CN103331036A - Chromatograph method for separating and concentrating target component from mixture - Google Patents
Chromatograph method for separating and concentrating target component from mixture Download PDFInfo
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Abstract
The invention provides a chromatograph method for separating and concentrating a target component from a mixture, and the method is a cycle operation process formed by a plurality of operating periods. The method comprises two circulating steps that: (1) absorption of the target component, namely absorbing the target component in the raw material into a chromatographic column, and separating weak reserve components; (2) desorption of the target component, namely injecting an eluent 2 into the chromatographic column, and eluting the target component in the chromatographic column; and (3) if strong reserve components exit in the raw material, desorbing the strong reserve components, namely injecting an eluent 3 into the chromatographic column, and eluting the strong reserve components in the chromatographic column. The method can be used for separating and concentrating the target component at the same time, namely, increasing the concentration of the target component in a product solution while improving the purity of the target component, thus reducing the burden of recovering the target component from the product solution. The method is especially suitable for separating and purifying of a trace component with very low content.
Description
Technical field
The present invention relates to a kind of chromatography separating method separated with the enrichment target components from mixture.
Background technology
Chromatography separating method be commonly use a kind of from mixture the method for separate targets component, in many fields extensive application such as medicine separation, Separation of Natural Products purifying.Usually, the just product solution that simple chromatographic isolation technique obtains, therefore also need to be aided with reclaimer operation, as evaporation or crystallization etc., could therefrom obtain pure target components.Obviously, product solution concentration is higher, more is conducive to reclaimer operation.
Yet, owing to having resistance to mass tranfer and axial non-ideal factors such as disperse, in most chromatographic separation process, target components always is diluted, its concentration in product solution will be lower than it concentration in material liquid.Therefore the reclaimer operation burden is increased the weight of, and makes it in total points, in cost, occupy very large proportion.In some separating technology, as the Separation of Natural Products purifying, because the content of target components is very low, reclaimer operation may determine the cost of whole separation process.Take from leaf of Japanese Yew and to extract and isolation of taxol is example, because content of taxol is very low, be only 3/10000ths left and right, also content can only be brought up to 3/1000ths left and right after extraction, now as adopted chromatographic isolation, medicinal extract is mixed with to material liquid, supposes concentration 50mg/mL, taxol concentration about 0.15mg/mL only in material liquid.Often be accompanied by the dilution of product solution when obtaining separation and purification because of product, in the product solution that chromatographic isolation obtains, taxol concentration will be much smaller than 0.15mg/mL, reclaim the taxol cost very large from so rare solution, therefore first take a plurality of pre-treatment operations, refining by chromatographic isolation again after being enriched to a certain degree by taxol, this method does not tackle the problem at its root, and just the burden of reclaimer operation is partly shifted in the pre-treatment operation.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of can be in the separation and purification target components, the concentrated and enrichment by target components, synchronously realizes the chromatography separating method of separation and enrichment target components from mixture.
In order to solve the problems of the technologies described above, first technical solution of the present invention is achieved in that
A kind of chromatography separating method separated from mixture with the enrichment target components, comprise by chromatographic column and two kinds of piece-rate systems that eluant, eluent forms; Be filled with the adsorbent that can adsorb target components in chromatographic column;
Described eluant, eluent comprises eluant, eluent 1 and eluant, eluent 2; There is the volumn concentration of solvent of strong eluting power in described eluant, eluent 2 higher than thering is the volumn concentration of the solvent of strong eluting power in eluant, eluent 1;
Described adsorbent is silica gel, polyamide, polymeric adsorbent or ion exchange resin.
Suppose that in raw material, the reservation of target components on chromatographic column is the strongest, the concrete separation and the cycling process of enrichment process for consisting of a plurality of operation cycles, first operation cycle comprises following two steps:
(1) absorption target components: raw material is dissolved in to eluant, eluent 1 and is mixed with material liquid, inject chromatographic column, at the chromatographic column afterbody, collect and obtain eluent 1.The purpose of this step operation is optionally the target components in material liquid to be adsorbed onto on chromatographic column, and the retention time weak retained fraction shorter than target components flows out chromatographic column.
(2) desorption target components: eluant, eluent 2 is injected to chromatographic column, the target components in the elution chromatography post, target components flows out from the chromatographic column afterbody, collects and obtains eluent 2.
Since second operation cycle, in the step (1) in each operation cycle, material liquid and eluent 2 collected within a upper operation cycle were merged to the injection chromatographic column, all the other are with first operation cycle;
In separation process, weak retained fraction can flow out chromatographic column and in entering eluent 1, therefore it can be separated with target components in the step (1) in each operation cycle; Target components in the step (2) in each operation cycle by desorption to eluent 2, then with new target components of supplementing in material liquid, merge, together be adsorbed to chromatographic column in the step (1) in next operation cycle.Therefore target components progressively is concentrated in eluent 2 or is enriched in chromatographic column, during meaning is eluted to n the eluent 2 in the operation cycle at the 1st to the target components in all material liquids that add in n operation cycle, or be adsorbed in interior chromatographic column of n operation cycle.If suppose, the material liquid volume added in each operation cycle is identical, is V
1, use eluant, eluent 2 volumes also identical, be V
2, to be equivalent to cumulative volume be nV in above-mentioned separation process
1material liquid in target components to be concentrated to a volume be V
2eluent 2 in, or be nV by cumulative volume
1material liquid in target components be enriched in a chromatographic column, thereby realize separating the purpose with the enrichment target components from mixture.Obviously, the operation cycle number is more, concentrated higher with enrichment degree.Usually, the component that will go out peak prior to target components is referred to as weak retained fraction, the component that is later than target components and goes out peak is called to strong retained fraction, and the retention time component shorter than target components is weak retained fraction, and the component that retention time is longer than target components is strong retained fraction.
Improvement as above-mentioned separation method, can in the step (2) in each operation cycle, be step (3) afterwards, use eluant, eluent 1 balance chromatographic column, detailed process is that eluant, eluent 1 is injected to chromatographic column, until chromatographic column outlet efflux forms identical with the composition of eluant, eluent 1; When in raw material, the reservation of target components is the strongest, three step cycle are carried out: step (1) absorption target components → step (2) desorption target components → step (3) balance chromatographic column → ...
The main points of above-mentioned isolation technics scheme are:
At first, the eluting power of eluant, eluent 2 is better than the eluting power of eluant, eluent 1, and this can realize by the composition that changes eluant, eluent 1, eluant, eluent 2.So, in the absorption target components stage in each operation cycle, because material liquid and the eluent in the upper operation cycle 2 are merged, therefore can weaken the liquid phase eluting power, when it flows through chromatographic column, inject volume by controlling material liquid, can guarantee that weak retained fraction flows out from chromatographic column, target components is not leaked from chromatographic column; In the desorption target components stage, because of the eluting power enhancing of eluant, eluent 2, only the eluant, eluent 2 of a small amount of volume of need use can be by the complete desorption of the target components on chromatographic column.Visible every through the once-through operation cycle, target components all can obtain enrichment to a certain degree and concentrate.
Secondly, collect the eluent 2 that obtains in each operation cycle not as final products solution, but itself and new supplementing after material liquid merges are added in chromatographic column again.The enrichment that this measure can obtain target components in cycling each time and the accumulation of concentrated effect, make the enrichment of target components and concentrating degree more and more higher.
In above-mentioned separation method, target components is progressively accumulation and enrichment in chromatographic column, finally must make chromatographic column reach capacity and can not adsorb more target components again.If now continue to supplement fresh feed liquid in step (1), the target components of newly supplementing in material liquid will be leaked from eluent 1, and this can not increase the utilization rate of chromatographic column, just loses time on the contrary and power; Conversely, if chromatographic column not yet reaches capacity, stop cycling, can not take full advantage of chromatographic column.Therefore should allow as far as possible chromatographic column just reach capacity, whether this can contain target components and judged by detecting eluent 1 in each operation cycle, if in the step (1) in some operation cycles while not yet finishing, target components leaks from eluent 1, shows that chromatographic column reaches capacity.Therefore, when target components in the inner constantly accumulation of chromatographic column, to such an extent as to until the saturated target components of chromatographic column while leaking from eluent 1, stops the cycling process.Now, in chromatographic column, mainly contain by the target components of enrichment, simultaneously also residual have a non-target components.If the purity requirement to target components is not high, but the target components that only need will be enriched in chromatographic column with the solvent of eluant, eluent 2 or other any wash-out target components elutes chromatographic column, collects the target components that obtains being concentrated at the chromatographic column afterbody; If high to the target components purity requirement, can be in the ensuing operation cycle, replace the new material liquid that supplements in step (1) with eluant, eluent 1, in step (1), eluent 2 no longer merges with supplementary material liquid, but eluent 2 is merged and injects chromatographic column with eluant, eluent 1, continue cycling.So, the non-target components in chromatographic column will be continued to elute chromatographic column, and target components still is concentrated in eluent 2 or is enriched in chromatographic column.
The present invention has the content of the solvent of strong eluting power by changing in eluant, eluent 1, eluant, eluent 2, thereby makes eluant, eluent 1 eluting power stronger than eluant, eluent 2 eluting powers.In the step (2) in each operation cycle, guarantee that eluant, eluent 2 consumptions are enough to reach the degree that makes target components whole desorptions from chromatographic column, step (2) finishes not contain target components in rear chromatographic column.As preferably, step (2) in each operation cycle increases step (3) afterwards, use eluant, eluent 1 balance chromatographic column, concrete steps are that eluant, eluent 1 is injected to chromatographic column, until chromatographic column outlet efflux forms identical with the composition of eluant, eluent 1.
Advantage of the present invention and beneficial effect: the present invention adopts a kind of the separation from mixture and the chromatography separating method of enrichment target components, separation and the enrichment of realize target component simultaneously, can also increase the concentration of target components in product solution when improving target components purity, thereby alleviate the burden that reclaims target components from product solution.The method is particularly suitable for separating and the very low microcomponent of purifying content.
Another technical solution of the present invention is:
A kind of chromatography separating method separated from mixture with the enrichment target components, is characterized in that: comprise by chromatographic column and at least three kinds of piece-rate systems that eluant, eluent forms; Be filled with the adsorbent that can adsorb target components in described chromatographic column;
Described eluant, eluent comprises eluant, eluent 1, eluant, eluent 2 and eluant, eluent 3; There is the volumn concentration of solvent of strong eluting power in described eluant, eluent 2 higher than thering is the volumn concentration of the solvent of strong eluting power in eluant, eluent 1; The volumn concentration that has the solvent of strong eluting power in described eluant, eluent 3 is not less than the volumn concentration that has the solvent of strong eluting power in eluant, eluent 2.
Described adsorbent is silica gel, polyamide, polymeric adsorbent or ion exchange resin;
Suppose that raw material contains weak retained fraction, target components and strong retained fraction, the reservation on chromatographic column strengthens successively, and concrete the separation and the cycling process of enrichment process for consisting of a plurality of operation cycles, first operation cycle comprises following three steps:
(1) absorption target components: raw material is dissolved in to eluant, eluent 1 and is mixed with material liquid, inject chromatographic column, at the chromatographic column afterbody, collect and obtain eluent 1.The purpose of this step operation is optionally the target components in raw material to be adsorbed onto on chromatographic column, and meanwhile, strong retained fraction also is adsorbed on chromatographic column, and the retention time weak retained fraction shorter than target components flows out chromatographic column.
(2) desorption target components: eluant, eluent 2 is injected to chromatographic column, the target components in the elution chromatography post, target components flows out from the chromatographic column afterbody, collects and obtains eluent 2; The strong retained fraction that retention time is longer than target components still is attracted in chromatographic column;
(3) the strong retained fraction of desorption: eluant, eluent 3 is injected to chromatographic column, the strong retained fraction in the elution chromatography post, strong retained fraction flows out from the chromatographic column afterbody, collects and obtains eluent 3;
Since second operation cycle, in the step (1) in each operation cycle, material liquid and eluent 2 collected within a upper operation cycle were merged to the injection chromatographic column, all the other are with first operation cycle;
In separation process, weak retained fraction is eluted chromatographic column in the step (1) in each operation cycle, and strong retained fraction is eluted chromatographic column in the step (3) in each operation cycle, therefore it is separated with target components; Target components in the step (2) in each operation cycle by desorption to eluent 2, then with new target components of supplementing in material liquid, merge, together be adsorbed to chromatographic column in the step (1) in next operation cycle, therefore target components progressively is concentrated in eluent 2 or is enriched in chromatographic column.
As the improvement of above-mentioned separation method, can within each operation cycle, add again a step, use eluant, eluent 1 balance chromatographic column, detailed process is that eluant, eluent 1 is injected to chromatographic column, until chromatographic column outlet efflux forms identical with the composition of eluant, eluent 1; Four step cycle are carried out: (1) absorption target components → (2) desorption target components → strong retained fraction of (3) desorption → (4) balance chromatographic column → ...
The main points of above-mentioned isolation technics scheme are:
At first, the eluting power of eluant, eluent 1, eluant, eluent 2 and eluant, eluent 3 strengthens successively, and this can realize by the composition that changes eluant, eluent 1, eluant, eluent 2 and eluant, eluent 3.So, in the absorption target components stage in each operation cycle, because material liquid and the eluent in the upper operation cycle 2 are merged, therefore can weaken the liquid phase eluting power, when it flows through chromatographic column, inject volume by controlling material liquid, can guarantee that weak retained fraction flows out from chromatographic column, target components is not leaked from chromatographic column; In the desorption target components stage, because of the eluting power enhancing of eluant, eluent 2, only the eluant, eluent 2 of a small amount of volume of need use can be by the complete desorption of the target components on chromatographic column.Visible every through the once-through operation cycle, target components all can obtain enrichment to a certain degree and concentrate.
Secondly, collect the eluent 2 that obtains in each operation cycle not as final products solution, but itself and new supplementing after material liquid merges are added in chromatographic column again.The enrichment that this measure can obtain target components in cycling each time and the accumulation of concentrated effect, make the enrichment of target components and concentrating degree more and more higher.
In above-mentioned separation method, target components is progressively accumulation and enrichment in chromatographic column, finally must make chromatographic column reach capacity and can not adsorb more target components again.If now continue to supplement fresh feed liquid in step (1), the target components of newly supplementing in material liquid will be leaked from eluent 1, and this can not increase the utilization rate of chromatographic column, just loses time on the contrary and power; Conversely, if chromatographic column not yet reaches capacity, stop cycling, can not take full advantage of chromatographic column.Therefore should allow as far as possible chromatographic column just reach capacity, whether this can contain target components and judged by detecting eluent 1 in each operation cycle, if when the step (1) in some operation cycles while not yet finishing, target components leaks from eluent 1, shows that chromatographic column reaches capacity.Therefore, when target components in the inner constantly accumulation of chromatographic column, to such an extent as to until the saturated target components of chromatographic column while leaking from eluent 1, stops the cycling process.Now, in chromatographic column, mainly contain by the target components of enrichment, simultaneously also residual have a non-target components.If the purity requirement to target components is not high, but the target components that only need will be enriched in chromatographic column with the solvent of eluant, eluent 2 or other any wash-out target components elutes chromatographic column, collect the eluent 2 obtained and be by the solution of the target components of enrichment; If high to the target components purity requirement, can be in the ensuing operation cycle, replace the new material liquid that supplements in step (1) with eluant, eluent 1, in step (1), eluent 2 no longer merges with supplementary material liquid, but eluent 2 is merged and injects chromatographic column with eluant, eluent 1, continue cycling.So, the non-target components in chromatographic column will be continued to elute chromatographic column, and target components still is concentrated in eluent 2 or is enriched in chromatographic column.
The present invention has the content of the solvent of strong eluting power by changing in eluant, eluent 1, eluant, eluent 2 and eluant, eluent 3, thus make eluant, eluent 1, eluant, eluent 2 and eluant, eluent 3 eluting power strengthen successively.In the step (2) in each operation cycle, guarantee that eluant, eluent 2 consumptions are enough to reach the degree that makes target components whole desorptions from chromatographic column, be not contain target components in chromatographic column after step (2) finishes, in the step (3) in each operation cycle, guarantee that eluant, eluent 3 consumptions are enough to reach the degree that makes strong retained fraction whole desorptions from chromatographic column, after step (3) finishes, in chromatographic column, do not contain strong retained fraction.As preferably, step (3) in each operation cycle increases step (4) afterwards, use eluant, eluent 1 balance chromatographic column, concrete steps are that eluant, eluent 1 is injected to chromatographic column, until chromatographic column outlet efflux forms identical with the composition of eluant, eluent 1.
Advantage of the present invention and beneficial effect: the present invention adopts a kind of the separation from mixture and the chromatography separating method of enrichment target components, separation and the enrichment of realize target component simultaneously, can also increase the concentration of target components in product solution when improving target components purity, thereby alleviate the burden that reclaims target components from product solution.The method is particularly suitable for separating and the very low microcomponent of purifying content.
The accompanying drawing explanation
Fig. 1 is that the present invention adopts the chromatography separating method schematic diagram separated from mixture with enrichment target components (retaining the strongest).
Fig. 2 is that the present invention adopts from mixture the chromatography separating method schematic diagram separated with enrichment target components (retaining the strongest), compared to Figure 1, and many steps of a balance chromatographic column.
Fig. 3 is that the present invention adopts the chromatography separating method schematic diagram separated from mixture with enrichment target components (it is not the strongest retaining).
Fig. 4 is that the present invention adopts from mixture the chromatography separating method schematic diagram separated with enrichment target components (it is not the strongest retaining), with Fig. 3, compares, many steps of a balance chromatographic column.
The liquid-phase chromatographic analysis spectrogram that Fig. 5 is Capsaicinoids.
The liquid-phase chromatographic analysis spectrogram that Fig. 6 is the dihydrocapsaicin solution that obtains of embodiment 1.
The liquid-phase chromatographic analysis spectrogram that Fig. 7 is the homocapsaicin solution that obtains of embodiment 3.
The specific embodiment
Below with from Capsaicinoids, separate respectively with enrichment dihydrocapsaicin and homocapsaicin as an example, describe technical scheme of the present invention in detail.The Capsaicinoids raw material is purchased from Zhengzhou Bei Baiou Bioisystech Co., Ltd.As shown in Figure 5, on carbon octadecyl silane post, in Capsaicinoids, the peak sequence of each monomer is followed successively by nordihydrocapsaicin, capsaicine, dihydrocapsaicin and homocapsaicin, and wherein dihydrocapsaicin and homocapsaicin content are followed successively by 28% and 1%.Employing the present invention proposition separates the chromatography separating method with the enrichment target components from mixture, from Capsaicinoids, separates respectively and enrichment dihydrocapsaicin and homocapsaicin.
chromatographic fractionation system
Chromatographic fractionation system mainly comprises infusion pump, chromatographic column (internal diameter 2cm) and 3 eluant, eluents.Eluant, eluent 1 and eluant, eluent 2 are methanol aqueous solution, methyl alcohol volumn concentration 60% in eluant, eluent 1 wherein, and methyl alcohol volumn concentration 80% in eluant, eluent 2, eluant, eluent 3 is pure methyl alcohol, therefore, the eluting power of eluant, eluent 1, eluant, eluent 2, eluant, eluent 3 strengthens successively.Capsaicinoids is dissolved in eluant, eluent 1 and is mixed with material liquid, total concentration 20mg/mL.
product inspection
The efficient liquid phase chromatographic analysis product purity.Highly effective liquid phase chromatographic system forms: German Knauer K501 pump, U.S. Agilent TC-C18 post (4.6 * 150mm, 5 μ m), German Knauer K2501 detector; The liquid-phase chromatographic analysis condition: mobile phase is methanol/water (volume ratio 70/30), and flow velocity 0.5mL/min detects wavelength 280nm, column temperature: 30 ℃.
Embodiment 1-employing carbon octadecyl silane separates and the enrichment dihydrocapsaicin from Capsaicinoids
Filling carbon octadecyl silane (15 microns of particle diameters) in chromatographic column, column length 15cm.
Concrete separation is a cycling process with enrichment process, and first operation cycle comprises following three steps:
Step 1-absorption dihydrocapsaicin: the 180mL material liquid is delivered to chromatographic column with pump, and flow 6mL/min, collect eluent 1 at the chromatographic column afterbody;
Step 2-desorption dihydrocapsaicin: 90mL eluant, eluent 2 use pumps are delivered to chromatographic column, and flow 6mL/min, collect eluent 2 at the chromatographic column afterbody, and now dihydrocapsaicin is eluted chromatographic column;
Step 3-desorption homocapsaicin: 40mL eluant, eluent 3 use pumps are delivered to chromatographic column, and flow 6mL/min, collect eluent 3 at the chromatographic column afterbody, homocapsaicin from chromatographic column by desorption.
From second operation cycle, in the step (1) in each operation cycle, the 90mL eluent 2 of collecting in 90mL material liquid and the step 2 in a upper operation cycle is merged, all the other are with first operation cycle.All only contain nordihydrocapsaicin and capsaicine in collected eluent 1 in each operation cycle in front 4 operation cycles, only contain homocapsaicin in eluent 3.Therefore in the material liquid (cumulative volume 450mL) added in front 4 cycles target components----dihydrocapsaicin all is eluted in the 90mL eluent 2 in the 4th cycle, and this is equivalent to target components and is concentrated 5 times.When the operation cycle, proceed, target components is leaked in the step 1 in the 5th operation cycle from chromatographic column, and this shows that chromatographic column has started to reach capacity.Now in chromatographic column except the target components by enrichment, also residual have a small amount of non-target components.As not high as the purity requirement to dihydrocapsaicin, the whole wash-outs of all components in chromatographic column are got final product.Obtain the high-purity dihydrocapsaicin as required, since the 5th operation cycle, only the 90mL material liquid in step 1 need be replaced by 90mL eluant, eluent 1 and get final product.Experimental result shows, when continuing to move to the 7th operation cycle, in chromatographic column, remaining non-target components is removed, and can obtain 99.5% dihydrocapsaicin (as Fig. 6).
Embodiment 2-adopts macroporous absorbent resin to separate from Capsaicinoids and the enrichment dihydrocapsaicin
Concrete implementation step is with embodiment 1, and different is filling nonpolar macroporous adsorption resin (ADS-8) in chromatographic column, column length 30cm; In addition, also within each operation cycle, add step 4 and use eluant, eluent 1 balance chromatographic column, four step cycle are carried out: the 1 absorption target components → 2 desorption target components → strong retained fraction of 3 desorption → 4 balance chromatographic columns ... through seven operation cycles, finally obtain 99.7% dihydrocapsaicin.
Embodiment 3-employing carbon octadecyl silane separates and the enrichment homocapsaicin from Capsaicinoids
Filling polyamide (15 microns of particle diameters) in chromatographic column, column length 15cm.
Concrete separation is a cycling process with enrichment process, and first operation cycle comprises following two steps:
Step 1-absorption homocapsaicin: the 130mL material liquid is delivered to chromatographic column with pump, and flow 6mL/min, collect eluent 1 at the chromatographic column afterbody.
Step 2-desorption homocapsaicin: 40mL eluant, eluent 3 use pumps are delivered to chromatographic column, at the chromatographic column afterbody, collect eluent 2, flow 6mL/min, now homocapsaicin is eluted chromatographic column.
From second operation cycle, in the step (1) in each operation cycle, the 40mL eluent of collecting in 90mL material liquid and the step 2 in a upper operation cycle is merged, all the other are with first operation cycle
.all only contain nordihydrocapsaicin, capsaicine and dihydrocapsaicin in the eluent 1 of collecting in each operation cycle in front 60 operation cycles.Therefore-homocapsaicin is concentrated 136 times to the target components in the material liquid (cumulative volume 5440mL) added in front 60 operation cycles----homocapsaicin all is eluted in the 40mL eluent 2 in the 60th cycle, and this is equivalent to target components---.When the operation cycle, proceed, homocapsaicin starts to leak from chromatographic column in the step 1 in the 61st operation cycle, and this shows that chromatographic column has started to reach capacity.Now in chromatographic column except the homocapsaicin by enrichment, also residual have other a small amount of component.As not high as the purity requirement to homocapsaicin, the whole wash-outs of all components in chromatographic column are got final product.Obtain highly purified homocapsaicin as required, since the 61st operation cycle, only the 90mL material liquid in step 1 need be replaced by 90mL eluant, eluent 1 and get final product.Experimental result shows, when continuing to move to the 66th operation cycle, in chromatographic column, remaining non-target components is removed, and can obtain 99.6% homocapsaicin (as Fig. 7).
Embodiment 4-employing ion exchange resin separates and the enrichment homocapsaicin from Capsaicinoids
Concrete implementation step is with embodiment 3, and different is to be filled with ion exchange resin in chromatographic column, column length 30cm; In addition, also add a step 3 after step 2, use eluant, eluent 1 balance chromatographic column, three step cycle are carried out: 1 absorption target components → 2 desorption target components → 3 balance chromatographic columns ... through 70 operation cycles, finally obtain 99.8% homocapsaicin.
Above embodiment is to explanation of the present invention and further explains, rather than limitation of the present invention, and any modification of making in spirit of the present invention and rights protection scope, all fall into protection scope of the present invention.
Claims (8)
1. the chromatography separating method separated from mixture with the enrichment target components, is characterized in that: comprise by chromatographic column and at least two kinds of piece-rate systems that eluant, eluent forms; Be filled with the adsorbent that can adsorb target components in described chromatographic column;
Described eluant, eluent comprises eluant, eluent 1 and eluant, eluent 2; There is the volumn concentration of solvent of strong eluting power in described eluant, eluent 2 higher than thering is the volumn concentration of the solvent of strong eluting power in eluant, eluent 1;
Described adsorbent is silica gel, polyamide, polymeric adsorbent or ion exchange resin;
The concrete separation and the cycling process of enrichment process for consisting of a plurality of operation cycles, first operation cycle comprises following two steps:
(1) absorption target components: raw material is dissolved in to eluant, eluent 1 and is mixed with material liquid, inject chromatographic column, target components is adsorbed onto on chromatographic column, the retention time weak retained fraction shorter than target components flows out chromatographic column, collects and obtains eluent 1;
(2) desorption target components: eluant, eluent 2 is injected to chromatographic column, the target components in the elution chromatography post, target components flows out from the chromatographic column afterbody, collects and obtains eluent 2;
Since second operation cycle, in the step (1) in each operation cycle, material liquid and eluent 2 collected within a upper operation cycle were merged to the injection chromatographic column, all the other are with first operation cycle;
In separation process, weak retained fraction flows out chromatographic column in the step (1) in each operation cycle, with target components, separates; Target components in the step (2) in each operation cycle by desorption to eluent 2, then be adsorbed to chromatographic column with new target components of supplementing in material liquid in the step (1) in next operation cycle, therefore target components progressively is concentrated in eluent 2 or is enriched in chromatographic column, thereby realization separates from mixture and the purpose of enrichment target components.
2. the chromatography separating method separated from mixture with the enrichment target components according to claim 1, it is characterized in that: the step (2) in described each operation cycle is step (3) afterwards, and described step (3) is for using eluant, eluent 1 balance chromatographic column; Separating with enrichment process is that step (1), step (2), step (3) loop.
3. the chromatography separating method separated from mixture with the enrichment target components according to claim 1 and 2, it is characterized in that: when target components constantly accumulates in chromatographic column inside, until to such an extent as to the saturated target components of chromatographic column while leaking from eluent 1, stop the cycling process, the target components that will be enriched in chromatographic column with eluant, eluent 2 elutes chromatographic column, collects the target components that obtains being concentrated at the chromatographic column afterbody.
4. the chromatography separating method separated from mixture with the enrichment target components according to claim 1 and 2, it is characterized in that: when target components constantly accumulates in chromatographic column inside, until to such an extent as to the saturated target components of chromatographic column while leaking from eluent 1, replace material liquid with eluant, eluent 1 in step (1), continue cycling, the non-target components remained in chromatographic column is removed, and target components continues to be concentrated in eluent 2 or is enriched in chromatographic column.
5. the chromatography separating method separated from mixture with the enrichment target components, is characterized in that: comprise by chromatographic column and at least three kinds of piece-rate systems that eluant, eluent forms; Be filled with the adsorbent that can adsorb target components in described chromatographic column;
Described eluant, eluent comprises eluant, eluent 1, eluant, eluent 2 and eluant, eluent 3; There is the volumn concentration of solvent of strong eluting power in described eluant, eluent 2 higher than thering is the volumn concentration of the solvent of strong eluting power in eluant, eluent 1; The volumn concentration that has the solvent of strong eluting power in described eluant, eluent 3 is not less than the volumn concentration that has the solvent of strong eluting power in eluant, eluent 2;
Described adsorbent is silica gel, polyamide, polymeric adsorbent or ion exchange resin;
Concrete separation is the cycling process formed in a plurality of operation cycles with enrichment process, and first operation cycle comprises following three steps:
(1) absorption target components: raw material is dissolved in to eluant, eluent 1 and is mixed with material liquid, inject chromatographic column, target components is adsorbed onto on chromatographic column, meanwhile, the strong retained fraction that retention time is longer than target components also is attracted on chromatographic column, the retention time weak retained fraction shorter than target components flows out chromatographic column, collects and obtains eluent 1;
(2) desorption target components: eluant, eluent 2 is injected to chromatographic column, the target components in the elution chromatography post, target components flows out from the chromatographic column afterbody, collects and obtains eluent 2; And the retention time strong retained fraction longer than target components still is attracted in chromatographic column;
(3) the strong retained fraction of desorption: eluant, eluent 3 is injected to chromatographic column, the strong retained fraction in the elution chromatography post, strong retained fraction flows out from the chromatographic column afterbody, collects and obtains eluent 3;
Since second operation cycle, in the step (1) in each operation cycle, material liquid and eluent 2 collected within a upper operation cycle were merged to the injection chromatographic column, all the other are with first operation cycle;
In separation process, weak retained fraction is eluted chromatographic column in the step (1) in each operation cycle, and strong retained fraction is eluted chromatographic column in the step (3) in each operation cycle, with target components, separates; Target components in the step (2) in each operation cycle by desorption to eluent 2, then together be adsorbed to chromatographic column with new target components of supplementing in material liquid in the step (1) in next operation cycle, therefore target components progressively is concentrated in eluent 2 or is enriched in chromatographic column, thereby realization separates from mixture and the purpose of enrichment target components.
6. the chromatography separating method separated from mixture with the enrichment target components according to claim 5, it is characterized in that: the step (3) in described each operation cycle is step (4) afterwards, and described step (4) is for using eluant, eluent 1 balance chromatographic column; Separating with enrichment process is that step (1), step (2), step (3), step (4) loop.
7. according to the described chromatography separating method separated from mixture with the enrichment target components of claim 5 or 6, it is characterized in that: when target components constantly accumulates in chromatographic column inside, until to such an extent as to the saturated target components of chromatographic column while leaking from eluent 1, stop cycling, the target components that will be enriched in chromatographic column with eluant, eluent 2 elutes chromatographic column, collects the target components that obtains being concentrated at the chromatographic column afterbody.
8. according to the described chromatography separating method separated from mixture with the enrichment target components of claim 5 or 6, it is characterized in that: when target components constantly accumulates in chromatographic column inside, until to such an extent as to the saturated target components of chromatographic column while leaking from eluent 1, replace material liquid with eluant, eluent 1 in step (1), continue cycling, the non-target components remained in chromatographic column is removed, and target components continues to be concentrated in eluent 2 or is enriched in chromatographic column.
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| CN109475789A (en) * | 2016-07-13 | 2019-03-15 | 克罗麦肯公司 | Purification process and application thereof |
| CN111551542A (en) * | 2019-12-31 | 2020-08-18 | 常德市正阳生物科技股份有限公司 | Switching detection method of eluent |
| CN112451996A (en) * | 2020-11-10 | 2021-03-09 | 浙江大学 | Optimization method for capturing protein by multi-column continuous flow chromatography |
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