CN102441295A - Separation and preparation chromatograph containing series dynamic axial compression (DAC) preparative chromatographic columns - Google Patents
Separation and preparation chromatograph containing series dynamic axial compression (DAC) preparative chromatographic columns Download PDFInfo
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Abstract
The invention provides a separation and preparation chromatograph containing series dynamic axial compression (DAC) preparative chromatographic columns and an application method thereof. The chromatograph comprises the following main components: a two-position three-way solenoid valve BV-3, a two-position six-way solenoid valve BV-6, a two-position four-way solenoid valve BV-4, a primary chromatographic separation column C1, a secondary chromatographic separation column 1 (C21), a secondary chromatographic separation column 2 (C22), a secondary chromatographic separation column 3 (C23), a secondary chromatographic separation column n (C2n), a manual injector (MI), a pre-column (PC), infusion pumps PA and PB, a detector, a fraction collector and the like. The key technology is as follows: a chromatographic working station completes program control on the two-position three-way solenoid valve BV-3, the two-position six-way solenoid valve BV-6 and the two-position four-way solenoid valve BV-4. In the invention, the DAC preparative chromatographic columns are applied to the separation and preparation process; and the primary chromatographic separation column is connected with the secondary chromatographic separation column group in series, and the secondary chromatographic separation columns are connected in parallel so as to independently separate and purify a compound monomer.
Description
Technical field:
The present invention relates to a kind of separation preparative chromatography instrument, the separation preparative chromatography instrument that two or more dynamic axial compression preparative chromatography splitters of particularly continuous use are combined into.
Background technology:
The research of natural activity compound or lead compound be unable to do without to extract and separates, along with the development of high throughput biological screening active ingredients technology (HTS); Native compound be separated into the research bottleneck.Preparative chromatography is as high efficient separation technology, and its clastotype comprises flushing, three kinds of patterns of displacement and forward position; Chromatographic system comprises all liquid-liquid chromatographys, liquid-solid chromatography, reverse-phase chromatography and supercritical fluid chromatography; Separating mechanism relates to absorption, distribution, ion-exchange, molecular exclusion, hydrophobic effect, affine and electronic micella etc.Particularly along with the development of chromatographic technique; Make the preparative chromatography technical application extensive day by day; Its separating power is high-tech sector in industries such as medicine, biotechnology, material, food, fine chemistry industry; The production application that particularly general isolation technics is difficult to realize in that purity requirement is high, added value is high; The field advantage such as medical product such as traditional Chinese medicine, autonomic drug, marine drug that particularly comes from natural resources at medicine is particularly outstanding, as (1) molecular mass be more or less the same, structure proximate, the approaching compound of physicochemical properties, like the separation of isomer, optical isomer or series structure analog; (2) under higher temperature, be easy to decompose, the separation of heat-sensitive substance of polycondensation or inactivation, bioactivator, albumen, enzyme etc.; And the fractionated of (3) natural or synthetic high polymer etc.Its growth is simultaneous growth with medicine, life science and development of biology; Preparative chromatography develops rapidly to be higher than 15% growth rate every year over nearly 10 years; Use in the industry very extensively, particularly almost ubiquitous in the preparation of activated monomer or compound products; But application is less in the system of natural products research separates, and main cause is that the chemical composition in the medicinal plant is too complicated, and preparative chromatography is used under study for action, trivial operations, and it is bothersome to waste time and energy, and its application is restricted.
China has the hugest chemical composition of Chinese materia medica and natural drug structural research troop; Mainly be engaged in the extraction separation and the structure identification research of natural products; Almost the work more than 80% all is in the separation of carrying out native compound; As if this research field becomes labor-intensive manual labor, and the separation and purification of native compound becomes the bottleneck of natural drug initiative or lead compound discovery, great amount of time and energy are wasted in separation and purification; And modern automation isolation technics equipment rapidly and efficiently remains the key of breakthrough bottleneck.So Combination application chromatogram new technology; The competition that designs various fast separation device equipment is also just very fierce; Many equipment and technical method occurring has a lot, but to preparing purifier apparatus efficient the separation fast of various compositions in a kind of medicinal plant of complete solution, actually rare; And complicated various in the face of the traditional Chinese medicine ingredients structure, satisfactory for result with regard to still less.Automatically in the separating natural application of compound equipment; The full-automatic high flux of the most famous is German Sepiatec GmbH company prepares the type piece-rate system; Wherein bigger model is Sepbox 2D-5000; This equipment main feature is within 24 hours, 0.5-5 to be restrained the cut that natural plant extracts automatically separates into 300 compounds, is comparatively effective equipment in the high-flux medicaments sifting.But this equipment is mainly used high speed adverse current chromatogram (HSCCC); Through a large amount of high speed adverse current chromatogram splitters, separative efficiency is limited, and a certain natural compounds content increases in the cut; Being separated into the less of high-purity unification compound, is some mixing cpd cuts mostly.
The 2D-HPLC analytical equipment has had remarkable progress at present, and research relatively fully and discussion have been arranged in theory, but in practical application, is still waiting to break through; Preparative chromatography is because compound component cut amount is very big; Cannot be mentioned in the same breath with analytical equipment on the technical difficulty; Thereby in the separation and purification preparation of a large amount of compounds, use the series connection chromatographic technique; The SMBC method can solve the demand of industrial preparation compound monomer at present, but wants efficient fast separating and purifying chemical compound lot monomer, still can not be as effective workaround.
Summary of the invention:
The object of the invention is intended to use solenoid control; Realize the simple switching of series connection dynamic axial compression preparative chromatography post; As the core technology of separating the preparative chromatography instrument, thereby solution natural products or the efficient fast separating and purifying of the field of Chinese medicines prepare the technical bottleneck of compound monomer.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Contain the separation preparative chromatography instrument of series connection dynamic axial compression preparative chromatography post, critical piece comprises: BV-3 two-position three way magnetic valve, two six three-way electromagnetic valves of BV-6, BV-4 two-position four-way solenoid valve; C1 one-level chromatography column, C21 secondary chromatography column 1, C22 secondary chromatography column 2, C23 secondary chromatography column 3; C2n secondary chromatography column n, MI manual injector, PC pre-column; PA, PB infusion pump, detector, fraction collection appearance; Said chromatography column is all used dynamic axial compression preparative chromatography post; Utilize one-level chromatography column C1 separate fraction F1-Fn continuously, one group of secondary chromatography column of difference sample introduction C21, C22, C23...C2n, separation and purification compound monomer; One-level chromatography column and secondary chromatography column group are passed through series system, and pass through parallel way between secondary chromatography column group, independent separate purifying compounds monomer; BV-3 two-position three way magnetic valve, two six three-way electromagnetic valves of BV-6, BV-4 two-position four-way solenoid valve are by the chromatographic work station programme-control.
Described separation preparative chromatography instrument, its two-position three way magnetic valve BV-3 and pre-column PC successively can exchange the position.
Described separation preparative chromatography instrument adopts three splitters: one-level chromatography column C1, secondary chromatography column C21, C22; The opposed polarity cut that one-level chromatography column C1 component obtains, the little cut of polarity be such as F1, F2, and F3 all separates with secondary chromatography column C21 successively; One-level chromatography column C1 component obtains the bigger cut of polarity such as F4, F5, and F6 all separates with secondary chromatography column C22 successively.So both having expanded the secondary chromatography column can the virtual C21-C26 of reaching, and has saved the secondary chromatography column, adopts the filler of different qualities, is easy to reach the purpose of separation and purification compound monomer.
Described separation preparative chromatography instrument, its one-level chromatography column and secondary chromatography column center pillar chromatotube material adopt stainless steel tube or polyfluortetraethylene pipe or titanium alloy tube; One-level chromatography column and secondary chromatography column generally adopt identical materials, and sized is identical or different; The column chromatography separating filler adopts various forward silica gel, C18, RP2-18, amino silica gel, nitro silica gel, alcohol radical silica gel, the derivatives resin of various sephadexes, polyethylene, polystyrene; The separation chromatography column pressure adopts medium-pressure or high pressure;
Described separation preparative chromatography instrument, its one-level chromatography column C1, secondary chromatography column C21, C22, C23 adopt radially dynamic compression preparative chromatography post in separation and purification compound monomer process.
Described separation preparative chromatography instrument, one-level chromatography column C1 be at eluting fraction F1, F2, and in the F3 process, the proportion of composing of eluent is identical or different; Secondary chromatography column C21; C22; C23 is in separation and purification compound monomer process, and the proportion of composing of eluent is identical or different, adopt methyl alcohol or/and chloroform or/and acetonitrile or/and ethyl acetate or/and water; Ratio is adjusted and controls the composition and the ratio of eluent from 0-100% according to separate object.
Described separation preparative chromatography instrument, the mobile process path of eluent is following in its secondary chromatography column C21 sample introduction and the separation process: 1) one-level splitter C1 chromatographic column separate fraction F1: two-position three way magnetic valve BV-3 → pre-column PC → one-level splitter C1 → two six three-way electromagnetic valves BV-6 → secondary splitter C21 → two-position four-way solenoid valve BV-4 → detector → waste liquid flows out; Be C21 post cut F1 sample introduction process; 2) secondary splitter C21 chromatographic column is separated: two-position three way magnetic valve BV-3 → two six a three-way electromagnetic valves BV-6 → secondary splitter C21 → two-position four-way solenoid valve BV-4 → detector → fraction collection instrument is collected compound monomer.
Described separation preparative chromatography instrument, the mobile process path of eluent is in its secondary chromatography column C22 sample introduction and the separation process: 1) one-level splitter C1 chromatographic column separate fraction F2: two-position three way magnetic valve BV-3 → pre-column PC → one-level splitter C1 → two six three-way electromagnetic valves BV-6 → secondary splitter C22 → two-position four-way solenoid valve BV-4 → detector → waste liquid flows out; Be C22 post cut F2 sample introduction process; 2) secondary splitter C22 chromatographic column is separated: two-position three way magnetic valve BV-3 → two six a three-way electromagnetic valves BV-6 → secondary splitter C22 → two-position four-way solenoid valve BV-4 → detector → fraction collection instrument is collected compound monomer.
Described separation preparative chromatography instrument, the mobile process path of eluent is in its secondary chromatography column C23 sample introduction and the separation process: 1) one-level splitter C1 chromatographic column separate fraction F3: two-position three way magnetic valve BV-3 → pre-column PC → one-level splitter C1 → two six three-way electromagnetic valves BV-6 → secondary splitter C22 → two-position four-way solenoid valve BV-4 → detector → waste liquid flows out; Be C23 post cut F3 sample introduction process; 2) secondary splitter C23 chromatographic column is separated: two-position three way magnetic valve BV-3 → two six a three-way electromagnetic valves BV-6 → secondary splitter C23 → two-position four-way solenoid valve BV-4 → detector → fraction collection appearance is collected compound monomer.
Described separation preparative chromatography instrument, the mobile process of eluent is similar in its secondary chromatography column C21-C23 sample introduction and the separation process, can expand to secondary chromatography column C2n.
The present invention has designed above-mentioned separation preparative chromatography apparatus (hereinafter to be referred as PAI) with series connection dynamic axial compression preparative chromatography post, and is core technology with this Equipment Design thinking, proposes a kind of method and thinking that solves the separation and purification compound monomer.The design and methods for using them of this equipment PAI is: the simple switching of application of dynamic axial compression preparative chromatography post; Realize the continuous series connection between the dynamic axial compression preparative chromatography post; To the separating repeatedly continuously of several preparative chromatography posts, reach the purpose of separation and purification compound monomer through one of a preparative chromatography post and other.To solve the technical bottleneck that natural products or chemical composition of Chinese materia medica separation and purification prepare pure article compound.
Description of drawings:
Fig. 1: be the present invention's equipment of separation preparative chromatography apparatus (PAI) of dynamic axial compression preparative chromatography post sketch map that is connected of connecting.
Among the figure:
The BV-3 two-position three way magnetic valve; Two six three-way electromagnetic valves of BV-6; The BV-4 two-position four-way solenoid valve; C1 one-level chromatography column; C21 secondary chromatography column 1; C22 secondary chromatography column 2; C23 secondary chromatography column 3; C2n secondary chromatography column n; The MI manual injector; The PC pre-column; PA, the PB infusion pump;
represents two six three-way electromagnetic valves.
The specific embodiment:
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with embodiments of the invention, but do not limit the present invention with this.
Embodiment 1:
The realization approach of content of the present invention and technical method, can represent through accompanying drawing 1, and detailed presentations is following:
1, go up appearance: test specimen with dissolution with solvents after, through manual injector MI sample introduction, wait to separate.If sample size is more, inconvenience also can promptly be used after the filling adsorption sample of pre-column through appearance on the pre-column dress post with 1-2 sample introduction of manual injector, adorns on the post kind.
2, separate:
1) one-level splitter C1 chromatographic column is separated: a certain proportion of eluent is with the test specimen wash-out; Two-position three way magnetic valve BV-3 control this moment eluent separates to one-level splitter C1 chromatographic column; One section cut F1 of while two six three-way electromagnetic valve BV-6 control eluents; This cut F1 makes F1 enter into secondary splitter C21 chromatographic column and separates with elution time T1 control, waits to separate.
The mobile process path of this separation phase eluent is: two-position three way magnetic valve BV-3 → pre-column PC → one-level splitter C1 → two six three-way electromagnetic valves BV-6 → secondary splitter C21 → two-position four-way solenoid valve BV-4 → detector → waste liquid flows out.
2) secondary splitter C21 chromatographic column sample introduction: separate simultaneously with one-level splitter C1 chromatographic column and to carry out.Separate T1 after the time from one-level splitter C1 chromatographic column, cut F1 enters into secondary splitter C21 chromatographic column and waits to separate through two six three-way electromagnetic valve BV-6 controls, forms secondary splitter C21 and goes up appearance.
The mobile process path of this separation phase eluent is: two-position three way magnetic valve BV-3 → pre-column PC → one-level splitter C1 → two six three-way electromagnetic valves BV-6 → secondary splitter C21 → two-position four-way solenoid valve BV-4 → detector → waste liquid flows out.
3) secondary splitter C21 chromatographic column is separated: after setting one-level splitter C1 chromatographic column separate fraction F1 gets on the secondary splitter C21 fully; Can control a certain proportion of eluent with the test specimen wash-out; Two-position three way magnetic valve BV-3 control this moment eluent is walked around one-level splitter C1 chromatographic column; Directly arrive and get into two six three-way electromagnetic valve BV-6 controls, eluent gets into secondary splitter C21 chromatographic column to be separated; The eluent separate fraction gets into detector, by different control methods, obtains the cut of unification compound absworption peak by detector, accomplishes secondary splitter C21 chromatographic column and separates.This separation process is the separation fully of separate fraction F1 that one-level splitter C1 is obtained, can collect all cpds monomer of separation and purification.
The mobile process path of this separation phase eluent is: two-position three way magnetic valve BV-3 → two six a three-way electromagnetic valves BV-6 → secondary splitter C21 → two-position four-way solenoid valve BV-4 → detector → fraction collection instrument (compound monomer).
4) the mobile path of eluent of step secondary splitter C22 chromatographic column sample introduction: after the secondary splitter C21 chromatographic column separation completion, repeat 1).A certain proportion of eluent is with the test specimen wash-out; Two-position three way magnetic valve BV-3 control this moment eluent separates to one-level splitter C1 chromatographic column; One section cut F2 of while two six three-way electromagnetic valve BV-6 control eluents; This cut F2 makes F2 enter into secondary splitter C22 chromatographic column and separates with elution time T2 control, waits to separate.This process is separated simultaneously with one-level splitter C1 chromatographic column and is carried out.Separate T1 after the time from one-level splitter C1 chromatographic column, cut F2 enters into secondary splitter C22 chromatographic column and waits to separate through two six three-way electromagnetic valve BV-6 controls, forms secondary splitter C22 and goes up appearance.
The moving process path of this sample introduction stepwise elution flow is: two-position three way magnetic valve BV-3 → pre-column PC → one-level splitter C1 → two six three-way electromagnetic valves BV-6 → secondary splitter C22 → two-position four-way solenoid valve BV-4 → detector → waste liquid flows out.
5) secondary splitter C22 chromatographic column is separated: after setting one-level splitter C1 chromatographic column separate fraction F2 gets on the secondary splitter C22 fully; Can control a certain proportion of eluent with the test specimen wash-out; Two-position three way magnetic valve BV-3 control this moment eluent is walked around one-level splitter C1 chromatographic column; Directly arrive and get into two six three-way electromagnetic valve BV-6 controls, eluent gets into secondary splitter C21 chromatographic column to be separated; The eluent separate fraction gets into detector, by different control methods, obtains the cut of unification compound absworption peak by detector, accomplishes secondary splitter C22 chromatographic column and separates.This separation process is the separation fully of separate fraction F1 that one-level splitter C1 is obtained, can collect all cpds monomer of separation and purification.
The mobile process path of this separation phase eluent is: two-position three way magnetic valve BV-3 → two six a three-way electromagnetic valves BV-6 → secondary splitter C22 → two-position four-way solenoid valve BV-4 → detector → fraction collection instrument (compound monomer).
Further carry out secondary splitter C23 chromatographic column and separate, control operation process and 4), 5) two step unanimities, that is:
6) the mobile path of eluent of step secondary splitter C23 chromatographic column sample introduction: after the secondary splitter C22 chromatographic column separation completion, repeat 1).A certain proportion of eluent is with the test specimen wash-out; Two-position three way magnetic valve BV-3 control this moment eluent separates to one-level splitter C1 chromatographic column; One section cut F3 of while two six three-way electromagnetic valve BV-6 control eluents; This cut F3 makes F3 enter into secondary splitter C22 chromatographic column and separates with elution time T3 control, waits to separate.This process is separated simultaneously with one-level splitter C1 chromatographic column and is carried out.Separate T3 after the time from one-level splitter C1 chromatographic column, cut F3 enters into secondary splitter C23 chromatographic column and waits to separate through two six three-way electromagnetic valve BV-6 controls, forms secondary splitter C23 and goes up appearance.
The moving process path of this sample introduction stepwise elution flow is: two-position three way magnetic valve BV-3 → pre-column PC → one-level splitter C1 → two six three-way electromagnetic valves BV-6 → secondary splitter C22 → two-position four-way solenoid valve BV-4 → detector → waste liquid flows out.
7) secondary splitter C23 chromatographic column is separated: after setting one-level splitter C1 chromatographic column separate fraction F3 gets on the secondary splitter C23 fully; Can control a certain proportion of eluent with the test specimen wash-out; Two-position three way magnetic valve BV-3 control this moment eluent is walked around one-level splitter C1 chromatographic column; Directly arrive and get into two six three-way electromagnetic valve BV-6 controls, eluent gets into secondary splitter C21 chromatographic column to be separated; The eluent separate fraction gets into detector, by different control methods, obtains the cut of unification compound absworption peak by detector, accomplishes secondary splitter C22 chromatographic column and separates.This separation process is the separation fully of separate fraction F1 that one-level splitter C1 is obtained, can collect all cpds monomer of separation and purification.
The mobile process path of this separation phase eluent is: two-position three way magnetic valve BV-3 → two six a three-way electromagnetic valves BV-6 → secondary splitter C23 → two-position four-way solenoid valve BV-4 → detector → fraction collection instrument (compound monomer).
The compound monomer structure of the test specimen of realization complex component is separated fully.The present invention also relates to secondary splitter C2n chromatographic column sample introduction, the mobile process path of eluent is: pre-column PC → two-position three way magnetic valve BV-3 → one-level splitter C1 → two six three-way electromagnetic valves BV-6 → secondary splitter C2n → two-position four-way solenoid valve BV-4 → detector → waste liquid flows out.It is the situation of two-position three way magnetic valve BV-3 and pre-column PC location swap.
If the component of initial test specimen is formed very complicated; Secondary splitter C21-C23 chromatographic column is separated and can't be separated fully in the inventive method; Just can control the time of one-level splitter C1 chromatographic column separate fraction F1-F3; Obtain more separate fraction F1-Fn, be connected in series more splitter, promptly can expand to the C2n splitter.Certainly if obtain good separating effect, should consider that one-level splitter C1 chromatographic column separates the effect of dividing cut F1-Fn, after carrying out various test specimen preliminary treatment, consider again to separate with this method, can obtain the result of getting twice the result with half the effort.
Said method of the present invention can be summarized as follows: as the separation preparative chromatography instrument (Fig. 1) of series connection dynamic axial compression preparative chromatography post; Utilize one-level chromatography column separate fraction F1-Fn continuously, one group of C21 of sample introduction secondary chromatography column, C22; C23...C2n, the separation and purification compound monomer; Form the one-level chromatography column and connect with secondary chromatography column group, and parallel form between secondary chromatography column group, independent separate purifying compounds monomer.This apparatus mainly comprises: the BV-3 two-position three way magnetic valve; Two six three-way electromagnetic valves of BV-6; The BV-4 two-position four-way solenoid valve; C1 one-level chromatography column; C21 secondary chromatography column 1; C22 secondary chromatography column 2; C23 secondary chromatography column 3; C2n secondary chromatography column n; The MI manual injector; The PC pre-column; PA, the PB infusion pump; Detector, the fraction collection appearance is formed by combining.And key technology is the BV-3 two-position three way magnetic valve; Two six three-way electromagnetic valves of BV-6; The programme-control of BV-4 two-position four-way solenoid valve is accomplished by chromatographic work station.
Above-mentioned one-level chromatography column and secondary chromatography column can have various selections in practical implementation:
The column chromatography tube material: materials such as stainless steel tube, polyfluortetraethylene pipe, titanium alloy tube are processed; Ordinary circumstance, one-level chromatography column and secondary chromatography column adopt same material.
Column chromatography separating filler: various forward silica gel, C18, RP2-18, amino silica gel, nitro silica gel, alcohol radical silica gel, derivatives resin of various sephadexes, polyethylene, polystyrene or the like.
The separation chromatography post is suitable for pressure: middle pressure, high pressure.
One-level chromatography column and secondary chromatography column sized are identical or different.
One-level chromatography column C1 is at eluting fraction F1, F2, and in the F3 process, the proportion of composing of eluent can be identical, also can be different; Secondary chromatography column C21, C22, C23 is in separation and purification compound monomer process, and the proportion of composing of eluent can be identical, also can be different; For example can adopt methyl alcohol, chloroform, acetonitrile, ethyl acetate, water or the like, ratio can be adjusted and control the composition and the ratio of eluent from 0-100% according to separate object.
The compound monomer that separation and purification obtains is mainly according to detector and fraction collection instrument, with the communications protocol of look shop work station to set control program, accomplish by chromatographic work station and to realize.
Embodiment 2:
One-level chromatography column C1 in this embodiment, secondary chromatography column C21, secondary chromatography column C22, secondary chromatography column C23 adopt the dynamic axial compression preparation splitter of stainless steel; Separating filler adopts: 1) four dynamic axial compression preparative chromatography posts are used a kind of material entirely; 2) according to the separating compound object, four dynamic axial compression preparative chromatography posts are used different materials; Need studying in great detail of separation condition in early stage.And use with I and II separation chromatography post and be complementary: the BV-3 two-position three way magnetic valve; Two six three-way electromagnetic valves of BV-6; The BV-4 two-position four-way solenoid valve; The MI manual injector; The PC pre-column; PA, the PB infusion pump; Detector, Instrument assemblies such as fraction collection appearance combine the separation preparative chromatography apparatus (PAI) that a cover series connection dynamic axial is compressed the preparative chromatography post according to accompanying schematic figure.According to above-mentioned: 1. go up appearance; 2. separation: 1)-7) detailed description of bar, the separation and purification operation for preparing compound monomer obtains target compound, realizes the separation preparation of test specimen.What adopt in this embodiment is the high pressure liquid chromatography clastotype.
Embodiment 3:
One-level chromatography column C1 in this embodiment, secondary chromatography column C21, secondary chromatography column C22, secondary chromatography column C23 adopt the dynamic axial compression preparation splitter of polytetrafluoroethylene (PTFE) system; Separating filler adopts: 1) four dynamic axial compression preparative chromatography posts are used a kind of material entirely; 2) according to the separating compound object, four dynamic axial compression preparative chromatography posts are used different materials; Need studying in great detail of separation condition in early stage.And use with I and II separation chromatography post and be complementary: the BV-3 two-position three way magnetic valve; Two six three-way electromagnetic valves of BV-6; The BV-4 two-position four-way solenoid valve; The MI manual injector; The PC pre-column; PA, the PB infusion pump; Detector, Instrument assemblies such as fraction collection appearance combine the separation preparative chromatography apparatus (PAI) that a cover series connection dynamic axial is compressed the preparative chromatography post according to accompanying schematic figure.According to above-mentioned: 1. go up appearance; 2. separation: 1)-7) detailed description of bar, the separation and purification operation for preparing compound monomer obtains target compound, realizes the separation preparation of test specimen.What adopt in this embodiment is the medium pressure liquid chromatography clastotype.
Embodiment 4:
One-level chromatography column C1 in this embodiment 1, secondary chromatography column C21, secondary chromatography column C22 adopts the dynamic axial compression of titanium alloy material system to prepare splitter; Separating filler adopts: 1) three dynamic axial compression preparative chromatography posts are used a kind of material entirely; 2) according to the separating compound object, three dynamic axial compression preparative chromatography posts are used different materials; Need studying in great detail of separation condition in early stage.And use with I and II separation chromatography post and be complementary: the BV-3 two-position three way magnetic valve; Two six three-way electromagnetic valves of BV-6; The BV-4 two-position four-way solenoid valve; The MI manual injector; The PC pre-column; PA, the PB infusion pump; Detector, Instrument assemblies such as fraction collection appearance combine the separation preparative chromatography apparatus (PAI) that a cover series connection dynamic axial is compressed the preparative chromatography post according to accompanying schematic figure.According to above-mentioned: 1. go up appearance; 2. separation: 1)-7) detailed description of bar is similar, prepares the separation and purification operation of compound monomer, obtains target compound, realizes the separation preparation of test specimen.But in this embodiment, with embodiment 1, what embodiment 2 was different is: the opposed polarity cut that one-level chromatography column C1 component obtains, the little cut of polarity be such as F1, F2, and F3 all separates with secondary chromatography column C21 successively; One-level chromatography column C1 component obtains the bigger cut of polarity such as F4, F5, and F6 all separates with secondary chromatography column C22 successively; So both having expanded the secondary chromatography column can the virtual C21-C26 of reaching, and has saved the secondary chromatography column, adopts the filler of different qualities, is easy to reach the purpose of separation and purification compound monomer.What adopt in this embodiment is the high pressure liquid chromatography clastotype.
The field that the present invention is suitable for the most is that the separation and purification with constituents prepares in chemical composition of Chinese materia medica or the natural medicine resource, particularly the bigger situation of compound monomer component requirement; Like extract at a medicinal plant; Or in the terpene extract; Always also can be mixed with all kinds such as flavones, diterpene, triterpene, steroidal, alkaloid, lignanoid, separate one-level splitter C1 chromatographic column separate fraction F1-Fn through PAI device of the present invention; At first can become dissimilar compound separation different compounds component cut, this cut section of drawing is superior to solvent and divides; Secondary splitter C21-C23 chromatographic column is separated from one-level splitter C1 chromatographic column separate fraction F1-Fn, makes structure similar compound composition cut section F1-Fn more, and the compound monomer of separation and purification homogeneous structure is precisely efficient more.And same component cut section such as triterpene set of segmentation F3; Then can utilize the physical property differences such as Acidity of Aikalinity of compound; On secondary splitter C21-C23 chromatographic column, adopt, the overall separation purifying is optimized to the filler carrier that separates the heterogeneity compound.This also is a big advantage of PAI device of the present invention.
The separation preparative chromatography instrument of series connection dynamic axial compression preparative chromatography post of the present invention; Be applicable to that the most natural drug extracts the separation and purification of compound monomer in the component; Because the complex compound system makes the filler of preparative chromatography subside easily, influence the separating effect of preparative chromatography post, and the dynamic axial that the present invention adopts compression preparative chromatography post; The post that then can dynamically improve the preparative chromatography post is at any time imitated, and guarantees separating effect.
The present invention is that the series connection of a multistage dynamic axial compression preparative chromatography post separates; To the compound system of complicacy, be applied to constitute in medicinal plant or the chemical composition of Chinese materia medica complicated miscellaneous mixed system, separating monomer compound structure especially; Highly versatile of the present invention; Simple and feasible, save the complex system lock out operation time in a large number, save the high amounts of solvents loss, have major application and be worth and dissemination.
Claims (10)
1. contain the separation preparative chromatography instrument of series connection dynamic axial compression preparative chromatography post, critical piece comprises: BV-3 two-position three way magnetic valve, two six three-way electromagnetic valves of BV-6, BV-4 two-position four-way solenoid valve; C1 one-level chromatography column, C21 secondary chromatography column 1, C22 secondary chromatography column 2, C23 secondary chromatography column 3; C2n secondary chromatography column n, MI manual injector, PC pre-column; PA, PB infusion pump, detector, fraction collection appearance; Said chromatography column is all used dynamic axial compression preparative chromatography post; Utilize one-level chromatography column C1 separate fraction F1-Fn continuously, one group of secondary chromatography column of difference sample introduction C21, C22, C23...C2n, separation and purification compound monomer; One-level chromatography column and secondary chromatography column group are passed through series system, and pass through parallel way between secondary chromatography column group, independent separate purifying compounds monomer; BV-3 two-position three way magnetic valve, two six three-way electromagnetic valves of BV-6, BV-4 two-position four-way solenoid valve are by the chromatographic work station programme-control.
2. separation preparative chromatography instrument according to claim 1, its two-position three way magnetic valve BV-3 and pre-column PC successively can exchange the position.
3. separation preparative chromatography instrument according to claim 1 adopts three splitters: one-level chromatography column C1, secondary chromatography column C21, C22; The opposed polarity cut that one-level chromatography column C1 component obtains, the little cut of polarity be such as F1, F2, and F3 all separates with secondary chromatography column C21 successively; One-level chromatography column C1 component obtains the bigger cut of polarity such as F4, F5, and F6 all separates with secondary chromatography column C22 successively.
4. separation preparative chromatography instrument according to claim 1, its one-level chromatography column and secondary chromatography column center pillar chromatotube material adopt stainless steel tube or polyfluortetraethylene pipe or titanium alloy tube; One-level chromatography column and secondary chromatography column generally adopt identical materials, and sized is identical or different; The column chromatography separating filler adopts various forward silica gel, C18, RP2-18, amino silica gel, nitro silica gel, alcohol radical silica gel, the derivatives resin of various sephadexes, polyethylene, polystyrene; The separation chromatography column pressure adopts medium-pressure or high pressure.
5. separation preparative chromatography instrument according to claim 1, its one-level chromatography column C1, secondary chromatography column C21, C22, C23 adopt radially dynamic compression preparative chromatography post in separation and purification compound monomer process.
6. separation preparative chromatography instrument according to claim 1, one-level chromatography column C1 be at eluting fraction F1, F2, and in the F3 process, the proportion of composing of eluent is identical or different; Secondary chromatography column C21; C22; C23 is in separation and purification compound monomer process, and the proportion of composing of eluent is identical or different, adopt methyl alcohol or/and chloroform or/and acetonitrile or/and ethyl acetate or/and water; Ratio is adjusted and controls the composition and the ratio of eluent from 0-100% according to separate object.
7. according to the described separation preparative chromatography of claim 1-6 instrument, the eluent process that flows is in its secondary chromatography column C21 sample introduction and the separation process: 1) one-level splitter C1 chromatographic column separate fraction F1: flow out through pre-column PC, one-level splitter C1, two six three-way electromagnetic valve BV-6, secondary splitter C21, two-position four-way solenoid valve BV-4, detector, waste liquids from two-position three way magnetic valve BV-3; Be C21 post cut F1 sample introduction process; 2) secondary splitter C21 chromatographic column is separated: collect compound monomer from two-position three way magnetic valve BV-3 through two six three-way electromagnetic valve BV-6, secondary splitter C21, two-position four-way solenoid valve BV-4, detector, fraction collection instrument.
8. according to the described separation preparative chromatography of claim 1-6 instrument, the eluent process that flows is in its secondary chromatography column C22 sample introduction and the separation process: 1) one-level splitter C1 chromatographic column separate fraction F2: flow out through pre-column PC, one-level splitter C1, two six three-way electromagnetic valve BV-6, secondary splitter C22, two-position four-way solenoid valve BV-4, detector, waste liquids from two-position three way magnetic valve BV-3; Be C22 post cut F2 sample introduction process; 2) secondary splitter C22 chromatographic column is separated: collect compound monomer from two-position three way magnetic valve BV-3 through two six three-way electromagnetic valve BV-6, secondary splitter C22, two-position four-way solenoid valve BV-4, detector, fraction collection instrument.
9. according to the described separation preparative chromatography of claim 1-6 instrument, the eluent process that flows is in its secondary chromatography column C23 sample introduction and the separation process: 1) one-level splitter C1 chromatographic column separate fraction F3: flow out through pre-column PC, one-level splitter C1, two six three-way electromagnetic valve BV-6, secondary splitter C22, two-position four-way solenoid valve BV-4, detector, waste liquids from two-position three way magnetic valve BV-3; Be C23 post cut F3 sample introduction process; 2) secondary splitter C23 chromatographic column is separated: collect compound monomer from two-position three way magnetic valve BV-3 through two six three-way electromagnetic valve BV-6, secondary splitter C23, two-position four-way solenoid valve BV-4, detector, fraction collection appearance.
10. according to the described separation preparative chromatography of claim 7-9 instrument, the mobile process of eluent is similar in its secondary chromatography column C21-C23 sample introduction and the separation process, can expand to secondary chromatography column C2n.
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Application publication date: 20120509 |