Column packing device and column packing method of C18 / phenyl mixed packing liquid chromatographic column

Abstract

The invention provides a column packing device and a column packing method of a C18 / phenyl mixed packing liquid chromatographic column. The column packing device comprises a column packing machine, a homogenizing tank, an ultrasonic device and a chromatographic column hollow pipe, the column packing machine is used for inputting displacing liquid into the chromatographic column hollow pipe and controlling the pressure and the flow speed of homogenizing liquid conveyed into the chromatographic column hollow pipe, the homogenizing tank is used for homogenizing raw materials comprising mixed packing and the homogenizing liquid, and the ultrasonic device is used for performing ultrasonic treatment on the homogenized raw materials. The ultrasonic device is used for carrying out real-time ultrasonic mixing on the mixed filler in the homogenate tank, the chromatographic column hollow pipe is used for filling the mixed filler in the homogenate tank, and homogenate comprises a mixture of trichloromethane, methanol and C3-C6 perhalogenated alkane. According to the invention, the column packing operation is simplified, the cost is low, the column packing effect is good, the transfer process and homogenization time of the filler are reduced, the column packing efficiency is improved, the service life of the chromatographic column is prolonged, the accuracy of an analysis result is improved, the relative standard deviation of the number of theoretical plates of the packed chromatographic column is less than 3%, the repeatability is high, and the stability of the chromatographic column is high.

Description

Technical Field

[0001] The invention relates to the technical field of liquid chromatography columns, and in particular to a column packing device and a column packing method for a C18 / phenyl mixed filler liquid chromatography column. Background Art

[0002] Liquid chromatography column technology is a key technology used in analytical chemistry to separate and identify components in mixtures. According to the type and purpose of the filler, chromatographic columns can be divided into many categories, including reversed phase chromatography columns (RPC), normal phase chromatography columns (NPC), ion exchange chromatography columns (IEC) and size exclusion chromatography columns (SEC). Reverse phase chromatography columns are the most commonly used type, suitable for the separation of most organic small molecules and biomacromolecules; normal phase chromatography columns are often used for the separation of polar substances; ion exchange chromatography columns are specifically used for the separation of ionic compounds; and size exclusion chromatography columns are used for molecular size separation and characterization. Among them, C18 fillers and phenyl fillers in reverse phase chromatography columns are the most commonly used. C18 fillers have the characteristics of good versatility and high resolution, while phenyl fillers have good separation ability for aromatic compounds. Actual samples often contain various compounds. Using C18 / phenyl mixed fillers can obtain better separation effects than single fillers in specific applications, such as improving the selectivity and resolution of separation, and providing better performance for liquid chromatography analysis.

[0003] Liquid chromatography column packing technology is a key step in the column preparation process, which involves how to evenly load the filler into the column to obtain a uniform and compact column bed. Using the same filler, different homogenates and packing conditions can significantly affect the final performance of the column. For example, the choice of homogenate will affect the suspension state and fluidity of the mixed filler, thereby affecting the uniformity of the distribution of the filler in the column. Different packing pressures and speeds will change the compactness of the filler. Too high or too low pressure may cause channelization or over-compaction of the filler, which will affect the stability and column efficiency of the finished column. Therefore, optimizing the type of homogenate and the packing device during the packing process is an important factor in ensuring that the column has high stability, high column efficiency and long life.

[0004] Appropriate column packing technology can significantly improve the performance of the chromatographic column, thereby ensuring the accuracy and reliability of liquid chromatography analysis. The Chinese patent with patent authorization announcement number CN101776668B describes the mixing of cyclohexanol and chloroform in a volume ratio of 9:11 to 13:7, and the addition of C18 silica gel to prepare a homogenate. The Chinese patent application with patent application publication number CN114217001A describes the preparation of a homogenate by adding C18 silica gel to tetrahydrofuran. The Chinese patent with patent authorization announcement number CN201803995U describes a new type of auxiliary column packing device, the main function of which is to use small but moderate frequency vibrations during the chromatographic column filling process to make the column bed of the dry column more compact, thereby improving the separation efficiency of the chromatographic column. Patent application publication number CN115078602A Chinese patent application describes a capillary high pressure liquid chromatography column ultrasonic preparation method, the main function of this ultrasonic device is to disperse the filler particles in the capillary, so that it can be more evenly accumulated and filled with capillary, which helps to improve the separation effect and stability of the capillary column, ensure the uniformity and compactness of the filler, thereby improving the service life and separation efficiency of the capillary column. However, conventional homogenate is difficult to make C18 / phenyl mixed filler uniformly dispersed, and sedimentation is prone to occur during column loading, resulting in uneven distribution of filler in the chromatographic column, and this unevenness can form channel effect or dead volume in the chromatographic column, thereby affecting the accuracy of column efficiency and analysis results. In addition, the sedimentation of filler may also cause the column head pressure to increase, reducing the service life of the column. Therefore, the homogenate and column loading method of the above patent are not suitable for the loading of C18 / phenyl mixed filler chromatographic column. Summary of the invention

[0005] The main purpose of the present invention is to provide a column packing device and a column packing method for a C18 / phenyl mixed filler liquid chromatography column, so as to solve the problems of uneven packing distribution and poor stability of the C18 / phenyl mixed filler chromatography column in the prior art.

[0006] In order to achieve the above-mentioned purpose, according to one aspect of the present invention, a column packing device for a C18 / phenyl mixed filler liquid chromatography column is provided, the column packing device comprising a column packing machine, a homogenization tank, an ultrasonic device and a chromatographic column empty tube, a column packing machine pump is arranged inside the column packing machine, the column packing machine pump has a liquid outlet end, the column packing machine is used to input a displacement liquid into the chromatographic column empty tube, and control the pressure and flow rate of the homogenate liquid transported into the chromatographic column empty tube; the homogenization tank has a tank feed port and a tank discharge port, the tank feed port is connected to the liquid outlet end, and the homogenization tank is used to homogenize a raw material including a mixed filler and a homogenate liquid; the ultrasonic device has a slot-shaped structure, which has a slot-shaped receiving cavity and a slot bottom surface, the slot-shaped receiving cavity is used to The invention relates to a device for containing ultrasonic medium, wherein the bottom surface of the groove is provided with an opening, the aperture of the opening matches the outer diameter of the homogenizing tank, at least a part of the tank body of the homogenizing tank is immersed in the ultrasonic medium in the groove-shaped containing cavity, and the homogenizing tank passes through the opening and extends out of the bottom surface of the groove; an ultrasonic generator is provided on the inner surface of the ultrasonic device, and the ultrasonic device is used for real-time ultrasonic mixing of the mixed filler in the homogenizing tank; the chromatographic column empty tube has a column feed port and a column discharge port, the column feed port is connected to the tank discharge port, and the chromatographic column empty tube is used for filling the mixed filler in the homogenizing tank; wherein the mixed filler comprises C18 filler and phenyl filler; and the homogenate comprises a mixture of chloroform, methanol and C3-C6 perhalogenated alkanes.

[0007] Furthermore, the probe of the ultrasonic generator is immersed in the ultrasonic medium in the groove-shaped accommodating cavity, and / or the ultrasonic medium is selected from any one or more of water, ethanol, methanol, acetonitrile, 50% ethanol aqueous solution, and 10% sodium chloride aqueous solution; and / or the ultrasonic generator is a cylindrical bottom ultrasonic generator or a ring-shaped probe ultrasonic generator.

[0008] Furthermore, a portion of the tank body of the homogenizing tank close to the tank discharge port is immersed in the ultrasonic medium in the trough-shaped containing cavity, and / or 10% to 90% of the volume of the homogenizing tank is immersed in the ultrasonic medium.

[0009] Furthermore, the vertical distance between the end of the homogenizing tank protruding from the bottom surface of the tank and the bottom surface of the tank is 1 to 10 cm.

[0010] Furthermore, a sealing ring is provided at the interface between the opening and the homogenization tank.

[0011] According to another aspect of the present invention, there is provided a method for packing a C18 / phenyl mixed filler liquid chromatography column using the aforementioned column packing device, the method comprising: step S1, mixing raw materials including a homogenate and a mixed filler in a homogenization tank and filling the homogenization tank; step S2, using an ultrasonic device to perform real-time ultrasonic mixing on the mixed filler in the homogenization tank; step S3, using a column packing machine to fill the filler in the homogenization tank into an empty tube of the chromatographic column; wherein the mixed filler comprises C18 filler and phenyl filler; and the homogenate comprises a mixture of chloroform, methanol and C3-C6 perhalogenated alkanes.

[0012] Furthermore, the halogen in the above-mentioned C3~C6 perhalogenated alkane is fluorine element and / or bromine element, and preferably the C3~C6 perhalogenated alkane is selected from any one or more of 1-bromonoperfluorohexane, 1-nonafluorobutyl bromide, and 1,6-dibromonoperfluorohexane; and / or the volume ratio of chloroform, methanol and C3~C6 perhalogenated alkane is 1~30:1~50:1~30, preferably 2~10:10:1~10.

[0013] Furthermore, the ultrasonic power of the ultrasonic mixing is 10 to 50 W, preferably 20 to 40 W.

[0014] Further, the above-mentioned step S3 includes: step S31, placing the sinker into the displacement liquid, turning on the switch of the column packing machine, adjusting the pump speed of the column packing machine pump until liquid is discharged from the outlet pipeline of the column packing machine pump, and stopping the column packing machine pump; step S32, adjusting the pump speed of the column packing machine pump to maintain its pressure at a stable P, controlling the total amount of liquid V flowing out of the outlet pipeline of the column packing machine pump, adjusting the column packing machine pump to a pump pressure of 0, and completing the column packing after standing for a certain period of time; characterized in that P is 20 to 80 MPa; and / or, the standing time is 5 to 60 min; and / or, for an empty tube of a chromatographic column with a specification of 250 mm×4.6 mm, the total amount of liquid V is 20 to 100 mL; and / or, for an empty tube of a chromatographic column with a specification of 150 mm×4.6 mm, the total amount of liquid V is 20 to 80 mL; and / or, for an empty tube of a chromatographic column with a specification of 100 mm×4.6 mm, the total amount of liquid V is 20 to 60 mL.

[0015] Furthermore, the ratio of the volume of the homogenate to the mass of the mixed filler is 5-50 mL: 1-5 g.

[0016] Using the technical solution of the present application, on the one hand, the present invention screens a homogenate of a mixed component, the density of the homogenate is adjustable, and the mixed filler of C18 filler and phenyl filler can be evenly distributed in the homogenization tank, and the selected solvent contains a halogenated carbon chain with good hydrophobicity, which can make the modified long-chain functional group on the filler in a stretched state, thereby improving the column packing effect. On the other hand, the homogenization tank of the present invention is matched with an ultrasonic device, and the ultrasonic device has a groove-type accommodating cavity, and the groove-type accommodating cavity contains an ultrasonic medium. The ultrasonic wave emitted by the ultrasonic generator can ensure the uniform suspension of the mixed filler in the whole column packing process through the transmission of the ultrasonic medium, effectively avoiding the common filler sedimentation problem in the traditional wet column packing, thereby ensuring the uniformity of the filler in the whole column packing process, obtaining a uniform and compact column bed, reducing the channel effect, dead volume and column head pressure inside the chromatographic column, and thus improving the resolution and stability of the chromatographic column. The present invention simplifies the column packing operation, has low device cost and good column packing effect, reduces the transfer process of filler and the homogenization time, improves the column packing efficiency, the service life of the chromatographic column and the accuracy of the analysis results, and the relative standard deviation of the theoretical plate number of the packed chromatographic column is less than 3%, with high repeatability and high stability of the chromatographic column. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 A schematic diagram of a column packing device for a C18 / phenyl mixed filler liquid chromatography column of the present invention is shown;

[0019] Figure 2 A typical chromatogram of a chromatographic column packed by a real-time ultrasonic homogenization method according to Example 1 of the present invention is shown;

[0020] Figure 3 A typical chromatogram of a conventional wet-packed column is shown;

[0021] Figure 4 The figure shows the repeatability test result of the real-time ultrasonic homogenization tank column of the present invention;

[0022] Figure 5 The chromatographic column performance stability test spectrum of the present invention is shown.

[0023] The above drawings include the following reference numerals:

[0024] 1. Homogenization tank; 2. Tank feed port; 3. Tank discharge port; 4. Ultrasonic device; 5. Groove-shaped receiving cavity; 6. Groove bottom surface; 7. Ultrasonic generator; 8. Ultrasonic medium liquid surface. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0026] As analyzed in the background technology, there are problems in the prior art of uneven filling distribution and poor stability of C18 / phenyl mixed filler chromatographic columns. In order to solve this problem, the present application provides a column filling device and a column filling method for a C18 / phenyl mixed filler liquid chromatography column.

[0027] In a typical embodiment of the present application, a column packing device for a C18 / phenyl mixed filler liquid chromatography column is provided, the column packing device comprises a column packing machine, a homogenization tank 1, an ultrasonic device 4 and a chromatographic column empty tube, a column packing machine pump is arranged inside the column packing machine, the column packing machine pump has a liquid outlet end, the column packing machine is used to input a displacement liquid into the chromatographic column empty tube, and control the pressure and flow rate of the homogenate liquid transported into the chromatographic column empty tube; the homogenization tank 1 has a tank feed port 2 and a tank discharge port 3, the tank feed port 2 is connected to the liquid outlet end, and the homogenization tank 1 is used to homogenize a raw material including a mixed filler and a homogenate liquid; the ultrasonic device 4 has a trough structure, which has a trough-type accommodating cavity 5 and a trough bottom surface 6, the trough-type accommodating cavity 5 is used to hold An ultrasonic medium is placed, and an opening is provided on the bottom surface 6 of the groove. The aperture of the opening matches the outer diameter of the homogenization tank 1. At least a part of the tank body of the homogenization tank 1 is immersed in the ultrasonic medium in the groove-shaped accommodating cavity 5, and the homogenization tank 1 passes through the opening and extends out of the bottom surface 6 of the groove; an ultrasonic generator 7 is provided on the inner surface of the ultrasonic device 4, and the ultrasonic device 4 is used for real-time ultrasonic mixing of the mixed filler in the homogenization tank 1; the chromatographic column empty tube has a column feed port and a column discharge port, and the column feed port is connected to the tank discharge port 3, and the chromatographic column empty tube is used for filling the mixed filler in the homogenization tank 1; wherein the mixed filler includes C18 filler and phenyl filler; the homogenate includes a mixture of chloroform, methanol and C3-C6 perhalogenated alkanes.

[0028] On the one hand, the present invention selects a homogenate of a mixed component, the density of the homogenate is adjustable, and the mixed filler of C18 filler and phenyl filler can be evenly distributed in the homogenate tank 1, and the selected solvent contains a halogenated carbon chain with good hydrophobicity, which can make the modified long-chain functional group on the filler in a stretched state, thereby improving the column packing effect. On the other hand, the homogenate tank 1 of the present invention is matched with an ultrasonic device 4, and the ultrasonic device 4 has a groove-type accommodating cavity 5, and the groove-type accommodating cavity 5 contains an ultrasonic medium. The ultrasonic wave emitted by the ultrasonic generator 7 can ensure the uniform suspension of the mixed filler in the whole column packing process through the transmission of the ultrasonic medium, effectively avoiding the common filler sedimentation problem in the traditional wet column packing, thereby ensuring the uniformity of the filler in the whole column packing process, obtaining a uniform and compact column bed, reducing the channel effect, dead volume and column head pressure inside the chromatographic column, and thus improving the resolution and stability of the chromatographic column. The present invention simplifies the column packing operation, has low device cost and good column packing effect, reduces the transfer process of filler and the homogenization time, improves the column packing efficiency, the service life of the chromatographic column and the accuracy of the analysis results, and the relative standard deviation of the theoretical plate number of the packed chromatographic column is less than 3%, with high repeatability and high stability of the chromatographic column.

[0029] In one embodiment of the present application, the probe of the ultrasonic generator 7 is immersed in the ultrasonic medium in the groove-shaped accommodating cavity 5, and / or the ultrasonic medium is selected from any one or more of water, ethanol, methanol, acetonitrile, 50% ethanol aqueous solution, and 10% sodium chloride aqueous solution; and / or the ultrasonic generator 7 is a cylindrical bottom ultrasonic generator or a ring-shaped probe ultrasonic generator.

[0030] The probe of the ultrasonic generator 7 is preferably immersed in the ultrasonic medium in the trough-shaped accommodating chamber 5, that is, the probe is located below the ultrasonic medium liquid level 8, which is conducive to further improving the uniformity of the ultrasonic waves transmitted to the homogenization tank 1. It is further preferred that the ultrasonic medium is water. Compared with other types of ultrasonic media, water is preferred as the ultrasonic medium, which is not only conducive to improving the good transmission of ultrasonic waves, but also helps to reduce its pollution to the environment and reduce its corrosion to the ultrasonic device 4. The cylindrical bottom ultrasonicator or the ring probe ultrasonicator is preferred to help select the direction of ultrasonic transmission according to different chromatographic column lengths and chromatographic column compactness and the conditions of the ultrasonic device 4.

[0031] In one embodiment of the present application, a portion of the tank body of the above-mentioned homogenization tank 1 close to the tank discharge port 3 is immersed in the ultrasonic medium in the trough-shaped containing cavity 5, and / or 10% to 90% of the volume of the homogenization tank 1 is immersed in the ultrasonic medium.

[0032] Preferably, 10% to 90% of the volume of the homogenization tank 1 is controlled to be immersed in the ultrasonic medium, which helps to further ensure the contact between the bottom of the homogenization tank and the medium, thereby improving the uniformity of the mixing of the filler at the bottom of the homogenization tank.

[0033] In one embodiment of the present application, when an ultrasonic medium is added to the trough-shaped holding cavity 5, it is preferred that the vertical distance between the end of the homogenization tank 1 that passes through the trough bottom surface 6 and the trough bottom surface 6 is 1 to 10 cm, which helps the ultrasonic medium to completely surround the homogenization tank 1 without flowing into the homogenization tank 1, and at the same time helps the filler at the bottom of the homogenization tank 1 to be fully acted upon by the ultrasonic wave.

[0034] In one embodiment of the present application, a sealing ring is provided at the interface between the above-mentioned opening and the homogenization tank 1 .

[0035] The setting of the sealing ring helps to reduce the risk of ultrasonic medium leakage while ensuring the effective transmission of ultrasonic energy. In addition, the sealing ring material should be selected with high elasticity and chemical stability, such as fluororubber or silicone rubber, to ensure that it can still maintain good sealing performance under high pressure and various solvent environments.

[0036] In another typical embodiment of the present application, a method for packing a C18 / phenyl mixed filler liquid chromatography column using the above-mentioned column packing device is provided, the method comprising: step S1, mixing raw materials including a homogenate and a mixed filler in a homogenization tank 1 and filling the homogenization tank 1; step S2, using an ultrasonic device 4 to perform real-time ultrasonic mixing on the mixed filler in the homogenization tank 1; step S3, using a column packing machine to fill the filler in the homogenization tank 1 into an empty tube of the chromatographic column; wherein the mixed filler comprises C18 filler and phenyl filler; and the homogenate comprises a mixture of chloroform, methanol and C3-C6 perhalogenated alkanes.

[0037] On the one hand, the present invention screens a homogenate of a mixed component, the density of the homogenate is adjustable, and the mixed filler of C18 filler and phenyl filler can be evenly distributed in the homogenization tank, and the selected solvent contains a halogenated carbon chain with good hydrophobicity, which can make the modified long-chain functional group on the filler in a stretched state, thereby improving the column packing effect. On the other hand, the homogenization tank 1 of the present invention matches the ultrasonic device 4, and the ultrasonic device 4 has a groove-type accommodating cavity 5, and the groove-type accommodating cavity 5 contains an ultrasonic medium. The ultrasonic wave emitted by the ultrasonic generator 7 can ensure the uniform suspension of the mixed filler in the whole column packing process through the transmission of the ultrasonic medium, effectively avoiding the common filler sedimentation problem in the traditional wet column packing, thereby ensuring the uniformity of the filler in the whole column packing process, obtaining a uniform and compact column bed, reducing the channel effect, dead volume and column head pressure inside the chromatographic column, and thus improving the resolution and stability of the chromatographic column. The method of the present invention has good column packing effect, reduces the transfer process of the filler and the homogenization time, improves the column packing efficiency, the service life of the chromatographic column and the accuracy of the analysis results, and the relative standard deviation of the theoretical plate number of the packed chromatographic column is less than 3%, with high repeatability and high stability of the chromatographic column.

[0038] In one embodiment of the present application, the halogen in the above-mentioned C3~C6 perhalogenated alkane is fluorine element and / or bromine element, and the C3~C6 perhalogenated alkane is preferably selected from any one or more of 1-bromonoperfluorohexane, 1-nonafluorobutyl bromide, and 1,6-dibromonoperfluorohexane; and / or, the volume ratio of chloroform, methanol and C3~C6 perhalogenated alkane is 1~30:1~50:1~30, preferably 2~10:10:1~10.

[0039] The preferred types of the above C3-C6 perhalogenated alkanes and the volume ratio of chloroform, methanol and C3-C6 perhalogenated alkanes help to improve the density adjustability of the homogenate and better disperse the mixed filler in the homogenate.

[0040] In one embodiment of the present application, the ultrasonic power of the ultrasonic mixing is preferably 10 to 50 W, preferably 20 to 40 W, which helps to keep the filler evenly suspended under the action of ultrasound and avoid sedimentation.

[0041] In one embodiment of the present application, the above-mentioned step S3 includes: step S31, placing the sinker into the displacement liquid, turning on the switch of the column packing machine, adjusting the pump speed of the column packing machine pump until liquid is discharged from the outlet pipeline of the column packing machine pump, and stopping the column packing machine pump; step S32, adjusting the pump speed of the column packing machine pump to maintain its pressure at a stable P, controlling the total amount of liquid V flowing out of the outlet pipeline of the column packing machine pump, adjusting the column packing machine pump to a pump pressure of 0, and completing the column packing after standing for a certain period of time; P is 20 to 80 MPa; and / or, the standing time is 5 to 60 min, and / or, for an empty tube of a chromatographic column with a specification of 250 mm×4.6 mm, the total amount of liquid V is 20 to 100 mL; and / or, for an empty tube of a chromatographic column with a specification of 150 mm×4.6 mm, the total amount of liquid V is 20 to 80 mL; and / or, for an empty tube of a chromatographic column with a specification of 100 mm×4.6 mm, the total amount of liquid V is 20 to 60 mL.

[0042] The purpose of step S31 is to exhaust bubbles. The presence of bubbles will affect the separation performance and stability of the chromatographic column, leading to pressure fluctuations, abnormal peak shape and other problems. Before filling the liquid chromatographic column with filler, the first exhaust operation with a displacement liquid helps to remove the bubbles remaining inside the chromatographic column and obtain a uniform, bubble-free column bed, thereby ensuring the accuracy and reliability of the chromatographic analysis. The properties of the displacement liquid should be similar to the chromatographic mobile phase so that the environment inside the chromatographic column can match the analysis conditions before using the chromatographic column for analysis. This can reduce the impact on the column bed caused by the sudden change of the mobile phase properties at the beginning of the analysis, affecting the performance of the chromatographic column. At the same time, the selection of the displacement liquid should take into account the properties of the filler and the final use of the chromatographic column. During the column loading process, the displacement liquid is introduced into the chromatographic column at a certain pressure and flow rate by the pump of the column loading machine, so that the uniformly dispersed filler is pushed into the chromatographic column from the homogenization tank to form a column bed. Therefore, the preferred displacement liquid of the present application is selected from any one or more of methanol, ethanol, acetonitrile, isopropanol, and acetone. It is preferred that P is controlled to be 20 to 80 MPa to help the filler to deposit uniformly. The preferred standing time is conducive to further uniform distribution and stability of the filler. Optimizing the matching of chromatographic column empty tubes of different specifications with the total amount of liquid V is more conducive to improving the column packing effect of the chromatographic column empty tube.

[0043] In addition, after the standing time is over, the chromatographic column is taken out of the homogenization tank, and then a frit plate and a column head are installed at both ends. The frit plate is used to prevent the packing from being lost, and the column head is used to connect other parts of the chromatograph.

[0044] In one embodiment of the present application, the ratio of the volume of the above-mentioned homogenate to the mass of the mixed filler is preferably 5-50 mL: 1-5 g, which is beneficial to improve the dispersion uniformity and stability of the mixed filler in the homogenate.

[0045] The beneficial effects of the present application will be described below in conjunction with specific embodiments and comparative examples.

[0046] (I) Experimental instruments and reagents

[0047] (1) The instruments used were a liquid chromatography column packing machine, a micro ultrasonic generator, a real-time ultrasonic homogenization tank, a liquid chromatography column empty column tube (250 mm × 4.6 mm), a chromatographic column 5 μm mixed filler (70% C18 silica gel filler and 30% phenyl filler), and an Agilent 1260 liquid chromatograph;

[0048] (2) Experimental reagents: methanol, tetrahydrofuran, isopropanol, chloroform, 1-bromoperfluorohexane, and ultrapure water.

[0049] (II) Experimental conditions

[0050] Filling the homogenization tank: connect the empty column tube to the outlet of the real-time ultrasonic homogenization tank, take 10 mL of homogenate and add it to the real-time ultrasonic homogenization tank, add 2.5 g of C18 / phenyl mixed filler, fill the real-time ultrasonic homogenization tank with the homogenate, and then tighten the inlet of the homogenization tank;

[0051] Ultrasound: Select water as the ultrasonic medium, place the micro ultrasonic generator probe into the ultrasonic medium of the real-time ultrasonic homogenization tank, and adjust the ultrasonic power to 30W;

[0052] Exhaust: put the sinker into the displacement liquid, turn on the column packing machine, adjust the pump speed until liquid is discharged from the pump outlet pipeline, and stop the pump;

[0053] Filling the column: Connect the solvent inlet of the homogenization tank to the outlet of the pump, ensuring that the connection is tight and there is no leakage. Turn on the pump, adjust the pump speed to raise the pressure to 40MPa, and adjust the pump speed to maintain a stable pressure. After the pressure stabilizes, when the total amount of liquid flowing out is 80mL, adjust the pump pressure to 40MPa. After stabilization, adjust the pump pressure to 0 and turn off the pump. After standing for 30 minutes, remove the chromatographic column and cover it with the sieve plate and column head.

[0054] (III) Chromatographic column performance test conditions

[0055] Column temperature: 25°C;

[0056] Detection wavelength: 254nm;

[0057] Flow rate: 1.0 mL / min;

[0058] Injection volume: 5.0 μL;

[0059] Isocratic elution: methanol: water volume ratio 85:15;

[0060] Column efficiency test standard: Use 85% methanol aqueous solution as solvent to prepare a mixed sample of 5.0 μg / mL uracil, 200.0 μg / mL phenol, 25.0 μg / mL 4-nitrochlorobenzene and 850.0 μg / mL toluene.

[0061] (I) Screening of homogenate types:

[0062] Homogenate 1: Mixed solution of cyclohexanol and chloroform:

[0063] Homogenization tank filling: Mix 2.5 g of C18 / phenyl mixed filler with 20 mL of a mixed solution of cyclohexanol and chloroform (volume ratio 9:11), mix by ultrasonic mixing for 5 min, pour into a homogenization tank with an empty column tube, and then tighten the inlet end of the homogenization tank;

[0064] Exhaust: put the sinker into the displacement liquid, turn on the column packing machine, adjust the pump speed until liquid is discharged from the pump outlet pipeline, and stop the pump;

[0065] Column filling: Connect the solvent inlet of the homogenization tank to the outlet of the column packing machine, and ensure that the connection is tight and there is no leakage. Turn on the pump, adjust the pump speed to raise the pressure to 40MPa, and adjust the pump speed to maintain a stable pressure. After the pressure stabilizes, when the total amount of liquid flowing out is 80mL, adjust the pump pressure to 40MPa. After stabilization, adjust the pump pressure to 0 and turn off the pump. After standing for 30 minutes, remove the chromatographic column and cover it with the sieve plate and column head;

[0066] Chromatographic column performance test: Connect the packed chromatographic column to the liquid chromatograph to test the chromatographic column performance. After analyzing the software performance report, the actual plate number and separation degree data are obtained.

[0067] Homogenate 2: Tetrahydrofuran

[0068] Homogenization tank filling: Mix 2.5 g of C18 / phenyl mixed filler with 20 mL of tetrahydrofuran solvent, mix by ultrasonic for 5 min, pour into the homogenization tank with an empty column tube, and then connect the homogenization tank to the column loading machine;

[0069] Exhaust: put the sinker into the displacement liquid, turn on the column packing machine, adjust the pump speed until liquid is discharged from the pump outlet pipeline, and stop the pump;

[0070] Column filling: Connect the solvent inlet of the homogenization tank to the outlet of the column packing machine, and ensure that the connection is tight and there is no leakage. Turn on the pump, adjust the pump speed to raise the pressure to 40MPa, and adjust the pump speed to maintain a stable pressure. After the pressure stabilizes, when the total amount of liquid flowing out is 80mL, adjust the pump pressure to 40MPa. After stabilization, adjust the pump pressure to 0 and turn off the pump. After standing for 30 minutes, remove the chromatographic column and cover it with the sieve plate and column head;

[0071] Chromatographic column performance test: Connect the packed chromatographic column to the liquid chromatograph to test the chromatographic column performance. After analyzing the software performance report, the actual plate number and separation degree data are obtained.

[0072] Homogenate 3: a mixture of chloroform, methanol and 1-bromoperfluorohexane

[0073] Filling the homogenization tank: Mix 2.5 g of C18 / phenyl mixed filler with 20 mL of homogenization solution (a mixture of chloroform, methanol and 1-bromoperfluorohexane), mix by ultrasonication for 5 min, pour into the homogenization tank with an empty column tube, and then tighten the inlet end of the homogenization tank;

[0074] Exhaust: put the sinker into the displacement liquid, turn on the column packing machine, adjust the pump speed until liquid is discharged from the pump outlet pipeline, and stop the pump;

[0075] Column filling: Connect the solvent inlet of the homogenization tank to the outlet of the column packing machine, and ensure that the connection is tight and there is no leakage. Turn on the pump, adjust the pump speed to raise the pressure to 40MPa, and adjust the pump speed to maintain a stable pressure. After the pressure stabilizes, when the total amount of liquid flowing out is 80mL, adjust the pump pressure to 40MPa. After stabilization, adjust the pump pressure to 0 and turn off the pump. After standing for 30 minutes, remove the chromatographic column and cover it with the sieve plate and column head;

[0076] Chromatographic column performance test: Connect the packed chromatographic column to the liquid chromatograph to test the chromatographic column performance. After analyzing the software performance report, the actual plate number and separation degree data are obtained.

[0077] Table 1 lists the column efficiency data of the packed chromatographic columns corresponding to different homogenates, wherein homogenate 1 is a mixture of cyclohexanol and chloroform, and the volume ratio of cyclohexanol to chloroform is 9:99; homogenate 2 is tetrahydrofuran; homogenate 3 is a mixture of chloroform, methanol and 1-bromoperfluorohexane, and the volume ratio of chloroform, methanol and 1-bromoperfluorohexane is 2:10:1.

[0078] Table 1

[0079] Homogenate Measured number of plates Separation Peak tailing factor Homogenate 1 14856 7.22 1.18 Homogenate 2 15632 7.38 1.14 Homogenate 3 18555 7.80 1.08

[0080] As can be seen from Table 1, the plate number and separation degree of the chromatographic column prepared using homogenate 3 as the homogenate are higher than those of the other two, and can provide higher column efficiency.

[0081] (II) Proportion screening of homogenate 3:

[0082] Filling the homogenization tank: Take 10 mL of the mixed homogenate of chloroform / methanol / 1-bromoperfluorohexane with a volume ratio of 10:10:1, 2:10:1, and 2:10:10, respectively, and add it to the real-time ultrasonic homogenization tank with an empty column tube, then add 2.5 g of C18 / phenyl mixed filler, and then tighten the inlet end of the homogenization tank;

[0083] Exhaust: put the sinker into the displacement liquid, turn on the column packing machine, adjust the pump speed until liquid is discharged from the pump outlet pipeline, and stop the pump;

[0084] Column filling: Connect the solvent inlet of the homogenization tank to the outlet of the column packing machine, and ensure that the connection is tight and there is no leakage. Turn on the pump, adjust the pump speed to raise the pressure to 40MPa, and adjust the pump speed to maintain a stable pressure. After the pressure stabilizes, when the total amount of liquid flowing out is 80mL, adjust the pump pressure to 40MPa. After stabilization, adjust the pump pressure to 0 and turn off the pump. After standing for 30 minutes, remove the chromatographic column and cover it with the sieve plate and column head;

[0085] Chromatographic column performance test: Connect the packed chromatographic column to the liquid chromatograph to test the chromatographic column performance. After analyzing the software performance report, the actual plate number and separation degree data are obtained.

[0086] Table 2 lists the column efficiency data of the chromatographic column packed with different proportions of the homogenate 3.

[0087] Table 2

[0088] Volume ratio of chloroform, methanol and 1-bromoperfluorohexane Measured number of plates Separation Peak tailing factor 1:1:1 14923 7.26 1.16 10:10:1 16856 7.48 1.10 2:10:1 18555 7.80 1.08 2:10:10 16555 7.45 1.10 30:50:30 15236 7.33 1.13

[0089] As can be seen from Table 2, the chromatographic column prepared by using a mixed slurry of chloroform, methanol and 1-bromoperfluorohexane in a volume ratio of 2:10:1 has a higher plate number and separation than the other two, and can provide higher column efficiency.

[0090] (III) Real-time ultrasonic homogenization method for filling chromatographic columns:

[0091] Example 1

[0092] according to Figure 1 The column is packed as shown in the schematic diagram of the column packing device of the C18 / phenyl mixed filler liquid chromatography column:

[0093] Filling the homogenization tank: Take 10 mL of chloroform, methanol and 1-bromoperfluorohexane mixed homogenate (volume ratio is 2:10:1) respectively and add them into the real-time ultrasonic homogenization tank 1 with an empty column tube (having a tank feed port 2 and a tank discharge port 3, and the tank feed port 2 is connected to the liquid outlet end), and then add 2.5 g of C18 / phenyl mixed filler respectively, fill the real-time ultrasonic homogenization tank with the homogenate, and then tighten the inlet end of the homogenization tank;

[0094] Ultrasound: An ultrasonic device 4 is specifically used, which has a trough structure, a trough-shaped receiving cavity 5 and a trough bottom surface 6. The trough-shaped receiving cavity 5 is used to contain ultrasonic medium. The trough bottom surface 6 is provided with an opening, and the aperture of the opening matches the outer diameter of the homogenizing tank 1. At least a part of the tank body of the homogenizing tank 1 is immersed in the ultrasonic medium in the trough-shaped receiving cavity 5, and the homogenizing tank 1 passes through the opening and extends out of the trough bottom surface 6. An ultrasonic generator 7 is provided on the inner surface of the ultrasonic device 4. The ultrasonic device 4 is used to perform real-time ultrasonic mixing of the mixed filler in the homogenizing tank 1. The probe of the ultrasonic generator 7 is immersed in the ultrasonic medium in the trough-shaped receiving cavity 5, that is, the probe is located below the ultrasonic medium liquid level 8. More specifically, water is selected as the ultrasonic medium, and the micro-ultrasonic generator probe is placed in the ultrasonic medium of the real-time ultrasonic homogenizing tank. The ultrasonic power is adjusted to 30W, 50% of the volume of the homogenizing tank 1 is immersed in the ultrasonic medium, and the vertical distance between the end of the homogenizing tank 1 that passes through the trough bottom surface 6 and the trough bottom surface 6 is 4cm;

[0095] Exhaust: put the sinker into the displacement liquid, turn on the column packing machine, adjust the pump speed until liquid is discharged from the pump outlet pipeline, and stop the pump;

[0096] Column filling: Connect the solvent inlet of the homogenization tank to the outlet of the column packing machine, and ensure that the connection is tight and there is no leakage. Turn on the pump, adjust the pump speed to raise the pressure to 40MPa, and adjust the pump speed to maintain a stable pressure. After the pressure stabilizes, when the total amount of liquid flowing out is 80mL, adjust the pump pressure to 40MPa. After stabilization, adjust the pump pressure to 0 and turn off the pump. After standing for 30 minutes, remove the chromatographic column and cover it with the sieve plate and column head;

[0097] Chromatographic column performance test: Connect the packed chromatographic column to the liquid chromatograph to test the chromatographic column performance. After analyzing the software performance report, the actual plate number and separation degree data are obtained.

[0098] Table 3 lists the column efficiency data of the chromatographic columns packed by the real-time ultrasonic homogenization method.

[0099] Table 3

[0100]

[0101] As can be seen from Table 3 and Table 2, the plate number and separation degree of the chromatographic column filled with the real-time ultrasonic homogenization tank are better, which can provide higher column efficiency, better separate the target compounds and improve the accuracy and sensitivity of the analysis. The typical chromatogram of the chromatographic column filled with the real-time ultrasonic homogenization method is shown in Figure 2 shown.

[0102] Example 2

[0103] Choice of ultrasound medium:

[0104] according to Figure 1 The column is packed as shown in the schematic diagram of the column packing device of the C18 / phenyl mixed filler liquid chromatography column:

[0105] Filling the homogenization tank: Take 10 mL of chloroform / methanol / 1-bromoperfluorohexane mixed homogenate (volume ratio is 2:10:1) and add it to the real-time ultrasonic homogenization tank with an empty column tube, then add 2.5 g of C18 / phenyl mixed filler, fill the real-time ultrasonic homogenization tank with the homogenate, and then tighten the inlet end of the homogenization tank;

[0106] Ultrasound: using water, ethanol, 50% ethanol aqueous solution and 10% sodium chloride aqueous solution as ultrasonic media respectively, placing the micro ultrasonic generator probe into the ultrasonic medium of the real-time ultrasonic homogenization tank, adjusting the ultrasonic power to 30W, 50% of the volume of the homogenization tank 1 is immersed in the ultrasonic medium, and the vertical distance between the end of the homogenization tank 1 that passes through the bottom surface 6 of the tank and the bottom surface 6 of the tank is 4 cm;

[0107] Exhaust: put the sinker into the displacement liquid, turn on the column packing machine, adjust the pump speed until liquid is discharged from the pump outlet pipeline, and stop the pump;

[0108] Column filling: Connect the solvent inlet of the homogenization tank to the outlet of the column packing machine, and ensure that the connection is tight and there is no leakage. Turn on the pump, adjust the pump speed to raise the pressure to 40MPa, and adjust the pump speed to maintain a stable pressure. After the pressure stabilizes, when the total amount of liquid flowing out is 80mL, adjust the pump pressure to 40MPa. After stabilization, adjust the pump pressure to 0 and turn off the pump. After standing for 30 minutes, remove the chromatographic column and cover it with the sieve plate and column head;

[0109] Chromatographic column performance test: Connect the packed chromatographic column to the liquid chromatograph to test the chromatographic column performance. After analyzing the software performance report, the actual plate number and separation degree data are obtained.

[0110] Table 4 lists the column efficiency data of the chromatographic columns packed with different ultrasonic media.

[0111] Table 4

[0112] Ultrasonic medium Measured number of plates Separation Peak tailing factor water 20995 8.13 1.01 Ethanol 18963 7.85 1.05 50% ethanol in water 19456 7.99 1.02 10% sodium chloride aqueous solution 19036 7.93 1.03

[0113] It can be seen from Table 4 that the plate number and separation degree of the chromatographic column made with water as the ultrasonic medium are higher than those of the other three media, and can provide higher column efficiency.

[0114] Example 3

[0115] Selection of ultrasonic power:

[0116] according to Figure 1 The column is packed as shown in the schematic diagram of the column packing device of the C18 / phenyl mixed filler liquid chromatography column:

[0117] Filling the homogenization tank: Take 10 mL of chloroform / methanol / 1-bromoperfluorohexane mixed homogenate (volume ratio is 2:10:1) and add it to the real-time ultrasonic homogenization tank with an empty column tube, then add 2.5 g of C18 / phenyl mixed filler, fill the real-time ultrasonic homogenization tank with the homogenate, and then tighten the inlet end of the homogenization tank;

[0118] Ultrasound: using water as the ultrasonic medium, placing the micro ultrasonic generator probe into the ultrasonic medium of the real-time ultrasonic homogenization tank, adjusting the ultrasonic power to change the ultrasonic power to 10W, 20W, 30W, 40W and 50W respectively, 50% of the volume of the homogenization tank 1 is immersed in the ultrasonic medium, and the vertical distance between the end of the homogenization tank 1 that passes through the bottom surface 6 of the tank and the bottom surface 6 of the tank is 4 cm;

[0119] Exhaust: put the sinker into the displacement liquid, turn on the column packing machine, adjust the pump speed until liquid is discharged from the pump outlet pipeline, and stop the pump;

[0120] Column filling: Connect the solvent inlet of the homogenization tank to the outlet of the column packing machine, and ensure that the connection is tight and there is no leakage. Turn on the pump, adjust the pump speed to raise the pressure to 40MPa, and adjust the pump speed to maintain a stable pressure. After the pressure stabilizes, when the total amount of liquid flowing out is 80mL, adjust the pump pressure to 40MPa. After stabilization, adjust the pump pressure to 0 and turn off the pump. After standing for 30 minutes, remove the chromatographic column and cover it with the sieve plate and column head;

[0121] Chromatographic column performance test: Connect the packed chromatographic column to the liquid chromatograph to test the chromatographic column performance. After analyzing the software performance report, the actual plate number and separation degree data are obtained.

[0122] Table 5 lists the column efficiency data of the packed chromatographic columns corresponding to different ultrasonic powers.

[0123] Table 5

[0124] Ultrasonic power / W Measured number of plates Separation Peak tailing factor 10 18563 7.83 1.06 20 19523 7.96 1.03 30 20995 8.13 1.01 40 19236 7.92 1.03 50 18023 7.79 1.08

[0125] It can be seen from Table 5 that the chromatographic column prepared with an ultrasonic power of 30 W has a better plate number and separation, and can provide a higher column efficiency.

[0126] Example 4

[0127] The difference from Example 1 is that 10% of the volume of the homogenization tank 1 is immersed in the ultrasonic medium.

[0128] Example 5

[0129] The difference from Example 1 is that 90% of the volume of the homogenization tank 1 is immersed in the ultrasonic medium.

[0130] Example 6

[0131] The difference from Example 1 is that 5% of the volume of the homogenization tank 1 is immersed in the ultrasonic medium.

[0132] Example 7

[0133] The difference from Example 1 is that the vertical distance between the end of the homogenizing tank 1 protruding from the bottom surface 6 of the tank and the bottom surface 6 of the tank is 1 cm.

[0134] Example 8

[0135] The difference from Example 1 is that the vertical distance between the end of the homogenizing tank 1 protruding from the bottom surface 6 of the tank and the bottom surface 6 of the tank is 10 cm.

[0136] Example 9

[0137] The difference from Example 1 is that the vertical distance between the end of the homogenizing tank 1 protruding from the bottom surface 6 of the tank and the bottom surface 6 of the tank is 15 cm.

[0138] Example 10

[0139] The difference from Example 1 is that the homogenization tank is filled: 5 mL of a mixed homogenate of chloroform, methanol and 1-bromoperfluorohexane (volume ratio of 2:10:1) is added to a real-time ultrasonic homogenization tank with an empty column tube, and then 2.0 g of C18 / phenyl mixed filler is added, the real-time ultrasonic homogenization tank is filled with the homogenate, and then the inlet end of the homogenization tank is tightened.

[0140] Embodiment 11

[0141] The difference from Example 1 is that the homogenization tank is filled: 20 mL of a mixed homogenate of chloroform, methanol and 1-bromoperfluorohexane (volume ratio of 2:10:1) are added to a real-time ultrasonic homogenization tank with an empty column tube, and then 5.0 g of C18 / phenyl mixed filler is added, and then the inlet end of the homogenization tank is tightened.

[0142] After the column packing was completed in the above Examples 4 to 11, the chromatographic column performance was tested respectively: the packed chromatographic column was connected to the liquid chromatograph, the chromatographic column performance was tested, and the measured plate number and separation degree data were obtained through software performance report analysis, as shown in Table 6.

[0143] Table 6

[0144] Example Measured number of plates Separation Peak tailing factor Example 4 18956 7.85 1.06 Example 5 19353 7.92 1.04 Example 6 16593 7.45 1.10 Example 7 19231 7.89 1.05 Example 8 18369 7.82 1.08 Example 9 15963 7.40 1.12 Example 10 17659 7.75 1.08 Embodiment 11 20865 8.13 1.02

[0145] (IV) Real-time ultrasonic homogenization tank column repeatability test:

[0146] The real-time ultrasonic homogenization tank in Example 1 was used to continuously load 10 chromatographic columns, and the performance of the chromatographic columns was tested. The results are as follows: Figure 4 As shown, the relative standard deviation of the measured plate number is 2.65%, and the real-time ultrasonic homogenization tank column has good repeatability.

[0147] (V) Chromatographic column stability test:

[0148] The chromatographic column was filled with the real-time ultrasonic homogenization tank in Example 1, and the chromatographic column performance was tested 500 times, wherein the chromatograms of the first, 100, 200 and 500 detections were substantially the same (e.g. Figure 5 This indicates that the chromatographic column packed in the real-time ultrasonic homogenization tank has good stability.

[0149] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0150] On the one hand, the present invention screens a homogenate of a mixed component, the density of the homogenate is adjustable, and the mixed filler of C18 filler and phenyl filler can be evenly distributed in the homogenization tank, and the selected solvent contains a halogenated carbon chain with good hydrophobicity, which can make the modified long-chain functional group on the filler in a stretched state, thereby improving the column packing effect. On the other hand, the homogenization tank of the present invention is matched with an ultrasonic device, and the ultrasonic device has a groove-type accommodating cavity, and the groove-type accommodating cavity contains an ultrasonic medium. The ultrasonic wave emitted by the ultrasonic generator can ensure the uniform suspension of the mixed filler in the whole column packing process through the transmission of the ultrasonic medium, effectively avoiding the common filler sedimentation problem in the traditional wet column packing, thereby ensuring the uniformity of the filler in the whole column packing process, obtaining a uniform and compact column bed, reducing the channel effect, dead volume and column head pressure inside the chromatographic column, and thus improving the resolution and stability of the chromatographic column. The present invention simplifies the column packing operation, has low device cost and good column packing effect, reduces the transfer process of filler and the homogenization time, improves the column packing efficiency, the service life of the chromatographic column and the accuracy of the analysis results, and the relative standard deviation of the theoretical plate number of the packed chromatographic column is less than 3%, with high repeatability and high stability of the chromatographic column.

[0151] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A column packing device for a C18 / phenyl mixed filler liquid chromatography column, characterized in that: The column packing device comprises: A column packing machine, wherein a column packing machine pump is arranged inside the column packing machine pump, the column packing machine pump has a liquid outlet end, the column packing machine is used to input displacement liquid into the empty tube of the chromatographic column, and control the pressure and flow rate of the homogenate liquid transported into the empty tube of the chromatographic column; A homogenizing tank (1) having a tank feed port (2) and a tank discharge port (3), wherein the tank feed port (2) is connected to the liquid outlet end, and the homogenizing tank (1) is used to homogenize raw materials including a mixed filler and a homogenizing liquid; The ultrasonic device (4) has a trough-shaped structure, comprising a trough-shaped receiving cavity (5) and a trough bottom surface (6), wherein the trough-shaped receiving cavity (5) is used to contain an ultrasonic medium, the trough bottom surface (6) is provided with an opening, the aperture of the opening matches the outer diameter of the homogenizing tank (1), at least a portion of the tank body of the homogenizing tank (1) is immersed in the ultrasonic medium in the trough-shaped receiving cavity (5), and the homogenizing tank (1) passes through the opening and extends out of the trough bottom surface (6); an ultrasonic generator (7) is provided on the inner surface of the ultrasonic device (4), and the ultrasonic device (4) is used to perform real-time ultrasonic mixing on the mixed filler in the homogenizing tank (1); The chromatographic column empty tube has a column feed port and a column discharge port, the column feed port is connected to the tank discharge port (3), and the chromatographic column empty tube is used to load the mixed filler in the homogenization tank (1); Wherein, the mixed filler includes C18 filler and phenyl filler; The homogenate comprises a mixture of chloroform, methanol and C3-C6 perhalogenated alkanes.

2. The column packing device according to claim 1, characterized in that: The probe of the ultrasonic generator (7) is immersed in the ultrasonic medium in the groove-shaped accommodating cavity (5), and / or the ultrasonic medium is selected from any one or more of water, ethanol, methanol, acetonitrile, 50% ethanol aqueous solution, and 10% sodium chloride aqueous solution; and / or the ultrasonic generator (7) is a cylindrical bottom ultrasonic generator or a ring-shaped probe ultrasonic generator.

3. The column packing device according to claim 1, characterized in that: A portion of the homogenization tank (1) near the tank discharge port (3) is immersed in the ultrasonic medium in the trough-shaped receiving cavity (5), and / or 10% to 90% of the volume of the homogenization tank (1) is immersed in the ultrasonic medium.

4. The column packing device according to any one of claims 1 to 3, characterized in that: The vertical distance between the end of the homogenizing tank (1) protruding from the bottom surface (6) of the tank and the bottom surface (6) of the tank is 1 to 10 cm.

5. The column packing device according to any one of claims 1 to 3, characterized in that: A sealing ring is provided at the interface between the opening and the homogenizing tank (1).

6. A method for packing a C18 / phenyl mixed filler liquid chromatography column using the column packing device according to any one of claims 1 to 5, characterized in that: The method comprises: Step S1, mixing raw materials including a homogenate and a mixed filler in a homogenization tank (1) and filling the homogenization tank (1); Step S2, using an ultrasonic device (4) to perform real-time ultrasonic mixing on the mixed filler in the homogenization tank (1); Step S3, using a column packing machine to fill the filler in the homogenization tank (1) into the empty tube of the chromatographic column; Wherein, the mixed filler includes C18 filler and phenyl filler; The homogenate comprises a mixture of chloroform, methanol and C3-C6 perhalogenated alkanes.

7. The method according to claim 6, characterized in that The halogen in the C3-C6 perhalogenated alkane is fluorine and / or bromine, and preferably the C3-C6 perhalogenated alkane is selected from any one or more of 1-bromoperfluorohexane, 1-nonafluorobutyl bromide, and 1,6-dibromoperfluorohexane; and / or the volume ratio of the chloroform, the methanol and the C3-C6 perhalogenated alkane is 1-30:1-50:1-30, preferably 2-10:10:1-10.

8. The method according to claim 6 or 7, characterized in that: The ultrasonic mixing has an ultrasonic power of 10 to 50 W, preferably 20 to 40 W.

9. The method according to any one of claims 6 to 8, wherein step S3 comprises: Step S31, placing the sinker into the displacement liquid, turning on the switch of the column packing machine, adjusting the pump speed of the column packing machine pump until liquid is discharged from the outlet pipeline of the column packing machine pump, and stopping the column packing machine pump; Step S32, adjusting the pump speed of the column packing machine pump to maintain its pressure at a stable P, controlling the total amount of liquid V flowing out of the outlet pipeline of the column packing machine pump, adjusting the column packing machine pump to a pump pressure of 0, and after standing for a certain period of time, the column packing is completed; Characterized in that the P is 20 to 80 MPa; and / or the standing time is 5 to 60 min; And / or, for an empty chromatographic column tube with a specification of 250 mm×4.6 mm, the total liquid volume V is 20 to 100 mL; And / or, for an empty chromatographic column tube with a specification of 150 mm×4.6 mm, the total liquid volume V is 20 to 80 mL; And / or, for an empty chromatographic column tube with a specification of 100 mm×4.6 mm, the total liquid volume V is 20 to 60 mL.

10. The method according to any one of claims 6 to 9, characterized in that The ratio of the volume of the homogenate to the mass of the mixed filler is 5-50 mL: 1-5 g.

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