CN1379243A - Efficient chromatographic separation method for industrial liquid phase - Google Patents
Efficient chromatographic separation method for industrial liquid phase Download PDFInfo
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- CN1379243A CN1379243A CN 01110373 CN01110373A CN1379243A CN 1379243 A CN1379243 A CN 1379243A CN 01110373 CN01110373 CN 01110373 CN 01110373 A CN01110373 A CN 01110373A CN 1379243 A CN1379243 A CN 1379243A
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Abstract
An efficient industrial liquid-phase chromatographic separation method for fine chemicals, food, health-care product, etc. features use of high-flow horizontal liquid supply, high-pressure efficient separating column without dead volume, in-line proportioning of solvent and in-line degassing, high-resolution split-flow detection, and in-line ultrafiltering for sterilizing.
Description
The present invention relates to a kind of efficient chromatographic separation method for industrial liquid phase, can be widely used in the industrial scale separation and purification process in fields such as natural products effective ingredient, fine chemicals, food and health products, electronic chemical product.
Any liquid chromatography separation equipment is formed by following major part:
Reservoir → pump → injector → separating column → detecting device → gatherer → registering instrument or workstation
Existing liquid chromatography is mainly analytic type, half preparation type and several classes of preparation type.Analytic type is used for the detection of sample, its objective is exactly the accurately qualitative or quantitative test of micro-example sample introduction, and does not obtain products of separated.Half preparation type is to utilize the analytic type instrument, only uses slightly bigger separating column instead and analyzes and obtain micro-product.The preparation type then partly preparing change part hardware on the basis, further increases separating column, is purpose to obtain a small amount of products of separated.Above-mentioned method for separating liquid phase chromatography is the simple amplification of analytic type liquid chromatography at hardware components, has following defective when being used for producing for purpose:
1, pump discharge is little: analysis and half preparative liquid chromatography pump maximum flow are generally 10ml/min, and preparative liquid chromatography pump maximum flow is generally less than 100ml/min.
2. column volume is little: the existing used separating column of method is generally in 4.6mmI.D. * 50mm to 100mmI, the D.*1000mm scope, has limited the sample charge capacity.
3. detection resolution is low: when the sample charge capacity increases, and effluent sample concentration height behind the post, the detecting device flow cell need amplify, and the sample peak is trapezoidal, can only carry out the peak cutting this moment roughly and collect, and the low productive rate that causes of resolution descends.
4. the leacheate degassing is loaded down with trivial details: the used moving phase of said method all needs the degassing in advance, and the leacheate preparation and the degassing are operated loaded down with trivial details often when the chance separation process is longer.And the existing online degassing of half preparation type generally adopts ultra filtration membrane to carry out, be unsuitable for than heavy traffic condition, and at this moment from efficient convenience, result of use and the no big-difference of the preceding degassing of use.
5. capacity gauge is little: said method adopts test-tube type, hyperchannel small electrical magnet valve or artificially collects mode.Be suitable for laboratory examination, and be not suitable for industrial-scale production.
6. be unsuitable for commercial production: above deficiency has determined said method to be difficult to be applied to large-scale industrial production, and it is little mainly to show as the sample charge capacity, is generally several micrograms to a few gram orders of magnitude; The peak cutting is rough, and increased separation progression: industrial scale can't realize the online degassing with a large amount of leacheates; Though adopt the fine grain stationary phase, small-sized post column loadability is low, generally can only adopt manually or a small amount of sample introduction of autovalve; Flow rate pump is little, can not be equipped with stake production; Above-mentioned factor directly causes output little, is generally the output of several micrograms to a few gram levels.
The industrial liquid chromatography of China is separated the classical low-pressure opening chromatographic columns that adopt more at present; leacheate adopts the head tank flow by gravity or adopts the pulse pump feed flow; artificially collect; online nothing detects; no automation control system; separation efficiency is low; long flow path; pollute bigger; operate loaded down with trivial detailsly, many stainless steel drum or the glass for chemical equipment-building pipelines with welding voluntarily of chromatographic column replace, and strictness is said and is similar to workshop-based production run; especially comparatively general at Chinese medicine and natural products industry, be unsuitable for scale, development of modernization requirement.
Adapt to the requirement of industrial separation purified product, lean on the simple amplification of said method can't realize that the present invention has overcome the deficiency of above-mentioned existing method, set up a kind of advanced modernized industrial scale method for separating liquid phase chromatography.Method of the present invention mainly comprises following content:
1. big flow advection feed flow: flow reaches 10ml/min-1500ml/min, and no pulse advection feed flow adopts to exchange fire proof motor, and pressure reaches 30MPa, can satisfy the high efficient, high pressure chromatographic resolution.
2. the automatically gradient dosing and the online degassing: the present invention adopts Single-chip Controlling to realize that leacheate is by gradient Automatic Program proportioning and adopt the online degasser of automatic ultrasonic.Accompanying drawing 2 is online degassing trap, and its underpart is a ultrasonic vibrator 1, liquid outlet 2; Level monitoring probe 3, inlet 4, high-pressure solenoid valve 5 automatic deflations are arranged at top.Its degassing rate has realized the online degassing of a large amount of mixed solvents under the high flow rate greater than 80%, need not to consider mixed liquor heat release situation and molecular size.
3. adopt large-scale high efficiency separation post: separating column is the core that liquid chromatography is separated.The simple amplification can not reach the requirement of industrial scale separating column on analytic type or half preparation and preparation type separating column basis.Reason mainly is: 1. above-mentioned small-sized separating column is easy to processing, and column wall smooth finish, same circularity, right alignment index are higher, and when being amplified to a certain degree, present manufacturing process can't keep These parameters not reduce, and post is imitated and can be descended.2. above-mentioned separating column post footpath is very little, and liquid is easy to evenly distribute in the cross section.When being amplified to 100mmI.D. with the upper prop footpath, leacheate is under high traffic condition, and the short time can't reach column section and evenly distribute, and dead volume increases, and turbulent flow, channel, wall effect cause post to imitate serious the reduction.3. the disposable filling prepacked column of the many employings of above-mentioned small-sized separating column needn't consider that the stationary phase bed changes.And the bed volume of industrial scale separating column stationary phase is big, the charge capacity height, and long flow path, the expansion of post bed or compression meeting obviously influence separating effect even can cause producing and can't normally carry out.The process-scale chromatography separating column that accompanying drawing 1 adopts for the present invention, its inwall is processed on request, and wall liner is moulded in it in case of necessity, improves its smooth finish and right alignment, same circularity; Column cap adopts embedded manual or automatic lifting column cap, the column cap inside surface is tapered fluid flow distributor 1, there is chamfering spray-hole 2 liquid porch in post, filter sieve plate and adopt stainless steel and ZrO2 nanometer piezoelectric ceramics spherical powder sintered plate 3, the pressure change value that this plate causes the variation of post bed is converted into electric signal and is delivered to control amplifier by the insulation signal wire, drive the column cap automatic lifting with the outer motor 4 of this drive post, or carry out hand-operated lifting by handle 5, keep post pressing weighing apparatus and post bed to contact with the post sieve plate all the time, reduce dead volume.Use above-mentioned design, separating column maximum of the present invention can be 400mmI.D.*4000mm, still keeps higher post to imitate.
4, adopt small circulation pond (8 μ l-20 μ l) shunting detecting device: its inlet carries out separating liquid, automatic adjusting flowmeter is all arranged on the distribution pipeline, automatic adjusting solenoid valve on the detection signal control distribution pipeline of this flowmeter, keep shunting pressure equilibrium and shunting back liquid to flow out time synchronized, make the detection resolution of enriched sample not reduce, realized online detection and peak cutting accurately.
5. the present invention adopts the disc type multi-channel electromagnetic valve to collect (5-40 passage) automatically, both can regularly collect separately, also can with connect at thread detector, collect automatically by the peak-to-peak signal that detects.
6. the present invention adopts automatic ultrafiltration instrument to connect with the fraction collection container, and the purifying substance that obtain can directly enter the ultrafiltration system sterilization, is suitable for the biological products and the medicament preparation of specific (special) requirements.
7. the present invention is equipped with temperature, pressure, flow, potential of hydrogen on-line monitoring instrument before separating column and behind the post, to realize robotization control and remote monitoring.
8. above assembly is connected with pipeline successively, set up a kind of efficient chromatographic separation method for industrial liquid phase, each assembly both can be operated separately, also can carry out the microcomputer master control.
The invention is characterized in the employing reasonable method, set up the plant-scale separation and purification of liquid chromatography, and kept high pressure, efficiently, the advantage changed fast and automatically, solved the problem that occurs when liquid chromatography is amplified, stationary phase can be selected 10 μ m-60 μ m particle size range for use, post is pressed and is reached as high as 30Mpa, and leacheate stream scooter 1500ml/min has realized the full-automation control of whole detachment process.The equipment rapidoprint can adopt that 1Cr18Ni9Ti, 316L, titanium, lining be moulded according to the needs of different product, glass etc.Its result's advantage is presented as and is suitable for setting up the modern production line, can carry out Site Design according to the requirement of various processes installs, charge capacity reaches several tens feather weight that restrain, output can reach a few gram to tens feather weight, post is imitated tens times that surpass common low-pressure opening chromatogram, and separation cycle is short, and is easy and simple to handle, manually expend and lack, production cost reduces.
Embodiment 1:
Get 5Kg buxenine crude product (total alkaloids purity is 20%), add 95% dissolve with methanol, add 2KG zeyssatite, stir, 40 ℃ of reduction vaporizations reclaim solvent, obtain zeyssatite sample loaded article.
This loaded article is added in the preposition sample column (200mmI.D.*500mm).Respectively with the solvent storage tank, ternary solenoid valve gradient controller, big flow advection solution feed pump (1500ml/min), the ultrasonic degas device, the sample pre-column, separating column (200mmI, D, * 2000mm, wall liner is moulded in the stainless steel, silica G 60), shunting evaporation luminescence detector, automatic fraction collector connects successively, use methenyl choloride: methyl alcohol: ammoniacal liquor=99: 1.0: 2.0,99: 5.0: 2.0,99: 10: 2.0,99: 15: 2.0 (V/V/V) gradient elutions, sampling is through the HPLC check analysis, take out ring dimension buxenine D and collect the liquid part, rotary evaporation goes out crystallization, and low temperature drying gets 712 gram white crystals, measure its purity greater than 98%, the single process isolated yield reaches 70%.
Embodiment 2:
Get pseudo-ginseng root alkali crude product extract (notoginseng total saponin purity 40%) 75Kg, the dissolving of 70% methanol aqueous solution adds 2Kg zeyssatite mixing and absorption, and evaporate to dryness reclaims methyl alcohol, obtains zeyssatite sample loaded article.Thereafter operation and equipment connect with example 1, but detecting device adopts the long ultraviolet-visible detecting device of shunting all-wave, and separating column is 200mmI.D. * 2000mm, 316L, particle diameter 30 μ m, aperture 200 , specific surface 400m
2The styrene tyle macroporous absorption stationary phase of/g, use methyl alcohol: water: HCL=75: 25: 0.2 (V/V/V) wash-outs, sampling is through the HPLC check analysis, taking-up contains the fraction of Rg3 and Rb1, the low temperature rotary evaporation concentrates, add 10% acetonitrile recrystallization, get Rg3 (160 gram) and Rb1 (60 gram) crystal respectively, product purity is all greater than 98%.
Embodiment 3: get methyl alcohol medicinal extract (the containing taxol 1%) 5Kg of leaf of Japanese Yew, 95% acetone solution adds the 4Kg diatomite adsorption, and evaporate to dryness reclaims acetone, obtains zeyssatite sample loaded article.Thereafter operation and equipment connect with embodiment 2, but separating column is 200mmI.D.*4000mm, particle diameter 30 μ m, aperture 200 , specific surface 400m
2The silica matrix acrylate of/g phase that is coated and fixed, use acetone: water=40: 60,50: 50,60: 40 (V/V) gradient elutions, sampling is taken out the cut low temperature rotation that contains taxol and is concentrated through the HPLC check analysis, obtains taxol white crystal (35.1 gram) after the straight empty drying, its purity is greater than 98% after measured, and the single process isolated yield reaches 70%.
Claims (10)
1. plant-scale high performance liquid chromatography separation method can be applied to the sample separation purifying of multiple industry, realizes efficient suitability for industrialized production.It comprises the gradient controller, and high pressure advection solution feed pump is online from the earthquake degasser, large-scale zero dead volume separating column, shunting detecting device, multi-channel electromagnetic valve disc type automatic fraction collector, online sterilization ultrafilter, technology controlling and process workstation.Above-mentioned part is set up plant-scale high performance liquid chromatography separation method with the pipeline connection successively.
2. the method for claim 1 is characterized in that adopting ternary solenoid valve gradient controller, and the automatic follow procedure online design of leacheate proportioning is provided with high pressure and low-voltage variation, and pressure and flow on-line monitoring return feedback automatically.
3. the method for claim 1 is characterized in that adopting the big flow solution feed pump of high pressure advection, and maximum flow can reach 1500ml/min, and top pressure can reach 30MPa.
4. the method for claim 1 is characterized in that adopting the online degasser of automatic ultrasonic, and degassing rate is greater than 80%, level monitoring, high-pressure solenoid valve automatic deflation.
5. the method for claim 1 is characterized in that adopting large-scale high efficiency separation post.The cylinder material can be glass, plastics, stainless steel, titanium, interior wall liner moulding material etc.Column cap adopts embedded movable column cap, and its inside surface is tapered fluid flow distributor, and the inlet of liquid in post has the chamfering spray-hole, and filtering sieve plate can be sintered plates such as stainless steel, engineering ceramics, glass; Outer motor of post internal pressure feedback signal drive post and the lifting of transmission column cap.
6. the method for claim 1 is characterized by and adopts the liquid road to divide current sensor, and distribution pipeline is equipped with the automatic flow of regulating and takes into account automatic adjusting solenoid valve.
7. the method for claim 1 is characterized by and adopts disc type multi-channel electromagnetic valve automatic fraction collector, the signal of detecting device can drive this gatherer to go out peak-to-peak signal by the component that detects and collect step by step.
8. the method for claim 1 is characterized by and adopts the online bactericidal unit of ultrafiltration behind the post, directly realizes the purified treatment of products of separated.
9. the method for claim 1 is characterized by organic coating sphere material, silica gel bonded alkyl sphere material, organic synthesis high polymer material, inorganic material that used stationary phase can be selected from silica gel, silica matrix.
10. the method for claim 1 is characterized by this method and is applicable to the sample of separation gram level to tens feather weight.
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| CN 01110373 CN1379243A (en) | 2001-04-09 | 2001-04-09 | Efficient chromatographic separation method for industrial liquid phase |
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| CN 01110373 CN1379243A (en) | 2001-04-09 | 2001-04-09 | Efficient chromatographic separation method for industrial liquid phase |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100384500C (en) * | 2006-03-23 | 2008-04-30 | 上海交通大学 | Quarternary low pressure gradient mixing device |
| CN115335696A (en) * | 2020-03-27 | 2022-11-11 | 积水医疗株式会社 | Liquid chromatography member |
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2001
- 2001-04-09 CN CN 01110373 patent/CN1379243A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100384500C (en) * | 2006-03-23 | 2008-04-30 | 上海交通大学 | Quarternary low pressure gradient mixing device |
| CN115335696A (en) * | 2020-03-27 | 2022-11-11 | 积水医疗株式会社 | Liquid chromatography member |
| CN115335696B (en) * | 2020-03-27 | 2024-04-30 | 积水医疗株式会社 | Member for liquid chromatography |
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