CN1301964A - Intelligent integrated automatic liquid phase chromatographic system - Google Patents
Intelligent integrated automatic liquid phase chromatographic system Download PDFInfo
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- CN1301964A CN1301964A CN 99127592 CN99127592A CN1301964A CN 1301964 A CN1301964 A CN 1301964A CN 99127592 CN99127592 CN 99127592 CN 99127592 A CN99127592 A CN 99127592A CN 1301964 A CN1301964 A CN 1301964A
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Abstract
The liquid phase chromatographic system designed based on settlement-filtration-thiophilic chromatography consists of settler, filter, thiophilic chromatograph and automatic controller. It has integrated sample pre-treatment and chromatographic separation in protein separation and computerized automatic control of whole technological process, so that it may be used to prepare egg specific antibody Ig Y in low cost, high purity and large scale. The system may be used in the separation of egg antibody as well as the separation of blood and milk antibody.
Description
The present invention relates to a kind of automatic liquid phase chromatographic system, be applicable to preparation gram level scale biological products, be particularly useful for making the egg specific antibody of gram level scale.
Egg antibody has broad application prospects in life science and biotechnology industry; the antibody partition method of having developed in the world at present; even if the partition method of the technical Japanese Sun Company Inc. that is in a leading position; basically be exactly classical Separation of Proteins method; as the polyelectrolyte post lipoprotein; ethanol precipitation; method such as gel filtration and ion chromatography; and usually sample pretreatment process in the Separation of Proteins and chromatographic separation process two big operations are separately carried out by the equipment of these method designs; it is many to make in the whole process of preparation ubiquity step; the process complexity; in batches little and the reagent consumption is big; problems such as equipment investment height; be difficult to realize suitability for industrialized production; and the total recovery of egg specific antibody IgY the highest only 65%; degreasing rate has only 93%; in a word, prior art all can't possess low cost simultaneously; high-purity; scale ground preparation egg specific antibody IgY.
The purpose of this invention is to provide a kind of intelligent integrated automatic liquid phase chromatographic system; it can be with sample pretreatment process in the Separation of Proteins and chromatographic separation process two big integrated processes, thereby can realize low cost, high-purity, scale ground preparation egg specific antibody IgY.
The objective of the invention is to realize by following technical proposals.
The present invention utilizes sedimentation-filtering-sulfurphilic chromatography process to design, and promptly it obtains supernatant to the pre-service of egg yolk liquid by citric acid-phosphate buffer one step sedimentation egg yolk liquid and realizes, thereby has simplified the yolk The pretreatment greatly; Adopt zeyssatite as the principal curvature agent, carry out membrane microfiltration one time, the grease removal rate is high; Sulphur adsorption chromatography material (T-gel) single step separation and purification egg antibody is had a liking in employing, can obtain highly purified egg antibody IgY.The present invention is by sedimentation device, filtration unit, thiophilic chromatography device and automaton are formed, sedimentation device is made up of slurry tank, filtration unit is by filtrator, head tank, air steel cylinder and high-pressure valve are formed, the thiophilic chromatography device is by mixing tank, chromatographic column, peristaltic pump, UV-detector, solution basin and product gatherer are formed, automaton is by industrial control computer, A/D converter, stirrer, volume pump, solenoid valve, combination valve, two liquid level gauges, two thermometers, conductivity meter and pressure gauge are formed, combination valve is made up of with solenoid valve and automatic liquid phase chromatographic Pneumatic valve gas control, it places the thiophilic chromatography device to flow into the conveyance conduit of the solution of chromatographic column, solenoid valve places each liquid and the gas delivery channels of sedimentation device and filtration unit, and it also places the thiophilic chromatography device to flow to the passage of mixing tank from the solution basin, exhaust ports and detecting device exit; But the liquid feed pressure jar in the slurry tank, the pressurized air in the air steel cylinder can act on head tank, makes that the liquid in the head tank can filter back inflow mixing tank through filtrator, and pressure gauge is installed on the head tank; Two liquid level gauges are installed in respectively on slurry tank and the mixing tank; Stirrer and conductivity meter all place in the mixing tank; Liquid in the solution basin flows into mixing tank or chromatographic column through volume pump or peristaltic pump respectively; Liquid in the mixing tank can flow into chromatographic column through peristaltic pump; There is an exhausr port at the input channel place of chromatographic column, output channel is connected with UV-detector, the UV-detector output channel is connected with product gatherer and waste liquid tank respectively, liquid level gauge, conductivity meter, UV-detector, temperature is taken into account pressure gauge the signal that collects is inputed to industrial control computer through A/D converter, and export to solenoid valve after the wherethrough reason, combination valve, volume pump, peristaltic pump, stirrer, slurry tank, head tank and filtrator place in 4 ℃ the refrigerator, the thiophilic chromatography device, volume pump, combination valve and solution basin all place in 20 ℃ of refrigerators, the air steel cylinder, high-pressure valve and industrial control computer place outside the refrigerator, and two thermometers are installed in respectively in above-mentioned two refrigerators.
The present invention has following advantage: (1) robotization: whole process flow can be all by computer controlled automatic; (2) oversimplify: the present invention can be with sample pretreatment process in the Separation of Proteins and chromatographic separation process two big integrated processes, thereby have simplified production stage greatly, can realize that low cost, high-purity, scale ground prepare IgY; (3) Highgrade integration: all processes height such as sedimentation, filtration, absorption are integrated in two refrigerators, have floor area little, be convenient to advantage such as automatically control; (4) maneuverability: the present invention can select arbitrarily automatically, the control of substep automatic or manual; (5) automatic data acquisition: the experimental data of whole automatic liquid phase chromatographic detachment process, all by computer-automatic collection, and record in the data file, be convenient to further carry out data analysis and processing with other softwares; (6) multi-usage: ⅰ) sedimentation, filtration preprocessing process can be used for the sedimentation and filtration pre-service of other suspending liquid; ⅱ) absorption, wash-out, the automatic chromatographic separation process of regeneration, both can be directly used in the automatic chromatography of different systems (as the immunoglobulin (Ig) in yolk, blood and the milk), also can be by changing chromatographic material, the native system expansion is used for the automatic chromatography (as the affinity chromatography separation of transgenosis effectiveness albumen etc.) of other system.
Below will the present invention is described in further detail by specific embodiment:
Fig. 1 is the process chart at band check point of the present invention, reference mark.
Fig. 2 is automaton circuit block diagram among the present invention.
Fig. 3 is a technological process flow chart of the present invention.
Fig. 4 is the structured flowchart of combination valve.
Fig. 5 is the structural drawing of automatic liquid phase chromatographic Pneumatic valve in the combination valve.
Fig. 6 is the detection curve figure of whole technological process.
Extremely shown in Figure 5 as Fig. 1, the present invention is by sedimentation device, filtration unit, thiophilic chromatography device and automaton are formed, sedimentation device is made up of slurry tank A, filtration unit is by filtrator C, head tank B, air steel cylinder and high-pressure valve are formed, the thiophilic chromatography device is by mixing tank D, chromatographic column J, peristaltic pump E1, UV-detector, five kinds of solution (are dense saline solution E, equilibrium liquid F, eluent G, regenerated liquid H, distilled water I) basin and product gatherer are formed, automaton is by industrial control computer (its model is IPC-610 P233), A/D converter (model is A-823), stirrer, volume pump E2, solenoid valve a, b, c, d, e, j, k, L, combination valve f, g, h, i, m, liquid level gauge Y1, Y2, conductivity meter Q, temperature sensor and pressure gauge P form, as shown in Figure 4 and Figure 5, combination valve is made up of with solenoid valve and automatic liquid phase chromatographic Pneumatic valve gas control, gas control is connected with the compressed air inlet of automatic liquid phase Pneumatic valve with the compressed air outlet of solenoid valve, in the thiophilic chromatography device, use combination valve can avoid the gas-liquid two-phase flow problem, combination valve g, h, i, m, f places the equilibrium liquid F basin of thiophilic chromatography device respectively, eluent G basin, regenerated liquid H basin, in the transfer passage of distilled water I basin and mixing tank D, solenoid valve a, b, c, d places each liquid of sedimentation device and filtration unit and the conveyance conduit of gas respectively, dense saline solution basin is connected by pipeline with mixing tank D, and between them, be equipped with volume pump E2 and solenoid valve e, liquid in the slurry tank can be through solenoid valve a feed pressure jar B, the compressed-air actuated effect in the air steel cylinder of liquid among the head tank B is dirty filters back inflow mixing tank D through filter C, liquid among the mixing tank D can flow into chromatographic column J through combination valve f and peristaltic pump E1, equilibrium liquid F, eluent G, regenerated liquid H and distilled water I all can flow into chromatographic column J through combination valve and peristaltic pump E1, on the output channel of near exhaust ports the chromatographic column J inlet and UV-detector, a solenoid valve L is arranged all, J, K, collected by the product gatherer by the flow through IgY of UV-detector of chromatographic column J, its waste liquid flows into waste liquid tank; Pressure gauge P is installed on the head tank B; Two liquid level gauge Y1 and Y2 place respectively on slurry tank A and the mixing tank D, stirrer R and conductivity meter Q all place in the mixing tank D, liquid level gauge Y1 and Y2, conductivity meter Q, UV-detector, temperature is taken into account pressure gauge P the signal that collects is inputed to industrial control computer through A/D converter, and export to solenoid valve a after the wherethrough reason, b, c, d, e, j, k, L, combination valve f, g, h, i, m, volume pump E2, peristaltic pump E1 and stirrer R, and control their and move, wherein AD_x represents A/D plate x analog input channel, DO_x represents A/D plate x digital quantity output channel, DA_x represents A/D plate x analog output channel, the air steel cylinder, high-pressure valve and industrial control computer place outside the refrigerator, remaining all places in the refrigerator, wherein sedimentation device and filtration unit place in 4 ℃ the refrigerator, the thiophilic chromatography device places in 20 ℃ the refrigerator, and two thermometers place respectively in above-mentioned two refrigerators.As Fig. 1, Fig. 3 and shown in Figure 6, with fully automatic working mode side example, it is as follows to describe the course of work of the present invention in detail: (1) is provided with working method and running parameter, be that working method is set to " automatically " mode, running parameter comprises level value, electric conductivity value, detected value, time or the like; (2) click " startup ", the beginning fully automatic working; (3) settling process: after the suspending liquid among the slurry tank A is deposited to a certain degree, (be generally 3～5 hours), open solenoid valve A and D and make supernatant feed pressure jar B; (4) filter process: when liquid level gauge Y1 displayed value equals setting value, and behind the whole feed pressure jar of the supernatant in the slurry tank B, under the control of computer, close solenoid valve A and D automatically, open high-pressure valve and solenoid valve b and c, by air pressure the liquid inflow filter C among the head tank B is filtered, its filtrate flows into mixing tank D; (5) thiophilic chromatography sample solution preprocessing process: when liquid level gauge Y2 displayed value reaches setting value, when promptly the liquid level of head tank B is zero, close high-pressure valve and solenoid valve b and c, start stirrer R, add about 90% solid Na
2SO
4, after waiting to dissolve, wait for and click " determining " button in the reminding window, open solenoid valve e, start volume pump E2, damping fluid is quantitatively mixed in mixing tank D with filtered fluid, when electric conductivity value equals setting value, close volume pump E2 and stirrer R; (6) thiophilic chromatography process: in this process, the liquid that flows out chromatographic column detects the concentration of protein through UV-detector, the detection wavelength is 280nm, the detection curve of whole process as shown in Figure 6, that is: 1), 1 → 2 is pre equilibrium process: open combination valve g and solenoid valve L, start peristaltic pump E1, get rid of the pipeline air, delayed time 1～2 minute, close solenoid valve L, open solenoid valve j, chromatographic column J is carried out pre-equilibration, required ionic strength when making ionic strength in the chromatographic column reach sample absorption with equilibrium liquid F, its waste liquid flows into waste liquid tank, behind the time-delay certain hour, close peristaltic pump E1, combination valve g and combination valve j; 2), 2 → 3 are last sample adsorption process: open combination valve f and combination valve L, start peristaltic pump E1, get rid of the pipeline air, delayed time 1～2 minute, and closed solenoid valve L, open solenoid valve j, allowing mixed liquor among the mixing tank D flow into chromatographic column regenerates, product (as IgY) is adsorbed on the chromatographic material (as T glue), and other component then flows through chromatographic column, is not attracted on the chromatographic material." 3 " some expression absorption saturation point among Fig. 6 when detected value rises to certain value (going up the sample stationary value) and stablizes, when promptly reaching the absorption saturation point, is closed combination valve f, solenoid valve j and peristaltic pump E1; 3), 3 → 4 are the balance flushing process: open combination valve g and solenoid valve L, start peristaltic pump E1, get rid of the pipeline air, delayed time 1～2 minute, close solenoid valve L, open solenoid valve j, with equilibrium liquid F chromatographic column J is washed, all drives away remaining among the chromatographic column J the not absorbed component in the bed space, so as not to these not absorbed component sneak in the wash-out product of back, when detected value drops to certain value (balance and stability value) and stablizes, close peristaltic pump E1, combination valve g and combination valve j; 4), 4 → 5 are elution process: open combination valve h and solenoid valve L, start peristaltic pump E1, get rid of the pipeline air, delayed time 1～2 minute, close solenoid valve L, open solenoid valve j, make eluent G flow into chromatographic column, be adsorbed on product (as IgY) on the chromatographic material (as T glue) with wash-out, behind the time-delay certain hour, if detected value begins rapid rising (gathering up initial value), then open solenoid valve k, close solenoid valve j, collect product, behind the time-delay certain hour, if detected value drops to certain value (wash-out stop value) and when stablizing, closes peristaltic pump E1, combination valve h and combination valve k are if chromatographic column needs regeneration, then carry out next step, otherwise completed product run or return step 1); 5), 5 → 6 are water-washing process: open combination valve m and solenoid valve L, start peristaltic pump E1, get rid of the pipeline air, delayed time 1～2 minute, close solenoid valve L, open solenoid valve j, make distilled water I flow into chromatographic column J, with the impurity on the flushing chromatographic column, so that save the regenerated liquid consumption, time-delay a period of time, close peristaltic pump E1, combination valve m and combination valve j; 6), 6 → 7 are regenerative process: open combination valve I and L, get rid of the pipeline air, delayed time 1～2 minute, close solenoid valve L, open solenoid valve j, make regenerated liquid H flow into chromatographic column J, the impurity that remains on the chromatographic column is all washed out, chromatographic column obtains regeneration, so that can recycle, behind the time-delay certain hour, when detected value drops to certain value (regeneration stop value), close peristaltic pump E1, combination valve i and solenoid valve j, completed product run or change regenerated liquid into distilled water, open combination valve m and solenoid valve L, start peristaltic pump E1, get rid of the pipeline air, delayed time 1～2 minute, close solenoid valve L, open solenoid valve j, make distilled water flow into chromatographic column J, behind the time-delay certain hour, close peristaltic pump E1, combination valve m and solenoid valve j return step 1), and so circulation finishes until producing.
Claims (1)
1. intelligent integrated automatic liquid phase chromatographic system, it is characterized in that it is by sedimentation device, filtration unit, thiophilic chromatography device and automaton are formed, sedimentation device is made up of slurry tank, filtration unit is by filtrator, head tank, air steel cylinder and high-pressure valve are formed, the thiophilic chromatography device is by mixing tank, chromatographic column, peristaltic pump, UV-detector, solution basin and product gatherer are formed, automaton is by industrial control computer, A/D converter, stirrer, volume pump, solenoid valve, combination valve, two liquid level gauges, two thermometers, conductivity meter and pressure gauge are formed, combination valve is made up of with solenoid valve and automatic liquid phase chromatographic Pneumatic valve gas control, it places the thiophilic chromatography device to flow into the conveyance conduit of the solution of chromatographic column, solenoid valve places each liquid and the gas delivery channels of sedimentation device and filtration unit, and it also places the thiophilic chromatography device to flow to the passage of mixing tank from the solution basin, exhaust ports and detecting device exit; But the liquid feed pressure jar in the slurry tank, the pressurized air in the air steel cylinder can act on head tank, makes that the liquid in the head tank can filter back inflow mixing tank through filtrator, and pressure gauge is installed on the head tank; Two liquid level gauges are installed in respectively on slurry tank and the mixing tank; Stirrer and conductivity meter all place in the mixing tank; Liquid in the solution basin flows into mixing tank or chromatographic column through volume pump or peristaltic pump respectively; Liquid in the mixing tank can flow into chromatographic column through peristaltic pump; There is an exhausr port at the input channel place of chromatographic column, and output channel is connected with UV-detector; The UV-detector output channel is connected with product gatherer and waste liquid tank respectively, liquid level gauge, conductivity meter, UV-detector, temperature is taken into account pressure gauge the signal that collects is inputed to industrial control computer through A/D converter, and export to solenoid valve after the wherethrough reason, combination valve, volume pump, peristaltic pump, stirrer, slurry tank, head tank and filtrator place in 4 ℃ the refrigerator, the thiophilic chromatography device, volume pump, combination valve and solution basin all place in 20 ℃ of refrigerators, the air steel cylinder, high-pressure valve and industrial control computer place outside the refrigerator, and two thermometers are installed in respectively in above-mentioned two refrigerators.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99127592 CN1117277C (en) | 1999-12-28 | 1999-12-28 | Intelligent integrated automatic liquid phase chromatographic system |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 99127592 CN1117277C (en) | 1999-12-28 | 1999-12-28 | Intelligent integrated automatic liquid phase chromatographic system |
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| CN1301964A true CN1301964A (en) | 2001-07-04 |
| CN1117277C CN1117277C (en) | 2003-08-06 |
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| CN 99127592 Expired - Fee Related CN1117277C (en) | 1999-12-28 | 1999-12-28 | Intelligent integrated automatic liquid phase chromatographic system |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101747407A (en) * | 2008-12-02 | 2010-06-23 | 杭州中肽生化有限公司 | High-efficient integrated system for separation and purification |
| CN101899110A (en) * | 2010-07-16 | 2010-12-01 | 浙江大学 | Method for separating immune globulin IgY(delta Fc) from goose blood |
| CN101113974B (en) * | 2007-09-04 | 2011-04-06 | 上海吴泾化工有限公司 | Liquid phase medium preprocess device of on-line chromatographic study and using method, and used thereof |
| CN102507764A (en) * | 2011-10-20 | 2012-06-20 | 安徽皖仪科技股份有限公司 | Automatic washing device and method for high-efficient liquid phase chromatograph |
| CN103293253A (en) * | 2013-05-24 | 2013-09-11 | 中国科学院过程工程研究所 | Efficient purification-analysis system for biotechnological medicine and medicine separation detection method thereof |
| CN103908800A (en) * | 2014-03-04 | 2014-07-09 | 中国科学院青海盐湖研究所 | Adsorption separation system |
| CN103946705A (en) * | 2011-11-10 | 2014-07-23 | 弗·哈夫曼-拉罗切有限公司 | Preparative column chromatography system |
| CN109012762A (en) * | 2018-09-28 | 2018-12-18 | 成都理工大学 | A kind of adjustable full automatic ion exchange column |
| CN109298734A (en) * | 2018-09-07 | 2019-02-01 | 上海出入境检验检疫局工业品与原材料检测技术中心 | The control method of intelligent temperature control detection device |
| CN110944729A (en) * | 2017-06-15 | 2020-03-31 | 通用电气健康护理生物科学股份公司 | Method and apparatus for determining one or more buffer composition formulations |
-
1999
- 1999-12-28 CN CN 99127592 patent/CN1117277C/en not_active Expired - Fee Related
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113974B (en) * | 2007-09-04 | 2011-04-06 | 上海吴泾化工有限公司 | Liquid phase medium preprocess device of on-line chromatographic study and using method, and used thereof |
| CN101747407A (en) * | 2008-12-02 | 2010-06-23 | 杭州中肽生化有限公司 | High-efficient integrated system for separation and purification |
| CN101899110A (en) * | 2010-07-16 | 2010-12-01 | 浙江大学 | Method for separating immune globulin IgY(delta Fc) from goose blood |
| CN101899110B (en) * | 2010-07-16 | 2012-08-08 | 浙江大学 | Method for separating immune globulin IgY(delta Fc) from goose blood |
| CN102507764A (en) * | 2011-10-20 | 2012-06-20 | 安徽皖仪科技股份有限公司 | Automatic washing device and method for high-efficient liquid phase chromatograph |
| CN103946705A (en) * | 2011-11-10 | 2014-07-23 | 弗·哈夫曼-拉罗切有限公司 | Preparative column chromatography system |
| CN103293253B (en) * | 2013-05-24 | 2015-04-01 | 中国科学院过程工程研究所 | Efficient purification-analysis system for biotechnological medicine and medicine separation detection method thereof |
| CN103293253A (en) * | 2013-05-24 | 2013-09-11 | 中国科学院过程工程研究所 | Efficient purification-analysis system for biotechnological medicine and medicine separation detection method thereof |
| CN103908800A (en) * | 2014-03-04 | 2014-07-09 | 中国科学院青海盐湖研究所 | Adsorption separation system |
| CN103908800B (en) * | 2014-03-04 | 2016-06-22 | 中国科学院青海盐湖研究所 | A kind of adsorption separation system |
| CN110944729A (en) * | 2017-06-15 | 2020-03-31 | 通用电气健康护理生物科学股份公司 | Method and apparatus for determining one or more buffer composition formulations |
| CN110944729B (en) * | 2017-06-15 | 2022-03-15 | 思拓凡瑞典有限公司 | Method, apparatus, computer program and computer program product for determining one or more buffered composition formulations |
| CN109298734A (en) * | 2018-09-07 | 2019-02-01 | 上海出入境检验检疫局工业品与原材料检测技术中心 | The control method of intelligent temperature control detection device |
| CN109012762A (en) * | 2018-09-28 | 2018-12-18 | 成都理工大学 | A kind of adjustable full automatic ion exchange column |
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