阅读说明：本技术 不同含碳量c18液相半制备色谱柱装填方法 (Different phosphorus content C18 liquid phases half prepare chromatography column packing method ) 是由 韩海峰 吴妹 徐飞 于 2018-05-25 设计创作，主要内容包括：本发明涉及一种装填不同含碳量C18液相半制备色谱柱的方法,主要解决半制备柱峰形易拖尾和前沿、柱效不高等问题。本发明装填方法包括以下步骤：1)配置顶替液：采用混合有机溶剂和去离子水2种顶替液分段使用；2)制备匀浆液：一定比例混合溶剂配置成匀浆液体,加入C18反相填料于锥形瓶中,超声搅拌配置成匀浆液。3)色谱柱装填：以压缩空气为气源,以气动放大泵为装填高压泵,把制备好的匀浆液倒入连接好半制备柱管的匀浆罐中,用顶替液将匀浆液替换出来,继续冲洗一段时间后关闭气源,待系统压力降至常压后,静置10～15分钟,卸下色谱柱。4)半制备柱评价。本发明实现了色谱柱快速、高效的装填。(The present invention relates to a kind of methods loaded different phosphorus content C18 liquid phases half and prepare chromatographic column, and mainly solution semi-preparative column peak shape is easily trailed and forward position, column imitate the problems such as not high.1) packing method of the present invention is the following steps are included: configure displacement fluid: being used using the 2 kinds of displacement fluid segmentations of mixed organic solvents and deionized water；2) prepare homogenate: certain proportion mixed solvent is configured to homogenate liquid, C18 reverse phase filler is added in conical flask, ultrasonic agitation is configured to homogenate.3) chromatographic column is loaded: using compressed air as gas source, it is filling high-pressure pump with pneumatic amplification pump, the homogenate prepared is poured into the homogenate tank for connecting semi-preparative column pipe, homogenate is replaced out with displacement fluid, continue to close gas source after rinsing a period of time, after system pressure is down to normal pressure, 10~15 minutes are stood, chromatographic column is unloaded.4) semi-preparative column is evaluated.The present invention realizes chromatographic column and fast and efficiently loads.)

1. difference phosphorus content C18 liquid phase half prepares chromatography column packing method and is broadly divided into following four step

1) it configures displacement fluid: being used using 2 kinds of displacement fluid segmentations, it is a kind of super with the volume ratio mixing of 2:1 with methanol and isopropanol Displacement fluid is configured to after sound 10 minutes or so, another kind makees displacement fluid with deionized water；

2) it prepares homogenate: taking tetrachloromethane and isopropanol with the different proportion volume mixture of 1:9~9:1, be configured to homogenate C18 reverse phase filler is added in conical flask in body, exhaust ultrasound 10 minutes, and ultrasonic and stirring is configured to homogenate in 3 minutes；

3) it fills column: intercolumniation is filled in no dust purification, after control dress column environment temperature is 25 DEG C, using compressed air as gas source, with gas Dynamic amplification pump is filling high-pressure pump, and the homogenate prepared is poured into the homogenate tank for connecting semi-preparative column pipe；Displacement fluid falls Enter in displacement fluid bottle, open packing column machine operated pneumatic valve, slowly improves dress column pressure, the displacement fluid for first mixing methanol and isopropanol It squeezes into homogenate tank, when flowing out certain volume mixed solvent in chromatographic column, packing column machine displacement fluid direction valve is adjusted, by deionization Water continues to squeeze into homogenate tank, until flowing out certain volume deionized water to chromatographic column, closes packing column machine operated pneumatic valve, stops adding Pressure stands 10~15 minutes, unloads chromatographic column after system pressure is down to normal pressure；

4) semi-preparative column is evaluated.

2. C18 liquid phase according to claim 1 half prepares chromatography column packing method, it is characterized in that organic molten using mixing Agent and deionized water are displacement fluid.

3. C18 liquid phase according to claim 1 half prepares chromatography column packing method, it is characterized in that being to generate air pressure The air compressor of 0.8 MPa is gas source, is in an enlarged scale filling high-pressure pump for the pneumatic amplification pump of 1:100.

4. C18 liquid phase according to claim 1 half prepares chromatography column packing method, it is characterized in that C18 column packing used When phosphorus content is 10%~25%, homogenate solvent used includes but is not limited to tetrachloromethane, isopropanol, chloroform, methanol The mixture of equal organic solvents, volume ratio are 1:9~9:1.

5. C18 liquid phase according to claim 1 half prepares chromatography column packing method, it is characterized in that mixed solvent used and Water is that displacement fluid segmentation uses, and mixed solvent is but is not limited to the mixture of the organic solvents such as methanol, isopropanol, volume ratio 1: 9~9:1.

6. C18 efficient liquid phase according to claim 1 half prepares chromatography column packing method, it is characterized in that half preparation chromatography Column column length is 50-300 mm, and column diameter is 10-500 mm.

7. C18 efficient liquid phase according to claim 1 half prepares chromatography column packing method, it is characterized in that different-diameter color It composes column dress column pressure and is no more than 400 bar.

8. C18 efficient liquid phase according to claim 1 half prepares chromatography column packing method, it is characterized in that the C18 of filling Chromatograph packing material partial size is at 5-100 μm.

Technical field

The method of the present invention is related to the dress column method of semi-preparative column, and in particular to a kind of half preparation of difference phosphorus content C18 liquid phase Column packing method.

Background technique

Chromatographic column is the main component of liquid chromatogram, and the packing method of chromatographic column, is to realize again to determining stationary phase Chromatographic column is high-effect, the key technology of good symmetry.For high-effect column, 5 μm of especially most-often used filler chromatographies Column must then use wet process, i.e. homogenate dress column method.Homogenate method dress column refers to that the one or several kinds of solvent works of selection disperse, and suspends and is situated between Matter, it is sonicated make particle high degree of dispersion and in the translucent homogenate of suspension in the medium, then homogenate is pressed under high pressure Enter in column tube, the chromatographic column with uniform close packed bed is made.The packing method of semi-preparative column is different from dynamic axial compression Column (DAC column), DAC column load repeatedly since its piston can move up and down, and use the single solvents such as isopropanol for homogenate. And general semi-preparative column both ends sieve plate is fixed, it is ensured that column cap both ends are smooth, use the examination of two kinds of opposed polarities thus Agent carries out being mixedly configured into homogenate, filler can be made to be suspended in liquid in this way, eliminate because between filler particles electrostatic interaction and The increasing of surface energy, it is intended to which the ill-effect assembled and cohered keeps the column bed of filling closely knit, and chromatography column life extends.

All bed can be caused different due to random accidentalia because filling column every time during filling, cause chromatographic column Forward position or rear dragging, carrying out chromatographic isolation or purifying to sample in the case where other experimental conditions are all constant in this way will obtain It is different as a result, reducing test repeatability and product stability.In view of the above problems, the present invention using mixed solvent and go from Son hydrolyzes determine chromatographic column forward position and tailing problem, realizes half preparation chromatography column packing method of C18 and stablizes, efficient repeatable.

Summary of the invention

In order to solve the above-mentioned technical problems, the present invention provides a kind of convenience, simple easily duplicate filling and preparation method, Intercolumniation is filled in non-dusting purification, control environment temperature is at 25 DEG C, side when homogenate and filler mixed configuration homogenate liquid Side ultrasound is stirred, filler can be made preferably to be suspended in homogenate in this way.And use is taken over by two kinds of displacement fluid segmentations, it solves Because of the forward position of chromatographic peak or rear dragging situation caused by difference in operation.

The present invention solves its technical problem by following technical proposals.

Different phosphorus content C18 liquid phases half prepare chromatography column packing method, carry out in the steps below.

1. configuring displacement fluid

It is used at times using 2 kinds of displacement fluids, it is a kind of 10 minutes or so ultrasonic with the volume ratio mixing of 2:1 with methanol and isopropanol After be configured to displacement fluid, another kind makees displacement fluid with deionized water.

2. preparing homogenate

It takes tetrachloromethane and isopropanol with the different proportion volume mixture of 1:9~9:1, is configured to homogenate liquid, exhaust ultrasound 10 Minute.The C18 reverse phase filler of partial size 5um is added in conical flask, ultrasonic and stirring is configured to homogenate in 3 minutes.

3. filling column

Intercolumniation is filled in no dust purification, after control dress column environment temperature is 25 DEG C, using compressed air as gas source, with pneumatic amplification pump To load high-pressure pump, the homogenate prepared is poured into the homogenate tank for connecting semi-preparative column pipe；Displacement fluid pours into displacement fluid In bottle, packing column machine operated pneumatic valve is opened, dress column pressure is slowly promoted to 300 bar, the top for first mixing methanol and isopropanol It is squeezed into homogenate tank for liquid, when flowing out 1000~1500ml of displacement fluid volume in chromatographic column, adjusts packing column machine displacement fluid direction Deionized water is continued to squeeze into homogenate tank by valve, until flowing out 1000~1200ml of deionized water displacement fluid volume to chromatographic column, Packing column machine operated pneumatic valve is closed, pressurization is stopped, after system pressure is down to normal pressure, 10~15 minutes is stood, unloads chromatographic column.

4. semi-preparative column is evaluated

The method of the present invention is at low cost, easy to operate, filling is close, column effect is high, chromatography peak symmetry is good.

Table one: the chromatographic column evaluation result that the C18 column packing that phosphorus content is 20% loads

Peak number	Retention time	Title	Theoretical cam curve	Tailing factor
					1	13.573	Naphthalene	15299	1.09

Chromatographic condition: chromatographic column, 10mm * 250mm;Mobile phase, methanol: water=85:15；Flow velocity: 3ml/min；

UV detector wavelength, 254nm.

Table two: the chromatographic column evaluation result that the C18 column packing that phosphorus content is 17% loads

Peak number	Retention time	Title	Theoretical cam curve	Tailing factor
					1	12.633	Naphthalene	18975	1.01

Chromatographic condition: chromatographic column, 10mm * 250mm；Mobile phase, methanol: water=85:15；Flow velocity: 3ml/min；

UV detector wavelength, 254nm.

Table three: the chromatographic column evaluation result that the C18 column packing that phosphorus content is 15% loads

Peak number	Retention time	Title	Theoretical cam curve	Tailing factor
					1	11.463	Naphthalene	22864	1.01

Chromatographic condition: chromatographic column, 10mm * 250mm;Mobile phase, methanol: water=85:15；Flow velocity: 3ml/min；UV detector Wavelength, 254nm.

Detailed description of the invention

Fig. 1 is the chromatographic peak evaluation figure that the C18 column packing that phosphorus content is 20% loads.

Fig. 2 is the chromatographic peak evaluation figure that the C18 column packing that phosphorus content is 17% loads.

Fig. 3 is the chromatographic peak evaluation figure that the C18 column packing that phosphorus content is 15% loads.

Specific embodiment

The present invention is described in further details below with reference to embodiment.