CN111548434B - Separation and purification method of gamma cyclodextrin - Google Patents
Separation and purification method of gamma cyclodextrin Download PDFInfo
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Abstract
The invention discloses a separation and purification method of gamma cyclodextrin. Firstly, dissolving a gamma cyclodextrin sample by using a mixed solution, and filtering insoluble substances; then loading the filtered sample solution to an ODS column; then, a single-column internal circulation mode or a double-column internal circulation mode is adopted for separation; and finally, carrying out gradient elution or isocratic elution by using a mixed solvent as a flowing relative sample, collecting target peaks in a segmented manner, and summarizing the components of which the target peaks meet the requirements to obtain a purified sample. The method has the advantages of mild separation conditions, high separation efficiency, high separation speed, strong universality and the like, well realizes the separation and purification of the gamma cyclodextrin sample, can realize enrichment in a short time, can realize purification by changing elution conditions and switching valves and even other types of compounds, and has good application prospect.
Description
Technical Field
The invention belongs to the field of medicine separation and purification, and particularly relates to a separation and purification method of gamma cyclodextrin, which utilizes assembled circulating equipment with a single-column or double-column internal circulation enhanced column effect mode and adopts 0.1% trifluoroacetic acid water and methanol or acetonitrile as mobile phases to obtain high-purity gamma cyclodextrin.
Background
Gamma-Cyclodextrin (gamma-Cyclodextrin) is cyclic oligosaccharide consisting of 8D-glucopyranose units, a large cylindrical cavity which is wide at the top and narrow at the bottom and has two openings at two ends can be formed in a gamma-Cyclodextrin host molecule, and the cavity can contain object molecules in a wider range, so that the solubility of the object molecules is improved, and the stability of the object molecules is further improved, therefore, the gamma-Cyclodextrin is widely applied to the fields of medicine, cosmetics, food, chemical industry and the like.
The gamma Cyclodextrin (γ -Cyclodextrin) has the following molecular formula:
since gamma cyclodextrin has a larger cavity and polarity, the separation difficulty of gamma cyclodextrin is greatly increased, so far, relevant journal documents, patent works and the like at home and abroad have only fresh reports on a separation and purification method of gamma cyclodextrin, and gamma cyclodextrin is mostly separated and purified by a solvent method in industry, namely, the gamma cyclodextrin is obtained by adding an organic solvent as a complex and selectively extracting. However, the solvent extraction method has high cost, and most solvents have toxicity and flammability, are not environment-friendly and have great potential danger. In addition, CN109400760A reports a method for purifying gamma-cyclodextrin by using cyclodextrin hydrolase, but the method requires a long time for screening of early stage hydrolase, and requires strict control over each operation, such as reaction substrate, reaction temperature and reaction time, otherwise the enzyme is easily inactivated, resulting in reaction failure, and is not suitable for industrial scale-up production. Gamma cyclodextrin
Although the above documents report methods for purifying gamma cyclodextrin, there is no obvious method for rapidly purifying cocoa and facilitating industrial amplification, and because gamma-cyclodextrin has large polarity, many structural analogs, complex structure and contains a plurality of chiral centers, the gamma-cyclodextrin is hardly reserved during the preparation of chromatography, and secondary purification cannot be realized after the completion of primary purification, so that the purification cost is high, the solvent consumption is large, and the methods have certain disadvantages and limitations. In order to meet the requirements of separating high-purity gamma cyclodextrin and realizing industrial scale-up production, a universal purification method suitable for the gamma cyclodextrin needs to be developed.
Disclosure of Invention
In order to solve the problems in the background technology, the invention provides a separation and purification method of gamma cyclodextrin, which utilizes a self-assembled circulating preparation device and adopts trifluoroacetic acid aqueous solution and methanol as mobile phases to obtain high-purity gamma cyclodextrin.
And a universal cyclic preparation, separation and purification device for gamma-cyclodextrin is designed, so that the gamma-cyclodextrin can be easily self-assembled in two cyclic modes, namely a single-column and a double-column mode, and high-purity gamma-cyclodextrin can be obtained.
In order to achieve the purpose, the technical scheme of the invention is as follows:
firstly, dissolving a gamma cyclodextrin sample by using a mixed solution, and filtering insoluble substances; then loading the filtered sample solution to an ODS column; then, a single-column internal circulation mode or a double-column internal circulation mode is adopted for separation; and finally, carrying out gradient elution or isocratic elution by using a mixed solvent as a flow relative sample, collecting target peaks in sections, and summarizing components of which the target peaks meet requirements (HPLC purity is more than or equal to 99%) to obtain a purified sample.
The mixed solution is composed of 1/1 vol% methanol and water, and the mixed solvent mainly comprises methanol and trifluoroacetic acid, and comprises 0.1% trifluoroacetic acid (TFA) aqueous solution as solution A and methanol as solution B.
The target peak is specifically a peak with the peak-off time of 65-75 min in an isocratic elution mode or a peak with the peak-off time of 115-140 min in a gradient elution mode.
The isocratic elution process is as follows: the mobile phase is a mixed solution of 77% volume fraction solution A and 23% volume fraction solution B within 0-80 min.
The gradient elution process is as follows: the mobile phase is a mixed solution of 95% volume fraction of solution A and 5% volume fraction of solution B within 0-15 min; the mobile phase is a mixed solution of 77 volume percent of solution A and 23 volume percent of solution B within 15.01-140 min; within 140.01-150min, the mobile phase is a mixed solution of 20% volume fraction of solution A and 80% volume fraction of solution B; in 150.01-173min, the mobile phase is only a B solution with 100% volume fraction. The gradient elution thus treated allows better purification and separation.
The mobile phase contains trifluoroacetic acid, which is known or determinable to those skilled in the art, and can be formic acid, acetic acid, phosphoric acid, trifluoroacetic acid, preferably trifluoroacetic acid.
Preferably, the trifluoroacetic acid in the 0.1% trifluoroacetic acid aqueous solution is 0.05-1%, preferably 0.1%.
Preferably, the ODS column is equilibrated before loading, wherein the equilibration time is ≥ 15min, preferably 15 min.
The method adopts the following cyclic preparation, separation and purification device which comprises a detector, a first pump, a second pump, a first column, a second column, a first switching valve, a second switching valve, a third switching valve and a fourth switching valve; the second mobile phase output end, the input end of the second pump, the fraction collecting port and the output port of the detector are respectively connected to three valve ports of the first switching valve, the first end of the second column, the output end of the second pump and the waste liquid collecting port are respectively connected to three valve ports of the second switching valve, the second end of the second column and the first end of the first column are respectively connected to two valve ports of the third switching valve, a sample of gamma cyclodextrin is contained in the first column, the first mobile phase output end, the input end of the first pump and the input port of the detector are respectively connected to three valve ports of the fourth switching valve, the output end of the first pump is connected to the first end of the first column, the second end of the second column, the output end of the second pump and the waste liquid collecting port are respectively connected to three valve ports of the second switching valve, meanwhile, one valve port of the second switching valve is directly communicated with one valve port of the third switching valve, and one valve port of the third switching valve is directly communicated with one valve port of the fourth switching valve.
The method is specifically performed under the control of the device as follows:
1) and (3) crude product loading: dissolving and filtering a gamma cyclodextrin sample by using the mixed solution, loading the filtered sample solution to a first column, and then controlling the following steps: the first pump is started to work, and the second pump is not started to work; opening a first switching valve to enable the output end of the detector to be communicated with the fraction collecting port; closing the second switching valve and not working; opening the third switching valve to enable the second end of the first column to be communicated with the input end of the detector after sequentially passing through the third switching valve and the fourth switching valve; opening a fourth switching valve to enable the first mobile phase output end to be communicated with the input end of the first pump; the first mobile phase enters a first pump through a flow channel of the fourth switching valve after being output, is pumped by the first pump, then sequentially enters a detector through the first column, a flow channel of the third switching valve and the other flow channel of the fourth switching valve, and enters a fraction collection port through a flow channel of the first switching valve after being output from the detector; most of samples in the first column are loaded into the detector, and the switching valve enters a single-column internal circulation mode or a double-column internal circulation mode after the detector to be detected detects a target peak;
2) separating by adopting a single-column internal circulation mode or a double-column internal circulation mode;
if the single-column internal circulation mode is adopted, the valve 1, the valve 2 and the valve 3 are switched in sequence after the target peak is detected by the detector, and then the single-column internal circulation mode is entered;
if the mode is a double-column internal circulation mode, the valve 1, the valve 2, the valve 3 and the valve 4 are switched in sequence after the detector detects a target peak, and then the double-column internal circulation mode is entered.
3) Performing gradient elution or isocratic elution by using a mixed solvent as a flow relative sample, switching a valve 1 when a detector to be detected detects that main components are completely separated, starting a second pump, and then performing target peak collection according to a target peak collection mode, wherein the target peak collection mode is divided into a single-column target peak collection mode and a double-column target peak collection mode, and summarizing to obtain a purified sample;
if the single-column internal circulation mode is adopted, a single-column target peak collection mode is adopted for collection;
and if the mode is a double-column internal circulation mode, adopting a double-column target peak collection mode for collection.
4) After collecting the sample, the first and second columns were equilibrated, and the valves switched back to step 1) for the next crude loading.
In the step 2), the single-column internal circulation mode specifically comprises the following steps: the first pump is not started and does not work, and the second pump is started and works; opening a first switching valve to communicate only the output end of the detector with the input end of the second pump; opening a second switching valve to only enable the output end of the second pump to be communicated with the first end of the second column; opening the third switching valve and the fourth switching valve, and only enabling the second end of the second column to be communicated with the input end of the detector after passing through the third switching valve and the fourth switching valve in sequence; the sample liquid in the detector is input into the second pump through a flow channel of the first switching valve, is pumped out from the second pump and then enters the second column through a flow channel of the second switching valve, and the second column flows out and then returns to the detector through a flow channel of the third switching valve and a flow channel of the fourth switching valve in sequence to complete circulation.
In the step 3), the single-column target peak collection mode specifically comprises the following steps: the first pump is not started and does not work, and the second pump is started and works; opening a first switching valve to enable the output end of the detector to be communicated with the fraction collecting port, and enabling the second flowing phase output end to be communicated with the input end of the second pump; opening a second switching valve to only enable the output end of the second pump to be communicated with the first end of the second column; opening the third switching valve and the fourth switching valve, and only enabling the second end of the second column to be communicated with the input end of the detector after passing through the third switching valve and the fourth switching valve in sequence; and the second mobile phase enters the second pump through a flow channel of the first switching valve after being output, is pumped by the second pump and then enters the second column through a flow channel of the second switching valve, flows out of the second column and then sequentially enters the detector through a flow channel of the third switching valve and a flow channel of the fourth switching valve, and enters the fraction collection port through the other flow channel of the first switching valve after being output from the detector.
In the step 2), the double-column internal circulation mode specifically comprises the following steps: the first pump and the second pump are started to work; opening a first switching valve to communicate only the output end of the detector with the input end of the second pump; opening a second switching valve to only enable the output end of the second pump to be communicated with the first end of the second column; opening the third switching valve to communicate only the second end of the second column with the second end of the first column; opening a fourth switching valve to enable only the input end of the first pump to be communicated with the input end of the detector, and enabling the first pump to work reversely; the sample liquid in the detector is input into the second pump through a circulation channel of the first switching valve, is pumped out from the second pump and then enters the second column through a circulation channel of the second switching valve, the second column flows out and then enters the first column through a circulation channel of the third switching valve in sequence, the first column and the second column flow out and then return to the detector through a circulation channel of the first pump and the fourth switching valve in sequence, and circulation is completed.
In the step 3), the double-column target peak collection mode specifically comprises the following steps: the first pump and the second pump are started to work; opening a first switching valve to enable the output end of the detector to be communicated with the fraction collecting port, and enabling the second flowing phase output end to be communicated with the input end of the second pump; opening a second switching valve to only enable the output end of the second pump to be communicated with the first end of the second column; opening the third switching valve to communicate only the second end of the second column with the second end of the first column; opening a fourth switching valve to enable only the input end of the first pump to be communicated with the input end of the detector, and enabling the first pump to work reversely; the second mobile phase enters the second pump through a flow channel of the first switching valve after being output, is pumped by the second pump and then sequentially enters the second column through a flow channel of the second switching valve, the second column flows out and then enters the first column through a flow channel of the third switching valve, the first column flows out and then enters the detector through a flow channel of the first pump and the fourth switching valve, and the first column flows out and then enters the fraction collection port through the other flow channel of the first switching valve after being output from the detector.
The ODS column is a self-assembled column. The filler type in the self-assembly column is SiliaSphere series ODS, the particle diameter is 50 mu m, and the pore diameter
The purity of the gamma cyclodextrin collected and purified in the invention can reach more than 99.0%, and the recovery rate is more than 90.0%.
The columns involved in the invention can be automatically assembled into columns with different inner diameters and different fillers according to personal requirements, and circulation can be realized outside the machine body, so that the limitation that the traditional liquid chromatography in colleges and universities is limited by chromatographic columns, overhigh pressure during operation and the like can be avoided.
Compared with the prior art, the invention has the beneficial effects that:
the invention can well separate and purify gamma-cyclodextrin, can realize the enrichment of high-purity cyclodextrin in a short time, can purify cyclodextrin and other compounds by changing elution conditions and switching valves, and has better market application prospect.
The method can purify the gamma cyclodextrin product, even other substances with extremely similar structures, has better universality and universality for the structures, and has the advantages of mild separation conditions, high separation efficiency, high separation speed and the like.
The invention can realize 2 circulation modes of single column and double column by adjusting the switching valve, is particularly suitable for samples with extremely large polarity, such as gamma-cyclodextrin, and does not realize separation after the common high performance liquid chromatography passes through the column once, but can realize separation once or for many times in the invention to obtain high-purity gamma-cyclodextrin.
In conclusion, the device can realize the cyclic preparation of two samples at the same time, ensure that the gamma-cyclodextrin can enter the column again without retention in the primary purification, realize the secondary purification, obtain the high-purity gamma-cyclodextrin, save the high instrument cost, be beneficial to the environmental protection of the solvent, realize the industrialization marketization and the like.
Drawings
FIG. 1 is a schematic diagram of a crude product loading pattern (pattern A);
FIG. 2 is a schematic view of a single column internal circulation mode structure (mode B);
FIG. 3 is a schematic diagram of a single-column target peak collection mode (mode C);
FIG. 4 is the HPLC chromatogram of gamma cyclodextrin after purification in example.
In the figure: a first switching valve 1, a second switching valve 2, a third switching valve 3, and a fourth switching valve 4.
Detailed Description
The technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiment of the present invention. The embodiments described in the present invention are only a part of the embodiments, and not all of the embodiments.
As shown in fig. 1 to 3, the experimental apparatus for implementation includes a detector, a first pump, a second pump, a first column, a second column, a first switching valve 1, a second switching valve 2, a third switching valve 3, and a fourth switching valve 4; the second mobile phase output end, the input end of the second pump, the fraction collecting port and the output port of the detector are respectively connected to three valve ports of the first switching valve 1, the first end of the second column, the output end of the second pump and the waste liquid collecting port are respectively connected to three valve ports of the second switching valve 2, the second end of the second column and the first end of the first column are respectively connected to two valve ports of the third switching valve 3, a sample of gamma cyclodextrin is filled in the first column, the first mobile phase output end, the input end of the first pump and the input port of the detector are respectively connected to three valve ports of the fourth switching valve 4, the output end of the first pump is connected to the first end of the first column, the second end of the second column, the output end of the second pump and the waste liquid collecting port are respectively connected to three valve ports of the second switching valve 2, and one valve port of the second switching valve 2 and one valve port of the third switching valve 3 are directly communicated, one port of the third switching valve 3 and one port of the fourth switching valve 4 are directly communicated.
Each of the first switching valve 1, the second switching valve 2, the third switching valve 3, and the fourth switching valve 4 has four ports, and the four ports are controlled to communicate with each other and switched by each switching valve 1.
The first column and the second column are both ODS columns, identical or different.
The specific implementation is that a drain port is arranged on a pipeline between the detector and the valve port of the first switching valve 1, and the drain port is connected with a drain valve. The evacuation process is performed with an evacuation port through an evacuation valve in the presence of air in the flow, typically during the crude loading process.
The detector specifically adopts an ultraviolet detector, the detector switches when the target peak appears about 85min after the sample is separated by the column, the valve 1 is switched, and the product collection mode is entered into fig. 3.
The fraction collecting port is connected with a fraction collector, the waste liquid collecting port is a waste liquid collector, and the fraction collector and the waste liquid collector are glassware.
The first pump and the second pump are both peristaltic pumps, the first pump is used for pumping the first mobile phase, and the second pump is used for pumping the second mobile phase.
In specific implementations, the circulation using the second column may be a single column circulation mode as shown in fig. 2, or a series circulation using both the first column and the second column may be a double column circulation mode.
The second switching valve 2 can realize the switching between the waste liquid and the second column, the third switching valve 3 can realize the switching between the second switching valve 2, the second column and the detector, and the first switching valve 1 can realize the switching between the second pump and the fraction collector.
In specific implementation, the mobile phase is stored in a liquid storage bottle, and 2 small holes are formed in the top of a bottle cap of the liquid storage bottle; the pump is a peristaltic pump and can also be matched with high performance liquid chromatography pumps of various brands for use. Before loading, the first column and the second column are balanced for about 15min, and the sample is loaded from the first pump. The switching valves related to the invention are all four-way switching valves.
The device is adopted to ensure that the circulation processing mode of the sample comprises single-column circulation and double-column circulation, wherein the switching valve 2 and the valve 1 are sequentially switched during the single-column circulation, and the switching valve 2, the valve 3 and the valve 1 are switched during the double-column circulation. The method comprises switching valves 2, 3 and 4 in order at 45min, and entering single-column internal circulation mode in FIG. 2. Or switching the valve 2 and the valve 1 in sequence at 75min, starting the second pump, and entering a double-column internal circulation mode C.
The specific implementation adopts the following cyclic preparation and separation purification devices, and the separation and purification control process is as follows:
1) and (3) crude product loading: and dissolving the sample by using the mixed solution, filtering, loading the filtered sample solution to a first column, and then loading according to a crude product loading mode.
Thus, most of the sample in the first column is loaded into the detector, and the switching valve enters a single-column internal circulation mode or a double-column internal circulation mode after the detector to be detected detects a target peak;
2) separating by adopting a single-column internal circulation mode or a double-column internal circulation mode;
if the single-column internal circulation mode is adopted, the valve 1, the valve 2 and the valve 3 are switched in sequence after the target peak is detected by the detector, and then the single-column internal circulation mode is entered;
if the mode is a double-column internal circulation mode, the valve 1, the valve 2, the valve 3 and the valve 4 are switched in sequence after the detector detects a target peak, and then the double-column internal circulation mode is entered.
3) Performing gradient elution or isocratic elution by using a mixed solvent as a flow relative sample, switching a valve 1 when a detector to be detected detects that main components are completely separated, starting a second pump, and then performing target peak collection according to a target peak collection mode, wherein the target peak collection mode is divided into a single-column target peak collection mode and a double-column target peak collection mode, and summarizing to obtain a purified sample;
if the single-column internal circulation mode is adopted, a single-column target peak collection mode is adopted for collection;
and if the mode is a double-column internal circulation mode, adopting a double-column target peak collection mode for collection.
4) After collecting the sample, the first and second columns were equilibrated, and the valves switched back to step 1) for the next crude loading.
In step 1), as shown in fig. 1, the crude product loading mode specifically comprises: dissolving and filtering a gamma cyclodextrin sample by using the mixed solution, loading the filtered sample solution to a first column, and then controlling the following steps: the first pump is started to work, and the second pump is not started to work; opening a first switching valve 1 to enable the output end of the detector to be communicated with a fraction collecting port; the second switching valve 2 is closed and does not work; opening the third switching valve 3 to enable the second end of the first column to sequentially pass through the third switching valve 3 and the fourth switching valve 4 and then to be communicated with the input end of the detector; opening the fourth switching valve 4 to communicate the first mobile phase output end with the input end of the first pump; the first mobile phase enters a first pump through a flow channel of the fourth switching valve 4 after being output, is pumped by the first pump, then sequentially enters a detector through the first column, a flow channel of the third switching valve 3 and the other flow channel of the fourth switching valve 4, and enters a fraction collection port through a flow channel of the first switching valve 1 after being output from the detector; so that most of the sample in the first column is loaded into the detector, and the switching valve enters a single-column internal circulation mode or a double-column internal circulation mode after the detector detects a target peak.
In step 2), as shown in fig. 2, the single-column internal circulation mode specifically includes: the first pump is not started and does not work, and the second pump is started and works; opening the first switching valve 1 to communicate only the output of the detector and the input of the second pump; opening the second switching valve 2 to only communicate the output end of the second pump with the first end of the second column; opening the third switching valve 3 and the fourth switching valve 4, and only enabling the second end of the second column to be communicated with the input end of the detector after passing through the third switching valve 3 and the fourth switching valve 4 in sequence; the sample liquid in the detector is input into the second pump through a flow channel of the first switching valve 1, is pumped out of the second pump and then enters the second column through a flow channel of the second switching valve 2, and the second column flows out and then returns to the detector through a flow channel of the third switching valve 3 and a flow channel of the fourth switching valve 4 in sequence to complete circulation; the single column internal circulation mode is to turn on the second pump, and only through single column internal circulation, increase column efficiency for separation, as shown in fig. 2 with a solid line labeled flow path.
In the step 2), the double-column internal circulation mode specifically comprises the following steps: the first pump and the second pump are started to work; opening the first switching valve 1 to communicate only the output of the detector and the input of the second pump; opening the second switching valve 2 to only communicate the output end of the second pump with the first end of the second column; opening the third switching valve 3 so that only the second end of the second column and the second end of the first column communicate; opening the fourth switching valve 4 to only enable the input end of the first pump and the input end of the detector to be communicated, and enabling the first pump to work reversely; the sample liquid in the detector is input into the second pump through a flow channel of the first switching valve 1, is pumped out from the second pump and then enters the second column through a flow channel of the second switching valve 2, the second column flows out and then enters the first column through a flow channel of the third switching valve 3 in sequence, and the first column and the second column flow out and then return to the detector through a flow channel of the first pump and the fourth switching valve 4 in sequence to complete circulation. The double-column internal circulation mode is a mode of increasing column effect through series connection of the first column and the second column with internal circulation, so that the purity of sample purification and separation can be better improved, and the yield is improved.
In step 3), as shown in fig. 3, the single-column target peak collection mode specifically includes: the first pump is not started and does not work, and the second pump is started and works; opening the first switching valve 1 so that the output end of the detector is communicated with the fraction collecting port, and the output end of the second mobile phase is communicated with the input end of the second pump; opening the second switching valve 2 to only communicate the output end of the second pump with the first end of the second column; opening the third switching valve 3 and the fourth switching valve 4, and only enabling the second end of the second column to be communicated with the input end of the detector after passing through the third switching valve 3 and the fourth switching valve 4 in sequence; the second mobile phase enters a second pump through a flow channel of the first switching valve 1 after being output, is pumped by the second pump and then enters a second column through a flow channel of the second switching valve 2, flows out of the second column and then sequentially enters a detector through a flow channel of the third switching valve 3 and a flow channel of the fourth switching valve 4, and enters a fraction collection port through the other flow channel of the first switching valve 1 after being output from the detector; this allows the sample in the detector to enter the fraction collection port for purification and collection, as indicated by the solid line labeled flow path in fig. 3.
In the step 3), the double-column target peak collection mode specifically comprises the following steps: the first pump and the second pump are started to work; opening the first switching valve 1 so that the output end of the detector is communicated with the fraction collecting port, and the output end of the second mobile phase is communicated with the input end of the second pump; opening the second switching valve 2 to only communicate the output end of the second pump with the first end of the second column; opening the third switching valve 3 so that only the second end of the second column and the second end of the first column communicate; opening the fourth switching valve 4 to only enable the input end of the first pump and the input end of the detector to be communicated, and enabling the first pump to work reversely; the second mobile phase enters the second pump through a flow channel of the first switching valve 1 after being output, is pumped by the second pump and then sequentially enters the second column through a flow channel of the second switching valve 2, the second column flows out and then enters the first column through a flow channel of the third switching valve 3, the first column flows out and then enters the detector through a flow channel of the first pump and the fourth switching valve 4, and the first column flows out and then enters the fraction collection port through the other flow channel of the first switching valve 1 after being output from the detector. So that the sample in the detector enters the fraction collecting port to be purified and collected.
The embodiment of the invention adopting the device is as follows:
example 1:
separation and purification of gamma cyclodextrin: preparing a solution with the concentration of 2g/mL by adding methanol/water (v/v, 1/1) into gamma cyclodextrin, and adjusting the pH value to 1-2 by using a 5% trifluoroacetic acid water solution. After the solution is clarified, the solution is filtered by a filter membrane with the aperture of 0.45 mu m, and the filtrate is collected for later use. Adopting a self-assembly column, wherein the type of column packing is SiliaSphere series ODS, the particle diameter is 50 mu m, and the pore diameter
0.1% trifluoroacetic acid water solution is used as a mobile phase A, methanol is used as a mobile phase B, and the flow rate is 80 mL/min. As shown in FIG. 1, the first and second columns are equilibrated for about 15min, from the first columnAnd (3) carrying out crude product loading by a pump (marked by a solid line in figure 1), sequentially switching the valve 1, the valve 2 and the valve 3 to be connected according to the solid line in figure 2 at 25min (namely, the detector detects a target peak), and entering a single-column internal circulation mode B shown in figure 2. And switching the valve 1 at 45min, starting a second pump, entering a product collection mode I according to the solid line mark of the figure 3, and collecting 65-75 min of target fraction liquid in a sectional manner. The elution time was 80min, isocratic elution pattern (phase A: 77%, phase B: 23%). The component solutions meeting the requirements are gathered, and the purity of the gamma cyclodextrin target component is more than 75.0 percent through high performance liquid chromatography analysis. The recovery rate is more than 75 percent.
Example 2:
separation and purification of gamma cyclodextrin: preparing a solution with the concentration of 2g/mL by adding methanol/water (v/v, 1/1) into gamma cyclodextrin, and adjusting the pH value to 1-2 by using a 5% trifluoroacetic acid water solution. After the solution is clarified, the solution is filtered by a filter membrane with the aperture of 0.45 mu m, and the filtrate is collected for later use. Adopting a self-assembly column, wherein the type of column packing is SiliaSphere series ODS, the particle diameter is 50 mu m, and the pore diameter
0.1% trifluoroacetic acid water solution is used as a mobile phase A, methanol is used as a mobile phase B, and the flow rate is 80 mL/min. As shown in FIG. 1, the first column and the second column are in equilibrium for about 15min, crude product is loaded by the first pump (indicated by the solid line in FIG. 1), and at 45min (the target peak is detected by the detector), the switching valves 1, 2 and 3 are sequentially connected according to the solid line in FIG. 2, and the single-column internal circulation mode B shown in FIG. 2 is entered. And when 75min, connecting each switching valve according to the solid line mark in the figure 3, starting a second pump, entering a product collection mode according to the solid line mark in the figure 3, and collecting 115-140 min of segmented collected target fraction liquid. The component solutions meeting the requirements are gathered, and the purity of the gamma cyclodextrin target component is more than 99.0 percent through high performance liquid chromatography analysis. The recovery rate is more than 90 percent. Gradient elution mode, elution procedure as in table 1.
TABLE 1 Gamma Cyclodextrin isolation and purification gradient elution procedure
The purity of the product was measured by HPLC using Agilent ZORBAX SB C18250X 4.6mm, 5 μm, 1mL/min flow rate, 30 deg.C column temperature, DAD detector, 203nm detection wavelength, 20 μ L sample size, and 70min running time. Mobile phase a was 0.05% phosphoric acid in water and mobile phase B was acetonitrile. The elution procedure is shown in table 2.
TABLE 2 Gamma Cyclodextrin HPLC elution procedure
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 89 | 11 |
| 25 | 89 | 11 |
| 45 | 70 | 30 |
| 58 | 25 | 75 |
| 59 | 10 | 90 |
| 70 | 10 | 90 |
The crude gamma cyclodextrin product used in the above example had a total HPLC purity of 61.80% and a total number of impurities of 32, as shown in Table 4.
TABLE 4. Gamma cyclodextrin crude product each component content table
The method adopts a single-column internal circulation mode or a double-column internal circulation mode for separation, greatly improves the separation column efficiency, has remarkable advantages in yield and purity, and can achieve the purity of the purified gamma cyclodextrin up to 99 percent and reduce the number of impurities to 3. As shown in figure 4 and table 5.
TABLE 5 content table of each component of the purified gamma cyclodextrin
| Serial number | Retention time (min) | Peak area (%) |
| 1 | 13.598 | 0.1002 |
| 2 | 16.292 | 0.0386 |
| 3 | 17.497 | 99.6535 |
| 4 | 31.918 | 0.2077 |
The result shows that the gamma cyclodextrin purification method improves the product purity to 99.6535%, greatly reduces the total amount of impurities, and is beneficial to reducing the toxicity of the gamma cyclodextrin so as to improve the medication safety of the gamma cyclodextrin.
Although preferred embodiments of the present invention have been described in detail, it will be apparent to those skilled in the relevant art that various changes, modifications and variations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A separation and purification method of gamma cyclodextrin is characterized in that a gamma cyclodextrin sample is dissolved by a mixed solution, and insoluble substances are filtered; then loading the filtered sample solution to an ODS column; then, a single-column internal circulation mode or a double-column internal circulation mode is adopted for separation; finally, gradient elution or isocratic elution is carried out by taking a mixed solvent as a flowing relative sample, a target peak is collected in sections, and components with the target peak meeting the requirements are gathered to obtain a purified sample;
the mixed solution is composed of 1/1 volume ratio of methanol and water, the mixed solvent is mainly composed of methanol and trifluoroacetic acid, and comprises 0.1% mass fraction trifluoroacetic acid (TFA) aqueous solution as A solution and methanol as B solution;
the target peak is specifically a peak with the peak-off time of 65-75 min in an isocratic elution mode or a peak with the peak-off time of 115-140 min in a gradient elution mode;
the isocratic elution process is as follows: within 0-80min, the mobile phase is a mixed solution of 77% volume fraction solution A and 23% volume fraction solution B;
the gradient elution process is as follows: the mobile phase is a mixed solution of 95% volume fraction of solution A and 5% volume fraction of solution B within 0-15 min; the mobile phase is a mixed solution of 77 volume percent of solution A and 23 volume percent of solution B within 15.01-140 min; within 140.01-150min, the mobile phase is a mixed solution of 20% volume fraction of solution A and 80% volume fraction of solution B; in 150.01-173min, the mobile phase is only the B solution with 100% volume fraction;
the method adopts the following cyclic preparation, separation and purification device which comprises a detector, a first pump, a second pump, a first column, a second column, a first switching valve (1), a second switching valve (2), a third switching valve (3) and a fourth switching valve (4); the second mobile phase output end, the input end of the second pump, the fraction collecting port and the output port of the detector are respectively connected to three valve ports of the first switching valve (1), the first end of the second column, the output end of the second pump and the waste liquid collecting port are respectively connected to three valve ports of the second switching valve (2), the second end of the second column and the first end of the first column are respectively connected to two valve ports of the third switching valve (3), the first mobile phase output end, the input end of the first pump and the input port of the detector are respectively connected to three valve ports of the fourth switching valve (4), the output end of the first pump is connected to the first end of the first column, the second end of the second column, the output end of the second pump and the waste liquid collecting port are respectively connected to three valve ports of the second switching valve (2), and one valve port of the second switching valve (2) and one valve port of the third switching valve (3) are directly communicated, one valve port of the third switching valve (3) is directly communicated with one valve port of the fourth switching valve (4);
the single-column internal circulation mode specifically comprises the following steps: the first pump is not started and does not work, and the second pump is started and works; opening a first switching valve (1) to communicate only the output of the detector and the input of the second pump; opening a second switching valve (2) to only enable the output end of the second pump to be communicated with the first end of the second column; opening the third switching valve (3) and the fourth switching valve (4) to enable only the second end of the second column to be communicated with the input end of the detector after passing through the third switching valve (3) and the fourth switching valve (4) in sequence; the sample liquid in the detector is input into a second pump through a flow channel of a first switching valve (1), is pumped out from the second pump and then enters a second column through a flow channel of a second switching valve (2), and the second column flows out and then returns to the detector through a flow channel of a third switching valve (3) and a flow channel of a fourth switching valve (4) in sequence to complete circulation;
the double-column internal circulation mode specifically comprises the following steps: the first pump and the second pump are started to work; opening a first switching valve (1) to communicate only the output of the detector and the input of the second pump; opening a second switching valve (2) to only enable the output end of the second pump to be communicated with the first end of the second column; opening a third switching valve (3) so that only the second end of the second column and the second end of the first column are communicated; opening a fourth switching valve (4) to enable the input end of the first pump and the input end of the detector to be communicated only, and enabling the first pump to work in the reverse direction; the sample liquid in the detector is input into the second pump through a flow channel of the first switching valve (1), is pumped out from the second pump and then enters the second column through a flow channel of the second switching valve (2), the second column flows out and then enters the first column through a flow channel of the third switching valve (3), and the first column and the second column flow out and then return to the detector through a flow channel of the first pump and the fourth switching valve (4) to finish circulation.
2. The method for separating and purifying gamma cyclodextrin as claimed in claim 1, wherein:
the method is specifically performed under the control of the device as follows:
1) and (3) crude product loading: dissolving and filtering a gamma cyclodextrin sample by using the mixed solution, loading the filtered sample solution to a first column, and then controlling the following steps: the first pump is started to work, and the second pump is not started to work; opening a first switching valve (1) to communicate only the output end of the detector with the fraction collection port; the second switching valve (2) is closed and does not work; opening the third switching valve (3) to enable the second end of the first column to be communicated with the input end of the detector after passing through the third switching valve (3) and the fourth switching valve (4) in sequence; opening a fourth switching valve (4) to enable the first mobile phase output end to be communicated with the input end of the first pump; the first mobile phase enters a first pump through a flow channel of a fourth switching valve (4) after being output, is pumped by the first pump, then sequentially enters a detector through a first column, a flow channel of the third switching valve (3) and the other flow channel of the fourth switching valve (4), and enters a fraction collection port through a flow channel of the first switching valve (1) after being output from the detector; most of samples in the first column are loaded into the detector, and the switching valve enters a single-column internal circulation mode or a double-column internal circulation mode after the detector to be detected detects a target peak;
2) separating by adopting a single-column internal circulation mode or a double-column internal circulation mode;
3) performing gradient elution or isocratic elution by using a mixed solvent as a flowing relative sample, collecting a target peak according to a target peak collecting mode after a detector to be detected detects that main components are completely separated, wherein the target peak collecting mode is divided into a single-column target peak collecting mode and a double-column target peak collecting mode, and summarizing to obtain a purified sample;
4) after collecting the sample, the first and second columns were equilibrated, and the valves switched back to step 1) for the next crude loading.
3. The method for separating and purifying gamma cyclodextrin as claimed in claim 2, wherein:
in the step 3), the single-column target peak collection mode specifically comprises the following steps: the first pump is not started and does not work, and the second pump is started and works; opening a first switching valve (1) so that the output of the detector is in communication with the fraction collection port and the second mobile phase output is in communication with the input of a second pump; opening a second switching valve (2) to only enable the output end of the second pump to be communicated with the first end of the second column; opening the third switching valve (3) and the fourth switching valve (4) to enable only the second end of the second column to be communicated with the input end of the detector after passing through the third switching valve (3) and the fourth switching valve (4) in sequence; and the second mobile phase enters a second pump through a flow channel of the first switching valve (1) after being output, is pumped by the second pump and then enters a second column through a flow channel of the second switching valve (2), flows out of the second column and then sequentially enters a detector through a flow channel of the third switching valve (3) and a flow channel of the fourth switching valve (4), and enters a fraction collection port through the other flow channel of the first switching valve (1) after being output from the detector.
4. The method for separating and purifying gamma cyclodextrin as claimed in claim 2, wherein:
in the step 3), the double-column target peak collection mode specifically comprises the following steps: the first pump and the second pump are started to work; opening a first switching valve (1) so that the output of the detector is in communication with the fraction collection port and the second mobile phase output is in communication with the input of a second pump; opening a second switching valve (2) to only enable the output end of the second pump to be communicated with the first end of the second column; opening a third switching valve (3) so that only the second end of the second column and the second end of the first column are communicated; opening a fourth switching valve (4) to enable the input end of the first pump and the input end of the detector to be communicated only, and enabling the first pump to work in the reverse direction; and the second mobile phase enters a second pump through a flow channel of the first switching valve (1) after being output, is pumped by the second pump and then sequentially enters a second column through a flow channel of the second switching valve (2), the second column flows out and then enters a first column through a flow channel of the third switching valve (3), the first column flows out and then enters a detector through a flow channel of the first pump and the fourth switching valve (4), and the second mobile phase enters a fraction collection port through the other flow channel of the first switching valve (1) after being output from the detector.
5. The method for separating and purifying gamma cyclodextrin as claimed in claim 1, wherein:
the ODS column is a self-assembled column.
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