CN104614458A - Recycling preparative HPLC (high performance liquid chromatograph) provided with multi-ported valve - Google Patents
Recycling preparative HPLC (high performance liquid chromatograph) provided with multi-ported valve Download PDFInfo
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Abstract
The invention discloses a recycling preparative HPLC (high performance liquid chromatograph) provided with a multi-ported valve as well as a method used for preparation, separation and purification. The recycling preparative HPLC provided with the multi-ported valve comprises a liquid accumulator M, a high-pressure pump P, a sampler S, chromatographic columns C1 and C2, the multi-ported valve, detectors D1 and D2, three-way switching valves T1 and T2, liquid pipelines and the like. The recycling preparative HPLC has the outstanding effects as follows: 1, to-be-separated mixed components are circularly separated only in the two chromatographic columns and are not required for circulation through the high-pressure pump, chromatographic peak broadening is light, the separation efficiency is high, and the separation effect is good; 2, a system is provided with two detectors, peak cutting and circular separation can be accurately controlled, and the separation effect is improved; 3, eluent without the separated components is completely recycled, the consumption of eluent is greatly reduced, and the separation cost is very low.
Description
Technical field
The present invention relates to a kind of preparative high-performance liquid chromatographic instrument and method for the preparation of separation and purification of circulating, belong to instrument and equipment manufacturing technology field, is a kind of high performance liquid chromatography separation and purification device.
Background technology
Common preparative high-performance liquid chromatographic instrument is made up of reservoir, high-pressure pump, injector, chromatographic column, detecting device, registering instrument etc., eluent in reservoir is carried by high-pressure pump, eluent in reservoir is conveyed through injector through high-pressure pump, sample in carrier band injector enters chromatographic column and is separated, separated component flows out from chromatographic column successively and enters detecting device, collects respective components according to detector signal after post.At present, high performance liquid chromatography has developed into the important isolation technics in field widespread uses such as chemistry, life science pharmacy.When carrying out sample separation, increase column length and can obtain better separating effect, but the increase of column length can increase SR, increase back-pressure, thus, the increase of column length can be subject to certain restrictions.In addition, when carrying out sample separation, need to use a large amount of eluents, this also makes separation costs increase.
Application publication number CN103157295A(application number 2013100876904) Chinese patent literature disclose a kind of cycling chromatographic separation system, comprise the first wash-out pump, the second wash-out pump and enter sample pump, making separated component by repeatedly circulation realization separation in twin columns.But its eluent does not recycle, need to use a large amount of eluents.In addition, need the first wash-out pump, the second wash-out pump in its operating process and enter sample pump and multiple six position three-way valves, logical six valves combinationally use, operation steps reaches 14 steps, very complicated.
Summary of the invention
For solving the defect existing for common preparative high-performance liquid chromatographic instrument, the invention provides a kind of twin columns circulation preparative high-performance liquid chromatographic instrument, by the switching over of valve, blending ingredients to be separated is made not flow through high-pressure pump and only be separated at two chromatographic column Inner eycle, the part separated is collected by the cutting of the switching over of valve, does not get back in reservoir recycling containing the eluent of separated component.
To achieve these goals, the technical solution used in the present invention is:
With the circulation preparative high-performance liquid chromatographic instrument of multiport valve and the method for the preparation of separation and purification, the circulation preparative high-performance liquid chromatographic instrument of band multiport valve is made up of reservoir M, high-pressure pump P, injector S, chromatographic column C1 and C2, multiport valve V, detecting device D1 and D2, three-way switch valve T1 and T2, fluid path pipeline etc.
Annexation between each parts is: No. 1 passage of multiport valve V is connected with M → P → S → pipeline, and No. 2 of multiport valve V are connected with the two ends of chromatographic column C1 respectively with No. 5 passages, and No. 7 of multiport valve V are connected with the two ends of chromatographic column C2 respectively with No. 10 passages.One end of flow-through detector D1 pipeline connects No. 6 passages of multiport valve V, and the other end is connected with T-valve T1, and the other two ends of T-valve T1 are connected with No. 9 expanding channels of multiport valve V and fraction collector F respectively; One end of flow-through detector D2 pipeline connects No. 4 passages of multiport valve V, and the other end is connected with T-valve T2, and the other two ends of T-valve T2 are connected with reservoir M and waste liquid bottle W respectively.No. 3 passages of multiport valve V are connected with No. 8 passage straight pipes.
Described multiport valve V channel number is not limited only to above-mentioned fixing channel number number, also comprises other connection situations utilizing multiport valve to realize situation of the present invention.
Described reservoir M can be the corrosion resistant glass container of organic solvent-resistant or organic material container etc.
Described high-pressure pump P can be the constant flow pump or constant pressure pump etc. that can provide steady pressure and flow velocity.
Described injector S can be the sampling valve or sampling pump etc. of fixed volume.
Described chromatographic column C1 and C2 can be glass, stainless steel, PEEK etc.The filler loaded can be normal-phase chromatography filler, reverse-phase chromatography filler, gel filler etc., and the amount of filler can be analytic type, semi-preparative, preparative etc.
Described multiport valve V can be two multiport valves that a port number (comprises 10) more than 10, utilizes two multiport valves of two port numbers below 10 also can realize object of the present invention.
Described detecting device D1 and D2 can be the flow-through detector etc. such as UV-detector, fluorescence detector, differential refraction detector.The detection cell of detecting device D1 is high pressure resistant detection cell.
Described three-way switch valve T1 and T2 can be manual three-way valve, electric T-shaped valve, Pneumatic three-way valve etc., also can replace with stop valve.
The invention allows for the isolation and purification method of described high performance liquid chromatograph: the eluent in reservoir M is conveyed through injector S through high-pressure pump P, sample in carrier band injector S enters chromatographic column C1 through multiport valve V and is separated, the efflux of chromatographic column C1 enters detecting device D1 through multiport valve V and detects, the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut, component lap enters by multiport valve V the separation that chromatographic column C2 carries out next step again, enter reservoir M recycle from entering after detecting device D2 detects through multiport valve V again of flowing out of chromatographic column C2.Now, eluent glide path is: M → P → S → V 1. 2. → C1 → V 5. 6. → D1 → T1 → V 9. 10. → C2 → V 7. 8. → V 3. 4. → D2 → T2 → M.
After whole overlapping mixing portion enters chromatographic column C2, by rotary multi-way valve V, change eluent fluid path, eluent glide path is: M → P → S → V 1. 10. → C2 → V 7. 6. → D1 → T1 → V 9. 8. → V 3. 2. → C1 → V 5. 4. → D2 → T2 → M.Eluent is introduced into chromatographic column C2, the efflux of chromatographic column C2 detects through detecting device D1, the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut, component lap enters the multi-cycle separation that chromatographic column C1 carries out next step, from chromatographic column C1 flow out not containing separated component eluent after testing device D2 enter reservoir and recycle after detecting.
Concrete detachment process is: the eluent in reservoir is conveyed through injector through high-pressure pump, sample in carrier band injector enters chromatographic column 1 and is separated, the efflux of chromatographic column 1 detects through detecting device 1, the component of purity requirement has been reached by collecting the cut extraction of transfer valve in its elution curve, component lap enters the separation that chromatographic column 2 carries out next step, from chromatographic column 2 flow out not containing separated component eluent after testing device 2 enter reservoir and recycle after detecting.After whole overlapping mixing portion enters chromatographic column 2, by the closedown of valve in flow channel switching valve group and unlatching and switching, change eluent fluid path, eluent is made to be introduced into chromatographic column 2, the efflux of chromatographic column 2 detects through detecting device 1, the component of purity requirement has been reached by collecting the cut extraction of transfer valve in its elution curve, component lap enters the multi-cycle separation that chromatographic column 1 carries out next step, from chromatographic column 1 flow out not containing separated component eluent after testing device 2 enter reservoir and recycle after detecting.Iterative cycles like this is separated, and just blending ingredients can be separated under the condition not increasing column length.A kind of twin columns circulation of the present invention preparative high-performance liquid chromatographic instrument separating power is strong, and can complete the separation task that common chromatograph has been difficult to, simultaneously because eluent recycles, separation costs is low.
The present invention is a kind of with the circulation preparative high-performance liquid chromatographic instrument of multiport valve and the method for the preparation of separation and purification, Instrument Design is simple, and have superpower separating power, eluent recycles, 1/10 of common liquid chromatography that the eluent consumed is not enough, practicality is extremely strong.
In addition, outstanding effect of the present invention is:
1. blending ingredients to be separated only carries out multi-cycle separation in two chromatographic columns, and do not need through high-pressure pump circulation, chromatographic peak broadening is light, and separation efficiency is high, good separating effect.
2. system is provided with two detecting devices, can ensure that peak cutting and multi-cycle separation accurately control, improve separating effect.
3. do not recycle completely containing the eluent of separated component, greatly reduce the consumption of eluent, separation costs is extremely low.
4. system can be extracted out by collecting transfer valve cutting the component reaching purity requirement, and overcome peak stretching overlapping phenomenon, sample size is large, and separation efficiency is high, and cost is low.
5. system is simple, and good separating effect, be applicable to the separation and purification of all kinds of complex mixture, practicality is extremely strong.
Accompanying drawing explanation
Fig. 1 is the structural representation of embodiment of the present invention circulation preparative high-performance liquid chromatographic instrument.
Fig. 2 is that embodiment 1 detecting device 1 outputs signal schematic diagram.
Fig. 3 is that embodiment 2 detecting device 1 outputs signal schematic diagram.
Fig. 4 is that embodiment 3 detecting device 1 outputs signal schematic diagram.
Fig. 5 is that embodiment 4 detecting device 1 outputs signal schematic diagram.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in detail, but protection domain is not by this restriction.
Embodiment 1 as shown in Figure 1, with the circulation preparative high-performance liquid chromatographic instrument of multiport valve and the method for the preparation of separation and purification, circulation preparative high-performance liquid chromatographic instrument is made up of reservoir M, high-pressure pump P, injector S, chromatographic column C1 and C2, multiport valve V, detecting device D1 and D2, three-way switch valve T1 and T2, fluid path pipeline etc.
Eluent in reservoir M is conveyed through injector S through high-pressure pump P, sample in carrier band injector S enters chromatographic column C1 through multiport valve V and is separated, the efflux of chromatographic column C1 enters detecting device D1 through multiport valve V and detects, the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut, component lap enters by multiport valve V the separation that chromatographic column C2 carries out next step again, enters reservoir M recycle from entering after detecting device D2 detects through multiport valve V again of flowing out of chromatographic column C2.Now, eluent glide path is: M → P → S → V 1. 2. → C1 → V 5. 6. → D1 → T1 → V 9. 10. → C2 → V 7. 8. → V 3. 4. → D2 → T2 → M.
After whole overlapping mixing portion enters chromatographic column C2, by rotary multi-way valve V, change eluent fluid path, eluent glide path is: M → P → S → V 1. 10. → C2 → V 7. 6. → D1 → T1 → V 9. 8. → V 3. 2. → C1 → V 5. 4. → D2 → T2 → M.Eluent is introduced into chromatographic column C2, the efflux of chromatographic column C2 detects through detecting device D1, the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut, component lap enters the multi-cycle separation that chromatographic column C1 carries out next step, from chromatographic column C1 flow out not containing separated component eluent after testing device D2 enter reservoir and recycle after detecting.
Iterative cycles like this is separated, and just blending ingredients can be separated under the condition not increasing column length.A kind of twin columns circulation of the present invention preparative high-performance liquid chromatographic instrument separating power is strong, and can complete the separation task that common chromatograph has been difficult to, simultaneously because eluent recycles, separation costs is low.
By above-mentioned connection and mode of operation, carry out normal-phase chromatography separation:
Normal phase chromatography is suitable for the separation of polar component, the polar component that the character for difficulty separation is similar, can use a kind of twin columns circulation of the present invention preparative high-performance liquid chromatographic instrument, effectively be separated.By above-mentioned connection and mode of operation, chromatographic column C1 and C2 diameter 25mm, the stainless steel column of long 200mm, 10mm purification on normal-phase silica gel is loaded in post, be eluent with isooctane-ethyl acetate mixed solvent that volume ratio is 85:15, can effectively be separated with Gamma-Tocopherol by the betatocopherol similar to structure, Fig. 2 is detecting device 1 recording of output signals in detachment process, leading peak is betatocopherol, postpeak is Gamma-Tocopherol, and the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut.After 4 circulations, can realize being separated completely.Due to the component of purity requirement will have been reached in elution curve by collecting the cut extraction of transfer valve, eliminate the impact of peak stretching in detachment process.In detachment process only cutting of components time consume eluent, other times eluent all recycles.
Embodiment 2 presses Instrument Design, connection and mode of operation described in embodiment 1, carries out reversed phase chromatography separation:
Reversed phase chromatography is suitable for being separated compound etc. that is nonpolar or middle polarity, the chemical composition of the nonpolar or middle polarity that the character for difficulty separation is similar, can use a kind of twin columns circulation of the present invention preparative high-performance liquid chromatographic instrument, effectively be separated.By above-mentioned connection and mode of operation, chromatographic column C1 and C2 diameter 25mm, the stainless steel column of long 200mm, the anti-phase C18 silica filler of 10mm is loaded in post, be eluent with the Methanol+Water that volume ratio is 80:20, Fig. 3 is detecting device 1 recording of output signals and component cutting schematic diagram in detachment process, and leading peak is daidzein, postpeak genistein, the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut.Can realize being separated completely through 4 circulations.
Embodiment 3 presses Instrument Design, connection and mode of operation described in embodiment 1, the chromatographic column in embodiment 2 and chromatographic condition is adopted to be separated, do not cut in detachment process, to be separated completely after regather corresponding peak, circulate and can effectively be separated with genistein by the daidzein similar to structure for 4 times.Fig. 4 is detecting device 1 recording of output signals schematic diagram in detachment process.Owing to only consuming eluent when collecting peak component, other times eluent all recycles, and the eluent consumed is considerably less.
Embodiment 4 presses Instrument Design described in embodiment 1, connect and mode of operation, carry out macromolecular separation: use a kind of twin columns circulation of the present invention preparative high-performance liquid chromatographic instrument, connect and mode of operation by described in embodiment 1, chromatographic column C1 and C2 diameter 25mm, long 300mm glass column, Butyl-S Sepharose gel filler is loaded in post, with ammonium sulfate buffer solution as eluent, lysozyme is separated with chymotrypsinogen, do not cut in detachment process, circulate and can realize being separated completely of lysozyme and chymotrypsinogen for 2 times, corresponding peak component is collected after being separated completely.Fig. 5 is detecting device 1 recording of output signals schematic diagram in detachment process.Owing to only consuming eluent when collecting peak component, other times eluent all recycles, and the eluent consumed is considerably less.
When embodiment of the present invention 1-4 is separated consume mobile phase and carry out the same conventional chromatography be separated and contrast, the inventive method sampling volume is 2ml, and conventional method sampling volume is 0.5ml.Related results sees the following form, from data in table, the inventive method sample size is large, consume 1/10 of the not enough conventional chromatography of mobile phase, this illustrates that separation efficiency of the present invention is high, separation and purification cost is low, practical, is particularly useful for the separation of the difficult separate substances such as chipal compounds, structure analogue compounds, character analogue compounds.
Claims (9)
1. the circulation preparative high-performance liquid chromatographic instrument with multiport valve, it is characterized in that, primarily of reservoir M, high-pressure pump P, injector S, chromatographic column C1 and C2, multiport valve V, detecting device D1 and D2, three-way switch valve T1 and T2, fluid path pipeline is formed, No. 1 passage of multiport valve V is connected with M → P → S → pipeline, No. 2 of multiport valve V are connected with the two ends of chromatographic column C1 respectively with No. 5 passages, No. 7 of multiport valve V are connected with the two ends of chromatographic column C2 respectively with No. 10 passages, one end of detecting device D1 pipeline connects No. 6 passages of multiport valve V, the other end is connected with three-way switch valve T1, the other two ends of three-way switch valve T1 are connected with No. 9 expanding channels of multiport valve V and fraction collector F respectively, one end of detecting device D2 pipeline connects No. 4 passages of multiport valve V, and the other end is connected with three-way switch valve T2, and the other two ends of three-way switch valve T2 are connected with reservoir M and waste liquid bottle W respectively, and No. 3 passages of multiport valve V are connected with No. 8 passage straight pipes.
2. circulation preparative high-performance liquid chromatographic instrument according to claim 1, it is characterized in that, described multiport valve V channel number be not limited only to above-mentioned fixing channel number number (can be two multiport valves that a port number (comprises 10) more than 10, utilize two multiport valves of two port numbers below 10 also can).
3. circulation preparative high-performance liquid chromatographic instrument according to claim 1, is characterized in that, described reservoir M can be the corrosion resistant glass container of organic solvent-resistant or organic material container etc.
4. circulation preparative high-performance liquid chromatographic instrument according to claim 1, is characterized in that, described high-pressure pump P can be the constant flow pump or constant pressure pump etc. that can provide steady pressure and flow velocity.
5. circulation preparative high-performance liquid chromatographic instrument according to claim 1, is characterized in that, described injector S can be the sampling valve or sampling pump etc. of fixed volume.
6. circulation preparative high-performance liquid chromatographic instrument according to claim 1, it is characterized in that, described chromatographic column C1 and C2 can be glass, stainless steel, PEEK etc. (filler of filling can be normal-phase chromatography filler, reverse-phase chromatography filler, gel filler etc., and the amount of filler can be analytic type, semi-preparative, preparative etc.).
7. circulation preparative high-performance liquid chromatographic instrument according to claim 1, is characterized in that, described detecting device D1 and D2 can be the flow-through detector etc. such as UV-detector, fluorescence detector, differential refraction detector; The detection cell of detecting device D1 is high pressure resistant detection cell.
8. circulation preparative high-performance liquid chromatographic instrument according to claim 1, is characterized in that, described three-way switch valve T1 and T2 can be manual three-way valve, electric T-shaped valve, Pneumatic three-way valve etc., also can replace with stop valve.
9. the separation method of circulation preparative high-performance liquid chromatographic instrument according to claim 1, it is characterized in that, eluent in reservoir M is conveyed through injector S through high-pressure pump P, sample in carrier band injector S enters chromatographic column C1 through multiport valve V and is separated, the efflux of chromatographic column C1 enters detecting device D1 through multiport valve V and detects, the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut, component lap enters by multiport valve V the separation that chromatographic column C2 carries out next step again, enter reservoir M recycle from entering after detecting device D2 detects through multiport valve V again of flowing out of chromatographic column C2, now, eluent glide path is: M → P → S → V 1. 2. → C1 → V 5. 6. → D1 → T1 → V 9. 10. → C2 → V 7. 8. → V 3. 4. → D2 → T2 → M,
After whole overlapping mixing portion enters chromatographic column C2, by rotary multi-way valve V, change eluent fluid path, eluent glide path is: M → P → S → V 1. 10. → C2 → V 7. 6. → D1 → T1 → V 9. 8. → V 3. 2. → C1 → V 5. 4. → D2 → T2 → M; Eluent is introduced into chromatographic column C2, the efflux of chromatographic column C2 detects through detecting device D1, the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut, component lap enters the multi-cycle separation that chromatographic column C1 carries out next step, from chromatographic column C1 flow out not containing separated component eluent after testing device D2 enter reservoir and recycle after detecting.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105664527A (en) * | 2016-01-27 | 2016-06-15 | 李广良 | Continuous chromatographic separation device and method using device |
| CN107158747A (en) * | 2017-05-19 | 2017-09-15 | 南京凯通粮食生化研究设计有限公司 | A kind of decolouring deionization and separating mixture device and operation method |
| CN108088937A (en) * | 2017-12-15 | 2018-05-29 | 徐洪平 | A kind of online flash purification system |
| CN111902196A (en) * | 2018-03-29 | 2020-11-06 | 沃特世科技公司 | Automated dual column cycle chromatography method for addressing challenging separation problems |
| CN112203737A (en) * | 2018-05-31 | 2021-01-08 | 沃特世科技公司 | High resolution and semi-preparative recycle chromatography system for separating and preparing unknown trace impurities in pharmaceutical products |
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| CN103157295A (en) * | 2013-03-19 | 2013-06-19 | 利穗科技(苏州)有限公司 | Double-column recycling chromatographic system |
| CN204439600U (en) * | 2015-01-21 | 2015-07-01 | 柳仁民 | A kind of circulation preparative high-performance liquid chromatographic instrument with multiport valve |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105664527A (en) * | 2016-01-27 | 2016-06-15 | 李广良 | Continuous chromatographic separation device and method using device |
| CN107158747A (en) * | 2017-05-19 | 2017-09-15 | 南京凯通粮食生化研究设计有限公司 | A kind of decolouring deionization and separating mixture device and operation method |
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| CN108088937A (en) * | 2017-12-15 | 2018-05-29 | 徐洪平 | A kind of online flash purification system |
| CN111902196A (en) * | 2018-03-29 | 2020-11-06 | 沃特世科技公司 | Automated dual column cycle chromatography method for addressing challenging separation problems |
| CN112203737A (en) * | 2018-05-31 | 2021-01-08 | 沃特世科技公司 | High resolution and semi-preparative recycle chromatography system for separating and preparing unknown trace impurities in pharmaceutical products |
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| CN104614458B (en) | 2016-06-22 |
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