A process for recovering high purity rebaudioside o and rebaudioside n using waste mother liquor of rebaudioside d

Abstract

The present application relates to the field of glycoside products, and particularly relates to a process method for recycling high-purity rebaudioside O and rebaudioside N by using waste mother liquor of rebaudioside D, comprising the following steps: (1) taking the waste sugar mother liquor, diluting and then entering a resin column, first eluting by using a first and a second desorbent, and collecting the first and the second desorption solution; (2) taking the first desorption solution, concentrating and drying to obtain a first dry matter, dissolving in ethanol and then entering a normal-phase medium-low pressure chromatographic column, performing gradient desorption by using ethanol, concentrating and drying the collected desorption solution, dissolving in methanol and then entering a reverse-phase high-pressure chromatographic column, performing desorption by using methanol, and collecting the first and the second desorption solution; (3) taking the first desorption solution, concentrating and drying to obtain rebaudioside O product; (4) taking the second desorption solution, concentrating and drying to obtain rebaudioside N product. The process method can recycle rebaudioside O and rebaudioside N in the waste mother liquor.

Description

A process for recovering high-purity rebaudioside O and rebaudioside N from the waste mother liquor of rebaudioside D. Technical Field

[0001] This invention relates to the field of glycoside products, and more particularly to a process for recovering high-purity rebaudioside O and rebaudioside N from the waste mother liquor of rebaudioside D. Background Technology

[0002] Steviosides are natural sweeteners isolated and extracted from the perennial shrub Stevia rebaudiana. Studies have shown that steviol glycosides include various compounds such as Rebaudioside A (RA), RB, RC, RD, and RM. Among them, steviol glycoside (ST) and Rebaudioside A (RA) account for the largest proportions (generally over 90%), while Rebaudioside C (RC), Dulcoside A (DA), and steviol disaccharides are present in smaller amounts. Although Rebaudioside A has potential positive health benefits and is relatively readily available, its lingering astringent taste makes it less than ideal as a sweetener.

[0003] Currently, most enterprises mainly use stevia as raw material to obtain high-content rebaudioside D, rebaudioside A, or rebaudioside C products through extraction and separation. However, the production of rebaudioside D generates a large amount of waste sugar mother liquor, which also contains rebaudioside O and rebaudioside N. If the waste sugar mother liquor is directly discarded, it will inevitably lead to a waste of raw materials. Therefore, to address the above technical problems, it is necessary to develop a process for recovering high-purity rebaudioside O and rebaudioside N from the waste sugar mother liquor produced in the production of rebaudioside D. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a process for recovering high-purity rebaudioside O and rebaudioside N from the waste mother liquor of rebaudioside D, which avoids the waste of resources.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] A process for recovering high-purity rebaudioside O and rebaudioside N from the waste mother liquor of rebaudioside D, the process comprising the following steps:

[0007] (1) Take the collected waste sugar mother liquor, add purified water to dilute it and then enter the resin column. First, use the first desorbent to elute the resin column and collect the first desorbed liquid; then use the second desorbent to elute the resin column and collect the second desorbed liquid.

[0008] (2) Take the first desorption liquid from step (1), concentrate and dry it to obtain the first dried product for later use; take the second desorption liquid from step (1), concentrate and dry it to obtain the second dried product for later use.

[0009] (3) Take the first dried substance from step (2), dissolve it in ethanol, and then enter it into a normal phase medium-low pressure chromatographic column. Use 90%-95% v / v, 80%-90% v / v and 70%-80% v / v ethanol to perform gradient desorption on the chromatographic column, and collect the desorbed chromatographic column of the above concentrations of ethanol respectively.

[0010] (4) Take the eluent from the chromatographic column in step (3) with 80%-90% v / v ethanol, concentrate and dry the dried product for later use.

[0011] (5) Take the dried material described in step (4), dissolve it in methanol, and then enter it into a reversed-phase high-pressure chromatography column. Use 40-70% v / v methanol for desorption. Collect the first desorption solution and the second desorption solution according to the spectrum.

[0012] (6) Take the first desorption solution from step (5), concentrate and dry it to obtain the Lebaudi glycoside O product;

[0013] (7) Take the second desorption solution from step (5), concentrate and dry it to obtain the Lebodiin N product.

[0014] As an improved technical solution, in step (1), the amount of purified water added is 5-10 times that of the waste sugar mother liquor; the diluted waste sugar mother liquor enters the resin column at a flow rate of 10-30 ml / min; the first desorbent is purified water, which elutes the resin column at a flow rate of 20-40 ml / min; the second desorbent is 50%-70% v / v methanol, which elutes the resin column at a flow rate of 50-100 ml / min.

[0015] As an improved technical solution, the resin in the resin column in step (1) is a macroporous adsorption resin, and the model of the macroporous adsorption resin is LX-T28, D001, T81, D354, or SPD-009.

[0016] As an improved technical solution, in step (3), the first dried substance is added to 80-90% v / v ethanol at a mass-volume ratio of 1:3-5 and stirred to dissolve. Then, it is introduced into a normal phase medium-low pressure chromatographic column at a flow rate of 20-40 ml / min. The packing material of the normal phase medium-low pressure chromatographic column is normal phase packing material Silica or NH2 with a particle size of 40-60 μm.

[0017] As an improved technical solution, in step (3), 70%-80% v / v, 80%-90% v / v and 90%-95% v / v ethanol are desorbed from the chromatographic column at a flow rate of 50-100 ml / min, and each ethanol concentration is desorbed at a volume of 1-4 BV of chromatographic column packing.

[0018] As an improved technical solution, the dried material in step (5) is added to 50%-70% v / v methanol at a mass-volume ratio of 1:8-12 and stirred to dissolve. The solution is then introduced into a reversed-phase high-pressure chromatography column at a flow rate of 40-60 ml / min. The packing material of the reversed-phase high-pressure chromatography column is C18 packing material or C8 (MOS) bonded with silica gel with a particle size of 10-20 μm.

[0019] As an improved technical solution, in step (5), the 40-70% v / v methanol is desorbed onto the chromatographic column at a flow rate of 40-60 ml / min.

[0020] After adopting the above technical solution, the beneficial effects of the present invention are:

[0021] This invention uses waste mother liquor of rebaudioside D as raw material. After dilution, it enters a resin column and is desorbed using purified water. The collected first eluent is concentrated and dried, and the resulting dried product is dissolved in ethanol and then introduced into a normal-phase medium-low pressure chromatography column. Gradient elution with ethanol is used, and the eluent obtained during the column desorption at 80%-90% v / v is collected. After concentration and drying, the dried product is dissolved in methanol and introduced into a reversed-phase high-pressure chromatography column. Desorption with methanol is performed, and the first and second eluents are collected. The first eluent is concentrated and dried to obtain rebaudioside O product. The dried product of the second eluent is concentrated and dried, and methanol is added. After cooling and crystallization, the product is filtered. The collected filter cake is added to methanol, stirred, cooled, crystallized, and filtered. The collected filter cake is dried to obtain rebaudioside N product. The above process can recover rebaudioside O and rebaudioside N from waste mother liquor and obtain high-purity and high-yield rebaudioside N and rebaudioside O. Attached Figure Description

[0022] Figure 1 is the liquid phase detection spectrum of rebaudioside O in Example 3 of the present invention;

[0023] Figure 2 is a liquid phase detection spectrum of rebaudioside N in Example 3 of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] Example 1

[0026] A process for recovering high-purity rebaudioside O and rebaudioside N from the waste mother liquor of rebaudioside D includes the following steps:

[0027] (1) Take 1000g of the collected waste sugar mother liquor (RO content is 4.7%, RN content is 6.1%), add 5L of purified water at a mass-volume ratio of 1:5, dilute it, and enter the resin column at a flow rate of 10ml / min. First, use 10L of the first desorbent (purified water, at a flow rate of 20ml / min) to elute the resin column and collect 10L of the first desorbent. Then, use 15L of the second desorbent (50% v / v methanol, at a flow rate of 50ml / min) to elute the resin column (the packing material is macroporous adsorption resin, model LX-T28) and collect the second desorbent.

[0028] (2) Take the first desorption liquid from step (1), concentrate and dry to obtain 100g of the first dried product for later use; take the second desorption liquid from step (1), concentrate and dry to obtain the second dried product for later use.

[0029] (3) Take 100g of the first dried material in step (2), add it to 300ml of 80% v / v ethanol at a mass-volume ratio of 1:3 and stir to dissolve. Then, enter the normal phase medium-low pressure chromatographic column with a packing volume of 5L at a flow rate of 20ml / min (the chromatographic column packing is Silica normal phase packing with a particle size of 40-60μm). Use 90% v / v, 80% v / v and 70% v / v ethanol with a packing volume of 1BV at a flow rate of 50ml / min to perform gradient desorption on the chromatographic column. Collect the desorption solution of each concentration when desorbing the chromatographic column.

[0030] (4) Take 5L of the desorption solution from the chromatographic column when desorbing 80% v / v ethanol in step (3), concentrate and dry the dried product for later use.

[0031] (5) Take 10g of the dried material from step (4), add 80ml of 50% v / v methanol at a mass-to-volume ratio of 1:8 to dissolve it, and then enter it into a reversed-phase high-pressure chromatography column with a packing volume of 1L at a flow rate of 40ml / min (the packing material is C18 packing material with silica gel bonded to a particle size of 10-20μm). Use 3L of 40% v / v methanol at a flow rate of 40ml / min for desorption. Collect 1L of the first desorption solution and 2L of the second desorption solution according to the chromatogram.

[0032] (6) Take 1L of the first desorption solution from step (5), concentrate and dry it to obtain the product of Rebaudine O;

[0033] (7) Take the second desorption solution from step (5), concentrate and dry it to obtain the Lebodiin N product.

[0034] Example 2

[0035] A process for recovering high-purity rebaudioside O and rebaudioside N from the waste mother liquor of rebaudioside D includes the following steps:

[0036] (1) Take 1000g of the collected waste sugar mother liquor (RO content is 4.7%, RN content is 6.1%), add 6L of purified water at a mass-volume ratio of 1:6 to dilute it, and enter the resin column at a flow rate of 15ml / min. First, use 10L of the first desorbent (purified water, at a flow rate of 25ml / min) to elute the resin column and collect 10L of the first desorbent. Then, use 15L of the second desorbent (55% v / v methanol, at a flow rate of 65ml / min) to elute the resin column (the packing material is macroporous adsorption resin, model D001) and collect the second desorbent.

[0037] (2) Take the first desorption liquid from step (1), concentrate and dry it to obtain the first dried product for later use; take the second desorption liquid from step (1), concentrate and dry it to obtain the second dried product for later use.

[0038] (3) Take 100g of the first dried material from step (2), add it to 350ml of 82% v / v ethanol at a mass-volume ratio of 1:3.5 and stir to dissolve. Then, enter the column into a normal phase medium-low pressure chromatography column with a packing volume of 5L at a flow rate of 25ml / min (the chromatographic column packing is normal phase packing NH2 with a particle size of 40-60μm). Use 93% v / v, 83% v / v and 72% v / v ethanol with a packing volume of 2BV at a flow rate of 65ml / min to perform gradient desorption on the chromatographic column. Collect the desorption solution of each concentration when desorbing the chromatographic column.

[0039] (4) Take 10L of the desorption solution from the column desorption of 83% v / v ethanol in step (3), concentrate and dry the dried product for later use.

[0040] (5) Take 10g of the dried material from step (4), add 90ml of 55% v / v methanol at a mass-volume ratio of 1:9 to dissolve it, and then enter it into a reversed-phase high-pressure chromatography column with a packing volume of 1L at a flow rate of 45ml / min (the packing material is C8 (MOS) with a particle size of 10-20μm). Use 3L of 50% v / v methanol at a flow rate of 45ml / min for desorption. Collect 1L of the first desorption solution and 2L of the second desorption solution according to the spectrum.

[0041] (6) Take 1L of the first desorption solution from step (5), concentrate and dry it to obtain the product of Rebaudine O;

[0042] (7) Take the second desorption solution from step (5), concentrate and dry it to obtain the dried product, which is the product of Rebaudio Glycoside N.

[0043] Example 3

[0044] A process for recovering high-purity rebaudioside O and rebaudioside N from the waste mother liquor of rebaudioside D includes the following steps:

[0045] (1) Take 1000g of the collected waste sugar mother liquor (RO content is 4.7%, RN content is 6.1%), add 8L of purified water at a mass-volume ratio of 1:8, dilute it, and enter the resin column at a flow rate of 20ml / min. First, use 10L of the first desorbent (purified water, at a flow rate of 30ml / min) to elute the resin column and collect 10L of the first desorbent. Then, use 15L of the second desorbent (60% v / v methanol, at a flow rate of 80ml / min) to elute the resin column (the packing material is macroporous adsorption resin, model T81) and collect the second desorbent.

[0046] (2) Take the first desorption liquid from step (1), concentrate and dry it to obtain the first dried product for later use; take the second desorption liquid from step (1), concentrate and dry it to obtain the second dried product for later use.

[0047] (3) Take 100g of the first dried material from step (2), add it to 400ml of 85% v / v ethanol at a mass-volume ratio of 1:4 and stir to dissolve. Then, enter the column into a normal phase medium-low pressure chromatography column with a packing volume of 5L at a flow rate of 30ml / min (the chromatographic column packing is Silica normal phase packing with a particle size of 40-60μm). Use 93% v / v, 85% v / v and 75% v / v ethanol with a packing volume of 3BV at a flow rate of 80ml / min to perform gradient desorption on the chromatographic column. Collect the desorption solution of each concentration when desorbing the chromatographic column.

[0048] (4) Take 15L of the desorption solution from the chromatographic column in step (3) with 85% v / v ethanol, concentrate and dry the dried product for later use.

[0049] (5) Take 10g of the dried material from step (4), add 100ml of 60% v / v methanol at a mass-to-volume ratio of 1:10 to dissolve it, and then enter it into a reversed-phase high-pressure chromatography column with a packing volume of 1L at a flow rate of 50ml / min (the packing material is C18 packing material with silica gel bonded to a particle size of 10-20μm). Use 3L of 60% v / v methanol at a flow rate of 50ml / min for desorption. Collect 1L of the first desorption solution and 2L of the second desorption solution according to the chromatogram.

[0050] (6) Take 1L of the first desorption solution from step (5), concentrate and dry it to obtain the product of Rebaudine O;

[0051] (7) Take the second desorption solution from step (5), concentrate and dry it to obtain the dried product, which is the product of Rebaudio Glycoside N.

[0052] Example 4

[0053] A process for recovering high-purity rebaudioside O and rebaudioside N from the waste mother liquor of rebaudioside D includes the following steps:

[0054] (1) Take 1000g of the collected waste sugar mother liquor (RO content is 4.7%, RN content is 6.1%), add 9L of purified water at a mass-volume ratio of 1:9 and dilute it. Then, enter the resin column at a flow rate of 25ml / min. First, use 10L of the first desorbent (purified water, at a flow rate of 40ml / min) to elute the resin column and collect 18L of the first desorbent. Then, use 15L of the second desorbent (65% v / v methanol, at a flow rate of 90ml / min) to elute the resin column (the packing material is macroporous adsorption resin, model D354) and collect the second desorbent.

[0055] (2) Take the first desorption liquid from step (1), concentrate and dry it to obtain the first dried product for later use; take the second desorption liquid from step (1), concentrate and dry it to obtain the second dried product for later use.

[0056] (3) Take 100g of the first dried material from step (2), add it to 450ml of 88% v / v ethanol at a mass-volume ratio of 1:4.5 and stir to dissolve. Then, enter the column into a normal phase medium-low pressure chromatographic column with a packing volume of 5L at a flow rate of 40ml / min (the chromatographic column packing is Silica normal phase packing with a particle size of 40-60μm). Use 93% v / v, 88% v / v and 78% v / v ethanol with a packing volume of 3.5BV at a flow rate of 90ml / min to perform gradient desorption on the chromatographic column. Collect the desorption solution of each concentration when desorbing the chromatographic column.

[0057] (4) Take 17.5 L of the desorption solution from the chromatographic column in step (3) with 88% v / v ethanol, concentrate and dry it to obtain the dried product for later use.

[0058] (5) Take 10g of the dried material from step (4), add 110ml of 65% v / v methanol at a mass-to-volume ratio of 1:11 to dissolve it, and then enter it into a reversed-phase high-pressure chromatography column with a packing volume of 1L at a flow rate of 55ml / min (the packing is C18 packing with silica gel bonded to a particle size of 10-20μm). Use 3L of 65% v / v methanol at a flow rate of 55ml / min for desorption. Collect 1L of the first desorption solution and 2L of the second desorption solution according to the chromatogram.

[0059] (6) Take 1L of the first desorption solution from step (5), concentrate and dry it to obtain the product of Rebaudine O;

[0060] (7) Take the second desorption solution from step (5), concentrate and dry it to obtain the dried product, which is the product of Rebaudio Glycoside N.

[0061] Example 5

[0062] A process for recovering high-purity rebaudioside O and rebaudioside N from the waste mother liquor of rebaudioside D includes the following steps:

[0063] (1) Take 1000g of the collected waste sugar mother liquor (RO content is 4.7%, RN content is 6.1%), add 10L of purified water at a mass-volume ratio of 1:10, dilute it, and enter the resin column at a flow rate of 30ml / min. First, use 10L of the first desorbent (purified water, at a flow rate of 40ml / min) to elute the resin column and collect 20L of the first desorbent. Then, use 15L of the second desorbent (70% v / v methanol, at a flow rate of 100ml / min) to elute the resin column (the packing material is macroporous adsorption resin, model SPD-009) and collect the second desorbent.

[0064] (2) Take the first desorption liquid from step (1), concentrate and dry it to obtain the first dried product for later use; take the second desorption liquid from step (1), concentrate and dry it to obtain the second dried product for later use.

[0065] (3) Take 100g of the first dried material in step (2), add it to 500ml of 90% v / v ethanol at a mass-volume ratio of 1:5 and stir to dissolve. Then, enter the normal phase medium-low pressure chromatographic column with a packing volume of 5L at a flow rate of 40ml / min (the chromatographic column packing is normal phase packing NH2 with a particle size of 40-60μm). Use 95% v / v, 90% v / v and 80% v / v ethanol with a packing volume of 4BV to perform gradient desorption on the chromatographic column at a flow rate of 100ml / min. Collect the desorption solution of each concentration when desorbing the chromatographic column.

[0066] (4) Take 20L of the desorption solution from the chromatographic column in step (3) with 90% v / v ethanol, concentrate and dry the dried product for later use.

[0067] (5) Take 10g of the dried material from step (4), add 120ml of 70% v / v methanol at a mass-to-volume ratio of 1:12 to dissolve it, and then enter it into a reversed-phase high-pressure chromatography column with a packing volume of 1L at a flow rate of 60ml / min (the packing material is C8 (MOS) bonded with silica gel with a particle size of 10-20μm). Use 3L of 70% v / v methanol at a flow rate of 60ml / min for desorption. Collect 1L of the first desorption solution and 2L of the second desorption solution according to the chromatogram.

[0068] (6) Take 1L of the first desorption solution from step (5), concentrate and dry it to obtain the product of Rebaudine O;

[0069] (7) Take the second desorption solution from step (5), concentrate and dry it to obtain the dried product, which is the product of Rebaudio Glycoside N.

[0070] To better demonstrate that the process of the present invention can obtain high purity and high content of rebaudioside O and rebaudioside N from waste mother liquor, six comparative examples are given with reference to Example 3. The yield and purity of rebaudioside O and rebaudioside N obtained in Examples 1-5 and Comparative Examples 1-8 are detailed in Table 1.

[0071] Comparative Example 1

[0072] Unlike Example 3, in step (1), the resin column packing material is macroporous adsorption resin, specifically model R203, and the rest of the operation is the same.

[0073] Comparative Example 2

[0074] The difference from the operation in Example 3 is that in step (3), the normal phase low-pressure chromatographic column (the packing material is APS) is used, but the rest of the operation is the same.

[0075] Comparative Example 3

[0076] The difference from the operation in Example 3 is that in step (5), the reversed-phase high-pressure chromatography column is filled with silica-bonded C4(B) with a particle size of 10-20 μm, while the rest of the operation is the same.

[0077] Comparative Example 4

[0078] The difference from the operation in Example 3 is that in step (3), an 80%-90% v / v ethanol desorption column is used directly, and the rest of the operation is the same.

[0079] Comparative Example 5

[0080] The difference from the operation in Example 3 is that the flow rate of the ethanol desorption column in step (3) is 40 ml / min, while the rest of the operation is the same.

[0081] Comparative Example 6

[0082] Unlike Example 3, step (5) uses 80% v / v methanol for desorption, while the rest of the operation is the same.

[0083] Comparative Example 7

[0084] Unlike Example 3, step (5) uses 30% v / v methanol for desorption, while the rest of the operation is the same.

[0085] Comparative Example 8

[0086] Unlike Example 3, the flow rate of methanol used for desorption in step (5) is 70 ml / min, while the rest of the operation is the same.

[0087] Table 1

[0088]

[0089]

[0090] As can be seen from Table 1, compared with Comparative Examples 1-8, the process method of the present invention can obtain high yield and high purity of rebaudioside O and rebaudioside N products from waste sugar mother liquor.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for recovering high-purity rebaudine O and rebaudine N from the waste mother liquor of rebaudine D, characterized in that, The process includes the following steps: (1) Take the collected waste sugar mother liquor, add purified water to dilute it and enter the resin column. First, use the first desorbent to elute the resin column and collect the first desorbed liquid; then use the second desorbent to elute the resin column and collect the second desorbed liquid; the resin in the resin column is a macroporous adsorption resin, and the model of the macroporous adsorption resin is LX-T28, D001, T81, D354; the first desorbent is purified water; (2) Take the first desorbed liquid in step (1), concentrate and dry it to obtain the first dried product for later use; take the second desorbed liquid in step (1), concentrate and dry it to obtain the second dried product for later use; (3) Take the first dried substance from step (2), dissolve it in ethanol, and then introduce it into a normal-phase medium-low pressure chromatographic column. Use 90%-95% v / v, 80%-90% v / v, and 70%-80% v / v ethanol to perform gradient desorption on the chromatographic column, and collect the eluent from the chromatographic column at the above concentrations of ethanol respectively. Add the first dried substance to 80-90% v / v ethanol at a mass-to-volume ratio of 1:3-5 and stir to dissolve. Then introduce it into a normal-phase medium-low pressure chromatographic column at a flow rate of 20-40 ml / min. The packing material of the normal-phase medium-low pressure chromatographic column is normal-phase packing material Silica or NH2 with a particle size of 40-60 μm. (3) Desorb the chromatographic column with 70%-80% v / v, 80%-90% v / v and 90%-95% v / v ethanol at a flow rate of 50-100 ml / min, and desorb each ethanol concentration at a volume of 1-4 BV of the chromatographic column packing; (4) Take the desorbed liquid from step (3) when desorbing the chromatographic column with 80%-90% v / v ethanol, concentrate and dry the dried product for later use; (5) Take the dried product from step (4), add methanol to dissolve it and enter it into the reversed-phase high-pressure chromatographic column, use 40-70% v / v methanol for desorption, and collect the first desorbed liquid and the second desorbed liquid according to the spectrum; The dried material is added to 50%-70% v / v methanol at a mass-volume ratio of 1:8-12 and stirred to dissolve. The solution is then introduced into a reversed-phase high-pressure chromatography column at a flow rate of 40-60 ml / min. The packing material of the reversed-phase high-pressure chromatography column is C18 packing material or C8 (MOS) bonded with silica gel with a particle size of 10-20 μm. The 40-70% v / v methanol is used to desorb the column at a flow rate of 40-60 ml / min. (6) The first desorbed solution from step (5) is taken, concentrated, and dried to obtain lebodiin O product. (7) The second desorbed solution from step (5) is taken, concentrated, and dried to obtain lebodiin N product.

2. The process for recovering high-purity rebaudine O and rebaudine N from the waste mother liquor of rebaudine D according to claim 1, characterized in that, In step (1), the amount of purified water added is 5-10 times that of the waste sugar mother liquor; the diluted waste sugar mother liquor is introduced into the resin column at a flow rate of 10-30 ml / min; the purified water is used to elute the resin column at a flow rate of 20-40 ml / min; the second desorbent is 50%-70% v / v methanol, and the methanol is used to elute the resin column at a flow rate of 50-100 ml / min.

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