阅读说明：本技术 一种色谱柱的序贯填充方法 (Sequential filling method of chromatographic column ) 是由 王峰 郁凯 吴开电 王志明 彭波 于 2021-08-30 设计创作，主要内容包括：本发明公开一种色谱柱的序贯填充方法,先制备匀浆悬液；把制备的匀浆液倒入已连接好色谱柱管的匀浆罐中；然后以多种不同的顶替液形成序贯顶替液的方式依次输入匀浆罐中,经匀浆罐注入到色谱柱管中；所述序贯顶替液包括第一顶替液、中间替换液和第二顶替液,所述第一顶替液、中间替换液和第二顶替液分别选自乙腈、丙腈、丁腈、甲醇、乙醇、丁醇、纯化水、异丙醇中的一种或几种。本发明采用序贯顶替液的填装方式,能加工出不同类型的色谱柱,且装填紧密、稳定性好、柱效高,同时降低人工成本。(The invention discloses a sequential filling method of a chromatographic column, which comprises the steps of firstly preparing homogenate suspension; pouring the prepared homogenate into a homogenate tank connected with a chromatographic column tube; then sequentially inputting the displacement liquid into a homogenate tank in a mode that a plurality of different displacement liquids form sequential displacement liquids, and injecting the displacement liquid into a chromatographic column tube through the homogenate tank; the sequential displacement liquid comprises a first displacement liquid, an intermediate displacement liquid and a second displacement liquid, wherein the first displacement liquid, the intermediate displacement liquid and the second displacement liquid are respectively selected from one or more of acetonitrile, propionitrile, butyronitrile, methanol, ethanol, butanol, purified water and isopropanol. The invention adopts the filling mode of sequential displacement liquid, can process different types of chromatographic columns, has compact filling, good stability and high column efficiency, and simultaneously reduces the labor cost.)

1. A sequential filling method of chromatographic column comprises preparing homogenate suspension; pouring the prepared homogenate suspension into a homogenate tank connected with a chromatographic column tube; the method is characterized in that the displacement liquid is sequentially input into a homogenate tank in a mode of forming sequential displacement liquid by a plurality of different displacement liquids, and is injected into a chromatographic column tube through the homogenate tank; the sequential displacement liquid comprises a first displacement liquid, an intermediate displacement liquid and a second displacement liquid, wherein the first displacement liquid, the intermediate displacement liquid and the second displacement liquid are respectively selected from one or more of acetonitrile, propionitrile, butyronitrile, methanol, ethanol, butanol, purified water and isopropanol;

the sequential displacement fluid comprises one of the following two forms:

the first method is as follows: the first displacing liquid is selected from 0.80mm²/s-1.30mm²A low viscosity displacement fluid in the viscosity range/s, the second displacement fluid being selected from 2.00mm²/s-3.00mm²A high-viscosity displacement fluid in the viscosity range per second, wherein the intermediate replacement fluid is selected from displacement fluids which are soluble in the first displacement fluid and the second displacement fluid, and the viscosity difference formed between the adjacent low-viscosity displacement fluid and the high-viscosity displacement fluid is 1.2mm²/s-2.2mm²The difference in viscosity between/s;

the second method comprises the following steps: the first displacement fluid is selected from displacement fluids capable of reducing the surface charge of the filler; the second displacement liquid is selected from displacement liquids which can enable the filler to be filled more densely, and the intermediate displacement liquid is selected from displacement liquids which are soluble in the first displacement liquid and the second displacement liquid.

2. A method of sequential packing of a chromatography column according to claim 1, comprising the steps of:

a) preparing a homogenate suspension; preparing a first displacement fluid, an intermediate displacement fluid and a second displacement fluid according to the principles of claim 1;

b) pouring the prepared homogenate suspension into a homogenate tank connected to a chromatographic column filling machine; respectively adding the first displacement liquid, the intermediate displacement liquid and the second displacement liquid into the displacement liquid bottle to form a related sequential displacement liquid form, and simultaneously setting the working time of the three displacement liquids; the chromatographic column filling machine comprises a pneumatic booster pump (2), a displacement liquid bottle (9), a homogenizing tank (14) and a pipeline for connection, wherein a gas input interface, a medium input interface and an output interface are arranged on the pneumatic booster pump (2); the gas input interface is connected with a gas inlet device; the output interface is connected with a homogenate tank (14), the medium input interface is connected with a displacement liquid selection mechanism (6) through a pipeline, the output end of the displacement liquid selection mechanism (6) is provided with at least three displacement liquid pipes, each displacement liquid pipe is connected with a displacement liquid bottle (9), and each displacement liquid pipe is respectively provided with a control device;

c) opening an air switch, starting gas to enter a pneumatic liquid pump, pushing liquid to move to realize pressurization, adjusting a pressure adjusting valve, stopping adjustment when the required pressure is reached, sequentially inputting three displacement liquids into a homogenate tank according to sequence and set time under the pressure of 60-80MPa, and entering a chromatographic column through the homogenate tank;

d, when the processing is finished, closing the stop valve to naturally reduce the pressure of the chromatographic column, standing, and when no liquid flows out from the lower end of the chromatographic column, dismounting the chromatographic column.

3. The method of claim 2, wherein the preparing the homogenate suspension comprises: adding the filler into a container filled with the homogenate, performing ultrasonic treatment for 1-5min, dispersing and shaking uniformly to form a homogenate suspension, wherein the material-liquid ratio of the homogenate suspension is maintained at 1: 10 and 1: 20, respectively.

4. A method according to claim 2, wherein the homogenate is selected from one or more of isopropanol, tetrahydrofuran, carbon tetrachloride, bromobenzene and dioxane.

5. A method of sequential packing of a chromatography column according to claim 2, wherein the working time parameters of three displacement fluids are simultaneously set as follows: the working time of the first displacement liquid is 5-15 min; working for 5-15min with intermediate replacement liquid; and the third displacement liquid works for 15-25 min.

6. A method for sequential packing of a chromatography column according to claim 2, wherein the control means comprises a solenoid valve (7) and a time relay (8), the solenoid valve (7) and the time relay (8) being electrically connected.

7. A method of sequential packing of a chromatography column according to claim 2, wherein: the output interface is connected with the pipeline branching mechanism (10) through a pipeline, the output end of the pipeline branching mechanism (10) is at least provided with two branches which are respectively connected with the pressure release valve (12) and the stop valve (13), and the rear end of the stop valve (13) is connected with the homogenate tank (14).

8. A method of sequential packing of a chromatography column according to claim 2, wherein: the gas input interface is connected with the pressure regulating valve (4) and the ventilation switch (5) in series through pipelines.

9. A method of sequential packing of a chromatography column according to claim 2, wherein: the pipeline branching mechanism is characterized by further comprising a first pressure gauge (11), wherein the first pressure gauge (11) is connected to the output end of the pipeline branching mechanism (10), and the measuring range of the first pressure gauge (11) is 0-160 MPa; and a second pressure gauge with the measuring range of 0-1.6MPa is arranged on the pressure regulating valve (4).

10. A method of sequential packing of a chromatography column according to any of claims 1 or 2, characterized in that: the combination of the first mode includes:

A) when the C18 column is packed, the mixture of acetonitrile and ethanol is used as low viscosity displacing liquid with viscosity of 1.13mm²S; selecting ethanol as a replacement fluid; selecting mixed solution of isopropanol and purified water as high-viscosity displacing liquid with viscosity of 2.73mm²/s；

B) When the naphthalene-based column is filled, a mixed solution of ethanol and acetonitrile is selected as a displacement solution with low viscosity, and the viscosity of the displacement solution is 1.15mm²S; selecting ethanol as a replacement fluid; selecting mixed solution of ethanol and purified water as high viscosity displacing liquid with viscosity of 2.72mm²/s；

C) When the bromobenzene column is filled, methanol and purified water are selected as displacement fluid with low viscosity of 0.96mm²(s) selecting ethanol as a replacement fluid; selecting mixed solution of ethanol and purified water as high viscosity displacing liquid with viscosity of 2.72mm²/s；

When the cation column is filled in the second mode, the mixed solution of methanol and acetonitrile is selected as the displacement solution with low viscosity, and the viscosity of the displacement solution is 1.14mm²S; selecting ethanol as a replacement fluid; selecting mixed solution of ethanol and purified water as high viscosity displacing liquid with viscosity of 2.72mm²/s。

Technical Field

The invention relates to the technical field of high performance liquid chromatography, in particular to a sequential filling method of a chromatographic column.

Background

Chromatography is a separation and analysis means, and separation is the core, so the chromatographic column which plays a role in separation is the heart of a chromatographic system. The requirements for chromatographic columns are high column efficiency, good selectivity, high analysis speed and the like. The method for filling the chromatographic column comprises a dry method and a wet method according to the size of a stationary phase. Fillers having a particle diameter of more than 20 μm are generally filled by a dry method, and fillers having a particle diameter of 10 μm or less are necessarily filled by a wet method (an equal density homogenization filling method).

Patent 201010250468 discloses a method for packing C18 high performance liquid chromatography column, mixing cyclohexanol and chloroform at a volume ratio of 9: 11-13: 7, adding C18 silica gel with a particle size of 5um to prepare a homogenate with a concentration of 14.5%, fully stirring and mixing the solution, and oscillating in an ultrasonic oscillator for 5-10 minutes to obtain colorless transparent liquid; methanol and isopropanol are mixed at a ratio of 1: 1 to be used as displacement liquid; pouring the prepared homogenate into a homogenate tank connected with a chromatographic column tube; adding the displacement liquid into a displacement tank, starting a column filling machine, inputting the displacement liquid into a homogenate tank under 6000psi pressure, entering a chromatographic column through the homogenate tank, closing the column filling machine until 70-100 ml of the displacement liquid flows out of the chromatographic column, stopping pressurizing, standing for 20-40 minutes when the system pressure is reduced to normal pressure, and discharging the chromatographic column.

The traditional wet chromatographic column packing filler is usually filled by single displacement liquid and cannot adapt to the problems of uniform implantation and dense packing formed by high-speed extrusion, so that the conditions of peak tailing, untight packing and poor consistency are easily presented, and the column effect is poor; and most of the traditional wet-process packed chromatographic columns are processed and researched by aiming at the conventional C18 column, and some special columns (such as a naphthalene-based column, a bromobenzene column, an ion column and the like) have no related processing technology support. Meanwhile, the traditional wet-process packed chromatographic column also needs manual monitoring, and the labor cost is increased.

Therefore, the chromatographic column filling device with the sequential liquid replacement system and the method are needed to be designed to improve the column efficiency, meet the production requirements of different types of fillers, do not need manual monitoring, save time and have important significance.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to solve the problem that the traditional single displacement liquid filling cannot adapt to uniform implantation and high-speed extrusion to form compact filling, so that a column bed is not compact.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

a sequential filling method of chromatographic column comprises preparing homogenate suspension; pouring the prepared homogenate into a homogenate tank connected with a chromatographic column tube; then sequentially inputting the displacement liquid into a homogenate tank in a mode that a plurality of different displacement liquids form sequential displacement liquids, and injecting the displacement liquid into a chromatographic column tube through the homogenate tank; the sequential displacement liquid comprises a first displacement liquid, an intermediate displacement liquid and a second displacement liquid, wherein the first displacement liquid, the intermediate displacement liquid and the second displacement liquid are respectively selected from one or more of acetonitrile, propionitrile, butyronitrile, methanol, ethanol, butanol, purified water and isopropanol;

the sequential displacement fluid comprises one of the following two forms:

the first method is as follows: the first displacing liquid is selected from 0.80mm²/s-1.30mm²A low viscosity displacement fluid in the viscosity range/s, the second displacement fluid being selected from 2.00mm²/s-3.00mm²A high-viscosity displacement fluid in the viscosity range per second, wherein the intermediate replacement fluid is selected from displacement fluids which are soluble in the first displacement fluid and the second displacement fluid, and the viscosity difference formed between the adjacent low-viscosity displacement fluid and the high-viscosity displacement fluid is 1.2mm²/s-2.2mm²The difference in viscosity between/s;

the second method comprises the following steps: the first displacement fluid is selected from displacement fluids capable of reducing the surface charge of the filler; the second displacement liquid is selected from displacement liquids which can enable the filler to be filled more densely, and the intermediate displacement liquid is selected from displacement liquids which are soluble in the first displacement liquid and the second displacement liquid.

Further, a method for sequential packing of a chromatography column, comprising the steps of:

a) preparing a homogenate suspension; preparing a first displacement liquid, an intermediate displacement liquid and a second displacement liquid according to the principle;

b) pouring the prepared homogenate suspension into a homogenate tank connected to a chromatographic column filling machine; respectively adding the first displacement liquid, the intermediate displacement liquid and the second displacement liquid into the displacement liquid bottle to form a form of associated sequential displacement liquid; setting working time parameters of three displacement liquids simultaneously; the chromatographic column assembling machine comprises a pneumatic booster pump, a displacement liquid bottle, a homogenizing tank and a pipeline for connection, wherein the pneumatic booster pump is provided with a gas input interface, a medium input interface and an output interface; the gas input interface is connected with a gas inlet device; the output interface is connected with the homogenizing tank, the medium input interface is connected with the displacement liquid selection mechanism through a pipeline, the output end of the displacement liquid selection mechanism is provided with at least three displacement liquid pipes, and each displacement liquid pipe is connected with a displacement liquid bottle;

c) opening an air switch, starting gas to enter a pneumatic liquid pump, pushing liquid to move to realize pressurization, adjusting a pressure adjusting valve, stopping adjustment when the required pressure is reached, sequentially inputting three displacement liquids into a homogenate tank according to sequence and set time under the pressure of 60-80MPa, and entering a chromatographic column through the homogenate tank;

d, when the processing is finished, closing the stop valve to naturally reduce the pressure of the chromatographic column, standing, and when no liquid flows out from the lower end of the chromatographic column, dismounting the chromatographic column.

The medium input interface is connected with the displacement liquid selection mechanism through a pipeline, the output end of the displacement liquid selection mechanism is provided with at least three displacement liquid pipes, and each displacement liquid pipe is connected with a displacement liquid bottle to form a sequential displacement liquid system so as to realize switching among different displacement liquids.

Further, the preparing a homogenate suspension comprises: adding the filler into a container filled with the homogenate, performing ultrasonic treatment for 1-5min, dispersing and shaking uniformly to form a homogenate suspension, wherein the material-liquid ratio of the homogenate suspension is maintained at 1: 10 and 1: 20, respectively.

Further, the homogenate is selected from one or more of isopropanol, tetrahydrofuran, carbon tetrachloride, bromobenzene and dioxane.

Further, the working time of the three displacement liquids is slightly different according to the chromatographic column to be filled, and it is generally preferred to set the working time parameters of the three displacement liquids as follows: the working time of the first displacement liquid is 5-15 min; working for 5-15min with intermediate replacement liquid; and the third displacement liquid works for 15-25 min.

Furthermore, the output interface is connected with the pipeline branching mechanism through a pipeline, the output end of the pipeline branching mechanism is at least provided with two branches which are respectively connected with the pressure release valve and the stop valve, and the rear end of the stop valve is connected with the homogenate tank.

Further, the gas input interface is connected with the pressure regulating valve and the ventilation switch in series through pipelines.

Further, the displacing liquid selecting mechanism and the pipeline branching mechanism are stainless steel cross joints.

Furthermore, check valves are arranged at the medium input interface and the medium output interface in the pneumatic booster pump, so that the gas in the pipeline keeps flowing in a single direction.

Furthermore, a second pressure gauge with the range of 0-1.6MPa is arranged on the pressure regulating valve and used for detecting the pressure in the pipe.

Further comprises a frame for placing a pneumatic booster pump, a displacement liquid bottle and a homogenate tank.

The pipeline branching mechanism is characterized by further comprising a first pressure gauge, wherein the first pressure gauge is connected to the output end of the pipeline branching mechanism, and the range of the first pressure gauge is 0-160 MPa.

In the first mode: the low-viscosity displacement liquid, the replacement liquid and the high-viscosity displacement liquid are one or more of the existing conventional displacement liquids, such as one or more of acetonitrile, propionitrile, butyronitrile, methanol, ethanol, butanol, purified water, isopropanol and the like, the selection of the displacement liquid is selected according to the type of the chromatographic column to be filled, and the displacement liquid can be the conventional displacement liquid, and the difference is that the viscosity difference is controlled, the low-viscosity displacement liquid is filled (aiming at facilitating the implantation of the filling material), the low-viscosity displacement liquid is replaced by the replacement liquid (the intermediate replacement liquid and the first and second replacement liquids meet the condition of phase dissolution), and then the high-viscosity displacement liquid is filled (the high-viscosity displacement liquid can enable the filling material to be more compact on the basis of the former), so as to form a mode of filling the chromatographic column by the sequential displacement liquid.

The first preferred combination is:

A) when the C18 column is packed, the mixture of acetonitrile and ethanol is used as low viscosity displacing liquid with viscosity of 1.13mm²S; selecting ethanol as a replacement fluid; selecting mixed solution of isopropanol and purified water as high-viscosity displacing liquid with viscosity of 2.73mm²/s；

B) When the naphthalene-based column is filled, a mixed solution of ethanol and acetonitrile is selected as a displacement solution with low viscosity, wherein the viscosity of the displacement solution is 1.15mm 2/s; selecting ethanol as a replacement fluid; selecting mixed solution of ethanol and purified water as high viscosity displacing liquid with viscosity of 2.72mm²/s。

C) When the bromobenzene column is filled, methanol and purified water are selected as displacement fluid with low viscosity of 0.96mm²Selecting ethanol as a replacement fluid; selecting ethanol and purified water as high viscosity displacing liquid with viscosity of 2.72mm²/s。

In the second mode: the first displacement fluid is selected from displacement fluids which can reduce the surface charge of the filler, so that the filler can be more easily implanted; the second displacement liquid is selected from displacement liquids capable of enabling the filler to be extruded more densely, and the intermediate displacement liquid is selected from displacement liquids which are soluble in the first displacement liquid and the second displacement liquid. The first displacement liquid, the intermediate displacement liquid and the second displacement liquid are one or more of the conventional displacement liquids, such as one or more of acetonitrile, propionitrile, butyronitrile, methanol, ethanol, butanol, purified water, isopropanol and the like.

The second preferred combination is as follows:

when the cation column is filled, the mixed solution of methanol and acetonitrile is selected as a displacement solution with low viscosity, and the viscosity of the displacement solution is 1.14mm²S; selecting ethanol as a replacement fluid; selecting mixed solution of ethanol and purified water as high viscosity displacing liquid with viscosity of 2.72mm²/s。

The viscosity of the invention is tested by using a Ubbelohde viscometer, and the invention writes the kinematic viscosity of the liquid; kinematic viscosity calculation formula: v-Ct (viscosity standard specified test temperature of 2)0.0 ℃ ± 0.1 ℃), where v — kinematic viscosity (unit: mm is²In/s), C-viscometer constants (one for each Ubbelohde viscometer, on a certificate of authenticity. The specifications of the viscometer currently used areConstant 0.009786mm²/s²) T is the time for the liquid to flow from line a to line b (unit: s, typically averaged over multiple recordings).

Compared with the prior art, the invention has the beneficial effects that:

1. the core technical point of the invention is that the problem that the traditional single displacement liquid filling cannot adapt to uniform implantation and high-speed extrusion to form compact filling is changed, and a plurality of different displacement liquid forming sequential displacement liquid modes are adopted for filling, so that different types of chromatographic columns can be processed, and the filling is tighter, the stability is good and the column efficiency is high.

2. The chromatographic column assembling machine is portable, simple in structure, convenient to assemble and disassemble a chromatographic column, convenient to maintain and use, and additionally provided with automatic control equipment, so that the automatic control of displacement liquid switching and displacement time is realized, manual operation is reduced, and the cost is saved.

Drawings

FIG. 1 is a connection diagram of the present invention;

FIG. 2 is a schematic structural view of the present invention;

FIG. 3 is a front view of the present invention;

FIG. 4 is a side view of the present invention;

FIG. 5 is a top view of the present invention;

FIG. 6 is a functional block diagram of the present invention;

FIG. 7 is a peak profile of a packed column of example 1 of the present invention;

FIG. 8 is a peak appearance spectrum of the conventional method for packing a column for comparative example 1;

FIG. 9 is a graph showing the comparison of the column efficiency of the packed column of example 1 of the present invention and the conventional method of comparative example 1.

FIG. 10 is a peak profile of a packed chromatography column of example 2 of the present invention;

FIG. 11 is a peak appearance spectrum of the packed column of comparative example 2;

FIG. 12 is a peak profile of the packed column of example 3;

FIG. 13 is a peak appearance spectrum of the packed column of comparative example 3;

FIG. 14 is a peak profile of the packed column of example 4;

FIG. 15 is a peak appearance spectrum of the packed column of comparative example 4;

specifically, the following description is provided: 7-15 are the contents of a set of drawings output simultaneously by the instrument, generally read and used together, without disassembly for placement;

the text labels in the figures are represented as: 1. a frame; 2. a pneumatic booster pump; 4. a pressure regulating valve; 5. a ventilation switch; 6. a displacement fluid selection mechanism; 7. an electromagnetic valve; 8. a time relay; 9. a displacement liquid bottle; 10. a pipeline branching mechanism; 11. a first pressure gauge; 12. a pressure relief valve; 13. a stop valve; 14. and (6) a homogenizing tank.

Detailed Description

The following detailed description of the present invention is given for the purpose of better understanding technical solutions of the present invention by those skilled in the art, and the present description is only exemplary and explanatory and should not be construed as limiting the scope of the present invention in any way.

As shown in fig. 1-6, a chromatographic column packing machine comprises a pneumatic booster pump 2, a displacement liquid bottle 9, a homogenizing tank 14, and a pipeline for connection, wherein the pneumatic booster pump 2 is provided with a gas input interface, a medium input interface, and an output interface; the gas input interface is connected with the pressure regulating valve 4 and the ventilation switch 5 in series through pipelines; the medium input interface is connected with a displacement liquid selection mechanism 6 through a pipeline, the output end of the displacement liquid selection mechanism 6 is provided with three displacement liquid pipes which are respectively a first displacement liquid bottle filled with low-viscosity displacement liquid, a second displacement liquid bottle filled with medium viscosity and a third displacement liquid bottle filled with high viscosity, each displacement liquid pipe is connected with a displacement liquid bottle 9, the displacement liquid pipes are provided with electromagnetic valves 7 and time relays 8, the electromagnetic valves 7 are electrically connected with the time relays 8, the electromagnetic valves 7 are used for controlling the connection and disconnection of the pipeline, and the time relays 8 are used for controlling the processing duration; the output interface is connected with the pipeline branching mechanism 10 through a pipeline, the output end of the pipeline branching mechanism 10 is provided with three branches which are respectively connected with a first pressure gauge 11, a pressure release valve 12 and a stop valve 13, the measuring range of the first pressure gauge 11 is 0-160MPa, and the rear end of the stop valve 13 is connected with the homogenate tank 14.

The displacing liquid selecting mechanism 6 and the pipeline branching mechanism 10 are stainless steel four-way joints.

And the medium input interface and the medium output interface in the pneumatic booster pump 2 are both provided with one-way valves, so that the gas in the pipeline 3 keeps flowing in a one-way mode.

And the pressure regulating valve 4 is also provided with a second pressure gauge with the range of 0-1.6MPa, and the second pressure gauge is used for detecting the pressure in the pipe.

Still including the frame 1 that is used for placing pneumatic booster pump 2, displacement liquid bottle 9, homogenate jar 14, frame 1 adopts the aluminium alloy material preparation, and the aluminium alloy quality is light, and possess good intensity, and wherein pipeline, gas input interface, medium input interface, output interface all adopt 316 stainless steel material preparation, strengthen its reliability.

When the device is used, the homogenizing tank 14 is connected with the output interface, the homogenizing tank is screwed down, the displacement liquid bottles 9 of the three flow paths are respectively added with required liquid, namely, a first displacement liquid bottle is filled with a first displacement liquid, a second displacement liquid bottle is filled with a displacement liquid, a third displacement liquid bottle is filled with a second displacement liquid with high viscosity, time is set on a time relay 8, the electromagnetic valve 7 is used for controlling the electrification and the outage, the displacement liquid bottles 9 in the multi-solvent system are switched, the ventilation switch 5 on the gas input interface and the pressure relief valve 12 on the output interface are closed without manual monitoring and manual switching, and the pressure regulating valve 4 on the gas input interface is set to be 0.2 MPa.

Starting a pressurization system: opening a ventilation switch 5, finely adjusting a pressure regulating valve 4, starting a pneumatic booster pump 2, inputting gas into the pneumatic booster pump 2, enabling the gas to generate power after the required output pressure is reached, guiding the displacement liquid in a displacement liquid bottle 9 into a homogenizing tank 14, enabling filler particles to flocculate in the homogenizing tank 14, setting the time for running, closing a stop valve 13, closing a time relay 8 switch, slowly opening a pressure release valve 12, and disassembling the homogenizing tank 14.

Example 1

A sequential filling method of chromatographic column comprises preparing homogenate suspension; pouring the prepared homogenate into a homogenate tank connected with a chromatographic column tube; sequentially inputting the displacement liquid into a homogenate tank in a mode of forming sequential displacement liquid by a plurality of different displacement liquids, and injecting the displacement liquid into a chromatographic column tube through the homogenate tank; selecting acetonitrile and ethanol mixed solution (low viscosity displacing liquid with viscosity of 1.13 mm)²S) displacement for 10min + ethanol (displacement liquid) for 10min + a mixture of isopropanol and purified water (high viscosity displacement liquid, viscosity 2.73mm²S) filling was performed in a manner of displacement for 20 min.

For example, a 5 μm silica gel 4.6X 250mm analytical column was packed: weighing 3.0-3.5g of filler (2.8 g of drier is required for the standard of 4.6X 250mm analytical column, and 3.2g of experience is proper); dissolving the filler by using 50-60mL of isopropanol homogenate, uniformly stirring by using a glass rod, and then using ultrasonic waves for over 1 minute to ensure the dispersibility of the filler; quickly pouring the mixture into a homogenizing tank (completing the process in as short a time as possible and ensuring no air bubbles in the homogenizing tank), quickly connecting the homogenizing tank and the column to a pump at a pressure of 70MPa, and mixing acetonitrile + ethanol (low viscosity displacing liquid with a viscosity of 1.13 mm)²S) displacement for 10min + ethanol (displacement liquid) for 10min + mixed solution of isopropanol and purified water (high viscosity displacement liquid, viscosity of 2.72mm²S) filling was performed in a manner of displacement for 20 min. Starting the column filling machine, keeping the column filling machine to work under the pressure of 70MPa, and working time of three flow paths: after the first displacement liquid is displaced for 10 minutes, the second displacement liquid is operated for 10 minutes and the third displacement liquid is operated for 20 minutes, the stop valve 13 is closed to naturally reduce the pressure of the chromatographic column until no liquid flows out from the lower end of the chromatographic column, the processed chromatographic column is disassembled, and whether the column head is flat or not is checked, if the column head is flat, the thimble is used for flattening.

The column was connected to a two-dimensional liquid chromatograph, run with methanol (chromatographic grade) for 30 minutes at a flow rate of 1.0mL/min, and the column performance was checked.

Comparative example 1

The specific procedure is the same as example 1, except that the method of mixing methanol and isopropanol at a ratio of 1: 1 as a displacement liquid in the background art is selected to replace the sequential displacement liquid filling method in example 1 of the present invention.

After multiple tests, the theoretical plate number and the separation degree of the chromatographic column filled in the example 1 are obviously higher than those of the chromatographic column filled with the single displacement liquid in the comparative example 1, and the peak width is obviously lower than that of the chromatographic column filled in the comparative example 1, and specific experimental comparison data are shown in a figure 7-a figure 9 (a chart is a complete set of data) and tables 1 and 2.

Table 1 data table of columns processed by the method of example 1

Table 2 data table of column processed in comparative example 1

Retention time	Number of theoretical plate	Tailing factor	Peak width (width 5%)	Degree of separation
					9.054	8422	0.895	0.596	--
11.201	8881	0.911	0.736	4.936

It should be noted that the theoretical plate number is one of column efficiency parameters of the chromatogram, and is used for quantitatively expressing the separation efficiency of the chromatographic column; the tailing factor is a parameter for evaluating the peak shape by calculating the ratio of the peak width at 5% of the peak height to the distance from the peak top to the front edge, and aims to ensure the chromatographic separation effect and the measurement precision; the peak width is the distance between two points of intersection of tangent lines and peak bottom at inflection points on two sides of the chromatographic peak; the degree of separation is used to determine the separation of a separated substance in a column, and is often used as an index of the total separation performance of the column.

According to the comparison graphs of peak spectra and column effects given in tables 1-2 and fig. 7-9, wherein data 1 is the column effect of the present application, and data 2 is the column effect of the conventional equipment, the column effect of the present application can be obtained to be significantly better than that of the packed chromatographic column of comparative example 1.

Example 2

The mechanical structure is the same as that of the embodiment 1, and the difference is that the packed chromatographic column and the sequential replacement liquid are different, specifically:

when packing a naphthalene-based column (4.6X 125mm as an example), a low-viscosity displacement fluid (ethanol and acetonitrile mixture) with a viscosity of 1.15mm was selected²A viscosity of 2.72mm is obtained by replacing 5 min/s with ethanol (replacement liquid) for 10min + high viscosity replacement liquid (mixed liquid of ethanol and water)²The pattern was replaced by 5 min/s.

TABLE 3 data table of chromatographic columns processed by the method of example 2, column effect chart see FIG. 10

Retention time	Number of theoretical plate	Tailing factor	Peak width (height 5%)
				6.730	5790	0.993	0.447

Comparative example 2, the specific procedure is the same as example 2, except that "ethanol and acetonitrile are mixed at a volume ratio of 1: 1 as the displacement liquid" is selected instead of the sequential displacement liquid filling manner of example 2 of the present invention.

TABLE 4 data table of columns processed by the method of comparative example 2, column effect chart please see FIG. 11

Retention time	Number of theoretical plate	Tailing factor	Peak width (height 5%)
				6.655	3926	1.187	0.584

It can be seen that the theoretical plate number and the separation degree of the chromatographic column packed in the example 2 are obviously higher than those of the chromatographic column packed by the single displacement liquid in the comparative example 2, and the peak width is obviously lower than that of the chromatographic column packed in the comparative example 2.

Example 3

The mechanical structure is the same as that of the embodiment 1, and the difference is that the packed chromatographic column and the sequential replacement liquid are different, specifically:

when the bromobenzene column is packed (taking 4.6X 125mm as an example), the displacement fluid (methanol and purified water) with low viscosity is selected, and the viscosity is 0.96mm²A viscosity of 2.72mm, which is obtained by replacing 5 min/s with ethanol (replacement liquid) and 5min + with high-viscosity replacement liquid (ethanol and purified water)²The pattern of 10min substitution/s.

TABLE 5 data table of chromatographic columns processed by the method of example 3, column effect chart see FIG. 12

Retention time	Number of theoretical plate	Tailing factor	Peak width (height 5%)
				6.637	5182	1.062	0.488

Comparative example 3, the procedure is the same as example 3, except that "methanol is selected as the displacement liquid" instead of the sequential displacement liquid filling method of example 3 of the present invention.

TABLE 6 data table of columns processed by the method of comparative example 3, column effect chart please see FIG. 13

Retention time	Number of theoretical plate	Tailing factor	Peak width (height 5%)
				6.511	5204	1.268	0.504

It can be seen that the theoretical plate number and the separation degree of the chromatographic column packed in the example 3 are obviously higher than those of the chromatographic column packed by the single displacement liquid in the comparative example 3, and the peak width is obviously lower than that of the chromatographic column packed in the comparative example 3.

Example 4

The mechanical structure is the same as that of the embodiment 1, and the difference is that the packed chromatographic column is different from the sequential replacement liquid, and the sequential replacement liquid is selected in a second mode, specifically:

when the cation column is filled, the mixed solution of methanol and acetonitrile is selected as a displacement solution with low viscosity, and the viscosity of the displacement solution is 1.14mm²S; selecting ethanol as a replacement fluid; selecting mixed solution of ethanol and purified water as high viscosity displacing liquid with viscosity of 2.72mm²/s。

TABLE 7 data table of columns processed by the method of example 4, column effect chart see FIG. 14

Retention time	Number of theoretical plate	Tailing factor	Peak width (height 5%)
				3.902	2060	1.048	0.449

Comparative example 4, the specific procedure is the same as example 4, except that "bromobenzene and isopropanol are mixed in a volume ratio of 1: 1 to be used as a displacement liquid" is selected to replace the sequential displacement liquid filling mode of example 4 of the present invention.

TABLE 8 data table for columns processed by the method of comparative example 4, column effect chart please see FIG. 15

Retention time	Number of theoretical plate	Tailing factor	Peak width (height 5%)
				4.673	1230	1.072	0.641

It can be seen that the theoretical plate number and the separation degree of the chromatographic column packed in the example 4 are obviously higher than those of the chromatographic column packed by the single displacement liquid in the comparative example 4, and the peak width is obviously lower than that of the chromatographic column packed in the comparative example 4.

The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts of the present invention. The foregoing is only a preferred embodiment of the present invention, and it should be noted that there are objectively infinite specific structures due to the limited character expressions, and it will be apparent to those skilled in the art that a plurality of modifications, decorations or changes may be made without departing from the principle of the present invention, and the technical features described above may be combined in a suitable manner; such modifications, variations, combinations, or adaptations of the invention using its spirit and scope, as defined by the claims, may be directed to other uses and embodiments.