CN1236308C - Method for preparing octyl group micro column liquid phase chromatograph integrate column - Google Patents
Method for preparing octyl group micro column liquid phase chromatograph integrate column Download PDFInfo
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Abstract
The present invention relates to a method for making a liquid chromatography integral column of an octyl microcolumn, which has the following steps: a silylation substance with alkenyl is pre-bonded at the inner wall of a capillary pipe; the integral column made of noctyl methacrylate-ethylene glycol dimethacrylate is made, reactive monomers are weighed and injected into the prebonding capillary pipe after the reactive monomers are mixed with a pore-forming agent, and mu-HPLC separating operation can be carried out after wash by using methanol as a mobile phase after reaction in a water bath. Noctyl methacrylate is used as the monomers, the capillary pipe is used as a column pipe, and organic alkali is added to the prebonding reaction of the capillary pipe to be used as a catalyst. Gamma-MAPS is easy to react with a silicon hydroxyl group of the inner wall of the capillary pipe, and the obtained integral column does not need to sinter filtration sieve plates at both ends of the column. The present invention has the advantages of excellent permeation performance, high spacial use ratio, low column pressure, strong stability and simple preparation. Integral columns with different performance can be obtained by simply adjusting the proportion of the monomers to a crosslinking agent and the proportion of a monomer mixture to the pore-forming agent, and at the same time, the length of the integral columns can be conveniently controlled according to actual demands.
Description
Technical field
The present invention relates to a kind of preparation method of micro column liquid chromatography integral post, especially a kind of preparation method of octyl group type micro column liquid chromatography integral post.
Background technology
(μ-HPLC) claims capillary liquid chromatography (c-HPLC) again to micro column high efficiency liquid phase chromatograph, be that a kind of chromatogram differential of growing up in recent years is from technology, it is low to have moving phase, stationary phase and sample consumption, environmental pollution is little, the separation efficiency height, be easy to and advantage such as other detection method on-line coupling, therefore will play an important role in fields such as biochemistry, medicine, environment.But the filling method is often adopted in the preparation of μ-HPLC chromatographic column, has very big post to press under the high flow condition, and this is to the serviceable life of chromatographic column and the maintenance of instrument having very big influence.If flow is too little, will cause the increase of analysis time; The filling difficulty of micro-dimension chromatographic column needs very high dress post skill simultaneously, and for capillary chromatographic column, need filter sieve plate at the two ends of post sintering, wastes time and energy.
Recent years development pass through the integral post stationary phase that in-situ polymerization forms, excellent transparent performance, high spatial utilization factor, preparation are simple, fast, the low post of mass transfer velocity is pressed and be need not sintering for capillary monolithic column and filters advantages such as sieve plate owing to have, and therefore developing micro column liquid chromatography integral post material is to solve μ-very desirable method of HPLC development bottleneck.
So far, the integral post major limitation is at conventional high performance liquid chromatography (internal diameter of selecting post usually is the conventional stainless-steel tube of 3.2～4.6mm) and capillary electric chromatogram (internal diameter of selecting post usually is the quartz capillary of 100～25 μ m), it is the quartz capillary of 320um that people such as Pavel C. have prepared internal diameter with butyl methacrylate, but its chromatogram capacity is still lower, and need higher technology of preparing, also to do corresponding transformation to the liquid chromatography instrument simultaneously.
Summary of the invention
It is monomer with the 2-Propenoic acid, 2-methyl-, octyl ester that the present invention aims to provide a kind of, ethylene glycol dimethacrylate is a crosslinking chemical, azobisisobutyronitrile or benzoyl peroxide are initiating agent, n-propanol, 1,4 butylene glycols and water tertiary mixture are cosolvent, the method for preparing capillary monolithic column, this integral post material can separate application under μ-HPLC.
Step of the present invention is as follows:
1: at the silanization material of the pre-bonding one deck of capillary tube inner wall band thiazolinyl;
2: preparation 2-Propenoic acid, 2-methyl-, octyl ester (MAOE)-ethylene glycol dimethacrylate (EDMA) integral post: the ratio between the reaction monomers potpourri is: the weight percent content of MAOE is 59.5%～44.5%, the EDMA weight percent content is 39.5%～54.5%, and initiating agent azobisisobutyronitrile (AIBN) or benzoyl peroxide are 0.5%～2%; Ratio between the pore-foaming agent potpourri is: propyl alcohol 50%～70%, 1,4-butylene glycol 20%～40%, water 5%～20%; Ratio between monomer mixture and the pore-foaming agent potpourri is 35%/65%～55%/45%; Take by weighing reaction monomers and pore-foaming agent according to the above ratio, mix in the kapillary that injects bonding in advance in the back, sealing two ends is immersed in 40～70 ℃ of water-baths and reacts 6～24h.After reaction was finished, the intercepting Len req was can carry out μ-HPLC lock out operation behind moving phase flushing 2～6h with methyl alcohol.Polymerization process is as follows:
The pre-bonding of said capillary tube inner wall can be by volume 1: (0.5～1.5) preparation γ-methacrylic acid oxygen propyl trimethoxy silicane (γ-MAPS) and methyl alcohol mixed solution, 0.1%～0.3% the organic base that adds above-mentioned mixed liquor volume then, in the injection kapillary, and with the kapillary sealing two ends, after placing 30～50 ℃ of reaction 6～24h, with nitrogen purging 2～6h, so far, on the capillary tube inner wall bonding silanization material of one deck band thiazolinyl; Pre-bonding reaction formula is as follows:
Said organic base is a pyridine, triethylamine or imidazoles etc.γ-methacrylic acid oxygen propyl trimethoxy silicane (before γ-MAPS) injects kapillary with the methyl alcohol mixed solution, best ultrasonic degas, its time can be 5～20min.
Reactant liquor is sucked in the capillary blank pipe of the certain-length that bonding is good in advance, seal with the little plug of rubber at two ends.
The present invention uses 2-Propenoic acid, 2-methyl-, octyl ester to be monomer, with the quartz capillary is column jecket, and in pre-bonding reaction capillaceous, add dry organic base (as pyridine, triethylamine, imidazoles etc.), can make the easier silicon hydroxyl reaction with capillary tube inner wall of γ-MAPS as catalyzer, therefore more two key and monomer mixture generation copolymerization are subsequently arranged, and the integral post material of gained has good stability like this.Prepared integral post need not to filter sieve plate at the two ends of post sintering, transparent performance, high spatial utilization factor, low post with excellence pressed, stability is strong, it is simple to prepare, can obtain the different integral post of performance by adjusting monomer with the ratio of crosslinking chemical ratio and monomer mixture and pore-foaming agent easily, can control the length of integral post simultaneously by actual needs easily.
Description of drawings
Fig. 1 is the electron-microscope scanning figure of embodiment 1 integral post.
Fig. 2 is the electron-microscope scanning figure of tube wall and polymkeric substance junction.
Fig. 3 is the electron-microscope scanning figure of embodiment 2 integral post.
Fig. 4 is embodiment 3 liquid chromatograms.
Fig. 5 is embodiment 4 liquid chromatograms.
Fig. 6 is the graph of a relation that flow velocity and post are pressed among the embodiment 5.
Embodiment
The present invention is further illustrated below by embodiment.
Embodiment 1:
1) inwall cleaning capillaceous: getting internal diameter is 530 μ m, the quartz glass capillary of long 5m, use the flow velocity flushing 0.5h of the HCl solution of 0.1moL/L earlier with 1.0mL/min, with deionized water with the flow velocity of 1.0mL/min wash to washing fluid be neutrality, use the flow velocity flushing 2.0h of the NaOH solution of 0.1moL/L again with 1.0mL/min, with deionized water with 1.0ml/min wash to washing fluid be neutrality, at last with methyl alcohol with the flow velocity of 1.0mL/min flushing 15min after, use nitrogen purging 2h.
2) the pre-bonding of capillary tube inner wall: preparation γ-methacrylic acid oxygen propyl trimethoxy silicane (γ-MAPS) and methyl alcohol mixed solution 3.0mL (γ-MAPS: MeOH=1: 0.5), the dry pyridine that adds 3 μ L then, ultrasonic degas 10min, inject in the pretreated kapillary with syringe, and the kapillary two ends are sealed with rubber plug, after placing 30 ℃ of reaction 6h, use nitrogen purging 6h, two ends are sealed standby with rubber plug.So far, on the capillary tube inner wall bonding silanization material of one deck band thiazolinyl.
3) preparation of integral post: by 2-Propenoic acid, 2-methyl-, octyl ester (MAOE): ethylene glycol dimethacrylate (EDMA): azobisisobutyronitrile (AIBN) is 59.5%: 39.5%: 0.5% a proportional arrangement reaction monomers potpourri, press propyl alcohol 60%, 1,4-butylene glycol 30%, the proportional arrangement pore-foaming agent potpourri of water 10%; The reaction monomers potpourri is mixed by 40%: 60% with the pore-foaming agent potpourri, and ultrasonic then 10min is towards nitrogen 5min, with syringe with reactant liquor sucked in advance bonding well length be 23cm, internal diameter is the empty kapillary of 530 μ m, seal with the little plug of rubber at two ends, respectively at 60 ℃ of reaction 6h, 12h, 24h, after having reacted, receive on the liquid chromatography pump, get final product with washed with methanol 6h, a among Fig. 1, b, c are respectively reaction 6h, 12h, the electron-microscope scanning figure of 24h.Fig. 2 is the electron-microscope scanning figure of tube wall and polymkeric substance junction.
Embodiment 2:
Inwall cleaning capillaceous is with embodiment 1, in the pre-bonding of capillary tube inner wall, preparation γ-methacrylic acid oxygen propyl trimethoxy silicane (γ-MAPS) and methyl alcohol mixed solution 3.0mL (γ-MAPS: MeOH=1: 1), the pyridine that adds 6 μ L then, inject in the pretreated kapillary, and with the kapillary sealing two ends, place 45 ℃ of reaction 12h after, use nitrogen purging 2h, sealing two ends is standby.Configuration reaction monomers potpourri, wherein MAOE: EDMA: AIBN=59.5%: 39.5%: 1%; Ratio between the pore-foaming agent potpourri is: propyl alcohol 60%, 1,4-butylene glycol 30%, water 10%; Ratio between reaction monomers potpourri and the pore-foaming agent potpourri was respectively 35%: 65%; 45%: 55%; 50%: 50%; 55%: 45%, mix the ultrasonic 10min in back, towards nitrogen 5min, then with syringe with reactant liquor sucked in advance bonding well length be 23cm, internal diameter is the empty kapillary of 250 μ m, seal with the little plug of rubber at two ends, respectively at 60 ℃ of reaction 12h, after having reacted, receive on the liquid chromatography pump, get final product with washed with methanol 3h, Fig. 3 a, 3b, 3c, 3d were respectively ratio 35%: 65%; 45%: 55%; 50%: 50%; 55%: 45% electron-microscope scanning figure.
Embodiment 3:
Get the kapillary that internal diameter is 50 μ m, the inwall cleaning is with embodiment 1, in the pre-bonding of capillary tube inner wall, preparation γ-methacrylic acid oxygen propyl trimethoxy silicane (γ-MAPS) and methyl alcohol mixed solution 3.0mL (γ-MAPS: MeOH=1: 1.5), add the triethylamine of 9 μ L then, inject in the pretreated kapillary, and with the kapillary sealing two ends, after placing 50 ℃ of reaction 24h, use nitrogen purging 4h, sealing two ends is standby.Configuration reaction monomers potpourri, wherein MAOE: EDMA: AIBN is 59.5%: 39.5%: 0.5%; Ratio between the pore-foaming agent potpourri is: propyl alcohol 70%, 1,4-butylene glycol 20%, water 10%; Ratio between reaction monomers potpourri and the pore-foaming agent potpourri is 40%: 60%, mix to inject internal diameter and be 50 μ m bonding is well long in advance to be 23cm sky kapillary, sealing two ends, respectively at 70 ℃ of reaction 12h, after having reacted, receive on the liquid chromatography pump, get final product with washed with methanol 6h.
Embodiment 4:
Inwall cleaning capillaceous is with embodiment 1, in the pre-bonding of capillary tube inner wall, and γ-MAPS: MeOH=1: 1.5, add the imidazoles of 7 μ L then, all the other are operated with embodiment 1.Configuration reaction monomers potpourri, wherein MAOE: EDMA: benzoyl peroxide=59.5%: 39.5%: 2%; Pore-foaming agent propyl alcohol, 1, the ratio between 4-butylene glycol and the aqueous mixtures is: 60%: 35%: 5%; Ratio between reaction monomers potpourri and the pore-foaming agent potpourri was respectively 40%: 60%, mix to inject internal diameter and be 320 μ m bonding is well long in advance to be 25cm sky kapillary, sealing two ends, respectively at 60 ℃ of reaction 24h, after having reacted, receive on the liquid chromatography pump, get final product with washed with methanol 4h.
Embodiment 5:
Configuration reaction monomers potpourri, wherein MAOE: EDMA: AIBN is 59.5%: 39.59%: 1%; Ratio between the pore-foaming agent potpourri is: propyl alcohol 60%, 1,4-butylene glycol 30%, water 10%; Ratio between reaction monomers potpourri and the pore-foaming agent potpourri is 40%: 60%, and all the other are operated with embodiment 2.The integral post that intercepting prepares is got long 20.1cm, and under μ-HPLC pattern, moving phase is acetonitrile/water=35/65, and flow velocity is 40 μ L/min, detects wavelength 254nm, and substituted benzene is separated, and its spectrogram as shown in Figure 4.
Embodiment 6:
With the integral post of embodiment 1 preparation, moving phase is by 5mmol/L oxine-5-sulfonic acid, 3mmol/L cetyl trimethyl ammonium bromide, 10mmol/L NaAc (pH=4.8) and 30% acetonitrile mixed solution composition; Flow velocity is 40 μ L/min; Fluorescence exciting wavelength and emission wavelength are respectively 388nm and 518nm, to Mg
2+, Zn
2+And Cd
2+Ion separates, and its spectrogram as shown in Figure 5.
Embodiment 7:
Configuration reaction monomers potpourri, wherein MAOE: EDMA: AIBN is 59.5%: 39.5%: 1%; Ratio between the pore-foaming agent potpourri is: propyl alcohol 60%, 1,4-butylene glycol 30%, water 10%; Ratio between reaction monomers potpourri and the pore-foaming agent potpourri was respectively 35%: 65% and 45%: 55%, column length then is respectively 19.7cm and 20.1cm, and moving phase is acetonitrile/water=60/40, and all the other are operated with embodiment 2, relation such as Fig. 6 a that their flow velocity and post are pressed are shown in the 6b.
Claims (4)
1. the preparation method of octyl group type micro column liquid chromatography integral post is characterized in that step is as follows:
1): at the silanization material of the pre-bonding one deck of capillary tube inner wall band thiazolinyl, pre-bonding by volume 1: 0.5～1.5 preparation γ-methacrylic acid oxygen propyl trimethoxy silicane and the methyl alcohol mixed solutions of said capillary tube inner wall, 0.1%～0.3% the organic base that adds above-mentioned mixed liquor volume then, in the injection kapillary, and with the kapillary sealing two ends, after placing 30～50 ℃ of reaction 6～24h, with nitrogen purging 2～6h, silanization material at capillary tube inner wall bonding last layer band thiazolinyl, said organic base is selected from pyridine, triethylamine or imidazoles;
2): preparation 2-Propenoic acid, 2-methyl-, octyl ester-ethylene glycol dimethacrylate integral post: the ratio between the reaction monomers potpourri is: the weight percent content of MAOE is 59.5%～44.5%, the EDMA weight percent content is 39.5%～54.5%, and initiating agent azobisisobutyronitrile or benzoyl peroxide are 0.5%～2%; Ratio between the pore-foaming agent potpourri is: propyl alcohol 50%～70%, 1,4-butylene glycol 20%～40%, water 5%～20%; Ratio between monomer mixture and the pore-foaming agent potpourri is 35%/65%～55%/45%; Take by weighing reaction monomers and pore-foaming agent in proportion, mix in the kapillary that injects bonding in advance in the back sealing two ends, be immersed in 40～70 ℃ of water-baths and react 6～24h, after reaction was finished, the intercepting Len req was can carry out μ-HPLC lock out operation behind moving phase flushing 2～6h with methyl alcohol.
2, the preparation method of octyl group type micro column liquid chromatography integral post as claimed in claim 1 is characterized in that the pre-bonding of said capillary tube inner wall was prepared γ-methacrylic acid oxygen propyl trimethoxy silicane and methyl alcohol mixed solution in by volume 1: 1.
3, the preparation method of octyl group type micro column liquid chromatography integral post as claimed in claim 1 is characterized in that before γ-methacrylic acid oxygen propyl trimethoxy silicane and methyl alcohol mixed solution injection kapillary ultrasonic degas.
4, the preparation method of octyl group type micro column liquid chromatography integral post as claimed in claim 3 is characterized in that the time of ultrasonic degas before γ-methacrylic acid oxygen propyl trimethoxy silicane and methyl alcohol mixed solution injection kapillary is 5～20min.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100494233C (en) * | 2005-12-14 | 2009-06-03 | 中国科学院大连化学物理研究所 | Porous integrated substrate of carrying protein solid and its preparation |
| CN100435935C (en) * | 2006-02-14 | 2008-11-26 | 厦门大学 | Method of preparing octadecyl type integral liquid chromatography micro-column |
| CN101362074B (en) * | 2007-08-08 | 2010-09-01 | 中国计量科学研究院 | Use of double-phenyl hybridization silica gel material monolithic column in chromatogram |
| CN101747478B (en) * | 2008-12-17 | 2012-02-01 | 中国科学院大连化学物理研究所 | 'One-pot' preparation of organic-inorganic hybridization porous monolithic material |
| CN102225249B (en) * | 2011-04-06 | 2013-04-17 | 福州大学 | Preparation method of organic-inorganic hybrid monolithic capillary column |
| CN103185678B (en) * | 2011-12-28 | 2014-12-10 | 华中师范大学 | Trace phosphorylated peptide desalting column |
| CN105797689B (en) * | 2016-03-15 | 2018-07-17 | 厦门大学 | A kind of preparation method based on two kinds of crosslinking agent porous adsorbents |
| CN111359587B (en) * | 2020-04-15 | 2021-05-14 | 厦门大学 | Preparation of quartz capillary hybridization monolithic column based on metal organic framework material |
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