CN104034830A - Method for filling high-temperature liquid-phase chromatographic column of padding with fine particle size and dedicated device for method - Google Patents
Method for filling high-temperature liquid-phase chromatographic column of padding with fine particle size and dedicated device for method Download PDFInfo
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- CN104034830A CN104034830A CN201410258961.2A CN201410258961A CN104034830A CN 104034830 A CN104034830 A CN 104034830A CN 201410258961 A CN201410258961 A CN 201410258961A CN 104034830 A CN104034830 A CN 104034830A
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Abstract
The invention discloses a method for filling a high-temperature liquid-phase chromatographic column of padding with the fine particle size. The method is characterized by comprising the following steps: firstly stirring and mixing the padding with a homogenate solvent, performing ultrasonic dispersing treatment to obtain homogenate, then heating a pressurized solvent pipeline, a homogenate tank and the chromatographic column to enable the temperatures of the pressurized solvent pipeline, the homogenate tank and the chromatographic column to reach 50-90 DEG C, pouring the homogenate into the homogenate tank, connecting the pressurized solvent pipeline, a column filling pump, the homogenate tank and the chromatographic column through a pipeline, sealing the homogenate tank, pressurizing the homogenate tank to 50-200MPa after the temperature of the homogenate is uniform with that of the homogenate tank, and filling the column filling pump with the homogenate and a pressurized solvent. The filling method has relatively low requirements on the high pressure sealing property of filling equipment. Meanwhile, the invention further relates to a dedicated filling device necessary for the filling method.
Description
Technical field
The present invention relates to a kind of chromatographic column packing method, and the special purpose device of this kind of required use of method.
Background technology
The filling of chromatographic column directly has influence on its separating property.Load bad chromatographic column, or post effect is not high, or after using in the short time, because the compression of packed bed causes column cap place to produce, subsides, chromatogram column efficiency can reduce suddenly.
The filling of liquid-phase chromatographic column, according to the size of the character of filler and chromatographic column, can adopt diverse ways.In principle, the filler filling large scale chromatographic column that particle diameter is greater than 20 μ m can adopt dry method filling.Filler is directly joined in gc column tube, by vibration, make its densification, obtain uniform packed bed.Chromatographic column for granule filler filling fine inner diameter, adopts high-pressure homogenization completion method, pneumatic completion method, centrifugal completion method, electronic completion method etc. more, adopts external motivating force that filler is packed in chromatographic column, forms uniform bed.
Slurry packing is the most frequently used liquid chromatography column packing method, and the method is generally used for the filler of the following particle diameter of 10 μ m.With one or several solvents, be mixed with the solution that density is close with filler, through ultrasonic processing, make filler particles high degree of dispersion in solution, present emulsion state.With pressurized solvent (hexane or methyl alcohol etc.), under high pressure homogenate is pressed in column jecket, makes the high efficiency chromatography post bed with even, tight packed bed.The back-pressure that bed produces is relevant to the length of packing material size, filling, therefore it is long that the chromatographic column that adopts particle diameter to be less than 2 μ m filler fillings is less than 10cm conventionally, in the time must loading longer chromatographic column, conventionally need larger dress column pressure, someone adopts the report of pressure filling 50cm length chromatographic column more than 300MPa.It loads hypertonia, to filling equipment require highly, and there is certain potential safety hazard, therefore need now a kind of chromatographic column packing method or device that can address the above problem.
Summary of the invention
The present invention is in order to solve the existing above-mentioned deficiency of prior art, proposes a kind of chromatographic column filling pressure that reduces, and the high pressure sealing performance of filling equipment is required to lower packing method, and this kind of filling special purpose device that method is used.
Technical solution of the present invention is: a kind of fine grain filler high temperature liquid chromatography column packing method, is characterized in that: described method is carried out according to following steps:
First filler is mixed with homogenate stirring solvent, and carry out ultrasonic dispersion treatment, obtain homogenate, then pressurized solvent pipeline, homogenate tank and chromatographic column are carried out to heat, make their temperature reach 50-90 ℃, homogenate is poured in homogenate tank, and pressurized solvent tank, dress post pump, homogenate tank are connected by pipeline with chromatographic column, the sealing of sealing homogenate tank, until homogenate temperature and homogenate fill with temperature consistent after, homogenate tank is forced into 50-200MPa, homogenate and pressurized solvent are packed in chromatographic column in the lump.
The special purpose device that fine grain filler high temperature liquid chromatography column packing method as above is used, is characterized in that: device comprises pressurized solvent tank 1; The homogenate tank 3 being connected by pipeline 2 with pressurized solvent tank 1; Homogenate tank 3 is connected with the chromatographic column 5 that is positioned at chromatogram column sleeve 4; On pipeline 2, be also provided with dress post pump 6 and tensimeter 7; Between pressurized solvent tank 1 and homogenate tank 3, be provided with heating chamber 8; In heating chamber 8, homogenate tank 3 and chromatogram column sleeve 4, be provided with heater strip; All heater strips are all unified to control by temperature control system.
The particle diameter of described filler is less than 3 μ m.
Described homogenate solvent is one or more in methyl alcohol, acetone, tetrahydrofuran.
Described chromatographic column is stainless steel column, quartz capillary column or Peek material post.
Compared with the existing technology, tool has the following advantages in the present invention:
Chromatographic column packing method disclosed in this invention, before filling, homogenate and pressurized solvent are heated and be incubated, under this temperature environment, carry out the filling of chromatographic column, can greatly reduce the filling pressure of chromatographic column, greatly reduce the high-pressure tightness requirement of filling equipment; Accurate control by temperature control system to each several part temperature, allows repeatability and the stability of chromatographic column filling greatly improve.It is particularly suitable for the filling that particle diameter is less than the filler of 3 μ m.Can say that it has possessed multiple advantage, possesses market outlook widely.
Accompanying drawing explanation
Fig. 1 is the structural representation of filling device used in the present invention.
Embodiment
Below in conjunction with accompanying drawing explanation the specific embodiment of the present invention.As shown in Figure 1: a kind of fine grain filler high temperature liquid chromatography column packing method, carries out according to following steps:
First the filler that particle diameter is less than to 3 μ m mixes with homogenate stirring solvent, and carry out ultrasonic dispersion treatment, obtain homogenate, then by temperature control system, unify to control heating wire works, to heating chamber 8, homogenate tank 3 heats with the chromatographic column 5 that is positioned at chromatogram column sleeve 4, make their temperature reach 50-90 ℃, homogenate is poured in homogenate tank 3, and by pressurized solvent tank 1, heating chamber 8, homogenate tank 3 is connected by pipeline with chromatographic column 4, make homogenate tank 3 seal and be forced into 50-200MPa, open dress post pump 6, pressurized solvent in pressurized solvent tank 1 is poured to heating in heating chamber 8, pressurized solvent after heating enters in homogenate tank 3, together with homogenate wherein, fill in the lump and enter in chromatographic column 5, here solvent used is methyl alcohol, acetone, one or more in tetrahydrofuran, chromatographic column used is stainless steel column, quartz capillary column or Peek material post.
Embodiment 1
By internal diameter 100 μ m, external diameter 360 μ m, the elastic quartz capillary chromatograph 5 that length is 15cm is placed in chromatogram column sleeve 4, is fixed on homogenate and fills with 3 bottoms; Take 50mg grain through being the C18 reverse-phase chromatography silica filler of 3 μ m, join in 10mL acetone and be prepared into the homogenate that concentration is 5mg/mL, vibration mixes, and ultrasonic dispersion is more than 10min; By temperature control system, control heating wire works, pressurized solvent (methyl alcohol), homogenate filling 3, chromatographic column 5 are heated to 50 ℃, get 200 μ L homogenates and pour in homogenate filling 3; Homogenate is filled with to 3 sealings, and pressurized solvent tank 1, homogenate tank 3 and chromatographic column 4 are connected by pipeline, open dress post pump 6, under 100MPa pressure, load 30min; Unload capillary chromatographic column, process entrance, complete dress post.
Chromatographic performance is evaluated: sample is the potpourri of uracil, acetophenone, toluene, ethylbenzene and fluorenes, sample size 10nL, and mobile phase is methanol/water (85/15) solution, flow is 400nL/min, detect wavelength 254nm, test result demonstration, the post effect of last component fluorenes is every meter of 6.0 ten thousand column plate.
Embodiment 2
By internal diameter 100 μ m, external diameter 360 μ m, the elastic quartz capillary chromatograph 5 that length is 15cm is placed in chromatogram column sleeve 4, is fixed on homogenate and fills with 3 bottoms; Take 50mg grain through being the C18 reverse-phase chromatography silica filler of 3 μ m, join in 10mL acetone and be prepared into the homogenate that concentration is 5mg/mL, vibration mixes, and ultrasonic dispersion is more than 10min; By temperature control system, control heating wire works, pressurized solvent (methyl alcohol), homogenate filling 3, chromatographic column 5 are heated to 70 ℃, get 200 μ L homogenates and pour in homogenate filling 3; Homogenate is filled with to 3 sealings, and pressurized solvent tank 1, homogenate tank 3 and chromatographic column 4 are connected by pipeline, open dress post pump 6, under 100MPa pressure, load 30min; Unload capillary chromatographic column, process entrance, complete dress post.
Chromatographic performance is evaluated: sample is the potpourri of uracil, acetophenone, toluene, ethylbenzene and fluorenes, sample size 10nL, and mobile phase is methanol/water (85/15) solution, flow is 400nL/min, detect wavelength 254nm, test result demonstration, the post effect of last component fluorenes is every meter of 7.2 ten thousand column plate.
Embodiment 3
By internal diameter 100 μ m, external diameter 360 μ m, the elastic quartz capillary chromatograph 5 that length is 15cm is placed in chromatogram column sleeve 4, is fixed on homogenate and fills with 3 bottoms; Take 50mg grain through being the C18 reverse-phase chromatography silica filler of 3 μ m, join in 10mL acetone and be prepared into the homogenate that concentration is 5mg/mL, vibration mixes, and ultrasonic dispersion is more than 10min; By temperature control system, control heating wire works, pressurized solvent (methyl alcohol), homogenate filling 3, chromatographic column 5 are heated to 90 ℃, get 200 μ L homogenates and pour in homogenate filling 3; Homogenate is filled with to 3 sealings, and pressurized solvent tank 1, homogenate tank 3 and chromatographic column 4 are connected by pipeline, open dress post pump 6, under 100MPa pressure, load 30min; Unload capillary chromatographic column, process entrance, complete dress post.
Chromatographic performance is evaluated: sample is the potpourri of uracil, acetophenone, toluene, ethylbenzene and fluorenes, sample size 10nL, and mobile phase is methanol/water (85/15) solution, flow is 400nL/min, detect wavelength 254nm, test result demonstration, the post effect of last component fluorenes is every meter of 8.7 ten thousand column plate.
Embodiment 4
By internal diameter 100 μ m, external diameter 360 μ m, the elastic quartz capillary chromatograph 5 that length is 15cm is placed in chromatogram column sleeve 4, is fixed on homogenate and fills with 3 bottoms; Take 50mg grain through being the C18 reverse-phase chromatography silica filler of 3 μ m, join in 10mL acetone and be prepared into the homogenate that concentration is 5mg/mL, vibration mixes, and ultrasonic dispersion is more than 10min; By temperature control system, control heating wire works, pressurized solvent (methyl alcohol), homogenate filling 3, chromatographic column 5 are heated to 70 ℃, get 200 μ L homogenates and pour in homogenate filling 3; Homogenate is filled with to 3 sealings, and pressurized solvent tank 1, homogenate tank 3 and chromatographic column 4 are connected by pipeline, open dress post pump 6, under 50MPa pressure, load 30min; Unload capillary chromatographic column, process entrance, complete dress post.
Chromatographic performance is evaluated: sample is the potpourri of uracil, acetophenone, toluene, ethylbenzene and fluorenes, sample size 10nL, and mobile phase is methanol/water (85/15) solution, flow is 400nL/min, detect wavelength 254nm, test result demonstration, the post effect of last component fluorenes is every meter of 6.5 ten thousand column plate.
Embodiment 5
By internal diameter 100 μ m, external diameter 360 μ m, the elastic quartz capillary chromatograph 5 that length is 15cm is placed in chromatogram column sleeve 4, is fixed on homogenate and fills with 3 bottoms; Take 50mg grain through being the C18 reverse-phase chromatography silica filler of 3 μ m, join in 10mL acetone and be prepared into the homogenate that concentration is 5mg/mL, vibration mixes, and ultrasonic dispersion is more than 10min; By temperature control system, control heating wire works, pressurized solvent (methyl alcohol), homogenate filling 3, chromatographic column 5 are heated to 70 ℃, get 200 μ L homogenates and pour in homogenate filling 3; Homogenate is filled with to 3 sealings, and pressurized solvent tank 1, homogenate tank 3 and chromatographic column 4 are connected by pipeline, open dress post pump 6, under 150MPa pressure, load 30min; Unload capillary chromatographic column, process entrance, complete dress post.
Chromatographic performance is evaluated: sample is the potpourri of uracil, acetophenone, toluene, ethylbenzene and fluorenes, sample size 10nL, and mobile phase is methanol/water (85/15) solution, flow is 400nL/min, detect wavelength 254nm, test result demonstration, the post effect of last component fluorenes is every meter of 9.0 ten thousand column plate.
Embodiment 6
By internal diameter 100 μ m, external diameter 360 μ m, the elastic quartz capillary chromatograph 5 that length is 15cm is placed in chromatogram column sleeve 4, is fixed on homogenate and fills with 3 bottoms; Take 50mg grain through being the C18 reverse-phase chromatography silica filler of 3 μ m, join in 10mL acetone and be prepared into the homogenate that concentration is 5mg/mL, vibration mixes, and ultrasonic dispersion is more than 10min; By temperature control system, control heating wire works, pressurized solvent (methyl alcohol), homogenate filling 3, chromatographic column 5 are heated to 70 ℃, get 200 μ L homogenates and pour in homogenate filling 3; Homogenate is filled with to 3 sealings, and pressurized solvent tank 1, homogenate tank 3 and chromatographic column 4 are connected by pipeline, open dress post pump 6, under 200MPa pressure, load 30min; Unload capillary chromatographic column, process entrance, complete dress post.
Chromatographic performance is evaluated: sample is the potpourri of uracil, acetophenone, toluene, ethylbenzene and fluorenes, sample size 10nL, mobile phase is methanol/water (85/15) solution, flow is 400nL/min, detect wavelength 254nm, test result demonstration, the post effect of last component fluorenes is every meter of 11.0 ten thousand column plate.
Claims (5)
1. a fine grain filler high temperature liquid chromatography column packing method, is characterized in that: described method is carried out according to following steps:
First filler is mixed with homogenate stirring solvent, and carry out ultrasonic dispersion treatment, obtain homogenate, then pressurized solvent pipeline, homogenate tank and chromatographic column are carried out to heat, make their temperature reach 50-90 ℃, homogenate is poured in homogenate tank, and pressurized solvent tank, dress post pump, homogenate tank are connected by pipeline with chromatographic column, the sealing of sealing homogenate tank, until homogenate temperature and homogenate fill with temperature consistent after, homogenate tank is forced into 50-200MPa, homogenate and pressurized solvent are packed in chromatographic column in the lump.
2. the special purpose device that fine grain filler high temperature liquid chromatography column packing method as claimed in claim 1 is used, is characterized in that: device comprises pressurized solvent tank (1); The homogenate tank (3) being connected by pipeline (2) with pressurized solvent tank (1); Homogenate tank (3) is connected with the chromatographic column (5) that is positioned at chromatogram column sleeve (4); On pipeline (2), be also provided with dress post pump (6) and tensimeter (7); Between pressurized solvent tank (1) and homogenate tank (3), be provided with heating chamber (8); In heating chamber (8), homogenate tank (3) and chromatogram column sleeve (4), be provided with heater strip; All heater strips are all unified to control by temperature control system.
3. fine grain filler high temperature liquid chromatography column packing method according to claim 1, is characterized in that: the particle diameter of described filler is less than 3 μ m.
4. fine grain filler high temperature liquid chromatography column packing method according to claim 1, is characterized in that: described homogenate solvent is one or more in methyl alcohol, acetone, tetrahydrofuran.
5. fine grain filler high temperature liquid chromatography column packing method according to claim 1, is characterized in that: described chromatographic column is stainless steel column, quartz capillary column or Peek material post.
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| CN201410258961.2A CN104034830A (en) | 2014-06-12 | 2014-06-12 | Method for filling high-temperature liquid-phase chromatographic column of padding with fine particle size and dedicated device for method |
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| CN201410258961.2A CN104034830A (en) | 2014-06-12 | 2014-06-12 | Method for filling high-temperature liquid-phase chromatographic column of padding with fine particle size and dedicated device for method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198831A (en) * | 2016-09-13 | 2016-12-07 | 吉尔生化（上海）有限公司 | A kind of method loading Reversed phase high performance liquid chromatography post |
| CN111089924A (en) * | 2019-12-21 | 2020-05-01 | 江苏奥迪康医学科技股份有限公司 | Preparation process of glycosylated hemoglobin high-pressure liquid chromatography column |
| CN113009056A (en) * | 2021-02-23 | 2021-06-22 | 晋江精纯科技有限公司 | Method and device for filling multi-column series-connected chromatographic column |
| CN117233303A (en) * | 2023-11-13 | 2023-12-15 | 维慕曦生物科技(常州)有限公司 | Automatic filling device for liquid chromatographic column |
-
2014
- 2014-06-12 CN CN201410258961.2A patent/CN104034830A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198831A (en) * | 2016-09-13 | 2016-12-07 | 吉尔生化（上海）有限公司 | A kind of method loading Reversed phase high performance liquid chromatography post |
| 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 |
| CN111089924A (en) * | 2019-12-21 | 2020-05-01 | 江苏奥迪康医学科技股份有限公司 | Preparation process of glycosylated hemoglobin high-pressure liquid chromatography column |
| CN113009056A (en) * | 2021-02-23 | 2021-06-22 | 晋江精纯科技有限公司 | Method and device for filling multi-column series-connected chromatographic column |
| CN117233303A (en) * | 2023-11-13 | 2023-12-15 | 维慕曦生物科技(常州)有限公司 | Automatic filling device for liquid chromatographic column |
| 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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Application publication date: 20140910 |