CN104713973B - There is two-dimentional preparative hplc instrument system and the application thereof of on-line preconcentration function - Google Patents
There is two-dimentional preparative hplc instrument system and the application thereof of on-line preconcentration function Download PDFInfo
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Abstract
The present invention relates to two-dimentional preparative hplc instrument system and the application thereof with on-line preconcentration function.Native system is made up of chromatograph flow channel switching valve, chromatographic column, solvent pump and detector.By Vavle switching and flowing phase dilution, trapping column is collected the first dimension chromatograph and is flowed out component, then through Vavle switching and flowing phase eluting, trapping component is imported the second dimension and prepares post, it is achieved Two way chromatograms separates.This instrument system can carry out two dimensional separation, and trapping column interface is effectively retained and enriched sample and one-dimensional separation component, and post effect loss is little, it is ensured that two dimensional separation effect.The expansible multi-cycle separation that carries out repeatedly of this instrument system separates with multidimensional, and separating effect will not increase with sample volume and separate number of times and increase and decline.Additionally, this instrument system is also equipped with the concentration and recovery function to separated final products, thus simplify sample drying process, be conducive to the degeneration avoiding sample in heat drying process.
Description
Technical field
The present invention relates to a set of two-dimentional preparative hplc instrument system with on-line preconcentration function.
Background technology
High activity chemical composition in natural drug is one of important sources of new drug, along with deepening continuously, greatly of research
Amount high-load composition is obtained by separation, and researcher is increasingly turned to the separation of microchemistry composition in natural drug, seeks
Look for new component therein.Compared with high-load composition, the micro constitutent in natural drug in separation process more susceptible to other
Composition interference effect, separating monitoring difficulty, conventional separation techniques uses raw material dosage big, and separation efficiency is low, it is therefore desirable to efficiently
Separating tool.
Preparative hplc has powerful separating power, is the main tool of natural drug separation.Natural product composition complexity,
Single chromatographic isolation effect is limited, needs through separating for several times, uses different chromatographic isolation pattern just can reach in separation process
To being satisfied with separating effect.Except considering the complementarity of different chromatogram mode, preparative hplc it is also contemplated that preparation in separation process
Scale issue, needs overload loading to ensure to prepare flux.The sample that first dimension preparative hplc was separated by online Two way chromatograms
Enter the second dimension preparative hplc after collecting directly or through special excuse to separate, separation process can be simplified, improve work
Efficiency.Shao Ping (Chinese patent, CN101791491A) devises the two-dimentional preparative hplc system that a set of online basic, normal, high pressure combines
Two preparing chromatography systems are coupled together by system by six-way valve and software, and during the first dimension preparative hplc separates, sample transships
Sample runs, and intercepts target flow point entrance the second dimension preparative hplc and separates further, is used for solving overload sample introduction and separation energy
Contradiction between power, improves chromatogram separating capacity.Zhang Weibing devises a kind of Quasi dynamic two dimension preparing chromatography system and material separates
Two preparative hplc posts are connected by three-way valve, pass through by method (Chinese patent, CN102914610A, CN202870047U)
The component that first dimension preparative hplc post separates is transferred to the second dimension preparative hplc post and is separated by Vavle switching, is equally used for solving
Contradiction between overload sample introduction and separating power.System above only has simple flow point forwarding function, big containing target component
Volume flow point is directly entered the second dimension preparing chromatography system can have a strong impact on its separating power, and system above does not have enrichment simultaneously
Function, needs to improve the first dimension preparative hplc applied sample amount and ensures to prepare flux, and micro constitutent is in heavy overload ruuning situation
Under, it is difficult to separate and monitor, affect separating effect.
Summary of the invention
The present invention relates to a set of two-dimentional preparative hplc instrument system with on-line preconcentration function.Native system is by chromatograph stream
Switching valve (two ten-way valves, two six-way valves or banked direction control valves), chromatographic column (prepares post, guard column and trapping column), solvent pump (bag
Include flowing phase pump and regulate pump mutually with flowing) and detector composition.It is characterized in that, by Vavle switching, flowing regulates mutually, and catches
The mode that clustered column three combines, it is achieved that cyclical chromatography interface and the interface function of Two way chromatograms, thus it is many to complete two peacekeepings
Dimension chromatographic isolation, the separating effect of simultaneity factor will not increase with loading volume and the carrying out and decline of separation process.Additionally, this
System is also equipped with the concentration and recovery function to separated final products, thus simplifies sample drying process, and is conducive to avoiding
Sample degeneration in heat drying process.
There is the two-dimentional preparing chromatography system of on-line preconcentration function, including a flowing phase pump, three three-way cut-off valves, one
Individual two ten-way valves, quantitative loop, guard column, trapping column, regulation pump, two two six-way valves, one
Threeway, chromatographic detector, the first dimension prepares post a, the second dimension prepares post b and other connect pipelines composition；
Wherein two six-way valve b, the first dimension prepares post a and the second dimension is prepared post b composition post and selected system；
Above-mentioned each parts order of connection is as described below: two ten-way valve interfaces the most successively with three-way cut-off valve a, prepare pump with
Prepare pump flowing to be connected, the 3rd the interface emptying outlet of three-way cut-off valve a；Two ten-way valve interfaces 2. with interface 3. phase
Connection；1. two ten-way valve interfaces 4. interface with two six-way valve I is connected；5. two ten-way valve interfaces sequentially pass through by three
2. logical stop valve b and two six-way valve II interfaces are connected, and the 3rd interface of three-way cut-off valve b is emptying outlet；Two ten lead to
6. valve interface is connected with guard column stigma interface；7. with 10. by quantitative loop two ten-way valve interfaces are connected；Two ten-way valves connect
8. mouth is sample injection port, and 9. interface is vented;
1. two six-way valve II interfaces are connected with the post tail interface of guard column；2. two six-way valve II interfaces are cut by threeway
Only valve b is connected with ten-way valve；3. the interface of two six-way valve II is connected with the post tail interface of trapping column；Two six-way valve II connect
4. mouth is connected with the entrance of chromatographic detector；4. the interface of two six-way valve II 5. interface with two six-way valve I is connected；Two
6. the interface of six-way valve II is connected with the stigma interface of trapping column by threeway, the other end of threeway successively with three-way cut-off valve c,
Regulation pump is connected with regulation solution, and the 3rd interface of three-way cut-off valve is emptying outlet;
4. 1. two six-way valve PCI interface be connected with two ten-way valve interfaces；3. 2. two six-way valve PCI interface distinguished with interface
The import and export preparing post b with the second dimension are connected；5. two six-way valve PCI interface 4. interface with two six-way valve II is connected；Two
The import and export that 4. six-way valve PCI interface prepares post a with the first dimension the most respectively with interface are connected.
Two-dimensional liquid chromatography sample transfer interface is a trapping column with reservation enrichment function, and regulation is by trapping column
Flowing phase eluting power makes target component adsorbing and trapping or eluting transfer.
Single trapping column can be by many trapping column system replacement.
Connecting two posts preparing post in system selects system can be selected system replacement by multicolumn.
By using many trapping column system and multicolumn to select system, multiple target components can be enriched with, and enter respectively
Prepared by the chromatographic isolation of row multidimensional.
The specifically used step of native system is as follows:
1) loading
After sample solution injects sample amounts ring, the first dimension preparative hplc flowing is used to be imported after guard column by sample
Trapping column, simultaneously to trapping column input regulation liquid, reduces flowing phase eluotropic strength, makes sample be retained in trapping column to be separated；
2) one-dimensional separation
Close flowing and regulates pump mutually, application the first dimension preparative hplc flowing phase, sample elution will be retained in trapping column extremely
First dimension is prepared post and is separated；
3) target component trapping
It is transferred to trapping column, simultaneously to trapping column by preparing the target component after post separates through the first dimension by Vavle switching
Input regulation liquid, reduces regulation strength of mobile phase, makes target peak be enriched in trapping column；
4) two dimensional separation
By Vavle switching, the second dimension is prepared post and is connected in flow path system, use the second dimension preparative hplc flowing mutually will
It is enriched in sample elution in trapping column and prepares post to the second dimension, it is achieved separate further;
5) sample reclaims
Prepare, through the second dimension, the subject component that post separates, i.e. can be collected according to separating situation shown in detector,
Also can be again trapped in trapping column by subject component, regulation pump input eluting power is strong, and the flowing phase of high volatility, makes mesh
Mark composition is dissolved in small size flowing mutually, it is simple to recovery sample.
Described 1) loading and 3) during target component trapping, regulation liquid is molten with the weak eluotropic strength dissolved each other mutually of flowing
Agent；In sample removal process, regulation liquid selects the solvent that eluotropic strength is big and volatility is high.
Described regulation liquid is beneficially to strengthen the solvent that sample retains in trapping column, including methanol, acetonitrile, acetone, second
Alcohol or the solvent that other eluotropic strengths are big and volatility is high.
Repeat step (2)～(3), can reach the effect separated with dimension chromatographic cycles；(1)～(3) process is repeated several times, can
Repeatedly carry out Two way chromatograms separation again after enrichment target component.
The present invention is used to have the advantage that compared with traditional method
1) this instrument system can carry out two dimensional separation, in the first dimension preparative hplc separation process, repeatedly goes up by carrying out
Sample and trapping, target component is enriched in trapping column, can improve the applied sample amount of two dimension preparation, remove the non-mesh trapped simultaneously
Mark composition, reduces the impurity interference to target component in the second dimension chromatographic separation process.Trapping column interface is effectively retained richness
Collection sample and one-dimensional separation component, be prevented effectively from sample volume overload and solvent effect problem, and post effect loss is little, it is ensured that two dimension point
From effect.
2) the expansible multi-cycle separation that carries out repeatedly of this instrument system separates with multidimensional, and separating effect will not increase with sample volume
Big and separation number of times increases and declines.
3) this instrument system is also equipped with the concentration and recovery function to separated final products, thus simplifies sample drying mistake
Journey, is conducive to the degeneration avoiding sample in heat drying process.
Accompanying drawing explanation
Fig. 1 is two dimension preparing chromatography system structure chart；
Fig. 2 is many trapping column system construction drawing；
Fig. 3 is that multicolumn selects system construction drawing；
Fig. 4 is that Two way chromatograms system runs schematic diagram.
Wherein, 1-flowing phase pump；2-three-way cut-off valve a；Two ten-way valves of 3-；4-three-way cut-off valve b；5-post selects system
System；6-quantitative loop；7-guard column；8-trapping column；9-regulates pump；Two six-way valve I of 10-；11-three-way cut-off valve c；12-threeway；
13-six-way valve II；14-chromatographic detector；15-many trapping column system；16-multicolumn selects system；A-the first dimension prepares post；B-
Two dimension prepares post.
Fig. 1 is the two-dimentional preparing chromatography system being made up of two ten-way valves and two six-way valves；Fig. 2 is replaced in Fig. 1
10, Fig. 3 replace in Fig. 1 12, and may make up can the preparing chromatography system that separates of multidimensional.
Detailed description of the invention
Being described in further details the present invention in conjunction with embodiment and accompanying drawing, embodiment is only limitted to illustrate the present invention, and
Non-limitation of the invention.
Embodiment 1 single loading is for the two dimension preparation of 40% content composition
This experiment plan prepares 40% content composition in natural plant extracts.First dimension preparative hplc post and trapping column
Being 10um Chromatorex C18 filler preparative hplc post, the second dimension preparative hplc post is that 5um Microsorb C18 fills out
Material preparative hplc post, the first dimension preparative hplc flowing is 60% methanol mutually, and the second dimension preparative hplc flowing is 45% acetonitrile mutually.Fixed
Sample solution in amount ring, enters trapping column after ten-way valve and guard column, is simultaneously entered water and reduces flowing phase eluting power,
Target component is made to be retained in trapping column.Use the first dimension preparative hplc flowing phase eluting trapping column, sample is brought into the first dimension
Preparative hplc post separates.By the flow point containing target component by Vavle switching, it is transferred in trapping column, is simultaneously entered water liquid
Reduce flowing phase eluting power, make target component be retained in trapping column.Change flowing phase, by Vavle switching, by the second dimension system
Standby chromatogram flow phase eluting trapping column, separates the target component sample elution of enrichment to second dimension preparative hplc post.Again
In the way of flowing regulates mutually, target component is enriched in trapping column, by regulation pump, product after purification is collected in corpusculum
Long-pending methanol, after testing, product purity is up to 98%.
More than 2 loading of embodiment is for the two dimension preparation of 0.2% content composition
This experiment plan prepares 0.2% content composition in natural plant extracts.First dimension preparative hplc post is 10um
Chromatorex C18 filler preparative hplc post, trapping column is 10um Calesil C18 filler chromatographic column, and the second dimension prepares color
Spectrum post is 5um Microsorb C18 filler preparative hplc post, and the first dimension preparative hplc flowing is 80% methanol mutually, the second dimension system
Standby chromatogram flow phase is 75% methanol.Sample solution in quantitative loop, enters trapping column after ten-way valve and guard column, with
Time input water reduce flowing phase eluting power, make target component be retained in trapping column.Use the first dimension preparative hplc flowing
Phase eluting trapping column, separates sample elution to first dimension preparative hplc post.Flow point containing target component is passed through valve
Switching, is transferred in trapping column, is simultaneously entered regulation liquid and reduces flowing phase eluting power, makes target component be retained in trapping column
In.Repeat loading, one-dimensional separation and the process of on-line preconcentration, be enriched in accumulative for target component in trapping column.Change flowing phase,
The second dimension preparative hplc flowing is made to flow through trapping column mutually by Vavle switching, by the target component sample elution of enrichment to the second dimension system
Standby chromatographic column separates.In the way of flowing regulates mutually, target component is enriched in trapping column again, by regulation pump by pure
Product after change is collected in small size methanol.After testing, product purity is up to 98%.
Claims (8)
1. there is the two-dimentional preparing chromatography system of on-line preconcentration function, it is characterised in that: include one flowing phase pump (1), three
Three-way cut-off valve (2,4,11), one two ten-way valves (3), quantitative loop (6), guard column (7), trapping column
(8), regulation pump (9), two two six-way valves (10,13), threeway (12), chromatographic detector (14), first dimension systems
Standby post a, the second dimension prepare post b and other connect pipeline composition；
Wherein two six-way valve I (10), the first dimension prepares post a and the second dimension is prepared post b composition post and selected system (5)；
Above-mentioned each parts order of connection is as described below: two ten-way valve (3) interfaces the most successively with three-way cut-off valve a (2), prepare pump
(1) with prepare pump flowing and be connected, the 3rd interface emptying outlet of three-way cut-off valve a (2)；Two ten-way valve (3) interfaces are 2.
3. it is connected with interface；1. two ten-way valve (3) interfaces 4. interface with two six-way valve I (10) is connected；Two ten-way valves (3)
5. interface is sequentially passed through and is 2. connected with two six-way valve II (13) interfaces by three-way cut-off valve b (4), three-way cut-off valve b's (4)
3rd interface is emptying outlet；6. two ten-way valve (3) interfaces are connected with guard column (7) stigma interface；Two ten-way valves (3)
7. with 10. by quantitative loop (6) interface is connected；8. two ten-way valve (3) interfaces are sample injection port, and 9. interface is vented；
1. two six-way valve II (13) interfaces are connected with the post tail interface of guard column (7)；2. two six-way valve II (13) interfaces lead to
Cross three-way cut-off valve b (4) to be connected with ten-way valve (3)；3. the interface of two six-way valve II is connected with the post tail interface of trapping column；Two
The interface of position six-way valve II (13) 4. entrance with chromatographic detector (14) is connected；The interface of two six-way valve II (13) is 5. with two
The interface of position six-way valve I (10) is 4. connected；The interface of two six-way valve II (13) is 6. by the post of threeway (12) with trapping column (8)
Head interface is connected, and successively with three-way cut-off valve c (11), regulation pump (9) and regulation liquid phase even, threeway cuts the other end of threeway (12)
Only the 3rd interface of valve c (11) is emptying outlet；
4. 1. two six-way valve I (10) interfaces be connected with two ten-way valve (3) interfaces；2. two six-way valve I (10) interfaces and connect
Mouth is prepared the import and export of post b the most respectively and is connected with the second dimension；Two six-way valve I (10) interfaces 4. with two six-way valve II (13)
5. interface is connected；The import and export that 5. two six-way valve I (10) interfaces prepare post a with the first dimension the most respectively with interface are connected.
The two-dimentional preparing chromatography system with on-line preconcentration function the most according to claim 1, it is characterised in that: Two-dimensional Liquid
Phase chromatographic sample transfer interface is a trapping column (8) with reservation enrichment function, regulates the flowing phase eluting by trapping column
Ability makes target component adsorbing and trapping or eluting transfer.
The two-dimentional preparing chromatography system with on-line preconcentration function the most according to claim 1, it is characterised in that: single catch
Clustered column (8) is replaced by many trapping column system (15).
The two-dimentional preparing chromatography system with on-line preconcentration function the most according to claim 1, it is characterised in that: in system
Connecting two posts preparing post selects system (5) to be selected system (16) to replace by multicolumn.
5., according to the two-dimentional preparing chromatography system with on-line preconcentration function described in any one of claim 1 or 3 or 4, it is special
Levy and be: by using the many trapping column system described in claim 3 and the multicolumn described in claim 4 to select system,
Multiple target components are enriched with, and are prepared by the chromatographic isolation carrying out multidimensional respectively.
6. having an application for the two-dimentional preparing chromatography system of on-line preconcentration function as claimed in claim 1, its feature exists
In: the specifically used step of native system is as follows:
1) loading
After sample solution injects sample amounts ring (6), the first dimension preparative hplc flowing is used to be led after guard column (7) by sample
Enter trapping column (8), simultaneously to trapping column (8) input regulation liquid, reduce flowing phase eluotropic strength, make sample be retained in trapping column
To be separated；
2) one-dimensional separation
Close flowing and regulate pump (9), application the first dimension preparative hplc flowing phase mutually, sample elution in trapping column (8) will be retained in
Prepare post (a) to the first dimension to separate；
3) target component trapping
It is transferred to trapping column (8), simultaneously to trapping by preparing the target component after post (a) separates through the first dimension by Vavle switching
Post (8) input regulation liquid, reduces strength of mobile phase, makes target peak be enriched in trapping column；
4) two dimensional separation
By Vavle switching, the second dimension is prepared post (b) and is connected in flow path system, use the second dimension preparative hplc to flow richness
Combine in sample elution in trapping column and prepare post (b) to the second dimension, it is achieved separate further；
5) sample reclaims
Prepare, through the second dimension, the subject component that post (b) separates, be collected according to separating situation shown in detector；Or by target
Composition is trapped in trapping column (8) again, and regulation pump input eluting power is strong, and the regulation liquid of high volatility, makes target component
It is dissolved in small size flowing mutually, it is simple to recovery sample.
Application the most according to claim 6, it is characterised in that:
Described 1) loading and 3) during target component trapping, regulation liquid is and the weak eluotropic strength solvent that dissolves each other mutually of flowing；
In sample removal process, regulation liquid selects the solvent that eluotropic strength is big and volatility is high.
Application the most according to claim 6, it is characterised in that:
Repeat step (2)～(3) in claim 6, reach with tieing up the effect that chromatographic cycles separates；Or right is repeated several times want
Seek (1)～(3) process in 6, repeatedly carry out Two way chromatograms separation again after enrichment target component.
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