CN103331036B - A Chromatographic Separation Method for Separating and Enriching Target Components from a Mixture - Google Patents

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

A Chromatographic Separation Method for Separating and Enriching Target Components from a Mixture

technical field

The invention relates to a chromatographic separation method for separating and enriching target components from a mixture.

Background technique

Chromatographic separation method is a commonly used method to separate target components from mixtures, and it is widely used in many fields such as drug separation, separation and purification of natural products, etc. Generally, the pure chromatographic separation process only obtains the product solution, so recovery operations, such as evaporation or crystallization, are required to obtain pure target components. Obviously, the higher the concentration of the product solution, the more favorable the recovery operation.

However, due to non-ideal factors such as mass transfer resistance and axial dispersion, in most chromatographic separation processes, the target component is always diluted, and its concentration in the product solution is lower than its concentration in the raw material solution . The recovery operation is thus burdened such that it accounts for a large proportion of the total separation cost. In some separation processes, such as the separation and purification of natural products, because the content of the target component is very low, the recovery operation may determine the cost of the entire separation process. Take the extraction and separation of paclitaxel from the leaves of yew as an example. Since the paclitaxel content is very low, only about 3/10000, the content can only be increased to about 3/1000 after solvent extraction. Separation, the extract is formulated into a raw material solution, assuming a concentration of 50 mg/mL, the concentration of paclitaxel in the raw material solution is only about 0.15 mg/mL. Because the product is often accompanied by the dilution of the product solution when it is separated and purified, the concentration of paclitaxel in the product solution obtained by chromatographic separation will be far less than 0.15 mg/mL, and the cost of recovering paclitaxel from such a dilute solution is very high. The pretreatment operation is to enrich paclitaxel to a certain extent and then use chromatographic separation and purification. This method does not fundamentally solve the problem, but only shifts the burden of the recovery operation to the pretreatment operation. the

Contents of the invention

The present invention aims at the deficiencies of the prior art, and provides a chromatographic separation method capable of concentrating and enriching the target component while separating and purifying the target component, that is, simultaneously realizing the separation and enrichment of the target component from the mixture.

In order to solve the above-mentioned technical problems, first technical solution of the present invention is realized like this:

A chromatographic separation method for separating and enriching a target component from a mixture, comprising a separation system composed of a chromatographic column and two eluents; the chromatographic column is filled with an adsorbent capable of adsorbing the target component;

Described eluent comprises eluent 1 and eluent 2; The volume percentage content of the solvent with strong eluting ability in described eluent 2 is higher than that of the solvent with strong eluting ability in eluent 1 volume percentage;

The adsorbent is silica gel, polyamide resin, adsorption resin or ion exchange resin.

Assuming that the target component in the raw material has the strongest retention on the chromatographic column, the specific separation and enrichment process is a cyclic operation process consisting of multiple operation cycles. The first operation cycle includes the following two steps:

(1) Adsorb the target component: Dissolve the raw material in eluent 1 to prepare a raw material solution, inject it into the chromatographic column, and collect the eluent 1 at the end of the chromatographic column. The purpose of this step is to selectively adsorb the target components in the raw material solution onto the chromatographic column, while the weakly retained components with shorter retention times than the target components flow out of the chromatographic column.

(2) Desorb the target component: inject the eluent 2 into the chromatographic column to elute the target component in the chromatographic column, the target component flows out from the tail of the chromatographic column, and collect the eluent 2.

Beginning from the second operation cycle, in the step (1) of each operation cycle, the raw material liquid and the eluent 2 collected in the last operation cycle are combined and injected into the chromatographic column, and the rest are the same as the first operation cycle;

During the separation process, the weakly retained components will flow out of the chromatographic column and enter the eluent 1 in step (1) of each operation cycle, so they can be separated from the target components; In the step (2) of the cycle, they are all desorbed into the eluent 2, and then combined with the target components in the newly supplemented raw material solution, and are adsorbed to the chromatographic column together in the step (1) of the next operation cycle. Therefore, the target components are gradually concentrated into the eluent 2 or enriched into the chromatographic column, which means that the target components in all the raw materials added in the first to nth operation cycles are eluted to the nth In the eluent 2 in the operating cycle, or adsorbed to the chromatographic column in the nth operating cycle. If it is assumed that the volume of the raw material solution added in each operation cycle is the same, both are V ₁ , and the volume of the eluent 2 used is also the same, both are V ₂ , then the above separation process is equivalent to adding the raw material solution with a total volume of n·V ₁ Concentrate the target components in an eluent 2 with a volume of V ₂ , or enrich the target components in a raw material solution with a total volume of n V ₁ into a chromatographic column, so as to achieve separation from the mixture with the purpose of enriching target components. Obviously, the more operating cycles, the higher the degree of concentration and enrichment. Generally, the components that erupt before the target component are collectively referred to as weakly retained components, and the components that elute later than the target component are called strongly retained components, that is, the components whose retention time is shorter than the target component are Weakly retentive components, components with a longer retention time than the target component are strongly retentive components.

As an improvement of the above separation method, step (3) can be followed by step (2) of each operation cycle, that is, to equilibrate the chromatographic column with eluent 1, and the specific process is to inject eluent 1 into the chromatographic column until the chromatographic column The composition of the outlet effluent is the same as the composition of eluent 1; then when the retention of the target component in the raw material is the strongest, three steps are cycled: step (1) adsorption of the target component → step (2) desorption of the target component Divide → step (3) equilibrate the chromatographic column →....

The main points of the above-mentioned separation technology scheme are:

First, the elution ability of eluent 2 is stronger than that of eluent 1, which can be achieved by changing the composition of eluent 1 and eluent 2. In this way, in the stage of adsorbing target components in each operation cycle, because the raw material liquid is combined with the eluent 2 in the previous operation cycle, the liquid phase elution ability will be weakened. When it flows through the chromatographic column, it will be controlled The injection volume of the raw material solution can ensure that the weakly retained components flow out of the chromatographic column, while the target component does not leak from the chromatographic column; in the stage of desorbing the target component, due to the enhanced elution ability of eluent 2, only a small amount of A volume of eluent 2 can completely desorb the target components on the chromatographic column. It can be seen that after each operation cycle, the target components can be enriched and concentrated to a certain extent.

Secondly, the eluent 2 collected in each operation cycle is not used as the final product solution, but it is combined with the new replenishment raw material solution and then added to the chromatographic column again. This measure can accumulate the enrichment and concentration effect obtained by the target component in each cycle operation, so that the enrichment and concentration of the target component become higher and higher.

In the above separation methods, the target components are gradually accumulated and enriched in the chromatographic column, and eventually the chromatographic column must be saturated and no more target components can be adsorbed. At this point, if you continue to add fresh raw material solution in step (1), the target components in the newly supplemented raw material solution will leak from eluent 1, which does not increase the utilization rate of the chromatographic column, but just wastes time and power; Conversely, if the cycle is stopped before the column reaches saturation, the column will not be fully utilized. Therefore, the chromatographic column should be saturated as far as possible. This can be judged by detecting whether the eluent 1 in each operation cycle contains the target component. If the step (1) of a certain operation cycle has not yet ended, the target Components have leaked from Eluent 1, indicating that the column is saturated. Therefore, when the target component continues to accumulate inside the column until the column becomes saturated so that the target component leaks from eluent 1, the cycle operation is stopped. At this time, the chromatographic column mainly contains the enriched target components, while non-target components remain. If the requirement for the purity of the target component is not high, then only the target component enriched in the chromatographic column is eluted out of the chromatographic column with eluent 2 or any other solvent that can elute the target component. The concentrated target component is collected at the end of the chromatographic column; if the purity of the target component is high, then in the next operation cycle, the new supplementary raw material solution in step (1) can be replaced with eluent 1, that is, in In step (1), the eluent 2 is no longer combined with the supplemented raw material solution, but the eluent 2 and the eluent 1 are combined and injected into the chromatographic column, and the cycle operation is continued. In this way, the non-target components in the chromatographic column will continue to be eluted out of the chromatographic column, while the target components are still concentrated in the eluent 2 or enriched in the chromatographic column.

In the present invention, by changing the content of the solvent with strong elution ability in the eluent 1 and the eluent 2, the elution ability of the eluent 1 is stronger than that of the eluent 2. In step (2) of each operation cycle, ensure that the amount of eluent 2 is sufficient to desorb the target component from the chromatographic column, that is, the chromatographic column does not contain the target component after step (2). As preferably, step (3) is added after step (2) of each operation cycle, promptly uses eluent 1 to equilibrate the chromatographic column, and concrete step is that eluent 1 is injected into chromatographic column, until chromatographic column outlet effluent composition and The composition of eluent 1 is the same so far.

Advantages and beneficial effects of the present invention: a chromatographic separation method for separating and enriching the target component from the mixture adopted in the present invention can realize the separation and enrichment of the target component at the same time, that is, while improving the purity of the target component It can also increase the concentration of the target component in the product solution, thereby reducing the burden of recovering the target component from the product solution. The method is especially suitable for the separation and purification of trace components in very low concentrations.

Another technical solution of the present invention is:

A chromatographic separation method for separating and enriching target components from a mixture, characterized in that: it includes a separation system composed of a chromatographic column and at least three eluents; the chromatographic column is filled with Adsorbent;

Described eluent comprises eluent 1, eluent 2 and eluent 3; The volume percent of the solvent with strong eluting ability in described eluent 2 is higher than eluent 1 and has strong eluent. The volume percentage of the solvent with strong eluting ability; the volume percentage of the solvent with strong eluting ability in the eluent 3 is not lower than the volume percent of the solvent with strong eluting ability in eluent 2.

The adsorbent is silica gel, polyamide resin, adsorption resin or ion exchange resin;

Assuming that the raw material contains weakly retained components, target components and strongly retained components, the retention on the chromatographic column is enhanced sequentially. The specific separation and enrichment process is a cyclic operation process consisting of multiple operating cycles. The first operating cycle includes Follow these three steps:

(1) Adsorb the target component: Dissolve the raw material in eluent 1 to prepare a raw material solution, inject it into the chromatographic column, and collect the eluent 1 at the end of the chromatographic column. The purpose of this step is to selectively adsorb the target components in the raw material to the chromatographic column. At the same time, the strongly retained components are also adsorbed to the chromatographic column, while the weakly retained components with shorter retention times than the target components are adsorbed on the chromatographic column. The fraction flows out of the column.

(2) Desorption of the target component: inject the eluent 2 into the chromatographic column to elute the target component in the chromatographic column, and the target component flows out from the tail of the chromatographic column, and the eluent 2 is collected; the retention time is longer than that of the target component Long strongly retained components are still adsorbed in the column;

(3) Desorb strongly retained components: inject the eluent 3 into the chromatographic column to elute the strongly retained components in the chromatographic column, the strongly retained components flow out from the tail of the chromatographic column, and collect the eluent 3;

Beginning from the second operation cycle, in the step (1) of each operation cycle, the raw material liquid and the eluent 2 collected in the last operation cycle are combined and injected into the chromatographic column, and the rest are the same as the first operation cycle;

During the separation process, the weakly retained components are eluted from the chromatographic column in step (1) of each operation cycle, and the strongly retained components are eluted from the chromatographic column in step (3) of each operation cycle, so its and the target component is separated; and the target component is desorbed into the eluent 2 in the step (2) of each operation cycle, and then combined with the target component in the new replenishment raw material solution, in the next operation In the step (1) of the cycle, they are adsorbed to the chromatographic column together, so the target components are gradually concentrated into the eluent 2 or enriched in the chromatographic column.

As an improvement to the above separation method, another step can be added in each operation cycle, that is, to equilibrate the chromatographic column with eluent 1. Until the composition of deagent 1 is the same; then four steps are cycled: (1) Adsorption of target components → (2) Desorption of target components → (3) Desorption of strongly retained components → (4) Equilibration of the chromatographic column →… …

The main points of the above-mentioned separation technology scheme are:

First, the elution abilities of eluent 1, eluent 2, and eluent 3 are sequentially enhanced, which can be achieved by changing the composition of eluent 1, eluent 2, and eluent 3. In this way, in the stage of adsorbing target components in each operation cycle, because the raw material liquid is combined with the eluent 2 in the previous operation cycle, the liquid phase elution ability will be weakened. When it flows through the chromatographic column, it will be controlled The injection volume of the raw material solution can ensure that the weakly retained components flow out of the chromatographic column, while the target component does not leak from the chromatographic column; in the stage of desorbing the target component, due to the enhanced elution ability of eluent 2, only a small amount of A volume of eluent 2 can completely desorb the target components on the chromatographic column. It can be seen that after each operation cycle, the target components can be enriched and concentrated to a certain extent.

Secondly, the eluent 2 collected in each operation cycle is not used as the final product solution, but it is combined with the new replenishment raw material solution and then added to the chromatographic column again. This measure can accumulate the enrichment and concentration effect obtained by the target component in each cycle operation, so that the enrichment and concentration of the target component become higher and higher.

In the above separation methods, the target components are gradually accumulated and enriched in the chromatographic column, and eventually the chromatographic column must be saturated and no more target components can be adsorbed. At this time, if you continue to add fresh raw material solution in step (1), the target components in the newly supplemented raw material solution will leak from eluent 1, which will not increase the utilization rate of the chromatographic column, but will just waste time and energy ; Conversely, if the chromatographic column has not reached saturation and stops the cycle operation, the chromatographic column cannot be fully utilized. Therefore, the chromatographic column should be saturated as much as possible. This can be judged by detecting whether the eluent 1 in each operation cycle contains the target component. If the step (1) of a certain operation cycle has not ended, the target Components have leaked from Eluent 1, indicating that the column is saturated. Therefore, when the target component continues to accumulate inside the column until the column becomes saturated so that the target component leaks from eluent 1, the cycle operation is stopped. At this time, the chromatographic column mainly contains the enriched target components, while non-target components remain. If the purity requirement of the target component is not high, it is only necessary to use eluent 2 or any other solvent that can elute the target component to elute the target component enriched in the chromatographic column from the chromatographic column, collect The obtained eluent 2 is the solution of the enriched target component; if the purity requirement of the target component is high, the new eluent 1 in step (1) can be replaced by eluent 1 in the next operation cycle. Replenish the raw material solution, that is, in step (1), the eluent 2 is no longer combined with the supplemented raw material solution, but the eluent 2 and the eluent 1 are combined and injected into the chromatographic column, and the cycle operation is continued. In this way, the non-target components in the chromatographic column will continue to be eluted out of the chromatographic column, while the target components are still concentrated in the eluent 2 or enriched in the chromatographic column.

The present invention makes the eluting ability of eluent 1, eluent 2 and eluent 3 by changing the content of the solvent with strong eluting ability in eluent 1, eluent 2 and eluent 3 Enhanced sequentially. In step (2) of each operation cycle, ensure that the amount of eluent 2 is sufficient to achieve the degree of desorption of the target component from the chromatographic column, that is, the chromatographic column does not contain the target component after step (2). In step (3) of each operation cycle, ensure that the amount of eluent 3 is sufficient to desorb all strongly retained components from the chromatographic column, and the chromatographic column does not contain strongly retained components after step (3). . As preferably, step (4) is added after step (3) of each operation cycle, promptly uses eluent 1 to equilibrate chromatographic column, and concrete steps is that eluent 1 is injected into chromatographic column, until chromatographic column outlet effluent composition and The composition of eluent 1 is the same so far.

Advantages and beneficial effects of the present invention: a chromatographic separation method for separating and enriching the target component from the mixture adopted in the present invention can realize the separation and enrichment of the target component at the same time, that is, while improving the purity of the target component It can also increase the concentration of the target component in the product solution, thereby reducing the burden of recovering the target component from the product solution. The method is especially suitable for the separation and purification of trace components in very low concentrations.

Description of drawings

Fig. 1 is a schematic diagram of a chromatographic separation method for separating and enriching target components (with the strongest retention) from a mixture in the present invention.

Fig. 2 is a schematic diagram of the chromatographic separation method adopting separation and enrichment of the target component (the strongest retention) from the mixture in the present invention. Compared with Fig. 1, there is one more step of equilibrating the chromatographic column.

Fig. 3 is a schematic diagram of the chromatographic separation method for separating and enriching the target component (not the strongest retention) from the mixture in the present invention.

Fig. 4 is a schematic diagram of the chromatographic separation method for separating and enriching the target component (not the strongest retention) from the mixture in the present invention. Compared with Fig. 3, there is an additional step of equilibrating the chromatographic column.

Fig. 5 is the liquid chromatography analysis spectrogram of total alkaloids of capsicum.

Fig. 6 is the liquid phase chromatographic analysis spectrogram of the dihydrocapsaicin solution that embodiment 1 obtains.

Fig. 7 is the liquid chromatography analysis spectrum of the homocapsaicin solution that embodiment 3 obtains.

Detailed ways

Taking the separation and enrichment of dihydrocapsaicin and homocapsaicin respectively from capsaicin as an example, the technical scheme of the present invention will be described in detail below. The raw material of total alkaloids of capsicum was purchased from Zhengzhou Beibaiou Biotechnology Co., Ltd. As shown in Figure 5, on the carbon octadecyl bonded silica gel column, the peak order of each monomer in total capsaicin is nordihydrocapsaicin, capsaicin, dihydrocapsaicin and homocapsaicin, wherein The contents of dihydrocapsaicin and homocapsaicin are 28% and 1% respectively. The chromatographic separation method for separating and enriching target components from the mixture proposed by the invention is used to separate and enrich dihydrocapsaicin and homocapsaicin from total capsaicin.

Chromatographic separation system

The chromatographic separation system mainly includes an infusion pump, a chromatographic column (2cm inner diameter) and 3 eluents. Eluent 1 and eluent 2 are aqueous methanol solutions, wherein the volume percentage of methanol in eluent 1 is 60%, the volume percentage of methanol in eluent 2 is 80%, and eluent 3 is pure methanol. Therefore, The elution ability of eluent 1, eluent 2, and eluent 3 increases sequentially. Dissolve total alkaloids of capsaicin in eluent 1 to prepare a raw material solution with a total concentration of 20 mg/mL.

Product testing

Product purity was analyzed by high performance liquid chromatography. High performance liquid chromatography system composition: German Knauer K501 pump, American Agilent TC-C18 column (4.6×150mm, 5μm), German Knauer K2501 detector; liquid chromatography analysis conditions: mobile phase is methanol/water (volume ratio 70/30 ), flow rate 0.5mL/min, detection wavelength 280nm, column temperature: 30°C.

Example 1—Using carbon octadecyl bonded silica gel to separate and enrich dihydrocapsaicin from total alkaloids in capsicum

The chromatographic column is filled with carbon octadecyl bonded silica gel (particle size 15 microns), and the column length is 15 cm.

The specific separation and enrichment process is a cyclic operation process, and the first operation cycle includes the following three steps:

Step 1—adsorption of dihydrocapsaicin: pump 180mL of raw material solution to the chromatographic column with a flow rate of 6mL/min, and collect eluent 1 at the end of the chromatographic column;

Step 2—Desorption of dihydrocapsaicin: pump 90mL of eluent 2 to the chromatographic column at a flow rate of 6mL/min, collect eluent 2 at the end of the chromatographic column, and dihydrocapsaicin is eluted from the chromatographic column ;

Step 3—desorption of homocapsaicin: transport 40 mL of eluent 3 to the chromatographic column with a pump at a flow rate of 6 mL/min, collect eluent 3 at the end of the chromatographic column, and desorb homocapsaicin from the chromatographic column.

From the second operating cycle, in step (1) of each operating cycle, 90 mL of raw material liquid is combined with 90 mL of eluent 2 collected in step 2 of the previous operating cycle, and the rest are the same as the first operating cycle. The eluate 1 collected in each of the first four operating cycles contained only nordihydrocapsaicin and capsaicin, while the eluate 3 contained only homocapsaicin. Therefore, the target component in the raw material solution (total volume 450mL) added in the first 4 cycles---dihydrocapsaicin is all eluted in the 90mL eluent 2 of the fourth cycle, which is equivalent to the target Fractions were concentrated 5-fold. As the cycle continued, the target component leaked from the column in Step 1 of the fifth cycle, indicating that the column had begun to saturate. At this time, in addition to the enriched target components, a small amount of non-target components remain in the chromatographic column. If the purity requirement of dihydrocapsaicin is not high, all the components in the chromatographic column can be eluted. Obtain high-purity dihydrocapsaicin as required, then start from the fifth operation cycle, only need to replace 90mL raw material solution in step 1 with 90mL eluent 1 and get final product. The experimental results show that when the operation continues to the seventh operation cycle, the remaining non-target components in the chromatographic column are removed, and 99.5% of dihydrocapsaicin can be obtained (as shown in Figure 6).

Embodiment 2-Use macroporous adsorption resin to separate and enrich dihydrocapsaicin from total alkaloids of capsicum

The specific implementation steps are the same as in Example 1, except that the non-polar macroporous adsorbent resin (ADS-8) is filled in the chromatographic column, and the length of the column is 30cm; Equilibrium chromatographic column, four steps cycle: 1 adsorption target component → 2 desorption target component → 3 desorption strong retention component → 4 balance chromatographic column... After seven operating cycles, 99.7% of the secondary Hydrocapsaicin.

Example 3—Separation and enrichment of homocapsaicin from total alkaloids of capsicum using carbon octadecyl bonded silica gel

The chromatographic column is filled with polyamide resin (particle size 15 microns), and the column length is 15 cm.

The specific separation and enrichment process is a cyclic operation process, and the first operation cycle includes the following two steps:

Step 1—adsorption of homocapsaicin: 130 mL of raw material liquid is pumped to the chromatographic column at a flow rate of 6 mL/min, and eluent 1 is collected at the end of the chromatographic column.

Step 2—desorption of homocapsaicin: pump 40 mL of eluent 3 to the chromatographic column, and collect eluent 2 at the end of the chromatographic column at a flow rate of 6 mL/min. At this time, homocapsaicin is eluted from the chromatographic column.

From the second operating cycle, in step (1) of each operating cycle, 90 mL of raw material liquid is combined with the 40 mL eluent collected in step 2 of the previous operating cycle, and the rest are the same as the first operating cycle . The eluate 1 collected in each of the first 60 operating cycles contained only nordihydrocapsaicin, capsaicin and dihydrocapsaicin. Therefore, the target component in the raw material solution (total volume 5440mL) added in the first 60 operating cycles---homocapsaicin is all eluted into the 40mL eluent 2 of the 60th cycle, which is equivalent to the target Components - Homocapsaicin is concentrated 136 times. As the cycle continued, homocapsaicin began leaking from the column in step 1 of cycle 61, indicating that the column had begun to saturate. At this time, in addition to the enriched homocapsaicin, a small amount of other components remain in the chromatographic column. If the purity requirement of homocapsaicin is not high, all the components in the chromatographic column can be eluted. If it is required to obtain high-purity homocapsaicin, then from the 61st operation cycle, it is only necessary to replace the 90mL raw material solution in step 1 with 90mL eluent 1. The experimental results show that when the operation continues to the 66th operation cycle, the remaining non-target components in the chromatographic column are removed, and 99.6% of homocapsaicin can be obtained (as shown in Figure 7).

Embodiment 4—Use ion exchange resin to separate and enrich homocapsaicin from total alkaloids of capsicum

The specific implementation steps are the same as in Example 3, except that the chromatographic column is filled with ion exchange resin, and the length of the column is 30 cm; in addition, after step 2, a step 3 is added, that is, the eluent 1 is used to balance the chromatographic column, and the three steps are circulated Carry out: 1 Adsorption of the target component → 2 Desorption of the target component → 3 Equilibration of the chromatographic column... After 70 operating cycles, 99.8% homocapsaicin is finally obtained.

The above embodiments are illustrations and further explanations of the present invention, rather than limitations of the present invention. Any modification made within the spirit and scope of protection of the present invention will fall into the protection scope of the present invention.

Claims (8)

1. a chromatography separating method for separation and enrichment target components from mixture, is characterized in that: comprise by chromatographic column and at least two kinds of piece-rate systems that eluant, eluent forms; In described chromatographic column, be filled with the adsorbent that can adsorb target components;

Described eluant, eluent comprises eluant, eluent 1 and eluant, eluent 2; In described eluant, eluent 2, there is the volumn concentration of solvent of strong eluting power higher than thering is the volumn concentration of the solvent of strong eluting power in eluant, eluent 1;

Described adsorbent is silica gel, polymeric adsorbent or ion exchange resin;

The concrete separated and cycling process of enrichment process for being formed by 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 in 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 elution chromatography post, target components flows out from 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 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, separated with target components; Target components in the step (2) in each operation cycle by desorption to eluent 2, then in the step (1) in next operation cycle, be adsorbed to chromatographic column with new target components of supplementing in material liquid, therefore target components is progressively concentrated in eluent 2 or is enriched in chromatographic column, thereby realize the object of separated and enrichment target components from mixture.

According to claim 1 from mixture the chromatography separating method of separated and enrichment target components, 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; Separated and enrichment process are that step (1), step (2), step (3) loop.

According to claim 1 and 2 from mixture the chromatography separating method of separated and enrichment target components, it is characterized in that: when target components is in the inner constantly accumulation of chromatographic column, when until chromatographic column is saturated, target components is leaked from eluent 1, stop cycling process, with eluant, eluent 2, the target components being enriched in chromatographic column is eluted to chromatographic column, at chromatographic column afterbody, collect the target components that obtains being concentrated.

According to claim 1 and 2 from mixture the chromatography separating method of separated and enrichment target components, it is characterized in that: when target components is in the inner constantly accumulation of chromatographic column, when until chromatographic column is saturated, target components is leaked from eluent 1, in step (1), with eluant, eluent 1, replace material liquid, continue cycling, the non-target components remaining in chromatographic column is removed, and target components continues to be concentrated in eluent 2 or is enriched in chromatographic column.

5. a chromatography separating method for separation and enrichment target components from mixture, is characterized in that: comprise by chromatographic column and at least three kinds of piece-rate systems that eluant, eluent forms; In described chromatographic column, be filled with the adsorbent that can adsorb target components;

Described eluant, eluent comprises eluant, eluent 1, eluant, eluent 2 and eluant, eluent 3; In described eluant, eluent 2, there is the volumn concentration of solvent of strong eluting power higher than thering is the volumn concentration of the solvent of strong eluting power in eluant, eluent 1; The volumn concentration in described eluant, eluent 3 with the solvent of strong eluting power is not less than the volumn concentration in eluant, eluent 2 with the solvent of strong eluting power;

Described adsorbent is silica gel, polymeric adsorbent or ion exchange resin;

Concrete separation is the cycling process forming 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 in chromatographic column, meanwhile, the strong retained fraction that retention time is longer than target components is also attracted in 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 elution chromatography post, target components flows out from chromatographic column afterbody, collects and obtains eluent 2; And the retention time strong retained fraction longer than target components is still attracted in chromatographic column;

(3) the strong retained fraction of desorption: eluant, eluent 3 is injected to chromatographic column, the strong retained fraction in elution chromatography post, strong retained fraction flows out from 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 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, separated with target components; Target components in the step (2) in each operation cycle by desorption to eluent 2, then in the step (1) in next operation cycle, be together adsorbed to chromatographic column with new target components of supplementing in material liquid, therefore target components is progressively concentrated in eluent 2 or is enriched in chromatographic column, thereby realize the object of separated and enrichment target components from mixture.

According to claim 5 from mixture the chromatography separating method of separated and enrichment target components, 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; Separated and enrichment process are that step (1), step (2), step (3), step (4) loop.

7. according to the separation from mixture described in claim 5 or 6 and the chromatography separating method of enrichment target components, it is characterized in that: when target components is in the inner constantly accumulation of chromatographic column, when until chromatographic column is saturated, target components is leaked from eluent 1, stop cycling, with eluant, eluent 2, the target components being enriched in chromatographic column is eluted to chromatographic column, at chromatographic column afterbody, collect the target components that obtains being concentrated.

8. according to the separation from mixture described in claim 5 or 6 and the chromatography separating method of enrichment target components, it is characterized in that: when target components is in the inner constantly accumulation of chromatographic column, when until chromatographic column is saturated, target components is leaked from eluent 1, in step (1), with eluant, eluent 1, replace material liquid, continue cycling, the non-target components remaining in chromatographic column is removed, and target components continues to be concentrated in eluent 2 or is enriched in chromatographic column.