JPWO2007021037A1 - INORGANIC POROUS BODY AND PROCESS FOR PRODUCING THE SAME - Google Patents

Abstract

The purpose is to easily manufacture monolithic structures which are inorganic porous materials, in particular, controlling so-called macropores made of Group 4 element compounds and nanometer-range pores, so-called mesopores, and combining them. A monolithic structure composed of a Group 4 element compound porous body, having a pore diameter of 100 nm or more and having so-called macropores that are continuous in a three-dimensional network, and is formed in a gel skeleton that forms these through-holes A so-called mesopore having a pore diameter of 2 to 100 nm, the volume ratio occupied by the pore is 10% or more with respect to the sum of the volume of the through-hole and the total pore size, and the gel The volume of the pores per 1 g of the skeleton is 10 cm 3 / g or less, and the volume ratio of the through holes is 20 to 90% with respect to the sum of the volume of the through holes and the pores.

Description

  The present invention relates to an inorganic porous material and a method for producing the same, and more particularly to an inorganic porous material prepared using a sol-gel method involving phase separation of a group 4 element alkoxide and a method for producing the same.

General chromatographic columns or adsorbents include those made of organic polymers such as styrene / divinylbenzene copolymer, those filled with an inorganic filler such as silica gel in a cylinder, and a cylindrical integrated type. A porous body is known in which the side surface is sealed so that liquid can be passed from both ends.
A column made of an organic material has problems such as low pressure resistance due to low strength, swelling / shrinking with a solvent, and inability to heat sterilize. Therefore, a column made of an inorganic material, particularly silica gel, is widely used as a column for solving such difficulties.
However, among the silica gel columns, those in which the packing material is packed in the cylinder have a drawback that the pressure required to circulate the sample in the column is high and it is not suitable for high-speed separation.
On the other hand, the formation of a pore structure and the control of a wide range of structures have already been realized in a silica-based porous body, and high performance as an integrated separation medium for liquid chromatography has been confirmed.
In particular, the inventor of the present invention, Nakanishi, in the sol-gel method by hydrolytic polycondensation of silicon alkoxide, induces phase separation by spinodal decomposition, and its excessive structure is fixed by gelation, and has a co-continuous structure. He has invented the macroporous material and has filed an international patent application. (WO2003 / 002458)
The column with an integral porous body has a low column pressure and can be separated at high speed. However, it has been found that the silica, which is a base material for both the filling type and the integral type, has a chemical property that the base material gradually dissolves and performance is impaired when used for a long time under strongly basic conditions. This is a common problem with conventional inorganic fillers based on silica.
In other words, silica is an acidic oxide and has high solubility in water. Therefore, when it comes into contact with a solvent containing water under strong basic conditions for a long period of time, the silicon-oxygen-silicon bond is cleaved from the surface. Dissolution occurs and the surface area required for material separation is lost.
Several inventions for producing a silica-titania porous body by a sol-gel method have been proposed. For example, there are JP-A-6-107461, JP-A-2001-172089, and the like.
The former is the production of titanium dioxide monolith, but heating at a high temperature is an essential condition for the production method. In such a case, it is difficult to form so-called macropores of macropores, and macroscopic fines are difficult to form. Simultaneous control of both the pores and the pores in the normal nanometer range, so-called mesopores, is extremely difficult.
The latter is a so-called silica-based porous body having a titania content of 10% or less, and the above-mentioned silica-based defects cannot be eliminated.
In addition, phosphorylation of proteins in cells has every influence on metabolism, and in order to investigate the process, it is important to analyze phosphorylated peptides as a structural analysis. The amount of phosphorylated peptide is very small compared to other mixed peptides. The sensitivity in the mass spectrometer is also low, and it is impossible to directly analyze a trace amount of phosphorylated peptide by HPLC. Therefore, a method of selectively concentrating and purifying only phosphorylated peptides by immobilized metal affinity chromatography is used.
However, this method requires a complicated process in which a phosphorylated peptide is finally obtained after immobilization of a metal, followed by an intermediate washing step, and takes time. On the other hand, a technique for analyzing these phosphorylated peptides by utilizing the property of selectively retaining the phosphate compound of titania gel has been devised.
However, the titania gel obtained from the conventional method is particulate and has few contact parts with the flowing liquid, and a titania gel having a higher selectivity has been required.

Thus, as a result of researches by the present inventors, it has been clarified that a porous body composed only of a group 4 element compound including titanium oxide can be formed by a chemical polymerization reaction. As a result, a non-silica inorganic porous body, particularly a group 4 element compound porous body, which is an integral type and whose pore diameter and pore distribution are appropriately controlled, has been completed. Conventional packed and integral silica columns Has found that it does not have the disadvantages.
The present invention has been made based on such knowledge. The object is to solve the problems of conventional silica-based porous materials and to provide an inorganic porous material, especially a Group 4 element compound porous material, which has a low pressure loss and is easy to handle. Another object is to provide an inorganic porous material that can be used for separation and adsorption without deterioration in performance even under strongly basic conditions.
Furthermore, another object of the present invention is to provide a method for producing an inorganic porous material such as titanium oxide, which is difficult to control the structure by the sol-gel method.
Furthermore, other objects of the present invention include energy conversion due to the properties peculiar to the Group 4 element porous body, provision of functional materials such as sensors, and titania generates active oxygen by ultraviolet rays to decompose organic substances. It can be used for odor and sterilization, and in chromatographs, it is used as a column to investigate the phosphorylation process of in vivo proteins by utilizing the specific adsorption property of phosphorous oxide, and zirconia is supported by sulfuric acid as a catalyst. Hafnia is to contribute to the expansion of the range of industrial use, such as its water repellency.
One of the means for achieving the above object is a group 4 element compound, having a pore diameter of 100 nm or more and having a three-dimensional network-like continuous through hole and a pore diameter of 2 to 100 nm formed in the gel skeleton forming the through hole. An inorganic porous material characterized in that the volume ratio of the pores is 10% or more with respect to the sum of the volume of the through-holes and the pores,
One is that the volume of the pores per gram of the gel skeleton is 10 cm ³ / g or less, and the volume ratio of the through holes is 20 to 90% with respect to the sum of the volumes of the through holes and the pores. Is the above-described inorganic porous body,
One of the inorganic porous materials is characterized in that the Group 4 element compound is mainly composed of titanium dioxide (TiO ₂ ), zirconium dioxide (ZrO ₂ ), or hafnium dioxide (HfO ₂ ). And
One is the inorganic porous body, wherein the group 4 element compound is titanium dioxide (TiO ₂ ),
One is the inorganic porous body, wherein the group 4 element compound is zirconium dioxide (ZrO ₂ ),
One is the inorganic porous body, wherein the group 4 element compound is made from a hydrolyzable metal compound as a raw material,
One is the above-mentioned inorganic porous body, wherein the hydrolyzable metal compound is a metal alkoxide,
One is the inorganic porous body, wherein the metal alkoxide is titanium alkoxide,
One is the inorganic porous body, wherein the metal alkoxide is zirconium alkoxide,
One is the inorganic porous material, wherein the titanium alkoxide is titanium n-propoxide,
One is the inorganic porous material, wherein the zirconium alkoxide is zirconium n-propoxide,
One is a chromatography column characterized by using the above-mentioned inorganic porous material,
One is an adsorption material for separating or concentrating a phosphoric acid compound characterized by using the above-mentioned inorganic porous material,
One is an adsorptive material for separation or concentration of a sulfuric acid compound characterized by using the above-mentioned inorganic porous material,
One of them includes a step of hydrolyzing a group 4 element compound in an acidic aqueous solution in the presence of a polar solvent, polymerizing and gelling the reaction solution, and a step of firing the generated gel. A method for producing an inorganic porous body comprising a group 4 element compound,
One is a step of hydrolyzing a group 4 element compound in an acidic aqueous solution in the presence of a polar solvent, polymerizing it to gel the reaction solution, and a step of subjecting the generated gel to a closed heat treatment. A method for producing an inorganic porous body comprising a Group 4 element compound,
One is a step of hydrolyzing a group 4 element compound in an acidic aqueous solution in the presence of a polar solvent, polymerizing and gelling the reaction solution, a step of subjecting the generated gel to a heat treatment, and A method for producing an inorganic porous body comprising a group 4 element compound, comprising a step of firing the porous gel obtained by the above steps,
One is a method for producing an inorganic porous body comprising the group 4 element compound described above, which includes a step of removing an organic component in the generated gel.
One is a method for producing an inorganic porous body comprising the group 4 element compound described above, wherein the through-hole diameter and the pore diameter are controlled by changing the concentration of water in the reaction solution,
One is that an inorganic porous body composed of the group 4 element compound described above, wherein a through-hole diameter and a pore diameter are controlled by dropping a strong acid into a reaction solution and controlling a gelation reaction rate. Manufacturing method,
One is a method for producing an inorganic porous body comprising the group 4 element compound described above, wherein the pore diameter is controlled by hermetic heat treatment,
One is a method for producing an inorganic porous body comprising the group 4 element compound described above, wherein the specific surface area of the porous body is controlled by hermetic heat treatment.
The inorganic porous material of the present invention and the column using the same have the following great effects.
Since it is an integrated column in which the inorganic porous body is partially sealed, there is no problem that the flow rate changes depending on the packing state, and the variation between lots is small.
In addition, the pressure loss is small because the through hole has a hole diameter and a volume ratio appropriately controlled. Therefore, if the inlet pressure is the same, the flow rate per unit time increases and analysis can be performed in a shorter time than in the past. In addition, since all the functional groups fixed to the pores react with the fluid, the consumption efficiency of the functional groups is high. Therefore, analysis can be performed with a short column.
In addition, it is easy to handle when used in a blood injection catheter or syringe.
Further, since the shape and size of the fluid flow path are highly uniform, the distribution of the analyte between the solution and the inner surface of the column does not vary depending on the location.
In addition, the performance of the inorganic porous material of the present invention even in basic conditions such as pH = 13 is lower than that of silica-based columns, which are deteriorated in performance under basic conditions. The performance as a separation medium and an adsorbent is not impaired.
In addition, the titania gel of the present invention has a strong selectivity for a phosphate compound, is chemically stable and is effective for sample purification and pretreatment.
Further, the zirconia gel of the present invention has a strong selectivity with respect to a sulfuric acid compound, is chemically stable and is effective for sample purification and pretreatment.
The inorganic porous material of the present invention carries enzymes such as trypsin, alkaline phosphatase and glucose isomerase, catalysts such as platinum and palladium, or functional groups such as octadecyl groups, and liquid chromatography, gas chromatography, etc. It can be suitably used for the chromatography column. Moreover, it can be used for separating or concentrating a specific phosphoric acid compound by passing the liquid while being fixed inside the syringe tip or inside the capillary. Moreover, it can be used for adsorbing and separating only those having specific properties by specific adsorption from a large number of protein mixtures.

  1 is a scanning electron micrograph of a porous titanium dioxide gel obtained according to the present invention, FIG. 2 is a mercury intrusion measurement diagram, and FIG. 3 is an adsorption-desorption isotherm diagram of a nitrogen adsorption measurement diagram of a sample. 4 is a pore size distribution diagram, FIG. 5 is a field emission scanning micrograph of the sample, FIG. 6 is a morphological change diagram due to a change in the amount of distilled water, and FIG. FIG. 8 is an X-ray diffraction pattern showing the heat treatment temperature dependency, and FIG. 9 is a scanning electron micrograph of the heat-treated porous titanium dioxide gel. 10 is a mercury intrusion measurement diagram of heat-treated porous titanium dioxide gel, FIG. 11 is a morphological change diagram due to a change in triblock polymer, and FIG. 12 is a mercury graph of the porous material obtained by the present invention. Press-fitting measurement FIG. 13 is an adsorption-desorption isotherm diagram in a nitrogen adsorption measurement diagram of a porous body, FIG. 14 is a pore size distribution diagram, (a) is a cumulative volume diagram, and (b) is a differential graph. FIG. 15 is a measurement table of nitrogen adsorption, FIG. 16 is a chromatogram showing complete separation of a phosphate compound by the monolith of the present invention, and FIG. 17 is a graph of the same substance by a titania membrane monolith. FIG. 18 is a chromatogram showing incomplete separation of the same substance by a titania membrane monolith, FIG. 19 is a chromatogram of an unpurified peptide mixture, and FIG. FIG. 21 is a chromatogram of a peptide mixture purified by the inventive monolith, FIG. 21 is a chromatogram of an unpurified peptide digest, and FIG. 22 is a chromatogram of a peptide digest purified by the monolith of the present invention. FIG. 23 is a chromatogram of a phenol / pyridine separation analysis using a silica gel packing material, FIG. 24 is a chromatogram of a phenol / pyridine separation analysis using a monolith of the present invention, and FIG. 25 is obtained according to the present invention. 26 is a scanning electron micrograph of the obtained porous body, FIG. 26 is a chromatogram of a phosphorylated sample by a column using the porous body obtained by the present invention, and FIG. 27 is obtained by the present invention. 28 is a scanning electron micrograph of a zirconium gel, FIG. 28 is a mercury intrusion measurement diagram, FIG. 29 is a crystal structure change diagram due to a change in firing temperature, and FIG. 30 shows a change in morphology due to a change in polar solvent. It is a scanning electron microscope photograph figure.

The present invention will be described in detail below. In principle, the present invention is based on the so-called sol-gel reaction of the above-mentioned Nakanishi invention.
The so-called sol-gel reaction refers to a reaction in which a low molecular weight compound that can be polymerized is produced and an aggregate or a polymer is finally obtained.
In the case of a general silicon alkoxide, the hydrolysis rate is slow, and an acid solution is also added to accelerate it, whereas the sol-gel method using a metal alkoxide causes hydrolysis of the metal alkoxide, Alcohol released reduces the proportion of water, causing polycondensation and gelation. Originally, Group 4 elemental compounds such as titanium alkoxide rapidly undergo hydrolysis and polycondensation with water alone, and solidify rapidly. Therefore, conventionally, a colloidal gel having no macropores was further condensed and gelled by sintering or the like, but the mechanical strength was weak.
Zirconium alkoxide is fired at a high temperature of 1000 ° C. or higher to create a stabilized crystal structure. However, high temperature firing causes cracks in the manufacturing process due to shrinkage. It was difficult to create.
The present invention, for example, hydrolyzes and polymerizes a group 4 element compound such as titanium alkoxide in an acidic solution to which a polar solvent is added, gels the reaction solution, and then forms an organic component in the generated porous gel. Is an inorganic porous body of a Group 4 element compound, specifically, a Group 4 element oxide, and a method for manufacturing the same.
The reaction solution is a solution containing a group 4 element compound, water, and an acidic solution to cause hydrolysis and polymerization reaction, and may be homogenized by adding a polar solvent.
Examples of the Group 4 element compound used in the present invention, specifically, the Group 4 element compound as a raw material of the inorganic porous body of the Group 4 element oxide include hydrolyzable metal compounds.
The inorganic porous body of the present invention is composed of one or more group 4 element compounds, more specifically, a group 4 element oxide, and one or more group 4 element compounds as the group 4 element compound as a raw material. In particular, a hydrolyzable metal compound can be used.
Examples of the hydrolyzable metal compound include metal alkoxide in which hydrogen of hydroxyl group of alcohol is substituted with a metal atom.
Examples of the metal alkoxide include titanium alkoxide, zirconium alkoxide, hafnium alkoxide and the like.
Examples of the titanium alkoxide include titanium n-propoxide, titanium isopropoxide, titanium n-butoxide, titanium s-butoxide, titanium t-butoxide, and the like.
Examples of the zirconium alkoxide include zirconium propoxide, zirconium isopropoxide, zirconium n-butoxide, zirconium s-butoxide, zirconium t-butoxide and the like.
Examples of the hafnium alkoxide include hafnium propoxide, hafnium isopropoxide, hafnium n-butoxide, hafnium s-butoxide, hafnium t-butoxide, and the like.
Furthermore, the mixture etc. which superposed | polymerized these suitably and raised the oxide content can be mentioned. In addition, oxychlorides of Group 4 elements such as zirconium oxychloride can be used similarly.
Moreover, Group 4 element halide compounds such as tetrachlorotitanium and tetrachlorozirconia can also be used. However, in these halide compounds, acid is generated by the progress of hydrolysis, so fine control of the acid to be added is required.
All the group 4 element compounds described so far have hydrolyzability, and serve as raw materials for the sol-gel reaction essential to the present invention, and the reaction proceeds and acts effectively. As a result, a porous body of a Group 4 element compound, specifically, a Group 4 element oxide can be produced.
In the present invention, the hydrolysis and polymerization reaction is performed under strong acid conditions in order to suppress the rapid polycondensation as described above. Under strong acid conditions, the hydrolyzed metal hydroxide becomes a relatively low-reactivity multimer and is ionized, nucleophilic attack is suppressed, and rapid polycondensation reaction is suppressed.
On the other hand, hydrolysis also occurs at the same time, and alcohol is produced, so that the acid suppressing effect is also reduced in the solution. Therefore, it is desirable to add the acidic solution dropwise so that the pH can be maintained at 3.5 or lower in order to always suppress it. In order to obtain a uniform solution, it is desirable to perform the reaction while stirring at a low temperature of −5 ° C. or more and 10 ° C. or less for 2 minutes or more.
As the acidic solution, mineral acids such as hydrochloric acid and nitric acid, organic acids such as acetic acid and citric acid and the like can be used, but a concentration of 10% by weight or more is desirable in order to obtain a sufficient suppression effect.
Next, hydrolysis is further advanced by adding water and stirring. The greater the amount of water added, the faster the hydrolysis proceeds, and the lower the acid concentration, the faster the polycondensation and the smaller the monolith through-holes. The hole diameter of the through hole can be controlled by the amount of water added.
The amount of water added depends on the volume of water contained in the initial acid solution, but the amount of water contained in the reaction solution is preferably about 2 to 100 times the molar amount of metal alkoxide in order to cause complete hydrolysis.
The addition of water while dropping is preferable because the change in the solution temperature decreases and the evaporation of the alcohol content is suppressed.
Further, in order to make the reaction solution uniform, a polar solvent such as formamide is added and stirred. Although it depends on the total amount, in order to make it uniform, stirring for 2 minutes or more while dropping is desirable. Under acidic conditions, hydrolysis of the reverse reaction tends to proceed with respect to polycondensation, and it takes a long time to solidify the reaction system by sol-gel transition, so it is desirable to neutralize with a compound generated from a polar solvent. For example, in the case of formamide, 0.5 mol or more of the added acid amount is preferable, and the acid is neutralized by ammonia generated by hydrolysis.
This homogenized reaction solution is put into a closed container according to the purpose and solidified to obtain a Group 4 element compound monolith porous body. As the shape of the sealed container, various shapes may be appropriately selected depending on the intended use, and any shape may be used. Moreover, what is necessary is just to select various materials suitably for the material of an airtight container. For example, when the monolithic porous body is used after being taken out from the sealed container, a polymer material that does not bond the inner surface of the sealed container and the monolithic porous body is preferable.
On the other hand, when a monolith porous body is formed inside the container, a container in which the inner surface of the container and the monolith porous body react, a fused silica capillary, or a container added with reactivity such as vitrification is preferable.
The through-hole and pore diameter and shape of the resulting monolith porous body can be controlled by the ratio of the alkoxide, acid, water, and polar solvent used, so the monolith porous body can be freely used according to the purpose. Can be made.
In the sol-gel reaction that solidifies by being held in a constant temperature water bath, transition and phase separation occur simultaneously, and a gel composed of a solvent-rich phase and a skeleton phase is generated. By using the acid as the reaction inhibitor, a co-continuous structure in which the through holes and the gel skeleton are continuous with each other is obtained. In the obtained gel, pores are generated without adding a polymer or the like to the gel skeleton. Although the reason for this is not clear, it is presumed that the size of the primary particles produced in the polymerization process is large.
Further, in this method, since the rate of hydrolysis and polycondensation changes depending on the ratio when a polymer is further added, the pore diameter, shape, etc. depending on the amount added from the case where no polymer is added. , And pore formation suitable for the purpose can be performed. The basic method is to create without adding polymer, but in this method of the invention, hydrolysis and polycondensation can occur simultaneously, so the skeleton formation time is predicted from the buffer effect of polymer addition, pore size, shape, etc. It becomes possible to control pore formation over a wide range.
After the formation of the gel, it is aged for about 24 hours and dried at a desired temperature to obtain a porous group 4 element oxide gel. The obtained porous group 4 element oxide gel has a structure in which through-holes having uniform pore diameters are entangled in a three-dimensional network. An organic component in the porous group 4 element oxide gel is removed and then fired to produce a group 4 element oxide porous body.
When a sealed heat treatment process in which a solidified porous gel is immersed in a basic aqueous solution and heated to 100 ° C. or higher under sealed conditions is added between the hydrolysis process and the firing process, It becomes easy to control the volume ratio. The basicity of water is not necessarily an essential condition, and is not limited to this as long as it is an aqueous solution that can fully utilize the characteristics of water.
Hydrothermal treatment, which is one of hermetic heat treatments, is known to have an erosion effect in porous materials such as silica gel, but is hardly eroded by the Group 4 element compound of the present invention. In the present invention, since hydrolysis and polycondensation occur at the same time, the skeleton is bonded in a loose state before firing, and the water is contained in the porous body or in a sealed state by adding water or the like. By performing the heat treatment, the state can be changed without changing the through hole, and the pore diameter can be freely controlled .
Since the desired pore size can be freely adjusted by selecting the type of solvent used, the heating temperature, and the reaction time, a monolith gel having a surface area according to the purpose can be produced as a result. This adjustment can be performed only by selecting the heating temperature, heating time, and water contained in the gel, but a solvent containing water can also be added, and any temperature can be used in the presence of the solvent containing water. However, it may be sealed. In addition, this process makes it possible to change the surface state and to control the interaction with the sample components.
In place of the polar solvent, a water-soluble organic polymer, a surfactant, or an amphiphilic substance may be used.
Furthermore, by adding a water-soluble organic polymer, a surfactant, an amphiphilic substance to a polar solvent, the gelation rate can be controlled by the buffering effect of these additive substances, and the pore diameter, shape, etc. Can be created in a wider range and freely.
In addition, as shown in FIG. 1, the inorganic porous body of the Group 4 element compound of the present invention has a μm-sized skeleton (gel skeleton) and a plurality of μm-sized through-holes periodically formed and entangled. The gel skeleton that forms the network structure has pores. Therefore, the inorganic porous body forms a double-hole structure having through-holes and pores serving as flow paths.
The through-hole of the present invention means a so-called macropore, and the pore of the present invention means a so-called mesopore, the through-hole opens at one end of the inorganic porous body, and is branched in the middle. It merges with other through holes and opens at the other end.
In addition, the inorganic porous body made of a Group 4 element compound of the present invention, more specifically a Group 4 element oxide, has a pore diameter and a total volume of pores formed in a through-hole and a gel skeleton that forms the through-hole. The volume ratio of the through holes with respect to the total volume when used as a column and the volume ratio of the pores with respect to the total volume of the pores and the through holes are important constituent requirements. The three-dimensional structure of the inorganic porous material or column varies depending on the reaction system composition, temperature, pH value, molecular weight of the organic polymer, and other conditions that affect the reactivity of the Group 4 element compound. Therefore, although it is difficult to uniformly describe the method of controlling the three-dimensional structure, an object having substantially the same pore diameter and the like can be provided with good reproducibility if the above-mentioned conditions are the same.
The removal of the organic polymer as the organic component from the porous gel produced as the intermediate substance can be achieved to some extent by washing the gel before drying. Further, depending on the removal component, the organic polymer as the organic component can also be removed by sealed heat treatment. However, it is more effective to completely remove the gel by heating it for a sufficiently long time to a temperature at which the organic polymer is decomposed or burned after the washing process.
The organic polymer used in the production of the inorganic porous material by the Group 4 element compound of the present invention, specifically the Group 4 element oxide, is a water-soluble organic polymer that can be theoretically formed into an aqueous solution having an appropriate concentration. In addition, it is sufficient that it can be uniformly dissolved in a reaction system containing an alcohol generated by hydrolysis of a group 4 element compound. Specifically, a sodium salt or potassium salt of polystyrenesulfonic acid which is a polymer metal salt Polyacrylic acid that is a salt, a polymer acid that dissociates to form a polyanion, a polymer base that produces a polycation in an aqueous solution, polyallylamine and polyethyleneimine, or a neutral polymer that has an ether bond to the main chain Polyethylene oxide having pyrrolidone, polyvinylpyrrolidone having a pyrrolidone ring in the side chain, and the like are suitable. In addition, diblock or triblock copolymers (pluronic F127, P123, F68, L122, L121, etc., which are composed of block chains of polyethylene glycol and polypropylene glycol containing polyether, for example, polyether BASF, all of which are German BASF Company products) are also preferably used. Furthermore, cationic surfactants such as alkylammonium halides, anionic surfactants such as sodium dodecyl sulfate, and amphoteric surfactants such as lauryldimethylaminoacetic acid (LDA) can be used in the same manner.
Instead of the organic polymer, polar solvents such as formamide, acetamide, N-methylformamide, and acid amides such as N-methylacetamide, polyhydric alcohols such as ethylene glycol and glycerin, sugar alcohols, and the like can also be used.
In addition, when using the inorganic porous body by the group 4 element compound of this invention as a column, if the three-dimensional structure satisfies said conditions, the effect mentioned later will be exhibited. Therefore, the manufacturing method is not limited.
The titania gel made by the method of the present invention is a monolithic structure having a three-dimensional network structure, has a property of selectively retaining a phosphate compound, has many contact portions that contribute to the separation, and has conventionally been used as a gel for chromatography. Stronger selectivity can be obtained. In addition, compared with conventional silica gel and titania-coated silica gel, the gel of the present invention is made of pure titania and has high chemical stability. Unlike immobilized metal affinity chromatography, it can be concentrated and eluted only with a buffer. The purity of the obtained phosphopeptide can be increased, and the eluate does not contain impurities, and is effective for sample purification and pretreatment.
The through-holes and pores of the inorganic porous body of the Group 4 element compound of the present invention will be described focusing on the action when the inorganic porous body is used as a column. A fluid such as a sample solution enters from one end of the column, passes through a three-dimensional continuous through-hole, and exits from the other end. During the passage, there is no extremely narrow flow path as in the case of using beads in a conventional packed column, and the hole diameter of the through hole is 100 nm or more, so the resistance to the fluid is small. Therefore, the pressure loss is small.
If the hole diameters of the through holes are the same, it is preferable that the volume ratio of the through holes with respect to the entire column is higher because the pressure loss becomes smaller. However, if the volume ratio exceeds 90%, the mechanical strength is significantly impaired, It becomes difficult to construct a column for chromatography. On the other hand, if the volume ratio is less than 20%, the pressure loss is larger than that of the packed column. The range of the volume ratio suitable for chromatography is 50 to 80%.
And since the pore is formed in the gel frame | skeleton which forms a through-hole, a specific surface area is high. Therefore, if a functional group such as an octadecyl group is fixed to the pore by chemical modification, or an enzyme such as glucose isomerase or trypsin, or a catalyst such as platinum or palladium is supported, the fluid passes through. Reacts efficiently with these molecules. Moreover, since the functional group is fixed in the pores, the functional group is not flowed even if the fluid flow is fast. In the case the inorganic porous material of the present invention is titanium dioxide as a main component Anata Ze crystalline phase can specifically adsorb a compound having a phosphoric acid group in a solution, liquid chromatography It is possible to selectively concentrate and detect the substance by applying the above.
However, the volume ratio of the pores in all pores including the through holes and the pores needs to be 10% or more. If it is less than 10%, even if the volume ratio of the through holes is increased to 90%, the functional groups can hardly be fixed. On the other hand, the pore volume per gram of gel skeleton needs to be 10 cm ³ / g or less. When the pore volume per gram of the gel skeleton exceeds 10 cm ³ / g, the mechanical strength is remarkably impaired, making it difficult to form a column for chromatography alone. The pore volume ratio and volume range suitable for chromatography are a volume ratio of 50% or more with respect to all pores and a pore volume of 2 cm ³ / g or more per gram of gel skeleton.
In addition, even if the volume ratio of the through-holes and the total volume of the pores with respect to the entire column are higher than the preferable upper limit value, within the scope of the present invention, mechanical reinforcement is performed by covering the outer periphery with a cylindrical body. Therefore, it can be applied to chromatography and the like.
Further, when the inorganic porous material of the present invention is a group 4 element compound, for example, titanium dioxide, the size of the through hole is set to approximately 10 μm or more, and the ratio is increased so that the body fluid circulates. By embedding in, it can be used as a drug sustained-release carrier that is harmless to the living body. Titanium oxide is very little eluted into body fluids under normal biological conditions and is often below the detection limit. When the surface crystal phase is appropriately controlled on the porous titanium oxide, an apatite layer, which is a bone component, is formed on the porous titanium oxide in the living body, and the living body attaches it to its own bone. To be regarded, it is compatible with living tissue without causing any rejection reaction. Therefore, this type of titanium oxide porous material can be used as a biological tissue-compatible substrate or a safe drug sustained-release carrier that does not cause an unfavorable rejection due to implantation in a living body.
Zirconium dioxide is effective for the separation and selective collection of sulfate compounds, but has the property of taking in and solidifying other metals, has high chemical stability, and can be effectively used as a waste treatment. Furthermore, in the method of the present invention, since the whole can be made in a free shape, a porous body corresponding to the application can be obtained.

<Example 1>
First, 2.645 mL of a 35 wt% aqueous hydrochloric acid solution was added dropwise to 17.404 g of titanium (IV) n-propoxide (manufactured by Aldrich) and stirred for 5 minutes. Distilled water (20.404 mL) was added dropwise thereto and stirred for 5 minutes. Further, 1.203 mL of formamide was added dropwise and stirred for 5 minutes. The obtained transparent solution was transferred to a closed container and kept in a constant temperature water bath at 30 ° C., and then solidified after about 1 hour. The solidified sample was aged for 24 hours and dried at 60 ° C. Thereby, a porous titanium dioxide gel was obtained.
Scanning electron microscope (Hitachi, S-2600N) diagram (5000 times) shows that the obtained porous titanium dioxide gel has through-holes of about 2 μm that are entangled in a three-dimensional network. 1) and a measurement diagram (FIG. 2) measured with a mercury intrusion device (Micromeritics, PORESIZER 9320). The porous titanium dioxide gel dried at 60 ° C. was heated to 300 ° C. at a temperature rising rate of 60 ° C./h. It was confirmed by nitrogen adsorption measurement (measurement of porous titanium dioxide gel heat-treated at 300 ° C.) that there were many pores of about 5 nm on the inner wall of the through-hole (adsorption-one-desorption isotherm shown in FIG. 3). FIG. 4 is a pore size distribution diagram). The pore volume and pore diameter of the porous titanium dioxide gel dried at 60 ° C. are almost the same as those of the porous titanium dioxide gel after the heat treatment, and the presence of the pores is determined by a field emission scanning electron microscope (JEOL, JSM-6700F) (FIG. 5).
It was confirmed by an X-ray diffractometer (Rigaku, RINT) that an anatase-phase titanium dioxide nanocrystal was formed on the porous titanium dioxide gel dried at 60 ° C. (FIG. 8). Applying the Scherrer equation using the half-width of the X-ray diffraction peak, the crystallite size of the nanocrystal was estimated to be about 10 nm. After heating to 300 ° C., 500 ° C., 700 ° C., and 900 ° C. at a rate of temperature increase of 60 ° C./h and holding at each temperature for 2 hours, X-ray diffraction measurement was performed. In the porous titanium dioxide gel heated at 300 ° C. and 500 ° C., anatase phase titanium dioxide was formed, but it was confirmed that the structure was transformed into the rutile phase by heat treatment at 700 ° C. and 900 ° C. or higher.
In the porous titanium dioxide gel heat-treated at 900 ° C., the through-holes entangled in a three-dimensional network are maintained, and the diameter of the through-hole is almost the same as that of the dried porous titanium dioxide gel. It was confirmed by (FIG. 9) and the figure (FIG. 10) which measured mercury intrusion.
<Example 1-a>
When distilled water is added to the reaction solution up to 21.214 mL and solidified, the larger the amount of water, the smaller the through-hole diameter of the porous body obtained, and this is continuously controlled to a minimum of about 0.5 μm. (Fig. 6).
FIG. 6A shows a case where 20.404 mL of distilled water is added, and through-holes having a thickness of about 2 μm are present in a three-dimensional network state. Fig. 6-b shows a distilled water amount of 20.944 mL, and Fig. 6-c shows a water amount of 21.214 mL. It can be seen that the through holes change in order.
<Example 1-b>
In addition, the amount of 35 wt% hydrochloric acid aqueous solution used was changed from a minimum of 2.436 mL to a maximum of 2.806 mL, and the through-hole diameter of the generated porous body could be controlled in a range from a maximum of 5 μm to a minimum of 0.2 μm. (FIG. 7 by a scanning electron microscope).
FIG. 7A shows a case where 2.754 mL of hydrochloric acid is added, and through-holes of about 3 μm are present in a three-dimensional network state. Fig. 7-b shows a case where the amount of hydrochloric acid is 2.645 mL, and Fig. 7-c shows a case where the amount of hydrochloric acid is 2.436 mL.
<Example 1-c>
Further, even if the reaction temperature is changed from 40 ° C. to 80 ° C., the through-hole diameter can be controlled within the same range as above by adjusting the concentration of distilled water and the concentration of 35 wt% hydrochloric acid aqueous solution. did it.
<Example 2>
First, 1.624 mL of 35% by weight aqueous hydrochloric acid solution was added dropwise to 11.602 g of titanium (IV) n-propoxide (manufactured by Aldrich) and stirred for 5 minutes. To this, 0.10 g of a triblock copolymer (F68, manufactured by BASF Germany) was added, and 14.071 mL of distilled water was added dropwise and stirred for 5 minutes. Further, 0.738 mL of formamide was added dropwise and stirred for 5 minutes. The obtained transparent solution was transferred to a sealed container and kept in a constant temperature water bath at 40 ° C., and solidified after about 1 hour. The solidified sample was aged for 24 hours and dried at 60 ° C. Thereby, a porous titanium dioxide gel was obtained.
It was confirmed by an electron microscope and mercury intrusion measurement that the obtained porous titanium dioxide gel had through-holes of about 3 μm in a structure entangled in a three-dimensional network. The sample dried at 60 ° C. was heated to 300 ° C. at a temperature increase rate of 60 ° C./h. It was confirmed by the measurement with a nitrogen adsorption device (Micromeritics, ASAP-2010) that there were many pores of about 5 nm on the inner wall of the through hole. The pore volume and pore diameter of the sample dried at 60 ° C. were almost the same as those of the porous titanium dioxide gel after heat treatment.
It was confirmed by X-ray diffraction measurement that anatase phase titanium dioxide nanocrystals were formed on the porous titanium dioxide gel dried at 60 ° C. Applying the Scherrer equation using the half-width of the X-ray diffraction peak, the crystallite size of the nanocrystal was estimated to be about 10 nm. After heating to 300 ° C., 500 ° C., 700 ° C., and 900 ° C. at a rate of temperature increase of 60 ° C./h and holding at each temperature for 2 hours, X-ray diffraction measurement was performed. In the samples heated at 300 ° C. and 500 ° C., anatase-phase titanium dioxide was formed, but it was confirmed that the heat treatment at 700 ° C. and 900 ° C. caused a structural transition to the rutile phase.
In the sample heat-treated at 900 ° C., it was confirmed by an electron microscope and mercury intrusion measurement that the through-holes entangled with the three-dimensional network were maintained and the diameter of the through-hole was almost the same as that of the dried sample.
<Example 2-1>
When distilled water was added to the reaction solution up to a maximum of 14.630 mL and solidified, the through-hole diameter of the resulting porous body was reduced and could be continuously controlled to a minimum of about 0.5 μm.
<Example 2-2>
Further, the amount of the 35 wt% hydrochloric acid aqueous solution used is changed from a minimum of 1.495 mL to a maximum of 1.691 mL, or the amount X of F68 (triblock copolymer) is changed from a minimum of 0.06 g to a maximum of 0.12 g. The morphological change due to the change in the amount of F68 produced is shown (FIG. 11). The through-hole diameter of the porous body could be controlled in the range from a maximum of 5 μm to a minimum of 0.2 μm. Furthermore, even if the reaction temperature is lowered to 30 ° C. or raised in the range of 50 ° C. to 80 ° C., the same as above can be achieved by adjusting the concentration of distilled water and the concentration of 35% by weight hydrochloric acid aqueous solution. The through hole diameter could be controlled within the range.
<Example 2-3>
A monolith gel was prepared by coexisting water-soluble organic polymer polyethylene oxide in a polar solvent.
First, 5.8265 mL of 35 wt% hydrochloric acid aqueous solution was added dropwise to 34.807 g of titanium (IV) n-propoxide (manufactured by Aldrich) and stirred for 5 minutes. To this, polyethylene oxide (manufactured by Aldrich) MW: 10,000, 1.05 g was added, and 1.39355 mL of distilled water was added dropwise, followed by stirring for 5 minutes. Further, the amount of N-methylformamide was added dropwise and stirred for 5 minutes. When the obtained transparent solution was transferred to a sealed container and kept in a constant temperature water bath at 40 ° C., it solidified after about 1 hour. The solidified sample was aged for 24 hours and dried at 60 ° C. Thereby, a porous titanium dioxide gel was obtained.
By changing the addition amount (X) of N-methylformamide, the through-hole diameter of the porous body can be controlled in the range from 5 μm at the maximum to 0.2 μm at the minimum. (Fig. 12).
The experimental conditions of the curves indicated by 1 to 5 in FIG. 12 are as follows.
1: X = 4.8206 mL (0.081 mol)
2: X = 4.9999 mL (0.084 mol)
3: X = 5.1776 mL (0.087 mol)
4: X = 5.3562 mL (0.090 mol)
5: X = 5.5347 mL (0.093 mol)
It was proved that pore size can be made freely even in the presence of water-soluble organic polymer.
<Example 2-4>
In the same manner as in Example 2-3, a titania porous body having through-holes with controlled pore diameters and the like was obtained, and further heat treatment (hereinafter referred to as “sealed heat treatment”) was performed in a sealed container before firing. By doing so, the pores were adjusted.
First, 1.942 mL of 35 wt% hydrochloric acid aqueous solution was dripped at 11.602 g of titanium (IV) n-propoxide (manufactured by Aldrich) and stirred for 5 minutes.
To this, polyethylene oxide (manufactured by Aldrich) MW: 10,000, 0.35 g was added, 1.39355 mL of distilled water was added dropwise, and the mixture was stirred for 5 minutes. Further, 1.7854 mL of N-methylformamide was added dropwise and stirred for 5 minutes. The obtained transparent solution was transferred to a sealed container and kept in a constant temperature water bath at 60 ° C., and gelled after about 1 hour. The gelled sample was aged for 24 hours and dried at 60 ° C.
Furthermore, this porous body was put into a pressure-resistant and sealable container without drying, and was sealed and heated. After aging, the porous body was subjected to sealed heat treatment at 100 ° C., 150 ° C., and 200 ° C., and each was baked at 400 C. After aging, the porous body was dried at 60 ° C. without performing the sealed heat treatment, aging Thereafter, a total of 8 types of porous bodies, which were dried at 60 ° C. and fired at 400 ° C., were not subjected to hermetic heat treatment.
Each porous body was measured with a nitrogen adsorption device (Micromeritics, ASAP-2010) and the surface area and pore diameter were measured with the nitrogen adsorption measurement device described above. As a result, an adsorption-desorption isotherm (FIG. 13), a pore size distribution diagram ( Cumulative volume diagram 14 (a), differential volume diagram 14 (b)) were obtained.
In the adsorption-desorption isotherm shown in FIG. 13, hysteresis is observed in the data of each porous body. This indicates that the pore size becomes larger as observed on the high pressure side, and that the pore size is more uniform as the increase in the adsorption isotherm (the lower curve of the two) becomes steep. It can be seen that when the sealed heat treatment is performed, the pore size increases and the sizes are relatively uniform. In addition, each data is 150cm in order from (1) because it is difficult to see if they are produced according to the obtained numerical value. ³ The display is shifted up by / g.
In the cumulative pore volume shown in FIG. 14A, the size and distribution of the pores can be evaluated by the rising position and the rising direction of the curve, and it can be seen how much volume the pores occupy at the final arrival point. The porous body (7) dried at 60 ° C. after hermetic heat treatment at 200 ° C. has a relatively sharp curve with a rising edge of about 10 nm. On the other hand, even when the pore size is reduced, Therefore, it is confirmed that there is a distribution although the pore sizes are uniform to some extent.
In addition, it is confirmed that the porous body (7) has the largest volume occupied by the pores among the eight porous bodies.
In addition, it can be seen from the data of (8) that heat treatment is performed at 400 ° C. after hermetic heat treatment at 200 ° C., and that the pores are hardly lost by the heat treatment.
Furthermore, it can be seen that the porous body (1) dried at 60 ° C. has almost no pores.
In the differential pore volume shown in FIG. 14-2, the average pore size is confirmed at the peak position.
Further, it is confirmed that the narrower the peak width, the more uniform the pore size and the smaller the distribution. In the porous body (7) dried at 60 ° C. after sealing heat treatment at 200 ° C. and the porous body (8) sealed at 200 ° C. and heat-treated at 400 ° C., the average pore size was 10 nm. is there.
The numerical values obtained from these figures are tabulated and shown in FIG.
Putting it all together,
In the dry state at 60 ° C., the pore diameter and specific surface area are scarcely present, but it can be seen that the specific surface area and pore diameter increase when the sealed heat treatment is performed. In order to enhance the effect peculiar to the Group 4 element compound, it is understood that a porous body having a large specific surface area, which increases the chance of contact with various samples, is suitable, and this hermetic heat treatment is effective. Furthermore, the pore diameter can be controlled according to the processing temperature.
Furthermore, the pore diameter can be increased by firing after the hermetic heat treatment.
When the firing heating temperature is changed, the pore diameter can be changed according to the purpose without greatly changing the through-holes. Although it is a well-known fact that the hermetic heat treatment has an effect of eroding the porous body, it has also been proved that the through-hole hardly changes, so that it is not an erosive action. Furthermore, by adding a firing step, the pore diameter can be increased, and it was proved that a porous body according to the purpose can be controlled and created.
After drying at 60 ° C., 20% by weight of ion-exchanged water was added to the weight of the porous body, and the same treatment was performed, but the same result was obtained. It was demonstrated that the specific surface area can be controlled.
<Example 3>
First, 2.645 mL of 35% by weight hydrochloric acid aqueous solution was added dropwise to 17.404 g of titanium (IV) n-propoxide (manufactured by Aldrich) and stirred for 5 minutes.
Distilled water (20.474 mL) was added dropwise thereto and stirred for 5 minutes, and then formamide (1.203 mL) was added dropwise and further stirred for 5 minutes to homogenize to obtain a sol-gel reaction solution. 5 mL of this reaction solution is placed in a cylindrical sealed container having a length of 200 mm and an inner diameter of 6 mm. The container is placed in a constant temperature water bath, and the temperature of the sol-gel reaction solution is maintained at 30 ° C. for 1 hour, followed by hydrolysis and polycondensation. And solidified, aged for 24 hours, and then dried at 60 ° C. to obtain a column-shaped sample.
<Example 4>
In Example 3, the obtained cylindrical monolith gel was heat-treated at 400 ° C. to obtain a hard monolith gel having an anatase phase of 2.1 mm in outer diameter. The sample was cut into a length of 150 mm, inserted into a polypropylene tube, heated at 150 ° C., and the polypropylene tube was contracted to form a column. Connection joints were attached to both ends, HPLC analysis was performed, and the retention of the phosphate compound was compared with that of a conventional type column.
As a comparative example, a titania-coated monolith was prepared by coating titanium monosilica (IV) isoproxide on a silica monolith having an outer diameter of 2.1 mm and a length of 150 mm with a pore size of 10 nm and heat-treating at 400 ° C. In the same manner as in Example 4. An HPLC column packed with 2.1 mm × 150 mm of a titania gel having a pore size of 10 nm and a particle diameter of 5 μm was prepared as a columnar column and a particle packed type column.
Adenosine without phosphate group, adenosine monophosphate with one phosphate group attached, adenosine diphosphate with two phosphate groups attached, and adenosine triphosphate with three phosphate groups attached in three columns The fade was analyzed and compared as a sample (each 1 μg / mL).
Experimental device:
Semi-micro HPLC compatible pump PU712,
UV-visible detector UV702 (semi-micro cell),
Data station EZChrom Elite (manufactured by GL Sciences Inc.),
Low diffusion stainless steel injector RHEODYNE 8125 (manufactured by Leodyne)
Experimental conditions:
Eluent 0.1M phosphate buffer (pH 7) / acetonitrile = 50/50
Flow rate 0.2mL / min
UV detection 260nm
Injection volume 0.1 μL
Column temperature Room temperature
The analysis result by Example 4 with this invention titania monolith is shown in FIG. 1-4 represent the following in the figure.
1: adenosine, 2: adenosine monophosphate, 3: adenosine diphosphate, 4: adenosine triphosphate
It was found that all four components were completely separated by the degree of phosphorylation and had high selectivity. This is probably because the contact with the sample is large because of the three-dimensional network structure.
The titania coating type of the comparative example has a property of retaining a phosphate compound, but adenosine monophosphate having a single adenosine and phosphate group does not separate into one peak (FIG. 17).
From this, it was found that even when a titania film having a similar structure was formed on the surface of a silica monolith, the inside of the pores was not coated and the selectivity was low.
In the titania particle type of another comparative example, although separation of four components was observed, the selectivity was lower than that of the monolith type of the present invention (FIG. 18).
It has been found that the titania monolith of the present invention is more selective than the silica coating type and the titania particle type, and has a large selective retention ability of the phosphate compound.
<Example 5>
To 17.404 g of titanium (IV) n-propoxide (manufactured by Aldrich), 2.645 mL of a 35 wt% hydrochloric acid aqueous solution was added dropwise and stirred for 5 minutes. Distilled water (20.404 mL) was added dropwise thereto and stirred for 5 minutes, and then formamide (1.203 mL) was added dropwise and further stirred for 5 minutes to homogenize to obtain a sol-gel reaction solution. 5 mL of this reaction solution is placed in a cylindrical sealed container having a length of 200 mm and an inner diameter of 6 mm, and the container is placed in a constant temperature water bath, and the temperature of the sol-gel reaction solution is maintained at 30 ° C. for 1 hour, followed by hydrolysis and condensation polymerization. And solidified, aged for 24 hours, and then dried at 60 ° C. to obtain a rod-shaped titania monolith having a through hole diameter of 10 μm and an outer diameter of 3.3 mm.
This monolith was cut to a thickness of 1.5 mm with a diamond cutter to obtain a disk-like monolith. Baking at 450 ° C. gave a filter-like hard titania monolith having a diameter of 2.8 mm and a thickness of 1.2 mm.
Further, a titania-coated monolith was prepared by coating titanium (IV) isoproxide on a disk-like silica monolith having the same through-hole diameter and heat-treating at 400 ° C.
When these monoliths were continuously stirred in a 0.1 M aqueous sodium hydroxide solution overnight, they were dissolved in the titania coating type.
The titania monolith of the present invention was proved to be stable in an alkaline solution with no change in weight.
<Example 6>
In Example 5, the filter-like monolith stirred overnight in an alkaline solution was fused to a pipette tip with an ultrasonic fuser.
Using a pipette tip fused with this titania monolith as a pipetter, it was examined whether the phosphorylated peptide could be selectively concentrated and purified.
The concentration and purification process is as follows.
1. Adjust the pipetter scale to 200 μL.
2. Pipet 3 times with 50% aqueous acetonitrile (0.1-1% formic acid) for conditioning.
3. Pipette sample 20-25 times for adsorption.
4). Pipette with 50% acetonitrile + 0.5 KCl aqueous solution (0.1% formic acid) for washing.
5. For elution, pipette 10 times with 0.2 M phosphate buffer (pH 7.0) to obtain a sample.
10 μL of a mixed sample of standard phosphorylated peptide (TRDIYpETDYYRK) (0.01 mg / mL) and non-phosphorylated peptide (TRDIYETDYYRK) (0.05 mg / mL) was added to Inertsil® WP300 C185 μm (150 × 2.1 mm I.D. D.) Using a column, a 15-minute gradient analysis from 10% acetonitrile with 0.1% trifluoroacetic acid to 40% acetonitrile with 0.1% trifluoroacetic acid was performed, and detection was performed at UV 210 nm.
Experimental device:
HPLC pump GL7411 (built-in low pressure gradient unit),
Column oven GL-7430,
Autosampler GL-7420,
UV detector GL-7450
Dedicated software EZChrom Elite (manufactured by GL Sciences Inc.)
FIG. 19 shows the analysis result of an unpurified mixed sample that is not concentrated and purified. FIG. 20 shows the analysis result of the mixed sample purified by the above pipetting method using the titania monolith gel of the present invention. In FIG. 20, 1 and 2 represent the following.
1: Phosphorylated peptide
2: Non-phosphorylated peptide
Although the non-phosphorylated peptide is detected as it is in the sample that is not purified, it can be seen in FIG. 20 that only the phosphorylated peptide is purified and concentrated. The recovery rate was about 70%.
It was confirmed that the titania monolith of the present invention can purify phosphorylated peptides efficiently and with a high recovery rate.
Furthermore, in the same manner, a peptide obtained by trypsin digesting 50 μg of a β-casein (0.5 mg / mL) solution, which is an actual protein, was prepared as a sample and subjected to HPLC analysis.
10 μL of sample was transferred from 10% acetonitrile with 0.1% trifluoroacetic acid to 90% acetonitrile with 0.1% trifluoroacetic acid using an Inertsil® WP300 C185 μm (150 × 2.1 mm ID) column. A 15 minute gradient analysis was performed and detected at UV 210 nm. The apparatus used for the experiment is the same as that used for the analysis of the standard product.
Analysis of casein peptide digests without purification does not allow detection of phosphorylated peptides due to the inclusion of other peptides (FIG. 21).
However, as a result of pretreatment and concentration with a pipette tip fused with the titania monolith of the present invention, two phosphorylated peptides FQpSEEEQQQTEDELQDK (molecular weight: 2061) in the digested peptide of β casein,
RELEELNVPGEIVEpSLpSpSpSpSEESLTR (molecular weight: 3122) could be detected and identified (FIG. 22). In FIG. 22, 1 and 2 represent the following.
1: FQpSEEEQQQTEDELQDK (molecular weight: 2061)
2: RELEELNVPGEIVEpSLpSpSpSpSEESLTR (molecular weight: 3122)
Selective purification and recovery of trace amounts of phosphorylated peptides in protein digests of actual samples But It has been proved that it can respond well to in vivo metabolism studies.
Similar results can be obtained with titania-coated monoliths, but in biological samples that have been subjected to alkaline hydrolysis or the like, the monolith is dissolved and purification after neutralization is required.
The purification using the monolith of the present invention has alkali durability, does not require a neutralization step, and does not require complicated steps such as immobilized metal affinity chromatography, and easily and selectively purifies phosphorylated peptides. it can.
A Hamilton microlabaster, which is a pretreatment automation device, was used, and the titania monolith of the present invention was fused to the dedicated chip in the same manner to constitute a pretreatment chip to be used. Further, the above-described manual pipetting protocol was input into the apparatus, and purification was performed automatically. As a result of analysis, an equivalent chromatogram could be obtained.
As described above, the pretreatment can be automated by incorporating the titania monolith of the present invention into the automation apparatus.
<Example 7>
To 1.7 kg of titanium (IV) n-propoxide (manufactured by Aldrich), 264 mL of a 35 wt% hydrochloric acid aqueous solution was added dropwise and stirred for 5 minutes. 2 L of distilled water was added dropwise thereto and stirred for 5 minutes, and then 150 mL of formamide was added dropwise and further stirred for 5 minutes to homogenize to obtain a sol-gel reaction solution. 500 mL of this reaction solution is placed in a cylindrical sealed container having a length of 200 mm and an inner diameter of 60 mm, the container is placed in a constant temperature water bath, and the temperature of the sol-gel reaction solution is maintained at 30 ° C. for 1 hour, followed by hydrolysis and condensation polymerization. And solidified, aged for 24 hours, and then dried at 60 ° C. to obtain a cylindrical monolith gel. Heat treatment was performed at 400 ° C. to obtain a hard monolith gel having an anatase phase and an outer diameter of 20 mm. The sample was cut into a length of 150 mm, inserted into a polypropylene tube, heated at 150 ° C., and the polypropylene tube was contracted to form a column.
This titania monolith column was chemically modified and compared with a conventional silica filler.
1 wt% butadiene monomer and 0.01 wt% azobisisobutyronitrile were dissolved in 1 L hexane based on the weight of titania monolith. While immersing the gel in this solution and reducing the pressure, the hexane solvent was evaporated and the reaction was carried out at 120 ° C. for 12 hours to chemically bond the butadiene polymer.
This chemically modified titania monolith was columned in the same manner as in Example 4.
As a comparative example, silica particles having a pore diameter of 10 nm were subjected to the same coating treatment and packed in a preparative column having an inner diameter of 20 mm and a length of 150 mm. 1 ml of a mixture of phenol, which is a weakly acidic sample, and pyridine, which is a basic sample, was injected, and fractionated with a 30% methanol eluent at a flow rate of 20 mL / min and detected at 300 nm UV.
Experimental device:
HPLC preparative system PLC561 (manufactured by GL Sciences Inc.)
1 and 2 shown in FIGS. 23 and 24 represent the following.
1: Phenol
2: Pyridine
FIG. 23 shows the analysis using the silica gel filler of the comparative example, but the pyridine which is a basic compound is adsorbed due to the influence of the silanol group and the peak is tailed. In the column chemically modified to the titania gel of the present invention, the reverse phase retention of phenol was obtained in the same manner, and further, there was no adsorption of pyridine and it was eluted quickly (FIG. 24).
The titania monolith of the present invention can add various characteristics by combining chemical species as in the conventional gel. There is no adsorption active site like silica gel, high chemical durability, no gel dissolution, and suitable for preparative chromatography used for purification.
<Example 8>
To 11.602 g of titanium (IV) n-propoxide (manufactured by Aldrich), 1.9422 mL of a 35 wt% hydrochloric acid aqueous solution was added dropwise and stirred for 5 minutes. To this, polyethylene oxide (manufactured by Aldrich) MW: 10,000, 0.35 g was added, 1.39355 mL of distilled water was added dropwise, and the mixture was stirred for 5 minutes. Further, 1.7854 mL of N-methylformamide was added dropwise, and the mixture was further stirred and homogenized for 5 minutes to obtain a sol-gel reaction solution. Place 5 mL of this reaction solution in a PFA (perfluoroalkoxyalkane) tube with a length of 150 mm and an inner diameter of 6 mm, and seal both ends. Put the tube in a constant temperature water bath and keep the temperature of the sol-gel reaction solution at 30 ° C. for 1 hour. Then, it was subjected to hydrolysis and polycondensation, gelled, put into a stainless steel pressure vessel having an inner diameter of 50 mm and a length of 150 mm, subjected to hermetic heat treatment at 200 ° C., and fired at 400 ° C.
Specific surface area 153m ² A controlled porous body of 3.9 mm × 50 mm having an average pore diameter of 13 nm and a through-hole diameter of 5 μm was obtained. A scanning electron micrograph of the obtained sample is shown in FIG.
The obtained porous body was inserted into a polypropylene tube, heated at 150 ° C., and the polypropylene tube was contracted to form a column. Attach connecting joints at both ends, perform HPLC analysis, adenosine monophosphate with one phosphate group, adenosine diphosphate with two phosphate groups, adenosine triphosphate with three phosphate groups Were analyzed and compared as samples (each 1 μg / mL). The apparatus used for the experiment is the same as that used in Example 4.
Experimental conditions:
Eluent 0.1M phosphate buffer (mixed with 1 potassium phosphate and 2 potassium phosphate) pH = 7 / ACN = 50/50
Detection wavelength 280nm
room temperature
Flow rate 0.5mL / min
Detection UV280nm
Injection volume 5μL
Column temperature Room temperature
FIG. 26 shows the analysis result of titania monolith in which the pore diameter is controlled by the hermetic heat treatment of the present invention. In FIG. 26, 1-3 represent the following.
1: Adenosine monophosphate
2: Adenosine diphosphate
3: Adenosine triphosphate
Since the pore size is appropriately controlled, the three components can be separated in a shorter time depending on the degree of phosphorylation. Compared to the packing type (Fig. 18), there is no tailing and the chromatogram has good objectivity. Obtained. It was demonstrated that the surface active state can be changed by hermetic heat treatment, and that a column applicable to high-speed analysis can be created by controlling the pore diameter.
<Example 9>
A zirconium dioxide monolith gel was prepared by coexisting polyethylene oxide, which is a water-soluble organic polymer, in a polar solvent in the same manner as titanium dioxide. First, 1.6742 mL of a 60 wt% nitric acid aqueous solution was dropped into 9.3594 g of zirconium (IV) n-propoxide (manufactured by Aldrich, 70 wt% in 1-propanol), and stirred for 5 minutes. Distilled water 6.9612mL was dripped at this, polyethylene oxide (made by Fluka company) MW: 35,000, 0.15g was added, and it stirred for 30 minutes. Further, 2.6781 mL of N-methylformamide was added dropwise and stirred for 5 minutes. When the obtained transparent solution was transferred to a sealed container and kept in a thermostat at 80 ° C., it solidified after about 1 hour. The solidified sample was aged for 24 hours and dried at 69 ° C. As a result, a porous zirconium dioxide gel was obtained (FIG. 27). By changing the amount of N-methylformamide added, the through-hole diameter of the porous body could be controlled in the range from 5 μm at the maximum to 1 μm at the minimum (FIG. 28). In FIG. 28, 1 and 2 represent the following.
1: N-methylformamide 2.66781 mL
2: N-methylformamide 2.9161 mL
It was demonstrated that the pore size can be made freely in the presence of water-soluble organic polymers.
Changes in the through-holes were also observed in the scanning electron micrograph (FIG. 27) of the example described in Example 9 and the scanning electron micrograph (FIG. 30) obtained by changing the amount of N-methylformamide added to 2.9161 mL. Observed.
<Example 9-1>
Furthermore, the change in crystal structure at the firing temperature was examined using the gel of Example 9. In the method of the present invention, it is amorphous at the time of solidification, but when heat-treated at a low temperature of 300 ° C., chemically stable tetragonal crystals or cubic crystals are obtained, and a porous structure is obtained. Furthermore, by firing at 1200 ° C., only the crystal structure can be changed without breaking the porous structure into a monoclinic single phase (FIG. 29). In FIG. 29, square plots represent monoclinic crystals, and round plots represent tetragonal crystals or cubic crystals.
Since the structure of the porous body is determined at the time of solidification of the gel, it can be freely selected according to the purpose depending on the shape of the sealed container. form Can be created. After that, it was proved that a crystal structure whose chemical properties change can be produced according to the purpose by firing.

  The present invention relates to an inorganic porous material that can be used as a chromatography column or an adsorbent. The inorganic porous material of the present invention carries an enzyme such as trypsin, alkaline phosphatase or glucose isomerase, a catalyst such as platinum or palladium, or a functional group such as octadecyl group, and is used for chromatography such as liquid chromatography and gas chromatography. It can be suitably used for a chromatography column. Moreover, it can be used for separating or concentrating a specific phosphoric acid compound by passing the liquid while being fixed inside the syringe tip or inside the capillary. Moreover, it can be used for adsorbing and separating only those having specific properties by specific adsorption from a large number of protein mixtures.

Claims (22)

A through-hole composed of a Group 4 element compound and having a pore diameter of 100 nm or more and continuous in a three-dimensional network, and a pore having a pore diameter of 2 to 100 nm formed in a gel skeleton forming the through-hole. An inorganic porous material, wherein a volume ratio occupied by pores is 10% or more with respect to a sum of pore volumes. The pore volume per gram of the gel skeleton is 10 cm ³ / g or less, and the volume ratio of the through-holes is 20 to 90% with respect to the sum of the through-hole and pore volumes. The inorganic porous body according to claim 1. The inorganic group according to claim 1 or ₂ , wherein the Group 4 element compound is mainly composed of titanium dioxide (TiO ₂ ), zirconium dioxide (ZrO ₂ ), or hafnium dioxide (HfO ₂ ). Porous body. The inorganic porous body according to claim 1, wherein the group 4 element compound is titanium dioxide (TiO ₂ ). The inorganic porous body according to claim 1, wherein the group 4 element compound is zirconium dioxide (ZrO ₂ ). The inorganic porous body according to any one of claims 1 to 5, wherein the Group 4 element compound uses a hydrolyzable metal compound as a raw material. The inorganic porous body according to claim 6, wherein the hydrolyzable metal compound is a metal alkoxide. The inorganic porous body according to claim 7, wherein the metal alkoxide is titanium alkoxide. 8. The inorganic porous body according to claim 7, wherein the metal alkoxide is zirconium alkoxide. The inorganic porous body according to claim 8, wherein the titanium alkoxide is titanium n-propoxide. The inorganic porous body according to claim 9, wherein the zirconium alkoxide is zirconium n-propoxide. A chromatography column using the inorganic porous material according to claim 1. An adsorption material for separating or concentrating a phosphoric acid compound, wherein the inorganic porous material according to any one of claims 1 to 11 is used. An adsorption material for separating or concentrating a sulfuric acid compound, wherein the inorganic porous material according to any one of claims 1 to 11 is used. The method comprises hydrolyzing a group 4 element compound in an acidic aqueous solution in the presence of a polar solvent, polymerizing it to gel the reaction solution, and firing the resulting gel. 4 A method for producing an inorganic porous body made of a group element compound. It comprises a step of hydrolyzing a group 4 element compound in an acidic aqueous solution in the presence of a polar solvent, polymerizing it to gel the reaction solution, and a step of subjecting the resulting gel to a closed heat treatment. The manufacturing method of the inorganic type porous body which consists of a group 4 element compound. In the acidic aqueous solution, in the presence of a polar solvent, the step of hydrolyzing the group 4 element compound and polymerizing it to gel the reaction solution, the step of subjecting the generated gel to a closed heat treatment, and the above steps The manufacturing method of the inorganic type porous body which consists of a group 4 element compound characterized by including the process of baking the obtained porous gel. The method for producing an inorganic porous body comprising a group 4 element compound according to any one of claims 15 to 17, further comprising a step of removing an organic component in the generated gel. The production of an inorganic porous body comprising a group 4 element compound according to any one of claims 15 to 18, wherein the through-hole diameter and the pore diameter are controlled by changing the concentration of water in the reaction solution. Method. The inorganic material comprising a group 4 element compound according to any one of claims 15 to 19, wherein a through-hole diameter and a pore diameter are controlled by dropping a strong acid into the reaction solution and controlling a gelation reaction rate. A method for producing a porous material. 21. The method for producing an inorganic porous body comprising a group 4 element compound according to any one of claims 16 to 20, wherein the pore diameter is controlled by hermetic heat treatment. The method for producing an inorganic porous body comprising a group 4 element compound according to any one of claims 16 to 21, wherein the specific surface area of the porous body is controlled by hermetic heat treatment.

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