing operation and the discharge are repeated, so that solid-liquid extraction can be repeatedly performed.
When the target substance to be recovered remains in the solid inside the apparatus, the solid containing the target substance can be recovered from the upper part of the apparatus after the extraction is sufficiently performed.

  Using the apparatus described in the second invention, solid-liquid extraction and high density liquid discharge operation can be performed by the method described in the tenth invention. In the operation of the tenth invention, in addition to the operation of the ninth invention, the opening at the upper part of the apparatus is opened when the solid and liquid are introduced from the upper part of the apparatus, and the opening is performed during the mixing operation for promoting the interface exchange reaction. It is characterized by closing. Thereby, mixing operation by operations such as upside down is possible.

By connecting a plurality of the devices of the first and second inventions, the device of the eleventh invention and the kit of the twenty-seventh invention are constituted, and the fifth and sixth inventions or The extraction operation according to the ninth and tenth inventions can be performed on a large number of samples in a short time, and high throughput for liquid-liquid extraction or solid-liquid extraction can be achieved.
Further, by connecting a plurality of the devices of the third and fourth inventions, a device of the twelfth invention and a kit of the twenty-seventh invention are formed, and the seventh and eighth devices using the device and the kit are used. The extraction operation according to the invention can be performed on a large number of samples in a short time, and high throughput for liquid-liquid extraction can be achieved.

To achieve high throughput, the device should be designed to a size that fits the size of a 96-well microplate, etc., the stoppers provided at the bottom and top of the column can be linked to multiple devices, The top end plug and the column can be manufactured separately, and only the column part can be discarded for each extraction step, and the bottom end and top end plug can be designed to be used multiple times (FIGS. 4A, B, C).
Also compatible with multiple types of components of the apparatus of the first invention, the second invention, the third invention, the fourth invention, the eleventh invention, the twelfth invention and the kit of the twenty-seventh invention. Unifying the standards with the characteristics makes it possible to make a kit of the device according to the present invention, which is a great merit and is easy to become familiar with mechanization and automation.

The thirteenth invention uses an apparatus according to any one of the first invention, the second invention, the third invention or the fourth invention to remove an excess of a measurement interfering substance such as a derivatization reagent. This is a method for purifying a labeled compound, characterized by separating and removing by liquid extraction.
In this case, when separating the excess derivatization reagent and the labeled compound, which is a labeled target, it is necessary to use the difference in distribution ratio between the two types of liquids that are not mixed with each other. Many derivatization reagents exhibit high detection sensitivity when the target substance is derivatized due to absorption or fluorescence in the visible / ultraviolet wavelength region, ionicity, and significantly increased electron accepting / donating ability. Intended to have. Specifically, since it often has a hydrophobic partial chemical structure such as a heterocyclic / aromatic ring structure, it is highly soluble in low polar liquids such as organic solvents. In liquid distribution, the distribution ratio is largely biased toward the organic solvent.
When the target substance to be analyzed to which the derivatizing reagent acts is highly hydrophilic, the labeled compound, which is the product after the derivatization reaction, is more hydrophilic than the derivatizing reagent and is a liquid of water and an organic solvent. -In liquid distribution, the distribution ratio is biased toward the water side compared to the derivatization reagent. Examples of such substances to be analyzed include carbohydrates, amino acids, organic acids, amines, lipids, proteins, secondary metabolites including antibiotics, and the like.

  In addition, as a derivatization reagent, it is labeled with a partial structure that reacts with a carbonyl group, a carboxyl group, an amino group, an alcohol hydroxyl group, a thiol group, etc. of a substance to be analyzed so far and improves the detection sensitivity of an analytical instrument. For this reason, derivatization reaction can be performed using a known technique.

Here, a detection / measurement technique using a carbohydrate fluorescent reagent is taken as an example. There are many known methods for labeling the carbonyl group at the reducing end of a carbohydrate, and most of them have stable fluorescence by forming a Schiff base with a fluorescent compound having an amino group and then reducing it with a reducing agent. It is characterized by a method of conversion to a derivative.
So far, many derivatization reagents such as PA, ABEE, ABOE, ANTS, etc. have been reported and kitted. However, these reactions require the addition of a large excess of derivatization reagent to the reaction system. In conventional analysis methods and kits, in order to separate the labeled compound after derivatization from a large excess of unreacted derivatization reagent, etc., liquid-liquid extraction must be repeated, and many containers and pipettes are used. As the process to do, it was a complicated and time-consuming work.
In addition, since there is a large excess of derivatization reagent to be removed, it is not practical to remove this by solid phase extraction because it requires a large retention capacity, and derivatization often has the same partial chemical structure Due to the problem that it is difficult to obtain a difference in retention between the reagent and the labeling compound, it is difficult to apply solid phase extraction. Therefore, there is still no known method for withstanding practical use. For example, in the “ABEE sugar composition analysis kit” of Seikagaku, 200 microliters of water and 200 microliters of chloroform are added after the reaction, and after vigorous stirring and centrifugation, the aqueous layer is subjected to high performance liquid chromatography analysis. Shows a method for detecting and measuring a derivative.

Thus, after performing liquid-liquid extraction once, it must be the method of analyzing by liquid chromatography. The reason is that a large excess of ABEE precipitates when only water is added, When recovering the aqueous layer, it may be mixed as fine particles. Therefore, it is considered to be necessary to add and dissolve an organic solvent such as chloroform.
If liquid-liquid extraction is repeated, the labeling substance ABEE moves to the chloroform layer, and the purity of the sugar-derived ABEE-labeled compound in the aqueous layer gradually increases. If the labeled compound can be removed to such an extent that the presence or absence of a sugar derivative can be detected and measured by directly analyzing the absorption or fluorescence in the visible / ultraviolet wavelength region of the aqueous layer, screening for investigating the production of the substance is significantly more efficient. Turn into.

The methods of the present invention according to the thirteenth and fourteenth aspects can also be used as a pretreatment step when performing sophisticated purification of a labeled compound using a commercially available solid phase extraction column or the like. In that case, by installing a solid phase with various selectivity on the surface inside the pores as the porous material of the device, pretreatment by liquid-liquid extraction and then advanced by solid phase extraction column It is also possible to construct an apparatus for performing the purification.
As the solid phase at this time, reverse phase (octadecyl (C18), octyl (C8), phenyl group, etc.), silica gel, aminopropyl, strong cation exchange phase, weak cation exchange phase, strong anion exchange phase, weak An anion exchange phase or the like can be used. Furthermore, after selecting only the sample for which the structure information of the substance should be obtained in detail in response to the screening results, by using a liquid chromatography / mass spectrometer (LC / MS, LC / MSMS, etc.) as appropriate, Rapid structural analysis and quantitative analysis can be performed.

  In the present invention, after performing a derivatization reaction of a substance and a liquid-liquid extraction reaction in a column space for performing an exchange partition reaction by appropriately combining known analytes, derivatization reagents, and analysis techniques. Detection and quantitative analysis can be performed with an instrument analyzer. It is possible to respond accurately and seamlessly to the need for efficient liquid-liquid extraction from such screening to subsequent substance identification and quantification.

Thus, the efficiency of the liquid-liquid extraction method is directly linked to the detection / measurement method of the reaction that generates or decomposes the substance. For example, the reducing sugar detection / measurement method can be used for detection / measurement of carbohydrate-related reactions characterized by the production or decomposition of reducing sugars. Therefore, when searching for useful enzymes in microorganisms and foods in the environment, a screening kit characterized by detecting and measuring the reaction product using the detection and measurement methods described in the nineteenth to twenty-sixth aspects of the invention. Can be built.
In addition, when exogenous gene fragments are put into a vector to make a library, which is expressed in microorganisms to express the protein encoded by each gene, a gene encoding an enzyme having the desired activity is screened. The screening kit characterized by detecting and measuring the reaction product using the detection and measurement methods described in the nineteenth to twenty-sixth inventions can be constructed.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Example 1
Plastic column (1 ml reservoir, made of polypropylene, inner diameter 5.7 mm, outer diameter 7.6 mm, length 57 mm, polyethylene frit (hole diameter 20 μm, thickness 1.5 mm) fixed at the bottom Or three layers of cellulose filter paper (Toyo filter paper, 5A) or five layers of glass filter paper (Whatman Japan Co., Ltd., GF / C) were prepared from the top of the column as appropriate. .
Next, 1 milliliter of water is added from the top, and this is pressurized using a plastic syringe and adapter to remove the total amount. Then, 500 microliters of the liquid shown in Table 1 (1) is added from the top, Then, pressure was applied using an adapter to discharge the liquid from the lower end, the pressurization was stopped at a level where the water surface was on the upper end of the bottom material, and a stopper (BioRad's female luer plug) was attached to the lower end opening.
Thereafter, from the upper end, 1.00 ml of the liquid shown in Table 1 (2) is added, the lower end plug is removed to open the lower end opening, and then left to stand. The drop in liquid level was measured. The ratio with the height of the liquid level before removing the stopper at the lower end was expressed as a percentage, and the measurement was performed on three apparatuses, and the average value and the standard deviation were shown (Table 1).

  As a result of experiments, it was found that cellulose filter paper or glass filter paper was placed and pretreated with water to block most of the tested organic solvents by 90% or more. The polyethylene frit used was 100% water blocked by pretreatment with an organic solvent, but when pretreated with water, the organic solvent was not sufficiently blocked, and the total amount was 10 minutes later. It was discharged.

(Example 2)
Plugged at the bottom of a plastic column (6ml reservoir, made of polypropylene, inner diameter 13mm, outer diameter 15mm, length 79mm, with PTFE porous filter (pore diameter 1.0 micrometer) or 1PS fixed on the bottom) (BioRad, female lure plug), the liquid combinations shown in (2) of Table 2 were added from the upper end of the column, stirred by a vortex mixer for several seconds, and then opened at the lower end. In the same manner, the liquid flow after 1 minute and 5 minutes was observed.
As a result, water was not discharged and the organic solvent was quickly discharged as shown in Table 2.

(Example 3)
As in Example 2, a plastic column (6 ml reservoir, made of polypropylene, inner diameter 13 mm, outer diameter 15 mm, length 79 mm, PTFE porous filter (pore diameter 1.0 micrometer) or 1 PS is fixed to the bottom. A stopper (Bio-Rad, female luer plug) was plugged at the lower end of the column, and the solvent combinations shown in (3) of Table 3 were added from the upper end of the column. Next, after stirring with a vortex mixer for several seconds, let the device stand upright for about 5 seconds, and then tilt the device at an angle of 45 degrees so that the ethyl acetate layer is in contact with the bottom material, and then open the lower end. The liquid flow after 1 minute and 5 minutes was observed. The water level was measured by standing the apparatus upright for about 1 second, and the angle was immediately returned to 45 degrees.
As a result, water was not discharged, and the organic solvent was quickly discharged as shown in Table 3.

Example 4
Plastic column (1 ml reservoir, made of polypropylene, inner diameter 5.7 mm, outer diameter 7.6 mm, length 57 mm, polyethylene porous filter at the bottom (pore size 20 micrometers, thickness 1.5 mm 2 sheets) 7) with the same material made of a polyethylene porous filter with a hole with a diameter of about 1.5 mm in the center, put from the top of the device, and put the bottom of the polyethylene porous filter and the top 7 The layers were stacked so that the porous filters were in contact with each other.
1 ml of hexane is added to this, and after pressurizing from the upper part of the apparatus using a syringe and an adapter to discharge the liquid, the stopper (Bio-Rad, female luer plug) attached to the lower end is closed, and water 10 is added from the upper part. Microliter was added, 500 microliters of hexane was further added, and a polypropylene stopper (a stopper made by improving a polypropylene chip) was formed on the upper portion, and then upside down was repeated five times.
Thereafter, the top end plug of the device and the bottom end plug of the device were opened in this order, and the liquid discharged from the lower end was collected. When the hexane in the column was reduced by 300 microliters or more, the lower end was closed, and further 500 microliters of hexane was added from the top, and then the upper end plug was closed and inverted upside down. During the upside down, water droplets were observed in hexane. After that, the upper end plug and the lower end plug were opened in order, and the liquid discharged from the lower end was collected. As a result of examining the collected liquid of 800 microliters by visual observation and addition of water, the collected liquid was composed of an organic layer (hexane), and water discharge was not confirmed.

(Example 5)
Plastic column (1 ml reservoir, made of polypropylene, inner diameter 5.7 millimeters, outer diameter 7.6 millimeters, length 57 millimeters, polyethylene porous filter at the bottom (2 pieces of pore diameter 20 micrometers, thickness 1.5 millimeters) 2), two cellulose filter papers (Toyo filter paper, 5A) cut into a circle so as to fit the column inner diameter were placed in contact with the bottom filter.
Next, the filter paper is cut out, folded twice into a cylinder with a height of 2.6 centimeters, cut into a few millimeters at the bottom and folded inside, put into the column from the top, and the bottom It was made to contact a cellulose filter paper. In addition, two pieces of the same filter paper cut into a circle to fit the inner diameter of the cylindrical filter paper put in advance are put from the top of the column to stabilize the cylindrical filter paper, and the bottom filter paper and the side cylindrical filter paper Strengthened contact with. 1 ml of water is added to this, and the liquid is discharged from the upper part of the apparatus using a syringe and an adapter. Then, a stopper (Bio-Rad, female luer plug) is closed at the lower end, and 200 microliters of chloroform from the upper part. 1 liter and 400 microliters of water were added, a pipette was inserted from the top, the liquid was taken out and mixed.

  Then, the stopper at the lower end was opened, and the liquid discharged from the lower end was collected. When 300 microliters of water was eluted, the lower end was closed, 400 microliters of water was added, pipetting was performed again, the lower end was opened, and the liquid discharged from the lower end was recovered. Repeated times. When the recovered liquid reached 800 microliters, the composition of the recovered liquid was confirmed by visual observation and addition of chloroform. As a result, the recovered liquid was water and no discharge of chloroform was confirmed.

(Example 6)
After putting 10.0 microliters of water into a plastic tube with a 1.5 ml screw cap, the ABEE reaction reagent (Biochemical “ABEE Sugar Composition Analysis Kit”) was preheated to about 40 ° C. to dissolve the contents. 40.0 microliters was added, the lid was closed, and the mixture was stirred with a vortex mixer for several seconds, and then reacted in a heat block at 80 ° C. for 30 minutes.
Plastic column (1 ml reservoir, made of polypropylene, inner diameter 5.7 millimeters, outer diameter 7.6 millimeters, length 57 millimeters, polyethylene porous filter at the bottom (2 pieces of pore diameter 20 micrometers, thickness 1.5 millimeters) After the flow of 500 microliters of dichloromethane, the lower end was closed with a Bio-Rad plug (female luer plug). Thereafter, 150 microliters of water was added to the reaction mixture for dilution, and the mixture was added together with 500 microliters of dichloromethane for extraction. After stirring for several seconds with a vortex mixer, the lower end was opened and the dichloromethane was discharged by natural flow. .

  Subsequently, 200 microliters of dichloromethane is added from the top to wash the polyethylene filter at the bottom, then the stopper at the bottom is closed, 500 microliters of dichloromethane is added, and then stirred for several seconds with a vortex mixer. The work which opened and discharged dichloromethane by natural flow was performed. After repeating this step a predetermined number of times (specifying samples as A (0 times), B (1 time), C (2 times) and D (3 times) in the order of the smallest number of steps), the column was tilted and the aqueous layer was removed. It was collected and 10.0 microliters was analyzed by high performance liquid chromatography (HPLC).

  When the ABEE concentration was estimated from the ABEE peak area in the analytical data, it was calculated using the following calibration curve and conversion factor.

Calibration curve
(ABEE concentration (micromolar concentration)) = 0.118 x (peak area of fluorescence detector) -1.53

Conversion factor
(Peak area in UV detector) = (Peak area in fluorescence detector) × 3.90 × 10 ⁻³

As a result, the ABEE concentrations of Samples A, B, C, and D were as follows: A = 2.91 mmol concentration (remaining rate 100%), B = 103 micromolar concentration (remaining rate when compared with Sample A) 54%), C = 9.67 micromolar concentration (residual rate when compared with sample A: 3.32 × 10 ⁻¹ %), D = 1.07 micromolar concentration (residual rate when compared with sample A) 3.68 × 10-2%).

HPLC apparatus / condition apparatus: Column: manufactured by Shiseido Co., Ltd. CAPCELL PAK C18 (UG120, 5
(Micrometer, column size: 1.0mmφ × 250mm)
Pump: PX-8020 manufactured by Tosoh Corporation
Pump controller: CCPM-II manufactured by Tosoh Corporation
Degassa: Tosoh Corporation SD-8022
Column oven: CO-8020 manufactured by Tosoh Corporation
Ultraviolet / visible light detector: Tosoh Corporation UV-8020
Fluorescence detector: FS-8020 manufactured by Tosoh Corporation
Analysis: PowerChrom v2.0.7 manufactured by AD Instruments

Condition: Column temperature 45 ° C
Flow rate 1ml / min Eluent A: 0.02% Trifluoroacetic acid aqueous solution Eluent B: Acetonitrile Gradient conditions
0 min B concentration 18%
5 minutes B concentration 20%
5.01 min B concentration 50%
10 minutes B concentration 50%
10.01 min B concentration 18%
20 minutes B concentration 18%

Detection: UV (305 nanometers)
Fluorescence (excitation 305 nanometers, fluorescence 360 nanometers)

(Example 7)
10.0 microliters of glucose were prepared as aqueous solutions of 0, 200, 400, 600 and 800 micromolar and each was placed in a separate plastic tube with a 1.5 ml screw cap. Then, add 40.0 microliters of ABEE reaction reagent (biochemical “ABEE sugar composition analysis kit”), which has been preheated to about 40 ° C. and dissolved the contents, close the lid and stir with a vortex mixer for several seconds. And it was made to react for 30 minutes in an 80 degreeC heat block.
Plastic column (1 ml reservoir, made of polypropylene, inner diameter 5.7 millimeters, outer diameter 7.6 millimeters, length 57 millimeters, polyethylene porous filter at the bottom (2 pieces of pore diameter 20 micrometers, thickness 1.5 millimeters) After 200 microliters of chloroform was flowed to the fixed one), the lower end was closed with a stopper (BioRad, female luer plug). Next, 100 microliters of water was added to the reaction mixture for dilution, and the mixture was added together with 500 microliters of chloroform for extraction. After stirring for several seconds with a vortex mixer, the lower end was opened, and chloroform was discharged under natural flow. Subsequently, 200 microliters of chloroform was added from the top to wash the bottom polyethylene filter. Thereafter, the stopper at the lower end was closed, 500 microliters of chloroform was added, the mixture was stirred for several seconds with a vortex mixer, the lower end was opened, and the chloroform was discharged by natural flow.

  After repeating this process three times, the upper part of the column was tilted to recover the aqueous layer, and 9 times the amount of acetonitrile was added to obtain a sample for solid phase extraction. After washing the solid phase extraction column (Varian's “Bond Elut NH2 aminopropyl (column size 50 mg / 1 ml)”) with 1 ml of acetonitrile, the sample was added, and a syringe and adapter were used from the top as appropriate. The column was passed through under pressure. Subsequently, the column was washed with 1 ml of 90% acetonitrile, and then the ABEE glucose derivative was eluted with 1.00 ml of water. Using this ultraviolet / visible spectrophotometer (Beckman, DU-600, cell length 1.00 cm), the absorbance at 305 nanometers was measured to prepare a calibration curve.

  As a result, the following calibration curve (1) was obtained, indicating that the labeled compound can be detected and measured. In addition, the same sample was diluted 4 times with water and analyzed with an excitation wavelength of 305 nanometers and a fluorescence wavelength of 360 nanometers using a fluorescence spectrophotometer (manufactured by Shimadzu Corporation, RF-5300PC). 2) was obtained, and it was shown that detection and measurement by a fluorescence spectrophotometer are possible.

Calibration curve (1)
(Micromolar concentration of ABEE glucose in stock solution)
= 2.17 x 10 ⁴ x (absorbance at 305 nanometers) -439

Calibration curve (2)
(Micromolar concentration of ABEE glucose in the stock solution) = 77.9 × (fluorescence intensity) −4.86

(Example 8)
In order to search for another enzyme activity (xylanase activity) mixed in a commercially available cellulase preparation, 10.0 mg of a commercially available cellulase preparation (Meiji Seika Co., Ltd., “Meicelase P-1”) was dissolved in 500 microliters of water, A liquid was used. 10.0 microliters of that was added to 390 microliters of reaction stock to make a reaction solution.
The reaction solution was 20 mM sodium acetate buffer (final concentration) (pH 5.5), 1.00% (final concentration) xylan (Birchwood Xylan (Sigma)), and 10.0 microliters of enzyme solution. including.
After mixing the reaction solution with a vortex mixer, the reaction was carried out at 37 ° C. for 1 hour. In addition, the control was left to stand at 37 ° C. for 1 hour before adding the enzyme solution, then ice-cooled, and 10.0 microliter of the enzyme solution was added. Each was ultrafiltered (Millipore, Ultrafree-MC, 10,000 NMWL Filter Unit, 4,700 × g, 4 ° C., 20 minutes), and the filtrate was recovered.

  Of these, 10.0 microliters was used for ABEE conversion and purification according to the method of Example 7, and the absorbance at 305 nanometers was analyzed. As a result, the control was 0.183, and the reacted sample was 0.371. Met. This suggested the release of reducing sugars that could be derivatized by ABEE. In addition, the sample was diluted 1.6 times with water and analyzed using a fluorescence spectrophotometer (manufactured by Shimadzu Corporation, RF-5300PC) at an excitation wavelength of 305 nanometers and a fluorescence wavelength of 360 nanometers. The value was 23.1 and the value of the reacted sample was 305.

A is a conceptual diagram of an apparatus for liquid-liquid extraction or solid-liquid extraction, and B is an explanatory view of an embodiment of a stopper provided in the apparatus. A is a conceptual diagram of an apparatus for liquid-liquid extraction, and B is an explanatory view of a mode of a stopper provided in the apparatus. The concept (cross-sectional view) of the arrangement of the material having pores fixed in the bottom part of the apparatus for liquid-liquid extraction and in the column height direction is shown. In all of (1), (2), (3) and (4) in the figure, the liquid located in the upper part is in contact with the material during liquid-liquid extraction. (6) shows a conceptual diagram when a combination of a high-density liquid and a low-density liquid that form two layers without being mixed with each other is added to (5). A, B, and C show the response to high throughput of liquid-liquid extraction or solid-liquid extraction apparatus.

Explanation of symbols

1: Column (space for interfacial exchange partition reaction)
2: Opening of the upper part of the apparatus for injecting the liquid 3: Opening of the lower part that can be closed 4a: Porous material 4b having high affinity with the liquid to be discharged from the lower part of the apparatus 4b: High affinity with the liquid to be discharged from the lower part of the apparatus, Layer 5 of porous material continuous from the lower part of the apparatus 5: Plug for closing the opening at the upper part of the apparatus and the opening at the lower part of the apparatus 6: Rotary plug for closing the opening at the lower part of the apparatus 7: Liquid in the upper layer 8: Layer of the lower layer liquid

Claims (27)

  A device for liquid-liquid exchange or solid-liquid exchange distribution extraction using a combination of a high-density liquid and a low-density liquid that do not mix with each other to form a two-layer liquid, and a space for performing a solute exchange distribution reaction at the liquid-liquid interface A column having a top, an upper opening for injecting both liquids into the space, and a lower pluggable opening for discharging one of the liquids, and a space for performing a solute exchange distribution reaction with the lower opening A device in which a porous material having a high affinity with the liquid to be discharged from the lower part of the device and having a low affinity with the liquid to be retained in the column space is mounted.   The device according to claim 1, which has a function of closing an opening at an upper portion of the device.   2. The apparatus according to claim 1, wherein the porous material is continuously attached from the front of the lower part of the apparatus to the side of the space of the column in which the solute exchange distribution reaction is performed.   The apparatus according to claim 3, wherein the apparatus has a function of closing an opening at an upper part of the apparatus.   In performing liquid-liquid exchange distribution extraction using the apparatus according to claim 1, if the liquid to be discharged from the lower part of the apparatus has a higher density than the liquid to be retained in the apparatus, the porous material attached to the apparatus A liquid-liquid exchange / distribution extraction method comprising impregnating only a high-density liquid used for liquid-liquid extraction and then performing liquid-liquid partition extraction from a low-density liquid with the high-density liquid.   6. The method according to claim 5, wherein an outflow of liquid from the upper part of the apparatus is prevented during the liquid-liquid distributed extraction operation by closing the opening at the upper part of the apparatus during the liquid-liquid distributed extraction operation. .   In performing liquid-liquid exchange distribution extraction using the apparatus according to claim 3, if the density of the liquid to be discharged from the lower part of the apparatus is lower than the liquid to be retained in the apparatus, the porous material attached to the apparatus A liquid-liquid exchange distribution extraction method comprising performing liquid-liquid partition extraction from a high-density liquid with a low-density liquid after impregnating with a low-density liquid used for liquid-liquid extraction.   8. The method according to claim 7, wherein the liquid is prevented from flowing out from the upper part of the apparatus during the liquid-liquid distributed extraction operation by closing the opening at the upper part of the apparatus during the liquid-liquid distributed extraction operation. .   When performing solid-liquid exchange distribution extraction using the apparatus according to claim 1, the solid-liquid extraction is performed by replacing the liquid to be retained in the apparatus space with a solid sample in which the porous material is not clogged. Solid-liquid exchange partition extraction method.   10. The method according to claim 9, wherein the liquid is prevented from flowing out from the upper part of the apparatus during the solid-liquid distributed extraction operation by closing the opening at the upper part of the apparatus during the solid-liquid exchange distribution extraction. .   A device for liquid-liquid exchange distribution extraction, wherein a plurality of devices according to claim 1 or 2 are connected in parallel.   5. A device for liquid-liquid exchange distribution extraction, wherein a plurality of devices according to claim 3 or 4 are connected in parallel.   An operation of removing a measurement interfering substance containing an unreacted derivatizing reagent present in a reaction mixture by liquid-liquid extraction after reacting the substance with a derivatization reagent to convert it into a labeled compound and performing a reaction. 4 and a method for purifying a labeled compound, which is performed using the apparatus according to any one of claims 11 to 12.   14. The method for derivatizing a substance according to claim 13, wherein a reaction for converting the substance into a labeled compound is performed inside the apparatus.   15. The method according to claim 13, wherein the liquid containing the labeled compound after liquid-liquid extraction is analyzed with an instrument analyzer while the liquid is retained inside the apparatus.   The detection / quantification method according to claim 15, wherein a derivatizing reagent characterized by imparting ultraviolet / visible absorption characteristics is used, and a labeled compound is detected / quantified by a spectrophotometer.   16. The detection / quantification method according to claim 15, wherein a derivatization reagent characterized by imparting fluorescence emission characteristics is used, and a labeled compound is detected / quantified by a fluorescence spectrophotometer.   A derivatization reagent characterized by providing a strong ionization characteristic and / or a characteristic that gives a characteristic product ion, and detecting and quantifying a labeled compound by a mass spectrometer. 15. The detection / quantification method according to 15.   The method for purifying a labeled compound according to claim 13, wherein the substance is a reducing sugar.   15. The method for derivatizing a substance according to claim 14, wherein the substance is a reducing sugar.   The analysis method according to claim 15, wherein the substance is a reducing sugar.   17. The detection / quantification method according to claim 16, wherein the substance is a reducing sugar.   18. The detection / quantification method according to claim 17, wherein the substance is a reducing sugar.   The detection / quantification method according to claim 18, wherein the substance is a reducing sugar.   The method according to any one of claims 19 to 24, wherein the derivatization reagent is a reductive amination reaction reagent.   26. The method according to claim 25, wherein 4-aminobenzoic acid ethyl ester and a reducing agent are used as a derivatizing reagent. A column according to any one of claims 1 to 4, a stopper having a function of closing the opening at the lower end of the column, and a device for connecting and assembling a plurality of these. High-throughput kit for liquid-liquid exchange or solid-liquid exchange partition extraction.

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