CN104069840B - A kind of preparation method of reverse-phase chromatography silica filler - Google Patents
A kind of preparation method of reverse-phase chromatography silica filler Download PDFInfo
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Abstract
A preparation method for reverse-phase chromatography silica filler, relates to silica filler.Silicon ball is placed in centrifuge tube, silicon ball-glass water repellent solution is obtained after adding glass waterproofing agent, the sealing centrifuge tube mouth of pipe, 1st vibration, centrifugal rear removing upper strata glass waterproofing agent, then in centrifuge tube, add ethanol, continue centrifugal removing supernatant after the 2nd vibration, after oven dry, namely obtain reverse-phase chromatography silica filler.Adopt glass waterproofing agent chemicalpiston method Rapid Modification silica matrix filler, the silicon ball filler after modification has the characteristic of anti-phase reservation, can be used for RPLC and is separated.This filler not only chromatographic separation performance is good, and excellent in stability, soaks under 24h and 80 DEG C cures the condition of 24h and still retain its original reverse-phase chromatography performance in 80% acetonitrile.
Description
Technical field
The present invention relates to silica filler, especially relate to a kind of preparation method of reverse-phase chromatography silica filler.
Background technology
In high performance liquid chromatography (HPLC), reverse-phase chromatography filler is most widely used anti-phase Stationary liquid, and it mainly contains two large classes: bonded silica gel microballoon and organic polymeric microspheres.Wherein, the Stationary liquid of silica matrix due to mechanical strength high, good stability, the size of microballoon and aperture easy-regulating, and be not usually used by the impact of various organic solvent.Take silica gel as the Stationary liquid of matrix, the quality of its chromatographic performance is directly related with the physicochemical characteristics of Silica Surface.
Silica Surface extensively also exists silicone hydroxyl group-Si-OH, because it has good reactivity, therefore modifies with diverse ways, can obtain dissimilar chromatographic stationary phases.Conventional method of modifying has two classes, i.e. chemical bonding and polymer-coated method.
In chemical bonding, usual use chlorosilane or alkoxy silane carry out Silanization reaction and introduce functional group, then Small molecular silylating reagent (as trim,ethylchlorosilane or hexamethyl three silicon amine) is utilized to carry out sealing process, to eliminate remaining silicone hydroxyl.For obtaining the Bonded Phase of monolayer, the silica gel used in course of reaction, silylating reagent and solvent must strictly dewater, and carry out bonding reaction at higher temperature.Meanwhile, due to the existence of space steric effect, the silylating reagent that molecular volume is larger, can not react with the silicone hydroxyl of Silica Surface comprehensively, usually only has 25% ~ 50% silicone hydroxyl generation bonding reaction.And the active portion branch of exposed Silica Surface to analyze thing particularly strong polarity and alkaline matter there is very strong absorption, cause distortion of peak, even Irreversible Adsorption occur.Further, for obtaining better separating effect, the buffer system of a lot of separation needs use acid, alkali, salt is as mobile phase, and under this elution requirement, silylating reagent is easy to run off, and column life also shortens thereupon.
The shortcoming of above-mentioned silica gel bonded phase can be overcome by the Stationary liquid that polymer-coated method modified silica-gel surface is obtained.This method mainly wraps up the thin polymer film of one deck on silica matrix, the contact effectively avoiding eluent and analysis thing and the non-specific adsorption analyzed between thing and silicone hydroxyl.
Summary of the invention
Object of the present invention is intended to overcome that the bonded silica stationary phase reaction time is long, condition is harsh, complex steps and reappearance is difficult to the problems such as guarantee, provides the preparation method of quick, easy and efficient a kind of reverse-phase chromatography silica filler.
Concrete steps of the present invention are as follows:
Silicon ball is placed in centrifuge tube, silicon ball-glass water repellent solution is obtained after adding glass waterproofing agent (Aquapel), the sealing centrifuge tube mouth of pipe, 1st vibration, centrifugal rear removing upper strata glass waterproofing agent, then in centrifuge tube, add ethanol, continue centrifugal removing supernatant after the 2nd vibration, after oven dry, namely obtain reverse-phase chromatography silica filler.
The particle diameter of described silicon ball can be 100nm ~ 30 μm; The proportioning of described silicon ball, glass waterproofing agent, ethanol can be (5 μ g ~ 100g): (5 μ L ~ 50mL): (5 μ L ~ 50mL), wherein, silicon ball is calculated in mass, glass waterproofing agent, ethanol are calculated by volume, all silicon balls are all immersed in glass waterproofing agent, and the submergence of all fillers equal energy in ethanol; Described 1st vibration can be placed in supersonic oscillations instrument vibration 5 ~ 120min; Described 5 ~ the 10min that can vibrate in supersonic oscillations instrument that vibrates for 2nd time; The temperature of described oven dry can be 40 ~ 80 DEG C, and the time of oven dry can be 30 ~ 180min.
The present invention adopts glass waterproofing agent (Aquapel) chemicalpiston method Rapid Modification silica matrix filler, and the silicon ball filler after modification has the characteristic of anti-phase reservation, can be used for RPLC and is separated.
The present invention utilizes the fast and convenient modified silica-gel matrix fill of polymer-coated method, utilize business-like glass waterproofing agent (Aquapel) (main chemical compositions: naphtha 90% ~ 100%, C10-12 alkane/cycloalkanes) process silica gel, thus obtain RPLC filler.This kind of filler not only chromatographic separation performance is good, and excellent in stability, soaks under 24h and 80 DEG C cures the condition of 24h and still retain its original reverse-phase chromatography performance in 80% acetonitrile.
Accompanying drawing explanation
Fig. 1 is glass waterproofing agent coating 350nmSiO
2filler pillar post bed ESEM (SEM) phenogram.
Fig. 2 is glass waterproofing agent coating 350nmSiO
2filler pillar enrichment three peptide species mixture chromatogram.
Fig. 3 is glass waterproofing agent coating 350nmSiO
2enrichment three peptide species mixture chromatogram after filler pillar 80%ACN immersion 24h.
Fig. 4 is glass waterproofing agent coating 350nmSiO
2filler pillar 80 DEG C cures enrichment three peptide species mixture chromatogram after 24h.
Fig. 5 is power spectrum (EDS) phenogram that glass waterproofing agent applies 10 μm of silica fillers.
Fig. 6 is that glass waterproofing agent applies 10 μm of silica filler chromatographic columns and is separated benzene series and looks for spectrogram.
Fig. 7 is that glass waterproofing agent applies 10 μm of silica filler chromatographic column protein isolate mixture chromatograms.
Detailed description of the invention
Embodiment 1:
Take the SiO that 150mg particle diameter is 350nm
2nano particle is placed in 5mL centrifuge tube, the centrifuge tube mouth of pipe is sealed after adding 1mLAquapel, centrifugal after being positioned in supersonic oscillations instrument the 20min that vibrates, removing supernatant, add 2mL EtOH Sonicate 5min, centrifugal, removing upper strata ethanolic solution, is positioned over sealing after curing 3h in baking oven under 50 DEG C of conditions and preserves.
The chromatogram retention property of filler is evaluated:
Get the SiO after appropriate above-mentioned Aquapel process
2nanoparticle dispersion is in ethanol, concentration controls as 10mg/mL, centrifugal filling preparation 2 length after 10min of vibrating in supersonic oscillations instrument are the solid phase extraction column of 5mm, and demonstrate SPE and concentration effect that it is the SRIF-A of 10 μ g/mL, acetic acid angiotensins and oxytocins three peptide species mixture to concentration.Fig. 1 is that Aquapel applies 350nmSiO
2post bed ESEM (SEM) figure, Fig. 2 of Nano filling pillar are that Aquapel applies 350nmSiO
2nano filling pillar enrichment three peptide species mixture chromatogram.As can be seen from Figure 1, Aquapel applies 350nmSiO
2do not change its original particle diameter and uniformity after nano particle, meanwhile, utilize the SiO after this modification
2pillar prepared by Nano filling has excellent concentration effect to three peptide species mixtures.
The chromatogram of filler retains estimation of stability:
1) in 2 SPE pillars of above-mentioned preparation, 80% acetonitrile (ACN) is injected, room temperature condition lower seal is preserved and to be tested SPE and the concentration effect that it is the SRIF-A of 10 μ g/mL, acetic acid angiotensins and oxytocins three peptide species mixture to concentration after 24h, and the chromatographic isolation spectrogram after enrichment is as Fig. 3.
2) 2 of above-mentioned preparation SPE pillars being positioned over temperature is cure 24h in the baking oven of 80 DEG C, then test SPE and the concentration effect of its SRIF-A to 10 μ g/mL, acetic acid angiotensins and oxytocins three peptide species mixture, the chromatographic isolation spectrogram after enrichment is as Fig. 4.
Comparison diagram 2 ~ 4 can prove, Aquapel applies 350nmSiO
2even if Nano filling pillar cures 24h soak 24h and 80 DEG C hot conditions under 80%ACN condition under, its reverse-phase chromatography performance still retains well.Prove that Aquapel applies 350nmSiO
2nano filling excellent in stability, effectively can avoid the problem of stationary phase bleeding.
Embodiment 2:
To take 300mg particle diameter be 10 μm of apertures is
silicon ball filler be placed in 5mL centrifuge tube, the centrifuge tube mouth of pipe is sealed after adding 1mLAquapel, centrifugal after being positioned in supersonic oscillations instrument the 1h that vibrates, removing supernatant, add 2mL EtOH Sonicate 5min, centrifugal, removing upper strata ethanolic solution, is positioned over sealing after curing 3h in baking oven under 60 DEG C of conditions and preserves.
The silicon ball filler got after appropriate above-mentioned Aquapel process is scattered in ethanol, concentration controls as 50mg/mL, it is 9cm that the high-pressure homogenization after 10min that vibrates in supersonic oscillations instrument fills preparation length, internal diameter is the capillary short column of 100 μm, and verifies its separating effect to benzene homologues (mixture of thiocarbamide, toluene, ethylbenzene and propyl benzene) and protein mixture (BSA, cromoci, transferrins and ovalbumin).Fig. 5 is power spectrum (EDS) phenogram that Aquapel applies 10 μm of silica fillers, Fig. 6 is the chromatogram that Aquapel applies that 10 μm of silica filler chromatographic columns are separated benzene homologues, and Fig. 7 is the chromatogram that Aquapel applies 10 μm of silica filler chromatographic column protein isolate mixtures.As can be seen from Figures 6 and 7,10 μm of silica filler chromatographic columns after utilizing Aquapel to apply can separating hydrophobicity is stronger well benzene homologues and protein mixture.Composition graphs 5 power spectrum characterizes known, 10 μm of silica fillers after Aquapel coating have higher carbon content, be not difficult to infer, prove that the hydrophobicity of the silica filler after modifying is the long chain alkane coming from the introducing of Aquapel coating effect, thus can carry out reversed phase chromatography separation.
Claims (6)
1. a preparation method for reverse-phase chromatography silica filler, is characterized in that concrete steps are as follows:
Silicon ball is placed in centrifuge tube, silicon ball-Aquapel glass water repellent solution is obtained after adding Aquapel glass waterproofing agent, the sealing centrifuge tube mouth of pipe, 1st vibration, centrifugal rear removing upper strata Aquapel glass waterproofing agent, then in centrifuge tube, add ethanol, continue centrifugal removing supernatant after the 2nd vibration, after oven dry, namely obtain reverse-phase chromatography silica filler.
2. the preparation method of a kind of reverse-phase chromatography silica filler as claimed in claim 1, is characterized in that the particle diameter of described silicon ball is 100nm ~ 30 μm.
3. the preparation method of a kind of reverse-phase chromatography silica filler as claimed in claim 1, it is characterized in that the proportioning of described silicon ball, Aquapel glass waterproofing agent, ethanol is for (5 μ g ~ 100g): (5 μ L ~ 50mL): (5 μ L ~ 50mL), wherein, silicon ball is calculated in mass, Aquapel glass waterproofing agent, ethanol are calculated by volume, all silicon balls are all immersed in Aquapel glass waterproofing agent, and the submergence of all fillers equal energy in ethanol.
4. the preparation method of a kind of reverse-phase chromatography silica filler as claimed in claim 1, is characterized in that described 1st vibration is for being placed in supersonic oscillations instrument vibration 5 ~ 120min.
5. the preparation method of a kind of reverse-phase chromatography silica filler as claimed in claim 1, is characterized in that described 2nd vibration is for the 5 ~ 10min that vibrates in supersonic oscillations instrument.
6. the preparation method of a kind of reverse-phase chromatography silica filler as claimed in claim 1, it is characterized in that the temperature of described oven dry is 40 ~ 80 DEG C, the time of oven dry is 30 ~ 180min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101191048A (en) * | 2006-11-21 | 2008-06-04 | 天津鑫泰利科技发展有限公司 | Method for preparing automobile windscreen rainproof agent |
| CN102391514A (en) * | 2011-09-26 | 2012-03-28 | 北京航空航天大学 | Ceramic precursor resin applicable to rainproof and self-cleaning coating of glass |
| CN102527454A (en) * | 2012-01-31 | 2012-07-04 | 复旦大学 | Micro-fluid control drop concentration device for sample enrichment |
| CN103041792A (en) * | 2013-01-23 | 2013-04-17 | 常州南京大学高新技术研究院 | Carbamic acid ester type liquid phase chromatogram stationary phase and preparation method thereof |
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| US7628915B2 (en) * | 2006-12-08 | 2009-12-08 | Varian, Inc. | Metal cyano bonded phases |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101191048A (en) * | 2006-11-21 | 2008-06-04 | 天津鑫泰利科技发展有限公司 | Method for preparing automobile windscreen rainproof agent |
| CN102391514A (en) * | 2011-09-26 | 2012-03-28 | 北京航空航天大学 | Ceramic precursor resin applicable to rainproof and self-cleaning coating of glass |
| CN102527454A (en) * | 2012-01-31 | 2012-07-04 | 复旦大学 | Micro-fluid control drop concentration device for sample enrichment |
| CN103041792A (en) * | 2013-01-23 | 2013-04-17 | 常州南京大学高新技术研究院 | Carbamic acid ester type liquid phase chromatogram stationary phase and preparation method thereof |
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Effective date of registration: 20180806 Address after: 361101 C fourteen floor, 2 tower, 1888 Hongxiang Road, Xiangan District, Xiamen, Fujian. Patentee after: Xiamen Hao Hao Scientific Instrument Co., Ltd. Address before: Xiamen City, Fujian Province, 361005 South Siming Road No. 422 Patentee before: Xiamen University |
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Effective date of registration: 20211231 Address after: 361101 floor 2, B20, No. 843, Lianting Road, digital economy industrial park, Xiang'an District, Xiamen City, Fujian Province Patentee after: XIAMEN CHROMATOGRAPHIC ANALYSIS INSTRUMENT Co.,Ltd. Address before: 361101 C fourteen floor, 2 tower, 1888 Hongxiang Road, Xiangan District, Xiamen, Fujian. Patentee before: XIAMEN HAOQI SCIENTIFIC INSTRUMENT CO.,LTD. |