CN101776668B - Filling method of C18 HPLC (High Performance Liquid Chromatography) column - Google Patents
Filling method of C18 HPLC (High Performance Liquid Chromatography) column Download PDFInfo
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- CN101776668B CN101776668B CN2010101250468A CN201010125046A CN101776668B CN 101776668 B CN101776668 B CN 101776668B CN 2010101250468 A CN2010101250468 A CN 2010101250468A CN 201010125046 A CN201010125046 A CN 201010125046A CN 101776668 B CN101776668 B CN 101776668B
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Abstract
The invention relates to a filling method of a C18 HPLC (High Performance Liquid Chromatography) column, which comprises the steps of: mixing cyclohexyl alcohol and chloroform in a volume ratio of 9:11-13:7; adding C18 silicone with grain diameter of 5mu m to prepare a homogenate liquid with the concentration of 14.5%; fully stirring and mixing the liquid, oscillating for 5-10 minutes in a ultrasonic oscillator till the homogenate liquid becomes transparent liquid; mixing methanol and isopropyl alcohol in ratio of 1:1 to prepare a displacing liquid; pouring the prepared homogenate liquid into a homogenate tank which is connected with a chromatographic column tube; adding the displacing liquid in a displacing tank, starting a column installation machine to transmit the displacing liquid into the homogenate tank at the pressure of 6000 psi and then to a chromatographic column, and shutting down the column installation machine and stopping pressurization till 70ml-100ml of the displacing liquid is flew out of the chromatographic column; and standing for 20-40 minutes when system pressure descends to constant pressure, and then dismantling the chromatographic column. The invention has the advantages of simple method, low cost, easy operation, tight and uniform filling, high column efficiency and good chromatographic peak symmetry and is suitable for the filling of an analysis type and preparation type liquid-phase chromatographic column.
Description
Technical field
The present invention relates to the packing method field of chromatographic column, is a kind of packing method of C18 performance liquid chromatographic column specifically.
Background technology
Chromatographic column is the critical piece of liquid chromatography, and the fill method of chromatographic column is again realize chromatographic column high-effect to the stationary phase of confirming, the good chromatographic peak symmetry and the gordian technique of permeate well.The method of filling liquid-phase chromatographic column has two kinds of dry method and wet methods.When the particle diameter of stationary phase during, generally adopt dry-packing greater than 20 microns.And be 3～10 microns stationary phase to particle diameter at present commonly used, then adopt the wet method filling.Wet method is also referred to as the homogenate method.Solvent used during according to making homogenate is different, and wet stowage can be divided into viscosimetry, three kinds of non-equilibrium densimetry and balance densimetrys, and displacement fluid adopts lower boiling normal hexane or methyl alcohol more.Chromatographic column tight ness rating or uniformity coefficient with preceding two kinds of methods filling are not ideal enough.When the equilibrium density legal system is equipped with homogenate, adopt halogenated hydrocarbons, like iodomethane, tetrabromoethane, zellon, phenixin equal solvent more.These solvents are toxic, and are prone on stationary phase to produce shortcoming such as irreversible adsorption.
Summary of the invention
Technical matters to be solved by this invention provide a kind of C18 high efficiency chromatography post separation efficiency (theoretical cam curve) and chromatographic peak symmetric easy and simple to handle, spend cheaply, be suitable for the fill method that use in the laboratory.
The present invention solves its technical matters through following technical proposals:
A kind of fill method of C18 performance liquid chromatographic column, undertaken by following step:
A. prepare homogenate
Getting cyclohexanol and methenyl choloride mixes with 9: 11～13: 7 volume ratio; It is 14.5% homogenate that the C18 preparation of silica gel that adds particle diameter and be 5um becomes concentration; Solution is fully mixed, in ultrasonic oscillator, vibrated 5～10 minutes, homogenate is colourless transparent liquid;
B. prepare displacement fluid
Adopt methyl alcohol and isopropyl alcohol to mix as displacement fluid with 1: 1 volume ratio;
C. adorn post
Pour the homogenate of preparation in the homogenate jar that connects gc column tube into; Displacement fluid is joined in the replacement jar, start packing column machine, under 6000psi pressure; Displacement fluid is imported in the homogenate jar, entered into chromatographic column, treat to flow out till displacement fluid 70ml～100ml in the chromatographic column through the homogenate jar; Close packing column machine, stop pressurization, when system pressure is reduced to normal pressure; Left standstill 20～40 minutes, and unloaded chromatographic column;
D. the chromatographic column of filling is estimated.
The inventive method is easy, cost is low, easy to operate, filling closely, evenly, post imitate high, the chromatographic peak symmetry is good, is applicable to the filling of analytic type and preparative liquid chromatography post.
Table one: carbon content is the column performance evaluation result of 20.9% C18 column packing filling
Chromatographic condition: chromatographic column, 250mm*4.6mm; Moving phase, methyl alcohol: water=85: 15 (V/V); Flow velocity, 1.0 ml/min, detecting device UV
254
Table two: carbon content is the column performance evaluation result of 13.5% C18 column packing filling.
Chromatographic condition: chromatographic column, 250mm*4.6mm; Moving phase, methyl alcohol: water=85: 15; Flow velocity, 1.0 ml/min, detecting device UV
254
Table three: carbon content is the column performance evaluation result of 17.3% C18 column packing filling.
Chromatographic condition: chromatographic column, 250mm*4.6mm; Moving phase, methyl alcohol: water=85: 15; Flow velocity, 1.0 ml/min, detecting device UV
254
Description of drawings
Fig. 1 is that carbon content is the column performance evaluation result chromatogram of 20.9% C18 column packing filling.
Fig. 2 is that carbon content is the column performance evaluation result chromatogram of 13.5% C18 column packing filling.
Fig. 3 is that carbon content is the column performance evaluation result chromatogram of 17.3% C18 column packing filling.
Embodiment
Below in conjunction with embodiment the present invention is explained further details.
Embodiment 1
The filling of the C18 column packing of carbon content 20.9%
1. preparation homogenate: each fully mixes to get 9ml cyclohexanol and 11ml methenyl choloride, and adding the 2.9g particle diameter is the silica gel bonded stationary phase of C18 of 5 μ m, the homogenate of configuration 20ml, and concussion is 5 minutes in ultrasonic oscillator, and homogenate is colourless transparent liquid.
2. preparation displacement fluid:
Get each 100ml of methyl alcohol and isopropyl alcohol and mix, configuration 200ml displacement fluid.
3. dress post: pour the homogenate for preparing rapidly in the homogenate jar that has connected gc column tube 250mm*4.6mm, be connected with packing column machine again.
4. start packing column machine: under 6000psi pressure, displacement fluid is input in the chromatographic column through the homogenate jar, treats till about 80 milliliters of the displacement fluid volume that flows out in the chromatographic column; Close packing column machine, stop pressurization, when system pressure is reduced to normal pressure; After leaving standstill 30 minutes, unload chromatographic column evaluation.
5. the chromatographic column of filling is estimated:
Chromatographic condition and the evaluation result estimated are seen table one explanation, and chromatogram is seen figure one.Show that the chromatographic column with the present invention's technology dress post has high post to imitate, theoretical cam curve is 6.4772 ten thousand～7.5868 ten thousand/rice, and the symmetry of chromatographic peak is good, and tailing factor is 0.99～1.09, and demonstrating the self-chambering post has good chromatographic performance.
The filling of the C18 column packing of carbon content 13.5%
1. preparation homogenate: get the 13ml cyclohexanol and the 7ml methenyl choloride fully mixes, adding the 2.9g particle diameter is the silica gel bonded stationary phase of C18 of 5 μ m, dispose 20 milliliters homogenate, in ultrasonic oscillator, shakes 5 minutes, and homogenate is colourless transparent liquid.
2. preparation displacement fluid:
Get each 100ml of methyl alcohol and isopropyl alcohol and mix, dispose 200 milliliters of displacement fluids;
3. dress post: pour the homogenate for preparing rapidly in the homogenate jar that has connected gc column tube 250mm*4.6mm, be connected with packing column machine again.
4. start packing column machine: under 6000psi pressure, displacement fluid is input in the chromatographic column through the homogenate jar, treats till about 80 milliliters of the displacement fluid volume that flows out in the chromatographic column; Close packing column machine, stop pressurization, when system pressure is reduced to normal pressure; After leaving standstill 30 minutes, unload chromatographic column evaluation.
5. the chromatographic column of filling is estimated:
Appreciation condition and evaluation result are seen table two explanation, and chromatogram is seen figure two.The chromatographic column that shows filling has good chromatographic performance.Theoretical cam curve is 7.0591 ten thousand～7.6520 ten thousand/rice, and the tailing factor of chromatographic peak is between 0.93～1.04.
The filling of the C18 column packing of carbon content 17.3%
1. preparation homogenate: cyclohexanol and methenyl choloride are respectively got 10ml fully mix, adding the 2.9g particle diameter is the silica gel bonded stationary phase of C18 of 5 μ m, dispose 20 milliliters homogenate, in ultrasonic oscillator, shakes 5 minutes, and homogenate is colourless transparent liquid.
2. preparation displacement fluid:
Get each 100ml of methyl alcohol and isopropyl alcohol and mix, dispose 200 milliliters of displacement fluids;
3. dress post: pour the homogenate for preparing rapidly in the homogenate jar that has connected gc column tube 250mm*4.6mm, be connected with packing column machine again.
4. start packing column machine: under 6000psi pressure, displacement fluid is input in the chromatographic column through the homogenate jar, treats till about 80 milliliters of the displacement fluid volume that flows out in the chromatographic column; Close packing column machine, stop pressurization, when system pressure is reduced to normal pressure; After leaving standstill 30 minutes, unload chromatographic column evaluation.
5. the chromatographic column of filling is estimated:
Appreciation condition and evaluation result are seen table three explanation, and chromatogram is seen figure three.The chromatographic column that shows filling has good chromatographic performance.Theoretical cam curve is 7.2044 ten thousand～7.6868 ten thousand/rice, and the tailing factor of chromatographic peak is between 0.95～1.04.
Claims (5)
1. the fill method of a C18 performance liquid chromatographic column is characterized in that preparing as follows:
A. prepare homogenate
Getting cyclohexanol and methenyl choloride mixes with 9: 11～13: 7 volume ratio; It is 14.5% homogenate that the C18 preparation of silica gel that adds particle diameter and be 5 μ m becomes concentration; Solution is fully mixed, in ultrasonic oscillator, vibrated 5～10 minutes, homogenate is colourless transparent liquid;
B. prepare displacement fluid
Adopt methyl alcohol and isopropyl alcohol to mix as displacement fluid with 1: 1 volume ratio;
C. adorn post
Pour the homogenate 20ml of preparation in the homogenate jar that connects gc column tube into, the specification of gc column tube is 250mm*4.6mm; Displacement fluid 200ml is joined in the replacement jar, start packing column machine, under 6000psi pressure; Displacement fluid is imported in the homogenate jar, entered into chromatographic column, treat to flow out till displacement fluid 70ml～100ml in the chromatographic column through the homogenate jar; Close packing column machine, stop pressurization, when system pressure is reduced to normal pressure; Left standstill 20～40 minutes, and unloaded chromatographic column;
D. the chromatographic column of filling is estimated.
2. the fill method of C18 performance liquid chromatographic column according to claim 1, the carbon content that it is characterized in that the C18 column packing is 20.9% o'clock, the proportioning of said cyclohexanol and methenyl choloride is 9: 11V/V.
3. the fill method of C18 performance liquid chromatographic column according to claim 1, the carbon content that it is characterized in that the C18 column packing is 17.3% o'clock, the proportioning of said cyclohexanol and methenyl choloride is 10: 10V/V.
4. according to the fill method of the said C18 performance liquid chromatographic column of claim 1, the carbon content that it is characterized in that the C18 column packing is 13.5% o'clock, and the proportioning of said cyclohexanol and methenyl choloride is 13: 7V/V.
5. according to the fill method of the said C18 performance liquid chromatographic column of claim 1, it is characterized in that chromatographic column flows out till the displacement fluid 80ml milliliter in the said c step, close packing column machine; Stop pressurization; When system pressure is reduced to normal pressure, left standstill 30 minutes, unload chromatographic column evaluation.
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| CN102323339B (en) * | 2011-05-19 | 2014-03-26 | 周鑫 | Method for detecting stability of liquid phase chromatographic column |
| CN102998401A (en) * | 2011-09-15 | 2013-03-27 | 上海安谱科学仪器有限公司 | Efficient environment-protection filling method for amino liquid chromatography column |
| CN103869007A (en) * | 2012-12-18 | 2014-06-18 | 中国科学院大连化学物理研究所 | Method for evaluating C18 reverse-phase filler preparative chromatographic column |
| CN106198831B (en) * | 2016-09-13 | 2017-11-28 | 吉尔生化（上海）有限公司 | A kind of method for loading Reversed phase high performance liquid chromatography post |
| CN110531012A (en) * | 2018-05-25 | 2019-12-03 | 中科院大连化学物理研究所淮安化工新材料研究中心 | Different phosphorus content C18 liquid phases half prepare chromatography column packing method |
| CN109613150B (en) * | 2018-11-09 | 2021-05-28 | 兰州中科安泰分析科技有限责任公司 | PLOT-Al2O3Chromatographic column high-pressure dynamic method coating process |
| CN113687007B (en) * | 2021-08-30 | 2023-10-27 | 湖南德米特仪器有限公司 | Sequential packing method of chromatographic column |
| CN113945652A (en) * | 2021-09-26 | 2022-01-18 | 浙江工业大学 | Method for filling high-column-efficiency agglomeration type ion chromatography stationary phase |
| CN117233303B (en) * | 2023-11-13 | 2024-01-19 | 维慕曦生物科技(常州)有限公司 | Automatic filling device for liquid chromatographic column |
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| CN1243761A (en) * | 1998-07-30 | 2000-02-09 | 武汉大学 | Process for preparing porous filler of regenerated cellulose for gel permeation chromatographic column |
| CN1424139A (en) * | 2002-12-20 | 2003-06-18 | 武汉大学 | Regenerated cellulose microporous filler and preparation and use thereof |
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| CN1243761A (en) * | 1998-07-30 | 2000-02-09 | 武汉大学 | Process for preparing porous filler of regenerated cellulose for gel permeation chromatographic column |
| CN1424139A (en) * | 2002-12-20 | 2003-06-18 | 武汉大学 | Regenerated cellulose microporous filler and preparation and use thereof |
| CN1532545A (en) * | 2003-03-21 | 2004-09-29 | 中国人民解放军军事医学科学院放射医 | Novel method for producing and packing capillary chromatographic column sieve plate |
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