CN103613622A - Method for separating and purifying rebaudioside A - Google Patents
Method for separating and purifying rebaudioside A Download PDFInfo
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- CN103613622A CN103613622A CN201310532268.5A CN201310532268A CN103613622A CN 103613622 A CN103613622 A CN 103613622A CN 201310532268 A CN201310532268 A CN 201310532268A CN 103613622 A CN103613622 A CN 103613622A
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Abstract
The invention discloses a method for separating and purifying rebaudioside A. In the method, a sulfonic chromatogram filling material is taken as the stationary phase; an organic solvent water solution is taken as the mobile phase, and then high purity rebaudioside A is obtained through the processes of balancing chromatogram column, sampling and eluting. In the method a quaternary amine group chromatogram filling material with a large absorption amount is adopted, the obtained product has a high purity, so the method is suitable for being applied to industrial production.
Description
Technical field
The present invention relates to a kind of method of separation and purification content rebaudioside-A, belong to food chemistry technical field.
Background technology
Stevioside is a kind of natural sweeteners extracting from feverfew sweet Stevia (Stevia rebaudiana Bertoni).Stevioside is glucoside mixture, content rebaudioside-A (Rebaudioside A wherein, RA) sugariness is 350 ~ 450 times and pure taste of sucrose, its high sugariness, the suitable especially obesity of feature, diabetes, hypertension, arteriosclerosis, eurodonticus low in calories eat, and are the best substitute of sucrose and other synthetic sweeteners.U.S. FDA in 2008 formally gives high purity content rebaudioside-A (content >=97%) GRAS (It is generally accepted safe material) status, so world market will be amplified rapidly the demand of high purity RA.
China is the country of global stevia rebaudianum cultivated area, stevioside output maximum, but take the lower RA crude product of purity as main because of Technology Restriction, product.Major impurity Steviosides (Stevioside, ST) in RA and rebaudioside C (Rebaudioside C, RC) are the analog of RA, and the latter's bitter taste is heavier, have a strong impact on quality and the price of stevioside.
At present, openly the preparation method of high purity RA has recrystallization method and chromatography.Recrystallization method be utilize each glucosides of stevioside organic solvent as methyl alcohol in solubleness difference and repeatedly crystalline go out highly purified RA, this method operation is loaded down with trivial details, yield is lower, solvent consumption is large and cost is higher.
Therefore, Many researchers attempts adopting chromatography to prepare high-purity RA.Hu Jing (macroporous resin D107 and the D108 Separation Research to SS in stevioside and RA, food research and development, 2008,29 (6): 1-4) adopt macroporous adsorbent resin D107, D108, with reverse chromatograms pattern, refine RA, but effect is not satisfactory.Chen Tianhong (the research of polymeric sorbent to each glucoside adsorption selectivity in sweet Stevia, Chinese science (B collects), 1998,28 (5): 460-465) on hydrophobic framework, introduce different groups as primary amino, ketone group, pyridyl, amide group, glucosyl, ester group etc., intention utilizes the different glycosyls of each component of stevioside and above-mentioned functions group to form the difference of hydrogen bond ability and increase the selectivity of adsorption medium to RA and ST.Research shows that the absorption of stevioside on above-mentioned medium is still mainly the physical adsorption between hydrophobicity glucoside unit and skeleton, does not reach initial design effect.
People (the Preparative purification of rebaudioside A from aqueous extracts using chromatography:a process idea such as Dominik Bergs, J. Verbr. Lebensm., 2012 (7): 295 – 303) adopt 3 step chromatogram series systems for RA, its final step treating process adopts primary amine groups (NH (CH
2)
2nH
2) silica matrix chromatogram packing, eluent is 80% acetonitrile solution, finally obtains purity to be
96.8%rA, this step yield is
97.5%.
US Patent No. 20120083593 discloses the process for purification of a kind of RA.Its core is to adopt primary amine groups (NH (CH
2)
2nH
2) resin, this resin belongs to weak base anion-exchange resin, the skeleton of resin is the superpolymer such as polystyrene-divinylbenzene or polymethacrylate, with water-miscible organic solvent, as the aqueous acetone solution wash-out of the combination of methyl alcohol, Virahol, acetone, acetonitrile or 5%-40%, through crystallization, obtain the RA component of purity > 98%.Because the skeleton of resin is superpolymer, under different concns organic solvent condition, there is volume change, during operation, post presses fluctuation larger, even cause the rough sledding such as moving phase is difficult to flow, filler fragmentation, and have to by secondary, mode that even three grades of Coupled columns reduce pressure to be step by step to overcome its deficiency, thereby must cause the drawbacks such as facility investment is large, technique is loaded down with trivial details, operation is inconvenient.
Above-mentioned two kinds adopt primary amine groups silica filler or the RA preparation method of primary amine groups resin and GB (the GB 8270-1999 of Steviosides, foodstuff additive Steviosides) detection method is basically identical, the functional group of filler is primary amine groups, and eluent is the aqueous solution of high-concentration water-soluble organic solvent.Different be that the stevioside detection method of GB adopts linear chromatography technology, main purpose is to analyze, and two kinds of methods in document adopt non-linear chromatography technology, main purpose to be preparation.Therefore belong to different technology categories.
At present, quaternary amine base strongly basic anion exchange resin is used widely in stevioside industry, is mainly used in decolouring (Shi Rongfu, the Shi Zuoqing of stevioside, Fan Yunge, Wang Chunhong, Lu Yanling, He Ping Lin, synthetic and the application in stevioside extraction separation of quaternary ammonium group polymeric adsorbent, ion-exchange and absorption, 2001,17 (2): 117-122).(Hydrophilic interaction chromatography is measured the main polar compound of stevioside to contriver's early-stage Study, Food science, 2013,34 (14): 280-284, Chen Bin, Li Shuguang, Ma Xiaoxun, Chen Guoliang, Li Rong) find that quaternary amine base silica gel chromatographic column filling material is better than primary amine groups silica filler to the detection successful of stevioside under linear chromatographic condition.
Summary of the invention
The object of the present invention is to provide a kind of novel method that is suitable for the separation and purification content rebaudioside-A of suitability for industrialized production, less to overcome prior art adsorptive capacity, the unsettled deficiency of separating medium skeleton.
Implementation procedure of the present invention is as follows:
A method for separation and purification content rebaudioside-A, it comprises the following steps:
(1) chromatographic column balance: pass into moving phase balance chromatographic column in chromatographic column;
(2) loading: the dissolving crude product that contains content rebaudioside-A, in moving phase, is pumped in chromatographic column or by by content rebaudioside-A crude product moving phase and chromatograph packing material mix, dried chromatograph packing material is put into the column cap of chromatographic column; Chromatograph packing material is quaternary amine base chromatograph packing material;
(3) wash-out: with ultraviolet or ELSD detector monitors, be eluted to content rebaudioside-A with moving phase and go out peak from chromatographic column and finish.
The skeleton of described quaternary amine base chromatograph packing material is silica gel, aluminum oxide or zirconium dioxide, is preferably silica gel; Functional group is quaternary amine base-CH
2n
+(CH
3)
3x
-or-CH
2n
+(CH
3)
2cH
2cH
2oHX
-; X wherein
-for OH
-, Cl
-, SO
4 2-, COO
-, NO
3 -or CH
3cOO
-, as commercial Sepra
tMsAX, ZORBAX SAX or Alltech Anion/S.
Above-mentioned moving phase is methanol aqueous solution, aqueous ethanolic solution, isopropanol water solution, aqueous acetone solution or acetonitrile solution.
In step (1), the moving phase of balance chromatographic column is preferably the aqueous acetone solution of volume ratio 85-95%.
In step (2), moving phase during loading is preferably the aqueous acetone solution of volume ratio 85-95%.
In step (3), moving phase during wash-out is isocratic elution or multistage isocratic elution or linear gradient elution, if adopt the moving phase of isocratic elution to be preferably the aqueous acetone solution of volume ratio 85-95%; If adopt the multistage during isocratic elution, first adopt the aqueous acetone solution of volume ratio 85-95% to be eluted to rebaudioside C and go out peak and finish, then switch to aqueous acetone solution or the pure water wash-out lower than volume ratio 85%; If the moving phase concentration while adopting linear gradient elution to be loading is to pure water solution.
The flow velocity of above-mentioned chromatographic column balance, loading, each step of wash-out is 50-600cm/hr, is preferably 200-250cm/hr.
Compared with prior art, the present invention has following useful technique effect:
1, the adsorptive capacity of quaternary amine base chromatograph packing material and selectivity are all obviously better than primary amine groups chromatograph packing material.Under same production task, adsorptive capacity is larger, and required chromatograph packing material is fewer, so the investment of chromatograph packing material and chromatographic equipment all significantly declines;
2, silica gel is rigid backbone, and volume stability in organic solvent has overcome that the superpolymer filler post that volume change causes in organic solvent is pressed the broken of fluctuation, filler and the drawback such as be difficult to flow;
3, the product purity that the inventive method obtains is high, is suitable for suitability for industrialized production.
Embodiment
Following examples are used for further illustrating the present invention, but the mode described in embodiment that do not represent is to implement unique channel of the present invention, does not also mean that any limitation of the invention.
The comparison of embodiment 1 quaternary amine based filler and primary amine groups filler
60% purity RA is so kind as to give by Qufu Sheng Ren pharmaceutical Co. Ltd, with 90% (v/v) acetone-water solution preparation, becomes 10 g/L sample solutions standby.
Respectively get 1.0 g quaternary amine based filler Sepra
tMsAX(Phenomenex) with primary amine groups filler Sepra
tMnH
2in 100 mL triangular flasks, respectively pipette the above-mentioned 60% purity RA sample solution of 7.5 mL, 7.5 mL deionized waters in triangular flask.Triangular flask is placed into shaking table, and 30 ℃, 150 rpm keep 12 h. test results as follows.
Adsorptive capacity in table 1
In formula,
q e, RA : adsorptive capacity (mg/g);
c 0 ,
c e : the sample concentration (mg/mL) during initial and balance,
v: adsorption liquid volume (mL),
w dry : chromatograph packing material butt quality (g)
Adsorption selectivity
α a-B : the adsorption selectivity of A material to B material;
s a ,
s b : after adsorption equilibrium, substance A, the B concentration in filler,
l a ,
l b : after adsorption equilibrium, substance A, the B concentration in adsorption liquid, A represents RA, B represents ST or RC.
Visible, quaternary amine base silica gel chromatographic column filling material to the adsorptive capacity of content rebaudioside-A (
q e ,
rA) and the selectivity (α to each material
rA-ST, α
rA-RC) all have significantly and improve.
Embodiment 2 isocratic elutions
Sample solution preparation is as embodiment 1.By Sepra
tMsAX silica matrix quaternary amine base chromatograph packing material homogenate method packs 10*250 mm stainless steel chromatogram post into, dress column pressure 100 bar.Then use 100 mL 90% (v/v) the acetone-water solution all identical therewith with the following step flow velocity of 225 cm/hr() stand-by after flow velocity balance chromatographic column.
First pump into the RA sample solution that 22.7 mL embodiment 1 prepare, then with 90% acetone-water eluant solution substep, collect elutriant and amount to 375 mL.After HPLC detects, merge the component that RA purity is higher, obtain RA purity 98.20% product 13.31g, yield is 97.72%.
Embodiment 3 gradient elutions
The preparation of sample solution is with embodiment 1, and the filling of chromatographic column and balance are with embodiment 2.
First pump into the RA sample solution that 22.7 mL embodiment 1 prepare, then with 90% acetone-water eluant solution and with HPLC, monitor, when RA starts to spill, moving phase switches to pure water, collects pure water elutriant 25 mL, obtain RA purity 97.56% product 13.39g, yield is 98.31%.
Embodiment 4
Contriver adopts and embodiment 2 and 3 similar elution processs, different is ZORBAX SAX (Agilent Technologies) quaternary amine based filler or Alltech Anion/S (Grace) the quaternary amine based filler of commodity in use, the RA purity obtaining is all greater than 97%, and yield is all greater than 97%.
Claims (10)
1. a method for separation and purification content rebaudioside-A, is characterized in that, comprises the following steps:
(1) chromatographic column balance: pass into moving phase balance chromatographic column in chromatographic column;
(2) loading: the dissolving crude product that contains content rebaudioside-A, in moving phase, is pumped in chromatographic column or by by content rebaudioside-A crude product moving phase and chromatograph packing material mix, dried chromatograph packing material is put into the column cap of chromatographic column; Chromatograph packing material is quaternary amine base chromatograph packing material;
(3) wash-out: with ultraviolet or ELSD detector monitors, be eluted to content rebaudioside-A with moving phase and go out peak from chromatographic column and finish.
2. the method for separation and purification content rebaudioside-A according to claim 1, is characterized in that: the skeleton of described quaternary amine base chromatograph packing material is silica gel, aluminum oxide or zirconium dioxide, and functional group is quaternary amine base-CH
2n
+(CH
3)
3x
-or-CH
2n
+(CH
3)
2cH
2cH
2oHX
-; X wherein
-for OH
-, Cl
-, SO
4 2-, COO
-, NO
3 -or CH
3cOO
-.
3. the method for separation and purification content rebaudioside-A according to claim 2, the skeleton that it is characterized in that quaternary amine base chromatograph packing material is silica gel.
4. the method for separation and purification content rebaudioside-A according to claim 2, is characterized in that quaternary amine base chromatograph packing material is commercial Sepra
tMsAX, ZORBAX SAX or Alltech Anion/S.
5. the method for separation and purification content rebaudioside-A according to claim 2, is characterized in that: moving phase is methanol aqueous solution, aqueous ethanolic solution, isopropanol water solution, aqueous acetone solution or acetonitrile solution.
6. the method for separation and purification content rebaudioside-A according to claim 2, is characterized in that: in step (1) and (2), the moving phase when moving phase of balance chromatographic column and loading is the aqueous acetone solution of volume ratio 85-95%.
7. the method for separation and purification content rebaudioside-A according to claim 2, is characterized in that: in step (3), moving phase during wash-out is isocratic elution or multistage isocratic elution or linear gradient elution.
8. the method for separation and purification content rebaudioside-A according to claim 7, is characterized in that: the moving phase of isocratic elution is the aqueous acetone solution of volume ratio 85-95%.
9. the method for separation and purification content rebaudioside-A according to claim 7, it is characterized in that: the multistage is during isocratic elution, first adopt the aqueous acetone solution of volume ratio 85-95% to be eluted to rebaudioside C and go out peak and finish, then switch to aqueous acetone solution or the pure water wash-out lower than volume ratio 85%.
10. the method for separation and purification content rebaudioside-A according to claim 7, is characterized in that: moving phase concentration when linear gradient elution is loading is to pure water solution.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897074A (en) * | 2019-03-07 | 2019-06-18 | 黑龙江八一农垦大学 | A kind of method that silica matrix strong anion exchange chromatographic isolates and purifies steviol glycoside |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101200480A (en) * | 2006-12-15 | 2008-06-18 | 成都华高药业有限公司 | Rebaudioside A and extraction method thereof |
| WO2009140394A1 (en) * | 2008-05-13 | 2009-11-19 | Cargill, Incorporated | Separation of rebaudioside a from stevia glycosides using chromatography |
| CN102050850A (en) * | 2009-10-30 | 2011-05-11 | 兴化格林生物制品有限公司 | New extraction technology of stevioside and high purity rebaudioside A |
| WO2012042508A1 (en) * | 2010-10-01 | 2012-04-05 | Shanghai Yongyou Bioscience Inc. | Separation and purification of stevioside and rebaudioside a |
-
2013
- 2013-11-04 CN CN201310532268.5A patent/CN103613622B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101200480A (en) * | 2006-12-15 | 2008-06-18 | 成都华高药业有限公司 | Rebaudioside A and extraction method thereof |
| WO2009140394A1 (en) * | 2008-05-13 | 2009-11-19 | Cargill, Incorporated | Separation of rebaudioside a from stevia glycosides using chromatography |
| CN102050850A (en) * | 2009-10-30 | 2011-05-11 | 兴化格林生物制品有限公司 | New extraction technology of stevioside and high purity rebaudioside A |
| WO2012042508A1 (en) * | 2010-10-01 | 2012-04-05 | Shanghai Yongyou Bioscience Inc. | Separation and purification of stevioside and rebaudioside a |
Non-Patent Citations (4)
| Title |
|---|
| DOMINIK BERGS等: "Preparative purification of rebaudioside A from aqueous extracts using chromatography: a process idea", 《J. VERBR. LEBENSM.》, vol. 7, no. 4, 14 August 2012 (2012-08-14), XP035144190, DOI: doi:10.1007/s00003-012-0784-x * |
| 施荣富等: "季铵基吸附树脂的合成及其在甜菊甙提取分离中的应用", 《离子交换与吸附》, vol. 17, no. 02, 28 April 2001 (2001-04-28), pages 117 - 122 * |
| 胡静等: "大孔树脂D107和D108对甜菊糖中SS和RA的分离研究", 《食品研究与开发》, vol. 29, no. 06, 5 June 2008 (2008-06-05), pages 1 - 4 * |
| 陈斌等: "亲水作用色谱法测定甜菊糖主要极性组分", 《食品科学》, vol. 34, no. 14, 5 January 2013 (2013-01-05) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897074A (en) * | 2019-03-07 | 2019-06-18 | 黑龙江八一农垦大学 | A kind of method that silica matrix strong anion exchange chromatographic isolates and purifies steviol glycoside |
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| CN103613622B (en) | 2016-08-17 |
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