WO2016082126A1

WO2016082126A1 - Device for enriching and purifying compounds and use method therefor

Info

Publication number: WO2016082126A1
Application number: PCT/CN2014/092286
Authority: WO
Inventors: 张洁; 张亮; 黄望
Original assignee: 四川九章生物科技有限公司
Priority date: 2014-11-26
Filing date: 2014-11-26
Publication date: 2016-06-02

Abstract

The present invention provides a device for enriching and purifying compounds. The present invention also provides a use method for the device and a purification method for chlorogenic acid. According to the present invention, extracted clear liquor or concentrated clear liquor of high and low-concentration chlorogenic acid is effectively enriched in an enrichment form of combining a single column with tandem columns, so that the dynamic adsorption capacity of resin per unit volume is increased, the problem of non-continuous sample loading adsorption of the single column due to dynamic adsorption leakage is solved, and the problem of reduced chlorogenic acid adsorption amount of the resin due to additional impurity adsorption caused by continuous penetration sample loading of the tandem columns is also solved; meanwhile, a mode of selective elution by the single column is adopted, so that the content of impurities in elution liquid is reduced, the product purity is improved, and the problem of reduced product purity due to continuous release of the impurities to the elution liquid by the resin during tandem elution is solved.

Description

Device for enriching purified compound and using method thereof

Technical field

The present invention relates to a device for enriching purified compounds, in particular for enriching and purifying chlorogenic acid.

Background technique

At present, the commonly used methods for compound purification include solvent extraction, metal ion precipitation, salting out, alcohol precipitation, membrane filtration, resin column chromatography, polyamide column chromatography, crystallization and recrystallization, etc. The compound is isolated and purified by a combination of one or several enrichment purification methods.

Chlorogenic acid has a wide range of antibacterial effects, but can be inactivated by proteins in the body. Similar to caffeic acid, oral excitability can be increased in rats when administered orally or intraperitoneally. It can increase the intestinal peristalsis of rats and mice and the tension of rat uterus. It has a beneficial effect on bile secretion in rats. It has a sensitizing effect on humans, and after inhaling plant dust containing this product, asthma, dermatitis, etc. may occur. At present, there are many methods for separation and purification of chlorogenic acid, such as: Application No.: 200810124130.0, invention name: a new method for adsorbing and separating high-purity chlorogenic acid, and the present invention provides a new method for adsorbing and separating high-purity chlorogenic acid. The method comprises the following steps: the adsorption process, the washing process, and the elution process, wherein the chlorogenic acid stock solution is dynamically adsorbed to the penetrating through at least two serial adsorption columns loaded with the macroporous resin, and the mixed solution is washed and mixed. Finally, the eluate was eluted until no chlorogenic acid was discharged, and the effluent was collected. The purity of the pure chlorogenic acid solution obtained by the method of the invention can reach 92% or more by HPLC, and the concentration can reach 0.1 mg/mL to 0.8 mg/mL, and can be widely used in the fields of food, medicine, cosmetics and the like. The patented method uses a two-column column and above series column to enrich and purify chlorogenic acid, which can significantly increase the adsorption capacity of the resin compared with a single column, but the single column and the multi-column series are not combined in the technical solution. The enriched form is loaded, so that the sample liquid which has been adsorbed without chlorogenic acid enters the next-stage resin column, causing the resin to re-adsorb the impurities which are not completely adsorbed in the sample liquid, thereby reducing the subsequent The adsorption of chlorogenic acid in the sample solution; in addition, in the elution process, due to the uniform use of a feed port, the elution solvent after the elution of chlorogenic acid in the resin column of the previous stage is continuously continued during the elution process. The column is passed through the resin column, whereby impurities in the column resin continue to be released into the elution solvent, resulting in a low product purity. Application No.: 200910029959.7, title: A new method for preparing high-purity chlorogenic acid, the present invention provides a new method for preparing high-purity chlorogenic acid, which is passed through a series adsorption of at least 4 macroporous resins. After column, washing and elution, the eluate is regenerated and subjected to continuous countercurrent extraction to obtain high-purity chlorogenic acid. In order to solve the shortcomings of the application No. 200810124130.0, the technical solution is completed by a combination of a chromatography column and a countercurrent extraction. The technical solution solves the problem of low purity of the product, but still cannot solve the problem of further increasing the adsorption capacity of the resin and reducing the production. The problem of cost.

Summary of the invention

In order to solve the above problems, the present invention provides an apparatus for enriching and purifying a compound, and a method for using the same, which is capable of effectively enriching a high-low concentration chlorogenic acid extraction clear liquid or a concentrated clear liquid, and improving the dynamics of the resin unit volume. The adsorption capacity solves the problem that the single column cannot continue to be sampled due to dynamic adsorption leakage, and also solves the problem that the series column continues to pass through the sample, so that the resin additionally adsorbs impurities and reduces the adsorption amount of chlorogenic acid; The method of sexual elution reduces the content of impurities in the eluent, improves the purity of the product, and solves the problem that the resin continuously releases impurities into the eluent during the series elution, thereby reducing the purity of the product.

The technical solution of the present invention is to provide an apparatus for enriching a purified compound. Another technical solution of the present invention is to provide a method of using the device, and a method of purifying chlorogenic acid.

The invention provides a device for enriching and purifying a compound, comprising a adsorption column of a macroporous resin, a pipeline, a raw liquid feed pipe connected to an inlet of the primary adsorption column (1) through a valve (11), and a primary adsorption column (1) The outlet of the secondary adsorption column (2) is connected to the inlet of the primary collection (13) and secondary adsorption column (2) through the tee and the valve (12, 14), and the outlet of the secondary adsorption column (2) is passed through the tee and the valve (22). 24) respectively connected to the inlet of the secondary collection irrigation (23) and the secondary adsorption column (3), and the outlet of the secondary adsorption column (3) through the tee and the valve (32, 34) respectively and above the secondary The inlet connection of the irrigation (33) and the lower adsorption column is collected, and the raw liquid feed pipe is also connected to the inlet of the secondary or secondary adsorption column through the valve (21, 31).

Further preferably, the adsorption column of each stage has an aspect ratio of 1:6 to 1:10.

Further preferably, the outer casing of the adsorption column of each stage and the resin support screen inside are made of stainless steel, or glass, or ceramic, or polyethylene, or polypropylene.

Wherein, the macroporous resin in the adsorption column of each stage has a particle size ranging from 0.2 to 1.5 mm and an aspect ratio of 1:4 to 1:8.

The invention also provides a method of using the device, comprising the steps of:

a. Wet packing:

Close valves

21, 31 and 14,

open valves

11 and 12, input resin water or ethanol to the primary column, and the effluent is collected by the primary collection tank until the resin is filled to the aspect ratio. 1:4-1:8, stop input,

close valves

12 and 11, complete the resin wet packing of the primary column; close the valve 24, open the

valves

21, 22, and input the resin water or ethanol solution to the secondary column. When the resin is filled until the aspect ratio is 1:4 to 1:8, the input is stopped, the

valves

22 and 21 are closed, and the resin is wet-packed for the secondary column; the wet packing of the columns of each stage is completed in sequence according to the above method. operating;

b. Pretreatment or regeneration treatment: Close the

valves

21, 31 and 12, 22, 32, open the

valves

11, 14, 24 and 34, input the pretreatment or regeneration treatment solvent or solution, and then wash with water until the effluent has no pretreatment. Or regenerating the solvent or solution, stopping the input, closing the

valves

34 and 11;

c. Loading:

Close valves

21, 31 and 14,

open valves

11 and 12, input the sample solution, and after the target compound is detected in the effluent at the outlet of valve 12,

open valves

14 and 22,

close Valves

12 and 24, after the target compound is detected in the effluent of the valve 22, the loading operation of each column is completed in accordance with the above method;

d, water washing: close the

valves

21, 31 and 12, 22, 32, open the

valves

14, 24 and 34, input water until the washing requirements are reached;

c. Elution:

Close valves

21, 31 and 12, 22, 32,

open valves

14, 24 and 34, input eluent, and open valve 12 sampling test from time to time. When no target compound is detected,

close valves

14 and 11 Open the valve 21; continue to input the eluent, open the valve 22 sampling test from time to time, when no target compound is detected, close the

valves

24 and 21, open the valve 31; follow the above method to complete the elution operation of each column .

The invention also provides a method for enriching and purifying chlorogenic acid, which is carried out by using the device, comprising the following steps:

a. Packing column: Packing the resin in a wet packing method with a diameter to height ratio of 1:4-1:8, and connecting the first and second ends of each single column in series, the number of the series columns is 2-10;

b. Pretreatment or regeneration treatment: Wash the column with a 1-3 times serial column volume pretreatment or regeneration treatment solvent or solution at a flow rate of 2-5 times the single column volume/hour through the column, and then wash with water at the same flow rate. The effluent has no pretreatment or regeneration treatment solvent or solution;

c. Loading: sequentially sorting according to the single column of the series column, loading from the inlet of the first stage single column, before the effluent is not detected by the sample effluent, and discharging from the discharge port of the column to the effluent When chlorogenic acid is detected, the loading effluent enters the next stage single column; the chlorogenic acid is detected in the single column loading effluent until the last stage according to the above method;

d, water washing: using 1-5 times the volume of a single column of water to wash the column through the series column at a flow rate of 2-5 times a single column volume / hour;

e, elution: sequentially separate and elute according to the single column of the series column, firstly input the elution solvent from the first-stage single-column feed port, and periodically open the outflow valve of the column for sampling and detection, to be washed When the chlorogenic acid is detected in the liquid-free detection, the elution solvent is further eluted from the single-stage feed port of the next stage. According to the above method, no chlorogenic acid is detected in the single-column elution effluent of the last stage. . The eluent is collected by the tail column discharge port;

f, concentration: collecting the elution effluent, recovering ethanol under low temperature and negative pressure conditions, and concentrating to a relative density of 1.3-1.5 (25 ° C);

g, lyophilization: The concentrate is dried in a freeze dryer.

Further preferably, the column packing resin described in step a is: a polystyrene macroporous adsorption resin having a particle size ranging from 0.2 to 1.5 mm; and the pretreatment or regeneration treatment solvent or solution described in step b: ethanol and a solution, Or one or more of methanol and solution, or acid water, or alkaline water, or water; the sample solution described in step c is a clear liquid with a chlorogenic acid content of 0.5-30 mg/ml, and the loading flow rate is 5 - 10 times single column volume / hour; the pH of the water in step d is 3-7; the elution solvent described in step e is 10-60% The ethanol solution or the methanol solution, the elution flow rate is 2-5 BV single column volume / hour; the concentration conditions described in the f step are: concentration temperature is 40-70 ° C, concentrated vacuum: greater than 0.04 Mp; g freeze-dried drying conditions For: pre-freezing temperature below -30 °C, pre-freezing time is more than 1h, drying temperature is raised from -30 °C to 30 °C, heating time is more than 8h, and drying time is more than 3h at 30 °C.

The invention adopts an enrichment form combining single column and multi-column series to effectively enrich the extraction liquid or concentrated clear liquid of high and low concentration chlorogenic acid, improve the dynamic adsorption capacity per unit volume of the resin, and solve the dynamic effect of the single column. The problem of adsorption leakage and the inability to continue the adsorption of the sample also solves the problem that the series column continues to pass through the sample, so that the resin additionally adsorbs impurities and reduces the adsorption amount of chlorogenic acid; and the single column selective elution method reduces the elution. The content of impurities in the liquid improves the purity of the product, and solves the problem that the resin continuously releases impurities into the eluent during the series elution, thereby reducing the purity of the product.

detailed description

EXAMPLES The apparatus for enriching purified compounds of the present invention (see Figure 1)

1. Packing column: Take several materials to prepare a number of columns with an inner diameter of 10 cm and an aspect ratio of 1:6-1:10, and connect the column to the series column in the first and last stages as shown in Fig. 1; The polystyrene resin is input into the column until the resin diameter ratio in each column of the column meets the requirements. The specific data are as follows:

Table 1 Packing Parameter Table

2, pre-treatment or regeneration treatment: 1-3 times the column volume of methanol or ethanol at a flow rate of 2-5 times a single column volume / hour through the series column wash column, and then washed with water at the same flow rate until the effluent without methanol or Ethanol solution: The specific process parameters are as follows:

Table 2 Pretreatment process parameter table

3. Loading: take the clear liquid with chlorogenic acid content of 0.5-50mg/ml, and load from the first-stage single-column feed port at a flow rate of 5-10 times single column volume/hour. The sampled effluent is not detected. Before chlorogenic acid, it is discharged from the discharge port of the column, and when chlorogenic acid is detected in the effluent, the sample effluent enters the next stage single column; the sample is loaded according to the above method until the last stage of the single column Chlorogenic acid was detected in the sample effluent, as shown in the following table:

Table 3 Loading process parameter table

4. Washing: Wash with 1-5 times the volume of single column column water at a flow rate of 2-5 times single column volume/hour through the series column. The specific parameters are as follows:

Table 4 Water washing process parameter table

5, elution: 10-60% ethanol or methanol solvent as the elution solvent, 2-5 times the single column volume / hour flow rate, in accordance with the single column sorting of the series column for single-stage column separation and elution; First order The column feed port is input with elution solvent elution, and the outflow valve of the column is opened periodically for sampling detection. When the eluent is detected without chlorogenic acid detection, the elution solvent is washed by the next-stage single-column feed port. With the above method, chlorogenic acid was not detected in the single column effluent of the last stage. The eluent is collected by the tail column discharge port; the specific parameters are as follows:

Table 5 Elution process parameter table

6. Concentration: collecting the elution effluent, recovering ethanol at 40-70 ° C, greater than 0.04 Mp under negative pressure, and concentrating to a relative density of 1.3-1.5 (25 ° C);

Table 6 Concentration process parameter table

7. Freeze-drying: The concentrated solution is freeze-dried under the following conditions: pre-freezing temperature below -30 °C, pre-freezing time is more than 1 h, drying temperature is raised from below -30 °C to 30 °C, heating time is above 8 h, at 30 °C The drying time is more than 3 hours.

Table 7 freeze-drying process parameter table

8. Results:

Table 8 Experimental results table

Comparative example:

1. Packing column: Prepare a column with a diameter ratio of 1:6 and 1:10 by using stainless steel and polypropylene; input the polystyrene resin into the column with water until the diameter of the resin in the single column is respectively For 1:4 and 1:8, the specific parameters are as follows:

Table 9 Packing Parameter Table

2, pretreatment: 1 times and 2 times the total column volume of ethanol, 2 column volume / hour flow rate through the series column wash column, and then washed with water at the same flow rate to the effluent without ethanol: the specific process parameters are shown in the table below :

Table 10 Pretreatment Process Parameter Table

3. Loading: Take the clarified liquid with chlorogenic acid content of 0.5 and 50mg/ml, and load them at 10 times and 5 times the volume of single column volume/hour respectively, and wait for the sample to flow out and add the ferric chloride test solution. In the case of light green or green reaction, stop loading. The specific process parameters are as follows:

Table 11 Sample process parameters

4. Washing: Wash with 5 times and 1 times the volume of single column column with 5 times and 2 times single column volume / hour flow rate. The specific parameters are as follows:

Table 12 Water washing process parameter table

5. Elution: elute with 60% ethanol and 10% methanol at a flow rate of 5 times and 2 times the volume of single column/hour. When the effluent is not detected by chlorogenic acid, the elution is stopped. The specific parameters are shown in the table below. :

Table 13 Elution Process Parameter Table

6. Concentration: Collect the elution effluent, recover the ethanol under the conditions of 40 ° C and 70 ° C, 0.08 Mp and 0.04 Mp under negative pressure, and concentrate to a relative density of 1.5 and 1.3 (25 ° C);

Table 14 Concentration process parameter table

Table 15 freeze-drying process parameter table

8. Results:

Table 16 Experimental Results Table

9. Conclusion

It can be seen from the experimental results that the adsorption capacity of the resin in the single column of the first column for chlorogenic acid is significantly lower than that of the specific example 1 of the present invention, and the sample purity and the chlorogenic acid transfer rate are significantly reduced; The tandem column was purified by conventional enrichment purification, and the adsorption capacity of the resin was significantly lower than that of the specific example 5 of the present invention, and the sample purity and the chlorogenic acid transfer rate were decreased.

Claims

The invention relates to a device for enriching and purifying a compound, comprising a adsorption column of a macroporous resin, a pipeline, characterized in that: a raw liquid feed pipe is connected to an inlet of a primary adsorption column (1) through a valve (11), and a primary adsorption column (1) The outlet of the secondary adsorption column (2) is connected to the inlet of the primary collection (13) and secondary adsorption column (2) through the tee and the valve (12, 14), and the outlet of the secondary adsorption column (2) is passed through the tee and the valve (22). 24) respectively connected to the inlet of the secondary collection irrigation (23) and the secondary adsorption column (3), and the outlet of the secondary adsorption column (3) through the tee and the valve (32, 34) respectively and above the secondary The inlet connection of the irrigation (33) and the lower adsorption column is collected, and the raw liquid feed pipe is also connected to the inlet of the secondary or secondary adsorption column through the valve (21, 31).
The apparatus for enriching and purifying a compound according to claim 1, wherein the adsorption column of each stage has an aspect ratio of 1:6 to 1:10.
The apparatus for enriching and purifying a compound according to claim 1, wherein the outer casing of the adsorption column and the resin support screen inside are made of stainless steel, or glass, or ceramic, or polyethylene, or polypropylene. production.
The apparatus for enriching and purifying a compound according to any one of claims 1 to 3, wherein the macroporous resin in the adsorption column has a particle size ranging from 0.2 to 1.5 mm and an aspect ratio of 1:4 to 1: 8.
A method of using the apparatus of any of claims 1-4, comprising the steps of:

a. Wet packing: Close valves 21, 31 and 14, open valves 11 and 12, input resin water or ethanol to the primary column, and the effluent is collected by the primary collection tank until the resin is filled to the aspect ratio. 1:4-1:8, stop input, close valves 12 and 11, complete the resin wet packing of the primary column; close the valve 24, open the valves 21, 22, and input the resin water or ethanol solution to the secondary column. When the resin is filled until the aspect ratio is 1:4 to 1:8, the input is stopped, the valves 22 and 21 are closed, and the resin is wet-packed for the secondary column; the wet packing of the columns of each stage is completed in sequence according to the above method. operating;

b, pre-treatment or regeneration treatment: close the valves 21, 31 and 12, 22, 32, open the valves 11, 14, 24 and 34, enter the solvent or solution, and then wash with water until the effluent has no solvent or solution, stop input, Closing valves 34 and 11;

c. Loading: Close the valves 21, 31 and 14, open the valves 11 and 12, input the sample liquid, and after the target compound is detected in the effluent at the outlet of the valve 12, open the valves 14 and 22, close the valve 12 and 24, after the target compound is detected in the effluent of the valve 22, the loading operation of each column is completed in accordance with the above method;

d, water washing: close the valves 21, 31 and 12, 22, 32, open the valves 14, 24 and 34, input water until the washing requirements are reached;

c. Elution: Close valves 21, 31 and 12, 22, 32, open valves 14, 24 and 34, input eluent, open valve 12 sampling test from time to time, when the target compound is not detected, close the valve 14 and 11, open the valve 21; continue to input the eluent, open the valve 22 sampling test from time to time, when the target compound is not detected, close the valves 24 and 21; in turn, according to the above method, complete the elution operation of each column.
A method for enriching and purifying chlorogenic acid, which comprises the apparatus according to any one of claims 1 to 4, comprising the steps of:

a. Packing column: Packing the resin in a wet packing method with a diameter to height ratio of 1:4-1:8, and connecting the first and second ends of each single column in series, the number of the series columns is 2-10;

b. Pretreatment or regeneration treatment: Wash the column with a 1-3 times serial column volume pretreatment or regeneration treatment solvent or solution at a flow rate of 2-5 times the single column volume/hour through the column, and then wash with water at the same flow rate. The effluent has no pretreatment or regeneration treatment solvent or solution;

c. Loading: sequentially sorting according to the single column of the series column, loading from the inlet of the first stage single column, before the effluent is not detected by the sample effluent, and discharging from the discharge port of the column to the effluent When chlorogenic acid is detected, the loading effluent enters the next stage single column; the chlorogenic acid is detected in the single column loading effluent until the last stage according to the above method;

d, water washing: using 1-5 times the volume of a single column of water to wash the column through the series column at a flow rate of 2-5 times a single column volume / hour;

e, elution: sequentially separate and elute according to the single column of the series column, firstly input the elution solvent from the first-stage single-column feed port, and periodically open the outflow valve of the column for sampling and detection, to be washed When the chlorogenic acid is detected in the liquid-free detection, the elution solvent is further eluted from the single-stage feed port of the next stage. According to the above method, no chlorogenic acid is detected in the single-column elution effluent of the last stage. The eluent is collected by the tail column discharge port;

f, concentration: collecting the elution effluent, recovering ethanol under low temperature and negative pressure conditions, and concentrating to a relative density of 1.3-1.5 (25 ° C);

g, lyophilization: The concentrate is dried in a freeze dryer.
The method of claim 6 wherein:

The packing resin described in step a is: a polystyrene macroporous adsorption resin having a particle size ranging from 0.2 to 1.5 mm;

The pretreatment or regeneration treatment solvent or solution described in step b: one or more of ethanol and a solution, or methanol and a solution, or acid water, or alkaline water, or water;

The sample solution described in step c is a clear liquid having a chlorogenic acid content of 0.5-30 mg/ml, and the loading flow rate is 5-10 times the single column volume/hour;

The pH of the water described in step d is 3-7;

The eluent solvent described in the e step is a 10-60% ethanol solution or a methanol solution, and the elution flow rate is 2-5 BV single column volume/hour;

The concentration conditions described in step f are: concentration temperature is 40-70 ° C, concentration vacuum degree: greater than 0.04Mp;

The lyophilization and drying conditions in the g step are: pre-freezing temperature below -30 ° C, pre-freezing time is more than 1 h, drying temperature is raised from -30 ° C to 30 ° C, heating time is more than 8 h, and drying time is 30 h at 30 ° C the above.