WO2016167379A2 - Capillaire de silice fixé à un polymère pour séparer des oligosaccharides ou des peptides et procédé pour le fabriquer - Google Patents

Capillaire de silice fixé à un polymère pour séparer des oligosaccharides ou des peptides et procédé pour le fabriquer Download PDF

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WO2016167379A2
WO2016167379A2 PCT/KR2015/003669 KR2015003669W WO2016167379A2 WO 2016167379 A2 WO2016167379 A2 WO 2016167379A2 KR 2015003669 W KR2015003669 W KR 2015003669W WO 2016167379 A2 WO2016167379 A2 WO 2016167379A2
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silica capillary
polymer
silica
capillary
represented
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PCT/KR2015/003669
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WO2016167379A3 (fr
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정원조
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인하대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification

Definitions

  • the present invention relates to a silica capillary tube with a polymer for separating ligosaccharides or peptides and a method for preparing the same.
  • the method of bonding the polymer membrane with the polymerization reaction is a well known technique.
  • Inorganic-organic hybrids prepared by such techniques can also be utilized as the stationary phase of chromatography.
  • the Atom Transfer Radical Polymerization (ATRP) method is mainly used to prepare organic-inorganic common stationary phase for chromatography, and the reversible addition-fragmentation chain transfer (RAFT) polymerization method is also partially used. Is being used.
  • atomic transfer radical polymerization In atomic transfer radical polymerization (ATRP), a catalyst mixture consisting of a halide of monovalent copper, a halide of divalent copper, and an amine base is used to induce a polymerization reaction on the silica surface to which a polymerization initiator including terminal halogen is attached.
  • the atomic transfer radical polymerization (ATRP) is known to form a polymer chain in the form of a brush having a large molecular weight and a low degree of dispersion.
  • Some atomic transfer radical polymerization (ATRP) studies have reported that the resulting stationary phase has superior selectivity compared to conventional C 18 stationary phases.
  • Reversible addition-fragmentation according to the chain transfer (RAFT) polymerization mechanism within the CS bond between the first ligand and the second ligand (diethyldithio carbamate) adhering to the surface of the polymer chain may be inserted, known to grow.
  • the necessary initial radicals arise from the thermal self-initialization of the monomers and no additional catalyst is required.
  • the chromatographic separation efficiency of the stationary phase obtained by reversible addition-fragmentation chain transfer (RAF) integration is considered to be significantly inferior to the conventional C 18 stationary phase. .
  • the present inventors have porous' 4 in the silica powder by the reaction of chloromethyl phenyl isocyanate and sodium diethyl dithiocarbamate in turn initiator prepared (S 1 type) attachment silica *, and by jeunghap styrene here polystyrene added silica stationary phase I have made and reported Korean Chem. Soc. 2009, Vol. 30, No. 3) While the stationary phase can give better separation efficiency than C 18, the solute peaks are densified by giving a significantly shorter retention time.
  • the polystyrene layer thus obtained did not form a uniform thickness on the silica surface, but large polystyrene masses were formed irregularly, and the retention time of the analyte solute was reduced by reducing the volume of the accessible pupil and the surface. There was a declining problem. The problem is believed to be the result of fast and uncontrolled polymerization reaction due to the generation of radicals upon very stable restart. Stable reactivation radicals are known to induce the production of polymers with large molecular weights and wide dispersions, ie, inhomogeneous polymer growth, by causing polymer hesitation to grow excessively before chain transfer or termination reactions occur.
  • the present inventors regarded the formation of highly stable polymer intermediate radicals and the growth of uncontrolled polymer chains due to the formation of polystyrene lumps in the above-mentioned studies, and the attachment of a new initiator (type S2) capable of controlling the growth rate of the polymer somewhat.
  • RAFT reversible addition-fragmentation chain transfer
  • the present inventors have found that even if the initiator addition silica of the S 1 form is sufficiently high, the adhesion density of the initiator ligand H is high enough to cause simultaneous and uniform growth of polymer chains in each initiator ligand and polymerization at the point when the chains are clustered together. If it is controlled to be terminated, it is thought that it is possible to further improve the separation efficiency by making a stationary phase to which polymer membrane of better properties is attached.
  • the present inventors react the semi-ung products with dithiocarbamate salt derivatives by using a catalyst which is well soluble in the dispersion solvent in the process of attaching isocyanate having a terminal halogen atom to the particles with isocyanate-hydroxysilica reaction.
  • a new method for producing silica with attached polymerization initiator was developed, and reversible addition-fragmentation chain transfer (RAFT) polymerization was carried out on the silica attached to the polymerization initiator to prepare a new polymer attached silica powder for liquid chromatography stationary phase with high separation efficiency. It was confirmed that it can be obtained and has applied for a patent. Meanwhile, as certain sugars are known as biomarkers of various diseases, sugar analysis has attracted much attention.
  • Capillary Electrophoresis is a very efficient method for the separation of biological materials.
  • the combined analysis of mass spectrometry and capillary electrophore sis (CE) has made it possible to dramatically speed up the process of selecting effective sugars in biomedical research.
  • the stationary phase is filled or attached inside the silica capillary, it is used for capillary
  • CEC Electrochromatography
  • HILIC stationary phase is a stationary phase with a large polarity. When using a water-soluble solvent with a mobile phase polarity, this is called HILIC.
  • the stationary phase is known to have better chromatographic resolution but inferior separation efficiency compared to the C 18 stationary phase, whereas the HILIC stationary phase is known to significantly increase the retention time of sugar due to the high polarity of the ground.
  • the present inventors have developed an open structure capillary ele ctrochromatography (CEC) capillary column having both porous graphite carbon (PGC) stationary phase and HILIC (Hydrophilic Interaction Liquid Chromatography) features.
  • a new open-structure capillary electrochromatography method is to copolymerize styrene of SP 2 structure and acrylamide having a large polarity by reversible addition-fragmentation chain transfer (RAFT) polymerization to form on capillary inner wall.
  • RAFT reversible addition-fragmentation chain transfer
  • Patent Document 1 Korean Registered Patent 10-1116566
  • Non-Patent Document 1 BuJJ. Korean Chew. Soc. 2009, Vol. 30, No. 3
  • An object of the present invention is to provide a method for preparing a silica-capillary tube with a polymer for separating many peptides in a hydrolyzate of a protein such as glucose or maltotriose or a protein such as cytochrome C. Another object of the present invention is to provide a high molecular weight attached silica capillary tube produced by the above production method. Still another object of the present invention is to provide a method for separating oligosaccharides using the above-described silica capillary with polymer. Another object of the present invention is to provide a method for separating peptides using the polymer attached silica capillary.
  • the present invention as shown in the following reaction formula 1, by reacting the hydroxyl group and the ligand represented by the formula (3) present on the inner surface of the silica capillary tube represented by the formula (2) in the presence of a catalyst, the ligand attached silica represented by the formula (4) Preparing a capillary tube (step 1);
  • the monomer was dissolved in a solvent in a silica capillary tube attached to the polymerization initiator represented by Chemical Formula 6 prepared in step 2, and a reversible addition-fragmentation chain transfer (RAFT) polymerization reaction was performed.
  • RAFT reversible addition-fragmentation chain transfer
  • Silica is located on the inner surface of the silica capillary in the reaction formula 1; C 6 -io aromatic ring;
  • R 1 and R 2 are independently hydrogen or alkyl of d- 4 ;
  • M is a monomer constituting the copolymer polymer chain formed in Step 3;
  • n is an integer from 1-10;
  • the present invention is characterized in that the manufacturing method Provides a silica capillary for separating ligosaccharides or peptides. Furthermore, the present invention comprises the steps of reacting oligosaccharides with derivatization and preparing oligosaccharides for structural isomers (step 1); And
  • step 2 of separating the structural isomers of the oligosaccharides prepared in step 1 through the silica capillary.
  • step 2 of separating the structural isomers of the oligosaccharides prepared in step 1 through the silica capillary.
  • the present invention is to hydrolyze the protein to prepare peptides (step 1);
  • step 2 Peptides prepared in the step 1, the step of separating through the silica capillary (step 2) provides a method for separating peptides comprising.
  • Silica capillary with polymer prepared by the manufacturing method according to the present invention has excellent affinity for the polymer film itself and is attached to the inside of the capillary tube in the form of a long, uniform polymer chain, while in a dry state, it shows a solid film shape, but aceto Oligosaccharides such as glucose or maltotriose, or cytokyl, are spread out in mobile phases with high nitrile content and act in the direction of narrowing peak band butterflies on both sides of analyte retention and mass transfer.
  • aceto Oligosaccharides such as glucose or maltotriose, or cytokyl
  • FIG. 1 is a schematic diagram illustrating the preparation of the ligand-attached silica capillary, the polymerization initiator-attached silica capillary, and the polymer-attached silica capillary described in Examples 1 and 2 step by step.
  • Figure 2 is a silica powder and a polymer attached sil prepared in accordance with the present invention A wide range of electron microscopy for the car capillary (scale bar 10 um, A) and a narrow range photograph (scale bar 2um, B).
  • FIG. 3 is a capillary electrochromatography (Capillary) for the maltotriose isomer stones (A, B, C) and D-glucose anomers (W, X, Y) as a silica capillary with polymer prepared according to the present invention
  • Electrochromatography (CEC) is an image showing the optimization process for the acetonitrile composition of the eluent in elution (the acetonitrile content is 95% for A and W, 90% for B and X, and 80% for C and Y, The CE voltage was 30 kV, the sample solution was injected at 12 kV and 5 seconds.
  • the optimized condition was 90/10 acetonitrile / 30 mM sodium acetate pH 6.6 obtained with B and X, and D and Z were obtained under the optimized conditions.
  • Acetone electrochromatogram, ⁇ and Xi are magnified electrochromograms of B and X, respectively).
  • CEC capillary electrochromatography for maltotriose isomers ( ⁇ , ⁇ ) and D-glucose anomers (W, X, Y) with a silica-capillary capillary with polymer prepared according to the present invention.
  • Ele ctrochromatogr.aphy, CEC Ele ctrochromatogr.aphy, CEC
  • the image shows the optimization process for the pH of the eluent (pH is 5.5 for A and W, 6.6 for B and X, 3 for C and Y, and CE voltage.
  • Silver 30kV, sample solution injection 8 kV, 5 seconds, the optimum conditions were 90/10 acetonitrile 3 ⁇ 4 / 30 mM sodium acetate ⁇ 6.
  • FIG. 5 is an electrochromatogram subjected to capillary electrochromatography (CEC) separation analysis of proteomic hydrolyzed samples using a polymer-captured silica capillary prepared in Example 2 (elution condition is a voltage of 30 kV) This was followed by eluting for 10 minutes in a 78/22 acetonitrile / 12.5 mM sodium phosphate pH 6.8 mobile phase and then eluting with a 65/35 acetonitrile / 12.5 mM sodium phosphate pH 6.8 mobile phase, the top of FIG. Separation electrochromatogram for Mick samples, The bottom is an electrochromatogram of acetone, an electroosmotic marker.
  • CEC capillary electrochromatography
  • Step 2 Preparing a silica capillary tube with a polymerization initiator represented by Chemical Formula 6 by reacting the ligand-attached silica capillary tube represented by Chemical Formula 4 with the polymerization initiator represented by Chemical Formula 5 (Step 2); and
  • R 1 and R 2 are independently hydrogen or alkyl of d- 4 ;
  • M is a monomer constituting the copolymerized polymer chainol formed in Step 3;
  • n is an integer from 1-10;
  • the step 1 is to react with a hydroxy group present on the inner surface of the silica capillary tube represented by the formula (2) in the presence of a catalyst and a ligand represented by the formula (3), This step is to prepare a ligand attached silica capillary.
  • the silica capillary tube is pretreated with NaOH solution according to a known method (Bulletin of the Korean Chemical Society, 2014, 35, 542) to activate the silanol groups on the inner wall of the capillary tube.
  • a solution containing 5-40 mg of the ligand represented by Formula 3, 5-35 mg of catalyst and 1-5 mL of anhydrous solvent was carried out at 60-15C C for 1-m silica silica capillary for 6-48 hours. It is preferred to stir, wash with anhydrous solvent and dry in a stream of nitrogen.
  • Ligand attachment is less than 5 mg for ligands represented by Formula 3 above for 1.0 m silica capillaries .
  • the catalyst is less than 5 mg for 1.0 m silica capillary When used as a catalytic effect .
  • the reaction does not progress because it does not come out, there is a problem that the treatment cost increases when it exceeds 35 mg.
  • the reaction anhydrous solvent is used in less than 1 mL with respect to 1.0 m silica capillary tube, there may be a problem that a heterogeneous reaction may occur, and when used in excess of 5 mL, there is a problem of decrease in reaction speed due to mass dilution.
  • reaction temperature is less than 60 ° C there is a problem that the reaction reaction is not easy to progress due to the slow reaction speed, if there is more than 150 ° C there is a problem of reaction.
  • the catalyst can be used as long as it can be dissolved in a nonpolar anhydrous reaction solvent and an organometallic compound having a catalytic effect on the isocyanate-hydroxy reaction.
  • compounds of transition metals having organic substituents are preferred in view of catalytic effect and solubility in solvents. Specifically, dibutyltindichloride, dibutyl tin diacetate
  • the silica capillary tube is usually coated with a polymer coating on the outer wall, a commercial silica capillary having an inner diameter of 25-200 ⁇ and a length of 0.5-5.0 m can be used.
  • a commercial silica capillary having an inner diameter of 25-200 ⁇ and a length of 0.5-5.0 m can be used.
  • the inner diameter of the capillary tube is less than 25 ⁇ , there is a problem that the column is easily clogged, and if more than 200 ym, there is a problem that the separation efficiency of the final column is lowered.
  • the length of the capillary tube is less than 0.5 m, there is a problem that the column separation efficiency is reduced, and if it is more than 5.0 m, there is a problem that the pressure of the column is increased.
  • C 6 - ring 10 may be a phenylene or naphthalene, most preferably phenylene.
  • ligand represented by the formula (3) 4 'chloromethyl phenyl isocyanate, 3-chloromethyl phenyl isocyanate, 2-chloromethyl phenyl isocyanate and the like can be used.
  • the anhydrous solvent may be any anhydrous nonpolar solvent, but a boiling point of 60 ° C. or higher is preferable in order to make the reaction temperature 60 ° C. or higher.
  • step 2 is performed by reacting the silica-attached silica capillary tube represented by Formula 4 prepared in Step 1 with a polymerization initiator represented by Formula 5.
  • silica capillary with attached polymerization initiator I a step.
  • a step For example, with respect to 1.0 m of the ligand-attached silica capillary represented by Formula 4 prepared in Step 1, 10-200 mg of a polymerization initiator represented by Formula 5 and a solution of 0.5-10.0 mL and anhydrous solvent were 25
  • the reaction is preferably carried out with 8 CTC for 4 to 24 hours, washed with anhydrous solvent, and dried under a stream of nitrogen.
  • the polymerization initiator represented by the formula (5) is less than 10 mg with respect to 1.0 m of the ligand-attached silica capillary represented by the formula (4), there is a problem of incomplete reaction completion, when exceeding 200 mg waste and There is a problem of occurrence of side reactions.
  • reaction temperature is less than 25 ° C there is a problem that the reaction rate is too slow, if the reaction temperature exceeds 80 ° C there is a problem that the thermal decomposition of the compound may occur.
  • the polymerization initiator represented by the formula (5) can be used as long as there is a dithio carbamate group, specifically, sodium dimethyldithiocarbamate, sodium dipropyldithiocarbamate, sodium dibutyldithiocarbamate , Sodium dimethyldithiocarbamate and the like can be used. It is preferable to use sodium diethyldithiocarbamate which is commercially available.
  • the semi-anhydrous solvent is polar enough to dissolve the polymerization initiator represented by Formula 5, and has a hydroxyl group, an amino group, Any solvent which does not have a carboxy group can be used. Among these, it is preferable to select a solvent having an ether group, a ketone group or an ester group and having a boiling point of 50 ° C. or more in order to keep the reaction temperature any longer and prevent side reactions. Specifically, the, or the like can be used in anhydrous tetrahydrofuran, diethyl Kerron ethyl acetate.
  • the washing solvent may be tetrahydrofuran, acecetone, methane / water mixed solvent, acetonitrile and the like.
  • the step 3 is a monomer dissolved in a solvent in a silica capillary tube with a polymerization initiator represented by Formula 6 prepared in step 2, and the reversible addition-fragmentation chain Reversible Addition-Fragmentation Chain Transfer (RAFT) is a step of preparing a polymer attached silica capillary represented by Chemical Formula 1 by performing polymerization reaction.
  • RAFT reversible addition-fragmentation chain Reversible Addition-Fragmentation Chain Transfer
  • the electroosmotic flow-induced monomer to the silica capillary tube with a polymerization initiator represented by the formula (6) prepared in step 2 to 1.0 m
  • the electroosmotic flow of the manufactured CEC column is too weak, and when used in excess of 150 mg, there is a problem of waste of material.
  • the retention time of the polar analyte is insufficient. 250 When used in excess of mg, the band or ratio of the polar analyte is too wide.
  • the reaction has a problem of incomplete reaction when the degree is less than 70 ° C, there is a problem of the reaction control impossible if it is more than 150 ° C.
  • reaction time is less than 6 hours, there is a problem of incomplete reaction, and when more than 36 hours, there is a problem of time wastage and side reactions.
  • the nonpolar monomer may be used without limitation as long as it is a compound having a benzene ring and having at least one double bond, but preferably has a molecular weight of 500 or less.
  • styrene, 4-methylstyrene, 2-chlorostyrene, 3-chlorostyrene, 4'chlorostyrene, 4-bromostyrene, 4-vinylbenzyl chloride, 4-vinylnaphthalene, etc. can be used. It is most preferable to use styrene because it is possible to obtain a stationary phase which is inexpensive and has excellent separation efficiency.
  • the polar monomer may be used without limitation as long as it has a benzene ring, a polar reactive group, and a compound having one or more double bonds, but preferably has a molecular weight of 500 or less.
  • N-phenylacrylamide, 4-amino styrene, 4- ⁇ N- (methylaminoethyl) amino methyl ⁇ styrene, 4-vinyl benzoic acid, 3, 4- dimethic styrene, etc. can be used.
  • the electroosmotic flow-inducing monomer has a carboxyl group or an amino group and can be used without limitation as long as it has a compound having one or more double bonds, but preferably has a molecular weight of 500 or less.
  • acrylic acid, methacrylic acid, methylmethacrylic acid, itaconic acid, allylamine, 4-amino styrene, 4-vinylpyridine, 2-vinylpyridine and the like can be used.
  • the nonpolar antisolvent can be used as long as it can dissolve the monomer well, but preferably has a benzene ring.
  • toluene, xylene, ethylbenzene and the like can be used.
  • the polar reaction solvent can dissolve the polar monomer well, and can be used as long as it does not have a hydroxyl group, amino group, carboxyl group. It is preferable that the boiling point is at least 80 ° C. Specifically, cyclopentanone, cyclonucleic acid temperature, methyl isobutyl ketone, 4-methyl-2-pentanone, or the like can be used.
  • the present invention provides a silica capillary for separation of oligosaccharides or peptides, characterized in that prepared by the above production method.
  • the inner diameter of the silica capillary is 25-200 um;
  • the length is 0.5-5 m;
  • the thickness of the resulting polymer film is 1-10 in dry state. It is good.
  • the present invention comprises the steps of reacting the oligosaccharides with the derivatization reagent to prepare the structural isomers of the oligosaccharides (step 1); And
  • step 1 is a step of preparing a structural isomer of the oligosaccharide by reacting the oligosaccharide with a derivatization reagent.
  • the ligosaccharide is preferably glucose or maltotriose, but is not limited thereto.
  • the derivatization reagent is preferably used para-aminobenzoic ethyl ester (aminobenzoic ethyl ester) #.
  • the structural isomer of the oligosaccharide prepared in Step 1 is reacted with a hydrogen reduction reagent to prepare the structural isomer of the oligosaccharide including the secondary amine before performing the step 2.
  • the hydrogen reduction reagent is preferably used cyanoborohydride (Nadium cyanoborohydride, NaBH 3 CN)-.
  • step 2 is a step of separating the structural isomers of the oligosaccharides prepared in step 1 through the silica capillary. Separation of the Oligosaccharides After that, the separation result can be confirmed by electrochromatogram. Further, the present invention is to hydrolyze the protein to prepare peptides (step 1); And
  • step 1 is a step of preparing a peptide by hydrolyzing the protein.
  • the protein is preferably Cytochrome C, but is not limited thereto.
  • step 2 is a step of separating the peptides prepared in step 1 through the silica capillary. After the separation of the oligosaccharides, the separation result is . This can be confirmed by electrochromatogram.
  • the silica capillary with polymer according to the present invention is characterized in that the peaks of analyte are accelerated as the mass transfer rate is accelerated as the CEC is in frostbite, especially in a solvent with a high content of organic solvent (acetonitrile). There is an effect that the band width is reduced and the separation efficiency is increased.
  • the capillary electro chromatography (CEC) using the silica-attached silica capillary prepared in Example 1 enables the separation of maltotriose co-isomers and D-glucose anomers according to acetonitrile content in the mobile phase.
  • CEC capillary electro chromatography
  • Step 1 Preparation of Silica Capillary Tube with Polymerization Initiator by Catalyzed Isocyanate-hydroxy Reaction
  • a solution was prepared in which 25 mg of 4-chloromethyl phenyl isocyanate and 20 mg of dibutyltin dichlo ride were dissolved in 2.5 mL of anhydrous toluene, which was prepared in a silica capillary with an internal diameter of 50 ⁇ and a length of 584 mm at 85 ° C. Over 20 hours (A process in FIG. 1). after. The capillary was washed with toluene for 10 hours at room temperature, washed for one day with aceron and then dried under nitrogen.
  • FIG. 2 shows a wide range (A) and a narrow range (B) photograph of an electron microscope with respect to a portion of the cross section of the polymer-capsulated silica capillary.
  • a solid polymer film is formed on the inner wall of the silica capillary.
  • the coating is composed of long polymer chains, which are expected to unfold when contacted with a mobile phase containing a large amount of organic solvent (acetonitrile).
  • Step 1 Preparation of Silica Moses 3 ⁇ 4 Attached to Polymerization Initiator by Catalyzed Isocyanate-hydroxy Reaction
  • Step 2 Preparation of Polymer Capillary Silica Tube by RAFT Polymerization Using Silica Capillary Tube with Initiator
  • silica capillary tube with a polymerization initiator prepared in step 1 0.6 mL of styrene, 70 uL methacrylic acid, 70 mg N-phenylacrylamide, dissolved in a mixed solvent of 2 mL toluene and 0.7 mL 4 -methyl-2 -pentanone at 14 ° C for 14 hours. I let go. Afterwards, Sangeun was transferred from to Ruen for 15 hours, 2-propane to 5 hours, 5 ⁇ / 50 (volume ratio) 2-propanol / water mixed solvent for 5 hours, and acetone for 1 hour. It was then dried under a nitrogen environment to prepare a silica attached capillary polymer. The capillary shell polymer was burned off to produce an ultraviolet light absorbing window 84 mm from one end of the silica capillary. Thus, the effective length of the prepared silica capillary column is 71 6 mm.
  • the reaction is carried out by reacting with an aldehyde group of a terminal sugar at one end of the sugar liposaccharide to form a Schiff base, which is reduced to become a secondary amine.
  • a hydrogen reduction reagent sodium cyanob orohydride (NaBH 3 CN) was used.
  • NaBH 3 CN sodium cyanob orohydride
  • D-glucose two anomers are observed when aminated, and only one substance is observed when reductively aminated.
  • maltotriose is D-glucose linked in 3 units It is a party.
  • FIG. 3 is a capillary electrochromatography (Capillary) for the maltotriose isomers (A, B, C) and D-glucose anomers (W, X, Y) as a silica capillary with a polymer prepared according to the present invention
  • Electrochromatography is an image showing the optimization of the acetonitrile composition of the eluent in elution (the acetonitrile content is 95% for A and W, 90% for B and X, 80% for C and Y, The CE voltage was 30 kV, the sample solution was injected at 12 kV, 5 seconds, and the optimum conditions were 90/10 acetonitrile / 30 mM sodium acetate pH 6.6 obtained with B and X, and D and Z were obtained under the optimized conditions.
  • Acetone electrochromatogram, ⁇ and) d are magnified electrochromograms of B and X, respectively). As shown in FIG. 3, when the volume content of acetonitrile was 90%, it was confirmed that maltotriose (B in FIG. 3) and D-glucose (X in FIG. 3) showed optimal separation.
  • CEC Capillary electrochro matography
  • the optimal mobile phase for the separation of maltotriose isomers and D-glucose anomers is 90/10 (volume ratio) acetonitrile / 30 mM sodium acetate pH 6.6.
  • the column performance and reproducibility at the optimum conditions are shown in Table 1 (specifically, Table 1 below shows the column-to-column and day-to-day data and reproducibility 3 of the column separation effect and retention time observed in the optimum mobile phase). to be).
  • Theoretical singular retention time Theoretical singular retention time Saturn / meter (minutes) / meter (minutes) Sieve average% average% average% average%.
  • % RSD is the relative standard deviation in%>.
  • a proteomic sample was prepared as follows as a product of hydrolyzing cytochrome C with trypsin. Mr. torque name seed 5 mg, trypsin 4 mg, 4 M urea aqueous solution 2 mL, 0.2 M ammonium bicarbonate (ammonium bic arbonate) 2 mL given a vigorous shake into a container together to create the solution, 37 ° water bath C Temperature Put in water for 48 hours Do it. After that, the filter was filtered using a 0.2 urn syringe filter, and stored in a 4 ° C. vault.
  • the prepared proteomic sample was subjected to capillary electrochromatography (CEC) separation analysis using the polymer-bonded silica capillary prepared in Example 2, and the results are shown in FIG. 5.
  • Elution conditions elute 10 min in a 78/22 (volume ratio) acetonitrile /12.5 mM clear phosphate (pH 6.8) mobile phase under a voltage of 30 kV and then 65/35 (volume ratio) acetonitrile / 12.5 mM sodium phosphate It was set as conditions eluting with the nitrate (pH 6.8) mobile phase.
  • Sample injection was injected for 5 seconds at a pressure of 8 mbar.
  • Example 5 is an electrochromatogram of capillary electrochromatography (CEC) separation analysis of proteomic hydrolysis samples using a silica-capillary capillary with polymer prepared in Example 2 (elution condition is a voltage of 30 kV) Eluted with 78/22 acetonitrile / 1 2.5 mM sodium phosphate pH 6.8 mobile phase for 10 minutes and then eluted with 65/35 acetonitrile / 12.5 mM digested phosphate pH 6.8 mobile phase, the top of FIG.
  • the separation electrochromogram for the theomic sample the lower part of which is the electrochromatogram of acetone, an electroosmotic flow marker. As shown in FIG.
  • the silica-capillary tube with the polymer prepared by the manufacturing method according to the present invention is prepared with excellent affinity for the polymer film itself, and thus adheres to the inside of the capillary tube in the form of a long, uniform polymer chain, and is dry. Although it has a solid film shape, it spreads widely in this frostbite with high acetonitrile content and acts to narrow the Bon-Ra-na-bavi on both sides of the analyte retention and mass transfer, which is useful for separating various kinds of sugar isomers. Can be used.
  • Silica capillary with polymer prepared by the manufacturing method according to the present invention has excellent affinity for the polymer film itself and is attached to the inside of the capillary tube in the form of a long, uniform polymer chain, while in a dry state, it shows a solid film shape, but aceto As it spreads wide in the mobile phase with high nitrile content and acts in the direction of narrowing peak bands on both sides of the analyte retention and mass transfer, it is possible to obtain sugars such as glucose and maltotriose.

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Abstract

La présente invention concerne un capillaire de silice fixé à un polymère pour séparer des oligosaccharides ou des peptides et un procédé pour le fabriquer. Le capillaire de silice fixé à un polymère fabriqué par le procédé selon la présente invention peut être avantageusement utilisé dans la séparation et l'analyse d'oligosaccharides, comme le glucose ou le maltotriose, et de nombreux peptides dans des hydrolysats de protéines, comme le cytochrome C, car un film polymère per se se forme, qui a une excellente affinité, et est donc fixé à l'intérieur du capillaire sous la forme d'une chaîne polymère longue et uniforme ; le film polymère présente la forme d'un film rigide dans un état sec mais s'étale largement sur une phase mobile ayant une teneur élevée en acétonitrile ; et le film polymère a un effet d'action dans une direction de rétrécissement de la largeur de bande de pic dans les deux aspects de rétention d'analyte et de transfert de masse.
PCT/KR2015/003669 2015-04-13 2015-04-13 Capillaire de silice fixé à un polymère pour séparer des oligosaccharides ou des peptides et procédé pour le fabriquer WO2016167379A2 (fr)

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