CN104614458B - A kind of circulation preparative high-performance liquid chromatographic instrument with multiple-way valve - Google Patents

A kind of circulation preparative high-performance liquid chromatographic instrument with multiple-way valve Download PDF

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CN104614458B
CN104614458B CN201510028536.9A CN201510028536A CN104614458B CN 104614458 B CN104614458 B CN 104614458B CN 201510028536 A CN201510028536 A CN 201510028536A CN 104614458 B CN104614458 B CN 104614458B
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way valve
detector
valve
chromatographic column
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CN104614458A (en
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柳仁民
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柳仁民
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Abstract

The invention discloses a kind of with the circulation preparative high-performance liquid chromatographic instrument of multiple-way valve and the method for preparative separation purification, the circulation preparative high-performance liquid chromatographic instrument with multiple-way valve is made up of reservoir M, high-pressure pump P, injector S, chromatographic column C1 and C2, multiple-way valve V, detector D1 and D2, three-way switch valve T1 and T2, fluid path pipeline etc.。The prominent effect of the present invention is: 1. blending ingredients to be separated is only circulated separation in two chromatographic columns, it is not necessary to circulate through high-pressure pump, and chromatographic peak broadening is light, and separation efficiency is high, good separating effect。2. system is provided with two detectors, it is ensured that peak cutting and multi-cycle separation accurately control, and improve separating effect。3. the eluent without separated component recycles completely, greatly reduces the consumption of eluent, and separation costs is extremely low。

Description

A kind of circulation preparative high-performance liquid chromatographic instrument with multiple-way valve
Technical field
The present invention relates to and a kind of circulate preparative high-performance liquid chromatographic instrument and the method for preparative separation purification, belong to instrument and equipment manufacturing technology field, be a kind of high performance liquid chromatography separation purification devices。
Background technology
Common preparative high-performance liquid chromatographic instrument is made up of reservoir, high-pressure pump, injector, chromatographic column, detector, monitor 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 easily separated, separated component exits into detector from chromatographic column successively, collects respective components according to detector signal after post。At present, high performance liquid chromatography has evolved in wide variety of important isolation technics in field such as chemistry, life sciences pharmacy。When carrying out sample and separating, increase column length and be obtained in that 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。It addition, when carrying out sample and separating, it is necessary to using substantial amounts of eluent, this also makes separation costs increase。
Application publication number CN103157295A(application number 2013100876904) Chinese patent literature disclose a kind of cycling chromatographic separation system, including the first eluting pump, the second eluting pump with enter sample pump, make separated component by twin columns repeatedly circulation realize separating。But its eluent does not recycle, it is necessary to use substantial amounts of eluent。It addition, need the first eluting pump, the second eluting pump in its operating process and enter sample pump and multiple six position three-way valves, logical six valves combination use, operating procedure is up to 14 steps, extremely complex。
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, switched by the switch of valve, blending ingredients to be separated is made to be not passed through high-pressure pump and only separate at two chromatographic column internal recycle, the separated part opened is collected by the switch switching cutting of valve, and the eluent without separated component returns in reservoir and recycles。
To achieve these goals, the technical solution used in the present invention is:
A kind of circulation preparative high-performance liquid chromatographic instrument with multiple-way valve and for the method for preparative separation purification, the circulation preparative high-performance liquid chromatographic instrument with multiple-way valve is made up of reservoir M, high-pressure pump P, injector S, chromatographic column C1 and C2, multiple-way valve V, detector D1 and D2, three-way switch valve T1 and T2, fluid path pipeline etc.。
Annexation between each parts is: No. 1 passage of multiple-way valve V is connected with M → P → S → pipeline, and No. 2 of multiple-way valve V and No. 5 passages are connected with the two ends of chromatographic column C1 respectively, and No. 7 of multiple-way valve V and No. 10 passages are connected with the two ends of chromatographic column C2 respectively。One end of flow-through detector D1 pipeline connects No. 6 passages of multiple-way valve V, and the other end is connected with three-way valve T1, and the other two ends of three-way valve T1 are connected with No. 9 passages of multiple-way valve V respectively and fraction collector F connects;One end of flow-through detector D2 pipeline connects No. 4 passages of multiple-way valve V, and the other end is connected with three-way valve T2, and the other two ends of three-way valve T2 are connected with reservoir M and waste liquid bottle W respectively。No. 3 passages and No. 8 passage straight pipes of multiple-way valve V connect。
Described multiple-way valve V channel number is not limited only to above-mentioned fixing passage count, also includes other connection situations utilizing multiple-way 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 constant flow pump or the constant pressure pump etc. that are provided that steady pressure and flow velocity。
Described injector S can be injection valve or the sampling pump etc. of fixed volume。
Described chromatographic column C1 and C2 can be glass, rustless steel, PEEK etc.。The filler loaded can be normal-phase chromatography filler, reversed phase chromatography filler, gel filler etc., and the amount of filler can be analytical type, semi-preparative, preparative etc.。
Described multiple-way valve V can be two multiple-way valves that a port number (includes 10) more than 10, utilizes two port numbers, two multiple-way valves below 10 can also realize the purpose of the present invention。
Described detector D1 and D2 can be the flow-through detector etc. such as UV-detector, fluorescence detector, differential refraction detector。The detection cell of detector 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., it is also possible to replaces 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 multiple-way valve V and is easily separated, the effluent of chromatographic column C1 enters detector D1 detection through multiple-way valve V, the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut, component lap enters chromatographic column C2 again through multiple-way valve V and carries out next step separation, entrance reservoir M recycling after detector D2 detects is entered then through multiple-way valve V from what chromatographic column C2 flowed out。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 all overlapping mixing portions enter chromatographic column C2, by rotary multi-way valve V, changing 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 effluent of chromatographic column C2 detects through detector 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 chromatographic column C1 and carries out next step multi-cycle separation, enters reservoir and recycle after the chromatographic column C1 device D2 after testing of the eluent without separated component flowed out detection。
Concrete separation 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 easily separated, the effluent of chromatographic column 1 detects through detector 1, its elution curve reaches the component of purity requirement by collecting the cut extraction of switching valve, component lap enters chromatographic column 2 and carries out next step separation, enters reservoir and recycle after the device 2 after testing of the eluent without the separated component detection that chromatographic column 2 flows out。After all 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 effluent of chromatographic column 2 detects through detector 1, its elution curve reaches the component of purity requirement by collecting the cut extraction of switching valve, component lap enters chromatographic column 1 and carries out next step multi-cycle separation, enters reservoir and recycle after the device 2 after testing of the eluent without the separated component detection that chromatographic column 1 flows out。Such iterative cycles is easily separated, and just can be separated by blending ingredients when not increasing column length。One twin columns of the present invention circulation preparative high-performance liquid chromatographic instrument separating power is strong, can complete the separation task that common chromatograph has been difficult to, simultaneously because eluent recycles, separation costs is low。
A kind of circulation preparative high-performance liquid chromatographic instrument with multiple-way valve of the present invention and for the method for preparative separation purification, instrument design is simple, has superpower separating power, and eluent recycles, the 1/10 of common liquid chromatograph that the eluent consumed is not enough, practicality is extremely strong。
In addition, the prominent effect of the present invention is:
1. blending ingredients to be separated is only circulated separation in two chromatographic columns, it is not necessary to circulate through high-pressure pump, and chromatographic peak broadening is light, and separation efficiency is high, good separating effect。
2. system is provided with two detectors, it is ensured that peak cutting and multi-cycle separation accurately control, and improve separating effect。
3. the eluent without separated component recycles completely, greatly reduces the consumption of eluent, and separation costs is extremely low。
4. the component reaching purity requirement can be extracted out by system by collecting switching valve cutting, overcomes peak stretching overlapping phenomenon, and sample size is big, and separation efficiency is high, and cost is low。
5. system design is simple, good separating effect, it is adaptable to the separation purification of all kinds of complex mixtures, 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 detector 1 exports signal schematic representation。
Fig. 3 is that embodiment 2 detector 1 exports signal schematic representation。
Fig. 4 is that embodiment 3 detector 1 exports signal schematic representation。
Fig. 5 is that embodiment 4 detector 1 exports signal schematic representation。
Detailed description of the invention
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 is as shown in Figure 1, a kind of circulation preparative high-performance liquid chromatographic instrument with multiple-way valve and for the method for preparative separation 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, multiple-way valve V, detector 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 multiple-way valve V and is easily separated, the effluent of chromatographic column C1 enters detector D1 detection through multiple-way valve V, the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut, component lap enters chromatographic column C2 again through multiple-way valve V and carries out next step separation, recycles from what chromatographic column C2 flowed out then through entrance reservoir M after multiple-way valve V entrance detector D2 detection。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 all overlapping mixing portions enter chromatographic column C2, by rotary multi-way valve V, changing 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 effluent of chromatographic column C2 detects through detector 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 chromatographic column C1 and carries out next step multi-cycle separation, enters reservoir and recycle after the chromatographic column C1 device D2 after testing of the eluent without separated component flowed out detection。
Such iterative cycles is easily separated, and just can be separated by blending ingredients when not increasing column length。One twin columns of the present invention circulation preparative high-performance liquid chromatographic instrument separating power is strong, 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, for the polar component that the character of difficulty separation is similar, can use one twin columns of the present invention circulation preparative high-performance liquid chromatographic instrument, efficiently separate。By above-mentioned connection and mode of operation, chromatographic column C1 and C2 diameter 25mm, the stainless steel column of long 200mm, post is loaded 10mm purification on normal-phase silica gel, it is eluent with the isobutyltrimethylmethane. that volume ratio is 85:15-ethyl acetate mixed solvent, the similar betatocopherol of structure and Gamma-Tocopherol can be effectively separated, Fig. 2 is detector 1 record output signal in separation 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 times circulate, it may be achieved be kept completely separate。Component owing to will reach purity requirement in elution curve is cut extraction by collecting switching valve, eliminates the impact of peak stretching in separation process。In separation 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:
Reverse-phase chromatography is suitable for separating nonpolar or middle polarity compound etc., for the chemical composition of the similar nonpolar or middle polarity of character that difficulty separates, can use one twin columns of the present invention circulation preparative high-performance liquid chromatographic instrument, efficiently separate。By above-mentioned connection and mode of operation, chromatographic column C1 and C2 diameter 25mm, the stainless steel column of long 200mm, post is loaded the anti-phase C18 silica filler of 10mm, being eluent with the Methanol+Water that volume ratio is 80:20, Fig. 3 is detector 1 record output signal and component cutting schematic diagram in separation 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 kept completely separate 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 easily separated, separation process is not cut, to be separated completely after regather corresponding peak, circulating 4 times can be effectively separated the similar daidzein of structure and genistein。Fig. 4 is detector 1 record output signal schematic representation in separation 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 the separation of macromole: use one twin columns of the present invention circulation 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, post is loaded Butyl-SSepharose gel filler, with ammonium sulfate buffer solution as eluent, lysozyme and chymotrypsinogen are easily separated, separation process is not cut, circulate and can realize being kept completely separate of lysozyme and chymotrypsinogen for 2 times, corresponding peak component is collected after being kept completely separate。Fig. 5 is detector 1 record output signal schematic representation in separation 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 easily separated, consumed mobile phase has contrasted with carrying out the same conventional chromatography separated, and the inventive method sampling volume is 2ml, and conventional method sampling volume is 0.5ml。Relevant result is shown in following table, from data in table, the inventive method sample size is big, consume the 1/10 of mobile phase deficiency conventional chromatography, this illustrates that separation efficiency of the present invention is high, separate purification cost low, practical, it is particularly suited for the separation of the difficult separate substance such as chipal compounds, structure analogue compounds, character analogue compounds。

Claims (8)

1. the circulation preparative high-performance liquid chromatographic instrument with multiple-way valve, it is characterized in that, main by reservoir M, high-pressure pump P, injector S, chromatographic column C1 and C2, multiple-way valve V, detector D1 and D2, three-way switch valve T1 and T2, fluid path pipeline is constituted, No. 1 passage of multiple-way valve V is connected with reservoir M → high-pressure pump P → injector S → pipeline, No. 2 of multiple-way valve V and No. 5 passages are connected with the two ends of chromatographic column C1 respectively, No. 7 of multiple-way valve V and No. 10 passages are connected with the two ends of chromatographic column C2 respectively, one end of detector D1 pipeline connects No. 6 passages of multiple-way 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 passages of multiple-way valve V respectively and fraction collector F connects;One end of detector D2 pipeline connects No. 4 passages of multiple-way 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 and No. 8 passage straight pipes of multiple-way valve V connect;Described reservoir M is the corrosion resistant glass container of organic solvent-resistant or organic material container;Described high-pressure pump P is the constant flow pump or the constant pressure pump that are provided that steady pressure and flow velocity;Described injector S is injection valve or the sampling pump of fixed volume;Described chromatographic column C1 and C2 is glass, rustless steel, PEEK;Described detector D1 and D2 is flow-through detector;The detection cell of detector D1 is high pressure resistant detection cell;Described three-way switch valve T1 and T2 is manual three-way valve, electric T-shaped valve, Pneumatic three-way valve。
2. circulation preparative high-performance liquid chromatographic instrument according to claim 1, is characterized in that, described multiple-way valve V channel number is not limited only to above-mentioned fixing passage count。
3. circulation preparative high-performance liquid chromatographic instrument according to claim 2, is characterized in that, described multiple-way valve V be a port number more than 10 or include 10 two multiple-way valves。
4. circulation preparative high-performance liquid chromatographic instrument according to claim 2, is characterized in that, described multiple-way valve V is two port numbers, two multiple-way valves below 10。
5. circulation preparative high-performance liquid chromatographic instrument according to claim 1, is characterized in that, the filler that described chromatographic column C1 and C2 loads is normal-phase chromatography filler, reversed phase chromatography filler, gel filler, and the amount of filler is analytical type, semi-preparative, preparative。
6. circulation preparative high-performance liquid chromatographic instrument according to claim 1, is characterized in that, described flow-through detector is UV-detector, fluorescence detector, differential refraction detector。
7. circulation preparative high-performance liquid chromatographic instrument according to claim 1, is characterized in that, described three-way switch valve T1 and T2 stop valve replaces。
8. the separation method of the circulation preparative high-performance liquid chromatographic instrument with multiple-way valve 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 multiple-way valve V and is easily separated, the effluent of chromatographic column C1 enters detector D1 detection through multiple-way valve V, the component having reached purity requirement in its elution curve is extracted out by three-way switch valve T1 is cut, component lap enters chromatographic column C2 again through multiple-way valve V and carries out next step separation, entrance reservoir M recycling after detector D2 detects is entered then through multiple-way valve V from what chromatographic column C2 flowed out;Now, eluent glide path is: reservoir M → high-pressure pump P → injector S → multiple-way valve V 1. 2. → chromatographic column C1 → multiple-way valve V 5. 6. → detector D1 → three-way switch valve T1 → multiple-way valve V 9. 10. → chromatographic column C2 → multiple-way valve V 7. 8. → multiple-way valve V 3. 4. → detector D2 → three-way switch valve T2 → reservoir M;
After all overlapping mixing portion enters chromatographic column C2, by rotary multi-way valve V, change eluent fluid path, eluent glide path is: reservoir M → high-pressure pump P → injector S → multiple-way valve V 1. 10. → chromatographic column C2 → multiple-way valve V 7. 6. → detector D1 → three-way switch valve T1 → multiple-way valve V 9. 8. → multiple-way valve V 3. 2. → chromatographic column C1 → multiple-way valve V 5. 4. → detector D2 → three-way switch valve T2 → reservoir M;Eluent is introduced into chromatographic column C2, the effluent of chromatographic column C2 detects through detector 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 chromatographic column C1 and carries out next step multi-cycle separation, enters reservoir and recycle after the chromatographic column C1 device D2 after testing of the eluent without separated component flowed out detection。
CN201510028536.9A 2015-01-21 2015-01-21 A kind of circulation preparative high-performance liquid chromatographic instrument with multiple-way valve Expired - Fee Related CN104614458B (en)

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CN105664527B (en) * 2016-01-27 2018-01-30 李广良 A kind of continuous chromatography separator and its method
CN108088937A (en) * 2017-12-15 2018-05-29 徐洪平 A kind of online flash purification system
WO2019186469A1 (en) * 2018-03-29 2019-10-03 Waters Technologies Corporation Automated two-column recycling chromatography method for unlocking challenging separation problems
WO2019229697A1 (en) * 2018-05-31 2019-12-05 Waters Technologies Corporation High resolution and semi-preparative recycling chromatography system for isolation and preparation of unknown trace impurities in pharmaceutical products

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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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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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