CN104865330A - Countercurrent chromatography column for separation and purification of macromolecule and nanoparticle products and method - Google Patents
Countercurrent chromatography column for separation and purification of macromolecule and nanoparticle products and method Download PDFInfo
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Abstract
The invention relates to the technical field of countercurrent chromatography, and provides a countercurrent chromatography column for the separation and purification of macromolecule and nanoparticle products and a method. The countercurrent chromatography column comprises a bobbin and a hollow circular pipe, wherein the hollow circular pipe has an inner diameter of 3-10 mm and a wall thickness of 1-3 mm and spirally winds the bobbin. The method comprises the following steps: the countercurrent chromatography column is arranged between a loading device and an ultraviolet absorbing device; a stationary phase is placed in a liquid storage bottle, and a flowing phase is placed in a syringe; after the data of a computer program is stable, the loading device is filled with the stationary phase and the flowing phase; the stationary phase and the flowing phase are mixed, and the mixture flows into the countercurrent chromatography column; the separated flowing phase is monitored by an ultraviolet signal in real time so as to obtain a final product; when the data is stable again, the detection is terminated, and the data is calculated; a conclusion is obtained: the improvement of the rotation speed and the acceleration of the flow rate of the flowing phase can improve the separation degree and contribute to the separation of the stationary phase. The countercurrent chromatography column is large in load capacity, high in sample separation degree, and beneficial to industrial production.
Description
Technical field
The present invention relates to countercurrent chromatography field, particularly for current chromatographic column and the method for large molecule and nanoparticle product separation and purification.
Background technology
Along with genomics and the birth of proteomics and the development of DNA recombinant technique, make to produce the protein and other with special bioactive functions and nanoparticle product becomes possibility.These biomacromolecules are present in complicated medium with the form of complexity usually, to structural characterization and the function cognition of these functional biomacromolecules, and further development and utilization, all need high efficiency separation and preparation technology as downstream support., as chromatography of ions, size exclusion chromatograph, reversed-phase high-performance liquid chromatography isochromatic spectrum technology, all there is the problems such as the absorption of the protein and other caused mutually by solid support and sex change and preparation amount is limited at present conventional protein separation technology.Therefore, one of development trend of current adverse current chromatogram in the world is exactly the advantage utilizing liquid luquid partition chromatography, high-speed countercurrent chromatography prepared by the more applicable separation of biopolymer of Study and Development and equipment.
Although adverse current chromatogram is a kind of preparative isolation technics, this technology does not reach industrialized production scale on separation and purification of biological macromolecule at present.Because this technology needs the centrifugal force field of transferring to produce with high-speed rotary to realize the reservation of Stationary liquid, in its amplification process, some technical matterss are had to require further study and solve.
Counter-current chromatograph is invented by Ito, the liquid-liquid diphase chromatographic system then grown up gradually.This technology is widely applied to a large amount of separation and the scale removal process of natural prodcuts.The system of counter-current chromatograph is special, does not need the existence of fixed leg, but utilizes the upper phase in solvent system or lower phase liquid to serve as Stationary liquid.Just because of this, do not have solid column to be just provided with the unexistent many advantages of other separation chromatography, comprise the consume of sample, inactivation and pollution etc.1970, the method obtained stable development in velocity of separation and efficiency.At the beginning of 1980, occurred cross-centennial significant breakthrough, that is exactly the generation of high-speed counter-current chromatograph, and it can a large amount of sample of quick separating within a short period of time.Due to the performance advantage that it is comparatively given prominence to, research emphasis almost all focuses on above high-speed counter-current chromatograph by current countercurrent chromatography.
Column type is the core of counter-current chromatograph, also be the core innovative point of this patent, its evolution experienced by circular column-snail post-tapered screw post-three-dimensional coiled column four kinds of patterns, wherein circular column structure is the simplest, analyze also relatively simple, can carry out various analysis and research as naive model, but its Stationary liquid retains, and can not popularize and come in actual production low.Biomacromolecule is used at present and the most general remains snail post.
Therefore, a kind of three-dimensional spiral post is badly in need of in countercurrent chromatography field, and charge capacity is large, and Separation of Solid and Liquid rate is high, is beneficial to the current chromatographic column for large molecule and nanoparticle product separation and purification and the method for suitability for industrialized production.
Summary of the invention
The invention provides the current chromatographic column for large molecule and nanoparticle product separation and purification and method, technical scheme is as follows:
For the current chromatographic column of large molecule and nanoparticle product separation and purification, comprising: bobbin and hollow circular-tube;
Hollow circular-tube internal diameter is 3-10mm, and wall thickness 1-3mm, spirally form is wrapped on bobbin.
Preferably, above-mentioned in the current chromatographic column of large molecule and nanoparticle product separation and purification, hollow circular-tube is stainless-steel tube.
Preferably, above-mentioned in the current chromatographic column of large molecule and nanoparticle product separation and purification, stainless-steel tube is the 316 type stainless-steel tubes of internal diameter 5mm, external diameter 6mm.
Preferably, above-mentioned in the current chromatographic column of large molecule and nanoparticle product separation and purification, stainless-steel tube support column carries out shaping by being wrapped in, and forms stainless steel helix tube, again the spiral pipe removing support column is wrapped on bobbin, forms three-dimensional stainless steel whirlwind post.
Preferably, above-mentioned in the current chromatographic column of large molecule and nanoparticle product separation and purification, also comprise: some joints, be connected by joint between every root stainless-steel tube.
Preferably, above-mentioned in the current chromatographic column of large molecule and nanoparticle product separation and purification, also comprise: 2 gears, a gear is fixedly installed on the central axis of bobbin, the support member coaxial position of bobbin is equipped with a size, the complete planet wheel identical with fixed gear of shape, and is mutually engaged.
For the method for testing of the current chromatographic column of large molecule and nanoparticle product separation and purification, comprise the steps:
Step one, is installed on support by the current chromatographic column for large molecule and nanoparticle product separation and purification, makes compactness stepless control coil, then is installed between charger and ultraviolet-absorption device by compactness stepless control coil;
Step 2, is loaded on Stationary liquid in liquid storage bottle, and mobile phase is loaded in syringe;
Step 3, opens COMPUTER DETECTION program;
Step 4, after computer program is presented at uv absorption data stabilization, is filled in charger by Stationary liquid and mobile phase;
Step 5, after Stationary liquid and mobile phase fully mix in charger, flows in current chromatographic column;
Step 6, the mobile phase after being separated by current chromatographic column, under UV signal Real-Time Monitoring, obtains final products;
Step 7, when computer program be presented at uv absorption data again stable after, illustrates and detect end, and observe final isolated sample, computing machine calculates the data obtained;
Step 8, draws the following conclusions: (1) slows down flow rate of mobile phase and mixing sample introduction contributes to the reservation of Stationary liquid; (2) improve rotating speed or/and accelerate flow rate of mobile phase can improve degree of separation, contribute to the separation of Stationary liquid.
Preferably in the method for testing of the above-mentioned current chromatographic column for large molecule and nanoparticle product separation and purification, improving rotating speed in step 6 can make degree of separation raise within the scope of 800-1000rpm, but rotating speed is very little on the impact of degree of separation after rotating speed is more than 1000rpm.
The invention has the beneficial effects as follows:
1. the present invention by by stainless-steel tube spirally form be wrapped on bobbin and make current chromatographic column, not fragile leakage, long service life, stability is higher.
2. the present invention adopts the hollow circular-tube of internal diameter 3-10mm, wall thickness 1-3mm as carrying container, and charge capacity is large, and every weekly production reaches 11g, is about 21 times of the equipment of existing report.
3. the present invention makes two-phase solvent in spiral pipe, present the distribution of single phase property by the centrifugal force field that the High Rotation Speed of three-dimensional spiral pipe changes, and makes Stationary liquid retention rate higher.
4. the flow velocity of mobile phase of the present invention reaches 20ml/min, the highest in the equipment for existing report.
5., in the present invention, upper and lower phase all can as mobile phase, and use the lower phase that viscosity is lower, such as: phosphoric acid salt face, the separating effect obtained as mobile phase is better.
6. the present invention is drawn by a large amount of experiments: slow down flow rate of mobile phase and mixing sample introduction contributes to the reservation of Stationary liquid; Improve rotating speed or/and accelerate flow rate of mobile phase degree of separation can be improved, contribute to the conclusion of the separation of Stationary liquid, be conducive to according to actual needs, suitable flow velocity is set, and then improve sample separation degree, can be used in the R and D of enzyme in laboratory, be beneficial to the suitability for industrialized production of feather weight protein formulation.
Accompanying drawing explanation
The present invention is described in detail below in conjunction with the drawings and specific embodiments:
Fig. 1 is the present invention for the manufacturing process structural representation of stainless steel helix tube in the current chromatographic column of large molecule and nanoparticle product separation and purification.
Fig. 2 is the present invention for the structural representation of the current chromatographic column of large molecule and nanoparticle product separation and purification.
Fig. 3 is the present invention for the rotation mode of the current chromatographic column of large molecule and nanoparticle product separation and purification and supporting piece structure schematic diagram thereof.
Fig. 4 structural representation that to be the present invention for the current chromatographic column of large molecule and nanoparticle product separation and purification be installed in separation and purification experiment detecting system.
Fig. 5-7 represents that changing input mode and flow rate of mobile phase affects chromatogram to Protein Separation experimental result.
Fig. 8-11 represents that different rotating speeds affects chromatogram to the Protein Separation experimental result of 12.5% aqueous two-phase system (ATPS).
Figure 12-14 represents that different rotating speeds affects chromatogram to the Protein Separation experimental result of 14%ATPS phase system.
Figure 15-16 represents that changing Stationary liquid flow velocity affects chromatogram to the dipeptides separating experiment under 12.5%ATPS phase system.
Wherein, the Reference numeral in Fig. 1-16 and the corresponding relation between component names are:
Bobbin 1, stainless-steel tube 2, gear 31,32, support column 4.
Embodiment
Core of the present invention, for providing a kind of current chromatographic column for large molecule and nanoparticle product separation and purification and method, solves load little, the problem of realization of industrialization difficulty.
The measure realized to make the technology of the present invention, creation characteristic, reaching object and effect is easy to understand, below in conjunction with concrete diagram, setting forth the present invention further.
Fig. 2 is the present invention for the structural representation of the current chromatographic column of large molecule and nanoparticle product separation and purification.
As shown in Figure 2, the invention provides the current chromatographic column for large molecule and nanoparticle product separation and purification, comprising: bobbin 1 and hollow circular-tube 2; Hollow circular-tube 2 internal diameter is 3-10mm, wall thickness 1-3mm, and spirally form is wrapped on bobbin 1.
In the present embodiment, hollow circular-tube 2 stainless-steel tubes, are preferably the 316 type stainless-steel tubes of internal diameter 5mm, external diameter 6mm.
Fig. 1 is the present invention for the manufacturing process structural representation of stainless steel helix tube in the current chromatographic column of large molecule and nanoparticle product separation and purification, as shown in Figure 1, in the present embodiment, stainless-steel tube 2 by be wrapped in support column 4 carries out shaping, form stainless steel helix tube, again the spiral pipe removing support column 4 is wrapped on bobbin 1, forms three-dimensional stainless steel whirlwind post.
Preferably, also comprise: some joints, be connected by joint between every root stainless-steel tube 2.
Fig. 3 is the present invention for the rotation mode of the current chromatographic column of large molecule and nanoparticle product separation and purification and supporting piece structure schematic diagram thereof, as shown in Figure 3, the present invention also comprises: 2 gears, a gear 31 is fixedly installed on the central axis of bobbin 1, the support member coaxial position of bobbin 1 is equipped with a size, the complete planet wheel 32 identical with fixed gear of shape, and is mutually engaged.
Fig. 4 structural representation that to be the present invention for the current chromatographic column of large molecule and nanoparticle product separation and purification be installed in separation and purification experiment detecting system.
As shown in Figure 4, for the method for testing of the current chromatographic column of large molecule and nanoparticle product separation and purification, comprise the steps:
Step one, is installed on support by the current chromatographic column for large molecule and nanoparticle product separation and purification, makes compactness stepless control coil, then is installed between charger and ultraviolet-absorption device by compactness stepless control coil;
Step 2, is loaded on Stationary liquid in liquid storage bottle, and mobile phase is loaded in syringe;
Step 3, opens COMPUTER DETECTION program;
Step 4, after computer program is presented at uv absorption data stabilization, Stationary liquid and mobile phase are filled in charger by peristaltic pump or other pump;
Step 5, after Stationary liquid and mobile phase fully mix in charger, flows in current chromatographic column;
Step 6, the mobile phase after being separated by current chromatographic column, under UV signal Real-Time Monitoring, obtains final products;
Step 7, when computer program be presented at uv absorption data again stable after, illustrates and detect end, and observe final isolated sample, computing machine calculates the data obtained;
Step 8, draws the following conclusions: (1) slows down flow rate of mobile phase and mixing sample introduction contributes to the reservation of Stationary liquid; (2) improve rotating speed or/and accelerate flow rate of mobile phase can improve degree of separation, contribute to the separation of Stationary liquid.
Further draw, improve rotating speed in step 6 and degree of separation can be made within the scope of 800-1000rpm to raise, but rotating speed is very little on the impact of degree of separation after rotating speed is more than 1000rpm.
In hydrodynamically balanced system, the centrifugal force field that the High Rotation Speed of spiral pipe changes makes two-phase solvent in spiral pipe, present the distribution of single phase property.A wherein light phase is always inclined to and is occupied head end, and heavy one occupies tail end mutually.When gently should introduce from the tail end of spiral tube as during mobile phase, through heavy phase Stationary liquid, finally flow out from head end, this mode is namely from tail type of elution to the end; When using heavy phase as mobile phase, then should taking contrary mode, carry out wash-out from the beginning to the end, namely introduce from the head end of spiral tube, through gently fixing phase, finally flowing out from tail end.Here head end and tail end are relative, relevant with the rotation direction of spiral tube.
The column system of CCC is made up of the liquid that the two-phase in the spiral pipe of high speed planetary motion is immiscible, and wherein one as Stationary liquid, and another is as mobile phase, and material realizes according to its partition factor difference in two-phase being separated.The selection of separating effect and selected dicyandiamide solution, Stationary liquid and mobile phase, type of elution, turning to of instrument have substantial connection with the concentration of rotating speed, sample and input mode and column temperature etc., therefore need to change relevant parameters and carry out compare test effect.
Below in conjunction with specific embodiment, concrete elaboration is carried out to the present invention:
Use myoglobins and lysozyme two kinds of protein as test sample, input mode is divided into mixing sample introduction and difference sample introduction.The two kinds of aqueous two-phase systems used are respectively: the first: accurately take 125 g PEG1000 and 250 g dipotassium hydrogen phosphates, add 1625 mL purified water and fully dissolve, called after 12.5% ATPS; The second: accurately take 280 g polyglycol PEG1000,221.35 g dipotassium hydrogen phosphates and 58.65 g potassium dihydrogen phosphates, add 1440 mL purified water and dissolve, called after 14% ATPS.The rotating speed of instrument is divided into 800rpm, 1000rpm, 1200rpm and 1400rpm.The flow velocity of mobile phase is divided into 3.75ml/min, 5ml/min, 10ml/min and 20ml/min.
Fig. 5-7 represents that changing input mode and flow rate of mobile phase affects chromatogram to Protein Separation experimental result, in 14%ATPS phase system, the mode of sample introduction is respectively adopted to carry out Protein Separation experiment with the flow rate of mobile phase of 10ml/min, Stationary liquid retention rate is about 36.7%, draws out elution curve as shown in Figure 5 in the chromatogram taking time as horizontal ordinate.Flow rate of mobile phase is changed into 20ml/min, as shown in Figure 6, although the separating effect that Stationary liquid retention rate has been reduced to 24%, two kinds of albumen does not decline too much.If by two kinds of albumen mixing sample introductions, flow rate of mobile phase is 10ml/min, as shown in Figure 7, Stationary liquid retention rate is 33.7%, and the degree of separation of two kinds of albumen is 1.24.As can be seen here, slow down flow rate of mobile phase and mixing sample introduction contributes to the reservation of Stationary liquid, and the separating effect of albumen can be better.
Fig. 8-11 represents that different rotating speeds affects chromatogram to the Protein Separation experimental result of 12.5% aqueous two-phase system (ATPS),
Protein Separation experiment is carried out with the rotating speed of 800rpm, 1000rpm, 1200rpm and 1400rpm respectively in 12.5%ATPS phase system, and result is compared, as illustrated in figs. 8-11, revolution-radius R=11cm in experiment, the scope of the β value axis of rotation radius that to be 0.52 ~ 0.86, β=r/R, r be in Fig. 3, R is the radius of hollow shaft in Fig. 3, and β value is a key factor affecting the immiscible solvent of two-phase and distribute in rotary helix pipe thin.Specific works is: hydro-extractor is inner drives rotating frame by a driven by motor by a pair toothed belt wheel and odontoid belt.Tubing string supporter and balance weight assembly are installed in centrifuge central shaft both sides by rotating frame symmetrically.The axis of each supporter is equipped with a planet wheel, and they are engaged mutually with the fixed gear be installed in the stationary pipes of center.In order to ensure mechanical stability, be installed in the free end of rotating frame with a short pipe coupling coaxially, the other end of pipe coupling is supported by the fixation side wall pieces of ball bearing by instrument.First pass the center pit of supporter rotating shaft from a pair runner pipe of the spiral pipe extraction be wound on supporter, then the side opening passing pipe coupling arrives the opening of center stationary pipes, then through this stationary pipes, draws from the opposite side of instrument.Grease lubrication and plastic casing safeguard measure should be adopted, to avoid it with metal parts direct friction outside runner pipe.
Result shows, increases rotating speed and Stationary liquid retention rate can not be made to reach a gratifying level, but degree of separation can be made to have sizable raising.With 800rpm rotating speed for datum-plane, each rotating speed number percent increased is respectively 25%, 20%, 16.7%, and the degree of separation that corresponding experiment draws is respectively 0.53,0.95,1.09.
Figure 12-14 represents that different rotating speeds affects look chromatogram to the Protein Separation experimental result of 14%ATPS phase system, ensure that the degree of separation of sample is a good level to improve sample loading volume, ensuing test is under 14%ATPS phase system, carry out the impact analysis of different rotating speeds to Protein Separation experimental result simultaneously.Use 14%ATPS phase system and 5% sample loading volume under the rotating speed of 800rpm, 1000rpm and 1200rpm, carry out Protein Separation experiment respectively, result is as shown in figs. 12-14.The change of Stationary liquid retention rate is very little.Protein Separation degree is 1.2 at 800rpm, is 1.53 at 1000rpm, is 1.54 at 1200rpm, obviously, improve rotating speed and degree of separation can be made within the scope of 800-1000rpm to raise, but rotating speed is very little on the impact of degree of separation after rotating speed is more than 1000rpm.
The problem that solve only enough Stationary liquid retention rates for protein separation be far from being enough.Be separated the phase mixing intensity that large molecule is as higher in protein needs.Mix fully if be have passed through by the large molecule of CCC system, so its final elute effect will be determined by K value.The partition factor of K value and protein or dipeptides, refer to material to be separated ATPS phase system upper and lower mutually in the ratio of concentration, be dimensionless number; The size of phase mixing intensity can be probed into by the chromatogram comparing dipeptides and protein macromolecule.Therefore we have selected the little dipeptides similar to macro-molecular protein water wettability.Dipeptides HIS-GLY His-Gly selected by us is almost identical with the K value of myoglobins in ATPS phase system.
Figure 15-16 represents that changing Stationary liquid flow velocity affects chromatogram to the dipeptides separating experiment under 12.5%ATPS phase system, as shown in figures 15-16, for under the rotating speed, 12.5%ATPS phase system of 800rpm, respectively with the flow rate of mobile phase of 3.75ml/min and 5ml/min, contrast with dipeptides, the i.e. dipeptides valine-tyrosine Val-Tyr of dipeptides HIS-GLY His-Gly and K=4.0 of K=1.9, carry out the result that separation elution experiments is measured, degree of separation is respectively 1.70 and 2.03.As can be seen here, its separating effect is fine, accelerates flow rate of mobile phase and can improve degree of separation.
The present invention by by stainless-steel tube spirally form be wrapped on bobbin and make current chromatographic column, not fragile leakage, long service life, stability is higher.
The present invention adopts the hollow circular-tube of internal diameter 3-10mm, wall thickness 1-3mm as carrying container, and charge capacity is large, and every weekly production reaches 11g, is about 21 times of the equipment of existing report.
The present invention makes two-phase solvent in spiral pipe, present the distribution of single phase property by the centrifugal force field that the High Rotation Speed of spiral pipe changes, and makes Stationary liquid retention rate higher.
The flow velocity of mobile phase of the present invention reaches 20ml/min, the highest in the equipment for existing report.
In the present invention, upper and lower phase all can as mobile phase, and use the lower phase that viscosity is lower, such as: phosphoric acid salt face, the separating effect obtained as mobile phase is better.
The present invention is drawn by a large amount of experiments: slow down flow rate of mobile phase and mixing sample introduction contributes to the reservation of Stationary liquid; Improve rotating speed or/and accelerate flow rate of mobile phase degree of separation can be improved, contribute to the conclusion of the separation of Stationary liquid, be conducive to according to actual needs, suitable flow velocity is set, and then improve sample separation degree, can be used in the R and D of enzyme in laboratory, be beneficial to the suitability for industrialized production of feather weight protein formulation.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and instructions just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (8)
1., for the current chromatographic column of large molecule and nanoparticle product separation and purification, it is characterized in that, comprising: bobbin and hollow circular-tube;
Hollow circular-tube internal diameter is 3-10mm, and wall thickness 1-3mm, spirally form is wrapped on bobbin.
2. the current chromatographic column for large molecule and nanoparticle product separation and purification according to claim 1, it is characterized in that, described hollow circular-tube is stainless-steel tube.
3. the current chromatographic column for large molecule and nanoparticle product separation and purification according to claim 2, it is characterized in that, described stainless-steel tube is the 316 type stainless-steel tubes of internal diameter 5mm, external diameter 6mm.
4. the current chromatographic column for large molecule and nanoparticle product separation and purification according to claim 2, it is characterized in that, described stainless-steel tube by be wrapped in support column carries out shaping, form stainless steel helix tube, again the spiral pipe removing support column is wrapped on described bobbin, forms three-dimensional stainless steel whirlwind post.
5. the current chromatographic column for large molecule and nanoparticle product separation and purification according to claim 1, be is characterized in that, also comprise: some joints, be connected described in every root between hollow circular-tube by joint.
6. the current chromatographic column for large molecule and nanoparticle product separation and purification according to claim 1, it is characterized in that, also comprise: 2 gears, a gear is fixedly installed on the central axis of bobbin, the support member coaxial position of bobbin is equipped with a size, the complete planet wheel identical with fixed gear of shape, and is mutually engaged.
7., for the method for testing of the current chromatographic column of large molecule and nanoparticle product separation and purification, comprise the steps:
Step one, is installed on support by the current chromatographic column for large molecule and nanoparticle product separation and purification, makes compactness stepless control coil, then is installed between charger and ultraviolet-absorption device by compactness stepless control coil;
Step 2, is loaded on Stationary liquid in liquid storage bottle, and mobile phase is loaded in syringe;
Step 3, opens COMPUTER DETECTION program;
Step 4, after computer program is presented at uv absorption data stabilization, is filled in charger by Stationary liquid and mobile phase;
Step 5, after Stationary liquid and mobile phase fully mix in charger, flows in current chromatographic column;
Step 6, the mobile phase after being separated by current chromatographic column, under UV signal Real-Time Monitoring, obtains final products;
Step 7, when computer program be presented at uv absorption data again stable after, illustrates and detect end, and observe final isolated sample, computing machine calculates the data obtained;
Step 8, draws the following conclusions: slow down flow rate of mobile phase and mixing sample introduction contributes to the reservation of Stationary liquid; Improve rotating speed or/and accelerate flow rate of mobile phase can improve degree of separation, contribute to the separation of Stationary liquid.
8. the method for testing of the current chromatographic column for large molecule and nanoparticle product separation and purification according to claim 7, it is characterized in that, improving rotating speed in described step 8 can make degree of separation raise within the scope of 800-1000rpm, but rotating speed is very little on the impact of degree of separation after rotating speed is more than 1000rpm.
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| CN105771317B (en) * | 2016-03-10 | 2020-06-02 | 山东省分析测试中心 | Countercurrent chromatography separation column and chromatograph |
| CN110462393A (en) * | 2017-03-28 | 2019-11-15 | 卓曼坦公司 | Continuous flow upstream helix chromatography |
| CN110462393B (en) * | 2017-03-28 | 2022-05-24 | 卓曼坦公司 | Continuous countercurrent spiral chromatography |
| CN116672916A (en) * | 2023-07-28 | 2023-09-01 | 北京凯莱谱生物科技有限公司 | Chromatograph with mixed wave reduction device and detection method thereof |
| CN116672916B (en) * | 2023-07-28 | 2023-11-17 | 北京凯莱谱生物科技有限公司 | Chromatograph with mixed wave reduction device and detection method thereof |
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