CN111841073B - Multi-column switching cycle chromatographic separation system and method for separating and concentrating target components from raw materials - Google Patents
Multi-column switching cycle chromatographic separation system and method for separating and concentrating target components from raw materials Download PDFInfo
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Abstract
A multi-column switching cycle chromatographic separation system and a method thereof for separating and concentrating target components from raw materials comprise: n chromatographic columns, wherein N is an integer more than or equal to 3; the N chromatographic columns are kept in series connection, and the series connection mode is switched at regular intervals; specifically, the method comprises the following steps: firstly, enabling eluent to flow from an inlet of a 1 st chromatographic column to an outlet of an Nth chromatographic column; after a certain time, the system is switched to the 2 nd chromatographic column, the … th chromatographic column, the Nth chromatographic column and the 1 st chromatographic column to be connected in series, and eluent flows from the inlet of the 2 nd chromatographic column to the outlet of the 1 st chromatographic column; in this way, after the system is switched for (N-1) times, the series connection mode is that the Nth, the 1 st, the 2 nd, … nd and the N-1 st chromatographs are connected in series, and eluent flows from the Nth inlet to the N-1 th outlet along the series connection sequence; after the system is switched for N times, the tandem mode is that the 1 st, the 2 nd, … th and the Nth chromatographs are connected in series; the modifier flows into the eluent from any chromatographic column inlet downstream of the eluent inlet, and the elution capacity of the modifier is weaker than that of the eluent.
Description
Technical Field
The invention relates to the technical field of chromatographic separation, in particular to a multi-column switching cycle chromatographic separation system and a method for separating and concentrating target components from raw materials.
Background
Cycle chromatography can extend the equivalent column length without increasing column pressure and is therefore particularly suitable for separating difficult separation systems such as enantiomers, isomers and natural homologues. However, the concentration bands of the components to be separated are continuously broadened during the separation process, so that the band front edge of the weakly retained component can easily catch up with the band rear edge of the strongly retained component, and the separation fails. To avoid this adverse effect, the feed rate can be reduced; or by using a peak cutting technique, the leading edge of the weakly retained component band and the trailing edge of the strongly retained component band, which have achieved separation, are cut out of the system in each cycle, with only the overlapping portions of the component bands circulating within the system. Although the measures avoid the front edge of the band of the weakly retained component from tracing the back edge of the band of the strongly retained component, the band expansion is not fundamentally solved, so that the concentration of the product fraction is low and the recovery is difficult.
In the prior art, a double-column solvent gradient circulation chromatographic technology is adopted, namely two chromatographic columns are always kept in series connection through valve switching, and a modifier is introduced between the two columns, so that a concentration spectral band contraction mechanism can be generated, and the spectral band expansion is prevented. However, such a two-column solvent gradient cycle chromatography has a problem that the stationary phase utilization rate is not high. As shown in fig. 1, in each switching cycle of the separation process, the concentration band (shaded in the figure) of the target component always migrates from the upstream chromatographic column to the downstream chromatographic column, and the stationary phase inside the upstream chromatographic column obviously does not perform the separation function for the region not covered by the target component band. Therefore, in such a dual-column solvent gradient cycle chromatography system, both columns are responsible for the separation, but in practice most of the stationary phase is not utilized. For example, in each switching cycle under optimal operating conditions, the tail of the band of the target component always leaves the upstream column and the leading edge reaches the outlet of the downstream column, and as can be seen from fig. 1, the unused stationary phase accounts for at least 50% of the total stationary phase, i.e., the stationary phase utilization rate is at most 50%.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a multi-column switching cycle chromatographic separation system capable of improving the utilization rate of a stationary phase and separating and concentrating target components from raw materials.
In order to solve the above technical problems, the technical solution provided by the present invention is realized as follows: a multi-column switching cycle chromatographic separation system for separating and concentrating a target component from a feed material, the system comprising: n chromatographic columns, wherein N is an integer more than or equal to 3, and a valve component; the N chromatographic columns are always connected in series through a valve assembly, and the series connection mode of the N chromatographic columns is switched at regular intervals; the specific switching process is as follows: firstly, enabling eluent to flow from an inlet of a 1 st chromatographic column to an outlet of an Nth chromatographic column; after a certain time, the system is switched to be connected with the 2 nd chromatographic column, the 3 rd chromatographic column, the … th chromatographic column, the Nth chromatographic column and the 1 st chromatographic column in series, and eluent flows from the inlet of the 2 nd chromatographic column to the outlet of the 1 st chromatographic column along the series sequence; after a certain time, the system is switched to the 3 rd chromatographic column, the 4 th chromatographic column, the … th chromatographic column, the Nth chromatographic column, the 1 st chromatographic column and the 2 nd chromatographic column in series, and the eluent flows from the inlet of the 3 rd chromatographic column to the outlet of the 2 nd chromatographic column along the series sequence; in this way, after the system is switched for (N-1) times, the serial connection mode is that the Nth chromatographic column, the 1 st chromatographic column, the 2 nd chromatographic column, … and the Nth-1 st chromatographic column are connected in series, and eluent flows from the inlet of the Nth chromatographic column to the outlet of the Nth-1 st chromatographic column along the serial connection sequence; after the system is switched for N times, the series connection mode is that the 1 st, the 2 nd, … th and the Nth chromatographs are connected in series, and eluent flows to the outlet of the Nth chromatographic column from the inlet of the 1 st chromatographic column; the system is switched back and forth according to the cycle described above, and during operation, the modifying agent flows into the system from the inlet of any one of the chromatographic columns located downstream of the eluent inlet, and wherein the eluting power of the modifying agent is weaker than the eluting power of the eluent.
Preferably, in order to make the elution capacity of the eluent stronger than the elution capacity of the modifying agent, the eluent can be heated and the modifying agent can be cooled to ensure that the temperature of the eluent is higher than that of the modifying agent, so that the elution capacity of the eluent is stronger than that of the modifying agent.
Preferably, in order to make the elution capacity of the eluent stronger than the elution capacity of the modifying agent, the eluent can be heated and the modifying agent can be cooled to ensure that the temperature of the eluent is higher than that of the modifying agent, so that the elution capacity of the eluent is stronger than that of the modifying agent.
Preferably, in order to make the elution ability of the eluent stronger than that of the modifier, a method may be employed in which the volume percentage of the solvent having a strong elution ability in the eluent is higher than that of the solvent having a strong elution ability in the modifier.
Or heating the eluent and cooling the modifier and the solvent with strong elution capacity in the eluent at the same time, wherein the volume percentage of the solvent with strong elution capacity in the modifier is higher than that of the solvent with strong elution capacity in the modifier, so that the elution capacity of the eluent is higher than that of the modifier.
The invention also provides a method for separating and concentrating target components by using the multi-column switching cycle chromatographic separation system, which comprises the following specific steps:
(1) feeding: dissolving the raw material to be separated into any solvent capable of dissolving the raw material to prepare a raw material solution; the initial state of the multi-column switching cycle chromatographic separation system is that the 1 st chromatographic column, the 2 nd chromatographic column, the … th chromatographic column and the Nth chromatographic column are sequentially connected in series, and then raw material liquid is added into the system from the inlet of any chromatographic column, so that a target object is adsorbed in the system;
(2) and (3) purification: the multi-column switching cycle chromatographic system is still in the state that 1 st, 2 nd, … th and Nth chromatographic columns are connected in series in sequence, then eluent flows into the system from the 1 st chromatographic column inlet, and modifier flows into the system from any chromatographic column inlet downstream of the eluent inlet; after a certain time, the system is switched, wherein the 2 nd chromatographic column, the 3 rd chromatographic column, the … th chromatographic column, the Nth chromatographic column and the 1 st chromatographic column are connected in series, eluent flows into the system from the 2 nd chromatographic column inlet, and a modifying agent flows into the system from any chromatographic column inlet downstream of the eluent inlet; after a certain time, the system is switched again, wherein 3 rd chromatographic column, 4 th chromatographic column, … th chromatographic column, Nth chromatographic column, 1 st chromatographic column and 2 nd chromatographic column are connected in series, eluent flows into the system from the 3 rd chromatographic column inlet, and modifier flows into the system from any chromatographic column inlet downstream of the eluent inlet; in this way, after the system is switched for (N-1) times, the Nth, the 1 st, the 2 nd, the … th and the N-1 st chromatographies are connected in series, eluent flows from the inlet of the Nth chromatographic column to the outlet of the N-1 st chromatographic column, and modifier is added into the system from any one of the inlets of the 1 st to the N-1 st chromatographic columns which are positioned at the downstream of the inlet of the eluent; after the system is switched for N times, connecting the 1 st, the 2 nd, the … th and the Nth chromatographs in series, enabling eluent to flow from the inlet of the 1 st chromatographic column to the outlet of the Nth chromatographic column, and adding a modifier into the system from any one of the inlets from the 2 nd to the Nth chromatographic column downstream of the eluent inlet; and (3) circularly and repeatedly switching the system according to the mode, namely, circularly and repeatedly changing the serial mode of the N chromatographic columns and the inlet positions of the eluent and the modifier, and finally obtaining the purified target product.
Preferably, in the system of the present invention, during the feeding process, neither the elution pump nor the finishing pump is operated, the valve assembly is not switched, the raw material is fed from the inlet of the 1 st chromatographic column, and when the target component flows out from the (N-1) th chromatographic column, the feeding is stopped, and the corresponding feeding amount is the optimum feeding amount.
The system switching of the present invention, i.e. the positions of the eluent inlet and effluent outlet are varied, and the time interval between any two adjacent switches depends on the specific separation system and the pressure resistance of the system, and may be equal or unequal.
The 1 st, 2 nd, … th and N th chromatographic columns of the invention are connected in series, or the 2 nd, 3 rd, … th, N th and 1 st chromatographic columns are connected in series, and are defined from the flowing direction of the medium flowing in the system: such as: the fluid flows from the 1 st chromatographic column to the Nth chromatographic column through the 2 nd chromatographic column, the 3 rd chromatographic column and the …, and the 1 st chromatographic column to the Nth chromatographic column are connected in series; and (3) the fluid flows from the 2 nd chromatographic column to the 1 st chromatographic column through the 3 rd chromatographic column, the 4 th chromatographic column and the … Nth chromatographic column, the 2 nd chromatographic column, the Nth chromatographic column and the 1 st chromatographic column are connected in series, and the rest series connection state is repeated.
The technical principle of the invention is as follows: in multi-column switching cycle chromatography, the concentration bands of the separated components move in a closed loop internal circulation formed by a plurality of columns, and in the band migration process, if the elution capacity of the mobile phase in the upstream column is stronger than that of the mobile phase in the downstream column, the migration rate of the back edge of each concentration band of the separated components can be faster than that of the front edge of the concentration band, so that a contraction effect is generated on the concentration bands to overcome and counteract the expansion of the concentration bands; in addition, more importantly, the chromatographic columns are designed into three or more than three chromatographic columns, the problem of low utilization rate of the stationary phase of the two chromatographic columns can be well solved, and the technical effect that the utilization rate is over 50 percent is realized by reasonably distributing the number of the chromatographic columns, the feeding and discharging positions, the feeding and discharging components and the like.
The invention is based on the recognition that the elution capacity of the mobile phase in the upstream column is stronger than that of the mobile phase in the downstream column by the following three measures, and the utilization rate of the chromatographic column reaches more than 50 percent:
(1) the volume percentage content of the solvent with strong elution capability in the eluent is higher than that of the solvent with strong elution capability in the modifier;
(2) the temperature of the eluent is higher than that of the modifier;
(3) simultaneously, the volume percentage content of the solvent with strong elution capability in the eluent is higher than that of the solvent with strong elution capability in the modifier, and the temperature of the eluent is higher than that of the modifier.
Compared with the existing double-column solvent gradient circulation chromatography, the stationary phase utilization rate of the technical scheme of the invention is higher. As shown in fig. 2, taking the six-column switching cycle chromatography as an example, in each switching cycle under the optimal operating conditions, the tail of the target component band always moves from the first column counted from the left in the figure to the second column, while the front of the band moves from the fifth column to the last column, the target component band always covers the stationary phase region 5 columns long, and the unused stationary phase is always located inside the first and last columns, accounting for 1/6 of the total stationary phase, i.e. the stationary phase utilization rate can be as high as 5/6, which is much higher than 1/2 utilization rate of the two-column system shown in fig. 1. Obviously, the more columns, the higher the stationary phase utilization. The stationary phase utilization rate of the multi-column system is improved, and the processing capacity of the multi-column system per unit column volume is improved accordingly.
The method of the invention also comprises a final target component recovery step, and can be realized by adopting the following two methods according to the requirement on the purity of the target product:
during the purification process, the concentrations in the system effluent and the feed solution are monitored to ensure that the ratio of the concentrations of the target component to the non-target component in the system effluent (i.e., the concentration of the target component divided by the concentration of the non-target component) is less than the ratio of the concentrations of the target component to the non-target component in the feed solution; the content ratio of the target component to the non-target component in the system (namely the concentration of the target component divided by the concentration of the non-target component) is gradually increased along with the progress of the purification process, when the content ratio of the target component to the non-target component in the system reaches the requirement, the purification operation is stopped, and the 1 st chromatographic column, the 2 nd chromatographic column, the …, the N-1 st chromatographic column and the Nth chromatographic column are eluted to recover the target component;
or
Monitoring the concentration of target and non-target components in the effluent of the system and the volume of the effluent during the purification process to ensure that the mass of target and non-target components in the effluent is less than the mass of target and non-target components added to the system during the feed stage; so as the purification process proceeds, the difference in spatial distribution of the concentration bands of the target component and the non-target component inside the system increases gradually; stopping the column switching operation when the separation of the target component from the non-target component concentration band meets the requirement; while the eluent continues to flow and collects the non-target component and the target component in sequence from the system outlet.
When the second recovery method of the present invention is adopted, a detector may be provided at the outlet of each column to monitor the separation of the target component and the non-target component concentration bands, to help qualitatively determine whether the separation requirement is met, for example, two bands are substantially separated and the overlapping part is not much.
The invention has the advantages and beneficial effects that: the invention provides a method for synchronously separating and concentrating a target component from a raw material by adopting multi-column switching cycle chromatography, which can improve the purity of the target component and increase the concentration of the target component in a product solution, thereby reducing the burden of recovering the target component from the product solution. The multi-column switching cycle chromatographic separation system provided by the invention has the advantages of high stationary phase utilization rate, low separation and purification cost and the like.
Drawings
Figure 1 is a graph of the shift of the target component band (shaded) within a prior art two-column solvent gradient cycle chromatogram within a certain switch.
Figure 2 is a graph showing the shift of the target component band (shaded) within a six-column solvent gradient cycle chromatogram of an example of the present invention within a certain switch.
FIG. 3 is a schematic diagram of the structure of the initial state of the multi-column switching cycle chromatographic separation system with 1 st to 6 th chromatographic columns connected in series in sequence.
FIG. 4 is a schematic diagram of the structure of the 2 nd to 1 st chromatographic columns connected in series in sequence after the first switching of the multi-column switching cycle chromatographic separation system of the present invention.
FIG. 5 is a schematic diagram of the 3 rd to 2 nd chromatographic columns of the multi-column switching cycle chromatographic separation system after the second switching in series.
FIG. 6 is a schematic diagram of the structure of the multi-column switching cycle chromatographic separation system in which the 4 th to 3 th chromatographic columns after the third switching are sequentially connected in series.
FIG. 7 is a schematic diagram of the structure of the multi-column switching cycle chromatographic separation system in which the 5 th to 4 th chromatographic columns after the fourth switching are connected in series in sequence.
Fig. 8 is a schematic structural diagram of the 6 th to 5 th chromatographic columns after the fifth switching in the multi-column switching cycle chromatographic separation system of the invention, which are connected in series in sequence.
FIG. 9 is a schematic structural diagram of the multi-column switching cycle chromatographic separation system of the present invention in which the 1 st to 6 th chromatographic columns after the sixth switching are sequentially connected in series.
FIG. 10 is a high performance liquid chromatogram (analysis volume 2. mu.L) of orlistat starting material.
FIG. 11 is a high performance liquid chromatogram enlargement of orlistat starting material (peak 4 is the target, peaks 1 and 3 are the pre-impurity, and peaks 2 and 5 are the post-impurity in the recycle chromatogram).
FIG. 12 is high performance liquid chromatogram of a recycle chromatography effluent a high performance liquid chromatogram (analysis volume 100. mu.L) of a recycle chromatography effluent.
FIG. 13 is a high performance liquid chromatogram of recycle chromatography fraction 1.
FIG. 14 is a high performance liquid chromatogram of recycle chromatography fraction 2.
FIG. 15 is a high performance liquid chromatogram of recycle chromatography fraction 3.
Detailed Description
The present invention is described in further detail below by way of examples, but the present invention is not limited to only the following examples.
The technical scheme of the invention is explained in detail below by taking the separation and purification of orlistat as an example. Orlistat starting material was purchased from biotechnology limited, north Hebei Haidede. On a carbon octadecyl bonding silica gel column, ethanol/water (88/12, v/v) is used as a mobile phase, and a plurality of front impurity peaks and rear impurity peaks are arranged before and after the main peak of orlistat, wherein the peak area percentage of each impurity is higher than 0.1%. The invention provides a multi-column switching cycle chromatographic separation method for synchronously separating and concentrating target components from raw materials, which is used for separating and concentrating orlistat serving as a main component from orlistat raw materials and ensuring that the peak area percentage of each impurity is lower than 0.1%. The timing of the switching of the eluent inlet and effluent outlet positions as described in the present invention depends on the specific separation system and system pressure capacity and may be equal or unequal.
The valve component of the invention is a plurality of valves which are conventional in the industry, such as three valves (three-way valves), six valves (six-way valves) and the like, and is connected with the system of the invention by combining a pipeline; can be reasonably equipped according to the switching requirement of an actual chromatographic column system, and the structure can be realized by a person skilled in the art according to the requirement without creative labor.
1. Multi-column switching circulating chromatographic separation system
The specific chromatographic columns designated by the first chromatographic column to the Nth chromatographic column are not changed, the first chromatographic column is always the first chromatographic column, the Nth chromatographic column is always the Nth chromatographic column, and only the serial connection mode of the chromatographic columns is changed (the specific column designated by each chromatographic column is not changed, and only the position (namely the serial connection mode) is changed).
The multi-column switching cycle chromatographic separation system of the invention comprises: n chromatographic columns, wherein N is an integer more than or equal to 3, and a valve component; the N chromatographic columns are always connected in series through a valve component, an eluent flows from an inlet of the 1 st chromatographic column to an outlet of the N (last) chromatographic column, and the series connection mode of the N chromatographic columns is switched at regular intervals; the specific switching process is as follows: firstly, the eluent flows from the inlet of the 1 st chromatographic column to the outlet of the Nth chromatographic column; after a certain time, the system is switched to be connected with the 2 nd chromatographic column, the 3 rd chromatographic column, the … th chromatographic column, the Nth chromatographic column and the 1 st chromatographic column in series, and eluent flows from the inlet of the 2 nd chromatographic column to the outlet of the 1 st chromatographic column along the series sequence; after a certain time, the system is switched to the 3 rd chromatographic column, the 4 th chromatographic column, the … th chromatographic column, the Nth chromatographic column, the 1 st chromatographic column and the 2 nd chromatographic column in series, and the eluent flows from the inlet of the 3 rd chromatographic column to the outlet of the 2 nd chromatographic column along the series sequence; in this way, after the system is switched for (N-1) times, the serial connection mode is that the Nth chromatographic column, the 1 st chromatographic column, the 2 nd chromatographic column, … and the Nth-1 st chromatographic column are connected in series, and eluent flows from the inlet of the Nth chromatographic column to the outlet of the Nth-1 st chromatographic column along the serial connection sequence; after the columns are switched for N times, the tandem mode is that the 1 st, the 2 nd, … th and the Nth chromatographs are connected in series, and eluent flows from the inlet of the 1 st chromatographic column to the outlet of the Nth chromatographic column; the system is switched back and forth according to the cycle described above, and during operation, the modifying agent flows into the system from the inlet of any one of the columns located downstream of the eluent inlet, and wherein the modifying agent has a weaker elution capacity than the eluent.
Feeding: dissolving the raw material to be separated into any solvent capable of dissolving the raw material to prepare a raw material solution; the initial state of the multi-column switching cycle chromatographic separation system is that the 1 st, the 2 nd, the 3 rd, the … th and the Nth chromatographic columns are connected in series, and then raw material liquid is added into the system from the inlet of any chromatographic column, so that a target object is adsorbed in the system;
in the system, the raw material liquid can be added into the system from any chromatographic column inlet in the feeding process, and the optimal feeding position is that the raw material liquid is added from the 1 st chromatographic column inlet, so that the target object is adsorbed in the system. During feeding, the elution pump and the modification pump are not operated, and the valve component is not switched.
In the system: the raw material liquid conveying pipeline and the eluent (eluent) conveying pipeline are connected through a three-way joint, and plunger pumps (the raw material liquid pipeline can be called a raw material pump, the eluent pipeline can be called an elution pump, and the modifier pipeline can be called a modification pump) are arranged on the pipelines of the raw material liquid, the eluent and the modifier to provide power for the circulating flow of the fluid in the system.
The multi-column switching cycle chromatographic separation system of the embodiment mainly comprises 3 plunger pumps for conveying raw material liquid, eluent and modifier, and 6 chromatographic columns (with the inner diameter of 1cm and the length of 10 cm) filled with C18 silica gel; the eluent is ethanol/water (88/12, v/v), and the water content of the modifying agent is higher than that of the eluent, so the elution capacity of the eluent is stronger than that of the modifying agent. Orlistat raw material was dissolved in ethanol/water (86/14, v/v) to make a raw material solution with a total concentration of 20 mg/mL.
2. Inspection of finished product
And analyzing the purity of the product by high performance liquid chromatography. The high performance liquid chromatography system comprises: a Dalian Eitide 1201 pump, a Phenomenex Knitex XB-18 column (4.6X 150mm, 5 μm), a Dalian Eitide 1201 detector; liquid chromatography conditions: the mobile phase is acetonitrile/water (volume ratio 90/10), the flow rate is 1.0mL/min, the detection wavelength is 210nm, and the column temperature is 30 ℃.
Example 1 separation and concentration of orlistat using a Multi-column switching cycle chromatographic separation System
The HPLC chromatogram (2. mu.L analysis volume) of the orlistat starting material is shown in FIG. 10, and the HPLC chromatogram of the orlistat starting material is enlarged (in the cycle chromatogram, peak 4 is the target, peaks 1 and 3 are the previous impurities, and peaks 2 and 5 are the next impurities) is shown in FIG. 11.
The initial series sequence of the six chromatographic columns of the separation system is shown in figure 3, and the 1 st chromatographic column, the 2 nd chromatographic column, the 3 rd chromatographic column … and the 6 th chromatographic column are connected in series in sequence; the initial feeding state is firstly: connecting the 1 st, 2 nd, … th and 6 th chromatographic columns in series, adding the raw material liquid into the system from the inlet of the 1 st chromatographic column at room temperature, when the volume of the added raw material liquid is 340mL, the separated components just flow out from the outlet of the 5 th chromatographic column, stopping feeding, wherein the feeding state is shown as the feeding state in the attached figure 2, and the shadow in the chromatographic columns represents the distribution condition of the raw material liquid; then, the elution pump and the finishing pump were started simultaneously, and eluent and finishing agent (water) were added to the system from the 1 st and 3 rd chromatography column inlets at 4mL/min and 0.2mL/min, respectively (as shown in FIG. 2 at the initial time and in the state of FIG. 3). After 10min, the system switches for the 1 st time, and at this time, as shown in fig. 4, the series mode is: the 2 nd chromatographic column, the 3 rd chromatographic column, the … th chromatographic column, the 6 th chromatographic column and the 1 st chromatographic column are sequentially connected in series, and an eluent and a modifier respectively flow into the system from inlets of the 2 nd chromatographic column and the 4 th chromatographic column at the flow rates of 4mL/min and 0.2 mL/min; after 10min, the system switches for the 2 nd time, and at this time, as shown in fig. 5, the serial mode is: the 3 rd chromatographic column, the 4 th chromatographic column, the 5 th chromatographic column, the 6 th chromatographic column, the 1 st chromatographic column and the 2 nd chromatographic column are sequentially connected in series, and an eluent and a modifier flow into the system from inlets of the 3 rd chromatographic column and the 5 th chromatographic column at the flow rates of 4mL/min and 0.2mL/min respectively; after 10min, the system switches for the 3 rd time, and at this time, as shown in fig. 6, the serial mode is: the 4 th chromatographic column, the 5 th chromatographic column, the 6 th chromatographic column, the 1 st chromatographic column, the 2 nd chromatographic column and the 3 rd chromatographic column are sequentially connected in series, and an eluent and a modifier flow into the system from inlets of the 4 th chromatographic column and the 6 th chromatographic column at 4mL/min and 0.2mL/min respectively; after 10min, the system is switched for the 4 th time, at this time, as shown in FIG. 7, the 5 th, 6 th, 1 st, 2 nd, 3 rd and 4 th chromatographic columns are sequentially connected in series, and the eluent and the modifier flow into the system from the inlets of the 5 th chromatographic column and the 1 st chromatographic column at the flow rates of 4mL/min and 0.2mL/min respectively; after 10min, the system is switched for the 5 th time, at this time, as shown in fig. 8, the 6 th chromatographic column, the 1 st chromatographic column, the 2 nd chromatographic column, the 3 rd chromatographic column, the 4 th chromatographic column and the 5 th chromatographic column are sequentially connected in series, and the eluent and the modifier flow into the system from the inlets of the 6 th chromatographic column and the 2 nd chromatographic column at the flow rates of 4mL/min and 0.2mL/min respectively; after 10min, the system is switched for the 6 th time, at this time, as shown in FIG. 9, the system returns to the initial state, the 1 st chromatographic column, the 2 nd chromatographic column, the … th chromatographic column and the 6 th chromatographic column are connected in series in sequence, and the eluent and the modifier flow into the system from the inlets of the 1 st chromatographic column and the 3 rd chromatographic column at the flow rates of 4mL/min and 0.2mL/min respectively.
The above steps are repeated in cycles, and the switching is carried out once every 10min for 13 times. In the separation and purification process, the effluent of the system contains impurities and trace orlistat (specifically shown in figure 12, which is a high performance liquid chromatogram (analysis volume 100 μ L) of the effluent of the circulation chromatography). And after the operation is carried out for 130min, the system stops switching, and the 2 nd chromatographic column, the 3 rd chromatographic column, the … th chromatographic column, the 6 th chromatographic column and the 1 st chromatographic column are connected in series in sequence. The dressing pump was then turned off and the elution pump was turned on separately to elute the system at a flow rate of 5mL/min, collecting three fractions from the system outlet in stages: fraction 1 mainly contains precursor impurities and a small amount of orlistat (specifically shown in figure 13, which is a high performance liquid chromatogram of fraction 1); fraction 2 contains orlistat and no pre-impurity and post-impurity (specifically as shown in figure 14, which is a high performance liquid chromatogram of fraction 2); fraction 3 mainly contains late impurities and a small amount of orlistat (specifically, as shown in fig. 15, namely, a high performance liquid chromatogram of fraction 3). Fraction 2 is the qualified product, and the yield is 95%.
Comparative example:
the dual column switching cycle chromatography shown in FIG. 1 consisted of two columns of 1cm internal diameter and 10cm column length, orlistat concentration was the same as in inventive example 1, but the sample volume was 60mL, the feed volume per column volume was 3.82mL, and the yield was 92%. The multi-column switching cycle chromatography (taking six columns as an example) provided by the invention has the sample volume of 340mL and the feed volume per column volume of 7.22 mL. In conclusion, it is because the present invention improves the stationary phase utilization, thereby increasing the throughput per unit column volume.
Example 2 separation and concentration of orlistat Using Multi-column switching cycle chromatographic separation System
The initial series sequence of the six chromatographic columns of the separation system is shown in figure 3, and the 1 st chromatographic column, the 2 nd chromatographic column, the 3 rd chromatographic column … and the 6 th chromatographic column are connected in series in sequence; the initial feeding state is firstly: sequentially connecting the 1 st chromatographic column, the 2 nd chromatographic column, … th chromatographic column and the 6 th chromatographic column in series, adding the raw material liquid into the system from the inlet of the 1 st chromatographic column at room temperature, and when the volume of the added raw material liquid is 410mL, enabling the separated components to just flow out from the outlet of the 6 th chromatographic column, and stopping feeding; then, the elution pump and the modification pump were simultaneously started, and eluent and a modifier (ethanol/water =20/80(v/v)) were added to the system at a flow rate of 4mL/min and 1.0mL/min from the 1 st and 2 nd chromatography column inlets, respectively. When the eluent and the modifier are delivered, the eluent is heated to 45 ℃ and the modifier is cooled to 5 ℃. After 10min, the system switches for the 1 st time, as shown in fig. 4: the 2 nd chromatographic column, the 3 rd chromatographic column, the … th chromatographic column, the 6 th chromatographic column and the 1 st chromatographic column are sequentially connected in series, and an eluent and a modifier respectively flow into the system from inlets of the 2 nd chromatographic column and the 4 th chromatographic column at the flow rates of 4mL/min and 0.2 mL/min; after 10min, the system is switched for the 2 nd time, at this time, as shown in fig. 5, the 3 rd chromatographic column, the 4 th chromatographic column, the 5 th chromatographic column, the 6 th chromatographic column, the 1 st chromatographic column and the 2 nd chromatographic column are sequentially connected in series, and the eluent and the modifier flow into the system from the inlets of the 3 rd chromatographic column and the 5 th chromatographic column at the flow rates of 4mL/min and 0.2mL/min respectively; after 10min, the system is switched for the 3 rd time, at this time, as shown in fig. 6, the 4 th chromatographic column, the 5 th chromatographic column, the 6 th chromatographic column, the 1 st chromatographic column, the 2 nd chromatographic column and the 3 rd chromatographic column are sequentially connected in series, and the eluent and the modifier flow into the system from the inlets of the 4 th chromatographic column and the 6 th chromatographic column at the flow rates of 4mL/min and 0.2mL/min respectively; after 10min, the system is switched for the 4 th time, at this time, as shown in FIG. 7, the 5 th, 6 th, 1 st, 2 nd, 3 rd and 4 th chromatographic columns are sequentially connected in series, and the eluent and the modifier flow into the system from the inlets of the 5 th chromatographic column and the 1 st chromatographic column at the flow rates of 4mL/min and 0.2mL/min respectively; after 10min, the system is switched for the 5 th time, at this time, as shown in fig. 8, the 6 th chromatographic column, the 1 st chromatographic column, the 2 nd chromatographic column, the 3 rd chromatographic column, the 4 th chromatographic column and the 5 th chromatographic column are sequentially connected in series, and the eluent and the modifier flow into the system from the inlets of the 6 th chromatographic column and the 2 nd chromatographic column at the flow rates of 4mL/min and 0.2mL/min respectively; after 10min, the system is switched for the 6 th time, at this time, as shown in FIG. 9, the system returns to the initial state, the 1 st chromatographic column, the 2 nd chromatographic column, the … th chromatographic column and the 6 th chromatographic column are connected in series in sequence, and the eluent and the modifier flow into the system from the inlets of the 1 st chromatographic column and the 3 rd chromatographic column at the flow rates of 4mL/min and 0.2mL/min respectively.
The above steps are repeated in cycles, and the switching is performed once every 10min for 11 times. Due to sample loading overload, in the separation and purification process, the effluent liquid of the system contains a certain amount of orlistat besides impurities. After 12 times of switching operation, the system stops switching, and the 1 st chromatographic column, the 2 nd chromatographic column, the 3 rd chromatographic column, the … th chromatographic column and the 6 th chromatographic column are sequentially connected in series. The dressing pump was then turned off and the elution pump was turned on separately to elute the system at a flow rate of 5mL/min, collecting three fractions from the system outlet in stages: fraction 1 contains mainly promiscuity and a small amount of orlistat; fraction 2 contains orlistat, does not contain pre-impurity and post-impurity, and the yield of orlistat is 85%; fraction 3 contains mainly late impurities and very dilute orlistat concentrations.
Through the embodiments of the present system described above, it can be seen that the separation system of the present invention can increase the concentration of the target component in the product solution while improving the purity of the target component, thereby reducing the burden of recovering the target component from the product solution. The double-column switching cycle chromatographic separation system provided by the invention has the advantages of simple structure, convenience in operation, simple treatment after separation and purification, and reduction of separation and purification costs.
The above examples are illustrative and further illustrative of the present invention and are not intended to be limiting, and any modification made within the spirit and scope of the present invention will fall within the scope of the present invention.
Claims (9)
1. A multi-column switching cycle chromatographic separation system for separating and concentrating target components from raw materials is characterized in that: the system comprises: n chromatographic columns, wherein N is an integer more than or equal to 3, and a valve component; the N chromatographic columns are always connected in series through a valve assembly, and the series connection mode of the N chromatographic columns is switched at regular intervals; the specific switching process is as follows: firstly, enabling eluent to flow from an inlet of a 1 st chromatographic column to an outlet of an Nth chromatographic column; after a certain time, the system is switched to be connected with the 2 nd chromatographic column, the 3 rd chromatographic column, the … th chromatographic column, the Nth chromatographic column and the 1 st chromatographic column in series, and eluent flows from the inlet of the 2 nd chromatographic column to the outlet of the 1 st chromatographic column along the series sequence; after a certain time, the system is switched to the 3 rd chromatographic column, the 4 th chromatographic column, the … th chromatographic column, the Nth chromatographic column, the 1 st chromatographic column and the 2 nd chromatographic column in series, and the eluent flows from the inlet of the 3 rd chromatographic column to the outlet of the 2 nd chromatographic column along the series sequence; in this way, after the system is switched for (N-1) times, the serial connection mode is that the Nth chromatographic column, the 1 st chromatographic column, the 2 nd chromatographic column, … and the Nth-1 st chromatographic column are connected in series, and eluent flows from the inlet of the Nth chromatographic column to the outlet of the Nth-1 st chromatographic column along the serial connection sequence; after the system is switched for N times, the series connection mode is that the 1 st, the 2 nd, … th and the Nth chromatographs are connected in series, and eluent flows to the outlet of the Nth chromatographic column from the inlet of the 1 st chromatographic column; the system is switched back and forth according to the cycle, and in the operation process, the modifier flows into the system from the inlet of any chromatographic column positioned at the downstream of the eluent inlet, and the elution capacity of the modifier is weaker than that of the eluent;
the method for separating and concentrating the target component by using the multi-column switching cycle chromatographic separation system for separating and concentrating the target component from the raw material comprises the following specific steps:
(1) feeding: dissolving the raw material to be separated into any solvent capable of dissolving the raw material to prepare a raw material solution; the initial state of the multi-column switching cycle chromatographic separation system is that the 1 st chromatographic column, the 2 nd chromatographic column, the … th chromatographic column and the Nth chromatographic column are sequentially connected in series, and then raw material liquid is added into the system from the inlet of any chromatographic column, so that a target object is adsorbed in the system;
(2) and (3) purification: the multi-column switching cycle chromatographic system is still in the state that 1 st, 2 nd, … th and Nth chromatographic columns are connected in series in sequence, then eluent flows into the system from the 1 st chromatographic column inlet, and modifier flows into the system from any chromatographic column inlet downstream of the eluent inlet; after a certain time, the system is switched, wherein the 2 nd chromatographic column, the 3 rd chromatographic column, the … th chromatographic column, the Nth chromatographic column and the 1 st chromatographic column are connected in series, eluent flows into the system from the 2 nd chromatographic column inlet, and a modifying agent flows into the system from any chromatographic column inlet downstream of the eluent inlet; after a certain time, the system is switched again, wherein 3 rd chromatographic column, 4 th chromatographic column, … th chromatographic column, Nth chromatographic column, 1 st chromatographic column and 2 nd chromatographic column are connected in series, eluent flows into the system from the 3 rd chromatographic column inlet, and modifier flows into the system from any chromatographic column inlet downstream of the eluent inlet; in this way, after the system is switched for (N-1) times, the Nth, the 1 st, the 2 nd, the … th and the N-1 st chromatographies are connected in series, eluent flows from the inlet of the Nth chromatographic column to the outlet of the N-1 st chromatographic column, and modifier is added into the system from any one of the inlets of the 1 st to the N-1 st chromatographic columns which are positioned at the downstream of the inlet of the eluent; after the system is switched for N times, connecting the 1 st, the 2 nd, the … th and the Nth chromatographs in series, enabling eluent to flow from the inlet of the 1 st chromatographic column to the outlet of the Nth chromatographic column, and adding a modifier into the system from any one of the inlets from the 2 nd to the Nth chromatographic column downstream of the eluent inlet; and (3) circularly and repeatedly switching the system according to the mode, namely, circularly and repeatedly changing the serial mode of the N chromatographic columns and the inlet positions of the eluent and the modifier, and finally obtaining the purified target product.
2. The multi-column switching cycle chromatographic separation system for separating and concentrating a target component from a feed material of claim 1, wherein: the elution capacity of the modifier is weaker than that of the eluent, and the method is realized by the following specific steps: the eluent is heated and the modifying agent is cooled to ensure that the temperature of the eluent is higher than the temperature of the modifying agent.
3. The multi-column switching cycle chromatographic separation system for separating and concentrating a target component from a feed material of claim 1, wherein: the elution capacity of the modifier is weaker than that of the eluent, and the method is realized by the following specific steps: the volume percentage content of the solvent with strong elution capacity in the eluent is higher than that of the solvent with strong elution capacity in the modifier.
4. The multi-column switching cycle chromatographic separation system for separating and concentrating target components from a feedstock of claim 2 or 3, wherein: the elution capacity of the modifier is weaker than that of the eluent, and the method is realized by the following specific steps: heating the eluent and cooling the modifying agent, wherein the volume percentage of the solvent with strong elution capacity in the eluent is higher than that of the solvent with strong elution capacity in the modifying agent.
5. The multi-column switching cycle chromatographic separation system for separating and concentrating a target component from a feed material of claim 1, wherein: the time intervals for the system switching are not necessarily equal.
6. The multi-column switching cycle chromatographic separation system for separating and concentrating a target component from a feed material of claim 5, wherein: the system for adding from the inlet of any chromatographic column in the step (1) is added from the inlet of the 1 st chromatographic column.
7. The multi-column switching cycle chromatographic separation system for separating and concentrating a target component from a feed material of claim 5, wherein: after a certain time, namely the switching time of the positions of the eluent and the modifier inlet and the effluent outlet is switched once for 2-20 min.
8. The multi-column switching cycle chromatographic separation system for separating and concentrating a target component from a feed material of claim 5, wherein: in the feeding process, neither the elution pump nor the modification pump is operated, the valve component is not switched, the raw material is added from the inlet of the chromatographic column 1, when the target component flows out of the (N-1) th chromatographic column, the feeding is stopped, and the corresponding feeding amount is the most suitable feeding;
in the system handover, the time intervals of any two adjacent handovers may be equal or unequal.
9. The multi-column switching cycle chromatographic separation system for separating and concentrating a target component from a feed material of claim 5, wherein: the 1 st, 2 nd, … th and Nth chromatographic columns are connected in series, or the 2 nd, 3 rd, … th, Nth and 1 st chromatographic columns are connected in series, and are defined sequentially from the sequential flowing direction of the medium flowing in the system.
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