CN114456168B - Preparation method of high-purity caffeine - Google Patents
Preparation method of high-purity caffeine Download PDFInfo
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- CN114456168B CN114456168B CN202210064519.0A CN202210064519A CN114456168B CN 114456168 B CN114456168 B CN 114456168B CN 202210064519 A CN202210064519 A CN 202210064519A CN 114456168 B CN114456168 B CN 114456168B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
- C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
- C07D473/06—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3
- C07D473/12—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3 with methyl radicals in positions 1, 3, and 7, e.g. caffeine
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0261—Solvent extraction of solids comprising vibrating mechanisms, e.g. mechanical, acoustical
- B01D11/0265—Applying ultrasound
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
- B01D15/203—Equilibration or regeneration
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
Abstract
The invention relates to a preparation method of high-purity caffeine, which combines two different reverse phase chromatography methods and obtains a caffeine standard substance with purity higher than 98% from a tea extract by optimizing the relation between sample loading amount and purity. Because the two chromatographs have different removal effects on different impurities, the combined use of the two chromatographs can remove different types of impurities, thereby increasing the sample loading capacity, improving the preparation efficiency and ensuring that the recovery rate can also reach 85 percent. The method is simple and economic, the obtained caffeine has high purity, can be used as a reference substance for quality standard research work of tea, can also be used for high-level plant raw materials, and provides effective assistance for related scientific research work.
Description
Technical Field
The invention relates to the technical field of tea extracts, in particular to a preparation method of high-purity caffeine.
Background
Chromatography is widely used in the fields of pharmacy, chemical industry and the like in modern society as a separation means. By utilizing the principle differences of adsorption force, partition coefficient, solubility and the like, the target compound can be continuously distributed on the chromatographic column to form a separable 'color band' (the colorless substance can show different absorption peaks under a specific detector). The application fields of the chromatographic technique are wide, and the requirements on the chromatographic technique under different requirements are greatly different. The chromatographic methods with different separation mechanisms are combined and combined, so that different types of impurities around the target compound can be removed, and the purification effect is more efficient than that of a single chromatographic method.
Caffeine, a xanthine alkaloid compound, is a central nervous stimulant that temporarily drives drowsiness and restores energy. In nature, tea is one of the main sources of caffeine. However, the extracts of tea leaves are often very complex, and the traditional sublimation method for extracting caffeine has the disadvantage of high energy consumption; the traditional chromatographic separation method has limited separation capacity, and the traditional chromatographic method CN 202110628342.8 has low energy consumption, but the purity of the caffeine extracted from coffee by only a single chromatographic method is only 81.32% -87.38%, and the caffeine standard product can not be developed. There are also studies (patent application No. 201811069217.2) to prepare caffeine from camellia pollen by combining high-speed counter-current chromatography and semi-preparative chromatography; however, since no reasonable combination separation scheme is constructed, when the sample loading of the semi-preparative chromatography is only 1mg, the separation of caffeine is overloaded, and the separation effect is affected. In addition, the purity of the caffeine prepared by the method is only 94.05%, and the caffeine cannot reach the standard development grade.
Disclosure of Invention
The invention aims to provide a preparation method of high-purity caffeine, which has high chromatographic sample and high purity of the prepared caffeine.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for preparing high-purity caffeine comprises the following steps:
s1, extracting dried tea leaves with ethanol, heating and refluxing, concentrating an obtained extraction solution to be dry, adding water, dissolving by ultrasonic waves, and filtering to obtain filtrate;
s2, pumping the filtrate into a chromatographic column filled with macroporous resin for adsorption, eluting the chromatographic column after adsorption saturation, collecting eluent, concentrating to dryness, adding water for dissolution and freeze-drying to obtain a tea extract, adding water into the tea extract and performing ultrasonic dissolution to prepare a solution with the concentration of 20-70mg/mL, and centrifuging to remove precipitates to serve as a first sample injection liquid for later use;
s3, connecting a reversed phase chromatographic column to a high performance liquid chromatograph equipped with a double-channel ultraviolet detector, adjusting the wavelength of the double-channel ultraviolet detector to be 254nm and 366nm respectively, and controlling the column temperature to be 20-50 ℃;
mixing 0.05-0.2wt% formic acid water solution and 30-35wt% methanol with solvent pump of high performance liquid chromatograph as first mobile phase, and balancing the reversed phase chromatographic column; injecting 600-1200 microliters of the first sample injection liquid into a balanced reversed phase chromatographic column for separation, collecting caffeine-containing components, concentrating to dryness, adding acetonitrile for redissolution, and using the caffeine-containing components as a second sample injection liquid for later use;
mixing formic acid water solution with concentration of 0.05-0.2wt% and acetonitrile with concentration of 12-16wt% by using a solvent pump of a high performance liquid chromatograph to obtain a second mobile phase, balancing the reversed phase chromatographic column, completely injecting the second injection solution into the balanced reversed phase chromatographic column for separation, collecting components containing caffeine, concentrating and freeze-drying to obtain the high-purity caffeine.
The preparation method of the high-purity caffeine is further improved as follows:
preferably, the ratio of the materials extracted by the tea leaves with ethanol in the step S1 is 1 (2-5).
Preferably, the step S2 is performed by eluting the column with ethanol.
Preferably, the macroporous resin is HPD 300.
Preferably, the reverse phase chromatographic column is C18, the chromatographic column has the length of 250 mm, the diameter of 4.6 mm and the grain size of a packing material of 10 microns.
Preferably, the time for equilibrating the reverse phase chromatography column in step S3 is 15min or more.
Preferably, the first mobile phase in step S3 is formed by mixing 0.1wt% aqueous formic acid solution and 32wt% methanol solution through a solvent pump of a high performance liquid chromatograph.
Preferably, the second mobile phase in step S3 is formed by mixing 0.1wt% formic acid solution and 14wt% acetonitrile solution through a solvent pump of a high performance liquid chromatograph.
Preferably, the concentration of the solution prepared by dissolving the tea leaf extract in water in step S2 is 50mg/mL.
Preferably, the column temperature of the chromatography column in step S2 is controlled at 30 ℃.
Compared with the prior art, the invention has the beneficial effects that:
the invention relates to a preparation method of high-purity caffeine, which takes tea leaves as a raw material, combines two different reverse phase chromatography methods into an orthogonal chromatography method, and prepares a caffeine standard substance with the purity higher than 98% from a tea leaf extract by optimizing the relation between the sample loading amount and the purity. In the present invention, impurities around caffeine, which are difficult to remove by the first reverse phase chromatography, can be completely removed by the second reverse phase chromatography by utilizing the difference in the degree of separation and the order between the impurities and caffeine in the two reverse phase chromatographs. Compared with a single reversed-phase chromatography, the two reversed-phase chromatography methods have complementary functions on the impurity removal capacity, so that the sample loading capacity of the tea extract on an analytical C18 chromatographic column (the specification is 250 mm long, the diameter is 4.6 mm, and the filler particle size is 10 microns) is up to 60 mg/time in the chromatographic preparation process, the preparation efficiency is greatly improved, and the recovery rate can also be up to 82.33%. The purity of the caffeine obtained by the method reaches more than 98 percent, meets the development level of standard products, can be used as a reference product for the quality standard research work of tea, can also be used for high-level plant raw materials, and provides effective assistance for related scientific research work. The method is simple and economical, has high sample loading capacity, high purity and high recovery rate, and has certain industrial value.
Drawings
FIG. 1 is a chromatogram identification chart of a tea extract in example 1 of the present invention, the identification method: mobile phase: the methanol volume fraction was 32wt% methanol and 0.1wt% formic acid water mixture.
FIG. 2 is a chromatogram identification chart of the tea extract in example 1 of the present invention, the identification method: mobile phase: acetonitrile volume fraction 14wt% acetonitrile and 0.1wt% aqueous formic acid mixture.
FIG. 3 is a schematic diagram of the chromatography during the first chromatographic purification in example 1 of the present invention.
FIG. 4 is a chromatogram identification chart of a fraction obtained by the first purification in example 1 of the present invention, the identification method: mobile phase: the methanol volume fraction was 32wt% methanol and 0.1wt% formic acid water mixture.
FIG. 5 is a chromatographic representation of a second sample solution during a second chromatographic purification process in accordance with example 1 of the present invention.
FIG. 6 is a high performance liquid chromatography purity chromatogram of caffeine obtained after two purifications according to example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any creative effort based on the embodiments of the present invention belong to the protection scope of the present invention.
Example 1
a. Taking 1 kg of dried tea leaves, adding ethanol according to the material-liquid ratio of 1. The resulting solution was concentrated to dryness by rotary evaporator. Adding 1L of water, performing ultrasonic dispersion and dissolution, and filtering to obtain a filtrate;
the obtained filtrate was loaded on a macroporous resin (inner diameter 10cm, column bed height 30 cm) chromatography column of HPD 300 for adsorption. And washing the column with water as a rinsing reagent until the eluate is colorless. Eluting the macroporous resin chromatographic column with ethanol as eluting solvent, concentrating the eluate with rotary evaporator to dryness, dissolving in water, and lyophilizing to obtain brown yellow tea extract. Dissolving 100mg of the tea extract in 2mL of water, ultrasonically dissolving, centrifuging to remove precipitate, and using as a first sample injection solution for later use;
a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 micron filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min.
A0.1 wt% formic acid aqueous solution and 32wt% methanol were mixed by a solvent pump of a high performance liquid chromatograph as mobile phases, and the column was equilibrated for 15min. And (c) taking 5 microliters of the first sample solution in the step a, injecting the sample into a well-balanced chromatographic column for analysis and identification, wherein an identification chromatogram is shown in figure 1. The chromatographic peak b in fig. 1 is caffeine, four significant impurities a, c, d and e are around caffeine, especially the content of the impurity a is the highest, and the peak area ratio of the impurity a to caffeine is as high as 1:2.
mixing 0.1wt% formic acid water solution and 14wt% acetonitrile by solvent pump of high performance liquid chromatograph as mobile phase, and balancing chromatographic column for 15min. And (c) taking 5 microliters of the first sample solution in the step a, injecting the sample into the well-balanced chromatographic column for analysis and identification, wherein an identification chromatogram is shown in figure 2. In fig. 2, caffeine, impurities a, c, d and e in the tea extract are also labeled. In fig. 1, the peak of impurity a appears in front of caffeine; whereas in fig. 2 the peak of impurity a appears behind caffeine. Fig. 1 and fig. 2 show different distributions of impurities, which lay the foundation for the establishment of an orthogonal separation method. The proportion of the impurity a to the caffeine chromatographic peak in the tea extract is 1.
b. A reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 μm filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. A0.1 wt% formic acid aqueous solution and 32wt% methanol were mixed by a solvent pump of a high performance liquid chromatograph as mobile phases, and the column was equilibrated for 15min. Feeding 1200. Mu.l of the first sample solution of step a to a well-balanced reversed phase chromatographic column for separation, wherein the chromatogram is shown in FIG. 3. The chromatogram in FIG. 3 shows that the tea extract is heavily overloaded on the C18 chromatographic column, and the ultraviolet absorption of the tea extract completely exceeds the absorption value range of the instrument. Collecting the components of 8.65min-11.15min, concentrating to dryness, and dissolving again with 1mL acetonitrile to obtain a second sample solution for later use; then, the second sample solution obtained in step b is sampled into a well-balanced chromatographic column for analysis and identification, and the identification chromatogram is shown in fig. 4. Comparing fig. 1 and fig. 4, the content of impurity a in the tea extract is significantly reduced by the purification of step b, and the ratio of the peak areas of impurity a and caffeine is 1:5.
c. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 μm filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. Mixing 0.1wt% formic acid aqueous solution and 14wt% acetonitrile via solvent pump of high performance liquid chromatograph as mobile phase, and balancing chromatographic column for 15min. And c, feeding the second sample injection liquid in the step b into a well-balanced chromatographic column for separation, wherein a chromatogram of the chromatogram is shown in fig. 5, and the chromatogram of fig. 5 shows that the second sample injection liquid is completely overloaded on the chromatographic column and all chromatographic peaks exceed the absorption range of the ultraviolet detector. Collecting the fractions of 7.5-11.5 min, concentrating, and lyophilizing to obtain white solid caffeine 15.6mg.
The purity of caffeine was identified using high performance liquid chromatography, and the results are shown in fig. 6, and the chromatogram of fig. 6 shows that the purity of caffeine is as high as 98.8%. The overall caffeine recovery was calculated to be 82.33%.
Example 2
a. Taking 1 kg of dried tea leaves, adding ethanol according to the material-liquid ratio of 1. The resulting solution was concentrated to dryness by rotary evaporator. Adding 1L of water, performing ultrasonic dispersion and dissolution, and filtering to obtain a filtrate;
the obtained filtrate was loaded on a macroporous resin (inner diameter 10cm, column bed height 30 cm) chromatography column of HPD 300 for adsorption. And washing the column with water as a rinsing agent until the eluate is colorless. Eluting the macroporous resin chromatographic column with ethanol as eluting solvent, concentrating the eluate with rotary evaporator to dryness, dissolving in water, and lyophilizing to obtain brown yellow tea extract. Dissolving 100mg of the tea extract in 5mL of water, ultrasonically dissolving, and centrifuging to remove precipitate to obtain a first sample injection solution for later use;
b. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 micron filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. A0.05 wt% formic acid aqueous solution and 32wt% methanol were mixed by a solvent pump of a high performance liquid chromatograph as mobile phases, and the column was equilibrated for 15min. B, feeding 600 microliters of the first sample solution obtained in the step a to a well-balanced reversed-phase chromatographic column for separation, collecting a component containing caffeine, concentrating to dryness, and redissolving with 1mL of acetonitrile to serve as a second sample solution for later use;
c. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 micron filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. A0.05 wt% formic acid aqueous solution and 14wt% acetonitrile were mixed by a solvent pump of a high performance liquid chromatograph as mobile phases, and the column was equilibrated for 15min. And c, injecting the second sample injection liquid in the step b into a well-balanced chromatographic column for separation, collecting components containing caffeine, concentrating, and freeze-drying to obtain 8.0mg of white solid caffeine.
The purity of the caffeine is identified by high performance liquid chromatography, and the purity is as high as 99%. The total caffeine recovery rate is calculated to reach 85%.
Example 3
a. Taking 1 kg of dried tea leaves, adding ethanol according to the material-liquid ratio of 1. The resulting solution was concentrated to dryness by rotary evaporator. Adding 1L of water, performing ultrasonic dispersion and dissolution, and filtering to obtain a filtrate;
the obtained filtrate was loaded on a macroporous resin (inner diameter 10cm, column bed height 30 cm) chromatography column of HPD 300 for adsorption. And washing the column with water as a rinsing reagent until the eluate is colorless. Eluting the macroporous resin chromatographic column with ethanol as eluting solvent, concentrating the eluate with rotary evaporator to dryness, dissolving in water, and lyophilizing to obtain brown yellow tea extract. Dissolving 100mg of the tea extract in 1.42mL of water, ultrasonically dissolving, and centrifuging to remove precipitate to obtain a first sample solution for later use;
b. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 μm filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. Mixing 0.2wt% formic acid water solution and 30wt% methanol with solvent pump of high performance liquid chromatograph as mobile phase, and balancing chromatographic column for 15min. Feeding 900 microliters of the first sample solution obtained in the step a to a well-balanced reversed-phase chromatographic column for separation, collecting a component containing caffeine, concentrating to dryness, and redissolving with 1mL of acetonitrile to serve as a second sample solution for later use;
c. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 μm filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. Mixing 0.2wt% formic acid water solution and 14wt% acetonitrile by solvent pump of high performance liquid chromatograph as mobile phase, and balancing chromatographic column for 15min. And c, injecting the second sample injection liquid in the step b into a well-balanced chromatographic column for separation, collecting components containing caffeine, concentrating, and freeze-drying to obtain 14.8mg of caffeine in white solid state.
The purity of the caffeine is identified by high performance liquid chromatography, and the purity is as high as 98.9%. The overall caffeine recovery was calculated to be 81.2%.
Example 4
a. Taking 1 kg of dried tea leaves, adding ethanol according to the material-liquid ratio of 1. The resulting solution was concentrated to dryness by rotary evaporator. Adding 1L of water, performing ultrasonic dispersion and dissolution, and filtering to obtain a filtrate;
the obtained filtrate was loaded on a macroporous resin (inner diameter 10cm, column bed height 30 cm) chromatography column of HPD 300 for adsorption. And washing the column with water as a rinsing agent until the eluate is colorless. Eluting the macroporous resin chromatographic column with ethanol as eluting solvent, concentrating the eluate with rotary evaporator to dryness, dissolving in water, and lyophilizing to obtain brown yellow tea extract. Dissolving 100mg of the tea extract in 2mL of water, ultrasonically dissolving, centrifuging to remove precipitate, and using as a first sample injection solution for later use;
b. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 μm filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. A0.1 wt% formic acid aqueous solution and a 30wt% methanol aqueous solution were mixed by a solvent pump of a high performance liquid chromatograph as mobile phases, and the column was equilibrated for 15min. Feeding 1200 microliters of the first sample solution of step a onto a well-balanced reverse phase chromatography column for separation, collecting a component containing caffeine, concentrating to dryness, re-dissolving with 1mL of acetonitrile, and using the re-dissolved component as a second sample solution for later use;
c. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 micron filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. Mixing 0.1wt% formic acid water solution and 12wt% acetonitrile by solvent pump of high performance liquid chromatograph as mobile phase, and balancing chromatographic column for 15min. And c, injecting the second sample injection liquid in the step b into a balanced chromatographic column for separation, collecting components containing caffeine, concentrating, and freeze-drying to obtain 14.0mg of caffeine in white solid.
The purity of the caffeine is identified by high performance liquid chromatography, and the purity is as high as 98.9%. The overall caffeine recovery was calculated to be 80.0%.
Example 5
a. Taking 1 kg of dried tea leaves, adding ethanol according to the material-liquid ratio of 1. The resulting solution was concentrated to dryness by rotary evaporator. Adding 1L of water, performing ultrasonic dispersion and dissolution, and filtering to obtain a filtrate;
the obtained filtrate was loaded on a macroporous resin (inner diameter 10cm, column bed height 30 cm) chromatography column of HPD 300 for adsorption. And washing the column with water as a rinsing agent until the eluate is colorless. Eluting the macroporous resin chromatographic column with ethanol as eluting solvent, concentrating the eluate with rotary evaporator to dryness, dissolving in water, and lyophilizing to obtain brown yellow tea extract. Dissolving 100mg of the tea extract in 2mL of water, ultrasonically dissolving, and centrifuging to remove precipitate to obtain a first sample injection solution for later use;
b. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 μm filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. Mixing 0.1wt% formic acid solution and 35wt% methanol solution with solvent pump of high performance liquid chromatograph as mobile phase, and balancing chromatographic column for 15min. Feeding 1200 microliters of the first sample solution of step a onto a well-balanced reverse phase chromatography column for separation, collecting a component containing caffeine, concentrating to dryness, re-dissolving with 1mL of acetonitrile, and using the re-dissolved component as a second sample solution for later use;
c. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 μm filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. Mixing 0.1wt% formic acid water solution and 12wt% acetonitrile by solvent pump of high performance liquid chromatograph as mobile phase, and balancing chromatographic column for 15min. And c, injecting the second sample injection liquid in the step b into a well-balanced chromatographic column for separation, collecting components containing caffeine, concentrating, and freeze-drying to obtain 11.0mg of caffeine in white solid state.
The purity of the caffeine is identified by high performance liquid chromatography, and the purity is as high as 98.5%. The overall caffeine recovery was calculated to be 75.0%.
Example 6
a. Taking 1 kg of dried tea leaves, adding ethanol according to the material-liquid ratio of 1. The resulting solution was concentrated to dryness by rotary evaporator. Adding 1L of water, performing ultrasonic dispersion and dissolution, and filtering to obtain a filtrate;
the obtained filtrate was loaded on a macroporous resin (inner diameter 10cm, column bed height 30 cm) chromatography column of HPD 300 for adsorption. And washing the column with water as a rinsing reagent until the eluate is colorless. Eluting macroporous resin chromatographic column with ethanol as eluting solvent, concentrating the eluate with rotary evaporator to dryness, dissolving in water, and lyophilizing to obtain brown yellow tea extract. Dissolving 100mg of the tea extract in 2mL of water, ultrasonically dissolving, and centrifuging to remove precipitate to obtain a first sample injection solution for later use;
b. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 μm filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. A0.1 wt% formic acid aqueous solution and 32wt% methanol were mixed by a solvent pump of a high performance liquid chromatograph as mobile phases, and the column was equilibrated for 15min. Feeding 1200 microliters of the first sample solution of step a onto a well-balanced reverse phase chromatography column for separation, collecting a component containing caffeine, concentrating to dryness, re-dissolving with 1mL of acetonitrile, and using the re-dissolved component as a second sample solution for later use;
c. a reversed phase C18 column (specification 250 mm long, 4.6 mm diameter, 10 μm filler particle size) was connected to a hplc equipped with a dual channel uv detector. The wavelength of the dual-channel ultraviolet detector is adjusted to 254nm and 366nm, the column temperature is controlled at 30 ℃, and the flow rate is controlled at 1.0mL/min. Mixing 0.1wt% formic acid aqueous solution and 16wt% acetonitrile via solvent pump of high performance liquid chromatograph as mobile phase, and balancing chromatographic column for 15min. And c, injecting the second sample injection liquid in the step b into a well-balanced chromatographic column for separation, collecting components containing caffeine, concentrating, and freeze-drying to obtain 11.0mg of caffeine in white solid state.
The purity of the caffeine is identified by high performance liquid chromatography, and the purity is as high as 98.8%. The overall caffeine recovery was calculated to be 76.0%.
As can be seen from the above examples, the recovery of caffeine is related to the amount of the tea extract loaded in step a, the methanol content in the mobile phase in step b and the acetonitrile content in the mobile phase in step c. The higher the content of methanol in the mobile phase in step b and acetonitrile in the mobile phase in step c, the higher the elution strength of the mobile phase and the lower the caffeine recovery rate.
It should be understood by those skilled in the art that the foregoing is only illustrative of several embodiments of the invention, and is not an exhaustive list. It should be noted that many variations and modifications are possible to those skilled in the art, and all variations and modifications that do not depart from the gist of the invention are intended to be within the scope of the invention as defined in the appended claims.
Claims (8)
1. A method for preparing high-purity caffeine, comprising the steps of:
s1, extracting dried tea leaves with ethanol, heating and refluxing, concentrating an obtained extraction solution to dryness, adding water, dissolving by ultrasonic waves, and filtering to obtain a filtrate;
s2, pumping the filtrate into a chromatographic column filled with macroporous resin for adsorption, eluting the chromatographic column by using ethanol after adsorption saturation, collecting eluent, concentrating to dry, adding water for dissolution and freeze-drying to obtain a tea extract, adding water into the tea extract and performing ultrasonic dissolution to prepare a solution with the concentration of 20-70mg/mL, and centrifuging to remove precipitates to serve as a first sample injection liquid for later use;
s3, connecting a reversed phase chromatographic column to a high performance liquid chromatograph provided with a dual-channel ultraviolet detector, adjusting the wavelengths of the dual-channel ultraviolet detector to 254nm and 366nm respectively, and controlling the column temperature to be 20-50 ℃; the model of the reversed phase chromatographic column is C18, the chromatographic column is 250 mm long, 4.6 mm in diameter and 10 microns in filler particle size;
mixing 0.05-0.2wt% formic acid water solution and 30-35wt% methanol with solvent pump of high performance liquid chromatograph as first mobile phase, and balancing the reversed phase chromatographic column; injecting 600-1200 microliter of first injection liquid into a balanced reversed phase chromatographic column for separation, collecting components containing caffeine, concentrating to dry, adding acetonitrile for redissolving, and using as second injection liquid for later use;
mixing formic acid water solution with concentration of 0.05-0.2wt% and acetonitrile with concentration of 12-16wt% by using a solvent pump of a high performance liquid chromatograph to obtain a second mobile phase, balancing the reversed phase chromatographic column, completely injecting the second injection solution into the balanced reversed phase chromatographic column for separation, collecting components containing caffeine, concentrating and freeze-drying to obtain the high-purity caffeine.
2. The method for preparing high-purity caffeine according to claim 1, wherein the ethanol-extraction ratio of the tea leaves in step S1 is 1 (2-5).
3. The method for preparing high purity caffeine according to claim 1, wherein the macroporous resin is HPD 300.
4. The method of claim 1, wherein the time for equilibrating the reverse phase column in step S3 is 15min or more.
5. The method for preparing high purity caffeine according to claim 1, wherein the first mobile phase in step S3 is formed by mixing an aqueous solution of formic acid having a concentration of 0.1wt% and methanol having a concentration of 32wt% using a solvent pump of a high performance liquid chromatograph.
6. The method of claim 1, wherein the second mobile phase in step S3 is prepared by mixing an aqueous solution of formic acid having a concentration of 0.1wt% and acetonitrile having a concentration of 14wt% by using a solvent pump of a high performance liquid chromatograph.
7. The method of preparing high purity caffeine according to claim 1, wherein the concentration of the aqueous solution of the tea extract prepared in step S2 is 50mg/mL.
8. The method for preparing high purity caffeine according to claim 1, wherein the column temperature of the chromatography column in step S2 is controlled to 30 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CA126623A (en) * | 1910-02-08 | 1910-06-28 | Robert Hubner | Process of extracting caffeine from coffee |
| ZA757059B (en) * | 1974-11-27 | 1976-11-24 | Nestle Sa | Decaffeination process |
| EP0010636A1 (en) * | 1978-11-06 | 1980-05-14 | Societe Des Produits Nestle S.A. | Caffeine extraction |
| CN1301252C (en) * | 2004-11-25 | 2007-02-21 | 三达膜科技（厦门）有限公司 | Production method of high purity tea polyphenol and caffeine |
| CN109111444B (en) * | 2018-09-13 | 2021-06-15 | 集美大学 | Method for separating and purifying caffeine from camellia pollen |
| CN113336755B (en) * | 2021-06-04 | 2023-03-28 | 北京理工大学 | Method for separating and purifying trigonelline, chlorogenic acid and caffeine |
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