JP2005292105A - Temperature responsive liquid chromatography carrier and liquid chromatography using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide liquid chromatography for making it possible to perform speration only on the basis of a temperature change. <P>SOLUTION: This temperature responsive liquid chromatography carrier to be utilized is constituted so that a dendritic polymer having a siloxane skeleton or a polymer obtained by grafting another polymer to the dendric polymer is fixed to the surface of a carrier in a membrane state. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention is an external signal of temperature, and a carrier for liquid chromatography that is carried out by controlling the interaction of solid substances with useful substances such as pharmaceuticals, biological substances (proteins, DNA, glycolipids, etc.) and cells, And a liquid chromatography method using the same.

  High-performance liquid chromatography (HPLC) has a wide variety of combinations of mobile phase liquids and stationary phases, and can be selected variously according to the sample. Therefore, it has been used in recent years for separation and purification of various substances. However, in the conventional chromatography, the surface structure of the stationary phase is not changed, and the interaction between the solute contained in the mobile phase and the stationary phase surface is mainly changed by changing the solvent of the mobile phase. Has been done. For example, in HPLC used in many fields, an organic solvent such as hexane, acetonitrile or chloroform is used as a mobile phase in a normal phase column using a carrier such as silica gel as a stationary phase, and an aqueous system is used. An organic solvent such as methanol or acetonitrile is used in the reverse phase column using the silica gel derivative separated in step 1 as a carrier.

  In ion exchange chromatography using an anion exchanger or a cation exchanger as a stationary phase, substance separation is performed by changing the external ion concentration or type. In recent years, due to rapid progress in genetic engineering and the like, bioactive peptides, proteins, DNA, and the like are expected to be widely used in various fields including pharmaceuticals, and their separation and purification are extremely important issues. In particular, there is an increasing need for a technique for separating and purifying a physiologically active substance without impairing its activity.

  However, since organic solvents, acids, alkalis, and surfactants used in conventional mobile phases impair the activity of physiologically active substances and at the same time become impurities, improvement of the system is expected. In addition, a separation / purification system that does not use these substances is required from the viewpoint of avoiding environmental pollution of such substances.

  Under such background, various studies have been made so far. Among them, the technology disclosed in Japanese Patent Publication No. 06-104061 particularly corresponds to those basic technologies. Here, cells are cultured at a temperature not higher than the upper critical lysis temperature or higher than the lower critical lysis temperature on a cell culture support in which the substrate surface is coated with a polymer having an upper or lower critical lysis temperature of 0 to 80 ° C. with respect to water. Thereafter, a technique is described in which the cultured cells are detached by setting the temperature to the upper critical solution temperature or lower or the lower critical solution temperature or lower. This is the first example of a temperature-responsive polymer used as a cell culture material in the biomedical field. In fact, when cells adhere to the surface of a substrate, the cells secrete adhesive proteins and attach through them. . Therefore, the phenomenon that cells peel from the surface of the substrate here also includes the separation of the adhesive protein secreted by the cells from the surface of the substrate. In fact, when cells obtained by this technique are replated or transplanted into a living tissue, the cells detached from the substrate adhere well to the substrate or tissue. This means that the detached cells retain the adhesive protein secreted during culture. That is, the technique here is the concept of the temperature-responsive chromatography technique in which the protein adsorbed by the temperature change in the present invention is desorbed.

  Under such circumstances, Japanese Patent Application Laid-Open No. 05-133947 has studied to immobilize on silica gel or polymer gel, which is usually used as a chromatography carrier. However, as far as the examples are concerned, the result of separation of the solute when actually using the carrier (separation chart) is not shown, and what kind of substance can be separated using this carrier, and more specifically There was no indication of any particular issues, and the details were unknown.

  On the other hand, Japanese Patent Application Laid-Open No. 07-318551 discloses an example of actually separating various steroids and further lymphocytes by immobilizing a temperature-responsive polymer on a silica gel surface and using the carrier. It is clearly shown that various steroids and lymphocytes can be separated due to the characteristics of the temperature-responsive polymer actually immobilized on the silica gel carrier surface. However, as far as the separation results shown here are seen, it is necessary to further increase the number of theoretical plates at the time of separation, and an innovative technology that greatly improves the conventional technology that can solve such problems is desired. It was rare.

  The present invention has been made with the intention of solving the problems of the prior art as described above. That is, an object of the present invention is to provide a novel temperature-responsive liquid chromatography carrier based on a completely different idea from the prior art. Another object of the present invention is to provide a liquid chromatography method using the same.

  In order to solve the above problems, the present inventors have studied and developed from various angles. As a result, it has been surprisingly found that when a dendritic polymer having a siloxane skeleton is immobilized on the surface of a chromatographic support in a thin layer, the chromatographic separation ability on the surface of the support suddenly changes at a certain temperature. At the beginning of the research, the present inventors paid attention to the stereoregularity of dendritic polymers and the functional groups existing at high density outside the molecular chain. It has been speculated that functional groups can be provided at a high density, and if the above-described temperature-responsive polymer is bound thereto, a higher performance than the temperature-responsive chromatographic carrier of the prior art can be obtained. The technology shown in the present invention was completely unpredictable from the prior art, and if the stereoregularity of the dendritic polymer structure itself is combined, it can be applied to a novel chromatography system that was not found in the prior art. Development is expected. The present invention has been completed based on such findings.

That is, the present invention provides a temperature-responsive liquid chromatography carrier characterized in that a dendritic polymer having a siloxane skeleton or a polymer grafted with another polymer is immobilized in a thin layer on the surface of the carrier. To do.
The present invention also provides a liquid chromatography method using the temperature-responsive liquid chromatography carrier.

  With the temperature-responsive liquid chromatography carrier described in the present invention, a novel separation system is proposed. With this system, peptides and proteins can be separated in a wide range. In addition, by utilizing the stereoregularity of the dendritic polymer itself, separation by the difference in the molecular structure of the solute becomes possible.

  As a result of various studies to satisfy the above-mentioned demands, the present inventors have determined that the surface structure of the stationary phase, for example, by changing external conditions such as temperature, the solute and the stationary phase surface without changing the mobile phase. Developed the technology to separate and purify by changing the interaction with, and completed the present invention. The purpose of the present invention is to reversibly change the surface properties of the stationary phase by changing external conditions. Thus, a chromatographic method which can be separated and purified by a single aqueous mobile phase and a packing material as a stationary phase used in the chromatography are provided.

  The gist of the present invention is a chromatographic method characterized in that the solute separation is performed using a filler capable of changing the properties of the stationary phase surface depending on the temperature while the mobile phase is fixed in the aqueous system. is there. Furthermore, the present invention shows a temperature-responsive chromatography method using the same. That is, by using the present invention, it is possible to separate biological elements such as peptides, proteins and cells by setting the external temperature to a critical temperature or higher. Therefore, at this time, since chemicals such as organic solvents, acids, alkalis and surfactants are not used at all, they can be prevented from becoming contaminants, and analysis while maintaining the functions of proteins and cells can be performed. The same can be used for separation.

  In the conventional chromatographic method, a sample in which various compounds are mixed in one type of mobile phase, especially when separating and analyzing a plurality of samples having greatly different polarities, it is difficult to separate and the time required for the separation becomes very long. End up. Therefore, when handling such a sample, separation is performed by a solvent gradient method in which the amount and type of organic solvent are continuously changed over time or a step gradient method in which steps are changed step by step. However, the temperature gradient according to the present invention is used. In the method or step gradient method, it is possible to achieve the same separation by changing the column temperature continuously or stepwise with a single mobile phase instead of using an organic solvent. Thus, contamination of the above-mentioned contaminants can be prevented, separation can be performed while maintaining the functions of proteins and cells, and desired components can be separated in a short time by controlling the temperature.

  The present invention is specifically shown below. The present invention is a liquid chromatography carrier in which a dendritic polymer having a siloxane skeleton or a polymer grafted with another polymer is immobilized in a thin layer on the surface of the carrier. Even if only this dendritic polymer is fixed in a thin layer, temperature responsiveness is developed on the surface of the carrier. The reason for this is not clear at the present time, but it is probably because the function of the dendritic polymer itself is immobilized in a thin layer on the surface of the carrier, and the dendritic polymer molecular chain is constrained and greatly changed. In the present invention, the hydrophilicity / hydrophobicity of the chromatography carrier, the degree of exposure of the hydrophobic group in the dendritic polymer molecular chain to the surface of the carrier, the fluctuation of the molecular chain, the molecular recognition ability of the molecular chain, the exclusion limit, the glass transition point, etc. Although it is considered that one or two or more factors overlap each other, this reason does not limit the technique of the present invention.

  The dendritic polymer shown in the present invention is not particularly limited as long as it is a siloxane skeleton, and examples thereof include those shown in Japanese Patent Application No. 2003-40251, specifically, bis (dimethylvinylsiloxane). ) Methylsilane, tris (dimethylvinylsiloxane) silane, bis (dimethylallylsiloxane) methylsilane, and tris (dimethylallylsiloxane) silane may be used alone or in combination of two or more.

  In the present invention, another polymer may be grafted to the dendritic polymer. At that time, the polymer to be grafted is not particularly limited, but the temperature responsiveness is expressed in the dendritic polymer fixed in a thin layer in the present invention. A temperature-responsive polymer is preferred. In addition, since the dendritic polymer has a structure in which hydrophobic groups are closely capped, it is preferable that a hydrophilic polymer opposite to the dendritic polymer is grafted. The graft polymer may contain one or both of a temperature-responsive polymer and a hydrophilic polymer.

  Examples of the temperature-responsive polymer used in the present invention include a polymer having a lower critical solution temperature (LCST) and a polymer having an upper critical solution temperature (UCST), and any of those homopolymers, copolymers or mixtures thereof may be used. Good. Examples of such a polymer include polymers described in Japanese Patent Publication No. 06-104061. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, vinyl ether derivatives, and polyvinyl alcohol partial acetylates. Of these, any two or more of them can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer. At that time, since the substance to be separated is a biological substance, the separation is performed in the range of 5 ° C. to 50 ° C. Therefore, as the temperature-responsive polymer, poly-Nn-propylacrylamide (lower limit of homopolymer) is used. Critical dissolution temperature 21 ° C), poly-Nn-propylmethacrylamide (27 ° C), poly-N-isopropylacrylamide (32 ° C), poly-N-isopropylmethacrylamide (43 ° C), poly-N -Cyclopropylacrylamide (at about 45 ° C), poly-N-ethoxyethylacrylamide (at about 35 ° C), poly-N-ethoxyethylmethacrylamide (at about 45 ° C), poly-N-tetrahydrofurfurylacrylamide (at about 45 ° C) 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (about 35 ° C.), poly-N, N-ethylmethylacrylate Amide (the same 56 ° C.), poly -N, N-diethyl acrylamide (the 32 ° C.), and the like.

  The hydrophilic polymer used in the present invention may be either a homopolymer or a copolymer. For example, polyacrylamide, poly-N, N-diethylacrylamide, poly-N, N-dimethylacrylamide, polyethylene oxide, polyacrylic acid and its salts, polyhydroxyethyl methacrylate, polyhydroxyethyl acrylate, polyvinyl alcohol, polyvinyl pyrrolidone, cellulose Examples thereof include water-containing polymers such as carboxymethyl cellulose, but are not particularly limited.

  The dendritic polymer shown in the present invention is one in which the terminal is capped with a hydrophobic group such as a methyl group, or a polymer different from the dendritic polymer is grafted to the dendritic polymer. In order to develop temperature responsiveness when a thin layer is immobilized on the support surface, the carbon atom concentration of the polymer needs to be 0.5 to 45%, preferably 1 to 30%, more preferably 3 ~ 25%. When the concentration is less than 0.5% or higher than 45%, the characteristics of the surface of the carrier are not changed within the range of 5 ° C. to 50 ° C., which is not preferable as the polymer of the present invention.

  The molecular weight of the polymer to be coated is not particularly limited, but is preferably in the range of 1000 to 100,000, preferably 1500 to 80000, and more preferably 2000 to 65000. When the molecular weight is 1000 or less, the molecular weight is too low, and thus a sufficient coating amount cannot be obtained even when the metal oxide is coated, which is not preferable. Further, if the molecular weight is 100,000 or more, the molecular weight of the polymer is too high this time, so that the molecule itself becomes bulky and the coating amount decreases, which is not preferable.

Further, the present invention is characterized in that a siloxane skeleton dendritic polymer or a polymer grafted with another polymer is immobilized in a thin layer on the surface of the carrier. When the coating amount of the polymer on the substrate is too large, the temperature responsiveness on the surface of the carrier is not expressed. Therefore, the coating amount of the polymer is good in the range of 0.05 to 8.0 mg / m ² , preferably 0.1 A range of ˜5.0 mg / m ² , more preferably a range of 0.5 to 3.0 mg / m ² is preferable. If it is 0.05 mg / m ² or less, temperature responsiveness is not recognized, and even if the value is higher than 8.0 mg / m ² , temperature responsiveness is not recognized. The measurement of the amount of immobilization may be in accordance with a conventional method, for example, elemental analysis, the amount of ESCA may be mentioned, and any method may be used.

  In the present invention, the above-described polymer is immobilized on a silica gel carrier. The immobilization method is not particularly limited. For example, the immobilization can be performed by dipping in a polymer solution at 50 to 80 ° C. At that time, the temperature of the reaction solution is not particularly limited, and the state of the reaction solution may be allowed to stand or be stirred, but the latter is preferable in view of being uniformly fixed on the support surface.

  The carrier used in the present invention is not particularly limited as long as it is a chromatographic carrier, but silica gel with which a siloxane skeleton dendritic polymer reacts efficiently is preferable. At that time, the pore diameter is not particularly limited, but is preferably 50 to 5000 mm, preferably 100 to 1000 mm, and more preferably 120 to 500 mm. If the molecular weight is less than 50 kg, only the solute having a considerably low molecular weight is targeted, and if it is more than 5000 mm, the surface area of the carrier is reduced and the separation is remarkably deteriorated.

  In the technique described in the present invention, a dendritic polymer having a siloxane skeleton or a polymer grafted with another polymer forms a strong bond on the surface of the carrier. By the binding, the temperature responsiveness shown in the present invention is expressed. The bonding mode may be a covalent bond, or may be adsorbed by a hydrogen bond, a hydrophobic bond or the like, or may be a combination thereof, and is not particularly limited.

  In the present invention, the temperature-responsive liquid chromatography carrier thus obtained is packed in a column and attached to a normal liquid chromatography apparatus, and used as a liquid chromatography system. In that case, the separation of the present invention is affected by the temperature of the support packed in the column. At that time, the method of applying the temperature to the carrier is not particularly limited. For example, it is possible to attach the whole or part of the column packed with the carrier to an aluminum block, water bath, air layer, jacket or the like having a predetermined temperature. Can be mentioned.

  Although the separation method is not particularly limited, an example is a method of separating a solute in a column packed with a carrier at a constant temperature. The surface of the carrier of the present invention varies depending on the temperature. Depending on the substance to be separated, it may be separated only by setting an appropriate constant temperature.

  As an example of another separation method, the temperature at which the characteristics of the support surface change may be confirmed in advance, and the solute may be separated while changing the temperature so as to sandwich the temperature. In this case, since the characteristics of the support surface are greatly changed only by the temperature change, it is expected that a large difference occurs in the time (holding time) in which the signal appears depending on the solute. In the case of the present invention, it is the most effective utilization method that the separation is performed so as to sandwich a temperature at which the characteristics of the carrier surface greatly change. Usually, when only the dendritic polymer is bound to the surface of the carrier, when a hydrophobic substance such as pharmaceuticals is used as the solute, the retention time is lower on the lower temperature side than on the higher temperature side, where the characteristics of the carrier surface change significantly. Becomes longer. This is presumed to mean that the dendritic polymer properties on the surface of the support mean that the lower temperature side is a more hydrophobic surface for the reasons described above.

  When changing the temperature, the temperature change may be changed intermittently once or more times after starting to flow the solute, or may be changed continuously. Moreover, you may combine those methods. The temperature change at that time may be performed manually, or an apparatus capable of automatically controlling the temperature according to a program may be used.

  Alternatively, as an example of another separation method, the adsorbed solute is released by once adsorbing the solute on the obtained temperature-responsive liquid chromatography carrier and then changing the temperature and changing the characteristics of the surface of the carrier. The method of using based on the catch and release method is mentioned. In this case, the mass to be adsorbed may or may not exceed the amount that can be adsorbed on the carrier. In any case, the adsorption is performed once, and then the temperature is changed to change the characteristics of the surface of the carrier to release the adsorbed solute.

  Furthermore, two or more types of temperature-responsive liquid chromatography carriers may be packed in the same column, and the solute may be separated while changing the temperature so as to sandwich the temperature at which the characteristics of the carrier surface change. In this case, for example, when two types of carriers are used, different temperature ranges are generated on the three surfaces of the carrier, and the temperature may be changed by the above-described method so as to sandwich these three temperatures. . This may be done by packing two or more types of temperature-responsive liquid chromatography carriers in two or more columns.

  As an example of another separation method, a temperature-responsive liquid chromatography carrier is used, the column inlet end temperature and the column outlet end temperature are set so as to sandwich the temperature at which the characteristics of the carrier surface change, and the temperature in the column is changed from the inlet end. A method of separating the solute by providing a temperature gradient to the outlet end can be mentioned. The method of changing the temperature step by step is not particularly limited. For example, the column inlet end temperature and the column outlet end temperature are monitored sufficiently to keep the entire column warm, and multiple aluminum blocks with different temperatures are connected to the column. The method of making it etc. is mentioned.

  In the present invention, as described above, the solute can be separated only by temperature while the moving bed is fixed. In this case, the mobile phase is preferably 100% aqueous. However, in the present invention, the composition of the mobile phase is not particularly limited because of the characteristics of the dendritic polymer immobilized on the support surface. It may contain a solvent, change the pH, or contain a salt. At that time, the carrier concentration of the present invention may be used in combination with a solvent gradient method by changing the solvent concentration. Further, the mobile phase may be 100% organic solvent.

  By using the temperature-responsive liquid chromatography carrier of the present invention described above and the chromatography method using the same, it is possible to separate pharmaceuticals and their metabolites, agricultural chemicals, peptides, and proteins. In that case, separation can be achieved by simple operation only by changing the temperature in the column.

  Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.

(Reference Example 1)
A 1 L three-necked flask equipped with a reflux tube for synthesis of dimethylvinylsilanol was purged with nitrogen, and then 700 ml of ethyl ether was put in an ice bath, and 8.38 g (0.09 mol) of aniline and 1.48 g (0.087 mol) of water were added. Added and stirred. 10 g (0.082 mol) of vinyldimethylchlorosilane previously dissolved in 50 ml of ethyl ether was slowly added dropwise and stirred at room temperature for 15 minutes. The reaction is as shown in Chemical formula 6. The generated salt was removed by filtration, followed by dehydration with anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain the desired product. The yield was 70%.

(Reference Example 2)
Synthesis of bis (dimethylvinylsiloxy) methylsilane A 1 L three-necked flask equipped with a reflux tube was purged with nitrogen, and then 500 ml of ethyl ether and 8.21 g (0.081 mol) of triethylamine were placed in an ice bath to obtain 7.54 g (0.25 g). 074 mol) of dimethylvinylsilanol was added and stirred. To this, 4.24 g (0.037 mol) of dimethylsilane dissolved in 50 ml of ethyl ether was slowly added dropwise and stirred at room temperature for 20 minutes. The reaction is as shown in Chemical formula 7. After the generated salt was removed by filtration, the low boiling point solvent and the like were removed by an evaporator. By distillation, colorless and transparent bis (dimethylvinylsiloxy) methylsilane was obtained. The yield was 69%. The boiling point (bp) was 65 to 68 ° C. (0.5 torr).

(Reference Example 3)
Synthesis of Dendritic Polymer Polymer A 100-ml three-flask flask equipped with a reflux tube was purged with nitrogen, and then 2.49 g (0.01 mol) of bis (dimethylvinylsiloxy) methylsilane was dissolved in 50 ml of THF in this flask. Add a few drops of Karstedt catalyst (platinum (0) -1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex 0.1M in xylene) and heat to reflux until the Si-H group disappears completely in the IR spectrum. And cooled to room temperature. After removing the low boiling point solvent and the like with an evaporator, the product was dropped into acetonitrile to obtain a colorless viscous liquid polymer. The yield was 93%. As a result of GPC content measurement using polystyrene as a standard and THF as a developing solvent, the weight average molecular weight was 6000.

  1.0 g of silica gel particles for column chromatography (average particle size 150 μm), 50 ml of hexane, and 0.1 g of the polymer of Reference Example 3 were mixed and stirred overnight. The silica gel particles were suction filtered, washed with hexane, and vacuum-dried in an oven at 100 ° C. to obtain treated silica gel particles. The XPS spectrum of the untreated silica gel used here and the XPS spectrum of the treated silica gel particles were compared, and the C1s peak was clearly increased, confirming that the polymer was supported on the surface.

Comparative Example 1

  The same silica gel particles for column chromatography (average particle size 150 μm) 1.0 g as in Example 1, 50 ml of hexane, and 0.1 g of allyltriethoxysilane were mixed and stirred overnight. The silica gel particles were suction filtered, washed with hexane, and vacuum-dried in an oven at 100 ° C. to obtain treated silica gel particles. Judging from the C1s peak in the XPS spectrum of the treated silica gel particles, it can be seen that the polymer was supported on the surface, but the degree was found to be smaller than in Example 1.

  1.0 g of silica gel particles for column chromatography (average particle size 3 μm), 50 ml of hexane, and 0.1 g of the polymer of Reference Example 3 were mixed and stirred overnight. The silica gel particles were filtered by suction, washed with hexane, and vacuum dried in an oven at 100 ° C. to obtain a treated silica gel. The C1s peak of the XPS spectrum of the treated silica gel particles was clearly larger than the XPS spectrum of the untreated silica gel used here, indicating that the polymer was supported on the surface.

(Segregation of steroid)
Two kinds of steroids were dissolved in purified water to prepare 10 ml of a steroid mixed solution having the following concentration. After the preparation, the mixed solution was filtered with a PTFE filter (0.2 μm).
Sample A
1. Hydrocortisone 0.03mg / ml
2. Testosterone 0.02mg / ml
20 μl of sample A was injected into a column packed with the above packing material (silica carrier chemically modified with the dendritic polymer prepared in Example 1). The column was connected to HPLC (high performance liquid chromatography), and the mobile phase was detected at a wavelength of 254 nm with a 66.7 mM phosphate buffer, a flow rate of 1 ml / min, and UV (ultraviolet-visible absorbance detector). The column was installed in a thermostat, and the resolution was compared by changing the column temperature. At 40 ° C., testosterone (peak 2 in FIG. 1) showed a retention time of 11.4 minutes, but at 15.7 minutes at 30 ° C., 21.3 minutes at 20 ° C. and 50.8 minutes at 10 ° C. . Thus, it was confirmed that by changing the column temperature, it was possible to change the hydrophobic interaction on the surface of the carrier and change the retention time of each steroid.

(Segregation of steroid)
Three kinds of steroids were dissolved in purified water to prepare 10 ml of a steroid mixed solution having the following concentration. After the preparation, the mixed solution was filtered with a PTFE filter (0.2 μm).
Sample B
1. Hydrocortisone 0.03mg / ml
2. Prednisolone acetate 0.03mg / ml
3. Testosterone 0.02mg / ml
20 μl of sample B was injected into a column packed with the above-described filler (a silica gel carrier obtained by chemically modifying a polymer obtained by grafting poly-N-isopropylacrylamide having a molecular weight of 3000 on the dendritic polymer prepared in Example 1). The column was connected to HPLC (high performance liquid chromatography), and the mobile phase was detected at a wavelength of 254 nm with a 66.7 mM phosphate buffer, a flow rate of 1 ml / min, and UV (ultraviolet-visible absorbance detector). The column was installed in a thermostat, and the resolution was compared by changing the column temperature. At 40 ° C. testosterone (peak 3 in FIG. 2) showed a retention time of 13.1 minutes, but at 20 ° C. it was 16.8 minutes and at 10 ° C. it was 24.5 minutes. Thus, it was confirmed that by changing the column temperature, it was possible to change the hydrophobic interaction on the surface of the carrier and change the retention time of each steroid.

(Separation of angiotensin I)
Angiotensin I was dissolved in purified water to prepare 5 ml of a solution having the following concentration. After preparation, the solution was filtered with a PTFE filter (0.2 μm).
Sample C
Angiotensin I 0.1mg / ml
20 μl of sample C was injected into the column described in Example 4. The column was connected to HPLC (high performance liquid chromatography), and the mobile phase was detected at a wavelength of 220 nm with 66.7 mM phosphate buffer, a flow rate of 1 ml / min, and UV (ultraviolet-visible absorbance detector). The column was installed in a thermostat, and the resolution was compared by changing the column temperature. The results are shown in FIG. The holding time was 5.3 minutes at 40 ° C., but a long holding time of 58.1 minutes was shown at 10 ° C. Therefore, when the analysis was started at 10 ° C. and the temperature was raised to 40 ° C. over 15 minutes at a heating rate of 2 ° C./min after 5 minutes, the holding time was 21.1 minutes. By changing the column temperature in this way, it is possible to change the hydrophobicity of the carrier surface and control the elution time.

(Separation of EGF)
EGF was dissolved in purified water to prepare 5 ml of a solution having the following concentration. After preparation, the solution was filtered with a PTFE filter (0.2 μm).
Sample D
EGF 0.1mg / ml
20 μl of sample D was injected into the column described in Example 4. The column was connected to HPLC (high performance liquid chromatography), and the mobile phase was detected at a wavelength of 220 nm with 66.7 mM phosphate buffer, a flow rate of 1 ml / min, and UV (ultraviolet-visible absorbance detector). The column was installed in a thermostat, and the resolution was compared by changing the column temperature. The results are shown in FIG. The retention time was 1.7 minutes at 40 ° C., but a long retention time of 7.1 minutes was exhibited at 10 ° C. In this way, it was confirmed that the holding time can be changed by changing the temperature.

(Pharmaceutical separation)
Four types of pharmaceutical ingredients were dissolved in purified water to prepare 10 ml of a mixed solution having the following concentration. After the preparation, the mixed solution was filtered with a PTFE filter (0.2 μm).
Sample E
1. Caffeine 0.05mg / ml
2. Ibuprofen 0.04mg / ml
3. o-Ethoxybenzamide 0.07mg / ml
4). Bromovaleryl urea 0.05mg / ml
20 μl of sample E was injected into the column described in Example 4. The column was connected to HPLC (high performance liquid chromatography), and the mobile phase was detected at a wavelength of 230 nm with 66.7 mM phosphate buffer, a flow rate of 1 ml / min, and UV (ultraviolet-visible absorbance detector). The column was installed in a thermostat, and the resolution was compared by changing the column temperature. At 10 ° C., the peaks overlap with a retention time of 5.5 minutes to 8.5 minutes, but at 40 ° C., the four components can be separated with a retention time of 3.2 minutes to 6.0 minutes. It was confirmed that the retention time of each substance can be changed by changing the column temperature in this manner, and that the separation can be optimized only by temperature control. The results are shown in FIG.

(Isolation of antiepileptic drugs)
The antiepileptic drugs primidone and carbamazepine were dissolved in purified water to prepare 10 ml of a mixed solution having the following concentration. After the preparation, the mixed solution was filtered with a PTFE filter (0.2 μm).
Sample F
1. Primidone 0.01mg / ml
2. Carbamazepine 0.01mg / ml
20 μl of sample F was injected into the column described in Example 4. The column was connected to HPLC (high performance liquid chromatography), and the mobile phase was detected at a wavelength of 220 nm with 66.7 mM phosphate buffer, a flow rate of 1 ml / min, and UV (ultraviolet-visible absorbance detector). The results are shown in FIG. The column was installed in a thermostat, and the resolution was compared by changing the column temperature. At 40 ° C., carbamazepine (peak 2 in FIG. 6) showed a retention time of 9.5 minutes, but at 10 ° C. it was 16.2 minutes. It was confirmed that the retention time of the antiepileptic drug can be changed by changing the column temperature.

(Measurement of exclusion molecular weight)
Seven types of polysaccharide polymers having different molecular weights were dissolved in purified water, and 10 ml of solutions having the following concentrations were prepared. After the preparation, the mixed solution was filtered with a PTFE filter (0.2 μm).
Sample G
Shodex Standard P-82 (manufactured by Shoko Tsusho)
P-400 Mw 404,000 Mw / Mn 1.13 2 mg / ml
P-200 Mw 212,000 Mw / Mn 1.13 2 mg / ml
P-100 Mw112,000 Mw / Mn 1.12 2 mg / ml
P-50 Mw 47,300 Mw / Mn 1.06 2 mg / ml
P-20 Mw 22,800 Mw / Mn 1.07 2 mg / ml
P-10 Mw 11,800 Mw / Mn 1.10 2 mg / ml
P-5 Mw 5,900 Mw / Mn 1.07 2 mg / ml
20 μl of sample G was injected into the column described in Example 4. The column was connected to HPLC (high performance liquid chromatography), and the mobile phase was detected with 66.7 mM phosphate buffer, flow rate 0.5 ml / min, RI (differential refractometer detector). Separation was performed at 10 ° C. The silica beads used were 120 mm and 5 μm. T ₀ = 2.0 ml (H ₂ O). The obtained results are shown in FIG. From this, the exclusion limit molecular weight seems to be about 100,000.

  With the method described in the present invention, peptides and proteins can be separated in a wide range only by changing the temperature of the chromatography carrier. As a result, the separation operation is simplified and the efficiency of the separation work is improved. Furthermore, by utilizing the stereoregularity of the dendritic polymer itself, separation by the difference in the molecular structure of the solute becomes possible. The separation operation obtained by this method is strongly expected to be used for drug development, for example. Therefore, the present invention is a very useful invention in fields such as medicine and biology.

  It is the figure which isolate | separated the steroid shown in Example 3. FIG.   It is the figure which isolate | separated the steroid shown in Example 4. FIG.   It is the figure which isolate | separated the angiotensin I shown in Example 5. FIG.   It is the figure which isolate | separated EGF shown in Example 6. FIG.   It is the figure which isolate | separated the pharmaceutical shown in Example 7. FIG.   It is the figure which isolate | separated the antiepileptic drug shown in Example 8. FIG.   It is the figure which showed the result of having measured the exclusion limit molecular weight shown in Example 9.

Claims (19)

A temperature-responsive liquid chromatography carrier, wherein a dendritic polymer having a siloxane skeleton or a polymer grafted with another polymer is immobilized in a thin layer on the surface of the carrier. A dendritic polymer obtained by polymerizing bis (dimethylvinylsiloxane) methylsilane, tris (dimethylvinylsiloxane) silane, bis (dimethylallylsiloxane) methylsilane, tris (dimethylallylsiloxane) silane alone or in combination of two or more. The temperature-responsive liquid chromatography carrier according to claim 1, wherein The temperature-responsive liquid chromatography carrier according to any one of claims 1 and 2, wherein the other polymer comprises one or both of a temperature-responsive polymer and a hydrophilic polymer. The polymer having temperature responsiveness is a poly-N-substituted acrylamide derivative, a poly-N-substituted methacrylamide derivative, a copolymer thereof, polyvinyl methyl ether, polyvinyl alcohol partially acetylated product, or two or more thereof. The temperature-responsive liquid chromatography carrier according to claim 3, comprising: The temperature-responsive liquid chromatography carrier according to claim 3, wherein the polymer having temperature responsiveness is poly-N-isopropylacrylamide. The temperature-responsive liquid chromatograph according to claim 3, wherein the hydrophilic polymer comprises one or more of polyacrylamide, poly-N, N-diethylacrylamide, poly-N, N-dimethylacrylamide, and polyethylene oxide. Graphy carrier. The temperature-responsive liquid chromatography carrier according to any one of claims 1 to 6, wherein the polymer has a carbon atom concentration of 0.5 to 45%. Coverage of the polymer onto the substrate is 0.05~8.0mg / ^{m 2,} claims 1 to 7 temperature-responsive liquid chromatography carrier according to any one. The temperature-responsive liquid chromatography carrier according to any one of claims 1 to 8, wherein the immobilization between the polymer and the substrate surface is by covalent bonding. The temperature-responsive liquid chromatography carrier according to any one of claims 1 to 9, wherein the carrier is silica gel. A liquid chromatography method using the temperature-responsive liquid chromatography carrier according to claim 1. 12. The liquid chromatography method according to claim 11, wherein the solute is separated at a constant temperature. 12. The liquid chromatography method according to claim 11, wherein the solute is separated while changing the temperature so as to sandwich the temperature at which the characteristics of the carrier surface change. The liquid chromatography method according to claim 13, wherein the temperature change is one of intermittent or continuous, or both. The solute is adsorbed on the temperature-responsive liquid chromatography carrier according to claim 1 and then the adsorbed solute is liberated by changing the temperature and changing the characteristics of the surface of the carrier. Liquid chromatography method. The solute separation is performed while the temperature is changed so that two or more kinds of temperature-responsive liquid chromatography carriers according to claims 1 to 10 are packed in the same column and the temperature at which the characteristics of the carrier surface change is sandwiched. 14. The liquid chromatography method according to 13. Using the temperature-responsive liquid chromatography carrier according to claim 1, the column inlet end temperature and the column outlet end temperature are set so as to sandwich the temperature at which the characteristics of the carrier surface change, and the temperature in the column is measured from the inlet end to the outlet end. 14. The liquid chromatography method according to claim 13, wherein the solute is separated by providing a gradient. The liquid chromatography method according to claim 11, wherein the mobile phase is an aqueous system. The liquid chromatography method according to claim 11, wherein a pharmaceutical product and a metabolite thereof, an agricultural chemical, a peptide, and a protein are separated.

Images