CN103755674B - The collaborative HSCCC of amination silica gel extracts the method for soybean isoflavone in soy sauce residues - Google Patents
The collaborative HSCCC of amination silica gel extracts the method for soybean isoflavone in soy sauce residues Download PDFInfo
- Publication number
- CN103755674B CN103755674B CN201410013130.9A CN201410013130A CN103755674B CN 103755674 B CN103755674 B CN 103755674B CN 201410013130 A CN201410013130 A CN 201410013130A CN 103755674 B CN103755674 B CN 103755674B
- Authority
- CN
- China
- Prior art keywords
- silica gel
- phase
- soybean isoflavone
- water
- soy sauce
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
- C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses the collaborative HSCCC of amination silica gel and extract the method for soybean isoflavone in soy sauce residues. The method by soy sauce residues extract through MPLC purification, eluting; Normal hexane, ethyl acetate, first alcohol and water are placed, with lower phase in separation; Above as fixing phase, lower phase is mobile phase, prepares and separates sample solution; Dehydrated alcohol distilled water adds positive esters of silicon acis, regulates pH value, reaction, add �� aminopropyl triethoxysilane, still aging, prepare �� Aminopropyl silica gel microsphere; Using upper as fixing phase, lower phase is that mobile phase pumps into high-speed counter-current chromatograph, injects the upper phase added with �� Aminopropyl silica gel microsphere and the mixed liquor of lower phase, carries out high speed adverse current chromatogram separation, obtain soybean isoflavone. The present invention utilizes the liquid liquid distribution of adverse current chromatogram and the synergism of silica gel microball modified group absorption thereof, improves the separating effect of highly polar natural product, effectively solves a difficult problem for highly polar separating substances in high speed adverse current chromatogram.
Description
Technical field
The present invention relates to soybean isoflavone, particularly relate to the collaborative HSCCC of a kind of amination silica gel and extract the method for soybean isoflavone in soy sauce residues, belong to processing of agriculture product and food industrial technical field.
Background technology
Soy sauce residues is the side-product that soy sauce produces, and production process has a large amount of soy sauce residues to generate. At present that its exploitation are limited, it is mainly used as the dispensing of feedstuff or fertilizer, added value is low, the extraction of its effective ingredient is separated and higher value application is the important directions that soy sauce residues develops. Therefore, extracting the soybean isoflavone isolating medical value, the research that soy sauce residues is recycled will have great importance. Even if the bean dregs after make soybean sauce still contain abundant soybean isoflavone composition, soybean isoflavone is a kind of natural plants estrogen in Semen sojae atricolor, there is various biological effect, closely related with health, except there is antitumor action, also aging, cardiovascular disease, osteoporosis and climacteric syndrome had preventive and therapeutic action. Therefore, in order to utilize soy sauce residues resource, extracting the soybean isoflavone isolating medical value, the research that soy sauce residues is recycled will have great importance.
Middle pressure chromatograph (MPLC) is relative to normal pressure chromatograph, and separation efficiency is higher; The ratio economy that high pressure preparative hplc is come, instrument requirements configuration is low; Use prepacked column, it is possible to improve efficiency. It is mainly used in the separation in substantial amounts of complicated organic mixture, combinatorial chemistry, new drug development or Natural products research process and purification, there is the features such as speed is fast, economize solvent, fractional dose is big and reproducible.
High speed adverse current chromatogram (HSCCC) is the liquid liquid partition chromatography isolation technics of a kind of continuous high-efficient that the eighties in 20th century grows up.Its principle is the change centrifugal force field that produces when doing planetary motion of serpentine pipe detached dowel mutually permanent to be retained in serpentine pipe by fixing for a part, makes two-phase solvent distribution in unipolarity in detached dowel, forms monophasic fluid dynamic equilibrium system. Mobile phase touches with fixing contrary multiple connection, and solute is by biphase repeatedly extraction, distribution, finally according to the difference of partition coefficient, it is achieved separate. It not only enables sample all reclaim, and the sample of recovery more can reflect its original characteristic, is particularly well-suited to the separation of natural bioactive ingredients.
Now soybean isoflavone being extracted, mainly based on heating and refluxing extraction, but heating and refluxing extraction is inapplicable to thermally labile component, and temperature is too high causes that part flavone compound decomposes; When being amplified to commercial production, ultrasonic extracting method is poor. High-speed countercurrent chromatography separate some simple systems and in, there is other isolation technics in low polarity sample system without superable advantage, but highly polar separating substances effect is subject to the restriction of dicyandiamide solution.
Summary of the invention
It is an object of the invention to overcome prior art Problems existing, it is provided that the collaborative HSCCC of a kind of amination silica gel extracts the method for soybean isoflavone in soy sauce residues.
The present invention utilizes the liquid liquid distribution of adverse current chromatogram and the synergism of silica gel microball modified group adsorbing separation thereof, improves the separating effect of adverse current chromatogram, improves separation efficiency. The present invention adds �� Aminopropyl silica gel microsphere in the process of high performance countercurrent chromatography separating natural product, utilizes the synergism of HSCCC solvent system and silica gel microball, improves the separating effect of natural product. The present invention is by the surface of highly polar amino bonded to silica gel microball, it is possible to resolve the separation problem of highly polar material, and hydrophilic Interaction Chromatography can meet the needs of this separation; The present invention will introduce �� Aminopropyl silica gel microsphere in current chromatographic column, utilize the liquid liquid distribution of adverse current chromatogram and the synergism of silica gel microball modified group absorption thereof, improve the separating effect of highly polar natural product; The present invention is not changing on the basis of adverse current chromatogram instrument, use for reference the Research Thinking of high performance liquid chromatography, adverse current chromatogram liquid liquid distribution principle is organically combined with the adsorptive separation function of �� Aminopropyl silica gel microsphere, current chromatographic column is inserted �� Aminopropyl silica gel microsphere and it is modified, introduce functionalization group, utilize the liquid liquid distribution of adverse current chromatogram and the synergism of silica gel microball modified group adsorbing separation thereof, improve the separating effect of adverse current chromatogram, improve separation efficiency.
The present invention to achieve these goals, adopts the following technical scheme that
The collaborative HSCCC of a kind of amination silica gel extracts the method for soybean isoflavone in soy sauce residues, comprises the following steps:
(1) preparation of crude extract: by soy sauce residues extract through MPLC purification, with ethanol distillation water for eluent, flow velocity is that 15 25mL/min carry out gradient elution, is then concentrated by solvent under reduced pressure, obtains soybean isoflavone crude extract, put Refrigerator store;
(2) preparation of sample solution: join in separatory funnel by normal hexane, ethyl acetate, first alcohol and water, fully places 12 24 hours after concussion, by upper phase with under be separated; By volume number meter, normal hexane is 100 parts, and ethyl acetate is 150 200 parts, and methanol is 150 250 parts, and water is 150 200 parts; Above as fixing phase, lower phase is mobile phase; To mix mutually with under 80 130 parts in 100 parts by volume, every milliliter above dissolves soybean isoflavone crude extract 3 15 milligrams with the mixture of lower phase, prepares and separate sample solution;
(3) preparation of �� Aminopropyl silica gel microsphere: by volume number meter, 100 120 parts of dehydrated alcohol distilled water add 13 15 parts of positive esters of silicon acis, regulate pH value to 79, after reacting 0.5 1.0h, add 23 parts of �� aminopropyl triethoxysilanes, still aging 24 48h, prepare �� Aminopropyl silica gel microsphere; The volume ratio of dehydrated alcohol and distilled water is 3:5 3:7;
(4) high-speed counter-current separates: pumps into high-speed counter-current chromatograph using upper mutually as fixing, is full of by the multilamellar sebific duct post of high-speed counter-current chromatograph; When high-speed counter-current chromatograph stable operation, it is mobile phase by lower phase, mobile phase is pumped into multilamellar sebific duct front end; When have 15 25mL fixing be pushed out mutually time, inject the mixed liquor of the 1 3mL upper phase added with �� Aminopropyl silica gel microsphere and lower phase, and every milliliter upper and addition �� Aminopropyl silica gel microsphere 100 300 milligrams in the mixture of lower phase; When there being mobile phase to flow out from multilamellar sebific duct tail end, 5 15mL sample solutions are injected from injection port, be supplemented with injecting the fixing phase of 10 20mL, carry out high speed adverse current chromatogram separation, obtain soybean isoflavone.
For realizing the object of the invention further, it is preferable that described gradient elution is in front 15min, carries out eluting with pure water; In 15 30min, eluent is 20% ethanol 80% water; In 30 45min, eluent is 40% ethanol 60% water; In 45 60min, eluent is 60% ethanol 40% water; In 60 75min, carry out eluting with 80% ethanol 20% water; In 75 90min, carry out eluting with 95% ethanol 5% water; Then solvent under reduced pressure is concentrated, obtain soybean isoflavone crude extract, put Refrigerator store;
Described using upper as the fixing high-speed counter-current chromatograph that pumps into mutually be mutually pump into high-speed counter-current chromatograph by upper as fixing using the flow velocity of 8.5 10.5mL/min. The described multilamellar sebific duct front end that is pumped into by mobile phase is that with the flow velocity of 1.2 1.8mL/min, mobile phase is pumped into multilamellar sebific duct front end. The speed of described stable operation is preferably 600 800rpm.
Continue to detect the effluent that high speed adverse current chromatogram separates at 254nm wavelength by UV-detector, obtain the HSCCC chromatogram of soy sauce residues extract, the appearance time according to peaks different in spectrogram, collect the effluent at each peak, obtain soybean isoflavone.
Compared with the prior art, there is advantages that
(1) present invention need not the liquid of any solid-state carrier, liquid chromatography technology, separation efficiency is high, and product purity is high.
(2) present invention is absent from carrier to the absorption of sample and pollution, and preparation amount is big and solvent consumption is few.
(3) invention significantly improves the added value of soy sauce residues, be conducive to the comprehensive development and utilization of soy sauce residues.
(4) it is suitable for the extraction of highly polar natural product in other, the adsorption of polarity sample is more notable.
Accompanying drawing explanation
Figure 1A, 1B, 1C, 1D are the HSCCC chromatogram of the soy sauce residues extract of �� Aminopropyl silica gel microsphere addition respectively 0mg, 200mg, 400mg and 600mg in comparative example and embodiment 1.
The HSCCC chromatogram of soy sauce residues extract when Fig. 2 A, 2B are embodiment 2 flow velocity respectively 1.2mL/min and 1.8mL/min.
Fig. 3 A, 3B, 3C are the HSCCC chromatogram of embodiment sample size respectively 50mg, 150mg and 200mg.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described, but present invention scope required for protection is not limited to the scope that embodiment is stated.
In high-speed countercurrent chromatography extraction soy sauce residues, the method for soybean isoflavone is for comparative example.
Comparative example
By pressure chromatograph (MPLC) purification in soy sauce residues extract warp, with ethanol distillation water for eluent, flow velocity is that 20mL/min carries out gradient elution. During 0 15min, carry out eluting with 100% water; During 15 30min, eluent is 20% ethanol, 80% water; During 30 45min, eluent is 40% ethanol, 60% water; During 45 60min, eluent is 60% ethanol, 40% water; During 60 75min, with 80% ethanol, 20% water carries out eluting; During 75 90min, with 95% ethanol, 5% water carries out eluting. Object soybean isoflavone is detected with wavelength 254nm. Then solvent under reduced pressure is concentrated, obtain soybean isoflavone crude extract, put Refrigerator store;
Normal hexane, ethyl acetate, first alcohol and water 1:2:2:1.8 by volume are joined in separatory funnel, fully place after concussion an evening, by upper phase with under be separated, above as stationarity, lower phase is mobile phase. Every milliliter above dissolves crude extract 5 milligrams with the mixture of lower phase (volume ratio is 1: 1), prepares and separate sample solution.
Input high-speed counter-current chromatograph with 9.9mL/min by infusion pump as fixing using upper, will multilamellar sebific duct be full of. Instrument is run with the velocity-stabilization of 700rpm, makes to be formed in instrument one-way fluid dynamical system, be that mobile phase is pumped into multilamellar sebific duct front end with the flow velocity of 1.2mL/min by lower phase, reach one-way fluid dynamic equilibrium state. When there being mobile phase to flow out from tail end, 2mL separating sample solution (every milliliter above dissolves 50mg sample with lower phase mixture) and injects from injection port, continuation mobile phase carries out eluting. Continuing to detect the effluent of sebific duct afterbody by UV-detector at 254nm, as shown in Figure 1A, the appearance time according to peaks different in spectrogram, from appearance time just to the effluent at each peak of manual collection, appearance time end, then through high-efficient liquid identification of phases purity. Using methanol-0.1% acetic acid water as mobile phase, the flow velocity of mobile phase is 0.8mL/min, carry out binary linear gradient eluting (methanol: l0 20min, 45 65%; 20 50min, 65%; 20 25min, 65 75%), detect object soybean isoflavone with wavelength 254nm. Finally record corresponding retention with graduated cylinder.
Embodiment 1
By soy sauce residues extract through MPLC purification, with ethanol distillation water for eluent, flow velocity is that 20mL/min carries out gradient elution. During 0 15min, carry out eluting with 100% water; During 15 30min, by volume, eluent is 20% ethanol, 80% water; During 30 45min, by volume, eluent is 40% ethanol, 60% water; During 45 60min, by volume, eluent is 60% ethanol, 40% water; During 60 75min, by volume, with 80% ethanol, 20% water carries out eluting; During 75 90min, with 95% ethanol, 5% water carries out eluting. Object soybean isoflavone is detected with wavelength 254nm. Then solvent under reduced pressure is concentrated, obtain soybean isoflavone crude extract, put Refrigerator store;
Normal hexane, ethyl acetate, first alcohol and water 1:2:2:1.8 by volume are joined in separatory funnel, fully place after concussion an evening, by upper phase with under be separated; Every milliliter above dissolves crude extract 5 milligrams with the mixture of lower phase (volume ratio is 1: 1), prepares and separate sample solution;
Add 100mL dehydrated alcohol/distilled water (v:v=3:7), add the positive esters of silicon acis of 15mL, regulate pH value to 9.After reaction 0.5h, add 3mL �� aminopropyl triethoxysilane, still aging 24h, prepare �� Aminopropyl silica gel microsphere.
High-speed countercurrent chromatography is the liquid liquid partition chromatography isolation technics of a kind of continuous high-efficient without solid carrier, adopt the spiral sebific duct post of multi-lay winding, the asymmetric centrifugal force field produced by the high speed planetary motion of cylinder realizes the high efficient mixed of two phase solvent system, distributes and be sufficiently reserved, formed and flow extraction continuously, thus realizing difference to dissolve the solute of partition coefficient high efficiency separation in two-phase solvent. Mutually pump into high-speed counter-current chromatograph by upper as fixing using the flow velocity of 9.8mL/min, multilamellar sebific duct post is full of; When instrument runs with the velocity-stabilization of 700rpm, it is the multilamellar sebific duct front end that mobile phase is pumped in high-speed counter-current chromatograph with the flow velocity of 1.2mL/min by lower phase; Observe that 20mL's fixes when being pushed out mutually (filling with), injected the phase mixed liquor up and down of 2mL �� Aminopropyl silica gel microsphere by six-way valve; When there being mobile phase to flow out from multilamellar sebific duct tail end, 10mL is separated sample solution and injects from injection port, be supplemented with following closely injecting the fixing phase of 15mL, carry out high speed adverse current chromatogram separation; Continue to detect effluent at 254nm wavelength by UV-detector, addition (situation that Figure 1A represents does not add �� Aminopropyl silica gel microsphere) according to �� Aminopropyl silica gel microspheres different as follows, respectively obtains the HSCCC chromatogram of the soy sauce residues extract that Figure 1B, 1C, 1D are shown �� Aminopropyl silica gel microsphere addition respectively 200mg, 400mg and 600mg. According to the appearance time of peak I and peak II in Figure 1A, 1B, 1C, 1D, manually collect the effluent at each peak respectively, obtain being dissolved in the soybean isoflavone of mobile phase, then purity is identified through high performance liquid chromatography, the HPLC measurement result comparison of the standard substance according to soybean isoflavone, showing that peak I is daidzein, peak II is genistein (being referred to as soybean isoflavone). Finally record corresponding retention with graduated cylinder. Testing result is as shown in table 1, and the amount of the �� Aminopropyl silica gel microsphere added in table 1 is 200mg, 400mg, 600mg, and namely every milliliter above adds �� Aminopropyl silica gel microsphere 100,200,300 milligrams with the mixture of lower phase (volume ratio is 1: 1). Comparative example does not add Aminopropyl silica gel microsphere.
HSCCC separation parameter is affected by the different amounts of �� Aminopropyl silica gel microsphere of table 1
In table, tR1For the retention time of peak I, tR2Retention time for peak II; Rs: the separating degree of peak I and peak II; Sf(%): fixing phase retention rate; N: theoretical cam curve.
Analyzed by table 1 and Figure 1A, 1B, 1C, 1D: when HSCCC separates sample, the separating degree Rs at peak is equal to the twice of the difference of adjacent chromatographic peak retention time and the ratio of two chromatographic peak peak sound stage width sums, and the separating degree Rs at peak increases along with the amount of �� Aminopropyl silica gel microsphere and is incrementally increased. While retention time increases, peak width also increases, and the increase degree of peak width becomes apparent from than the increase of retention time, causes that theoretical cam curve decreases on the contrary. Equally, not there is obvious change because of adding silica gel microball in system in the retention rate of fixing phase.
This embodiment shows, when HSCCC separates the soybean isoflavone of middle polarity in soy sauce residues, the �� Aminopropyl silica gel microsphere of addition and solvent system interact. Add 200 600mg �� Aminopropyl silica gel microspheres, change peak I and the retention time of peak II so that it is the difference of retention time increases, so that separating degree all has increase in various degree.Separating degree shows the separation case in chromatography, often makes the index of over-all resolution efficiency. Separating degree is more big, it was shown that separating effect is more good, and object separates more thorough with other components. The polar group of sample is attracted to the polar stationary phase surface containing amino. In elution process, non-polar solven (weak polar solvent) replaces sample polar group to be attracted to fixing phase surface. Under same chromatographic condition, in comparative example, separating degree Rs is only 1.57, adds �� Aminopropyl silica gel microsphere and separating degree can be made to significantly increase, and adds 200mg �� Aminopropyl silica gel microsphere and makes separating degree increase to 1.67. Along with the �� Aminopropyl silica gel microsphere added increases, when adding 400mg �� Aminopropyl silica gel microsphere, separating degree Rs reaches 1.75, embodies the extraction efficiency of the collaborative high speed adverse current chromatogram of �� Aminopropyl silica gel microsphere higher than high-speed countercurrent chromatography. Utilize the liquid liquid distribution of adverse current chromatogram and the synergism of silica gel microball modified group adsorbing separation thereof, adverse current chromatogram liquid liquid distribution principle is organically combined with the adsorptive separation function of functional silica gel microsphere, the separating effect of adverse current chromatogram can be improved, improve separation efficiency.
Embodiment 2
By soy sauce residues extract through MPLC purification, with ethanol distillation water for eluent, flow velocity is that 15 25mL/min carry out gradient elution. During 0 15min, carry out eluting with 100% water; During 15 30min, by volume, eluent is 20% ethanol, 80% water; During 30 45min, by volume, eluent is 40% ethanol, 60% water; During 45 60min, by volume, eluent is 60% ethanol, 40% water; During 60 75min, with 80% ethanol, 20% water carries out eluting; During 75 90min, with 95% ethanol, 5% water carries out eluting. Object soybean isoflavone is detected with wavelength 254nm. Then solvent under reduced pressure is concentrated, obtain soybean isoflavone crude extract, put Refrigerator store;
Normal hexane, ethyl acetate, first alcohol and water 1:2:2:1.8 by volume are joined in separatory funnel, fully place after concussion an evening, by upper phase with under be separated; Every milliliter above dissolves crude extract 5 milligrams with the mixture of lower phase (volume ratio is 1: 1), prepares and separate sample solution;
100mL dehydrated alcohol/distilled water (v:v=3:5) adds the positive esters of silicon acis of 13mL, regulates pH value to 9. After reaction 0.5h, add 2.5mL �� aminopropyl triethoxysilane, still aging 24h, prepare �� Aminopropyl silica gel microsphere.
Input high-speed counter-current chromatograph with 9.9mL/min by infusion pump as fixing using upper, will multilamellar sebific duct be full of. instrument is run with the velocity-stabilization of 700rpm, make to be formed in instrument one-way fluid dynamical system, it is mobile phase by lower phase, flow rate of mobile phase is 1.2mL/min respectively, 1.5mL/min, 1.8mL/min, it was observed that when having 20mL fixing to be pushed out mutually, injecting 2mL by six-way valve and add the phase mixed liquor up and down of �� Aminopropyl silica gel microsphere, every milliliter above adds �� Aminopropyl silica gel microsphere 200 milligrams with the mixture of lower phase (volume ratio is 1: 1), when there being mobile phase to flow out from multilamellar sebific duct tail end, 10mL is separated sample solution and injects from injection port, be supplemented with injecting that 15mL is fixing carries out high speed adverse current chromatogram separation mutually, continue to detect effluent at 254nm wavelength by UV-detector, according to flow velocitys different as follows, obtain Fig. 2 A, the HSCCC chromatogram of soy sauce residues extract during 2B flow velocity respectively 1.2mL/min and 1.8mL/min, according to Fig. 2 A, the appearance time of peak I and peak II in 2B, the effluent at each peak of manual collection respectively, obtain being dissolved in the soybean isoflavone of mobile phase, then purity is identified through high performance liquid chromatography, the HPLC measurement result comparison of the standard substance according to soybean isoflavone, show that peak I is daidzein, peak II is genistein (being referred to as soybean isoflavone).Finally record corresponding retention with graduated cylinder. Testing result is as shown in table 2.
HSCCC separation parameter is affected by table 2 different in flow rate
In table, tR1For the retention time of peak I, tR2Retention time for peak II; Rs: the separating degree of peak I and peak II; Sf(%): fixing phase retention rate; N: theoretical cam curve.
It is shown that add silica gel microball and HSCCC generation synergism, in separation process, retention time, separating degree and theoretical cam curve are all produced impact. Sample is mainly produced adsorption by �� Aminopropyl silica gel microsphere action principle in HSCCC in separation process. Amino bonded silica gel is most commonly used in normal-phase chromatography, and when normal-phase chromatography separate substance, the polar group of sample is attracted to the polar stationary phase surface containing amino. In elution process, non-polar solven (weak polar solvent) replaces sample polar group to be attracted to fixing phase surface.
After adding �� Aminopropyl silica gel microsphere, separating degree Rs increases. When flow velocity increases, retention time diminishes, and peak-to-peak separating degree Rs all has increase in various degree compared with comparative example. Showing to add �� Aminopropyl silica gel microsphere, in different in flow rate situation, separating effect all increases.
Embodiment 3
By soy sauce residues extract through MPLC purification, with ethanol distillation water for eluent, flow velocity is that 15 25mL/min carry out gradient elution. During 0 15min, carry out eluting with 100% water; During 15 30min, by volume, eluent is 20% ethanol, 80% water; During 30 45min, by volume, eluent is 40% ethanol, 60% water; During 45 60min, by volume, eluent is 60% ethanol, 40% water; During 60 75min, by volume, with 80% ethanol, 20% water carries out eluting; During 75 90min, by volume, with 95% ethanol, 5% water carries out eluting. Object soybean isoflavone is detected with wavelength 254nm. Then solvent under reduced pressure is concentrated, obtain soybean isoflavone crude extract, put Refrigerator store;
Normal hexane, ethyl acetate, first alcohol and water 1:2:2:1.8 by volume are joined in separatory funnel, fully place after concussion an evening, by upper phase with under be separated; Soybean isoflavone crude extract is dissolved in the mixture of upper and lower phase (volume ratio is 1: 1), prepares and separate sample solution. The sample size separating sample is 50mg, 100mg, 150mg, 200mg, and namely every milliliter above separates sample 5mg, 10mg, 15mg, 20mg with adding in lower phase mixture.
Add 100mL dehydrated alcohol/distilled water (v:v=3:7), add the positive esters of silicon acis of 13mL, regulate pH value to 9. After reaction 0.5h, add 2.5mL �� aminopropyl triethoxysilane, still aging 24h, prepare �� Aminopropyl silica gel microsphere.
Input high-speed counter-current chromatograph with 9.9mL/min by infusion pump as fixing using upper, will multilamellar sebific duct be full of. Instrument is run with the velocity-stabilization of 700rpm, makes to be formed in instrument one-way fluid dynamical system, be that mobile phase is pumped into sebific duct front end with the flow velocity of 1.2mL/min by lower phase, reach one-way fluid dynamic equilibrium state. Observe time 20mL fixing is pushed out mutually, inject the mixed liquor of phase up and down (with addition �� Aminopropyl silica gel microsphere 200 milligrams in the mixture of lower phase (volume ratio is 1: 1) every milliliter) of 2mL �� Aminopropyl silica gel microsphere from injection port. When there being mobile phase to flow out from the afterbody of tail end, 10mL separating sample solution and injects from injection port, be supplemented with injecting the fixing phase of 15mL, continuation mobile phase carries out eluting, carries out high speed adverse current chromatogram separation.Continue to detect effluent at 254nm wavelength by UV-detector, according to sample sizes different as follows, obtain the HSCCC chromatogram of the soy sauce residues extract of the respectively 50mg of the sample size shown in Fig. 3 A, 3B, 3C, 150mg and 200mg; According to the appearance time of peak I and peak II in Fig. 3 A, 3B, 3C, manually collect the effluent at each peak respectively, the HPLC measurement result comparison of the standard substance according to soybean isoflavone, it was shown that peak I is daidzein, peak II is genistein (being referred to as soybean isoflavone). Obtain being dissolved in the soybean isoflavone of mobile phase, finally record corresponding retention with graduated cylinder. Testing result is as shown in table 3.
HSCCC separation parameter is affected by the different sample size of table 3
In table, tR1For the retention time of peak I, tR2Retention time for peak II; Rs: the separating degree of peak I and peak II; Sf(%): fixing phase retention rate; N: theoretical cam curve.
In the HSCCC piece-rate system adding 400mg �� Aminopropyl silica gel microsphere, separate the sample size of sample in the scope of 50mg to 200mg, increase along with sample size, peak I and peak II is all advanced by out the time at peak, and separating degree also reduces therewith, the change of fixing phase retention rate is not maintained at more than 60% very much. Along with the increase of sample size, separating degree reduces, but still relatively the separating degree in comparative example 1 is high. Illustrate that, in the piece-rate system of the collaborative HSCCC of �� Aminopropyl silica gel microsphere, separating effect is better.
Claims (5)
1. the collaborative HSCCC of amination silica gel extracts the method for soybean isoflavone in soy sauce residues, it is characterised in that comprise the following steps:
(1) preparation of crude extract: by soy sauce residues extract through MPLC purification, with ethanol distillation water for eluent, flow velocity is that 15 25mL/min carry out gradient elution, is then concentrated by solvent under reduced pressure, obtains soybean isoflavone crude extract, put Refrigerator store; Described gradient elution is in front 15min, carries out eluting with pure water; With volume percentage, in 15 30min, eluent is 20% ethanol 80% water; In 30 45min, eluent is 40% ethanol 60% water; In 45 60min, eluent is 60% ethanol 40% water; In 60 75min, carry out eluting with 80% ethanol 20% water; In 75 90min, carry out eluting with 95% ethanol 5% water;
(2) preparation of sample solution: join in separatory funnel by normal hexane, ethyl acetate, first alcohol and water, fully places 12 24 hours after concussion, by upper phase with under be separated; By volume number meter, normal hexane is 100 parts, and ethyl acetate is 150 200 parts, and methanol is 150 250 parts, and water is 150 200 parts; Above as fixing phase, lower phase is mobile phase; To mix mutually with under 80 130 parts in 100 parts by volume, every milliliter above dissolves soybean isoflavone crude extract 3 15 milligrams with the mixture of lower phase, prepares and separate sample solution;
(3) preparation of �� Aminopropyl silica gel microsphere: by volume number meter, 100 120 parts of dehydrated alcohol distilled water add 13 15 parts of positive esters of silicon acis, regulate pH value to 79, after reacting 0.5 1.0h, add 23 parts of �� aminopropyl triethoxysilanes, still aging 24 48h, prepare �� Aminopropyl silica gel microsphere; The volume ratio of dehydrated alcohol and distilled water is 3:5 3:7;
(4) high-speed counter-current separates: pumps into high-speed counter-current chromatograph using upper mutually as fixing, is full of by the multilamellar sebific duct post of high-speed counter-current chromatograph; When high-speed counter-current chromatograph stable operation, it is mobile phase by lower phase, mobile phase is pumped into multilamellar sebific duct front end; When have 15 25mL fixing be pushed out mutually time, inject the mixed liquor of the 1 3mL upper phase added with �� Aminopropyl silica gel microsphere and lower phase, and every milliliter upper and addition �� Aminopropyl silica gel microsphere 100 300 milligrams in the mixture of lower phase;When there being mobile phase to flow out from multilamellar sebific duct tail end, 5 15mL sample solutions are injected from injection port, be supplemented with injecting the fixing phase of 10 20mL, carry out high speed adverse current chromatogram separation, obtain soybean isoflavone.
2. the collaborative HSCCC of amination silica gel according to claim 1 extracts the method for soybean isoflavone in soy sauce residues, it is characterised in that: described using upper as the fixing high-speed counter-current chromatograph that pumps into mutually be mutually pump into high-speed counter-current chromatograph by upper as fixing using the flow velocity of 8.5 10.5mL/min.
3. the collaborative HSCCC of amination silica gel according to claim 1 extracts the method for soybean isoflavone in soy sauce residues, it is characterised in that: the speed of described stable operation is 600 800rpm.
4. the collaborative HSCCC of amination silica gel according to claim 1 extracts the method for soybean isoflavone in soy sauce residues, it is characterised in that: the described multilamellar sebific duct front end that is pumped into by mobile phase is that with the flow velocity of 1.2 1.8mL/min, mobile phase is pumped into multilamellar sebific duct front end.
5. the collaborative HSCCC of amination silica gel according to claim 1 extracts the method for soybean isoflavone in soy sauce residues, it is characterized in that: continue to detect the effluent that high speed adverse current chromatogram separates at 254nm wavelength by UV-detector, obtain the HSCCC chromatogram of soy sauce residues extract, appearance time according to peaks different in spectrogram, collect the effluent at each peak, obtain soybean isoflavone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410013130.9A CN103755674B (en) | 2014-01-10 | 2014-01-10 | The collaborative HSCCC of amination silica gel extracts the method for soybean isoflavone in soy sauce residues |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410013130.9A CN103755674B (en) | 2014-01-10 | 2014-01-10 | The collaborative HSCCC of amination silica gel extracts the method for soybean isoflavone in soy sauce residues |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103755674A CN103755674A (en) | 2014-04-30 |
| CN103755674B true CN103755674B (en) | 2016-06-08 |
Family
ID=50523034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410013130.9A Active CN103755674B (en) | 2014-01-10 | 2014-01-10 | The collaborative HSCCC of amination silica gel extracts the method for soybean isoflavone in soy sauce residues |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103755674B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107913686A (en) * | 2017-11-17 | 2018-04-17 | 安徽师范大学 | Affine thiophilic silicon ball chromatographic material and its preparation method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103223001A (en) * | 2013-04-25 | 2013-07-31 | 徐州工程学院 | Microwave-supersonic wave cooperation method for extracting soy isoflavone in soy sauce residue |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013242284A (en) * | 2012-05-21 | 2013-12-05 | Brooks Holdings:Kk | Trigonelline separation method by high-speed counter-current chromatography |
-
2014
- 2014-01-10 CN CN201410013130.9A patent/CN103755674B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103223001A (en) * | 2013-04-25 | 2013-07-31 | 徐州工程学院 | Microwave-supersonic wave cooperation method for extracting soy isoflavone in soy sauce residue |
Non-Patent Citations (2)
| Title |
|---|
| 酱油渣中大豆异黄酮的动态逆流提取及分离纯化研究;何隽婷，等;《华南师范大学学报（自然科学版）》;20101130(第4期);第81-82页 * |
| 高速逆流色谱法分离大豆异黄酮中的大豆苷和染料木苷;钱俊青，等;《浙江工业大学学报》;20121031;第40卷(第5期);第494页右栏 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103755674A (en) | 2014-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhao et al. | Preliminary separation and purification of rutin and quercetin from Euonymus alatus (Thunb.) Siebold extracts by macroporous resins | |
| Ma et al. | Separation of four flavonoids from Rhodiola rosea by on-line combination of sample preparation and counter-current chromatography | |
| CN106866602A (en) | A kind of method that application high speed adverse current chromatogram separates flavone compound in Hericium erinaceus | |
| CN107652260B (en) | Method for preparing natural flavanone compound by high-speed countercurrent chromatography rapid separation | |
| CN107823237A (en) | A kind of extracting method of burdock root total flavone | |
| Zhang et al. | Separation and purification of flavonoid from ginkgo extract by polyamide resin | |
| CN107011308A (en) | The method that polymethoxyflavone class compound is isolated and purified from bowl mandarin orange fruit | |
| CN106349301B (en) | Method for separating and purifying punicalagin in pomegranate peel | |
| Zhu et al. | Isolation and enrichment of Ginkgo biloba extract by a continuous chromatography system | |
| CN103142685B (en) | Method for extraction of total flavonoid aglycones from hickory leaves | |
| CN109293509B (en) | Method for preparing high-purity chlorogenic acid from bamboo leaf extract | |
| Qu et al. | Combined application of macroporous resin and high speed counter‐current chromatography for preparative separation of three flavonoid triglycosides from the leaves of A ctinidia valvata D unn | |
| Wang et al. | An Efficient Strategy Based on Liquid–Liquid Extraction With Acid Condition and HSCCC for Rapid Enrichment and Preparative Separation of Three Caffeoylquinic Acid Isomers From Mulberry Leaves | |
| CN100528892C (en) | Method for separating 4-methylsulphinyl-3-cyclobutenyl glucosinolates | |
| CN103755674B (en) | The collaborative HSCCC of amination silica gel extracts the method for soybean isoflavone in soy sauce residues | |
| CN102115467A (en) | Method for preparing catechin monomers | |
| Hui et al. | Enrichment and purification of total chlorogenic acids from tobacco waste extract with macroporous resins | |
| Liu et al. | Simultaneous detoxification and preparative separation of chlorogenic acid from Eupatorium adenophorum by combined column chromatography | |
| CN103739583B (en) | The collaborative HSCCC of a kind of ODS extracts the method for soybean isoflavone in soy sauce residues | |
| CN110922439A (en) | Method for separating and preparing gram-grade high-purity natural product | |
| CN104645667A (en) | Expanded bed chromatography and counter-current chromatography on-line combination method, application and apparatus thereof | |
| CN110240585B (en) | Preparation method of agilawood tetraol | |
| CN104130299B (en) | The extraction separation method of rhamnetin-3-O-β-D-6-O-.alpha.-L-rhamnosyl-D-glucose. in Flos Caraganae Sinicae alabastrum | |
| CN104497632B (en) | A kind of method utilizing the red wood carthamin that continues of macroporous resin-active carbon compound adsorbent purification | |
| CN109369382B (en) | A method for preparing ginkgolic acids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |