CN104069840A - Preparation method of reversed phase chromatography silica gel filler - Google Patents
Preparation method of reversed phase chromatography silica gel filler Download PDFInfo
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- CN104069840A CN104069840A CN201410326763.5A CN201410326763A CN104069840A CN 104069840 A CN104069840 A CN 104069840A CN 201410326763 A CN201410326763 A CN 201410326763A CN 104069840 A CN104069840 A CN 104069840A
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Abstract
The invention discloses a preparation method of a reversed phase chromatography silica gel filler, and relates to a silica gel filler. The preparation method comprises the steps: putting a silicon ball in a centrifuge tube, adding a glass waterproof agent to obtain a silicon ball-glass waterproof agent solution, sealing a mouth of the centrifuge tube, oscillating for the first time, removing the glass waterproof agent at the upper layer after centrifuging, then adding ethyl alcohol in the centrifuge tube, continuously centrifuging to remove supernate after oscillating for the second time, and drying to obtain the reversed phase chromatography silica gel filler. A silica gel matrix filer is rapidly modified by adopting a glass waterproof agent chemical coating method, the modified silicon ball filler has a characteristic of reversed phase retention, and can be used for reversed phase high-performance liquid chromatography. The reversed phase chromatography silica gel filler is good in chromatography separation performance, and excellent in stability; the original reversed chromatography performance of the reversed phase chromatography silica gel filler is reserved under the condition that the reversed phase chromatography silica gel filler is soaked in 80% acetonitrile for 24h and baked at a temperature of 80 DEG C for 24h.
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 fixedly phase, and it mainly contains two large classes: bonded silica gel microballoon and organic polymer microballoon.Wherein, silica matrix fixedly because mechanical strength is high, good stability, the size of microballoon and aperture easy-regulating, and be not subject to the impact of various organic solvents and be usually used.The fixedly phase that the silica gel of take is matrix, the quality of its chromatographic performance is directly related with the physicochemical characteristics of Silica Surface.
Silica Surface extensively exists silicon hydroxyl groups-Si-OH, because it has good reactivity, therefore with diverse ways, modifies, and can obtain dissimilar chromatographic stationary phases.Conventional method of modifying has two classes, i.e. chemical bonding and polymer-coated method.
In chemical bonding, conventionally use chlorosilane or alkoxy silane to carry out Silanization reaction and introduce functional group, then utilize little molecule silylating reagent (as trim,ethylchlorosilane or hexamethyl three silicon amine) to carry out sealing processing, to eliminate remaining silicon hydroxyl.For obtaining the Bonded Phase of monolayer, the silica gel using 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 silicon hydroxyl of Silica Surface comprehensively, conventionally only has 25%～50% silicon hydroxyl generation bonding reaction.And the active portion branch of exposed Silica Surface is to particularly strong polarity and the very strong absorption of alkaline matter generation of analyte, cause distortion of peak, even there is Irreversible Adsorption.And for obtaining better separating effect, a lot of separated buffer systems of use acid, alkali, salt that need are as mobile phase, under this elution requirement, silylating reagent is easy to run off, and column life also shortens thereupon.
Fixing of making by polymer-coated method modified silica-gel surface can overcome the shortcoming of above-mentioned silica gel bonded phase mutually.This method is mainly on silica matrix, to wrap up the polymer film that one deck is thin, effectively avoid eluent and analyte contact and analyte and silicon hydroxyl between non-specific adsorption.
Summary of the invention
Object of the present invention is intended to overcome that the silica gel bonded fixedly phase reaction time is long, condition is harsh, complex steps and reappearance are difficult to the problems such as assurance, and the preparation method of quick, easy and efficient a kind of reverse-phase chromatography silica filler is provided.
Concrete steps of the present invention are as follows:
Silicon ball is placed in to centrifuge tube, after adding glass waterproofing agent (Aquapel), obtain silicon ball-glass water repellent solution, the sealing centrifuge tube mouth of pipe, the 1st vibration, after centrifugal, remove upper strata glass waterproofing agent, then in centrifuge tube, add ethanol, after the 2nd vibration, continue the centrifugal supernatant of removing, after oven dry, obtain reverse-phase chromatography silica filler.
The particle diameter of described silicon ball can be 100nm～30 μ m; 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 all fillers all can be immersed in ethanol; Described the 1st vibration can be placed in the supersonic oscillations instrument 5～120min that vibrates; Described 5～the 10min that can vibrate in supersonic oscillations instrument that vibrates for the 2nd time; The temperature of described oven dry can be 40～80 ℃, 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 separation.
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, thereby obtain RPLC filler.This class filler not only chromatographic separation performance is good, and excellent in stability, soaks 24h and 80 ℃ and cure under 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 350nm SiO
2filler pillar post bed ESEM (SEM) phenogram.
Fig. 2 is glass waterproofing agent coating 350nm SiO
2filler pillar enrichment three peptide species mixture chromatograms.
Fig. 3 is glass waterproofing agent coating 350nm SiO
2enrichment three peptide species mixture chromatograms after filler pillar 80%ACN immersion 24h.
Fig. 4 is glass waterproofing agent coating 350nm SiO
280 ℃ of filler pillars cure enrichment three peptide species mixture chromatograms after 24h.
Fig. 5 is power spectrum (EDS) phenogram that glass waterproofing agent applies 10 μ m silica fillers.
Fig. 6 is that glass waterproofing agent applies the separated benzene series of 10 μ m silica filler chromatographic columns and looks for spectrogram.
Fig. 7 is that glass waterproofing agent applies 10 μ m silica filler chromatographic column protein isolate mixture chromatograms.
The specific embodiment
Embodiment 1:
Take the SiO that 150mg particle diameter is 350nm
2nano particle is placed in 5mL centrifuge tube, after adding 1mL Aquapel, seal the centrifuge tube mouth of pipe, be positioned over after the 20min that vibrates in supersonic oscillations instrument centrifugal, remove supernatant, add the ultrasonic 5min of 2mL ethanol, centrifugal, remove upper strata ethanolic solution, be positioned in baking oven, under 50 ℃ of conditions, cure 3h after sealing preserve.
The chromatogram retention property of filler is evaluated:
Get the SiO after appropriate above-mentioned Aquapel processes
2nano particle is scattered in ethanol, concentration is controlled as 10mg/mL, the solid phase extraction column that 2 length are 5mm is prepared in the centrifugal filling after 10min of vibrating in supersonic oscillations instrument, and to demonstrate it be SPE and the concentration effect of SRIF-A, acetic acid angiotensins and the oxytocins three peptide species mixtures of 10 μ g/mL to concentration.Fig. 1 is Aquapel coating 350nm SiO
2post bed ESEM (SEM) figure of Nano filling pillar, Fig. 2 is Aquapel coating 350nm SiO
2nano filling pillar enrichment three peptide species mixture chromatograms.As can be seen from Figure 1, Aquapel coating 350nm SiO
2after nano particle, do not change its original particle diameter and all once, meanwhile, utilize the SiO after this modification
2pillar prepared by Nano filling has good concentration effect to three peptide species mixtures.
The chromatogram of filler retains estimation of stability:
1) in 2 SPE pillars of above-mentioned preparation, inject 80% acetonitrile (ACN), after room temperature condition lower seal preservation 24h, testing it is SPE and the concentration effect of SRIF-A, acetic acid angiotensins and the oxytocins three peptide species mixtures of 10 μ g/mL to concentration, and the chromatographic isolation spectrogram after enrichment is as Fig. 3.
2) 2 SPE pillars of above-mentioned preparation being positioned over to temperature is to cure 24h in the baking oven of 80 ℃, then test its SPE and concentration effect to the SRIF-A of 10 μ g/mL, acetic acid angiotensins and oxytocins three peptide species mixtures, the chromatographic isolation spectrogram after enrichment is as Fig. 4.
Comparison diagram 2～4 can prove, Aquapel coating 350nm SiO
2even if Nano filling pillar soaks under 24h and 80 ℃ of hot conditions under 80%ACN condition, cure 24h, its reverse-phase chromatography performance still retains well.Proof Aquapel coating 350nmSiO
2nano filling excellent in stability, can effectively avoid the problem of stationary phase bleeding.
Embodiment 2:
Taking 300mg particle diameter is that 10 μ m apertures are
silicon ball filler be placed in 5mL centrifuge tube, after adding 1mL Aquapel, seal the centrifuge tube mouth of pipe, be positioned over after the 1h that vibrates in supersonic oscillations instrument centrifugal, remove supernatant, add the ultrasonic 5min of 2mL ethanol, centrifugal, remove upper strata ethanolic solution, be positioned in baking oven, under 60 ℃ of conditions, cure 3h after sealing preserve.
Get silicon ball fillers dispersed after appropriate above-mentioned Aquapel processes in ethanol, concentration is controlled as 50mg/mL, it is 9cm that the high-pressure homogenization that vibrates in supersonic oscillations instrument after 10min is filled preparation length, internal diameter is the capillary short column of 100 μ m, 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 μ m silica fillers, Fig. 6 is the chromatogram that Aquapel applies the separated benzene homologues of 10 μ m silica filler chromatographic columns, and Fig. 7 is the chromatogram that Aquapel applies 10 μ m silica filler chromatographic column protein isolate mixtures.As can be seen from Figures 6 and 7, utilize 10 μ m silica filler chromatographic column separating hydrophobicity is stronger well benzene homologues and protein mixtures after Aquapel coating.In conjunction with Fig. 5 power spectrum, characterize known, 10 μ m silica fillers after Aquapel coating have higher carbon content, be not difficult to infer, the hydrophobicity that proves the silica filler after modifying is to come from the long chain alkane that Aquapel coating effect is introduced, thereby 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 to centrifuge tube, after adding glass waterproofing agent, obtain silicon ball-glass water repellent solution, the sealing centrifuge tube mouth of pipe, the 1st vibration, after centrifugal, remove upper strata glass waterproofing agent, then in centrifuge tube, add ethanol, after the 2nd vibration, continue the centrifugal supernatant of removing, after oven dry, obtain reverse-phase chromatography silica filler.
2. a kind of preparation method of reverse-phase chromatography silica filler as claimed in claim 1, the particle diameter that it is characterized in that described silicon ball is 100nm～30 μ m.
3. a kind of preparation method of reverse-phase chromatography silica filler as claimed in claim 1, the proportioning that it is characterized in that described silicon ball, glass waterproofing agent, ethanol is (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 all fillers all can be immersed in ethanol.
4. a kind of preparation method of reverse-phase chromatography silica filler as claimed in claim 1, is characterized in that described the 1st vibration is for being placed in the supersonic oscillations instrument 5～120min that vibrates.
5. a kind of preparation method of reverse-phase chromatography silica filler as claimed in claim 1, is characterized in that described the 2nd vibration for vibrating 5～10min in supersonic oscillations instrument.
6. a kind of preparation method of reverse-phase chromatography silica filler as claimed in claim 1, the temperature that it is characterized in that described oven dry is 40～80 ℃, the time of oven dry is 30～180min.
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Citations (5)
| 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 |
| US20080135485A1 (en) * | 2006-12-08 | 2008-06-12 | Varian, Inc. | Metal cyano bonded phases |
| 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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- 2014-07-10 CN CN201410326763.5A patent/CN104069840B/en active Active
Patent Citations (5)
| 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 |
| US20080135485A1 (en) * | 2006-12-08 | 2008-06-12 | Varian, Inc. | Metal cyano bonded phases |
| 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. |
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