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Method for separating and purifying stevioside by using silica gel substrate strong anion exchange chromatography

A strong anion, exchange chromatography technology, applied in the field of extraction and purification of natural products, can solve the problems of inability to quickly prepare high-purity glycoside monomers, etc., and achieve the effects of good mechanical stability, long service life and strong operability

Pending Publication Date: 2019-06-18
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the extraction and purification methods of steviol glycosides mainly include macroporous adsorption resin method, membrane separation method, solvent crystallization method and chromatographic separation method, etc. These methods cannot quickly prepare high-purity glycoside monomers, so a method for preparing high-purity glycoside monomers method, to be further explored
[0004] HP-SAX bonded stationary phase is based on high-purity silica gel with good mechanical stability. It has a mixed chemical structure of quaternary ammonium and phenyl functional groups by using high-purity bonding reagents to bond quaternary ammonium strong anion exchange groups on silica gel. , the mixing mode of this strong anion exchange phase and hydrophobic phase is especially suitable for the efficient and highly selective separation of hydrophilic compounds with special structures. The chromatographic column can use a variety of mobile phases, including organic solvents, water and organic solvents (such as methanol and acetonitrile), buffer (such as phosphate), etc., at present, there is no report on the simultaneous separation and purification of high-purity steviol glycosides by using silica gel matrix strong anion exchange chromatography

Method used

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  • Method for separating and purifying stevioside by using silica gel substrate strong anion exchange chromatography
  • Method for separating and purifying stevioside by using silica gel substrate strong anion exchange chromatography
  • Method for separating and purifying stevioside by using silica gel substrate strong anion exchange chromatography

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Embodiment 1

[0032] Step 1. Pretreatment of samples: Take the dry leaves of Stevia rebaudiana obtained during the harvest period, crush the dry leaves through a 200-mesh sieve, add 10 times the mass of hot water at 70°C to the sample after the sieve, and extract the glycosides with the assistance of ultrasonic waves. Add 1 / 8 of its volume to lime milk suspension and mix to remove impurities. Centrifuge the mixture at 16000rpm for 10min. Add phosphoric acid to the supernatant to neutralize. Concentrate the soluble solid content (Brix) to 10% to obtain a treatment solution for later use; the crude steviol glycoside treatment method is to directly dissolve it with water to form an aqueous solution with a soluble solid content of 10% as the treatment solution for later use.

[0033] Step 2. Separation and purification of steviol glycosides: (a) Hardware preparation: Pack HP-SAX silica gel-based strong anion exchange chromatography filler into the chromatographic column by wet method and test wh...

Embodiment 2

[0040] Step 1. Pretreatment of samples: directly dissolving the crude steviol glycosides in water into a 10% (Brix) aqueous solution as a treatment solution for later use;

[0041]Step 2. Separation and purification of steviol glycosides: (a) Hardware preparation: Pack HP-SAX silica gel-based strong anion exchange chromatography filler into the chromatographic column by wet method and test whether its separation performance is up to standard. After reaching the standard, install the chromatographic column, or put The purchased HP-SAX finished chromatographic column is directly installed on the preparative liquid chromatograph, the column size is 21.2mm×250 mm, 5μm, Start the chromatograph;

[0042] (b) Activation and equilibrium of the chromatographic column: activate the chromatographic column with 150mmol / L sodium phosphate (pH 7.0) solution of 30 times the column volume successively, wash the chromatographic column with 30 times the column volume of pure water, and then us...

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Abstract

The invention relates to a method for separating and purifying stevioside by using silica gel substrate strong anion exchange chromatography. Stevia leaves or stevioside crude products are used as rawmaterials, HP-SAX silica gel substrate strong anion exchange chromatography filler or a homotype finished product chromatographic column is used as a separation medium, separation and purification are performed with liquid chromatography, and three steviosides including stevioside, rebaudioside C and rebaudioside A are obtained simultaneously. The invention also relates to an application of the method in preparation of the stevioside, the rebaudioside C and the rebaudioside A by separation and purification. The method is convenient and rapid, single separation can be completed within 15 min,the purity of purified products is higher than 99.0%, repeatability is good, operability is high, an organic solvent aqueous solution in any proportion and even 100% water can be used as a chromatographic separation medium to wash away impurities such as inorganic salt and pigment caused by samples on the head of the chromatographic column, the separation medium is not prone to hydrolysis or collapse in the washing process, and the separation medium has long service life and can be reused for a long time.

Description

technical field [0001] The invention belongs to the technical field of extraction and purification of natural products, in particular to a method for separating and purifying steviol glycosides by strong anion exchange chromatography on a silica gel matrix. Background technique [0002] Stevioside is a new type of natural sweetener extracted from the leaves of Stevia rebaudiana, a plant of Compositae. The sweetness of stevioside is 250-450 times that of sucrose, and the sweetness of stevioside (St) is about 300 times that of sucrose. , The sweetness of rebaudioside A (RA) is about 450 times that of sucrose, and the taste is better. Steviol glycoside is an important sweet substitute and flavor enhancer because it is not absorbed and does not produce heat after eating. It is widely used in the production of food, beverages and condiments. [0003] At present, the extraction and purification methods of steviol glycosides mainly include macroporous adsorption resin method, memb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H1/06C07H1/08C07H15/256
Inventor 贾鹏禹孙蕊郑殿峰冯乃杰冯胜杰
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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