WO2016167379A2

WO2016167379A2 - Polymer-attached silica capillary for separating oligosaccharides or peptides and method for manufacturing same

Info

Publication number: WO2016167379A2
Application number: PCT/KR2015/003669
Authority: WO
Inventors: 정원조
Original assignee: 인하대학교 산학협력단
Priority date: 2015-04-13
Filing date: 2015-04-13
Publication date: 2016-10-20
Also published as: WO2016167379A3

Abstract

The present invention relates to a polymer-attached silica capillary for separating oligosaccharides or peptides and a method for manufacturing the same. The polymer-attached silica capillary manufactured by the method according to the present invention can be favorably used in the separation and analysis of oligosaccharides, such as glucose or maltotriose, and many peptides in hydrolysates of proteins, such as cytochrome C, since a polymer film per se is formed to have excellent affinity and thus is attached to the inside of the capillary in the form of a long and uniform polymer chain; the polymer film shows the shape of a rigid film in a dried state but widely spreads on a mobile phase having a high content of acetonitrile; and the polymer film has an effect of acting in a direction of narrowing the peak band width in both aspects of analyte retention and mass transfer.

Description

[Specification】

[Name of invention]

Oligosaccharides and Separation Silica Capillary Tubes with Polymers

Technical Field

The present invention relates to a silica capillary tube with a polymer for separating ligosaccharides or peptides and a method for preparing the same. [Background]

After attaching the polymerization initiator to the surface of the inorganic structure, 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. 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. But atomic transfer The separation efficiency (theoretical number) of radical polymerization (ATRP) stationary phases has been reported to be significantly inferior to conventional C 18 stationary phases. In reversible addition-fragmentation chain transfer (RAFT) polymerization, a ligand having a terminal halogen is first bound to the silica surface (see A in FIG. 1), and then reacted with a dithiocarbamate salt derivative to form a polymerization initiator attached silica (FIG. 1). B), surface polymerization proceeds here (see C of FIG. 1). 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. However, 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. . Thus, 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. In addition, 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. By preparing silica and proceeding reversible addition-fragmentation chain transfer (RAFT) polymerization, a new liquid chromatography stationary phase with a thin and even polymer membrane was developed and a patent application was filed (Korea Patent 10—1 1 16566). .

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.

In the present invention, 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. Field of structural analysis of sugar hitting a ^'full due to the party's diversity and structural complexity is extremely challenging. Capillary Electrophoresis (CE) 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. When the stationary phase is filled or attached inside the silica capillary, it is used for capillary

Electrochromatography (CEC), the separation selectivity is significantly improved compared to CE using a simple silica capillary. In other words, CEC

High-Performance Liquid Chromatography (HPLC) Recently, open tubular capillary electrochromatography (T-CEC), which uses thin polymer membranes attached to the inner wall of capillaries, has attracted much attention due to its ease of manufacture. OT-CEC is a useful method for the isolation and analysis of various kinds of biological materials such as proteins, peptides and glycoproteins. Stationary phases known to be effective for the analysis of sugar isomers include porous graphite carbide (PGC) and HIdrop (Hydrophilic Interaction Liquid Chromatography). 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. PGC in the Analysis of Sugar Structural Isomers 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.

(Open Tubular Capillary Electrochromatography, OT-CEC) Two D-glucose Anomers Prepared and Derivatized with Absorption Chromophore

We have reported the separation of several isomers of anomers and oligosaccharides with a high column separation efficiency (30, 000 / m theoretical) (Bulletin of the Korean Chemical Society, 2014, 35, 539). However, in the above study, the selection of the reaction solvent according to the high polarity difference between styrene and acrylamide; Due to the difficulty in self-compatibility of copolymerized polymers, problems such as reproducibility of column production and limitations of improving column separation efficiency were revealed. Accordingly, the inventors of the present invention introduced a new concept in the selection of the reaction reaction monomer while changing the reaction solvent, while improving the affinity of the produced copolymerized polymer while producing a capillary tube in which the above problems were improved. In addition to improving the reproducibility of the present invention, a silica capillary tube with a polymer and a method for preparing the same have been developed and the column separation efficiency is remarkably improved.

Prior Art Documents [Patent Documents]

(Patent Document 1) Korean Registered Patent 10-1116566

[Non-Patent Documents]

(Non-Patent Document 1) BuJJ. Korean Chew. Soc. 2009, Vol. 30, No. 3

[Detailed Description of the Invention]

[Technical Challenges]

Disclosure of Invention 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.

Technical Solution

In order to achieve the above object ，

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 ligand-attached silica capillary represented by Formula 4 prepared in Step 1 was reacted with the polymerization initiator represented by Formula 5. Preparing a silica capillary tube with a polymerization initiator represented by

2); And

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. It provides a method for producing a silica capillary tube comprising the step (step 3) of preparing a silica capillary tube with a co-polymer polymer represented by.

[Scheme]

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;

m is an integer from 1-1000. In addition, 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

It provides a separation method of the oligosaccharides comprising; step (step 2) of separating the structural isomers of the oligosaccharides prepared in step 1 through the silica capillary. In addition, the present invention is to hydrolyze the protein to prepare peptides (step 1); And

Peptides prepared in the step 1, the step of separating through the silica capillary (step 2) provides a method for separating peptides comprising.

Advantageous Effects

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.

It can be useful for separating and analyzing many peptides in hydrolysates of proteins such as CCCytochrome C). [Brief Description of Drawings]

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).

Figure 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).

4 is a CEC capillary electrochromatography (Capillary) for maltotriose isomers (Α, ΒΧ) and D-glucose anomers (W, X, Y) with a silica-capillary capillary with polymer prepared according to the present invention.

Ele ctrochromatogr.aphy, CEC) In the elution, 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 ¾ / 30 mM sodium acetate ρΗ 6. ^· 6 obtained with B and X, D and 에서 under the optimized conditions Electrochromatogram of aceron obtained).

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.

[Best form for implementation of the invention]

Hereinafter, the present invention will be described in detail. In the present invention, as shown in Scheme 1, the ligand attached silica represented by the formula (4) by reacting with the hydroxyl group represented by the formula (3) and the hydroxyl group present on the inner surface of the silica capillary tube represented by the formula (2) in the presence of a catalyst Preparing a capillary tube (step 1);

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

Formula by performing wave pyeonhwa ^{'principalities} transmission (Reversible Addition ^{^} Fragmentation Chain Transfer, RAFT) polymerization-filled melt the monomers in complex silica capillary inside a polymerization initiator unit represented by a formula (6) prepared in Step 2 in a solvent, reversible addition It provides a method for producing a silica capillary tube comprising the step (step 3) of preparing a silica capillary tube with a copolymerized polymer represented by 1.

Scheme 1]

In the reaction 1 Silica is located on the inner surface of the silica capillary

An aromatic ring of C _{6 10} ;

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;

m is an integer from 1-1000. Hereinafter, a method for preparing a silica capillary tube with a polymer according to the present invention will be described in detail step by step. In the method of preparing a silica capillary with polymer according to the present invention, 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. First, 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. As an example, 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 _. There is a problem of lowering, there is a problem of material waste when used in excess of 40 mg. In addition, the catalyst is less than 5 mg for 1.0 m silica capillary When used as a catalytic effect _. There is a problem that 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. Furthermore, when 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.

In addition, when the 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.

Furthermore, if the reaction time is less than 6 hours, there is a problem of incomplete reaction completion, if more than 48 hours there is a problem of waste time and side reactions. 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. In particular, compounds of transition metals having organic substituents are preferred in view of catalytic effect and solubility in solvents. Specifically, dibutyltindichloride, dibutyl tin diacetate

(dibutyltindiacetate), dibutyltindilaurate, triphenyltinacetate, tributyltin acetate

(tributyltinacetate), zinc diacetate, titanium tetra-acetate, cobalt tris (2-ethylhexanoate), bismuth tris (2-ethylnucleonos) Bismuth tris (2-ethylhexanoate), zinc di (2-ethylhexanoate), cobalt tris (2,4-pentadione) 2,4-pentadionate)), titanium tetra (2,4-pentadionate), manganese di (2,4-pentadioneate) pentadionate)), nickel di (2,4-pentadioneate) (nickel di (2,4-pentadionate), zirconium tetra (2,4-pentadione) (Zirc onium tetra (2,4-pentadionate)) and the like, and dibutyltindichlo ride is most preferred. 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 μπι and a length of 0.5-5.0 m can be used. Here, if the inner diameter of the capillary tube is less than 25 μπι, 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. In addition, when 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.

In Chemical Formula 3

C ₆ - ring ₁₀ may be a phenylene or naphthalene, most preferably phenylene. As the 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. Specifically, anhydrous toluene, anhydrous xylene, anhydrous methyl isobutyl ketone, anhydrous methyl isopropyl keron, cyclopentanone, butyrolactone and the like can be used. The anhydrous solvent in the washing process may be anhydrous toluene, acetone, tetrahydrofuran, acetonitrile and the like. In the method for preparing a polymer-attached silica capillary tube according to the present invention, 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. To prepare the silica capillary with attached polymerization initiator Is 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. Here, if 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.

In addition, when the anhydrous solvent is used in less than 0.5 mg for the ligand-attached silica capillary 1.0 m represented by the formula (4) there is a problem of incomplete semi-finished, if the excess exceeds 10.0 mL depending on the material dilution There is a problem of decrease in reaction speed.

Furthermore, if the 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.

In addition, if the reaction time is less than 4 hours, there is a problem of incomplete reaction completion, if more than 24 hours there is a problem of occurrence of side reactions. 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.

In the method of preparing a silica capillary tube with a polymer according to the present invention, 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. Here, with respect to 1.0 m of the silica capillary tube with a polymerization initiator represented by Chemical Formula 6 prepared in Step 2, 0.2-1 .5 mL of a nonpolar monomer, 30—150 mg of an electroosmotic flow-induced monomer, 30-250 mg of a polar monomer. A semi-aqueous solution consisting of 0.5-5.5 mL of a nonpolar solvent and 0.3-2.5 mL of a polar solvent was poured into 70-15 C C for 6-36 hours to proceed with RAFT polymerization reaction, washed with a non-polar and polar solvent, and washed with acelon. After that, drying in a nitrogen stream is preferable. Herein, when the nonpolar monomer is used in an amount less than 0.2 mL with respect to 1.0 m of the silica capillary with the polymerization initiator represented by Chemical Formula 6 prepared in Step 2, there is a problem of incomplete semi-finished, and in excess of 1.5 mL When used, there is a problem of impossibility of polymerization control.

In addition, 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 In case of using less than 30 mg, 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.

Furthermore, when the polar monomer is used in an amount less than 30 mg with respect to the polymerization initiator-attached silica capillary 1.0 m represented by Chemical Formula 6 prepared in Step 2, 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.

In addition, when the nonpolar semi-aqueous solvent is used in less than 0.5 mL with respect to 1.0 m of the silica capillary tube with the polymerization initiator represented by Chemical Formula 6 prepared in Step 2, there is a problem of heterogeneous reaction, and more than 5.5 mL is used. In this case, there is a problem of reducing the reaction rate due to mass lime.

Furthermore, when the polar reaction solvent is used in less than 0.3 mL with respect to 1.0 m of the polymerization initiator-attached silica capillary represented by Chemical Formula 6, there is a problem in dissolving the polar monomers, Affinity may be limited, and when used in excess of 2.5 mL, there is a problem of decrease in reaction speed due to dilution of material.

In addition, 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.

Furthermore, when the 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. Specifically, 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. Specifically, 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. Specifically, 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. Specifically, 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.

In the washing, a solvent such as toluene, 2-propanol, acetonitrile, acetone may be used. In addition, the present invention provides a silica capillary for separation of oligosaccharides or peptides, characterized in that prepared by the above production method. Wherein 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. Furthermore, 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

It provides a separation method of the oligosaccharide comprising the; step (step 2) of separating the structural isomer of the oligosaccharide prepared in step 1 through the silica capillary. Hereinafter, the step of separating the oligosaccharides according to the present invention will be described in detail. In the separation method of the oligosaccharide according to the present invention, step 1 is a step of preparing a structural isomer of the oligosaccharide by reacting the oligosaccharide with a derivatization reagent.

At this time, the ligosaccharide is preferably glucose or maltotriose, but is not limited thereto.

In addition, the derivatization reagent is preferably used para-aminobenzoic ethyl ester (aminobenzoic ethyl ester) #. Here, in the separation method, 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. It may further include, and the hydrogen reduction reagent is preferably used cyanoborohydride (Nadium cyanoborohydride, NaBH ₃ CN)-. In the method for separating oligosaccharides according to the present invention, 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

Peptides prepared in the step 1, the step of separating through the silica capillary (step 2) provides a separation method of the peptide comprising a. Hereinafter, the step of separating the oligosaccharides according to the present invention will be described in detail. In the method for isolating saccharides according to the present invention, step 1 is a step of preparing a peptide by hydrolyzing the protein.

In this case, the protein is preferably Cytochrome C, but is not limited thereto.

In addition, the hydrolysis of the protein is preferably trypsin (Trypsin), but is not limited thereto. In the method for separating oligosaccharides according to the present invention, 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. Therefore, 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. Experiments were conducted to evaluate whether the volume content of acetonitrile was 90%, indicating that both malto triose (B in FIG. 3) and D-glucose (X in FIG. 3) showed optimal separation. (See FIG. 3 of Experimental Example 1). In addition, the separation of maltotraose structural isomers and D-glucose anomers according to the pH of the mobile phase through capillary electrochromatography (CEC) using the polymer-attached silica capillary prepared in Example 1 Experiments were conducted to evaluate the feasibility of the experiments, indicating that both maltotriose (D in Fig. 4) and D-glucose (X in Fig. 4) showed optimal separation when the pH of the mobile phase buffer was 6.6. (See FIG. 4 of Experimental Example 2). Furthermore, as a result of the experiment for separating and analyzing the proteomic sample using the polymer-attached silica capillary prepared in Example 2, more than 20 peaks (peptides) were separated from the proteomic sample and the theory of each peak It was confirmed that the singular shows good results ranging from several hundred thousand to one million (see FIG. 5 of Experimental Example 3).

[Mode for carrying out the invention]

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto. Example 1 Preparation of Silica Capillary Tube with Polymer 1

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 μηι 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. In the dry capillary, a solution of 100 mg of sodium diethyl dithiocarbamate dissolved in 3.0 mL of anhydrous tetrahydrofuran was poured at 55 ° C. for 12 hours (B process in FIG. 1). After the reaction, the capillary was immediately washed with tetrahydrofuran at room temperature for 6 hours, followed by methanol for 2 hours. Finally, the mixture was washed with acetone for 2 hours and dried for 30 minutes in a nitrogen environment to prepare a silica capillary tube with a polymerization initiator. Step 2: Preparation of Polymer Capillary Silica Tube by RAFT Polymerization Using Silica Capillary Tube with Initiator

In the silica capillary with the polymerization initiator prepared in step 1, 0.6 mL styrene, 50 uL methacrylic acid, 80 mg N _ phenylacrylamide, 2 mL anhydrous and 0.8 mL 4 -methyl-2 -pentanone The solution dissolved in the mixed solvent was held at 10C C for 15 hours (C process of FIG. 1). Banung solution to ultrasonic vibration with a nitrogen purge go through each 10 minutes, and filtering using a syringe filter of 0.2 μι ^η pupil. Immediately after the reaction, the following washing process was performed. Specifically, 5 hours at room temperature with toluene, 5 hours at 50 ^° C, and 5 hours at 4-methyl-2-pentanone at Sangeun and finally 2 hours at acetone. Thereafter, the mixture was dried in a nitrogen environment to prepare a silica-attached silica capillary tube. The capillary shell polymer was burned off to produce a UV absorbing window 84 mm from one end of the silica capillary. Thus, the effective length of the silica capillary column is 500 mm. 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. As shown in Figure 2, it can be seen that 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).

Example 2 Preparation of Silica Capillary Tube with Polymer 2

Step 1: Preparation of Silica Moses ¾ Attached to Polymerization Initiator by Catalyzed Isocyanate-hydroxy Reaction

30 mg of 4-chloromethyl phenyl isocyanate and dibutyl dichloride

(dib utyltin dichloride) 40 mg was dissolved in 2.5 mL of anhydrous toluene to make a solution, which was poured into a silica capillary tube having an internal diameter of 50 μιη and a length of 800 mm at 85 ^° C. over 6 hours. The capillary was then washed with toluene at room temperature for one day, with acetone for one hour, and then dried under nitrogen. In the dry capillary, a solution of 100 mg of sodium diethyl dithiocarbamate dissolved in 3.0 mL of anhydrous tetrahydrofuran was poured at 55 ^° C. for 6.5 hours. After the reaction, the capillaries were immediately poured into tetrahydrofuran at room temperature for one day, washed with methanol for 5 hours, finally washed with acetone for 2 hours, dried under nitrogen environment for 30 minutes, and then subjected to polymerization initiator-attached silica. Capillary tubes were prepared. Step 2: Preparation of Polymer Capillary Silica Tube by RAFT Polymerization Using Silica Capillary Tube with Initiator

In the 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 Ό / 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.

Experimental Example 1 Evaluation of Separation Ability of Maltotriose Structural Isomers and D-glucose Anomers (Effect of Acetonitrile Content in Mobile Phase)

Separation of maltotriose sphere isomers and D-glycose anomers according to acetonitrile content in the mobile phase through capillary electrochromatography (CEC) using the polymer-attached silica capillary prepared in Example 1 An experiment was performed to evaluate the possibility, and the results are shown in FIG. 3. At this time, the sugar sample was derivatized in order to be detected by an ultraviolet absorbance detector. The derivatization reagent was subjected to an amination reaction using para-aminobenzoic acid ethyl ester. Subsequently, 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. When hydrogen reduction is done, this is called reductive amination. As a hydrogen reduction reagent, sodium cyanob orohydride (NaBH ₃ CN) was used. In the case of D-glucose, two anomers are observed when aminated, and only one substance is observed when reductively aminated. Thus D-glucose was aminated and maltotriose was reduced amine. Here, maltotriose is D-glucose linked in 3 units It is a party. Figure 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 (CEC) 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.

Experimental Example 2 Evaluation of Separation Capability of Maltotraose Structural Isomer and D-Glucose Anomer (Phase Effect of Mobile Phase)

Capillary electrochro matography (CEC) using the silica-attached silica capillary prepared in Example 1, the separation of maltotriose structural isomers and D-glucose anomer according to the pH of the mobile phase An experiment was conducted to evaluate whether the result was shown in FIG. 4. At this time, the acetonitrile volume content was fixed at 90% in the mobile phase, except that the mobile phase at various pHs was used, and was carried out in the same manner as in Experimental Example 1. 4 is a CEC capillary electrochromatography method for maltotriose isomers (A, B, C) and D-glucose anomers (W, X, Y) with a silica-attached silica capillary prepared according to the present invention. Capillary

In Electrochromatography, CEC) elution, the image showing the optimization process for the pH of the eluting solution (pH is 7.3 with respect to 5.5, and for the B and X 6.6, C and Y, with respect to A and W, _CE. Voltage is 30kV, Sample solution injection was performed at 8 kV and 5 seconds, and the optimization conditions were 90/10 acetonitrile / 30 mM sodium acetate pH 6.6 obtained with B and X, and D and Z are the electrochromograms of acetone obtained under the optimized conditions. ). As shown in FIG. 4, when the pH value of the mobile phase buffer was 6.6, it was confirmed that maltotriose (B in FIG. 4) and D-glucose (X in FIG. 4) showed optimal separation. Thus, 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).

a. Three batches of capillary columns were prepared for column-to-column reproducibility investigations and three consecutive days were measured with one of them for day-to-day reproducibility investigation. Table 1

Substance column vs column blade vs blade

Theoretical singular retention time Theoretical singular retention time Saturn / meter (minutes) / meter (minutes) Sieve average% average% average% average%. Lee RSD RSD RSD RSD Oh 1,364,000 4.6% 15.11 2.3% 1,337,000 1.4% 15.02 1.1% Switch 1

Μ 1,473,000 4.9% 15.40 2.4% 1,490,000 1.7% 15.39 1.2%

2

Μ 1,358,000 4.5% 15.80 2.5% 1,381,000 1.1% 15.77 0.8%

3

Μ 1,280,000 3.8% 16.20 2.6% 1,305,000 1.5% 16.13 0.5%

4

Μ 1,264,000 4.0% 16.5 2.8% 1,240,000 0.9% 16.44 0.9%

5

D Gi 1,008,000 3.9% 12.85 1 6% 1,023,000 1.0% 12.93 1.2%

G ₂ 976,000 4.3% 13.13 1.7% 965,000 1.2% 13.19 0.8%

τ

nose

Five

S

In Table 1 above,

% RSD is the relative standard deviation in%>.

. As shown in Table 1, the theoretical number of columns exceeds one million per meter, and it can be seen that the reproducibility of the column is very excellent. Two anomers of D glucose were completely separated, and five isomers were observed for maltotriose. Excellent column separation efficiency and peaks As the bandana is narrow, the chromatographic resolution between two adjacent peaks is excellent, and as shown in Table 2, the value is about 6.2-7.5.

Table 2

This result is remarkably superior to any isomeric separation of sugars reported in the prior art. The strengths of the present ^{invention, "structured} polymer attachment silica capillary column open according to is stick to the capillary inside to be prepared in excellent polymer film itself affinity formed in the manufacturing conditions, a long and uniform polymer principalities aspect of the present invention, dry In the state of A ', it shows the shape of a solid film, but it spreads widely in the mobile phase with high acetonitrile and acts in the direction of narrowing peak band butterfly on both analyte retention and mass transfer.

Experimental Example 3 Evaluation of Separation Analysis of Proteomic Samples

In order to separate and analyze a proteomic sample using the silica-attached silica capillary prepared in Example 2, the following experiment was performed. First, 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. FIG. 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. 5, more than 20 peaks (peptides) were separated from the proteomic sample, and the theoretical number of peaks of each peak showed good results ranging from several hundred thousand to one million. Therefore, 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.

[Available industrially]

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.

It may be useful for the separation and analysis of many peptides in the hydrolysis products of proteins such as CCCytochrome C).

Claims

[Range of request]

【Claim 1】.

As shown in Reaction Formula 1 below, reacting the hydroxyl group present on the inner surface of the silica capillary tube represented by Formula 2 with the ligand represented by Formula 3 in the presence of a catalyst to prepare a ligand-attached silica capillary represented by Formula 4 (Step 1);

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.

The monomer was dissolved in a solvent in a silica capillary tube with a polymerization initiator represented by Chemical Formula 6 prepared in step 2, and a reversible addition-fragmentation "Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization reaction was performed. A method for preparing a silica capillary tube comprising the step (step 3) of preparing a silica capillary tube with a co-condensation polymer represented by Formula 1:

[Scheme 1

(In the above reaction l,

Silica located on the inner surface of the silica capillary;

0 _6-ring of the _10;

R ¹ and R ² are independently hydrogen or alkyl of Ci- ₄ ;

M is a monomer constituting the co-polymer chain formed in Step 3;

n is an integer from 1-10;

m is an integer from 1-1000).

[Claim 2]

The method of claim 1,

The catalyst of step 1 is a manufacturing method characterized in that the boiling point is dissolved in a nonpolar anhydrous solvent of 60 ^° C or more, including a transition metal, an organic metal compound having an organic substituent. _

Γclaim 3

The method of claim 2

And said anhydrous solvent is at least one selected from the group consisting of anhydrous leuene, anhydrous xylene, anhydrous methyl isobutyl keron, anhydrous methyl isopropyl ketone, cyclopentanone and butyrolactone.

[Claim 4]

The method of claim 1,

The catalyst of step 1 is dibutyltindichloride (dibutyltindichloride), dibutyltin diacetate (clibutyltindiacetate), dibutyltindilaurate (di_butyltindilaurate), triphenyltinacetate (triphenyltinacetate), tributyltinacetate (tributyltinacetate), zinc acetate (zinc diacetate), titanium tetra-acetate, cobalt tris (2-ethylhexanoate), bismuth tris (2-ethylnucleonoate) (bismuth tris (2-ethylhexanoate)), zinc di (2-ethylnucleonoate) (zinc di (2-ethylhexanoate)), cobalt tris (2,4 pentadionate) ( cobalt tris (2,4-pentadionate)), titanium tetra (2,4-pentadionate), manganese di (2,4-pentadionate) , 4-pentadionate), nickel di (2,4-pentadionate) (nickel di (2,4-pentadionate) and zirconium tetra (2,4-pentadiionate) (zirconium tetra (2,4-pentadionate) 1) at least one member selected from the group consisting of

[Claim 5]

The method of claim 1,

The monomer of step 3 is characterized in that it comprises a non-polar monomer, an electroosmotic flow induced monomer and a polar monomer.

[Claim 6]

The method of claim 5,

The non-polar monomer is a benzene ring, consisting of styrene, 4-methylstyrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 4—bromostyrene, 4—vinylbenzyl chloride and 4-vinylnaphthalene; At least one selected from the group of compounds having a double bond;

The electroosmotic flow-inducing monomer is composed of acrylic acid, methacrylic acid, methyl methacrylate, itaconic acid, allylamine, 4-aminostyrene, 4-vinylpyridine and 2-vinylpyridine, carboxyna amino functional group and double bond At least one selected from the group of compounds having; And

The polar monomer is composed of N-phenylacrylamide, 4-aminostyrene, 4- {N- (methylaminoethyl) aminomethyl} styrene, 4-vinylbenzoic acid and ^' 3,4-dimethoxystyrene, polar with benzene ring At least one member selected from the group of compounds having a functional group and a double bond Manufacturing method

[Claim 7]

The method of claim 1,

The solvent of step 3 is at least one member selected from the group consisting of a polar solvent and a non-polar solvent.

[Claim 8]

In accordance with paragraph 7,

Wherein the polar solvent is cyclo pentane, the ^"cycle on the nucleic acid, methyl isobutoxy butyl Kerron and 4, and one or more selected from the group consisting of methyl-2-pentanone; and"

The nonpolar solvent is at least one selected from the group consisting of toluene, xylene and ethylbenzene.

[Claim 9]

Silica capillary for separation of oligosaccharides or peptides, characterized in that prepared by the method of claim 1.

[Claim 10]

Reacting the oligosaccharide with the derivatization reagent to prepare a structural isomer of the oligosaccharide (step 1); And

Separating the structural isomers of the oligosaccharides prepared in step 1, through the silica capillary tube of claim 9 (step 2); Separation method of oligosaccharides comprising a.

[Claim 11]

Hydrolyzing the protein to prepare peptides (step 1); And Peptides prepared in step 1, the step of separating the silica capillary of claim 9 (step 2); separation method of the peptides comprising a.