CN102940980B - Preparation method and application for hydrophilic organic polymer liquid phase monolithic chromatographic column - Google Patents
Preparation method and application for hydrophilic organic polymer liquid phase monolithic chromatographic column Download PDFInfo
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Abstract
The invention belongs to the research field of organic polymeric materials, and discloses a preparation method and an application for a hydrophilic organic polymer liquid phase monolithic chromatographic column. The method comprises the following steps of employing monomers of 3-[N,N-dimethyl-[2-(2-enoyloxy)ethyl]butylamin]propane-1-sulfonate as functional monomers, mixing the functional monomers with a cross-linking agent, a pore-forming agent and an initiator, performing a thermocatalytic reaction, and carrying out in-situ polymerization in a quartz capillary to form the polymer liquid phase monolithic chromatographic column. The obtained chromatographic column has larger hydrophilic interaction area than a common hydrophilic chromatographic column. The monolithic chromatographic column is small in usage amount of the organic mobile phase, has little environmental pollution, is simple in preparation process and low in cost, has low column pressure, and is suitable for rapid separation of polar compounds.
Description
Technical field
The invention belongs to the chromatogram research field of organic polymer materials, particularly a kind of method and application of preparing hydrophily Organic Polymer Monolithic Columns by thermal polymerization technology original position.
Background technology
As everyone knows, liquid chromatogram and GC-MS have been widely used in the fields such as environment, medical science and life science, also day by day go deep into about the research of its core parts chromatography separation media and analytic function.In recent years, novel chromatographic stationary phases integral material, because having, high-permeability, low post are pressed, mass transfer velocity is fast, prepare the advantages such as simple, become one of study hotspot, and success is applied in the clastotypes such as reverse-phase chromatography, ion-exchange chromatography, affinity chromatography, has effectively separated the multiclass compounds such as amino acid, polypeptide, protein, polypeptide and steroids.Prepare the difference of function monomer according to integral material; mainly be divided into three major types: Organic Polymer Monolithic Columns, monolithic silica column and hybrid integral post [Zou H; Huang X; Ye M, Luo Q.Monolithic stationary phases for liquid chromatography and capillary electrochromatography.J Chromatogr is A.2002; 954 (1-2): 5-32.].Because Organic Polymer Monolithic Columns preparation only has a phase conventionally, therefore its preparation is the easiest, select nature more, expand widely range of application, especially aspect bioanalysis.Hydrophilic liquid phase chromatographic column is the core component of hydrophilic liquid phase chromatogram, has directly determined the separating effect of chromatogram.From nineteen ninety hydrophilic liquid phase chromatogram (Hydrophilic interaction chromatography; HILIC) rear [Alpert AJ.Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds.J Chromatogr.1990 are proposed first; 499:177-96.], become one of study hotspot of international chromatographic field for the research of the polyalcohol integral pole of HILIC.As Lin etc. passes through in-situ polymerization 3-sulfopropyl methacrylate (3-sulfopropyl methacrylate, and pentaerythritol triacrylate (pentaerythritol triacrylate SPMA), PETA), be prepared into the integral post of surface band sulfonic group and hydroxyl, capillary liquid chromatography with under pressure-actuated capillary electric chromatogram pattern, the little neutrality of polarity is successfully separated to [Lin J with charged molecule, Huang G, Lin X, Xie Z.Methacrylate-based monolithic column with mixed-mode hydrophilic interaction/strong cation-exchange stationary phase for capillary liquid chromatography and pressure-assisted CEC.Electrophoresis.2008, 29 (19): 4055-65.], in order to strengthen its hydrophilic interaction, author by adding long chain quaternary-softex kw (cetyltrimethylammonium bromide in mobile phase, CTAB), shielding strong cation exchange site, set up the strong cation integral post of dynamic embellishment, realize rp mode centering, alkaline and acid hydrophilic compounds successfully separate [Lin J, Lin J, Lin X, Xie Z.Capillary liquid chromatography using a hydrophilic/cation-exchange monolithic column with a dynamically modified cationic surfactant.J Chromatogr A.2009, 1216 (45): 7728-31.], Lin etc. also use 2-(methylacryoyloxyethyl) alkyl trimethyl sulfuric acid (2-(methacryloyloxy) ethyl trimethylammonium methyl sulfate, META) be ion-exchange site and polarity aglucon donor, with triethylene monomer PETA combined polymerization, by adjusting the ratio of pore generating agent and META, successfully prepare the integral post with strong cation and hydrophilic mechanism, centering, acid have well selective with separating of alkaline polar compound, and can avoid smearing [the Lin J of alkali compounds, Lin J, Lin X, Xie Z.Preparation of a mixed-mode hydrophilic interaction/anion-exchange polymeric monolithic stationary phase for capillary liquid chromatography of polar analytes.J Chromatogr A.2009, 1216 (5): 801-6.], under photoinduction, N, N-dimethyl-N-first propylene ethyl-N-(3-sulfonic acid) ammonium betaine (N, N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine) and PEG diacrylate (poly (ethylene glycol) diacrylate) combined polymerization be prepared into the porous amphion integral post of Hydrophilic Mechanism, to amino, phenols and benzic acid compounds have good separation function [Chen X, Tolley HD, Lee ML.Preparation of zwitterionic polymeric monolithic columns for hydrophilic interaction capillary liquid chromatography.J Sep Sci.2011Jun 22.doi:10.1002/jssc.201100155.].Feng YQ seminar utilizes N-acryloyl three (methylol) aminomethane (poly (N-acryloyltris (hydroxymethyl) aminomethane, and PETA NAHAM), methacrylic acid (methacrylic acid, and ethylene glycol dimethyl (ethylene glycol dimethacrylate MAA), EDMA) integral post that prepared by combined polymerization is respectively successfully for nucleoside compound [Chen ML, Wei SS, Yuan BF, Feng YQ.Preparation of methacrylate-based monolith for capillary hydrophilic interaction chromatography and its application in determination of nucleosides in urine.J Chromatogr A.2012, 1228:183-92.] and analysis [the Chen ML of bovine serum albumin(BSA) enzymolysis polypeptide product, Li LM, Yuan BF, Ma Q, Feng YQ.Preparation and characterization of methacrylate-based monolith for capillary hydrophilic interaction chromatography.J Chromatogr is A.2012, 1230:54-60.].(tetramethoxy-silicane (tetramethoxysilane such as alkoxy silane for Wang etc., and vinyl-trimethoxy silane (vinyl-trimethoxysilane TMOS), VTMS) aldolisation), at water soluble starter 2, two (the 2-methyl-propyl amidine) hydrochlorides (2 of 2 '-azo, 2 '-azobis (2-methylpropionamidine) dihydrochloride, AIBA) under effect, with acrylamide monomer with quaternary ammonium group ([2-(acryloyl-oxy) ethyl] trimethyl ammonium sulfate ([2-(acryloyloxy) ethyl] trimethyl ammonium methyl sulfate, AETA) copolyreaction, success preparation has affine and organic inorganic hybridization integral post strong cation exchange effect, and under pressure capillary electric chromatogram pattern, polarity is carried out separating [Wang X with alkaline nucleic acid base and nucleosides, Zheng Y, Zhang C, Yang Y, Lin X, Huang G, Xie Z.Preparation and characterization of hybrid-silica monolithic column with mixed-mode of hydrophilic and strong anion-exchange interactions for pressurized capillary electrochromatography.J Chromatogr A.2012, 1239:56-63.], recently, there is one kettle way strategy (one-pot) successfully to prepare to have the zwitterionic silica hybrid integral post of hydrophilic separating mechanism, use 3-methoxy silane acrylamide (3-methacryloxypropyltrimethoxysilane, γ-MAPS) be crosslinking agent, synthesize two kinds of Iwitterionic siloxane hybrid integral posts ([2-(methacryloxypropyl) ethyl] dimethyl (3-sulfonic acid propyl group) ammonium hydroxide ([2-(methacryloyloxy) ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide, and 2-methacryl ethoxy Phosphorylcholine (2-methacryloyloxyethyl phosphorylcholine MSA), MPC) be function monomer), its permeability and mechanical strength are fine, MSA integral post can centering, alkalescence separates with acid compound and little peptide, simultaneously, coupling tandem mass spectrum can successfully be identified BSA enzymolysis polypeptide [Lin H, Ou J, Zhang Z, Dong J, Wu M, Zou H.Facile preparation of zwitterionic organic-silica hybrid monolithic capillary column with an improved " one-pot " approach for hydrophilic-interaction liquid chromatography (HILIC) .Anal Chem.2012, 84 (6): 2721-8.].The director of this seminar has also carried out the work of a series of hydrophilic interaction integral post, as used 4-SPV(1-(3-sulphopropyl)-4-vinylpyridinium-betaine) and methyl bisacrylamide (N, N '-methylenebisacrylamide, MBA) integral post of copolymerization, purine and pyrimidine [Jiang Z in 1min, are successfully separated, Smith NW, Ferguson PD, Taylor MR.Novel highly hydrophilic zwitterionic monolithic column for hydrophilic interaction chromatography.J Sep Sci.2009, 32 (15-16): 2544-55.], the organic polymer film that MPC and EDMA polymerization are prepared into is separating polar compound and little peptide [Jiang Z effectively, Reilly J, Everatt B, Smith NW.Novel zwitterionic polyphosphorylcholine monolithic column for hydrophilic interaction chromatography.J Chromatogr is A.2009, 1216 (12): 2439-48.], taking methacrylate as function monomer, successfully prepare SPE-co-EDMA integral post, centering, acidity and alkaline polar micromolecules compound have good selective [Jiang Z, Smith NW, Ferguson PD, Taylor MR.Hydrophilic interaction chromatography using methacrylate-based monolithic capillary column for the separation of polar analytes.Anal Chem.2007, 79 (3): 1243-50.].Although the research of hydrophilic interaction mechanism solid chromatography column is many, these chromatographic column polarity are limited, and solvent range of choice is narrow, mostly can only be applied to the condition of high organic phase (>60%), limited its optimizing application scope.Therefore, how further to improve the polarity of hydrophilic solid chromatography column, to effective enrichment, the separation of strong polar compound, remain separation science problem in science in the urgent need to address.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art part, primary and foremost purpose of the present invention is to provide a kind of preparation method of hydrophily organic polymer liquid phase solid chromatography column; The method is prepared by thermal polymerization technology.
The hydrophily organic polymer liquid phase solid chromatography column that provides said method to prepare is provided.
A further object of the present invention is to provide the application of above-mentioned hydrophily organic polymer liquid phase solid chromatography column.
Object of the present invention is achieved through the following technical solutions: a kind of preparation method of hydrophily organic polymer liquid phase solid chromatography column, comprise following operating procedure: with monomer 3-[N, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt (N, N-dimethyl-N-acryloyloxyethyl-N-(3-sulfopropyl) ammonium betaine, SPDA, No. CAS is 88992-91-0) be function monomer, by itself and crosslinking agent, pore generating agent and initator mix, carry out heat catalysis, in the pretreated quartz capillary of process, in-situ polymerization becomes hydrophily organic polymer liquid phase solid chromatography column.
The pretreated quartz capillary of described process is to prepare by following operating procedure: with the NaOH solution flushing quartz capillary 15min of 1mol/L, then quartz capillary sealing two ends is placed on to 100 DEG C of water-baths reaction 2h; Then use deionized water scouring stone English capillary, until the liquid pH flowing out is 7; Then with using nitrogen drying 4h after washed with methanol quartz capillary; After dry, the methyl alcohol that is 1:1 volume ratio and 3-(isobutene acyl-oxygen) propyl trimethoxy silicane (γ-MAPs) mixed liquor throws in quartz capillary, sealing two ends and be placed on reaction 12h in 60 DEG C of water-baths; Finally respectively rinse 30min with first alcohol and water respectively, nitrogen drying 12h, obtains through pretreated quartz capillary; By aforesaid operations, at silanol base and γ-MAPs(3-(isobutene acyl-oxygen on quartz capillary surface) propyl trimethoxy silicane) react, γ-MAPs is bonded on silanol.
The preparation method of above-mentioned hydrophily organic polymer liquid phase solid chromatography column, specifically comprise following operating procedure: by monomer 3-[N, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt, crosslinking agent, pore generating agent and initator mix, after ultrasonic degas, pour into through in pretreated quartz capillary column, then by quartz capillary sealing two ends, putting into water-bath reacts, remove after completion of the reaction the unreacted reactant in quartz capillary, obtain hydrophily organic polymer liquid phase solid chromatography column.
Described crosslinking agent is N, N-methylene-bisacrylamide (N, N '-methylenebisacrylamide, MBA), described pore generating agent is that mass ratio is the mixture of the first alcohol and water of 67:33～70:30, and described initator is azodiisobutyronitrile (Azobisisobutyronitrile, AIBN).
Described pore generating agent is that mass ratio is the mixture of the first alcohol and water of 68.5:31.5.
The temperature of described reaction is 45～75 DEG C, and the time is 2～24h; Described monomer 3-[N, the mass ratio of N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt and crosslinking agent is 70:30～85:15; The quality of described initator is monomer 3-[N, 0.5%～1% of N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt and crosslinking agent gross mass; Described monomer 3-[N, the quality sum of N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt and crosslinking agent, with the mass ratio of pore generating agent be 25:75～35:65.
Be more preferably, the temperature of described reaction is 60 DEG C, and the time is 12h; Described monomer 3-[N, the mass ratio of N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt and crosslinking agent is 85:15; The quality of described initator is monomer 3-[N, 1% of N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt and crosslinking agent gross mass; Described monomer 3-[N, the quality sum of N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt and crosslinking agent, with the mass ratio of pore generating agent be 30:70.
The described unreacted reactant of removing in quartz capillary is to operate according to following steps: one end of quartz capillary is connected with high-pressure pump, rinses with organic solvent, described organic solvent is at least one in methyl alcohol, acetonitrile and acetone.
The hydrophily organic polymer liquid phase solid chromatography column preparing according to said method, this chromatographic column has continuous loose structure (SPDA-co-MBA).It is by the monomer 3-[N of end band hydrophily sulfonic acid group, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt (SPDA) successively order connects N, N-methylene-bisacrylamide (MBA), γ-MAPs(3-(isobutene acyl-oxygen) propyl trimethoxy silicane) and the silanol base formation of capillary wall.
The application of above-mentioned hydrophily organic polymer liquid phase solid chromatography column in the compartment analysis research of polar substances; Described polar substances is base, nucleosides and little peptide.
The present invention compared with prior art, tool has the following advantages and beneficial effect: solid chromatography column of the present invention (v/v) under 5% acetonitrile concentration is still hydrophily interaction, and the hydrophily chromatographic column of general bibliographical information be greater than under 60% acetonitrile, just express hydrophily interaction (can bibliography [Jiang Z, Smith NW, Ferguson PD, Taylor MR
hydrophilic interaction chromatography using methacrylate-based monolithic capillary column for the separation of polar analytes.anal Chem.2007; 79 (3): 1243-50.]).Therefore, this chromatographic column hydrophilic interaction interval is larger than general hydrophily chromatographic column; Organic mobile phase consumption is few, low in the pollution of the environment; Preparation technology is simple, and cost is low; Post forces down, and is applicable to polar compound (as the urea in vitamin, cosmetics in fruit and allantoin etc.) and separates fast.
Brief description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph that embodiment 1 prepares gained SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column.
Fig. 2 is the retention behavior figure that embodiment 1 prepares gained SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column.
Fig. 3 is that embodiment 1 prepares gained SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column separation base chromatogram.
Fig. 4 is the nucleosides separate colors spectrogram that embodiment 1 prepares gained SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column.
Fig. 5 is that embodiment 1 prepares the separating resulting figure of gained SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column to little peptide.
Fig. 6 is that embodiment 2 prepares the testing result figure of gained SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column to urea and allantoin standard items.
Detailed description of the invention
Below in conjunction withspecific examples and drawings the present invention is described in further detail, but implementation method of the present invention is flexible, is not limited only to the described concrete operations mode of this example.
The pretreated quartz capillary of process that embodiment 1～6 uses is to prepare by following operating procedure: with the NaOH solution flushing quartz capillary 15min of 1mol/L, then quartz capillary sealing two ends is placed on to 100 DEG C of water-baths reaction 2h; Then use deionized water scouring stone English capillary, until the liquid pH flowing out is 7; Then with using nitrogen drying 4h after washed with methanol quartz capillary; After dry, the methyl alcohol that is 1:1 volume ratio and 3-(isobutene acyl-oxygen) propyl trimethoxy silicane (γ-MAPs) mixed liquor throws in quartz capillary, sealing two ends and be placed on reaction 12h in 60 DEG C of water-baths; Finally respectively rinse 30min with first alcohol and water respectively, nitrogen drying 12h, obtains through pretreated quartz capillary.
Embodiment 1
By monomer 3-[N, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt 0.0600g, crosslinking agent N, N-methylene-bisacrylamide 0.0106g, pore generating agent (comprising methyl alcohol 0.1128g and water 0.0519g) and initator azodiisobutyronitrile 0.0010g, be mixed with reaction mixture, after ultrasonic degas, pour into through in pretreated quartz capillary, then by quartz capillary sealing two ends, put into 60 DEG C of water-baths and react 12 hours; After completion of the reaction quartz capillary is connected with high-pressure pump, falls unreacted reactant by washed with methanol, obtain SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column, in post, the electron scanning Electronic Speculum result of organic polymer as shown in Figure 1.
Embodiment 2
By monomer 3-[N, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt 0.0600g, crosslinking agent N, N-methylene-bisacrylamide 0.0257g, pore generating agent (methyl alcohol 0.1370g and water 0.0630g) and initator azodiisobutyronitrile 0.0010g, be mixed with reaction mixture, after ultrasonic degas, pour into through in pretreated quartz capillary, then by quartz capillary sealing two ends, putting into 60 DEG C of water-baths reacts 12 hours, after completion of the reaction quartz capillary is connected with high-pressure pump, fall unreacted reactant by washed with methanol, obtain SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column.
Embodiment 3
By monomer 3-[N, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt 0.0600g, crosslinking agent N, N-methylene-bisacrylamide 0.0132g, pore generating agent (methyl alcohol 0.1170g and water 0.0538g) and initator azodiisobutyronitrile 0.0010g, be mixed with reaction mixture, after ultrasonic degas, pour into through in pretreated quartz capillary, then by quartz capillary sealing two ends, putting into 60 DEG C of water-baths reacts 12 hours, after completion of the reaction quartz capillary is connected with high-pressure pump, fall unreacted reactant with acetone rinsing, obtain SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column.
Embodiment 4
By monomer 3-[N, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt 0.0600g, crosslinking agent N, N-methylene-bisacrylamide 0.0150g, pore generating agent (methyl alcohol 0.1541g and water 0.0709g) and initator azodiisobutyronitrile 0.0010g, be mixed with reaction mixture, after ultrasonic degas, pour into through in pretreated quartz capillary, then by quartz capillary sealing two ends, put into 60 DEG C of water-baths and react 12 hours; After completion of the reaction quartz capillary is connected with high-pressure pump, falls unreacted reactant with acetone rinsing, obtain SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column.
Embodiment 5
By monomer 3-[N, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt 0.0600g, crosslinking agent N, N-methylene-bisacrylamide 0.0150g, pore generating agent (methyl alcohol 0.0954g and water 0.0439g) and initator azodiisobutyronitrile 0.0010g, be mixed with reaction mixture, after ultrasonic degas, pour into through in pretreated quartz capillary, then by quartz capillary sealing two ends, put into 60 DEG C of water-baths and react 12 hours; After completion of the reaction quartz capillary is connected with high-pressure pump, rinses out unreacted reactant with acetonitrile, obtain SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column.
Embodiment 6
By monomer 3-[N, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt 0.0600g, crosslinking agent N, N-methylene-bisacrylamide 0.0150g, pore generating agent (methyl alcohol 0.1199g and water 0.0551g) and initator azodiisobutyronitrile 0.0010g, be mixed with reaction mixture, after ultrasonic degas, pour into through in pretreated quartz capillary, then by quartz capillary sealing two ends, put into 60 DEG C of water-baths and react 12 hours; After completion of the reaction quartz capillary is connected with high-pressure pump, rinses out unreacted reactant with acetonitrile, obtain SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column.
Embodiment 7
The liquid phase retention behavior of SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column:
With the SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column of embodiment 1 gained, acetonitrile and water are mobile phase, and toluene, uracil and thiocarbamide are test compounds, test their retention behaviors under the following conditions, and result is as shown shown in 2.
Test condition:
Sample: toluene, uracil, thiocarbamide
Mobile phase: acetonitrile/water
Flow velocity: 400nL/min
Detect wavelength: 254nm
Result: result shown in Fig. 2 shows that (5%, v/v) three curves have no crossing under low acetonitrile concentration, hydrophily interacts and still exists, large between aqueous favoring interaction region.Than the document [Jiang Z, Smith NW, Ferguson PD, Taylor MR
hydrophilic interaction chromatography using methacrylate-based monolithic capillary column for the separation of polar analytes.anal Chem.2007; 79 (3): 1243-50.] much bigger between the aqueous favoring interaction region of report.
Embodiment 8
SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column separates base:
With the SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column of embodiment 1 gained, isocratic elution, tests the separating effect of a series of bases in integral post, and result as shown in Figure 3.
Test condition:
Sample: toluene, thymidine, adenine, thiocarbamide, uracil, cytimidine
Mobile phase: 95% acetonitrile and 5% water (v/v)
Flow velocity: 400nL/min
Detect wavelength: 254nm
Result: result shown in Fig. 3 shows that above 6 biased samples utilize SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column of the present invention to separate, and can reach good separating effect.
Embodiment 9
SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column separates nucleosides:
With the SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column of embodiment 1 gained, isocratic elution, tests the separating effect of a series of nucleosides in integral post, and result as shown in Figure 4.
Test condition:
Sample: toluene, cytidine, adenosine, thymidine, uridine, inosine
Mobile phase: 90% acetonitrile and 10% water (v/v)
Flow velocity: 800nL/min
Detect wavelength: 254nm
Result: result shown in Fig. 4 shows that above 6 biased samples utilize SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column of the present invention to separate, and can reach good separating effect.
Embodiment 10
SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column separates little peptide:
With the SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column of embodiment 1 gained, isocratic elution, tests the separating effect of a series of little peptides in integral post, and result as shown in Figure 5.
Test condition:
Sample: Leu-Leu, Gly-Leu, Ala-Tyr, Gly-Gly-Gly, Phc-Gly-Trp-Mer-OH
Mobile phase: 90% acetonitrile and 10% water (v/v)
Flow velocity: 800nL/min
Detect wavelength: 254nm
Result: result shown in Fig. 5 shows that above 5 biased samples utilize SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column of the present invention to separate, and can reach good separating effect.
Embodiment 11
The detection of SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column to urea and allantoin standard items:
With the SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column of embodiment 1 gained, isocratic elution, test urea and the separating effect of allantoin standard items in integral post, result is as shown in Figure 6.
Test condition:
Sample: urea, allantoin
Mobile phase: 75% acetonitrile and 25% water (v/v)
Flow velocity: 800nL/min
Detect wavelength: 200nm
Result: result shown in Fig. 6 shows that above 2 biased samples utilize SPDA-co-MBA hydrophily organic polymer liquid phase solid chromatography column of the present invention to separate, and can reach good separating effect.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (4)
1. the preparation method of a hydrophily organic polymer liquid phase solid chromatography column, it is characterized in that, comprise following operating procedure: with monomer 3-[N, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt is function monomer, it is mixed with crosslinking agent, pore generating agent and initator, carry out heat catalysis, in the pretreated quartz capillary of process, in-situ polymerization becomes hydrophily organic polymer liquid phase solid chromatography column; Specifically comprise following operating procedure: by monomer 3-[N, N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt, crosslinking agent, pore generating agent and initator mix, after ultrasonic degas, pour into through in pretreated quartz capillary column, then by quartz capillary sealing two ends, put into water-bath and react, remove after completion of the reaction the unreacted reactant in quartz capillary, obtain hydrophily organic polymer liquid phase solid chromatography column; Described crosslinking agent is N, N-methylene-bisacrylamide, and described initator is azodiisobutyronitrile; Described pore generating agent is that mass ratio is the mixture of the first alcohol and water of 68.5:31.5; The temperature of described reaction is 60 DEG C, and the time is 12h; Described monomer 3-[N, the mass ratio of N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt and crosslinking agent is 70:30～85:15; The quality of described initator is monomer 3-[N, 1% of N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt and crosslinking agent gross mass; Described monomer 3-[N, the quality sum of N-dimethyl-[2-(2-alkene acyloxy) ethyl fourth ammonium] propane-1-acid inner salt and crosslinking agent, with the mass ratio of pore generating agent be 25:75～35:65.
2. the preparation method of a kind of hydrophily organic polymer liquid phase solid chromatography column according to claim 1, it is characterized in that: the pretreated quartz capillary of described process is to prepare by following operating procedure: with the NaOH solution flushing quartz capillary 15min of 1mol/L, then quartz capillary sealing two ends is placed on to 100 DEG C of water-baths reaction 2h; Then use deionized water scouring stone English capillary, until the liquid pH flowing out is 7; Then with using nitrogen drying 4h after washed with methanol quartz capillary; After dry, the methyl alcohol that is 1:1 volume ratio and 3-(isobutene acyl-oxygen) propyl trimethoxy silicane mixed liquor is thrown in quartz capillary, sealing two ends and be placed on reaction 12h in 60 DEG C of water-baths; Finally respectively rinse 30min with first alcohol and water respectively, nitrogen drying 12h, obtains through pretreated quartz capillary.
3. according to the preparation method of a kind of hydrophily organic polymer liquid phase solid chromatography column described in claims 1, it is characterized in that: described in the unreacted reactant removed in quartz capillary be to operate according to following steps: one end of quartz capillary is connected with high-pressure pump, rinse with organic solvent, described organic solvent is at least one in methyl alcohol, acetonitrile and acetone.
4. the hydrophily organic polymer liquid phase solid chromatography column preparing according to method described in claim 1-3 any one, is characterized in that: this chromatographic column has continuous loose structure.
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| CN115197465B (en) * | 2022-07-27 | 2023-05-30 | 佛山科学技术学院 | Preparation method and application of betulinic acid polymer monolithic column |
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