JP3145505U - Liquid chromatography column - Google Patents

Abstract

The present invention provides an inorganic porous continuous column having pressure resistance and solvent resistance for use in liquid chromatography that separates components such as proteins and DNA from samples such as body fluid and culture fluid.
A heat-shrinkable tube 2 made of a fluororesin is disposed around an inorganic porous continuous body 1, an epoxy resin 3 is disposed around the heat-shrinkable tube 2, and a metal tube 4 is disposed around the heat-shrinkable tube 2. By using an epoxy resin that cures at 200 ° C. or lower, the functional group chemically modified on the silica surface is not decomposed, and the pressure resistance can be 30 MPa or more.
[Selection] Figure 1

Description

  The present invention relates to a liquid chromatography column for separating components such as proteins and DNA from samples such as body fluids and culture fluids.

As a column for liquid chromatography (HPLC), a particle packed column in which spherical porous particles are uniformly packed in a tube having pressure resistance and solvent resistance such as a SUS tube has been used. In the particle-packed column, the smaller the particle size, the better the resolution, but the higher the load pressure for passing the solution through the column. In addition, since various solvents are used in liquid chromatography, pressure resistance and solvent resistance are required for tubes filled with particles, and SUS tubes are mainly used.
On the other hand, an inorganic porous continuum having micrometer-sized continuous pores and nanometer-sized mesopores can produce a column with a smaller load pressure than a particle-packed column. It is possible to produce a solid-phase extraction column with resolution (for example, Patent Document 1).
The inorganic porous continuum has a rod-like shape, and in order to use it as an HPLC column, the periphery of the inorganic porous continuum must be covered with a material having pressure resistance and solvent resistance with good adhesion.

Regarding an HPLC column using an inorganic porous continuum, a PEEK (polyetheretherketone) resin reinforced with carbon fiber around the inorganic porous continuum is molded (Patent Document 2), inorganic The porous continuum is covered with a heat-shrinkable tube having a two-layer structure of a non-molten fluororesin layer provided outside the melt-type fluororesin layer. There are a molded one (Patent Document 3) and a column holder in which a fluororubber is arranged around an inorganic porous continuous body (Patent Document 4).

JP-A-11-287791 Patent Publication 2003-530571 JP-A-10-197508 JP-A-11-287791

Among these, the one using fiber reinforced PEEK resin has a molding temperature of about 400 ° C. for PEEK resin. When using an inorganic porous continuum gel as an HPLC column, in many cases, the silica surface is chemically modified with a functional group such as an octadecylsilyl group, but at 400 ° C., this modified functional group is decomposed, It must be chemically modified after PEEK molding. Further, the pressure resistance of the fiber reinforced PEEK resin is about 20 MPa, and sufficient pressure resistance cannot be obtained. In the case of a polyimide resin, the adhesion between the heat shrinkable tube and the polyimide is not sufficient, and a gap is formed between them, and the liquid flows through the gap, so that sufficient characteristics as a column cannot be obtained. When using fluororubber, the pressure resistance is about 5 MPa, and the pressure resistance is not sufficient for use as an HPLC column.

The present invention overcomes the above-described drawbacks and provides an inorganic porous continuous column that is made of a solvent-resistant product and a pressure-resistant product of 30 MPa or more and can be molded at a low temperature.
The inorganic porous continuum column of the present invention has a heat shrinkable tube made of fluororesin around the inorganic porous continuum, an epoxy resin around it, and a metal tube around it. By using a heat-shrinkable tube made of a fluororesin that shrinks at 200 ° C. or less, the functional group chemically modified on the silica surface is not decomposed and the solvent resistance can be ensured. Epoxy resins have a characteristic that they have a small volume change upon curing, good adhesion to plastics and metals, and cure at 200 ° C. or lower. By using this epoxy resin, the functional group chemically modified on the surface of the silica is not decomposed, and it is brought into close contact with the metal and the fluorine resin, so that the gap between the fluorine resin and the metal tube can be molded without any gap. Furthermore, pressure resistance can obtain 30 Mpa or more by arrange | positioning a metal tube in the circumference | surroundings.

The inorganic porous continuum is preferably prepared by a sol-gel method using phase separation, and the inorganic porous continuum in the present invention has a macropore having a diameter of 100 nm to 10,000 nm and a skeleton having a co-continuous structure. It is an inorganic porous continuum, and mesopores with a diameter of 2 nm to 100 nm exist in the skeleton.
The inorganic porous continuum can be obtained by causing a reaction solution mainly composed of silica to undergo a sol-gel transition accompanied by phase separation. Examples of the precursor of the network component that causes gel formation used in the sol-gel reaction include metal alkoxides, complexes, metal salts, organically modified metal alkoxides, organic cross-linked metal alkoxides, and partial hydrolysis products and partial polymerization products thereof. Can be used. Sol-gel transition by changing the pH of water glass and other silicate aqueous solutions can be used as well.

  More specifically, the means for achieving the above-mentioned object is to generate a water-soluble polymer and a compound that thermally decomposes in an acidic aqueous solution, add a metal compound having a hydrolyzable functional group, and perform a hydrolysis reaction. After the product has solidified, the wet gel is preferably heated to thermally decompose the low molecular weight compound previously dissolved at the time of gel preparation, and then dried and heated.

Here, the water-soluble polymer is theoretically a water-soluble organic polymer that can be formed into an aqueous solution having an appropriate concentration, and in a reaction system including an alcohol generated by a metal compound having a hydrolyzable functional group. The polymer metal salt is a sodium salt or potassium salt of polystyrene sulfonic acid, which is a polymer metal salt, polyacrylic acid which is a polymer acid and dissociates to form a polyanion, Polyallylamine and polyethyleneimine, which are molecular bases that generate polycations in aqueous solution, or neutral polymers, such as polyethylene oxide having an ether bond in the main chain and polyvinylpyrrolidone having a carbonyl group in the side chain are suitable. . In addition, formamide, polyhydric alcohol, or surfactant may be used in place of the organic polymer. In this case, glycerin is optimal as the polyhydric alcohol, and polyoxyethylene alkyl ethers are optimal as the surfactant.

  As the metal compound having a hydrolyzable functional group, a metal alkoxide or an oligomer thereof can be used, and those having a small number of carbon atoms such as a methoxy group, an ethoxy group, and a propoxy group are preferable. Further, as the metal, an oxide metal finally formed, for example, Si, Ti, Zr, or Al is used. The metal may be one type or two or more types. On the other hand, any oligomer can be used as long as it can be uniformly dissolved and dispersed in alcohol.

  Moreover, as acidic aqueous solution, the thing of 0.001 mol concentration or more of mineral acids, such as hydrochloric acid and nitric acid, or 0.01 mol concentration or more of organic acids, such as an acetic acid and a formic acid, is preferable normally.

The phase separation and gelation can be achieved by storing the solution at room temperature of 40 to 80 ° C. for 0.5 to 5 hours. Phase separation / gelation is a process in which an initially transparent solution becomes cloudy, causing phase separation between a silica phase and an aqueous phase and finally gelling. By this phase separation / gelation, the water-soluble polymers are in a dispersed state and their precipitation does not substantially occur.

Specific examples of the thermally decomposable compound that coexists in advance include urea or hexamethylenetetramine, formamide, N-methylformamide, N, N-dimethylformamide, acetamide, N-methylacetamide, N, N-dimethylacetamide and the like. However, since the pH value of the solvent after heating is an important condition, there is no particular limitation as long as it is a compound that makes the solvent basic after thermal decomposition.
Although the thermally decomposable compound to coexist depends on the kind of the compound, for example, in the case of urea, it is 0.05 to 0.8 g, preferably 0.1 to 0.7 g, with respect to 10 g of the reaction solution. The heating temperature is preferably 40 to 200 ° C. in the case of urea, for example, and the pH value of the solvent after heating is preferably 6.0 to 12.0.
Moreover, what produces the compound with the property to melt | dissolve a silica like hydrofluoric acid by thermal decomposition can be utilized similarly.

In the above method, when a water-soluble polymer is dissolved in an acidic aqueous solution, and a hydrolysis reaction is performed by adding a metal compound having a hydrolyzable functional group thereto, a gel separated into a solvent-rich phase and a skeleton phase is generated. . After the product (gel) has solidified, an appropriate aging time has passed, and then the wet gel is heated to thermally decompose the amide compound dissolved in the reaction solution in advance. The pH of the solvent in contact with increases. The solvent erodes the inner wall surface, and gradually changes the pore size by changing the uneven state of the inner wall surface.
In the case of gels containing silica as the main component, the degree of change is very small in the acidic or neutral region, but as thermal decomposition becomes more vigorous and the basicity of the aqueous solution increases, the part constituting the pore dissolves. Then, by reprecipitation in a flatter portion, a reaction in which the average pore diameter becomes large occurs remarkably.

  In gels that have only three-dimensionally confined pores without huge pores, the elution substance cannot be diffused to the external solution even if it can be dissolved as an equilibrium condition. Remains in a considerable proportion. On the other hand, in a gel having a solvent-rich phase that becomes giant pores, there are many pores that are restricted only two-dimensionally, and exchange of substances with an external aqueous solution occurs frequently enough. In parallel with the development, small pores disappear and the entire pore size distribution does not spread significantly.

  In the heating process, it is effective to place the gel in a hermetically sealed condition so that the vapor pressure of the pyrolysis product is saturated and the pH of the solvent quickly takes a steady value.

  The heat treatment time required for the dissolution / reprecipitation reaction to reach a steady state and to obtain the corresponding pore structure varies depending on the size of the huge pores and the volume of the sample. It is necessary to determine the shortest processing time at which the pore structure does not change.

The gel after the heat treatment becomes a dry gel that is in close contact with the tube wall in the groove by vaporizing the solvent. Since the coexisting substances in the starting solution may remain in this dry gel, the target inorganic porous material can be obtained by heat-treating at an appropriate temperature and thermally decomposing organic matter. it can. In addition, drying is performed by leaving at 30-80 degreeC for several hours-several dozen hours, and heat processing is heated at about 200-800 degreeC.

Any heat-shrinkable tube may be used as long as it shrinks at 200 ° C. or less and has solvent resistance, and a heat-shrinkable tube made of a fluorine resin such as FEP can be used. The surface of the fluororesin is subjected to surface modification so as to be in close contact with the thermosetting resin.

The epoxy resin only needs to have a small volume change when cured and cured at 200 ° C. or less, has solvent resistance, and is in close contact with a metal and a fluorine resin. Epoxy resin mainly composed of bisphenol A, urethane-modified epoxy resin Etc. can be used.

The metal tube should just have pressure resistance and solvent resistance, and metal tubes, such as SUS304, SUS316, titanium, can be used.

Example 1
An HPLC column as shown in FIG. 1 was prepared using a porous silica continuum having a diameter of 2.3 mm, a macropore diameter of 1.5 μm, and a mesopore diameter of 11 nm. The silica porous continuum 1 was placed in a heat shrinkable tube (inner diameter 3.2 mm before shrinkage) 2 made of FEP, and the heat shrinkable tube 2 and the silica porous continuum 1 were brought into close contact with each other by being shrunk at 110 ° C. The surface of the heat shrinkable tube 2 made of FEP was previously sandblasted so as to improve the adhesion to the resin.
Next, the porous silica continuous body 1 covered with the heat-shrinkable tube 2 is placed in the SUS316 tube 4 having an outer diameter of 1/4 inch and an inner diameter of 4 mm so as to come to the center of the SUS316 tube 4, and urethane-modified epoxy resin in the gap. 3 (main agent: product name Adeka Resin EPU-17T-6, curing agent: product name Adeka Hardener EH-3895) was injected and cured at 25 ° C. for 7 days.

The prepared product was cut into a length of 85 mm, and end fittings to which pipes could be connected were fixed at both ends, and the performance as an HPLC column was evaluated.
FIG. 2 shows the chromatogram obtained when 80% methanol and 20% water are used as the mobile phase, alkylbenzene C6H5 (CH2) nH (n = 0 to 6) is used as the sample, and the flow rate is 1.2 ml / min. .
The number of stages of amylbenzene (n = 5) was 8,366, and the symmetry was 1.03. From this result, it can be seen that the liquid has a sufficient number of theoretical plates and symmetry, and no liquid flows between the layers. At this time, the load pressure was 15.4 MPa.
FIG. 3 shows the result of a similar test performed at a flow rate of 2.5 ml / min. This case also shows a good number of theoretical plates and symmetry. The load pressure at this time was 32.0 MPa, and the pressure resistance was sufficient.

Next, FIG. 4 shows a chromatogram measured after continuously flowing a solvent of 60% acetolyl / 40% water containing 1% trifluoroacetic acid for 5 hours at a flow rate of 0.8 ml / min. At this time, the mobile phase was 60% acetonitrile and 40% water. When the flow rate was 0.4 ml / min, the number of stages of amylbenzene was 10,260, the symmetry was 1.11, no deterioration was observed, and sufficient acid resistance was observed.

Next, 100% tetrahydroxyfuran was added to this column at a flow rate of 0.8 ml / min. FIG. 5 shows a chromatogram measured after flowing for 5 hours continuously. When the mobile phase is 60% acetonitrile, 40% water, and the flow rate is 0.4 ml / min., The number of amylbenzene stages is 9,603 and the symmetry is 1.10. It had organic solvent properties.

Industrial application fields

  The present invention can be used as a chromatographic separation column, a blood and plasma component separation column, or a reactor column.

Schematic diagram of the solid-phase extraction column of the present invention Chromatogram obtained with the integrated column of the present invention Chromatogram obtained with the integrated column of the present invention Chromatogram obtained with the integrated column of the present invention Chromatogram obtained with the integrated column of the present invention

Explanation of symbols

1: Silica porous continuous body 2: FEP heat shrinkable tube 3: Urethane modified epoxy resin 4: SUS316 tube

Claims (1)

A heat-shrinkable tube made of fluororesin, an epoxy resin around it, and a metal around it, around an inorganic porous continuum mainly composed of silica prepared by causing a sol-gel transition with phase separation of the reaction solution A column for liquid chromatography, characterized in that a tube is arranged.

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