A method and device for extracting and purifying organic matter from a mixture

Abstract

The present invention provides a method for online extraction and purification of organic matter from a mixture, comprising the following steps: S1, solid phase extraction of organic matter: inputting a sample solution into a solid phase extraction column for pre-purification to obtain a pre-purified fraction; S2, judging the purity of the pre-purified fraction, and replacing the solid phase extraction column according to the purity of the fraction; S3, storing qualified fractions in the pre-purified fraction in an intermediate tank or injecting them into a chromatographic column; S4, chromatographic separation and purification of qualified pre-purified fractions in the intermediate tank or chromatographic column from S3; passing a sample solution prepared with a non-polar or low-polar solvent through a solid phase extraction column, and replacing the target from the filler in the solid phase extraction column by strongly retained impurities with stronger adsorption than the target. The method described in the present invention first performs solid phase extraction pre-purification before formal purification, and ensures sufficient pre-purification through full-wavelength online detection, reduces the filling frequency of the chromatographic column, minimizes the generation of solid waste, and saves solvent.

Description

Technical Field

[0001] The invention belongs to the field of separation and purification, and in particular relates to a method and a device for extracting and purifying organic matter from a mixture. Background Art

[0002] At present, the normal phase chromatography method for separating and purifying organic molecular mixtures has the following defects and problems:

[0003] 1. The life of chromatographic packing is short and it can usually only be used once or twice. It not only produces a large amount of solid waste, but also requires constant disassembly and assembly of the chromatographic column during the entire purification process, which greatly reduces work efficiency and increases safety risks.

[0004] 2. The entire purification process involves numerous manual operations, and there are many unavoidable exposures of organic solvents in the air, which not only poses great safety risks and harms to personnel health, but also causes product quality problems due to human errors, as well as a large amount of solvent loss and environmental problems.

[0005] 3. The ratio of the purified substance to silica gel and the consumed solvent is too low, resulting in excessively high costs and a large amount of solid waste.

[0006] 4. Many organic compounds extracted from natural products or fermented have complex components and low target component content. It is difficult to achieve effective online monitoring of components in solid phase extraction or chromatographic separation fractions by simple UV-visible detectors. Summary of the invention

[0007] In view of this, the present invention aims to provide a method and device for extracting and purifying organic matter from a mixture, in order to solve at least one of the above technical problems.

[0008] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0009] In one aspect, a method for purifying an organic substance from a mixture by solid phase extraction followed by liquid chromatography is provided, comprising the steps of:

[0010] S1. Solid phase extraction of organic matter: input the sample solution into a solid phase extraction column for pre-purification to obtain a pre-purified fraction;

[0011] S2, determining the purity of the pre-purified fraction and replacing the solid phase extraction column according to the purity of the fraction;

[0012] S3. The qualified fractions in the pre-purification fractions are stored in an intermediate tank or injected into a chromatographic column;

[0013] Among them, the method used for solid phase extraction is: the sample solution prepared with a non-polar or low-polar solvent is passed through a solid phase extraction column, and the target object is displaced from the filler in the solid phase extraction column by a strongly retained impurity with stronger adsorption than the target object, so that the target object flows out of the solid phase extraction column.

[0014] Among them, solid phase extraction uses a non-polar or low-polarity solvent (the overall polarity is lower than the eluent used in chromatographic column purification to ensure that the target in the pre-purification fraction does not diffuse on the chromatographic column or the diffusion of the target in the chromatographic column is less than 30% of the length of the chromatographic column) to prepare a sample solution that directly passes through the solid phase extraction column, and the target is displaced from the packing in the extraction component by strongly retained impurities with stronger adsorption than the target, so that the target flows out of the extraction component, while the strongly retained impurities are retained on the packing of the extraction component.

[0015] S4, performing chromatographic separation and purification on the qualified pre-purified fraction in the intermediate tank or chromatographic column from S3: the qualified pre-purified fraction is transported to a chromatographic column filled with silica, and enters a collection tank after being purified by the chromatographic column;

[0016] The extraction component in S1 may include one or more solid phase extraction columns, and the full-wavelength online detection system detects whether the fraction flowing out of the solid phase extraction column is qualified; specifically:

[0017] If the outflowing fraction is found to be qualified, the second branch is opened to allow the qualified fraction to be stored in the intermediate tank;

[0018] If it is detected that the fraction flowing out of the first solid phase extraction column contains a high content of strongly retained impurities, the first solid phase extraction column is connected in series with the second solid phase extraction column, and the fraction flowing out of the first solid phase extraction column flows into the second solid phase extraction column until the content of strongly retained impurities in the fraction of the first solid phase extraction column approaches or reaches the content of strongly retained impurities in the sample solution, and then the sample solution enters the second solid phase extraction column, and the first solid phase extraction column is removed. The first solid phase extraction column that is removed is replaced with fillers and then added to the extraction assembly;

[0019] Similarly, the sample solution enters the Nth solid phase extraction column, where N is an integer greater than or equal to 1;

[0020] Until all the samples enter the solid phase extraction column for pre-purification.

[0021] That is: the full-wavelength online detection system detects the fractions flowing out of the first solid phase extraction column. If the detected fractions flowing out are mainly weakly retained front fractions, that is, the content of the target substance in the fraction is low at this time, and there is no need for further chromatographic purification, this part of the fraction will be transferred to the waste liquid tank; if the fraction contains more target substances, that is, the content of strongly retained impurities in the fraction is very small at this time, and the chromatographic column can be effectively avoided from being contaminated, then the fraction will enter the intermediate tank or directly enter the chromatographic column; when the content of the target substance in the fraction is between the above two situations, it can enter the intermediate tank or directly enter the chromatographic column, or the recovery tank, or the waste liquid tank according to the specific standard requirements of the project. The solution in the recovery tank will be mixed with the original material solution and enter the extraction process again. When the measured fraction contains more than a certain content of post-impurities, the fraction of the first solid phase extraction column is connected to the second solid phase extraction column connected to the first solid phase extraction column, and so on, until all samples enter the solid phase extraction column for pre-purification. The "certain content" in "certain content of subsequent impurities" is determined according to different project requirements. "Subsequent impurities" refer to strongly retained impurities that are more strongly retained than the target substance.

[0022] Further, there are N solid phase extraction columns, where N is an integer greater than or equal to 1;

[0023] Also included is a full-wavelength online detection system for detecting the pre-purified fraction;

[0024] The organic substance solid phase extraction is specifically:

[0025] If the pre-purification fraction discharged is detected to be a qualified fraction, the qualified fraction is stored in an intermediate tank or injected into a chromatographic column;

[0026] If it is detected that the pre-purified fraction discharged from the first solid phase extraction column contains a high content of strongly retained impurities, the first solid phase extraction column and the second solid phase extraction column are connected in series, and the pre-purified fraction discharged from the first solid phase extraction column flows into the second solid phase extraction column until the content of strongly retained impurities in the pre-purified fraction discharged from the first solid phase extraction column approaches or reaches the content of strongly retained impurities in the sample solution, and then the sample solution enters the second solid phase extraction column, and the first solid phase extraction column is removed;

[0027] And so on, the sample solution enters the Nth solid phase extraction column to complete the pre-purification of all samples.

[0028] Furthermore, the S2 is specifically the following steps:

[0029] S21: Before extraction, detecting the absorbance of each substance to be pre-purified in the mixture over the entire wavelength range;

[0030] S22: During extraction, click the start button. At this time, the absorbance a1 of substance A in the mixture under the first set wavelength condition is greater than the absorbance b1 of substance B in the mixture under the first set wavelength condition. As the experiment progresses, the solid phase extraction column is replaced in the following two situations:

[0031] Case 1: a1 decreases, b1 increases. When a1<b1 and a1 is lower than the first set value, it is judged that substance A is completely separated. When b1 increases to the second set value, it is judged that impurity penetration occurs in the solid phase extraction column, and the solid phase extraction column is replaced at this time.

[0032] Case 2: a1 decreases, b1 decreases, and a1>b1. At this time, start to compare the absorbance a2 of substance A in the mixture under the second set wavelength condition and the absorbance b2 of substance B in the mixture under the second set wavelength condition, and compare with the initial value. When the trend of a2 and b2 is still that a2 decreases, b2 decreases, and a2>b2, start to compare the absorbance a3 of substance A in the mixture under the third set wavelength condition and the absorbance b3 of substance B in the mixture under the third set wavelength condition until a difference appears, so as to judge that substance A is completely separated; judge whether impurity penetration occurs in the solid phase extraction column according to the absorbance of substance B in the mixture, that is, after the absorbance of b rises to the set value, it is judged that impurity penetration occurs in the solid phase extraction column, and the solid phase extraction column is replaced at this time;

[0033] And so on, using the same conditions, compare substances (B, C), (C, D)…(X-1, X).

[0034] Furthermore, the full wavelength range is 200nm-800nm.

[0035] Furthermore, the first set value in step 2 is 100mv-50000mv.

[0036] On the other hand, a device for extracting and purifying organic matter from a mixture is also provided, comprising a solid phase extraction device and a chromatographic column connected in series with the solid phase extraction device, wherein the chromatographic column is a forward chromatographic column.

[0037] Among them, solid phase extraction uses non-polar or low-polarity eluents, and the target object is displaced from the filler in the solid phase extraction device by strongly retained impurities with stronger adsorption than the target object, so that the target object flows out of the solid phase extraction device, while the strongly retained impurities are retained on the filler of the solid phase extraction device until the impurities penetrate the filler, and then the solid phase extraction column is replaced.

[0038] Further, the solid phase extraction device includes a full-wavelength online detection system, an extraction component and a controller;

[0039] The full-wavelength online detection system includes a light source, a flow cell, and a light energy signal receiver. The light source is connected to a straight optical fiber. The light source and the light energy signal receiver are located on both sides of the flow cell, and the line connecting the two is perpendicular to the sample channel in the flow cell. The light energy signal receiver is a full-wavelength diode array UV-visible detector or a triode array UV-visible detector.

[0040] The extraction component includes N solid phase extraction columns, N is an integer greater than or equal to 1, the input end of each solid phase extraction column is connected to the sample tank through an injection pipeline, each injection pipeline is provided with a first valve, the output end of each solid phase extraction column is connected to a liquid outlet pipeline, the first branch of the liquid outlet pipeline is connected to a flow cell of a full-wavelength online detection system, the first branch is provided with a third valve, the second branch of the liquid outlet pipeline is connected to a forward chromatographic column, and the second branch is provided with a second valve;

[0041] The second valve of the first solid phase extraction column is also connected to the input end of the second solid phase extraction column through a third branch;

[0042] The second valve of the second solid phase extraction column is also connected to the input end of the third solid phase extraction column through a third branch;

[0043] By analogy, the second valve of the N-1th solid phase extraction column is also connected to the input end of the Nth solid phase extraction column through the third branch;

[0044] The controller is configured to control the switching of each valve according to the detection results of the full-wavelength online detection system.

[0045] Furthermore, an intermediate tank is provided between the solid phase extraction device and the chromatographic column.

[0046] Furthermore, the number of the solid phase extraction columns is 2-10.

[0047] Further, the collecting tank includes a first collecting tank and a second collecting tank, the input ends of the first collecting tank and the second collecting tank are connected to the output end of the chromatographic column through a pipeline, and are used to collect the fractions from the chromatographic column, the first collecting tank and the second collecting tank are connected to a qualified product tank and an unqualified product tank, and the second online detection system is used to detect whether the fractions in the first collecting tank or the second collecting tank are qualified;

[0048] Also included is a solvent recovery system for recovering solvent from the acceptable product tank and the rejected product tank fractions.

[0049] Furthermore, the output end of the chromatographic column is connected to the input end of the first collection tank and the second collection tank through pipelines, respectively. An ultraviolet detector and a valve are provided on the pipeline, and the ultraviolet detector is used to detect whether there is a chromatographic peak in the fraction of the main pipeline;

[0050] The first collection tank and the second collection tank are connected to a second online detection system, and the second online detection system is used to detect the purity of the target substance in the fractions of the first collection tank and the second collection tank;

[0051] The output ends of the first collecting tank and the second collecting tank are respectively connected to the input ends of the qualified product tank and the unqualified product tank through pipelines, and valves are arranged on the pipelines;

[0052] The ultraviolet detector and the second online detection system are also electrically connected to the controller to control the opening and closing of the plurality of valves.

[0053] Further, the solvent recovery system includes a mixer and a solvent tank;

[0054] The output end of the unqualified product tank is connected to the input end of the mixer through a first pipeline, and the first pipeline is provided with a second valve, a fourth infusion pump, a second pressure sensor and a second mass flow meter;

[0055] The output end of the qualified product tank is connected to the input end of the mixer through a second pipeline, and the structure of the second pipeline is the same as that of the first pipeline;

[0056] The output end of the solvent tank is connected to the input end of the mixer through a third pipeline, and the pipeline is provided with a first valve, a third infusion pump, a first pressure sensor and a first mass flow meter;

[0057] The output end of the mixer is connected to the input end of the eluent tank through a fourth pipeline, a third mass flow meter is arranged on the pipeline, and the output end of the eluent tank is connected to the liquid inlet of the chromatographic column through a pipeline;

[0058] A plurality of mass flow meters are electrically connected to the controller, and the controller controls the opening and closing of corresponding solvent tank valves according to the detection results of the mass flow meters.

[0059] Furthermore, the filler filled in the solid phase extraction column in S1 is spherical or amorphous mesoporous silica gel with a particle size of 20-200 um.

[0060] Furthermore, the mass ratio of the sample to be separated in S1 (calculated by solid content) to the filler in the solid phase extraction column is related to the content of the strongly retained impurities in the sample and the saturated adsorption amount of the strongly retained impurities on the solid phase extraction filler, and is generally between 10% and 300%.

[0061] Furthermore, the sample solution to be separated in S1 can be added continuously or in batches to the solid phase extraction column until the collected fractions contain strongly retained impurities that are more strongly retained than the target substance; then the solid phase extraction column is connected in series with the next solid phase extraction column, and the sample solution is continued to be added until the ratio of the strongly retained impurities to the target substance detected at the end of the first solid phase extraction column has reached or is close to the ratio in the sample solution.

[0062] Furthermore, a low-polarity solvent is used to prepare the sample solution during solid phase extraction in S1, and a high-polarity solvent is used as the eluent in S4.

[0063] Furthermore, the low polarity solvent is usually at least one of n-hexane, n-heptane, petroleum ether, or a mixture with a small amount of a high polarity solvent. The high polarity solvent is usually at least one of ethyl acetate, dichloromethane, methanol, ethanol, acetonitrile, isopropanol, acetone, or a mixture with a low polarity solvent.

[0064] The most important thing is that the polarity of the eluent in S4 (or the proportion of polar components) is higher than the polarity of the solvent used to prepare the sample solution in S1 (or the proportion of polar components); further, after the sample solution collected in S1 is added to the chromatographic column, the diffusion of the target in the chromatographic column should be less than 30% of the length of the chromatographic column.

[0065] Further, the length of the chromatographic column in S4 is 25cm-500cm;

[0066] The characteristics of the packing material filled in the chromatographic column of S4 are:

[0067] The silicon dioxide is spherical silicon dioxide;

[0068] The particle size of the silicon dioxide is 8 μm-200 μm, the mesopore size is 5 nm-20 nm, and the porosity is 0.8-1.2 cm 3 / g; specific surface area is 50-600m 2 / g;

[0069] The silica gel is high-purity mesoporous silica; the total content of calcium, magnesium, aluminum, iron, zinc, chromium and manganese in the silica is less than 100 μg / g;

[0070] The porosity of the silica gel is between 0.8 and 1.2 cm 3 / g, with a specific surface area of ​​50-600m 2 / g;

[0071] The average particle size distribution of the spherical silicon dioxide satisfies: D90 / D10≤1.5;

[0072] The spherical silica is modified silica, including silica with surface chemical modification using organic molecules or silica gel containing metal ions; the organic molecules used for surface chemical modification are silanes containing polar molecular functional groups, and the polar molecular functional groups include amino groups, amide groups, alcoholic hydroxyl groups, urea groups, cyano groups, carboxyl groups, heterocycles, amino acids, monosaccharides or polysaccharides.

[0073] More preferably, the modified silica is one or a combination of two or more of aminosilane-modified, amidesilane-modified, carboxylsilane-modified, ureasilane, heterocyclic-substituted silane, alcohol-based silane, aminocarbonate- or amino acid-substituted silane, and saccharide-substituted silane.

[0074] Furthermore, the temperature of the eluent, solid phase extraction column and chromatographic column is controlled at 10°C-60°C, preferably 20°C-30°C.

[0075] This method has general applicability for chromatographic purification of organic compounds, especially organic compounds prepared by biological methods. The present invention provides corresponding cases of purification of coenzyme Q10, liposomes, and phosphoramidite, but it does not mean that the invention is only applicable to the above applications.

[0076] Compared with the prior art, the present invention has the following beneficial effects:

[0077] 1. The present invention adopts the "displacement desorption" method to pre-purify the sample to be purified through a series of solid phase extraction devices, which can achieve two effects at the same time:

[0078] First, it can retain strongly retained impurities in the solid phase extraction column to avoid contamination of the chromatographic column; and by using this principle, the use of elution solvents can be minimized and the saturation adsorption of silica gel extraction fillers can be maximized to reduce solid waste. Through multiple columns in series and cross-switching, the extraction fillers can be further fully saturated and adsorbed, and strongly retained impurities can be prevented from entering the chromatographic column with the collected effluent; and through cross-switching, the work efficiency is further improved.

[0079] Secondly, the solvent in the fraction after the solid phase extraction column is a non-polar or low-polar solvent, so the fraction can be directly injected into the chromatographic column without concentration to ensure that the target substance will not diffuse (or diffuses in a limited manner). This is another important innovation of the present invention, which greatly simplifies the process, improves efficiency, saves solvent and energy consumption, and at the same time increases the life of the chromatographic column filler.

[0080] 2. Full-wavelength UV monitoring is carried out through logical operation processing of the UV absorbance of the detected main strongly retained impurities and target objects at each wavelength and the absorbance change, and further comparison and logical operation processing of the absorbance of the main strongly retained impurities and target objects at each wavelength. Finally, the proportion and content of the front impurities (weakly retained impurities), target objects and the back impurities (strongly retained impurities) in the fractions flowing out of the solid phase extraction column can be calculated, so as to determine the timing of column replacement and improve the utilization rate of the solid phase extraction column.

[0081] 3. The chromatographic purification in the present invention uses high-purity silica gel and functionalized silica gel, which can not only further extend the life of the filler in the chromatographic column (reduce solid waste and reduce costs), but also improve the yield of purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] The accompanying drawings constituting a part of the present invention 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 accompanying drawings:

[0083] Figure 1 It is a schematic diagram of the overall structure of the device described in the present invention.

[0084] Figure 2 This is a schematic diagram of the structure of the full-wavelength online detection system described in the present invention.

[0085] Figure 3 This is a sample detection diagram before rough classification in Example 1 of the present invention.

[0086] Figure 4 This is a sample detection diagram after rough separation by solid phase extraction column in Example 1 of the present invention.

[0087] Figure 5 This is a sample detection diagram after fine separation by chromatographic column 7 in Example 1 of the present invention.

[0088] Figure 6 This is the crude product analysis spectrum of Example 2 of the present invention.

[0089] Figure 7 This is the spectrum of the sample after rough separation in Example 2 of the present invention.

[0090] Figure 8 This is the precise sample spectrum of Example 2 of the present invention.

[0091] Fig. 9 A fitting curve diagram is drawn for the refined preparation fractions of Example 3 of the present invention.

[0092] Fig.10 A fitting curve diagram is drawn for the separated fractions of Comparative Example 4 of the present invention.

[0093] Fig.11 This is a fitting curve diagram of phosphoramidite purification in Example 4 of the present invention.

[0094] Fig.12 The silica gel prepared in Example 2 of the present invention;

[0095] Fig.13 It is conventional amorphous silica gel;

[0096] Fig.14 It is conventional spherical silica gel;

[0097] Fig.15 The selectivity test results of the silica gel prepared in Example 2 of the present invention;

[0098] Fig.16 This is the selectivity test result of conventional amorphous silica gel;

[0099] Fig.17 This is the selectivity test result of conventional spherical silica gel.

[0100] Reference numerals:

[0101] 1. Sample tank; 2. Sample injection pipeline; 3. Solid phase extraction column; 4. Intermediate tank; 5-1. First valve; 5-2. Second valve; 5-3. Third valve; 6. Full wavelength online detection system; 6-1. Light source; 6-2. Circulation cell; 6-3. Light energy signal receiver; 7. Chromatographic column; 8. Liquid outlet pipeline; 8-1. First branch; 8-2. Second branch; 8-3. Third branch; 9-1. First collecting tank; 9-2. Second collecting tank; 10. Qualified product tank; 11. Unqualified product tank; 12. Second online detection system; 13. Mixer; 14. Solvent tank; 15. Ultraviolet detector. DETAILED DESCRIPTION

[0102] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0103] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings.

[0104] This embodiment provides a method for online extraction and purification of organic matter from a mixture, comprising the following steps:

[0105] S1, solid phase extraction of organic matter: input the sample solution into the solid phase extraction column 3 for pre-purification to obtain a pre-purified fraction;

[0106] S2, determining the purity of the pre-purified fraction, and replacing the solid phase extraction column 3 according to the purity of the fraction;

[0107] S3, the qualified fractions in the pre-purification fractions are stored in the intermediate tank 4 or injected into the chromatographic column 7;

[0108] S4, performing chromatographic separation and purification on the qualified pre-purified fraction in the intermediate tank 4 or the chromatographic column 7 from S3: the qualified pre-purified fraction is transported to the chromatographic column 7 filled with silica, and enters the collection tank after being purified by the chromatographic column 7;

[0109] Among them, the method adopted for solid phase extraction is: the sample solution prepared with a non-polar or low-polar solvent is passed through the solid phase extraction column 3, and the target object is displaced from the filler in the solid phase extraction column 3 by the strongly retained impurities with stronger adsorption than the target object, so that the target object flows out of the solid phase extraction column 3, while the strongly retained impurities are retained on the filler of the solid phase extraction column 3 until the impurities penetrate the filler, and then the solid phase extraction column 3 is replaced.

[0110] It is understandable that the sample solution is prepared using a non-polar or low-polar solvent, and the non-polar or low-polar solvent refers to a solvent with a polarity lower than that of the chromatographic column 7, so as to ensure that the target substance in the pre-purification fraction does not diffuse on the chromatographic column 7 or the diffusion of the target substance in the chromatographic column 7 is less than 30% of the length of the chromatographic column 7.

[0111] Specifically, the qualified fraction detected by the full-wavelength online detection system 6 in S2 is stored in the intermediate tank 4 or injected into the chromatographic column 7; specifically: the full-wavelength online detection system 6 detects the fraction flowing out of the first solid phase extraction column 3. If the detected fraction flowing out is mainly the weakly retained front fraction, that is, the target content in the fraction is low at this time, and there is no need for further purification, this part of the fraction is transferred to the waste liquid tank; if the fraction contains more target substances, that is, the content of strongly retained impurities in the fraction is very small at this time, and the chromatographic column can be effectively avoided from being contaminated, then the fraction enters the intermediate tank or directly enters the chromatographic column; when the target content in the fraction is between the above two situations, it can enter the intermediate tank or directly enter the chromatographic column, or the recovery tank, or the waste liquid tank according to the specific standard requirements of the project. The solution in the recovery tank will be mixed with the original material solution and enter the extraction process again. When the measured fraction contains more than a certain amount of impurities, the fraction from the first solid phase extraction column is connected to the second solid phase extraction column connected to the first solid phase extraction column, and so on, until all samples enter the solid phase extraction column for pre-purification. The "certain amount" in "certain amount of impurities" is determined according to different project requirements, and "impurities" refer to strongly retained impurities that are more strongly retained than the target substance.

[0112] If the sample loading amount is too low, the proportion of the target substance is too low, and there are not enough strongly retained impurities to push out the target substance adsorbed on the solid phase extraction column 3, resulting in a large amount of target substance remaining on the filler; if the proportion is too high, many strongly retained impurities will be entrained in the collected fractions and enter the chromatographic column 7, thereby contaminating the chromatographic column 7 and causing the separation performance of the chromatographic filler to deteriorate.

[0113] The core principle of the present invention at this point is "displacement desorption", in which the target object is displaced from the silica gel by a strongly retained impurity with stronger adsorption than the target object, which not only allows the strongly retained impurities that may cause contamination of the chromatographic column 7 to be retained on the solid phase extraction column 3, but also saves a large amount of elution solvent.

[0114] Further, the number of solid phase extraction columns 3 is N, where N is an integer greater than or equal to 1;

[0115] It also includes a full-wavelength online detection system 6 for detecting the pre-purified fraction;

[0116] The organic substance solid phase extraction is specifically:

[0117] If the pre-purification fraction that flows out is detected to be a qualified fraction, the qualified fraction is stored in the intermediate tank 4 or injected into the chromatographic column 7;

[0118] If it is detected that the pre-purified fraction flowing out of the first solid phase extraction column 3 contains a high content of strongly retained impurities, the first solid phase extraction column 3 and the second solid phase extraction column 3 are connected in series, and the pre-purified fraction flowing out of the first solid phase extraction column 3 flows into the second solid phase extraction column 3 until the content of strongly retained impurities in the pre-purified fraction flowing out of the first solid phase extraction column 3 approaches or reaches the content of strongly retained impurities in the sample solution, and then the sample solution enters the second solid phase extraction column 3, and the first solid phase extraction column 3 is removed;

[0119] By analogy, the sample solution enters the Nth solid phase extraction column 3 to complete the pre-purification of all samples.

[0120] It can be understood that when it is detected that the strongly retained impurities flow out of the first solid phase extraction column 3, the adsorption capacity of the first solid phase extraction column 3 for the strongly retained impurities is not saturated. At this time, the sample solution continues to enter the first solid phase extraction column 3, and at this time, the fraction flowing out of the first solid phase extraction column 3 already contains strongly retained impurities and cannot enter the chromatographic column 7, but enters the second solid phase extraction column 3 through the third liquid outlet pipeline 8, until the content of strongly retained impurities in the fraction flowing out of the first solid phase extraction column 3 is close to or reaches the content of strongly retained impurities in the sample solution, which indicates that the adsorption capacity of the first solid phase extraction column 3 for the strongly retained impurities has been saturated. At this time, the sample solution is allowed to enter the second solid phase extraction column 3, and the first solid phase extraction column 3 is removed.

[0121] Such operation not only prevents the strongly retained impurities from entering the chromatographic column 7 along with the collected fractions and contaminating the filler in the chromatographic column 7 , but also avoids wasting the adsorption capacity of the solid phase extraction column 3 for the strongly retained impurities that has not yet been saturated.

[0122] Specifically, when the measured fraction contains more than a certain amount of post-impurities (strongly retained impurities that are more strongly retained than the target substance), the sample is connected to the second solid phase extraction column 3 connected in parallel with the first solid phase extraction column 3, and the first solid phase extraction column 3 is removed, and so on. If the outflowing fraction is qualified, no treatment is performed until all the samples enter the solid phase extraction column 3 for pre-purification. The "certain amount" in "certain amount of post-impurities" is determined according to different project requirements, and "post-impurities" refer to strongly retained impurities that are more strongly retained than the target substance.

[0123] Furthermore, when in use, the light emitted by the light source 6-1 is directed toward the light energy signal receiver 6-3 perpendicular to the flow direction of the sample in the flow cell 6-2. The absorbance of substance A under different wavelength conditions is defined as: a1, a2, a3...a n .

[0124] The absorbance of substance B at different wavelengths is defined as: b1, b2, b3…b n .

[0125] The absorbance of substance C at different wavelengths is defined as: c1, c2, c3…c n .

[0126] You can set many substances, supporting up to 24 substances, and define the maximum value as x.

[0127] The absorbance of substance X at different wavelengths is defined as: x1, x2, x3…x n .

[0128] The maximum value of n is 800.

[0129] According to the definition of absorbance: it refers to the logarithm with L0 as the base of the ratio of the incident light intensity before the light passes through a solution or a substance to the transmitted light intensity after the light passes through the solution or the substance (i.e. lg(L0 / L1)), where L0 is the incident light intensity and L1 is the transmitted light intensity. Factors that affect it include solvent, concentration, temperature, etc.

[0130] L0 is the incident light intensity: the detector is read under detector parameters with no absorption in air or pure water, or at a specific wavelength.

[0131] L1 is the transmitted light intensity: the transmitted light intensity of the detection target.

[0132] In addition, when testing L0 and L1, we use the same temperature to avoid errors caused by temperature effects.

[0133] As the separation and purification process progresses, samples of different concentrations will pass through the detector and different values ​​of a1, a2…a will be obtained. n , different values ​​of b1, b2…b3.

[0134] The S2 is specifically the following steps:

[0135] S21: Before extraction, detecting the absorbance of each substance to be pre-purified in the mixture over the entire wavelength range;

[0136] S22: During extraction, click the start button. At this time, the absorbance a1 of substance A in the mixture under the first set wavelength condition is greater than the absorbance b1 of substance B in the mixture under the first set wavelength condition. As the experiment progresses, the solid phase extraction column is replaced in the following two situations:

[0137] Case 1: a1 decreases, b1 increases. When a1<b1 and a1 is lower than the first set value, it is judged that substance A is completely separated. When b1 increases to the second set value, it is judged that impurity penetration occurs in the solid phase extraction column, and the solid phase extraction column is replaced at this time.

[0138] Case 2: a1 decreases, b1 decreases, and a1>b1. At this time, start to compare the absorbance a2 of substance A in the mixture under the second set wavelength condition and the absorbance b2 of substance B in the mixture under the second set wavelength condition, and compare with the initial value. When the trend of a2 and b2 is still that a2 decreases, b2 decreases, and a2>b2, start to compare the absorbance a3 of substance A in the mixture under the third set wavelength condition and the absorbance b3 of substance B in the mixture under the third set wavelength condition until a difference appears, so as to judge that substance A is completely separated; judge whether impurity penetration occurs in the solid phase extraction column according to the absorbance of substance B in the mixture, that is, after the absorbance of b rises to the set value, it is judged that impurity penetration occurs in the solid phase extraction column, and the solid phase extraction column is replaced at this time;

[0139] By analogy, using the same conditions, compare substances (B, C), (C, D) ... (X-1, X)

[0140] The above-mentioned design of the present invention adopts a full-wavelength online detection system 6 which is a full-wavelength diode array UV-visible detector or a triode array UV-visible detector, matched with an online monitoring software for judging the main component in the fraction by using the difference in the change of the secondary and tertiary functions of the absorbance of each component at different wavelengths of the UV-visible spectrum, and is used to judge and automatically collect the target object, and to judge whether a large amount of strongly retained components flow out of the solid phase extraction column 3, that is, to judge whether impurity penetration occurs in the solid phase extraction column 3; in order to fully utilize the adsorption capacity of the solid phase extraction filler, while preventing too many strongly retained components from entering the chromatographic column 7.

[0141] In the present invention, when the full-wavelength online detection system 6 detects that the fraction already contains a high content of strongly retained impurities, it automatically switches to be connected in series with another solid phase extraction column 3 that has not been loaded with samples; or when it is detected that the content of the strongly retained component has basically tended to be constant, the sample loading port can be switched to the second solid phase extraction column 3. The solid phase extraction column 3 can be switched automatically or manually, and the first solid phase extraction column 3 that has reached saturated adsorption will be removed for processing, and this cycle is repeated.

[0142] After solid phase extraction adsorption, the solution containing the target object with high purity directly enters the chromatographic column 7; during the whole process, the solid phase extraction filler is fully saturated with adsorption, which minimizes the generation of solid waste; at the same time, a high concentration and a high ratio of the object to be separated relative to the filler are adopted, and the target component is "pushed out" by relying on strongly retained impurities, thereby obtaining a high yield of the target object and minimizing the use of solid phase extraction fillers and solvents.

[0143] This embodiment provides a method for online extraction and purification of organic matter from a mixture, wherein the sample is pre-purified by a solid phase extraction component, thereby preventing strongly retained impurities from entering the chromatographic column 7 along with the collected effluent and contaminating the filler in the chromatographic column 7, as shown in the following embodiment:

[0144] Example 1: Figure 3-5 As shown, solid phase tandem extraction-chromatographic separation and purification of fermentation product coenzyme Q10.

[0145] 1) Weigh 2 kg of bacterial powder, extract with n-hexane, concentrate, saponify with alkali solution, wash with water, and then concentrate to obtain 67 g of extract. The purity of the sample determined by reverse phase chromatography was 81.7%. Dissolve the extract with n-hexane to 85 g / L, which is the sample solution before solid phase extraction.

[0146] 2) Fill 55 g of 100-200 mesh amorphous silica gel into a 24×200 mm solid phase extraction column. The average pore size of the silica gel is 6 nm. Fill 3 solid phase extraction columns of this specification and connect them in parallel to the extraction component. Depending on the usage, use a single solid phase extraction column for each separation. The interface is a 1 / 8 Luer connector. The sieve plate is made of polytetrafluoroethylene with a pore size of 20 μm and a plate thickness of 3.5 mm.

[0147] 3) n-Hexane was infused into the solid phase extraction column through an infusion pump at a flow rate of 15 ml / min. After flushing the column for 5 minutes, the full wavelength detector was set to 276 nm and 365 nm. At this time, the absorbance at wavelengths of 276 nm and 365 nm was 0 mv; the sample solution of coenzyme Q10 was infused into the solid phase extraction column through an infusion pump at a flow rate of 15 ml / min. As the target flow in the sample flowed out, the absorbance at a wavelength of 276 nm increased. When it reached 450 mv, the absorbance at 365 nm was 0 mv, and the target was collected in the intermediate tank.

[0148] 4) When the absorbance at a wavelength of 365 nm gradually increases from 0 mv, it indicates that a small amount of strongly retained impurities have been detected and eluted; when the absorbance at a wavelength of 365 nm gradually increases to 50 mv, the fraction flowing out of the outlet of the first solid phase extraction column is connected to another new solid phase extraction column through valve switching.

[0149] 5) Repeat the operation steps in step 3; when the detector at the outlet of the first solid phase extraction column detects that the absorbance at a wavelength of 365nm reaches 340mv or more, it can be determined that the first solid phase extraction column has reached saturated adsorption; at this time, the flow path is switched by the valve, and the sample is directly pumped into the second solid phase extraction column, and the first solid phase extraction column is removed from the system.

[0150] 6) Repeat steps 2), 3), 4), and 5) to achieve saturated adsorption of strongly retained impurities by the filler in the solid phase extraction column.

[0151] 7) The qualified fractions obtained by the solid phase extraction column are further purified by a chromatographic column to meet the qualified standards. The specific steps are as follows:

[0152] a. Fill 132 g of microsphere silica gel prepared in Example 2 into a 32 x 235 mm column, the silica gel having a diameter of 30 μm and an average pore size of 6 nm, and connect the column to a chromatography system for fine separation.

[0153] b. Use n-hexane as the balancing agent and use an infusion pump to pump it into the chromatographic column at a flow rate of 32mL / min for 20min to balance the system. Then pump the qualified fraction of solid phase extraction into it at the same flow rate for 5min. That is, the injection volume is 160mL, the injection amount is 13.2g, and the injection amount is 10% of the amount of silica gel.

[0154] c. After the injection, the infusion pump pumps the eluent at the same flow rate. The eluent is a mixed solution of n-hexane and ethyl acetate in a volume ratio of 9:1. The refined fraction is judged by the second online detection system for the interference of impurities, and is collected and divided into qualified product fractions and unqualified product fractions. The wavelengths of the second online detection system are set to 276nm and 315nm. When the ratio of the absorption intensity of 315nm to 276nm is not greater than 0.15%, it is a qualified product fraction; otherwise, it is an unqualified fraction, and the second online detection system automatically switches the fraction collection according to the set value; when the ratio of the absorption intensity of the two wavelengths is greater than 0.45%, the fraction is switched to waste liquid, and the eluent is changed to a mixed solution of n-hexane and ethyl acetate in a volume ratio of 5:5 for flushing. The flow rate remains unchanged, and the flushing is carried out for about 20 minutes until the total ultraviolet absorbance is less than 0.15Au, and the flushing is stopped, and n-hexane is used as the balancing agent to balance for 20 minutes at the same flow rate to complete a refined process.

[0155] d. The qualified fraction 40min (1280ml), the unqualified fraction 24min (768ml) and the waste liquid 28min (896ml) were concentrated under reduced pressure at 5°C to remove the solvent, with a pressure range of 0.8-0.9kpa, to obtain 10.73g, 1.35g and 1.12g of solids. The purity of the qualified product was 99.1% and the yield was 81.3%; the purity of the unqualified fraction was 92.3% and the yield was 10.2%; the solvents recovered from the three parts were The liquid is introduced into the eluent preparation tank through the online preparation system. The online preparation system automatically detects the density of n-hexane and ethyl acetate as 0.693 to 0.780 g / mL through the mass flow meter, and the density of the eluent is 0.683 g / mL. When the density is less than 0.683 g / mL, it means that the n-hexane content is too high, and the online preparation will automatically add ethyl acetate to the density of 0.683 g / mL. Similarly, when it is greater than 0.683 g / mL, n-hexane will be added. After mixing, it can be recycled as an eluent.

[0156] The fermented bacteria powder is extracted with n-hexane and concentrated into an extract, which is then saponified, washed, and tested. Figure 3 As shown in Figure 1, although the purity has been improved, it still contains a lot of impurities such as protein and sugar. Then solid phase extraction was performed using a solid phase extraction column filled with silica gel. The obtained fractions were tested, such as Figure 4 As shown in Figure 2, the content of strongly retained impurities is significantly reduced. Then, after a further step of chromatographic column purification, the obtained fractions are tested, as shown in Figure 2. Figure 5 As shown, the purity can reach more than 97%, meeting the needs of health care product preparations. The biggest feature of the solid phase extraction part of this process is the extremely large sample loading. In terms of silica gel, the solid loading can reach 100% to 150% depending on the sample situation, and the process hardly requires the addition of additional mobile phase, so the solution concentration is not diluted; due to the removal of highly polar components, the life of the chromatographic column is increased from 1 time to 40-50 times, effectively reducing the generation of silica gel solid waste and saving time and labor for repeated filling.

[0157] As a possible implementation method, the solvent is concentrated and recovered by means of reduced pressure rotary evaporation or the like.

[0158] Comparative Example 1: Coenzyme Q10 was purified directly by one-step chromatography without solid phase extraction. The specific steps are as follows:

[0159] The n-hexane solution obtained in step 1) of Example 1 is directly subjected to the a / b / c operation of step 7);

[0160] The qualified fraction of step d was only 480 ml of qualified fraction, while the unqualified fraction reached 2112 ml and the waste liquid was 1984 ml; the three parts were concentrated under reduced pressure at 5°C to remove the solvent, and the pressure range was 0.8-0.9 kPa to obtain 3.48 g, 6.55 g and 3.17 g of solids. The purity of the qualified product was 99.1% after testing, and the yield was only 26.3%; the purity of the unqualified fraction was 87.2%, and the yield reached 49.5%; the chromatographic column was subjected to re-sampling and separation, and no qualified product could be obtained, indicating that the silica gel can only be used once.

[0161] The results are compared in Table 1.

[0162] Table 1

[0163]

[0164] As shown in Table 1, the separation of coenzyme Q10 by solid phase extraction to adsorb strongly retained impurities and then chromatographic separation can greatly improve the yield and service life of silica gel compared with a single chromatographic separation method when the sample load and the purity of qualified samples are the same.

[0165] Furthermore, the full wavelength range is 200nm-800nm.

[0166] That is to say, the wavelength range covers the ultraviolet-visible spectrum range, and the components in the fraction can be detected and judged by utilizing the difference in absorbance of each component at different wavelengths of the ultraviolet-visible spectrum.

[0167] Furthermore, the first set value in step 2 is 100mv-5000mv.

[0168] It can be understood that according to the formula: A=lg(1 / T), A is absorbance, T is transmittance, which is the ratio of the outgoing light intensity (I) to the incident light intensity (I0), T=I / I0, the light transmittance T can be calculated. The absorbance range is 100mv-5000mv, and the light transmittance is 0~79%, which means that the light is absorbed by 21%~100%. When the light absorption is less than 21%, it means that the content of the target in the fraction is very low and there is no need to retain it. Therefore, a1 should be set within the range of 100mv-5000mv according to the needs.

[0169] On the other hand, the present invention also provides a device for extracting and purifying organic matter from a mixture, which is used to perform the above-mentioned method of online extraction and purification of organic matter from a mixture, including a solid phase extraction device and a chromatographic column 7 connected in series with the solid phase extraction device, wherein the chromatographic column 7 is a normal phase chromatographic column.

[0170] The solid phase extraction device includes a full wavelength online detection system 6, an extraction component and a controller;

[0171] Among them, the full-wavelength online detection system 6 includes a light source 6-1, a circulation cell 6-2, and a light energy signal receiver 6-3. The light source 6-1 is connected to a straight optical fiber. The light source 6-1 and the light energy signal receiver 6-3 are located on both sides of the circulation cell 6-2, and the line connecting the two is perpendicular to the sample channel in the circulation cell 6-2; the light energy signal receiver 6-3 is a full-wavelength diode array ultraviolet-visible detector or a triode array ultraviolet-visible detector;

[0172] The extraction component includes N solid phase extraction columns 3, N is an integer greater than or equal to 1, the input end of each solid phase extraction column 3 is connected to the sample tank 1 through the injection pipeline 2, each injection pipeline 2 is provided with a first valve 5-1, the output end of each solid phase extraction column 3 is connected to the liquid outlet pipeline 8, the first branch 8-1 of the liquid outlet pipeline 8 is connected to the flow cell 6-2 of the full-wavelength online detection system 6, the first branch 8-1 is provided with a third valve 5-3, the second branch 8-2 ​​of the liquid outlet pipeline is connected to the forward chromatographic column 7, and the second branch 8-2 ​​is provided with a second valve 5-2;

[0173] The second valve 5-2 of the first solid phase extraction column 3 is also connected to the input end of the second solid phase extraction column 3 through the third branch 8-3;

[0174] The second valve 5-2 of the second solid phase extraction column 3 is also connected to the input end of the third solid phase extraction column 3 through the third branch 8-3;

[0175] By analogy, the second valve 5 - 2 of the N-1th solid phase extraction column 3 is also connected to the input end of the Nth solid phase extraction column 3 through the third branch 8 - 3;

[0176] The controller is configured to control the opening and closing of valves on each pipeline according to the detection results of the full-wavelength online detection system 6 .

[0177] In the specific implementation process of this embodiment, once strongly retained impurities are detected, the third branch 8-3 is opened to switch the fraction of the first solid phase extraction column 3 to enter the second solid phase extraction column 3, that is, the first two solid phase extraction columns 3 are connected in series. When the content of strongly retained impurities in the fraction flowing out of the first solid phase extraction column 3 approaches or reaches the content of strongly retained impurities in the sample solution, the third branch 8-3 is closed, and the first valve 5-1 of the second solid phase extraction column 3 is opened to allow the sample solution to enter the second solid phase extraction column 3, and the first solid phase extraction column 3 is removed at the same time, and so on.

[0178] Such operation not only prevents the strongly retained impurities from entering the chromatographic column 7 along with the collected fractions and contaminating the filler in the chromatographic column 7 , but also avoids wasting the adsorption capacity of the solid phase extraction column 3 for the strongly retained impurities that has not yet been saturated.

[0179] That is to say, the connection method of the solid phase extraction column 3 in the above embodiment can fully utilize the filler of the solid phase extraction column 3 to saturate the strongly retained impurities, and can also prevent the strongly retained impurities from contaminating the filler in the chromatographic column 7.

[0180] Furthermore, an intermediate tank 4 is provided between the solid phase extraction device and the chromatographic column 7 .

[0181] It is understandable that the intermediate tank 4 can be used to temporarily store the qualified fractions flowing out of the solid phase extraction column 3, and then perform chromatographic separation on the qualified fractions according to process requirements, time arrangements, etc.

[0182] Furthermore, the number of the solid phase extraction columns 3 is 2-10.

[0183] It can be understood that the above-mentioned design of the present invention adopts a full-wavelength online detection system 6 which is a full-wavelength diode array UV-visible detector or a triode array UV-visible detector, matched with an online monitoring software for judging the main component in the fraction by using the difference in the change of the secondary and tertiary functions of the absorbance of each component at different wavelengths of the UV-visible spectrum, and is used to judge and automatically collect the target object, and to judge whether a large amount of strongly retained components flows out of the solid phase extraction column 3, that is, to judge whether impurity penetration occurs in the solid phase extraction column 3; in order to fully utilize the adsorption capacity of the solid phase extraction filler, while preventing too many strongly retained components from entering the chromatographic column 7.

[0184] When the full-wavelength online detection system 6 detects that the fraction already contains a high content of strongly retained impurities, it automatically switches to be connected in series with another solid phase extraction column 3 that has not been loaded with samples; when it is further detected that the content of the strongly retained component has basically tended to be constant, the loading port can be switched to the second solid phase extraction column 3, and the solid phase extraction column 3 can be switched automatically or manually, and the first solid phase extraction column 3 that has reached saturated adsorption will be removed for treatment; this cycle is repeated; the solution containing a higher purity target after solid phase extraction adsorption directly enters the chromatographic column 7; during the whole process, the solid phase extraction filler is fully saturated with adsorption, thereby minimizing the generation of solid waste; at the same time, a high concentration and a high ratio of the object to be separated to the filler are used, and the target component is "pushed out" by relying on strongly retained impurities, thereby obtaining a high yield of the target and minimizing the use of solid phase extraction fillers and solvents.

[0185] It is understandable that the contents of strongly retained impurities in sample solutions are different, and the most suitable number of solid phase extraction columns 3 can be selected according to different sample solutions and different contents of strongly retained impurities.

[0186] Further, the collecting tank includes a first collecting tank 9-1 and a second collecting tank 9-2, the input ends of the first collecting tank 9-1 and the second collecting tank 9-2 are connected to the output end of the chromatographic column 7 through a pipeline, and are used to collect the fractions from the chromatographic column 7, the first collecting tank 9-1 and the second collecting tank 9-2 are connected to a qualified product tank 10 and an unqualified product tank 11, and the second online detection system 12 is used to detect whether the fractions in the first collecting tank 9-1 or the second collecting tank 9-2 are qualified;

[0187] It also includes a solvent recovery system for recovering the solvents in the fractions of the qualified product tank 10 and the unqualified product tank 11.

[0188] Furthermore, the output end of the chromatographic column 7 is connected to the input end of the first collection tank 9-1 and the second collection tank 9-2 through pipelines, and an ultraviolet detector 15 and a valve are provided on the pipeline. The ultraviolet detector 15 is used to detect whether there is a chromatographic peak in the fraction flowing out of the chromatographic column;

[0189] The first collection tank 9-1 and the second collection tank 9-2 are connected to a second online detection system 12, and the second online detection system 12 is used to detect the purity of the target substance in the fractions of the first collection tank 9-1 and the second collection tank 9-2;

[0190] The output ends of the first collection tank 9-1 and the second collection tank 9-2 are respectively connected to the input ends of the qualified product tank 10 and the unqualified product tank 11 through pipelines, and valves are provided on the pipelines;

[0191] The ultraviolet detector 15 and the second online detection system 12 are also electrically connected to the controller to control the opening and closing of the plurality of valves.

[0192] It is understood that the fractions of the chromatographic column 7 are detected by the ultraviolet detector 15. The fractions with chromatographic peaks can be considered to contain the target substance to be purified, and this part of the fractions needs to be further processed. During the processing, the controller controls the corresponding valve on the pipeline to open, and the first collection tank 9-1 or the second collection tank 9-2 collects the fractions with chromatographic peaks.

[0193] The fractions entering the first collection tank 9-1 and the second collection tank 9-2 may be fractions with target purity that meet the standard or fractions with target purity that do not meet the standard; the fractions with target purity that meet the standard need to be collected using the qualified product tank 10, while the fractions with target purity that do not meet the standard need to be collected using the unqualified product tank 11. Therefore, the output ends of the first collection tank 9-1 and the second collection tank 9-2 need to be connected to the qualified product tank 10 and the unqualified product tank 11, so that the corresponding tanks can be used to collect the fractions as needed.

[0194] The specific method is: first, after the valve of the first collecting tank 9-1 is opened, the distillate enters the first collecting tank 9-1, and the first collecting tank 9-1 collects the distillate, and the second collecting tank 9-2 is empty at this time; after the collection of the first collecting tank 9-1 is completed, the distillate is controlled by the valve and switched to flow into the second collecting tank 9-2.

[0195] Of course, the distillate may also flow into the second collection tank 9-2 first, and the second collection tank 9-2 collects the distillate, while the first collection tank 9-1 is empty; after the second collection tank 9-2 completes the collection, the distillate is controlled by the valve and switched to flow into the first collection tank 9-1.

[0196] It can be understood that after the operation is stable, when the first collecting tank 9-1 collects the distillate, the second online detection system 12 performs target purity detection on the distillate of the second collecting tank 9-2. When the purity of the target in the distillate meets the standard, the controller controls the valve of the third branch 8-3 to open, and the distillate flows to the qualified product tank 10; when the purity of the target in the distillate does not meet the standard, the controller controls the valve of the fourth branch to open, and the distillate flows to the unqualified product tank 11.

[0197] When the second collecting tank 9-2 collects the distillate, the second online detection system 12 performs a target purity detection on the distillate of the first collecting tank 9-1. When the purity of the target in the distillate meets the standard, the controller controls the valve of the first branch 8-1 to open, and the distillate flows to the qualified product tank 10; when the purity of the target in the distillate does not meet the standard, the controller controls the valve of the second branch 8-2 ​​to open, and the distillate flows to the unqualified product tank 11.

[0198] Therefore, the first collecting tank 9-1 and the second collecting tank 9-2 cyclically perform fraction collection and detection work, and only two tanks are used to realize the collection of fractions with chromatographic peaks, and the qualified product tank 10 can further collect the fractions with target purity that meet the standard, and the unqualified product tank 11 can collect the fractions with unqualified target purity; while the traditional liquid chromatography needs to collect the fractions of the entire flat-top peak. In order to improve the accuracy of fraction collection, a small-volume fraction collection tube is required. In other words, the higher the accuracy, the smaller the volume of the fraction collected by each fraction collection tube, the more fraction collection tubes are needed, and the more workbench space is needed; subsequently, the target content and concentration in the fractions of each collection tube must be tested offline to determine the qualified range of the collected fractions for combined tube processing.

[0199] Further, the solvent recovery system includes a mixer 13 and a solvent tank 14;

[0200] The output end of the unqualified product tank 11 is connected to the input end of the mixer 13 through a first pipeline, and the first pipeline is provided with a second valve 5-2, a fourth infusion pump, a second pressure sensor and a second mass flow meter;

[0201] The output end of the qualified product tank 10 is connected to the input end of the mixer 13 through a second pipeline, and the structure of the second pipeline is the same as that of the first pipeline;

[0202] The output end of the solvent tank 14 is connected to the input end of the mixer 13 through a third pipeline, and the pipeline is provided with a first valve 5-1, a third infusion pump, a first pressure sensor and a first mass flow meter;

[0203] The output end of the mixer 13 is connected to the input end of the eluent tank through a fourth pipeline, a third mass flow meter is provided on the pipeline, and the output end of the eluent tank is connected to the liquid inlet of the chromatographic column 7 through a pipeline;

[0204] A plurality of mass flow meters are electrically connected to the controller, and the controller controls the opening and closing of the valves of the corresponding solvent tanks 14 according to the detection results of the mass flow meters.

[0205] It can be understood that the solvent recovery system is used to separate the eluent in the qualified product tank 10 and the unqualified product tank 11 , and the separated eluent enters the mixer 13 .

[0206] Specifically, the fractions in the qualified product tank 10 and the unqualified product tank 11 are processed by a flash tank or a rotary evaporator, and the solvent is rapidly boiled and vaporized, separated, and further cooled and liquefied before entering the mixer 13.

[0207] Specifically, the mass flow meter automatically detects the density of the recovered solvent and compares it with the density of the eluent; based on the density difference, the corresponding solvent is automatically replenished, the density is compared again, and the corresponding solvent is further replenished until the density of the solvent after preparation is consistent with the density of the eluent, which means that the ratio of the recovered solvent after preparation is now completely consistent with the ratio of the eluent and can be recycled as an eluent.

[0208] Furthermore, the filler filled in the solid phase extraction column 3 in S1 is spherical or amorphous mesoporous silica gel with a particle size of 20-200 um.

[0209] Further, the chromatographic filler filled in the chromatographic column is spherical silica;

[0210] The particle size of the spherical silica is 8 μm-200 μm; the mesopore diameter is 5 nm-20 nm; and the porosity is 0.8-1.2 cm 3 / g; specific surface area is 50-600m 2 / g;

[0211] The total content of calcium, magnesium, aluminum, chromium, iron and manganese in the spherical silicon dioxide is less than 100 μg / g;

[0212] The average particle size distribution of the spherical silicon dioxide satisfies: D90 / D10≤1.5;

[0213] The spherical silica is modified silica, including silica with surface chemical modification using organic molecules or silica gel containing metal ions; the organic molecules used for surface chemical modification are silanes containing polar molecular functional groups, and the polar molecular functional groups include amino groups, amide groups, alcoholic hydroxyl groups, urea groups, cyano groups, carboxyl groups, heterocycles, amino acids, monosaccharides or polysaccharides.

[0214] Coenzyme Q10 was separated by first solid phase extraction to adsorb strongly retained impurities and then chromatographic separation. In the chromatographic separation step, amorphous silica gel, ordinary spherical silica gel and silica gel microspheres prepared in Example 2 were compared as fillers.

[0215] Comparative Example 2: The same operation as in Example 1 was adopted, except that the microsphere silica gel prepared in Example 2 in step 7) a. was replaced with ordinary spherical silica gel with a particle size of 40-75 μm. Other operations were the same as in Example 1. The results are shown in Table 2.

[0216] Comparative Example 3: The same operation as in Example 1 was adopted, except that the microsphere silica gel prepared in Example 2 in step 7) a. was replaced with amorphous silica gel with a particle size of 200-400 mesh. Other operations were the same as in Example 1. The results are shown in Table 2.

[0217] Table 2

[0218]

[0219] In the chromatographic separation step, we compared the separation of coenzyme Q10 using amorphous silica gel, ordinary spherical silica gel and silica gel microspheres prepared in Example 2 as fillers, and the yields were 51.42%, 60.84% ​​and 81.30% respectively; the service life of silica gel was also increased from 10 times and 12 times to 15 times; this shows that the high-purity silica gel microspheres prepared in Example 2, with their low metal content, uniform pore size and large specific surface area, reduced the dead adsorption of the sample during separation, improved the selectivity of separation, reduced the cost of separation, and improved the separation efficiency.

[0220] Example 2: A method for preparing silica microsphere filler.

[0221] The preparation method of silica microsphere filler is as follows: 50 kg of n-decanol, 3750 g of 2% polyvinyl pyrrolidone aqueous solution, 350 mL of concentrated ammonia and 500 g of n-decanol dissolved with 10% hexamethylenetetramine are added to a 100 L glass reactor, and stirred until a clear, transparent and uniform solution is obtained; 12.5 kg of 8 nm silica sol is added, and the mixture is stirred rapidly and vigorously at 500 rpm until a dispersed emulsion of silica sol in n-decanol with a particle size of 30-50 μm is obtained. This step can be observed under a microscope, and stirring is stopped; the dispersed emulsion is allowed to stand for 10 minutes, 30 mL of acetic acid solution is added, stirred for 2 minutes, switched to low-speed stirring, heated to 75°C, and about 15 L of water is distilled out under negative pressure, and then distillation is stopped; the temperature is cooled to below 30°C and then filtered, washed three times with 20 L of methanol, and then calcined at 700°C in a muffle furnace for 10 hours to obtain a pore size of 100A, a pore volume of 0.95 mL / g, and a specific surface area of ​​450 m 2 / g of silica microspheres.

[0222] The silica gel prepared by high-purity water glass in the present invention and the amorphous and spherical silica gel prepared by conventional water glass were compared and tested, and the results are shown in Table 3 below:

[0223] Table 3 Comparison of silica gel prepared by the present invention and conventional silica gel

[0224]

[0225] From Table 3 and Figure 12-14 It can be seen that the silica gel particles prepared in this embodiment are uniform, have a larger specific surface area and better surface properties based on the same pore size, and therefore have a larger sample loading capacity and purification effect during purification; at the same time, the heavy metal content of the silica gel prepared in this embodiment is lower, so the sample dead adsorption is lower and the tailing phenomenon is correspondingly reduced.

[0226] pass Figure 15-17 The selectivity test shows that the silica gel prepared in this example has better selectivity and separation.

[0227] Embodiment 3, as Figure 9-10 As shown, one implementation method of preparing high-purity liposome monomers using the silica microspheres prepared in Example 2 is:

[0228] 1) Sample dissolution: 150 g sample + 1500 mL n-hexane, shake well and sonicate.

[0229] 2) Filling of solid phase extraction column: Take a solid phase extraction column with a diameter-to-height ratio of about 1:6, and fill it with 66g of silica gel with a particle size of 100-200 mesh and an average pore size of 6nm under dry vibration. The sieve plate is made of polytetrafluoroethylene with a pore size of 20μm and a thickness of 3.5mm.

[0230] 3) Pre-purification Sampling and pre-purification: Pump n-hexane at a flow rate of 15 mL / min for 20 minutes. After the column is conditioned, pump all the solution containing 150 g of sample at the same flow rate. The sample loading amount is 226.9% based on the mass of silica gel. Collect for 110 minutes to complete the crude fraction.

[0231] 4) The solid phase extraction separation fraction was sampled and tested by HPLC-CAD. The purity was 61.38%; calculated by the product of the peak area converted concentration and the total volume of the fraction, 127.5 g of the sample was obtained, and the yield was 85% (127.5 g / 150 g); the sample concentration in the fraction was 12.94 mg / ml.

[0232] 5) Chromatographic column filling: A 20×250 stainless steel preparative column was filled with 40 g of 30 μm silica gel prepared in Example 2 under dry vibration. The sieve plate was made of stainless steel with a pore size of 10 μm and a thickness of 2.5 mm.

[0233] 6) Sampling and separation: After the column was conditioned by pumping n-hexane at a flow rate of 12 mL / min for about 20 min, the sample solution was pumped in at the same flow rate for 14.5 min (injection volume was 2.8 g). The mobile phase was then eluted with a mixture of n-hexane containing 0.1% triethylamine and ethyl acetate in a volume ratio of 95:5 at a flow rate of 12 mL / min. The fractions were collected at 36 mL / tube.

[0234] 7) The fractions were tested by HPLC-CAD, and the qualified fractions were combined, concentrated, and weighed to obtain 0.98 g of qualified product with a yield of 35% (0.98 g / 2.8 g).

[0235] 8) Repeat steps 6) and 7) 20 times to obtain qualified products with a yield of 32-36% and a sample area normalized purity of 97.5%.

[0236] Comparative Example 4: Comparison of results of direct purification of liposomes by chromatography (without solid phase extraction, different solid phase extraction sample to silica gel ratio, etc.) Comparison of separation effect, yield, silica gel life, etc.

[0237] Fill the silica gel glass column, elute with a low polar organic solvent, receive the effluent and confirm the basic purity through the spot plate, detect the qualified fraction through HPLC-CAD, and concentrate it. In order to obtain a purer monomer, the silica gel column is usually filled with a length of more than 3000mm, the column time is 70-100 hours, and the silica gel column loses its separation ability after 1 to 2 separations. If you want to get more monomers, you need to constantly disassemble and assemble the chromatographic column. Therefore, this process has the disadvantages of more solid waste, heavy pollution, long working hours, large amount of solvent, and low efficiency. The operation steps and data are as follows:

[0238] Sample dissolution: 6.7 g sample + 67 mL n-hexane, shake well and sonicate.

[0239] Chromatographic column filling: Take a glass preparation column with a diameter-to-height ratio of about 1:20, and fill it with 67 g of 100-200 mesh silica gel under dry vibration. The sieve plate is made of polytetrafluoroethylene with a pore size of 20 μm and a thickness of 3.5 mm.

[0240] Sample loading and separation: Pump n-hexane at a flow rate of 5 mL / min for 20 min. After the column is conditioned, pump all the solution containing 6.7 g of sample at the same flow rate. The sample loading amount is 10.0% based on the mass of silica gel. Collect for 95 h to complete the separation.

[0241] Detection and concentration: The fractions from 33h to 56h were combined and concentrated to obtain 1.07g of solid with a purity of 97.6% according to HPLC-CAD. The remaining fractions were combined and concentrated to obtain 1.47g with a total yield of 38%.

[0242] Repeat 1) to 4) and inject the sample again. The amount of solid with a purity of 97.5% is 0.87 g, which is 18.7% lower than that of the first purification. In terms of comprehensive cost, silica gel does not need to be separated for the third time.

[0243] Comparative Example 5: The same operation as in Example 3 was adopted, except that the 30 μm silica gel prepared in Example 2 in step 6) was replaced with ordinary spherical silica gel with a particle size of 200-300 mesh. The other operations were the same as in Example 3. The results are shown in Table 4.

[0244] Table 4

[0245]

[0246] It can be seen from Table 4 that the comparison of the common chromatographic silica gel and the spherical silica gel prepared in this embodiment in the purification of liposomes shows that the loading amount of the common silica gel is 10%, the qualified product yield is 15-18%, the total sample yield is 38%, the silica gel is used twice, the separation time is 70-100h, and the solvent dosage is 100-150 times the column volume. In this embodiment, solid phase extraction is used to remove most of the matrix and enrich the target, and then the chromatographic column is used for precise separation. Compared with the chromatographic column, the target is obtained by one separation. On the one hand, this method greatly increases the number of times the column separation silica gel is used, and because the solid phase extraction effectively removes most of the interference of the matrix, the loading amount of the chromatographic separation is also increased from 10% to 15%, an increase of 50%, which effectively increases the separation flux; on the other hand, the precise separation adopts Example 2 Prepared spherical silica gel, the sample loading of the prepared spherical silica gel is 15%, the qualified product yield is 26-35%, the total sample yield is 42-58%, the number of silica gel use times is 20-30 times, the separation time is 1-2h, and the solvent consumption is 8-10 times of column volume;And the fraction after solid phase extraction can be directly sampled without concentration for chromatographic separation, without the loss of heat energy consumption and solvent, and is simple to operate;Comparative Example 5 is compared with the second chromatographic separation of Example 3, after being replaced by the high-purity silica gel microspheres prepared by Example 2 by ordinary spherical silica gel, the same sample loading is kept for separation, and the qualified product yield, silica gel life and total sample yield are all greatly improved. Therefore, the spherical silica gel prepared by the present embodiment can significantly improve the sample loading, qualified product yield, total sample yield and the number of silica gel use times, and can significantly reduce the separation time and solvent consumption, thereby improving purification efficiency and reducing costs.

[0247] Depend on Fig. 9 and Fig.10 The comparison shows that the separation of liposomes by solid phase extraction combined with chromatographic separation can remove most of the strongly retained impurities at 14.7min, 15.2min, and 15.4min, and the separation selectivity is greatly increased.

[0248] Example 4, as shown in Table 5, a comparison of phosphoramidite purification using the silica microspheres prepared in Example 2 and different conventional silica gel fillers.

[0249] The purification of phosphoramidite was compared using a 12 g silica gel column loaded with high purity spherical silica gel prepared in Example 2 and conventional amorphous and spherical silica gel (Comparative Example 6 and Comparative Example 7). Mobile phase: dichloromethane (with 0.3% diisopropylamine), flow rate: 5 ml / min, wavelength 254 nm. The fitted spectrum after purification is shown in Fig.11 , the loading amount, yield and life span are shown in the following table:

[0250] Table 5

[0251]

[0252] By comparing Comparative Example 6 with Comparative Example 7, it can be seen that in the comparison of the purification experiment of phosphoramidite using silica microspheres with a particle size of 40-75 μm in Example 2 and spherical silica gel with a conventional particle size of 75-150 μm, the loading amount is increased from 10% to 20%; the yield is also increased from 45-55% to 50-60%, and the number of times it can be used is also increased from 2-5 times to 20-30 times;

[0253] By comparing Example 7 with Example 2, it can be seen that after using solid phase extraction to remove most of the matrix interference, the same spherical silica gel is used for precise separation, and the sample load, yield and number of times the silica gel is used are greatly improved: the sample load is increased from 20% to 30%, the yield is further increased from 50-60% to 80-85%, and the number of times the silica gel is used is greatly increased from 20-30 times to 90-120 times.

[0254] In other words, the silica microspheres of this embodiment have a smaller particle size and a longer lifespan, and the sample loading amount and sample yield during purification of phosphoramidite are higher. It is generally known by those skilled in the art that small particles can increase the number of plates and resolution, thereby improving the purification effect. The increase in sample loading amount and yield as well as the extension of lifespan can save costs. Therefore, the silica microspheres of this embodiment have greater advantages than conventional silica gel.

[0255] The silicon dioxide is modified silicon dioxide or unmodified silicon dioxide.

[0256] More preferably, the modified silica is one or a combination of two or more of amino-modified, ester-modified, carbonyl-modified, amide-modified, carboxyl-modified, carbonate-modified, and aminocarbonate-modified.

[0257] The solid phase extraction principle of this embodiment is based on the "displacement desorption" method. The sample to be purified is pre-purified by a solid phase extraction device connected in series or in parallel, so that the strongly retained impurities in the phosphoramidite matrix can be retained in the solid phase extraction column 3 to avoid contamination of the chromatographic silica gel for precise separation; and after purification by solid phase extraction, the sample loading amount entering the precise separation can be greatly increased, the content of strongly retained impurities is reduced, the corresponding separation effect will also be improved, the yield is increased, and the number of times the silica gel is used is also increased.

[0258] The polar groups modified on the surface of silica gel have better similarity with polypeptides in composition and structure, and according to the principle of like dissolves like, it will be beneficial to increase the sample loading amount; the polar groups modified on the surface of silica gel can seal the exposed sites of unbonded silanol groups remaining on the surface of silica gel, as well as impurity metals such as Al, Fe, Mn, Cr, etc., to reduce excessive adsorption of polypeptides by fillers and improve the recovery rate; -NHR / -NH2 groups are embedded on the surface of silica, that is, two pairs of lone pairs of electrons and NH bonds on N are spatially introduced into the stationary phase, and the lone pairs of electrons can produce P-π effects with polar groups such as double bonds or conjugated double bonds in the separated sample molecules, and can form hydrogen bonds with fluorine atoms, oxygen atoms or nitrogen atoms; due to the existence of spatial effects, P-π effects and hydrogen bonds may exist at the same time, thereby strengthening the difference in spatial forces of different samples and improving the selectivity of separation, as shown in the following embodiments:

[0259] Example 5: Preparation of urea-modified silica gel

[0260] 100 g of silica gel obtained in Example 2, 60 g of ureapropyltrimethoxysilane, 500 ml of toluene, refluxed at about 110°C for 16 hours. The obtained solid was filtered, washed with toluene 3 times, 50 ml each time; washed with methanol 6 times, 50 ml each time; and vacuum dried at 100°C for 8 hours. C% = 7.1%.

[0261] Example 6: The urea-modified silica gel prepared in Example 5 is used in the purification method of phosphoramidite using the following steps:

[0262] One-time preparation:

[0263] Sample dissolution: Weigh 240 g of sample, dissolve in 1 L of mobile phase (petroleum ether: ethyl acetate = 25:75), filter, and the sample concentration is 240 mg / mL.

[0264] Chromatographic column filling: 800 g of 20-45 μm urea-modified silica gel was filled into a solid phase extraction column with a diameter-to-height ratio of 1:8.

[0265] Loading and separation: Pump the mobile phase (petroleum ether: ethyl acetate = 25:75) at a flow rate of 180 mL / min for 15 minutes to moisten the column; then pump the sample solution into the solid phase extraction device at the same flow rate, elute with the mobile phase at the same flow rate for 110 minutes, mix the 19.5-64.1 min fractions in a 10 L bucket, concentrate with 5 L rotary evaporation, and add 300 mL of ethyl acetate to dissolve.

[0266] Secondary preparation

[0267] Column balancing: After the first preparation, the column was flushed with ethyl acetate at a flow rate of 180 mL / min for 10 min, then replaced with the mobile phase (petroleum ether: ethyl acetate = 72:28), and balanced at the same flow rate for 15 min to complete the balancing.

[0268] Loading and separation: The sample solution was pumped into the solid phase extraction device at a flow rate of 180 mL / min, and the mobile phase (petroleum ether: ethyl acetate = 72:28) was used for elution for 75 min at the same flow rate, and then 100% ethyl acetate was used to wash the column for 25 min; the 41.0-74.0 min fraction was collected.

[0269] Concentration and detection: The fraction was concentrated to dryness under reduced pressure to obtain 13.7 g. A sample was taken for liquid chromatography detection, and the purity was 98.9%.

[0270] Phosphoramidite monomers are essential for gene synthesis, and mass production and purification of high-purity phosphoramidite monomers provide a guarantee for the synthesis of nucleic acid drugs. Compared with traditional silica gel column separation, urea-modified silica gel as the stationary phase for separating phosphoramidite synthesis products has obvious advantages (see Table 6 for details):

[0271] Time-saving. The whole process is divided into two purifications. The first purification removes impurities other than 2'-OMe-U phosphoramidite monomer and 3'-OMe-U phosphoramidite monomer, which takes about 2 hours. The second separation obtains the pure 2'-OMe-U phosphoramidite monomer target, which is injected twice and takes about 2.5 hours (using cross injection method). Including rotary evaporation, the whole cycle can be completed in 8 hours; at the same time, the side reactions caused by the long separation time of the sample are reduced; the same separation process using silica gel takes about 50-60 hours.

[0272] 2) High recovery rate. During the whole process, the target 2'-OMe-U phosphoramidite monomer is collected in an almost lossless manner. At the same time, the separation of 3'-OMe-U phosphoramidite monomer is relatively high, and the recovery rate of two separations can reach nearly 85%; the total recovery rate is about 95%; while the separation based on silica gel has obvious dead adsorption, and the product recovery rate is 60-75%.

[0273] 3) Simple operation and labor saving. This process, when extended to process production, can adopt the characteristics of pump loading, automatic collection, automatic mixing of mobile phase, etc. 2-3 workers can be competent for the entire process after simple training, which greatly reduces the cumbersome factors of manual operation and makes the full automation of the process possible; while silica gel separation has poor reproducibility and relies more on the experience of separation personnel, so the requirements for separation personnel are high, which is not conducive to the realization of automation.

[0274] 4) Solvent saving. Since the entire separation process can be detected by the UV detector, the process is intuitive and easy to control each part of the process. The mobile phase adopts the isocratic method, which is convenient for cross injection, making solvent saving possible. Of course, this is only a small test process, and the use of solvents cannot be optimized to the extreme. If the production is scaled up, I believe that solvents will be greatly saved.

[0275] 5) Less pollution, saving resources. Ordinary silica gel has serious adsorption problems and is almost impossible to reuse. The repeated filling of silica gel causes huge consumption of manpower and material resources, and the pollution to the environment is also quite prominent. Urea-modified silica gel reduces the activity of silica gel and greatly reduces the dead adsorption of polar compounds by silanol groups. Therefore, it can be used repeatedly, eliminating the trouble of repeated filling. More importantly, the whole system adopts a fully enclosed operation, which greatly reduces the pollution caused by solvent volatilization and minimizes the harm to operators.

[0276] Table 6

[0277]

[0278] Furthermore, the sample solution to be separated in S1 can be added continuously or in batches to the solid phase extraction column 3 until the collected fraction contains strongly retained impurities that are more strongly retained than the target substance; then the solid phase extraction column 3 is connected in series with the next solid phase extraction column 3, and the sample solution is continued to be added until the ratio of strongly retained impurities to the target substance detected at the end of the first solid phase extraction column 3 has reached or exceeded the ratio in the sample solution.

[0279] When the fraction from the solid phase extraction column 3 contains strongly retained impurities that are more strongly retained than the target substance, in order to avoid contamination of the chromatographic column 7 by the strongly retained impurities, the fraction should not enter the chromatographic column 7 at this time. However, the adsorption capacity of the solid phase extraction column 3 is not saturated at this time, and it will be a waste if the solid phase extraction column 3 is replaced. At this time, the fraction can enter the second solid phase extraction column 3. On the one hand, the first solid phase extraction column 3 can continue to adsorb the strongly retained impurities in the sample solution, and the second solid phase extraction column 3 can adsorb the strongly retained impurities in the fraction from the first solid phase extraction column 3, ensuring that the qualified fraction enters the chromatographic column 7. This not only makes full use of the filler of the solid phase extraction column 3, but also avoids contamination of the chromatographic column 7.

[0280] Furthermore, the mass ratio of the sample to be separated in S1 (calculated by solid content) to the filler in the solid phase extraction column 3 is related to the content of the strongly retained impurities in the sample and the saturated adsorption amount of the strongly retained impurities on the solid phase extraction filler, and is generally between 10% and 300%.

[0281] It can be understood that the spherical silica gel used in this embodiment has a larger specific surface area and better surface properties based on the same pore size.2 / g increased to 450m 2 / g, so it also has a larger sample loading and purification effect in purification. For details, please refer to the introduction of the preparation method of silica below.

[0282] Furthermore, a low-polarity solvent is used to prepare the sample solution during solid phase extraction in S1, and a high-polarity solvent is used as the eluent in S4.

[0283] Further, the low polarity solvent is at least one of n-hexane, n-heptane and petroleum ether, or a mixture of one of n-hexane, n-heptane and petroleum ether and a small amount of a high polarity solvent;

[0284] The high polarity solvent is at least one of ethyl acetate, dichloromethane, methanol, ethanol, acetonitrile, isopropanol and acetone, or a mixture of one of ethyl acetate, dichloromethane, methanol, ethanol, acetonitrile, isopropanol and acetone and a low polarity solvent.

[0285] It can be understood that after the sample solution enters the solid phase extraction column 3, the target object is first adsorbed on the filler in the solid phase extraction column 3. By using a low-polarity eluent, the target object can be displaced from the filler in the extraction component by a strongly retained impurity with a stronger adsorption than the target object, so that the target flow flows out of the extraction component, while the strongly retained impurity is retained on the filler of the extraction component until the impurity penetrates the filler, and then the solid phase extraction column 3 is replaced; and by using a low-polarity eluent, there is no need to concentrate the qualified fraction flowing out of the solid phase extraction column 3 at a later stage, and the fraction can directly enter the chromatographic column 7 for fine separation.

[0286] Furthermore, the length of the chromatographic column 7 in S4 is 25 cm-500 cm.

[0287] Furthermore, the temperature of the eluent, the solid phase extraction column 3 and the chromatographic column 7 is controlled at 1°C-60°C, preferably 2°C-30°C.

[0288] It is understandable that high temperature is an important factor causing instability of the chromatographic column 7 , and therefore it is necessary to control the temperature of the eluent, the solid phase extraction column 3 , and the chromatographic column 7 during the purification process to maintain the stability of the solid phase extraction column 3 and the chromatographic column 7 .

[0289] Furthermore, the device for extracting and purifying organic matter from a mixture is applied to the purification of organic matter produced by biological methods.

[0290] The organic substances include the purification of amides, alcohols, sugars, lipids, macrolides, cyclosporine, polypeptides, vitamins, phospholipids, heterocyclics, and corticosteroids.

[0291] Preferably, the organic substance is coenzyme Q10, liposome and phosphoramidite.

[0292] Coenzyme Q 10 The market demand for liposomes and phosphoramidites is relatively large. For example, liposomes are used for genetic modification or drug preparation. The liposomes can be fused with cell membranes to deliver drugs into cells. Liposomes used for drug conjugates are usually small molecule mixtures with molecular weights of 500 to 4000. High-quality drugs require liposomes to be monomers with a single molecular weight. The traditional separation process of liposomes is usually silica gel chromatography: fill silica gel glass columns, elute with low-polarity organic solvents, receive the effluent and confirm the basic purity through spot plates, and then detect qualified fractions through HPLC-CAD and concentrate. In order to obtain purer monomers, the silica gel column is usually filled with a length of more than 3000mm, and the column time is 70-100 hours. The silica gel column loses its separation ability completely after 1 to 2 separations. If you want to get more monomers, you need to constantly disassemble and assemble the chromatographic column. Therefore, this process has the disadvantages of more solid waste, heavy pollution, long working hours, large amount of solvent, and low efficiency.

[0293] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for online extraction and purification of organic matter from a mixture, characterized in that: The steps include: S1. Solid phase extraction of organic matter: input the sample solution into a solid phase extraction column for pre-purification to obtain a pre-purified fraction; S2, determining the purity of the pre-purified fraction and replacing the solid phase extraction column according to the purity of the fraction; S3. The qualified fractions in the pre-purification fractions are stored in an intermediate tank or injected into a chromatographic column; S4, performing chromatographic separation and purification on the qualified pre-purified fraction flowing out of the solid phase extraction column or the qualified pre-purified fraction stored in the intermediate tank: the qualified pre-purified fraction is transported to a chromatographic column filled with silica, and enters a collection tank after being purified by the chromatographic column; The characteristics of solid phase extraction are as follows: a sample solution prepared with a non-polar or low-polar solvent is passed through a solid phase extraction column, and the target substance is displaced from the filler in the solid phase extraction column by a strongly retained impurity with stronger adsorption than the target substance, so that the target substance flows out of the solid phase extraction column, and the non-polar or low-polar solvent has a lower overall polarity than the eluent used for column purification; There are N solid phase extraction columns, where N is an integer greater than or equal to 1; Also included is a full-wavelength online detection system for detecting the pre-purified fraction; The organic substance solid phase extraction is specifically: If the pre-purification fraction discharged is detected to be a qualified fraction, the qualified fraction is stored in an intermediate tank or injected into a chromatographic column; If it is detected that the pre-purified fraction discharged from the first solid phase extraction column contains a high content of strongly retained impurities, the first solid phase extraction column and the second solid phase extraction column are connected in series, and the pre-purified fraction discharged from the first solid phase extraction column flows into the second solid phase extraction column until the content of strongly retained impurities in the pre-purified fraction discharged from the first solid phase extraction column approaches or reaches the content of strongly retained impurities in the sample solution, and then the sample solution enters the second solid phase extraction column, and the first solid phase extraction column is removed; And so on, the sample solution enters the Nth solid phase extraction column to complete the pre-purification of all samples.

2. The method for online extraction and purification of organic matter from a mixture according to claim 1, characterized in that: The S2 is specifically the following steps: S21: Before extraction, detecting the absorbance of each substance to be pre-purified in the mixture over the entire wavelength range; S22: During extraction, click the start button. At this time, the absorbance a1 of substance A in the mixture under the first set wavelength condition is greater than the absorbance b1 of substance B in the mixture under the first set wavelength condition. As the experiment progresses, the solid phase extraction column is replaced in the following two situations: Case 1: a1 decreases, b1 increases. When a1<b1 and a1 is lower than the first set value, it is judged that substance A is completely separated. When b1 increases to the second set value, it is judged that impurity penetration occurs in the solid phase extraction column, and the solid phase extraction column is replaced at this time. Case 2: a1 decreases, b1 decreases, and a1>b1. At this time, start to compare the absorbance a2 of substance A in the mixture under the second set wavelength condition and the absorbance b2 of substance B in the mixture under the second set wavelength condition, and compare with the initial value. When the trend of a2 and b2 is still that a2 decreases, b2 decreases, and a2>b2, start to compare the absorbance a3 of substance A in the mixture under the third set wavelength condition and the absorbance b3 of substance B in the mixture under the third set wavelength condition until a difference appears, so as to judge that substance A is completely separated; judge whether impurity penetration occurs in the solid phase extraction column according to the absorbance of substance B in the mixture, that is, after the absorbance of b rises to the set value, it is judged that impurity penetration occurs in the solid phase extraction column, and the solid phase extraction column is replaced at this time; And so on, using the same conditions, compare substances (B, C), (C, D)…(X-1, X).

3. The method for online extraction and purification of organic matter from a mixture according to claim 2, characterized in that: The full wavelength range is 200nm-800nm.

4. The method for online extraction and purification of organic matter from a mixture according to claim 2, characterized in that: The first set value in S22 is 100mv-5000mv.

5. A device for extracting and purifying organic matter from a mixture, characterized in that: The method for online extraction and purification of organic matter from a mixture according to any one of claims 1 to 4 comprises a solid phase extraction device and a chromatographic column connected in series with the solid phase extraction device, wherein the chromatographic column is a forward chromatographic column.

6. The device for extracting and purifying organic matter from a mixture according to claim 5, characterized in that The solid phase extraction device includes a full-wavelength online detection system, an extraction component and a controller; Among them, the full-wavelength online detection system includes a light source, a flow cell, and a light energy signal receiver. The light source is connected to a straight optical fiber. The light source and the light energy signal receiver are located on both sides of the flow cell, and the line connecting the two is perpendicular to the sample channel in the flow cell. The light energy signal receiver is a full-wavelength diode array ultraviolet-visible detector or a triode array ultraviolet-visible detector. The extraction component includes N solid phase extraction columns, N is an integer greater than or equal to 1, the input end of each solid phase extraction column is connected to the sample tank through an injection pipeline, each injection pipeline is provided with a first valve, the output end of each solid phase extraction column is connected to a liquid outlet pipeline, the first branch of the liquid outlet pipeline is connected to a flow cell of a full-wavelength online detection system, the first branch is provided with a third valve, the second branch of the liquid outlet pipeline is connected to a forward chromatographic column, and the second branch is provided with a second valve; The second valve of the first solid phase extraction column is also connected to the input end of the second solid phase extraction column through a third branch; The second valve of the second solid phase extraction column is also connected to the input end of the third solid phase extraction column through a third branch; By analogy, the second valve of the N-1th solid phase extraction column is also connected to the input end of the Nth solid phase extraction column through the third branch; The controller is configured to control the switching of each valve according to the detection results of the full-wavelength online detection system.

7. The device for extracting and purifying organic matter from a mixture according to claim 6, characterized in that: An intermediate tank is arranged between the solid phase extraction device and the chromatographic column.

8. The device for extracting and purifying organic matter from a mixture according to claim 7, characterized in that: The number of the solid phase extraction columns is 2-10.

9. The device for extracting and purifying organic matter from a mixture according to claim 8, characterized in that: The collecting tank comprises a first collecting tank and a second collecting tank, the input ends of the first collecting tank and the second collecting tank are connected to the output end of the chromatographic column through a pipeline, and are used to collect fractions from the chromatographic column, and the first collecting tank and the second collecting tank are both connected to a qualified product tank and an unqualified product tank; Also included is a solvent recovery system for recovering solvent from the acceptable product tank and the rejected product tank fractions.

10. The device for extracting and purifying organic matter from a mixture according to claim 9, characterized in that: The output end of the chromatographic column is connected to the input end of the first collection tank and the input end of the second collection tank through pipelines, and an ultraviolet detector and a valve are arranged on the pipeline. The ultraviolet detector is used to detect whether there is a chromatographic peak in the fraction flowing out of the chromatographic column; The first collection tank and the second collection tank are both connected to a second online detection system, and the second online detection system is used to detect the purity of the target substance in the fractions of the first collection tank and the second collection tank; The ultraviolet detector and the second online detection system are also electrically connected to the controller to control the opening and closing of the plurality of valves.

11. The device for extracting and purifying organic matter from a mixture according to claim 10, characterized in that: The solvent recovery system includes a mixer and a solvent tank; The output end of the unqualified product tank is connected to the input end of the mixer through a pipeline, and a valve, an infusion pump, a pressure sensor and a mass flow meter are arranged on the pipeline; The output end of the qualified product tank is connected to the input end of the mixer through a pipeline, and a valve, an infusion pump, a pressure sensor and a mass flow meter are arranged on the pipeline; The output end of the solvent tank is connected to the input end of the mixer through a pipeline, and a valve, an infusion pump, a pressure sensor and a mass flow meter are arranged on the pipeline; The output end of the mixer is connected to the input end of the eluent tank through a pipeline, a mass flow meter is arranged on the pipeline, and the output end of the eluent tank is connected to the liquid inlet of the chromatographic column through a pipeline; A plurality of mass flow meters are electrically connected to the controller, and the controller controls the opening and closing of corresponding solvent tank valves according to the detection results of the mass flow meters.

12. The device for extracting and purifying organic matter from a mixture according to claim 5, characterized in that: The filler filled in the solid phase extraction column in S1 is spherical or amorphous mesoporous silica gel with a particle size of 20-200 um.

13. The device for extracting and purifying organic matter from a mixture according to claim 5, characterized in that: The chromatographic filler filled in the chromatographic column is spherical silica; The particle size of the spherical silica is 8 μm-200 μm; the mesopore diameter is 5 nm-20 nm; and the porosity is 0.8-1.2 cm 3 / g; specific surface area is 50-600m 2 / g; The total content of calcium, magnesium, aluminum, chromium, iron and manganese in the spherical silicon dioxide is less than 100 μg / g; The average particle size distribution of the spherical silicon dioxide satisfies: D90 / D10≤1.5; The spherical silica is modified silica, including silica with surface chemical modification using organic molecules or silica gel containing metal ions; the organic molecules used for surface chemical modification are silanes containing polar molecular functional groups, and the polar molecular functional groups include amino groups, amide groups, alcoholic hydroxyl groups, urea groups, cyano groups, carboxyl groups, heterocycles, amino acids, monosaccharides or polysaccharides.

14. The device for extracting and purifying organic matter from a mixture according to claim 13, characterized in that: The sample solution to be separated in S1 can be added to the solid phase extraction column continuously or in batches until the collected fraction contains strongly retained impurities that are more strongly retained than the target substance; then the solid phase extraction column is connected in series with the next solid phase extraction column, and the sample solution is continued to be added until the ratio of the strongly retained impurities to the target substance detected at the end of the first solid phase extraction column has reached or is close to the ratio in the sample solution.

15. The device for extracting and purifying organic matter from a mixture according to claim 5, characterized in that: In the S1, a low-polarity solvent is used to prepare the sample solution during solid phase extraction, and in the S4, a high-polarity solvent is used as the eluent.

16. The device for extracting and purifying organic matter from a mixture according to claim 15, characterized in that: The low polarity solvent is at least one of n-hexane, n-heptane and petroleum ether, or a mixture of one of n-hexane, n-heptane and petroleum ether and a small amount of a high polarity solvent; The high polarity solvent is at least one of ethyl acetate, dichloromethane, methanol, ethanol, acetonitrile, isopropanol and acetone, or a mixture of one of ethyl acetate, dichloromethane, methanol, ethanol, acetonitrile, isopropanol and acetone and a low polarity solvent.

17. The device for extracting and purifying organic matter from a mixture according to claim 5, characterized in that: The length of the chromatographic column in S4 is 25cm-500cm.

18. The device for extracting and purifying organic matter from a mixture according to claim 15, characterized in that: The temperature of the eluent, solid phase extraction column and chromatographic column is controlled at 1°C-60°C.

19. The device for extracting and purifying organic matter from a mixture according to claim 5, characterized in that: Applied to the purification of organic substances produced by biological methods.

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