A method for separating and purifying anthocyanins by using a continuous macroporous resin chromatography system

Abstract

This invention provides a method for separating and purifying anthocyanins using a continuous macroporous resin chromatography system, belonging to the field of separation and purification technology. This invention utilizes a continuous macroporous resin chromatography system for the separation and purification of anthocyanins. The sample injection zone, water washing zone, ethanol stripping zone, resin regeneration zone, and water wash solution recovery zone do not interfere with each other and can operate simultaneously at room temperature, avoiding anthocyanin decomposition and significantly improving the separation and purification efficiency. It enables continuous, large-scale separation and purification of anthocyanins, achieving good separation and purification effects, high purity enhancement, and high yield. This invention uses ethanol and water as eluents, which are non-destructive to the compounds to be analyzed and purified, exhibiting low reactivity and toxicity, and are abundant and inexpensive solvents. Furthermore, this invention allows for the rapid processing of large quantities of anthocyanin samples while recovering ethanol, achieving greater efficiency with lower costs.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology, specifically to a method for separating and purifying anthocyanins using a continuous macroporous resin chromatography system. Background Technology

[0002] Anthocyanins are glycoside derivatives with the structural formula shown in Formula I. They possess antioxidant properties and can help prevent cancer, making them widely used in food, dyes, pharmaceuticals, and cosmetics. However, anthocyanins often exist as a mixture with impurities, making them difficult to separate.

[0003]

[0004] Purifying anthocyanins using macroporous resins is a common method, as seen in studies such as "Sun Qianyi, Cai Enbo, Xia Yuting, et al. Study on the purification process of anthocyanins from blueberries using macroporous resins [J]. Chinese Traditional and Herbal Drugs, 2017, 48(22):7.", "Wu Fan, Wang Zhong. Study on the extraction and purification process of anthocyanins from dried blueberries [J]. Shandong Chemical Industry, 2021.", and "Zhang Panpan, Jia Chengtong, Wang Li. Study on the purification of anthocyanins from blueberry pomace using macroporous resins [J]. Journal of Anhui University of Science and Technology, 2017, 31(2):7.". However, existing methods for purifying anthocyanins using macroporous resins rely on single macroporous resin columns, resulting in low purification efficiency and hindering continuous, large-scale purification of anthocyanins. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for separating and purifying anthocyanins using a continuous macroporous resin chromatography system. The method provided by this invention enables continuous, large-scale purification of anthocyanins with high purification efficiency.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for separating and purifying anthocyanins using a continuous macroporous resin chromatography system. The continuous macroporous resin chromatography system includes: an injection zone, a water washing zone, an ethanol elution zone, a resin regeneration zone, and a water wash recovery zone. The injection zone consists of three columns connected in series; the water washing zone consists of two columns connected in series; the ethanol elution zone consists of two columns connected in series; the resin regeneration zone consists of two columns connected in series; and the water wash recovery zone consists of three columns. The injection zone, water washing zone, ethanol elution zone, resin regeneration zone, and water wash recovery zone can be operated continuously and simultaneously by switching the columns. Each column is a macroporous resin column.

[0008] The method includes the following steps: loading anthocyanin sample solution, washing with water to remove impurities, ethanol analysis, resin regeneration, and recycling of the washing solution; the ethanol analysis yields anthocyanins; the recycling of the washing solution involves purifying the washing solution obtained from the water washing process to obtain anthocyanins.

[0009] Preferably, the continuous macroporous resin chromatography system includes 12 columns, numbered 1 to 12, wherein columns 1 to 9 are used in a cycle as the injection zone, water washing zone, ethanol desorption zone and resin regeneration zone, and columns 10 to 12 are used as the water washing solution recovery zone.

[0010] The method specifically includes the following steps:

[0011] (1) Use a pump to pump the anthocyanin raw material solution into columns No. 1, No. 2 and No. 3 connected in series until column No. 1 is saturated with sample, and columns No. 2 and No. 3 are used as guard columns.

[0012] (2) Water is pumped into column 1 for washing, and the washing solution is directly introduced into column 10; columns 2, 3 and 4 are injected in series until column 2 is saturated with sample.

[0013] (3) Columns 1 and 2 are washed in series with water, and the washing solution is directly introduced into column 10; columns 3, 4 and 5 are injected in series until column 3 is saturated with sample.

[0014] (4) Ethanol was used to elute column 1 and anthocyanins were collected at the bottom of column 1; columns 2 and 3 were washed in series with water and the washing solution was directly introduced into column 11; columns 4, 5 and 6 were injected in series and column 10 was washed with pure water.

[0015] (5) Columns 1 and 2 are connected in series and eluted with ethanol to obtain anthocyanins; Columns 3 and 4 are connected in series and washed with water, and the washing solution is directly introduced into column 11; Columns 5, 6 and 7 are connected in series and injected, and column 10 is eluted with ethanol, and anthocyanins are collected at the bottom of column 10.

[0016] (6) Regenerate column 1 by rinsing with water, desorb column 2 and column 3 in series with ethanol to obtain anthocyanins; wash column 4 and column 5 in series with water, and the washing solution directly enters column 12; inject column 6, column 7 and column 8 in series, wash column 11 with water, wash column 10 with ethanol, and recover and reuse the ethanol solution collected at the bottom of column 10.

[0017] (7) Columns 1 and 2 are regenerated by water washing, columns 3 and 4 are desorbed by ethanol in series, columns 5 and 6 are washed by water in series, the washing solution is directly introduced into column 12, columns 7, 8 and 9 are injected in series, column 1 is regenerated when the injection is finished, column 10 is regenerated by water, column 11 is desorbed by ethanol, and anthocyanins are collected at the bottom of column 11.

[0018] (8) Columns 2 and 3 are regenerated in series, columns 4 and 5 are eluted in series, columns 6 and 7 are washed with water in series, the water wash solution is reintroduced into column 10, column 11 is washed with ethanol and the ethanol wash solution collected at the bottom of column 11 is recovered and reused, column 12 is washed with ethanol to obtain anthocyanins, and columns 8, 9 and 1 are injected in series.

[0019] Preferably, the macroporous resin column comprises a DM21D macroporous resin column;

[0020] Preferably, the pressure of the continuous macroporous resin chromatography system is <0.5 MPa.

[0021] Preferably, the purification includes sample loading, water washing to remove impurities, ethanol elution, and resin regeneration.

[0022] Preferably, the flow rate for each loading is 3–4 BV / h.

[0023] Preferably, the anthocyanin purity in the anthocyanin sample solution is <5%, and the solvent is water.

[0024] Preferably, the flow rate of the washing material in each wash is 0.6 to 1 BV / h.

[0025] Preferably, the flow rate of each ethanol elution is 1.3 to 1.7 BV / h, the volume fraction of the ethanol solution used in the ethanol elution is 70 to 80%, and the amount of pure ethanol used per liter of resin per hour during each ethanol elution process is 0.12 to 0.16 L.

[0026] Preferably, the resin regeneration method is water washing; the flow rate for each resin regeneration is 0.6 to 1 BV / h.

[0027] This invention utilizes a continuous macroporous resin chromatography system for the separation and purification of anthocyanins. The sample injection zone, water washing zone, ethanol stripping zone, resin regeneration zone, and water wash solution recovery zone operate simultaneously at room temperature without interference, preventing anthocyanin decomposition and significantly improving the separation and purification efficiency. It enables continuous, large-scale separation and purification of anthocyanins, achieving excellent separation and purification results with a high purity increase and high yield. This invention uses ethanol and water as eluents, which are non-destructive to the compounds being analyzed and purified, exhibiting low reactivity and toxicity, and are abundant and inexpensive solvents. Furthermore, this invention allows for the rapid processing of large quantities of anthocyanin samples while simultaneously recovering ethanol, achieving greater efficiency with lower costs.

[0028] Furthermore, this invention employs a DM21D macroporous resin column for continuous chromatographic separation and purification of anthocyanins. The DM21D macroporous resin column can be saturated with sample loading, and the DM21D macroporous resin has high utilization. The continuous macroporous resin chromatography system has low pressure and a high safety factor, which further improves the separation and purification efficiency, purity, and yield of anthocyanins, while reducing costs. Attached Figure Description

[0029] Figure 1 The overall process flow diagram for separating and purifying anthocyanins using a continuous macroporous resin chromatography system;

[0030] Figure 2 The flowchart is for step (1) of continuous chromatographic separation and purification.

[0031] Figure 3 The flowchart is for step (2) of continuous chromatographic separation and purification.

[0032] Figure 4 The flowchart is for step (3) of continuous chromatographic separation and purification.

[0033] Figure 5 The flowchart is for step (4) of continuous chromatographic separation and purification.

[0034] Figure 6 The flowchart is for step (5) of continuous chromatographic separation and purification.

[0035] Figure 7 The flowchart is for the continuous chromatographic separation and purification step (6);

[0036] Figure 8 The flowchart is for the continuous chromatographic separation and purification step (7);

[0037] Figure 9 The HPLC chromatogram is for anthocyanin standards.

[0038] Figure 10 The HPLC chromatogram of the anthocyanin raw material solution used in Example 1;

[0039] Figure 11 The image shows the HPLC chromatogram of the anthocyanin desorption solution obtained from the separation and purification in Example 1. Detailed Implementation

[0040] This invention provides a method for separating and purifying anthocyanins using a continuous macroporous resin chromatography system. The continuous macroporous resin chromatography system includes: an injection zone, a water washing zone, an ethanol elution zone, a resin regeneration zone, and a water wash recovery zone. The injection zone consists of three columns connected in series; the water washing zone consists of two columns connected in series; the ethanol elution zone consists of two columns connected in series; the resin regeneration zone consists of two columns connected in series; and the water wash recovery zone consists of three columns. The injection zone, water washing zone, ethanol elution zone, resin regeneration zone, and water wash recovery zone can be operated continuously and simultaneously by switching the columns. Each column is a macroporous resin column.

[0041] The method includes the following steps: loading anthocyanin sample solution, washing with water to remove impurities, ethanol analysis, resin regeneration, and recycling of the washing solution; the ethanol analysis yields anthocyanins; the recycling of the washing solution involves purifying the washing solution obtained from the water washing process to obtain anthocyanins.

[0042] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0043] The process flow diagram of the method for separating and purifying anthocyanins using a continuous macroporous resin chromatography system provided by this invention is as follows: Figure 1 As shown, the specific steps are as follows: Figures 2-8 As shown below, in conjunction with Figures 1 to 8 A detailed description is provided of the method for separating and purifying anthocyanins using a continuous macroporous resin chromatography system.

[0044] In this invention, the continuous macroporous resin chromatography system includes 12 columns, numbered 1 to 12. Columns 1 to 9 are used in rotation as the sample injection zone, water washing zone, ethanol desorption zone, and resin regeneration zone, while columns 10 to 12 are used as the water washing solution recovery zone.

[0045] The method for separating and purifying anthocyanins using a continuous macroporous resin chromatography system provided by this invention specifically includes the following steps:

[0046] (1) Use a pump to draw the anthocyanin raw material solution into columns 1, 2, and 3 connected in series until column 1 is saturated. Columns 2 and 3 serve as guard columns. Figure 2 As shown;

[0047] (2) Water is pumped into column 1 for washing, and the washing solution is directly introduced into column 10; columns 2, 3, and 4 are injected in series until column 2 is saturated with sample, such as... Figure 3 As shown;

[0048] (3) Columns 1 and 2 are washed in series with water, and the washing solution is directly introduced into column 10; columns 3, 4, and 5 are injected in series until column 3 is saturated with sample, such as... Figure 4 As shown;

[0049] (4) Ethanol was used to elute the sample from column 1, and anthocyanins were collected at the bottom of column 1; columns 2 and 3 were washed in series with water, and the washing solution was directly introduced into column 11; columns 4, 5, and 6 were injected in series, and column 10 was washed with pure water. Figure 5 As shown;

[0050] (5) Columns 1 and 2 were connected in series and elute with ethanol to obtain anthocyanins; columns 3 and 4 were connected in series and washed with water, the washing solution was directly introduced into column 11; columns 5, 6, and 7 were connected in series for injection, and column 10 was elute with ethanol. Anthocyanins were collected at the bottom of column 10. Figure 6 As shown;

[0051] (6) Regenerate column 1 by rinsing with water; elute columns 2 and 3 in series with ethanol to obtain anthocyanins; wash columns 4 and 5 in series with water, and directly inject the wash solution into column 12; inject columns 6, 7, and 8 in series; wash column 11 with water; wash column 10 with ethanol; recover and reuse the ethanol solution collected at the bottom of column 10, such as... Figure 7 As shown;

[0052] (7) Columns 1 and 2 are regenerated by water washing in series; columns 3 and 4 are regenerated by ethanol elution in series; columns 5 and 6 are regenerated by water washing in series, and the washing solution is directly introduced into column 12. Columns 7, 8, and 9 are injected in series. At the end of the injection, column 1 is regenerated. Column 10 is regenerated by water, and column 11 is eluted with ethanol. Anthocyanins are collected at the bottom of column 11. Figure 8 As shown;

[0053] (8) Columns 2 and 3 are regenerated in series, columns 4 and 5 are eluted in series, columns 6 and 7 are washed with water in series, the washing solution is re-entered into column 10, column 11 is washed with ethanol and desorbed, the ethanol solution collected at the bottom of column 11 (almost no impurities and anthocyanins) is recovered and reused, column 12 is desorbed with ethanol to obtain anthocyanins, and columns 8, 9 and 1 are injected in series.

[0054] In this invention, the macroporous resin column preferably includes a DM21D macroporous resin column. This invention does not have a special limitation on the specifications of the macroporous resin column, which can be determined according to actual needs. In a specific embodiment of this invention, the specifications of the macroporous resin column are 2.4×75cm.

[0055] In this invention, the pressure of the continuous macroporous resin chromatography system is preferably <0.5 MPa.

[0056] In this invention, the purification preferably includes sample loading, water washing to remove impurities, ethanol elution, and resin regeneration.

[0057] In this invention, the purity of the anthocyanin sample solution is preferably <5%, more preferably 1-4%, and even more preferably 2-3%; the solvent in the anthocyanin sample solution is preferably water.

[0058] In this invention, the flow rate for each sample loading is preferably 3 to 4 BV / h, more preferably 3.3 to 3.7 BV / h; the sample loading amount for each loading is preferably 2 to 2.4 L, more preferably 2 to 2.2 L.

[0059] In this invention, the flow rate of each water wash is preferably 0.6 to 1 BV / h, more preferably 0.75 to 0.85 BV / h; the water volume of each water wash is preferably 0.4 to 0.55 L, more preferably 0.45 L.

[0060] In this invention, the flow rate of each ethanol eluent is preferably 1.3–1.7 BV / h, more preferably 1.4–1.5 BV / h; the volume fraction of the ethanol solution used in the ethanol eluent is preferably 70–80%, more preferably 75%; the amount of pure ethanol used per liter of resin per day during the ethanol eluent process is preferably 2.9–3.8 L, more preferably 3.1–3.5 L; and the amount of pure ethanol used per liter of resin per hour during each ethanol eluent process is preferably 0.12–0.16 L, more preferably 0.13–0.15 L.

[0061] In this invention, the resin regeneration method is preferably water washing, and the flow rate for each resin regeneration is preferably 0.6 to 1 BV / h, more preferably 0.75 to 0.85 BV / h; the water volume for each resin regeneration is preferably 0.4 to 0.55 L, more preferably 0.5 L.

[0062] To further illustrate the present invention, the method for separating and purifying anthocyanins using a continuous macroporous resin chromatography system is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] Continuous macroporous resin chromatography system: 12 DM21D macroporous resin columns (2.4×75cm) were used, designated as columns 1 to 12. Among them, 3 DM21D macroporous resin columns in series were used for sample loading, 2 DM21D macroporous resin columns in series were used for water washing to remove impurities, 2 DM21D macroporous resin columns in series were used for ethanol elution, 2 DM21D macroporous resin columns in series were used for resin regeneration, and 3 DM21D macroporous resin columns in series were used for water washing and recovery. Throughout the separation and purification process, the pressure of the continuous macroporous resin chromatography system was <0.5MPa.

[0065] The purification flowchart of anthocyanins is as follows: Figure 1As shown, the process involves a cycle of injection-washing-elution-regeneration, with reprocessing after washing. The flow rate for each injection is 3.67 BV / h, the flow rate for each wash is 0.75 BV / h, the flow rate for each alcohol wash (regeneration) is 0.834 BV / h, and the flow rate for each ethanol eluent eluent eluent is 1.5 BV / h. The daily water consumption per liter of macroporous resin is 5.875 L (including pure water in the ethanol solution, calculated based on 75% ethanol solution at a flow rate of 1.5 BV / h), and the hourly water consumption per liter of macroporous resin is 0.245 L (including pure water in the ethanol solution, calculated based on 75% ethanol solution at a flow rate of 1.5 BV / h). The washing solution from the washing material enters the recovery zone (i.e., the washing solution is loaded onto the column in the recovery zone, 900 mL) for a total of 4 hours. The sample loading zone only runs for 2 hours. This means the sample loading zone can run 12 times a day, the washing zone 12 times, the elution zone 12 times, and the regeneration zone 12 times. However, the sample loading (washing solution enters the recovery zone) in the recovery zone takes 4 hours and only runs 6 times a day. The washing, elution, and regeneration processes in the recovery zone also run 6 times a day. (The number of sample loadings per day is the same as the number of washing, elution, and regeneration processes per day. If there are 12 sample loadings per day, then there are also 12 washing, elution, and regeneration processes per day. If there are 6 sample loadings per day in the recovery zone, then there are also 6 washing, elution, and regeneration processes per day in the recovery zone.) The specific steps are as follows:

[0066] (1) Use a pump to draw the anthocyanin raw material solution (purity 3%) into columns 1, 2, and 3 connected in series until column 1 is saturated with the sample, thus improving resin utilization. A total of 2.2 L of material was fed. Columns 2 and 3 were used as guard columns. See [link to instructions]. Figure 2 As shown.

[0067] (2) 0.45 L of water is pumped into column 1 for washing. This wash solution contains a large amount of impurities and some anthocyanins. To improve the yield, this wash solution is directly flushed into column 10 for further anthocyanin recovery. This process is required for all subsequent wash solutions. Columns 2, 3, and 4 are injected in series until column 2 is saturated with sample. 2.2 L of sample is injected. See [link to relevant documentation]. Figure 3 As shown.

[0068] (3) Columns 1 and 2 were washed in series with water. 0.45 L of the washing solution was directly injected into column 10. Columns 3, 4, and 5 were injected in series until column 3 was saturated with sample. See [link to documentation]. Figure 4 As shown.

[0069] (4) Anthocyanins were elute in column 1 with 0.9 L of ethanol. Qualified anthocyanins were collected at the bottom of column 1. Columns 2 and 3 were washed in series with water, and the wash solution was directly injected into column 11. Columns 4, 5, and 6 were injected in series with 2.2 L of ethanol. Column 10 was rinsed with 0.45 L of pure water. See [link to article]. Figure 5 As shown.

[0070] (5) Columns 1 and 2 were connected in series, and qualified anthocyanins were extracted using 0.9 L of ethanol. Columns 3 and 4 were connected in series, and 0.45 L of water was used for washing. The washing solution was directly injected into column 11. Columns 5, 6, and 7 were connected in series, and 2.2 L of sample was injected. Anthocyanins in column 10 were extracted using 0.9 L of ethanol. The eluent was collected at the bottom of column 10. See [link to table]. Figure 6 As shown.

[0071] (6) Rinse and regenerate column 1 with 0.5L of pure water. Columns 2 and 3 are connected in series. Extract anthocyanins with 0.9L of ethanol. Wash columns 4 and 5 in series with 0.45L of water. The wash solution is directly injected into column 12. Inject 2.2L of the solution into columns 6, 7, and 8 in series. Wash column 11 with 0.45L of pure water and column 10 with 0.9L of ethanol. The ethanol solution collected at the bottom of column 10 is recycled to reduce ethanol consumption. See [link to relevant documentation]. Figure 7 Show.

[0072] (7) Columns 1 and 2 were regenerated by water washing in series; columns 3 and 4 were elute with ethanol in series; columns 5 and 6 were washed with 0.45 L of water in series, and the washing solution was directly injected into column 12; columns 7, 8, and 9 were injected in series. At the end of the injection, column 1 was also regenerated. Column 10 was regenerated with 0.5 L of pure water; anthocyanins were eluted from column 11 with 0.9 L of ethanol, and the anthocyanins were collected at the bottom of column 11. (See...) Figure 8 As shown.

[0073] (8) Columns 2 and 3 are regenerated in series, columns 4 and 5 are eluted in series, columns 6 and 7 are washed with water in series, the water wash solution is injected into column 10 again, and 0.9L of ethanol is washed into column 11. The resulting ethanol wash solution contains almost no impurities and anthocyanins and can be recycled; column 12 is eluted with ethanol to remove anthocyanins, and columns 8, 9 and 1 are injected in series.

[0074] Table 1 shows the solution volume used in each zone during the continuous purification of anthocyanins.

[0075] Table 1 Solution usage for each zone of continuous chromatography

[0076]

[0077] Note:

[0078] Washing material: 1 ～ Water consumption during the washing of column No. 9;

[0079] Water-washed alcohol: 1 ～ Water consumption during the regeneration of column No. 9;

[0080] Protects against water washing: 10 ～ Water consumption during the washing of column No. 12;

[0081] Ethanol analysis of guard column: 10 ～ Ethanol usage during column 12 analysis;

[0082] Guard column washed with ethanol: 10 ～ Water consumption during regeneration of column 12.

[0083] Continuous purification of anthocyanins involves switching macroporous resin columns as described above, continuously operating five zones (sample loading, water washing for impurity removal, ethanol elution, resin regeneration, and water washing solution recovery). Each zone operates independently and can be performed simultaneously.

[0084] Anthocyanin standards and anthocyanins prepared according to this invention were detected using HPLC. The HPLC detection conditions are as follows:

[0085] Mobile phase A: 10% (v / v) formic acid aqueous solution; Mobile phase B: formic acid: purified water: acetonitrile: methanol (v / v) = 40:160:90:90; Mobile phase flow rate: 1 mL / min; Column: Nucifera C185um 4.6×250 mm; Detection wavelength: 535 nm; Gradient elution program is shown in Table 2.

[0086] Table 2 Gradient elution program

[0087]

[0088] Note: HPLC can only detect anthocyanin spectra in the raw material; the content of other solid impurities is detected by lyophilization. The solution is placed in a freeze dryer, and the solid obtained after lyophilization is the solid content. Weighing this solid using an electronic balance gives the solid content in the solution.

[0089] The HPLC chromatogram of anthocyanin standards is shown below. Figure 9 As shown, the HPLC chromatogram of the anthocyanin raw material solution is as follows. Figure 10 As shown, the purity and yield of the anthocyanin eluent obtained by separation and purification in this embodiment are as follows: Figure 11 As shown in Table 3.

[0090] Table 3. Composition of the eluent and re-eluent for loading onto the sample column.

[0091]

[0092] Note: The eluent for loading the sample column is 1. ～ The liquid (high-purity anthocyanins) precipitated from ethanol on column 9 was recovered as 10. ～ The liquid (high-purity anthocyanins) that was precipitated from ethanol on column 12.

[0093] Anthocyanin purity calculation:

[0094] Anthocyanin purity = Total anthocyanin content / Total solids content × 100%.

[0095] Depend on Figures 9-11As shown in Table 3, the peak shapes of the eluent and the raw material spectra are similar, reproducing all components of anthocyanins without significant loss. Using the method provided by this invention, the purity of anthocyanins increased from 3% to 32.75%, a purity increase of more than 10 times, with a yield of over 97% (79.42% + 17.72%). The anthocyanin spectrum in the eluent obtained by the method provided by this invention is similar to that of the anthocyanin standard, indicating that the anthocyanins obtained by this invention have high purity.

[0096] Comparative Example 1

[0097] The anthocyanin raw material solution in Example 1 was separated and purified using a DM21D macroporous resin column. The specific steps are as follows: the anthocyanin raw material solution was loaded into the DM21D macroporous resin column at a flow rate of 3.34 BV / h, washed with water at a flow rate of 0.75 BV / h to remove impurities, and then eluted with ethanol at a flow rate of 1.5 BV / h to obtain the anthocyanin eluent.

[0098] Example 1: The calculation of the throughput for continuous chromatography purification scaled up to industrial-grade production is shown in Table 4.

[0099] Table 5 shows a comparison of the solvent usage in Example 1 and Comparative Example 1, and Table 6 shows a comparison of the costs.

[0100] Table 4 Calculation of Industrial-Grade Purification Data for Continuous Chromatography Schemes

[0101] Daily processing capacity (raw material purity <5%) 30-36 tons Single-column injection 1200~1500L / h Daily cyclic column count 24 × 60 / 120 = 12 times Column volume 500L (20% expansion factor) If the required column size is small, try increasing the scale. 1667 times

[0102] Table 5 Comparison of solvent usage for purifying anthocyanins in Comparative Example 1 (single column) and Example 1 (continuous chromatography)

[0103] <![CDATA[Single column (m 3 / day)]]> <![CDATA[Continuous chromatography (m 3 / day)]]> Annual savings (tons / day) Macroporous resin amount 10 4.9 Washing material 12 7.4 1380 Ethanol analysis 24 14.7 2790 Water-washed alcohol 16.2 8.2 2400 Protective column water washing 3 3.7 -210 Ethanol analysis of guard column 6 7.4 -420 ethanol washed with water from the guard column 4 4 0

[0104] Note:

[0105] Washing material: 1 ～ Water consumption during the washing of column No. 9;

[0106] Water-washed alcohol: 1 ～ Water consumption during the regeneration of column No. 9;

[0107] Protects against water washing: 10 ～ Water consumption during the washing of column No. 12;

[0108] Ethanol analysis of guard column: 10 ～ Ethanol usage during column 12 analysis;

[0109] Guard column washed with ethanol: 10 ～ Water consumption during regeneration of column 12.

[0110] Table 6. Cost Comparison of Anthocyanin Purification Methods Using Single-Column and Continuous Chromatography

[0111]

[0112] Note: Steam 1 (concentration of washing solution): The washing solution contains anthocyanins and needs to be concentrated.

[0113] Steam 2 (concentration of ethanol eluent): The eluent contains anthocyanins and needs to be concentrated.

[0114] As shown in Tables 5 and 6, compared with single-column separation and purification of anthocyanins, the continuous chromatography method of this invention saves approximately 6,000 tons of solvent per year, resulting in annual cost savings of nearly 1.7 million yuan.

[0115] Comparative Example 2

[0116] Three LAK30 macroporous resin columns (2.4×75cm) were connected in series to separate and purify the anthocyanin raw material solution in Example 1. The specific steps are as follows: the sample was loaded at a flow rate of 3.34 BV / h. When 1000 mL of sample was loaded, the column pressure rose to 1 MPa, which was not conducive to the process and the sample loading volume was low.

[0117] Compared with Comparative Example 2, Example 1 shows that the continuous macroporous resin chromatography system using DM21D macroporous resin can load 2L of sample, which is larger than that of LKA30 resin, and there is no increase in column pressure.

[0118] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for separating and purifying anthocyanins using a continuous macroporous resin chromatography system, wherein the continuous macroporous resin chromatography system comprises: The system comprises an injection zone, a washing zone, an ethanol analysis zone, a resin regeneration zone, and a washing solution recovery zone. The injection zone consists of three columns connected in series; the washing zone consists of two columns connected in series; the ethanol analysis zone consists of two columns connected in series; the resin regeneration zone consists of two columns connected in series; and the washing solution recovery zone consists of three columns. The continuous macroporous resin chromatography system includes 12 columns, numbered 1 to 12. Columns 1 to 9 are used cyclically as the injection zone, washing zone, ethanol analysis zone, and resin regeneration zone, while columns 10 to 12 serve as the washing solution recovery zone. The system allows for continuous and simultaneous operation of the injection zone, washing zone, ethanol analysis zone, resin regeneration zone, and washing solution recovery zone by switching columns. All columns are macroporous resin columns, specifically DM21D macroporous resin columns. The method includes the following steps: loading anthocyanin sample solution, washing with water to remove impurities, ethanol elution, resin regeneration, and recovery of the washing solution; the ethanol elution yields anthocyanins; the recovery of the washing solution involves purifying the washing solution obtained from the water elution process to obtain anthocyanins; the ethanol elution uses an ethanol solution with a volume fraction of 70-80%. The method specifically includes the following steps: (1) Use a pump to pump the anthocyanin raw material solution into columns No. 1, No. 2 and No. 3 connected in series until column No. 1 is saturated with sample, and columns No. 2 and No. 3 are used as guard columns. (2) Water is pumped into column 1 for washing, and the washing solution is directly introduced into column 10; columns 2, 3 and 4 are injected in series until column 2 is saturated with sample. (3) Columns 1 and 2 are washed in series with water, and the washing solution is directly introduced into column 10; columns 3, 4 and 5 are injected in series until column 3 is saturated with sample. (4) Ethanol was used to elute column 1 and anthocyanins were collected at the bottom of column 1; columns 2 and 3 were washed in series with water and the washing solution was directly introduced into column 11; columns 4, 5 and 6 were injected in series and column 10 was washed with pure water. (5) Columns 1 and 2 are connected in series and eluted with ethanol to obtain anthocyanins; Columns 3 and 4 are connected in series and washed with water, and the washing solution is directly introduced into column 11; Columns 5, 6 and 7 are connected in series and injected, and column 10 is eluted with ethanol, and anthocyanins are collected at the bottom of column 10. (6) Regenerate column 1 by rinsing with water, desorb column 2 and column 3 in series with ethanol to obtain anthocyanins; wash column 4 and column 5 in series with water, and the washing solution directly enters column 12; inject column 6, column 7 and column 8 in series, wash column 11 with water, wash column 10 with ethanol, and recover and reuse the ethanol solution collected at the bottom of column 10. (7) Columns 1 and 2 are regenerated by water washing, columns 3 and 4 are desorbed by ethanol in series, columns 5 and 6 are washed by water in series, the washing solution is directly introduced into column 12, columns 7, 8 and 9 are injected in series, column 1 is regenerated when the injection is finished, column 10 is regenerated by water, column 11 is desorbed by ethanol, and anthocyanins are collected at the bottom of column 11. (8) Columns 2 and 3 are regenerated in series, columns 4 and 5 are eluted in series, columns 6 and 7 are washed with water in series, the washing solution is reintroduced into column 10, column 11 is washed with ethanol and desorbed, the ethanol solution collected at the bottom of column 11 is recovered and reused, column 12 is desorbed with ethanol to obtain anthocyanins, and columns 8, 9 and 1 are injected in series.

2. The method according to claim 1, characterized in that, The pressure of the continuous macroporous resin chromatography system is <0.5 MPa.

3. The method according to claim 1, characterized in that, The purification process includes sample loading, water washing to remove impurities, ethanol elution, and resin regeneration.

4. The method according to any one of claims 1 to 3, characterized in that, The flow rate for each loading is 3~4 BV / h.

5. The method according to any one of claims 1 to 3, characterized in that, The anthocyanin purity in the anthocyanin sample solution is <5%, and the solvent is water.

6. The method according to any one of claims 1 to 3, characterized in that, The flow rate of the washing material is 0.6~1 BV / h each time.

7. The method according to any one of claims 1 to 3, characterized in that, The flow rate for each ethanol elution was 1.3~1.7 BV / h, and the amount of pure ethanol used per liter of resin per hour during each ethanol elution process was 0.12~0.16 L.

8. The method according to any one of claims 1 to 3, characterized in that, The resin is regenerated by water washing, and the flow rate for each regeneration is 0.6~1 BV / h.

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