CN101619090A - Column-switching circulating volume exclusion chromatography system for biomacromolecule separation and purification - Google Patents
Column-switching circulating volume exclusion chromatography system for biomacromolecule separation and purification Download PDFInfo
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Abstract
The invention relates to a column-switching circulating volume exclusion chromatography system for biomacromolecule separation and purification, which comprises a liquid chromatography pump, a sampling valve, a three-way or four-way connector, a switching valve A, a switching valve B, a volume exclusion column A, a volume exclusion column B and a detector. The pump, the sampling valve, the volume exclusion column A, the switching valve A, the volume exclusion column B, the detector, the switching valve B and the three-way or four-way connector are connected with one another in turn through pipelines, the number of the biomacromolecules getting into a circulation and separation system can be controlled by adjusting the switching frequency or mode of the valve A, and the separation and the collection of the biomacromolecule components can be controlled by adjusting the switching of the valve B. Compared with the conventional volume exclusion chromatography, the system has the characteristics of strong separating power, high resolution and the like, and can be applied to the separation and the purification of biomacromolecule samples. Simultaneously, the system also has good utility value in constructing a continuous biomacromolecule multi-dimensional separation platform.
Description
Technical field
The present invention relates to the separation and purification of protein system, specifically a kind of post switches circulation Size Exclusion Chromatograph SEC separation system.
Background technology
In the separation of biomacromolecule, ion-exchange chromatography and Size Exclusion Chromatograph SEC are two kinds of the most frequently used isolation technique.And Size Exclusion Chromatograph SEC is preserved the activity of biomolecules easily owing to the separation condition gentleness, therefore often is used to the isolation and purification of biomolecules.Compare with other clastotype, Size Exclusion Chromatograph SEC is considered to the low liquid-phase chromatography method of a kind of resolving power, has therefore limited its application in biomacromolecule separates.In order to improve resolving power, can adopt longer chromatographic column usually or use the more chromatographic column of the filler filling of fine grain size, yet post presses too high this method that also makes to be difficult to be committed to practice.The circulation Size Exclusion Chromatograph SEC is a kind of liquid-phase chromatography method that improves resolving power, and sample can carry out multiple separation many times in a root chromatogram column, and therefore can be described as unrestrictedly increases column length, thereby can obtain very high resolving power.Yet along with the growth of disengaging time, spectrum peak broadening thereupon, the chromatographic peak of separating (the wide albumen of molecular weight ranges, nucleic acid etc.) may also can be overlapping after repeatedly circulation separates.
Summary of the invention
In order to overcome this defective, the applicant has set up post switches the circulation volume exclusion chromatography system, this method is to be captured on second Size Exclusion Chromatograph SEC post presorting the biomacromolecule that leaves, switch by control valve, separate in the biomacromolecule incision recycle system that molecular weight is similar, after obtaining satisfied resolution, the component of separating is collected.Yet, cut out the sub-fraction separation that circulates from trapping column again, and the like.This method is suitable for the separation and purification of actual complex sample, has higher use value.
The object of the present invention is to provide a kind of is the biomacromolecule tripping device of clastotype with the Size Exclusion Chromatograph SEC, utilize this device can the separation and purification biomacromolecule, obtain more highly purified biomacromolecule, also make up continuous biomacromolecule multidimensional analysis platform simultaneously.This installs simplicity of design, the resolving power height, and good separating effect, practical.
To achieve these goals, the technical solution used in the present invention is:
A kind of post that is used for separation and purification of biological macromolecule switches the circulation volume exclusion chromatography system, it is characterized in that: comprise liquid chromatography pump, switching valve, Size Exclusion Chromatograph SEC post A, Size Exclusion Chromatograph SEC post B, detector, they link to each other by the form of pipeline with closed circuit;
Size Exclusion Chromatograph SEC post A one end links to each other with sampling valve, the other end links to each other with Size Exclusion Chromatograph SEC post B, detector respectively by a switching valve A, and the outer end of sampling valve is connected with the mobile phase source and the detector outlet in the external world respectively through Y-junction by pump,
The connecting pipeline of Y-junction and detector is provided with switching valve B, and detector links to each other with receiving flask or waste liquid bottle by switching valve B.
Described switching valve A is six-way valve or ten-way valve, and it links to each other with detector with Size Exclusion Chromatograph SEC post A, Size Exclusion Chromatograph SEC post B respectively.
Described sampling valve is made up of quantitative ring and six-way valve or ten-way valve, and sampling valve is connected in series with in the connecting pipeline of Size Exclusion Chromatograph SEC post A and pump by six-way valve or ten-way valve; Described detector is UV-detector, mass spectrum or other types detector.
Pump, sampling valve, volume-exclusion post A, switching valve A, Size Exclusion Chromatograph SEC post B, detector, switching valve B link to each other by pipeline successively with threeway or four-pass connecting joint, by controlling the quantity that switching valve A can control the biomacromolecule that enters the circulation separation system, the switching of control valve B can be controlled the separation and the collection of biomacromolecule component.
The concrete analysis process is as follows: before circulation separates, by valve A two Size Exclusion Chromatograph SEC posts are cascaded.The biomacromolecule sample at first separates by Size Exclusion Chromatograph SEC post A, be captured in then among the Size Exclusion Chromatograph SEC post B, when the sub-fraction biomacromolecule is eluted from Size Exclusion Chromatograph SEC post B, switching valve A and valve B, pump, Size Exclusion Chromatograph SEC post A, detector form in the closed circuit by a threeway or four-way jointing at this moment, this moment, the biomacromolecule sample can constantly separate until reaching satisfied separating resulting in this system, and other sample then is retained in second root chromatogram column.After the first part biomacromolecule was finished separation, the biomacromolecule component was cut out systematic collection and is got off.Another small portion biomacromolecule cuts out from second root chromatogram column, the separation that circulates, and the like.
Biomacromolecule is after Size Exclusion Chromatograph SEC post A, Size Exclusion Chromatograph SEC post B separate successively, by the control switching valve, the sample of similar molecular weight size is successively from the Size Exclusion Chromatograph SEC post B incision recycle system, and be the separator column separation that circulates with Size Exclusion Chromatograph SEC post A, until reaching satisfied resolution.
With Size Exclusion Chromatograph SEC post A is separator column, and the biomacromolecule sample separates in the isolating mode that circulates.The column volume of Size Exclusion Chromatograph SEC post A is less than Size Exclusion Chromatograph SEC post B.Biomacromolecule enters the recycle system in the mode of peak cutting from Size Exclusion Chromatograph SEC post B.Size Exclusion Chromatograph SEC post B has the dual function of separating and capturing.
Biomacromolecule of the present invention can be protein or nucleic acid; The present invention can be used for the isolation and purification of biomacromolecule, can be used as the vitals that makes up continuous biomacromolecule multidimensional separation platform simultaneously.This system compares with traditional Size Exclusion Chromatograph SEC method that to have separating power strong, and characteristics such as resolving power height can be used for the separation and purification of biomacromolecule sample.Simultaneously, this system also has good practical value in making up continuous biomacromolecule multidimensional separation platform.
The invention has the beneficial effects as follows:
1. this system adopts the isolating mode of circulation, and not needing increases column length or reduce particle diameter, just can greatly improve resolving power, improves separating effect.
2. owing to adopt the Size Exclusion Chromatograph SEC clastotype, be eluent with low salt buffer solution, therefore, it is active that albumen still can keep after separation and purification.
3. this system adopts two Size Exclusion Chromatograph SEC posts, and wherein Size Exclusion Chromatograph SEC post A is a separator column, and Size Exclusion Chromatograph SEC post B is the sample trapping column.Protein sample is captured among the Size Exclusion Chromatograph SEC post B after separating by Size Exclusion Chromatograph SEC post A, enters the recycle system by the albumen that cuts out small portion from Size Exclusion Chromatograph SEC post B, can realize the proteinic separation and purification very wide to molecular weight ranges.
4. when making up multiple-biological macromole separation platform, adopt this system can guarantee the isolating continuity of bidimensional as isolating first dimension of biomacromolecule.
5. this system design is simple, the resolving power height, and good separating effect, practical, have excellent popularization and be worth.
Description of drawings
Fig. 1, post switch circulation Size Exclusion Chromatograph SEC setting drawing, (1), Y-junction; (2), pump; (3), sampling valve; (4), Size Exclusion Chromatograph SEC post A; (5), switching valve A; (6), Size Exclusion Chromatograph SEC post B; (7), detector, (8), switching valve B, (9), collector or waste liquid bottle, (10), moving phase C, (11), sample introduction needle or sampler, (12), waste liquid bottle A.
Fig. 2, series connection Size Exclusion Chromatograph SEC (a) and post switch the circulation Size Exclusion Chromatograph SEC, and (b, c d) separate five kinds of standard proteins.Separation condition: chromatographic column: SEC SRT-150
With SEC nanofilm-250
Moving phase C:10mM Tris-HCl+150mM NaCl+5%ACN, flow: 150 μ L/min; Detect: 215nm.
Fig. 3, post switch Size Exclusion Chromatograph SEC-reversed phase chromatography separation stage apparatus figure, (1), Y-junction; (2), pump; (3), sampling valve; (4), Size Exclusion Chromatograph SEC post A; (5), switching valve A; (6), Size Exclusion Chromatograph SEC post B; (7), detector A; (8), switching valve B; (9), switching valve C; (10), moving phase C; (11), sample introduction needle or sampler; (12); Waste liquid bottle A; (13), C8 trapping column A; (14) C8 trapping column B; (15) reverse-phase chromatographic column; (16), detector B, (17), pump B, (18), moving phase D, (19), waste liquid bottle B, (20), waste liquid bottle C, (21), pump C, (22), pump D.
The two dimensional separation figure of Fig. 4, seven kinds of standard proteins.(a) figure is the first dimension post cutting circulation Size Exclusion Chromatograph SEC separation graph.(b) figure is the second dimension reversed phase chromatography separation figure.The first dimension separation condition: chromatographic column: SEC SRT-150
With SEC nanofilm-250
, moving phase C:10mM Tris-HCl+150mMNaCl+5%ACN, moving phase D:50mM Tris-HCl+2%ACN, flow: 150 μ L/min; Detect: the 215nm. second dimension separation condition: moving phase: A:2%ACN+0.1%TFA, B:98%ACN+0.1%TFA; Gradient: 0-1min, 0%B, 1-3min, 0%-20%B, 3-14min, 20%-80%B, 14-15min, 80%B, 15-15.01min, 80%-0%B, 15.01-20min, 0%B; Flow: 2mL/min; Detect: 225nm
Embodiment
As shown in Figure 1, a kind of post that is used for separation and purification of biological macromolecule switches the circulation volume exclusion chromatography system, this installs by Y-junction 1, pump 2, sampling valve 3, Size Exclusion Chromatograph SEC post A4, switching valve A5, Size Exclusion Chromatograph SEC post B6, detector 7, switching valve B8 forms, and they link to each other by the form of pipeline with closed circuit.
Size Exclusion Chromatograph SEC post A4 one end links to each other with sampling valve 3, the other end links to each other with Size Exclusion Chromatograph SEC post B6, detector 7 respectively by a switching valve A5; The outer end opening for feed of sampling valve 3 is connected with the mobile phase source and detector 7 outlets in the external world respectively through Y-junction 1 by pump 2; Y-junction 1 is provided with switching valve B8 with the connecting pipeline of detector 7, and detector 7 links to each other with receiving flask or waste liquid bottle by switching valve B8.
Described switching valve A5 is a ten-way valve, with the interface that is connected with Size Exclusion Chromatograph SEC post A4 for 1., be followed successively by 2. 3. 4. 5. 6. 7. 8. 9. 10. by clockwise all the other interfaces, 1. interface links to each other with Size Exclusion Chromatograph SEC post A4,2. with 5. interface links to each other with the two ends of Size Exclusion Chromatograph SEC post B6 respectively, 6. interface links to each other with detector 7,7. and 10. interface is connected.
Described switching valve B8 is a ten-way valve, with the interface that is connected with detector 7 for 1., be followed successively by 2. 3. 4. 5. 6. 7. 8. 9. 10. by clockwise all the other interfaces, 1. interface links to each other with detector 7,2. interface links to each other with receiving flask or waste liquid bottle, 10. interface links to each other with an interface of Y-junction 1;
Described sampling valve 3 is made up of quantitative ring and ten-way valve, with the interface that is connected with Size Exclusion Chromatograph SEC post A4 for 1., be followed successively by 2. 3. 4. 5. 6. 7. 8. 9. 10. by clockwise all the other interfaces, 1. interface links to each other with the end of Size Exclusion Chromatograph SEC post A4,2. with 9. interface links to each other with the two ends of quantitative ring respectively, 10. interface links to each other with pump 2,3. interface is injection port, the 8. external waste liquid bottle of interface; Sampling valve is connected in series with in the connecting pipeline of Size Exclusion Chromatograph SEC post A4 and pump 2 by ten-way valve.
The protein sample that will be flow in the quantitative ring by pump 2 transport flow phase C10 pours among the Size Exclusion Chromatograph SEC post A4, and this moment, switching valve A5 switched, and Size Exclusion Chromatograph SEC post A4 is linked to each other with Size Exclusion Chromatograph SEC post B6.Sample obtains separating by series connection Size Exclusion Chromatograph SEC post.When sample was eluted to the post tail of Size Exclusion Chromatograph SEC post B6, the clipping time of control switching valve A8 was in sub-fraction sample incision circulating system.This moment, Size Exclusion Chromatograph SEC post A4 linked to each other with UV-detector 7, and switching valve B8 is in the position that UV-detector 7 is linked to each other with Y-junction 1.By the sample cut, separate according to the complexity Size Exclusion Chromatograph SEC that in circulating system, repeatedly circulates.When reaching the optimal separation degree, switching valve B8 switches, and separated sample obtains collecting.Fig. 2 (a) is that series connection separates five kinds of standard protein mixtures to Size Exclusion Chromatograph SEC post A4 with Size Exclusion Chromatograph SEC post B6; Fig. 2 (b), (c), (d) for using post switching circulation volume exclusion chromatography system five kinds of standard proteins to be carried out the color atlas that obtains after the separation detection.
As shown in Figure 3, this installs by Y-junction 1, pump 2, sampling valve 3, Size Exclusion Chromatograph SEC post A4, switching valve A5, Size Exclusion Chromatograph SEC post B6, UV-detector 7, valve B8, pump B17, pump C21, pump D22, switching valve C9, C8 trapping column A13, C8 trapping column B14, reverse-phase chromatographic column 15, UV-detector B16 forms.
Difference from Example 1
Described switching valve B8 is a ten-way valve, with the interface that is connected with detector 7 for 1., be followed successively by 2. 3. 4. 5. 6. 7. 8. 9. 10. by clockwise all the other interfaces, 1. interface links to each other with detector 7,10. interface links to each other with an interface of Y- junction 1,3. interface links to each other with extraneous moving phase D18 by pump B17, and 2. interface links to each other with a switching valve C9;
Switching valve C9 is a ten-way valve, with the interface that is connected with switching valve B8 for 1., be followed successively by 2. 3. 4. 5. 6. 7. 8. 9. 10. by clockwise all the other interfaces, 2. with 5. interface links to each other with the two ends of C8 trapping column A13, and 3. with 8. interface is connected, and 4. interface is connected by pipeline with pump D22 with parallel pumps C21, the 6. external waste liquid bottle of interface, 7. with 10. interface links to each other with the two ends of C8 trapping column B14, and interface is 9. outer to be connected with reverse-phase chromatographic column 15 and UV-detector B16 in turn, and UV-detector B16 is circumscribed with waste liquid bottle.
The protein sample that will be flow in the quantitative ring by pump 2 transport flow phase C pours among the Size Exclusion Chromatograph SEC post A4, and this moment, switching valve A5 switched, and Size Exclusion Chromatograph SEC post A4 is linked to each other with Size Exclusion Chromatograph SEC post B6.Sample obtains separating by series connection Size Exclusion Chromatograph SEC post.When sample was eluted to the post tail of Size Exclusion Chromatograph SEC post B6, the clipping time of control switching valve A8 was in sub-fraction sample incision circulating system.This moment, Size Exclusion Chromatograph SEC post A4 linked to each other with UV-detector 7, the switching time of control switching valve B8 a part of albumen (cut 1) was entered in the second dimension reversed phase chromatography separation system, and another part enters the Size Exclusion Chromatograph SEC that circulates in the circulating system to be separated.Cut enters among the C8 trapping column A13 by pump B17 transport flow phase D, carries Flow Injection Chemiluminescence Method to carry out anti-phase wash-out mutually by pump C21, pump D22.Sample in the recycle system is through after a while separation, part protein sample (cut 2) is also cut out the recycle system and is entered among the C8 trapping column B14 by pump B transport flow phase D, after cut 1 is finished reversed phase chromatography separation, switching valve C12 switches, and pump C21, pump D22 conveying Flow Injection Chemiluminescence Method is sent into the protein sample among the C8 trapping column B14 (cut 2) and carried out reversed phase chromatography separation in the reverse-phase chromatographic column 15.And the like.Fig. 4 switches the chromatographic fractionation figure of circulation Size Exclusion Chromatograph SEC-reversed phase chromatography separation platform to seven kinds of standard proteins for post, wherein Fig. 4 (a) is the separate colors spectrogram that the first dimension post switches the circulation Size Exclusion Chromatograph SEC, Fraction1, Fraction2 ... .Fraction13 is the albumen cut of cutting.Fig. 4 (b) is the second dimension reversed phase chromatography separation figure.
Claims (5)
1, a kind of post that is used for separation and purification of biological macromolecule switches the circulation volume exclusion chromatography system, it is characterized in that: comprise liquid chromatography pump, switching valve, Size Exclusion Chromatograph SEC post A, Size Exclusion Chromatograph SEC post B, detector, they link to each other by the form of pipeline with closed circuit;
Size Exclusion Chromatograph SEC post A (4) one ends link to each other with sampling valve (3), the other end links to each other with Size Exclusion Chromatograph SEC post B (6), detector (7) respectively by a switching valve A (5),
The outer end of sampling valve (3) is connected with the mobile phase source and detector (7) outlet in the external world respectively through Y-junction (1) or four-way connection by pump (2),
The connecting pipeline of Y-junction (1) or four-way connection and detector (7) is provided with switching valve B (8), and detector (7) links to each other with receiving flask or waste liquid bottle by switching valve B (8).
2, according to the described system of claim 1, it is characterized in that: described switching valve A (5) is six-way valve or ten-way valve, and it links to each other with detector (7) with Size Exclusion Chromatograph SEC post A (4), Size Exclusion Chromatograph SEC post B (6) respectively.
3, according to the described system of claim 1, it is characterized in that: described sampling valve (3) is made up of quantitative ring and six-way valve or ten-way valve, and sampling valve is connected in series with in the connecting pipeline of Size Exclusion Chromatograph SEC post A (4) and pump (2) by six-way valve or ten-way valve.
4, according to the described system of claim 1, it is characterized in that: described detector (7) is UV-detector, differential refraction detector, mass detector or other types detector.
5, according to the described system of claim 1, it is characterized in that: described switching valve B (8) is six-way valve or ten-way valve, it links to each other with Y-junction (1), detector (7) respectively, an interface therein links to each other with moving phase D18 by pump B (17), and an interface links to each other with a switching valve C (9);
Switching valve C (9) is a ten-way valve, with the interface that is connected with switching valve B (8) for 1., be followed successively by 2. 3. 4. 5. 6. 7. 8. 9. 10. by clockwise all the other interfaces, 2. with 5. interface links to each other with the two ends of C (8) trapping column A (13), 3. with 8. interface is connected, 4. interface is connected by pipeline with pump D (22) with parallel pumps C (21), the 6. external waste liquid bottle of interface, 7. with 10. interface links to each other with the two ends of C8 trapping column B (14), 9. outer reverse-phase chromatographic column (15) and the detector B (16), the external waste liquid bottle of detector B (16) of being connected with in turn of interface.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108088933A (en) * | 2016-11-21 | 2018-05-29 | 中国科学院大连化学物理研究所 | A kind of high throughput body fluid albumen quality sample pretreatment unit and its application |
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| CN101982769A (en) * | 2010-09-07 | 2011-03-02 | 安徽皖仪科技股份有限公司 | Cutting, sub-sampling and purifying device and method for gel chromatography purifying system |
| CN101982769B (en) * | 2010-09-07 | 2013-05-01 | 安徽皖仪科技股份有限公司 | Cutting, sub-sampling and purifying device and method for gel chromatography purifying system |
| CN103842045A (en) * | 2011-10-04 | 2014-06-04 | 默克专利股份公司 | Method and apparatus for chromatographic purification |
| CN103842045B (en) * | 2011-10-04 | 2016-08-17 | 默克专利股份公司 | Chromatographic purification method and equipment |
| CN107073358A (en) * | 2014-10-27 | 2017-08-18 | 豪夫迈·罗氏有限公司 | For two to RPLC SFC chromatograms system and method |
| CN108088933A (en) * | 2016-11-21 | 2018-05-29 | 中国科学院大连化学物理研究所 | A kind of high throughput body fluid albumen quality sample pretreatment unit and its application |
| CN111530124A (en) * | 2020-05-12 | 2020-08-14 | 杭州泽邦科技有限公司 | Universal circulating preparation, separation and purification device for gamma cyclodextrin and sugammadex |
| CN115112782A (en) * | 2021-03-18 | 2022-09-27 | 株式会社岛津制作所 | Preparative liquid chromatograph |
| CN115112782B (en) * | 2021-03-18 | 2024-05-03 | 株式会社岛津制作所 | Preparation of liquid chromatograph |
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