A method for the separation and purification of a chiral compound

Abstract

The present invention belongs to the technical field of separation and analysis, and particularly relates to a method for separating and purifying chiral compounds. The present invention provides a method for separating and purifying chiral compounds, which uses an axial electromagnetic field chromatography device to separate a chiral compound sample to obtain a target chiral compound and a racemic impurity; the axial electromagnetic field chromatography device includes a separation system, and the separation system includes a chromatographic column and an electromagnetic field system; the electromagnetic field system includes a conductive spiral coil, the conductive spiral coil is arranged around the outer periphery of the chromatographic column, and the conductive spiral coil can generate an electromagnetic field along the axial direction of the chromatographic column. The present invention can effectively separate chiral biomacromolecules, and has the characteristics of high separation efficiency, good selectivity, high detection sensitivity, automated operation and wide application.

Description

Technical Field

[0001] The present invention belongs to the technical field of separation and analysis, and particularly relates to a method for separating and purifying chiral compounds. Background Art

[0002] Currently, liquid chromatography uses a liquid as the mobile phase, and utilizes the differences in the chemical interactions of different substances with the stationary phase, including differences in partitioning or adsorption and some other interactions, so as to form differences in elution on the chromatogram to achieve separation. Its essence is to utilize the chemical interactions between substances. However, traditional liquid chromatography has a single separation mechanism, and it is difficult to effectively separate, analyze, and prepare and amplify different compounds, especially chiral compounds with similar structures and physical and chemical properties.

[0003] Capillary electro chromatography (CEC) is a newly emerging high-efficiency separation and analysis technology in recent years. It uses electroosmotic flow or electroosmotic flow combined with pressure flow to drive the mobile phase, and has the dual separation mechanisms of capillary electrophoresis and high-performance liquid chromatography. It can separate both charged substances and neutral substances.

[0004] However, traditional electrochromatography devices are composed of a high-voltage power supply, a capillary chromatographic column (packed column) with packing, and detection equipment. As the particle size of the packing in the packed column becomes smaller and smaller, the tiny particle size packing is more likely to aggregate and block the column when separating biological chiral macromolecules, thus greatly reducing the service life of the analyzer. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for separating and purifying chiral compounds. The method for separating and purifying chiral compounds provided by the present invention can effectively separate chiral biological macromolecules, and has the characteristics of high separation efficiency, good selectivity, high detection sensitivity, automated operation, and wide application.

[0006] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a method for separating and purifying chiral compounds, which uses an axial electromagnetic field chromatography device to separate a chiral compound sample to obtain a target chiral compound and a racemic impurity; the axial electromagnetic field chromatography device includes a separation system, and the separation system includes a chromatographic column and an electromagnetic field system; the electromagnetic field system includes a conductive spiral coil, the conductive spiral coil is wound around the outer periphery of the chromatographic column, and the conductive spiral coil can generate an electromagnetic field along the axial direction of the chromatographic column.

[0008] Preferably, the south pole of the electromagnetic field generated by the conductive spiral coil is located at the inlet end of the chromatographic column, and the north pole of the electromagnetic field generated by the conductive spiral coil is located at the outlet end of the chromatographic column.

[0009] Preferably, it includes the following steps:

[0010] Start the electromagnetic field system to generate an electromagnetic field along the axial direction of the chromatographic column within the chromatographic column; introduce the mobile phase and the chiral compound sample together into the chromatographic column from the inlet end of the chromatographic column, and obtain the target chiral compound and racemic impurities respectively from the outlet end of the chromatographic column.

[0011] Preferably, the chiral compound sample is a chiral polypeptide drug.

[0012] Preferably, the chemical structure of the target chiral compound is:

[0013] 。

[0014] Preferably, the mobile phase used for the separation includes phase A and phase B; phase A is an aqueous acetic acid solution, and the volume content of acetic acid in the aqueous acetic acid solution is 0.1%; phase B is acetonitrile;

[0015] The gradient elution program for the separation is as follows: phase B is 5% (v:v) - 5% (v:v) within 5 min; from 5.1 min to 25 min, phase B rises from 5% (v:v) to 35% (v:v), and phase B at 35% (v:v) is maintained for at least T min; from 25.1 + T min to 36 min, phase B rises from 35% (v:v) to 95% (v:v), and phase B at 95% (v:v) is maintained for at least 6 min.

[0016] Preferably, the intensity of the electromagnetic field is 0 - 12 mT and is not 0.

[0017] Preferably, the electromagnetic field system further includes a DC power supply, and the voltage of the DC power supply is 0 - 36 V and is not 0.

[0018] Preferably, the axial electromagnetic field chromatographic device further includes an infusion system, a quantitative injection system, a detection system, and a data processing system; the output end of the infusion system is connected to the quantitative injection system, the downstream of the quantitative injection system is connected to the inlet end of the chromatographic column, the outlet end of the chromatographic column is connected to the detection system, and the detection system is connected to the data processing system; the quantitative injection system includes a quantitative injection valve.

[0019] Preferably, the column tube material of the chromatographic column includes glass, plastic, or resin.

[0020] The present invention provides a method for separating and purifying chiral compounds. The chiral compound sample is separated by using an axial electromagnetic field chromatography device to obtain the target chiral compound and racemic impurities. The axial electromagnetic field chromatography device includes a separation system, and the separation system includes a chromatographic column and an electromagnetic field system. The electromagnetic field system includes a conductive helical coil, which is wound around the outer periphery of the chromatographic column, and the conductive helical coil can generate an electromagnetic field along the axis of the chromatographic column. The axial electromagnetic field chromatography device provided by the present invention utilizes the large-scale and efficient application of liquid chromatography in analysis and separation. On the basis of achieving separation by relying on the chemical forces between substances, an electromagnetic field is applied to the chromatographic column by using the electromagnetic field formed by the conductive helical coil, so that the chiral compound sample is subjected to a magnetic field with a certain magnitude and direction during the chromatographic separation process. Due to the differences in the structures of the chiral compound samples themselves and the polarity differences of the chiral compound samples, the directions and magnitudes of the magnetic forces exerted by the electromagnetic field on the target chiral compound and racemic impurities in the chiral compound sample are different. Therefore, the effective separation of the chiral compound sample is achieved by superimposing the differences in the moving speeds of the chiral compound samples under the action of the electromagnetic field on the differences in the chemical forces of the conventional liquid chromatography. Moreover, the axial electromagnetic field chromatography device provided by the present invention adopts an external electromagnetic field system, and the conductive helical coil is wound around the outer periphery of the chromatographic column, and the magnitude and direction of the electromagnetic field intensity can be adjusted and controlled, that is, the stepless adjustment of the direction and magnitude of the electromagnetic field is realized. The axial electromagnetic field chromatography device provided by the present invention superimposes the separation effect of the electromagnetic field force on the basis of a single chromatographic separation mechanism for different compounds, thereby showing a more efficient separation ability than a single chromatographic separation mechanism. The method for separating and purifying chiral compounds provided by the present invention has the following beneficial effects:

[0021] The axial electromagnetic field chromatography device used in the present invention is simple, has good versatility, is suitable for industrialization, has good compatibility with the procedures of ordinary preparative chromatography, and only makes minor adjustments to the chromatographic column, adding an external electromagnetic field system, and the injection system, detection system, infusion system, etc. of the rest of the chromatographic devices are all general equipment.

[0022] Since the magnetic force of the electromagnetic field acts on the chiral compound in the present invention, this electromagnetic field driving force is superimposed on the fluid driving force, so that the fluid required for the chiral compound to flow out of the liquid chromatography is less than that of the conventional liquid chromatography, and the speed of achieving effective separation will be faster. That is, when dealing with the same mixed compound, due to the increase in the separation driving force and the increase in the dimension of the separation driving force, under the premise of combining two separation mechanisms, the substance separation driving is simultaneously affected by multiple cross dimensions, and its separation effect has a stronger separation ability than a single separation mechanism. At the same time, less solvent and reagent are required, and the required time is shorter, that is, it has better economy and higher working efficiency.

[0023] Furthermore, in the present invention, the electromagnetic field system further includes a DC power supply, the voltage of the DC power supply is 0 to 36V and not 0. The axial electromagnetic field chromatography device provided by the present invention has high safety, and the voltage of the DC power supply of the electromagnetic field system does not exceed 36V, which does not involve high voltage situations and belongs to the range of human safe voltage.

[0024] In summary, the separation and purification method of chiral compounds provided by the present invention has high separation efficiency, good selectivity, high detection sensitivity, automated operation, wide application, and also has the characteristics of electrochromatography. It has a significant resolution, separation, and enrichment effect on chiral compounds due to the difference in the size of the electromagnetic field. At the same time, the present invention realizes the change of the direction and size of the electromagnetic field in the axial direction of the fluid direction through an external electromagnetic field system. For different compounds, especially those with similar structures and physical and chemical properties, due to the difference in the cis and trans magnetic fields of the compounds themselves, high-efficiency separation is achieved under the dual action mechanism of magnetic field driving and chromatographic separation mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the analysis and detection spectrum of the crude polypeptide (EYH008-002-0801);

[0026] Figure 2 It is the purification spectrum of conventional liquid chromatography in Comparative Example 1;

[0027] Figure 3 It is the analysis and detection spectrum after purification by conventional liquid chromatography in Comparative Example 1;

[0028] Figure 4 It is the purification spectrum of axial electromagnetic field chromatography in Example 1;

[0029] Figure 5 It is the analysis and detection spectrum after purification by axial electromagnetic field chromatography in Example 1;

[0030] Figure 6 It is the structural schematic diagram of the axial electromagnetic field chromatography device provided by the present invention;

[0031] Figure 7 It is the partial enlarged structural schematic diagram of the axial electromagnetic field chromatography device provided by the present invention;

[0032] In the figure: 1 is a liquid storage device, 2 is a liquid delivery pump, 3 is a sample bottle, 4 is a quantitative sampling system, 5 is a separation system, 6 is a detection system, 7 is a waste liquid bottle, and 8 is a data processing system. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention provides a method for separating and purifying chiral compounds. The chiral compound sample is separated by an axial electromagnetic field chromatography device to obtain the target chiral compound and racemic impurities. The axial electromagnetic field chromatography device includes a separation system, and the separation system includes a chromatographic column and an electromagnetic field system. The electromagnetic field system includes a conductive spiral coil, which is wound around the outer periphery of the chromatographic column, and the conductive spiral coil can generate an electromagnetic field along the axial direction of the chromatographic column.

[0034] Figure 6 It is a schematic structural diagram of the axial electromagnetic field chromatography device provided by the present invention; Figure 7 It is a partially enlarged schematic structural diagram of the axial electromagnetic field chromatography device provided by the present invention. The following will be combined with Figure 6 and Figure 7 , and the axial electromagnetic field chromatography device provided by the present invention will be described in detail.

[0035] The axial electromagnetic field chromatography device provided by the present invention includes a separation system 5. The separation system includes a chromatographic column and an electromagnetic field system. The electromagnetic field system includes a conductive spiral coil, which is wound around the outer periphery of the chromatographic column, and the conductive spiral coil can generate an electromagnetic field along the axial direction of the chromatographic column.

[0036] As one or more embodiments of the present invention, the south pole of the electromagnetic field generated by the conductive spiral coil is located at the inlet end of the chromatographic column, and the north pole of the electromagnetic field generated by the conductive spiral coil is located at the outlet end of the chromatographic column.

[0037] As one or more embodiments of the present invention, the intensity of the electromagnetic field is 0 - 12 mT and not 0.

[0038] As one or more embodiments of the present invention, the electromagnetic field system further includes a DC power supply, and the voltage of the DC power supply is 0 - 36 V and not 0.

[0039] As one or more embodiments of the present invention, the column tube material of the chromatographic column includes glass, plastic or resin.

[0040] As one or more embodiments of the present invention, the specifications of the chromatographic column are 250 mm × 10 mm, 250 mm × 20 mm, 250 mm × 21.2 mm, 250 mm × 30 mm or 250 mm × 50 mm, and the packing is C18.

[0041] As one or more embodiments of the present invention, the particle size of the C18 is 7 - 20 μm, specifically it can be 7 μm, 8 μm, 10 μm or 20 μm.

[0042] As one or more embodiments of the present invention, the pore diameter of the filler is preferably 100-200 Å, and the embodiments can be 100 Å, 120 Å, 150 Å or 200 Å.

[0043] The axial electromagnetic field chromatography device provided by the present invention preferably further includes an infusion system. The infusion system includes a reservoir 1 and an infusion pump 2. The reservoir 1 is used to store the mobile phase.

[0044] The axial electromagnetic field chromatography device provided by the present invention preferably further includes a quantitative injection system 4. The output end of the infusion system is connected to the quantitative injection system 4, and the downstream of the quantitative injection system 4 is connected to the inlet end of the chromatographic column.

[0045] As one or more embodiments of the present invention, the quantitative injection system 4 includes a quantitative injection valve.

[0046] The axial electromagnetic field chromatography device provided by the present invention preferably further includes a detection system 6. The outlet end of the chromatographic column is connected to the detection system 6, and the detection system 6 is connected to the data processing system.

[0047] As one or more embodiments of the present invention, the detection system 6 includes a detector.

[0048] As one or more embodiments of the present invention, the detection system 6 is connected to a waste liquid bottle 7.

[0049] The axial electromagnetic field chromatography device provided by the present invention preferably further includes a data processing system 8.

[0050] The axial electromagnetic field chromatography device provided by the present invention utilizes a conductive spiral coil to form an electromagnetic field and applies an electromagnetic field to the chromatographic column, so that compounds are subjected to a magnetic field of a certain magnitude and direction during the chromatographic separation process. Due to the structural differences and polarity differences of chiral compounds themselves, under the conditions of a certain direction and a certain magnetic field intensity, due to the difference in paramagnetic and diamagnetic properties with the electromagnetic field, the difference in the moving speeds of different chiral compounds under the action of the electromagnetic field is superimposed on the difference in the chemical forces of liquid chromatography, realizing the separation between different compounds. Since different chiral compounds have a certain spin polarization direction themselves, they will form a small magnetic field at the microscopic level, with a magnetic field direction and magnitude. Under the action of an external magnetic field, chiral compounds will show obvious paramagnetism or diamagnetism due to their own magnetic field characteristics. For a pair of chiral compounds, due to the chiral symmetry of their molecular configurations, their own magnetic field directions are opposite. Therefore, the racemic compound is in a racemic state in terms of optical rotation and is also in a demagnetized state due to the opposite symmetry and mutual cancellation in the magnetic field. However, under the external electromagnetic field device, due to the uneven force caused by the paramagnetic and diamagnetic properties of the self-magnetic field and the external magnetic field of the racemic mixture of chiral compounds, the chiral compound consistent with the direction of the external magnetic field is subjected to a force in the direction of the magnetic field, while its enantiomer is subjected to a force in the opposite direction of the magnetic field due to the opposite magnetic field direction. Thus, after passing through the magnetic field, the racemic mixture (i.e., the sample) has a difference in the force magnitude of at least 2∆F between a pair of enantiomers. 磁 , when simultaneously subjected to other chromatographic forces, the racemic enantiomers can be effectively separated after passing through this special electromagnetic field liquid chromatography, and this separation difference can be achieved by adjusting chromatographic parameters or magnetic field parameters. For example, adjusting the magnitude of the external magnetic field can significantly change it without the need for special design of the chromatographic column, and the magnitude and direction of the electromagnetic field intensity can be controlled, that is, the adjustment of the direction and magnitude of the electromagnetic field intensity is realized by the stepless adjustment of the electromagnetic field direction and magnitude, so that different parameters can be adjusted for different compounds to achieve effective separation.

[0051] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well-known to those skilled in the art.

[0052] In the present invention, the chiral compound sample is a racemic chiral compound.

[0053] In the present invention, the separation and purification method of the chiral compound preferably includes the following steps:

[0054] Start the electromagnetic field system to generate an electromagnetic field along the axial direction of the chromatographic column within the chromatographic column; introduce the mobile phase and the chiral compound sample together into the chromatographic column from the inlet end of the chromatographic column, and obtain the target chiral compound and the racemic impurity respectively from the outlet end of the chromatographic column.

[0055] In the present invention, while starting the electromagnetic field system, the mobile phase and the chiral compound sample are introduced together into the chromatographic column from the inlet end of the chromatographic column.

[0056] In an embodiment of the present invention, the method for separating and purifying the chiral compound specifically includes the following steps: Start the electromagnetic field system to generate an electromagnetic field along the axial direction of the chromatographic column within the chromatographic column; use an infusion system to deliver the mobile phase to the quantitative injection system 4, and introduce it into the chromatographic column together with the sample from the inlet end of the chromatographic column, and start adsorption, elution, and separation, and obtain the target chiral compound and the racemic impurity respectively from the outlet end of the chromatographic column.

[0057] In the present invention, the chiral compound sample is preferably a chiral polypeptide drug.

[0058] In an embodiment of the present invention, the chemical structure of the target chiral compound is:

[0059] 。

[0060] In an embodiment of the present invention, the chemical structure of the racemic impurity is:

[0061] 。

[0062] In the present invention, the mobile phase includes phase A and phase B. Phase A is preferably an aqueous acetic acid solution, and the volume content of acetic acid in the aqueous acetic acid solution is preferably 0.1%. Phase B is preferably acetonitrile. The injection flow rate is preferably 15 mL / min.

[0063] In the present invention, before the elution, the present invention preferably equilibrates the chromatographic column with the mobile phase. The volume ratio of phase A and phase B used for equilibrating the chromatographic column is preferably 95:5. The time for equilibrating the chromatographic column is preferably 10 - 15 min.

[0064] In the present invention, the elution preferably adopts gradient elution. The elution program is preferably as follows: phase B is 5% (v:v) - 5% (v:v) within 5 min; from 5.1 min to 25 min, phase B is increased from 5% (v:v) to 35% (v:v), and phase B at 35% (v:v) is maintained for at least T min; from 25.1 + T min to 36 min, phase B is increased from 35% (v:v) to 95% (v:v), and phase B at 95% (v:v) is maintained for at least 6 min. The T min is preferably 3 min, and 25.1 + T min is preferably 28.1 min.

[0065] In the present invention, the wavelength of the detector is preferably dual wavelength, and the dual wavelength is preferably 220 nm and 254 nm.

[0066] In the present invention, the voltage of the DC power supply is preferably 20 V, and the intensity of the electromagnetic field is preferably 10 mT. In the present invention, the sample moves from the inlet end of the chromatographic column to the outlet end along with the fluid elution, and is simultaneously affected by the conventional chromatographic force and the electromagnetic field. Due to the difference in the charged properties of different compounds in the sample, an external magnetic field parallel to the fluid direction exerts different magnetic field forces on different compounds in the chromatographic column, and the mixture in the sample gradually changes from a mass to separate movements with differences, and finally the sequential elution of each compound in the sample is realized.

[0067] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0068] In the following, the raw materials in Example 1 and Comparative Example 1 are crude polypeptide products;

[0069] Among them, the chemical structure of the target product in the crude polypeptide product is:

[0070] .

[0071] The chemical structure of the main impurity (racemic impurity) in the crude polypeptide product is:

[0072] .

[0073] Comparative Example 1 (conventional liquid chromatography cannot effectively separate):

[0074] (1) Sample treatment: A crude chiral polypeptide drug product (EYH008 - 002 - 0801) with a purity (area normalization method) of 40.97% and a weight of about 300 mg was obtained by solid-phase synthesis. The analysis and detection chromatogram of the crude polypeptide product (EYH008 - 002 - 0801) is as Figure 1 shown, and the analysis and detection method of the crude polypeptide product (EYH008 - 002 - 0801) is shown in Table 1.

[0075] Table 1 Analytical and Detection Methods for Crude Polypeptide (EYH008-002-0801)

[0076]

[0077] Dissolve the crude polypeptide in methanol at a concentration of 2 g / L, and then filter it through a 0.45-μm organic filter membrane to obtain the filtrate of the crude polypeptide.

[0078] (2) Chromatographic Separation: About 60 mL (about 120 mg) of the above crude polypeptide solution was purified and separated using a liquid chromatography system. The specifications of the chromatographic column were 250 mm × 20 mm, the packing was C18, 20 μm, 100 Å. The mobile phase A was: 0.1% (v:v) acetic acid aqueous solution; the mobile phase B was: acetonitrile; the flow rate of the mobile phase was 15 mL / min; the chromatographic column was equilibrated with 5% (v:v) of B phase and 95% (v:v) of A phase for 10 min. Elution gradient: 5% (v:v) of B phase from 5% (v:v) to 5% (v:v) within 5 min, from 5.1 min to 30 min, B phase increased from 5% (v:v) to 45% (v:v) in gradient, and maintained the 45% (v:v) gradient of B phase for 5 min; from 35.1 min to 40 min, B phase increased from 45% (v:v) to 95% (v:v) in gradient, and maintained the 95% (v:v) gradient for 8 min. The detection system wavelength (dual wavelength) was: 220 nm, 254 nm.

[0079] Figure 2 For Comparative Example 1, it was the purification chromatogram during the preparation by conventional liquid chromatography, Figure 3 For Comparative Example 1, it was the analytical and detection chromatogram after purification by conventional liquid chromatography (the analytical and detection methods are shown in Table 1). It can be seen on the analytical instrument that the chromatographic retention behaviors of the polypeptide compound and the impurities were relatively close, Figure 2 it can be seen that the main component and the impurities overlapped, and there was a lack of good resolution in the analysis and detection. During the purification and preparation, due to the increase in the sample treatment amount, the separation performance of the chromatographic column was inferior to the analytical level. It was significantly observed on the preparative chromatography that both the main peak and its impurity peak had a large broadening and overlapped with each other ( Figure 2 ), and it was difficult to effectively separate. It can be Figure 3 seen that after the separation by the conventional liquid chromatography of Comparative Example 1, the content of the target polypeptide compound in the obtained product was only 55.79%.

[0080] Example 1 (Electromagnetic Liquid Chromatographic Separation):

[0081] This example used the axial electromagnetic field chromatographic device shown in Figure 6 and Figure 7 for separation and purification.

[0082] (1)Sample: The crude product solution (about 90 mL) dissolved in Example 1 was used.

[0083] (2)Chromatographic separation: Approximately 90 mL (about 180 mg) of the above-mentioned crude product solution was purified and separated using an axial electromagnetic field chromatography device. The specifications of the chromatographic column (high-pressure resistant resin column) were 250 mm × 20 mm, and the packing material was C18, 20 μm, 100 Å. Phase A in the mobile phase was: 0.1% (v:v) acetic acid aqueous solution; Phase B in the mobile phase was: acetonitrile; the volume flow rate of the mobile phase was: 15 mL / min; for the external electromagnetic field system settings, the externally applied electromagnetic field was set with a DC voltage of 20 V, a magnetic field strength of 10 mT, the magnetic field direction was the axial direction of fluid flow, the south pole of the magnetic field was the inlet end of the chromatographic column, and the north pole of the magnetic field was the outlet end of the chromatographic column. The electromagnetic field was turned on at the start of elution. The mobile phase used 5% (v:v) of Phase B and 95% (v:v) of Phase A to equilibrate the chromatographic column for 10 min. Elution gradient: From 5% (v:v) to 5% (v:v) of Phase B within 5 min, from 5% (v:v) to 35% (v:v) of Phase B from 5.1 min to 25 min with a gradient, and maintaining a 35% (v:v) gradient of Phase B for 3 min, from 35% (v:v) to 95% (v:v) of Phase B from 28.1 min to 36 min with a gradient, and maintaining a 95% (v:v) gradient of Phase B for 6 min. The detection system wavelength (dual wavelength) was: 220 nm, 254 nm.

[0084] Due to the addition of the externally applied electromagnetic field, the overall separation time of the polypeptide compound was shortened. Due to the introduction of the electromagnetic field strength, the overall original chromatographic elution gradient was reduced (using less organic phase), that is, a similar elution effect was achieved, and the resolution in the purification and preparation was significantly improved overall.

[0085] Figure 4 It is the chromatogram during the purification in the preparation process of Example 1 using an axial electromagnetic field chromatography device. Figure 5 It is the analytical detection chromatogram after purification using an axial electromagnetic field chromatography device in Example 1 (the analytical detection method is shown in Table 1). On the analytical instrument, it can be seen that the chromatographic retention behaviors of the polypeptide compound and the impurities are still relatively close. However, during the purification by electromagnetic chromatography, due to the improvement of the elution resolution, it can be significantly observed on the preparative chromatogram that the mutual cross-over and overlap of the main peak and its impurity peaks are reduced, and the compound is better separated and purified, and the content of the main impurity peaks has been effectively controlled. Figure 5 It can be known that after the electromagnetic chromatography separation in Example 1, a target polypeptide compound with a total impurity not greater than 2.0% and a main peak purity greater than 98% was obtained.

[0086] After calculation: The maximum impurity of the finished product in this example is 1.91%, the purity is 98.09%, and the purification yield of the main component is 58.2%.

[0087] As can be seen from the above embodiments, the axial electromagnetic field chromatography method and apparatus provided by the present invention can be used in all fields of liquid chromatography applications, and can also utilize the capabilities of electromagnetic chromatography to achieve certain applications and separate compounds in fields where liquid chromatography has certain limitations. The axial electromagnetic field chromatography apparatus provided by the present invention has a simple structure, wide generality, is easy to implement analytical, preparative and industrial applications, and uses an externally applied electromagnetic field, and the magnitude and direction of the electromagnetic field can be arbitrarily adjusted according to the characteristics of the compound.

[0088] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments can also be obtained according to this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for separating and purifying a chiral compound, characterized in that: An axial electromagnetic field chromatography device is used to separate a chiral compound sample to obtain a target chiral compound and a racemic impurity; the axial electromagnetic field chromatography device comprises a separation system, the separation system comprises a chromatographic column and an electromagnetic field system; the electromagnetic field system comprises a conductive spiral coil, the conductive spiral coil is arranged around the periphery of the chromatographic column, and the conductive spiral coil can generate an electromagnetic field along the axial direction of the chromatographic column; The chemical structure of the target chiral compound is: ； The mobile phase used for separation includes phase A and phase B; the phase A is an acetic acid aqueous solution, and the volume content of acetic acid in the acetic acid aqueous solution is 0.1%; the phase B is acetonitrile; The gradient elution procedure for the separation is: phase B 5% (v:v) to 5% (v:v) within 5 min; from 5.1 min to 25 min, phase B increases from 5% (v:v) to 35% (v:v), and phase B is maintained at 35% (v:v) for at least Tmin; from 25.1+Tmin to 36 min, phase B increases from 35% (v:v) to 95% (v:v), and phase B is maintained at 95% (v:v) for at least 6 min.

2. The separation and purification method according to claim 1, characterized in that The south pole of the electromagnetic field generated by the conductive spiral coil is located at the inlet end of the chromatographic column, and the north pole of the electromagnetic field generated by the conductive spiral coil is located at the outlet end of the chromatographic column.

3. The separation and purification method according to claim 1 or 2, characterized in that: The following steps are involved: Starting the electromagnetic field system to generate an electromagnetic field along the axial direction of the chromatographic column in the chromatographic column; The mobile phase and the chiral compound sample enter the chromatographic column from the inlet end of the chromatographic column, and the target chiral compound and the racemic impurity are respectively obtained from the outlet end of the chromatographic column.

4. The separation and purification method according to claim 1 or 2, characterized in that: The intensity of the electromagnetic field is 0~12mT and is not 0.

5. The separation and purification method according to claim 1 or 2, characterized in that: The electromagnetic field system also includes a DC power supply, and the voltage of the DC power supply is 0~36V and is not 0.

6. The separation and purification method according to claim 1, characterized in that: The axial electromagnetic field chromatography device also includes an infusion system, a quantitative injection system, a detection system and a data processing system; the output end of the infusion system is connected to the quantitative injection system, the downstream of the quantitative injection system is connected to the inlet end of the chromatographic column, the outlet end of the chromatographic column is connected to the detection system, and the detection system is connected to the data processing system; the quantitative injection system includes a quantitative injection valve.

7. The separation and purification method according to claim 1, characterized in that: The column tube material of the chromatographic column includes glass, plastic or resin.

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