CN110229089A - A kind of solvent pairs combination medium pressure liquid chromatography divides the method for purification of high-purity canthaxanthin - Google Patents
A kind of solvent pairs combination medium pressure liquid chromatography divides the method for purification of high-purity canthaxanthin Download PDFInfo
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- CN110229089A CN110229089A CN201910481317.4A CN201910481317A CN110229089A CN 110229089 A CN110229089 A CN 110229089A CN 201910481317 A CN201910481317 A CN 201910481317A CN 110229089 A CN110229089 A CN 110229089A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C403/00—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone
- C07C403/24—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by six-membered non-aromatic rings, e.g. beta-carotene
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
Abstract
The invention discloses a kind of methods that solvent pairs combination medium pressure liquid chromatography divides purification of high-purity canthaxanthin, it can be extracted from canthaxanthin feed addictive from single solvent, it is loaded to the middle pressure glass column for having loaded silica filler, with double base mixed solvent isocratic elution, collect high pure fraction, can a step crude samples concentration is increased to 99% or more by 10%, sample stable yield is 90% or more after purification.The method of the present invention product purity is high, study sample applied sample amount is big, is greatly saved purifying cost；Linear flow rate is high in chromatogram purification, can quickly and efficiently be purified into canthaxanthin, and yield is high, saves the time.In addition, the method for the present invention is easy to operate, reliable and stable, have it is stronger can amplification, be suitable as the method that preparation of industrialization solvent pairs combination medium pressure liquid chromatography divides purification of high-purity canthaxanthin.
Description
Technical field
The invention belongs to field of food safety, and in particular to a kind of purification process of high-purity canthaxanthin reference substance.
Background technique
Canthaxanthin (or canthaxanthin, No. CAS: 514-78-3) of the present invention is a kind of vitamin a source carotenoids
Element, entitled 4, the 4 '-diketone of beta carotene-of chemistry.As shown in Figure 1, canthaxanthin crystalline solid is mainly based on alltrans structure, spot
Chinese blister beetle Huang is darkviolet crystal or crystalline powder, and fusing point is about 210 DEG C, to oxygen and photo-labile, need to be stored in inert gas
In light-proofness container.Canthaxanthin dissolves in chloroform (10%), is slightly soluble in vegetable oil (0.005%), acetone (0.03%), does not dissolve in
Water, ethyl alcohol, propylene glycol.
Canthaxanthin is used in food and feed industry frequently as pigment, and product makes animal and food color field
With more than 40 years, for improving the poultry colour of skin and yolk color, to increase the commercial value of poultry and egg.Canthaxanthin still ties up life
The raw material of plain A can play the role of replenishing vitamins A.Natural canthaxanthin be widely present in shellfish, poultry,
In algae, has the function of anti-oxidant, elimination free radical, but its content in vivo is little.It is closed with artificial
At the industrialization of canthaxanthin, it is widely used in industries such as feed, food, chemical industry, medicine.1984, FDA/WHO
Approval canthaxanthin is included in food additives and has formulated quality standard.
Currently, canthaxanthin mainly passes through natural animal-plant extraction, chemical synthesis, microculture fermentation and beta carotene
Four kinds of approach are aoxidized to obtain.Natural animal-plant canthaxanthin source is limited, content is lower and biological fermentation process bacterial screening is difficult,
The problems such as process is complicated, extraction efficiency is lower limits industrial scale production.It is feasible that chemical synthesis prepares canthaxanthin theory,
But since the isomer of canthaxanthin is relatively more, and it easily occurs to go bad without being commonly applied under the conditions of photo-thermal is oxygen-containing
In industrially prepared.And microculture fermentation is relatively simple in synthesis in the lab, but is applied in industrial production and has
Some insoluble technical problems.
The patent of existing industrially prepared canthaxanthin is aoxidized by beta carotene.Such as Publication No. CN
The Chinese patent of 108101817A, the Chinese patent of Publication No. CN 1793098A, in Publication No. CN 105777599A
State's patent all discloses the method for preparing canthaxanthin by beta carotene oxidation.But the above method does not generally use largely and not
Recyclable organic reagent, oxidant etc. cause the waste of reagent, and yield is low.The China of Publication No. CN 105777599A
Patent extracts canthaxanthin using halogenated hydrocarbons, since halogenated hydrocarbons reagent has toxicity, for the application in food, it is necessary to strict guarantee
The content of halogenated hydrocarbons must not exceed maximum magnitude.Though and halogenated hydrocarbons reagent have high volatility, for remove canthaxanthin in
Contaminated with halogenated hydrocarbons object it is more difficult.The Chinese patent report halate of Publication No. CN 1793098A is as oxidizing
The method of beta carotene, but the hydracid salt waste after is not easy to handle, and certain pollution is caused to environment.It is even more important
, the canthaxanthin purity of beta carotene oxidizing process preparation cannot be up to state standards the requirement of substance, and above method is obtained
The sample purity of canthaxanthin is respectively less than 90%, cannot function as food, in feed assay reference substance.For this purpose, establishing one
The feasible and purification process suitable for industrially prepared high-purity canthaxanthin of kind theory is particularly significant.
Summary of the invention
It is an object of the present invention to provide a kind of method that solvent pairs combination medium pressure liquid chromatography divides purification of high-purity canthaxanthin, solutions
The certainly above problem.
The technical scheme is that
A kind of solvent pairs combination medium pressure liquid chromatography divides the method for purification of high-purity canthaxanthin, which is characterized in that including step
It is rapid:
(1) canthaxanthin is extracted from raw material using single solvent；
(2) middle pressure glass column is loaded to be chromatographed；
(3) double base solvent isocratic elution is utilized；
(4) subject component is collected up to sterling.
Further, in step (1) extractant be acetonitrile, chloroform, ethyl acetate, in acetone any one or it is more
Kind.
Further, the aglucon of chromatographic stuffing is any in phenyl, pentafluorophenyl group, penta-bromophenyl or C18 in step (2)
One or two kinds of combination.
Further, the aglucon of chromatographic stuffing is that C18 and two kinds of pentafluorophenyl group combine in step (2), the C18 and five fluorine
The ratio of phenyl is 2:8.
Further, the average particle size of chromatographic stuffing is 15-35 μm in step (2), and the aperture of the chromatographic stuffing isOr
Further, the average particle size of chromatographic stuffing is 15 μm in step (2), and the aperture of the chromatographic stuffing is
Silica matrix filler.
Further, the A phase with B phase of double base solvent described in step (3) are methanol, acetonitrile, tetrahydrofuran, ethyl acetate
In any two kinds.
Further, the A phase with B phase of double base solvent described in step (3) are acetonitrile and tetrahydrofuran, and mixed proportion is
35:65.
Further, it is 1~5 times of column volume of elution that the fraction period is collected in step (4).
Further, it is 1.5 times~2.0 times column volumes of elution that the fraction period is collected in step (4).
The present invention provides a kind of method that solvent pairs combination medium pressure liquid chromatography divides purification of high-purity canthaxanthin, advantages
Are as follows:
(1) present invention is used as chromatography stationary phase by using mixed fillers, can be from 10% canthaxanthin crude product, once
Preparation is purified into 99% or more canthaxanthin, and sample stable yield is 85% or more after purification, sample purity and receipts after purification
Rate is all much higher than prior art achievement obtained, this is particularly important for preparing for reference substance and standard items；
(2) without heating, and when concentration, reduced vacuum is concentrated the present invention, is protected from light when dry, available by these conditions
The stable canthaxanthin being unlikely to deteriorate, and existing oxidizing process prepares canthaxanthin, all needing heated or illumination just can be carried out, greatly
To increase canthaxanthin rotten or obtain the probability of other isomers；
(3) applied sample amount of study sample to be purified is big in the present invention, has been greatly saved cost, and existing some oxidations
The amount and organic reagent dosage of oxidant needed for method prepares canthaxanthin are big, and at high cost, environmental pollution is serious.To environmental protection
It is required that and in the case where producers' health growing interest, the consumption for reducing solvent is particularly important；
(4) chromatographic purification process of the present invention is quick, just can be collected into canthaxanthin eluent within ten minutes or so, preferably avoid
Purpose product has an adverse effect in purification process, such as generates isomer, to influence sample purity and yield.
Detailed description of the invention
Fig. 1 is the molecular structure of canthaxanthin；
Fig. 2 be using C18-5 μm-The analysis chromatogram purification map of (4.6mm × 250mm) analysis chromatographic column；
Fig. 3 be using PFP-C18-15 μm-(15mm × 460mm) prepares the middle compacting of chromatographic column for chromatogram purification figure
Spectrum；
Fig. 4 be using PFP-C18-15 μm-The color for continuously preparing canthaxanthin of (15mm × 460mm) preparation chromatographic column
Spectrogram；
Fig. 5 is the analysis chromatogram of the canthaxanthin sterling after pressing chromatogram purification in preparation；
Fig. 6 is the mass spectrogram of the canthaxanthin sterling after pressing chromatogram purification in preparation；
Fig. 7 is the hydrogen spectrogram of the canthaxanthin sterling after pressing chromatogram purification in preparation.
Specific embodiment
The technical scheme is that
(1) canthaxanthin is extracted from raw material using single solvent；
(2) middle pressure glass column is loaded to be chromatographed；
(3) double base solvent isocratic elution is utilized；
(4) subject component is collected up to sterling.
Chromatographic purification method of the present invention, extractant described in step (1) be acetonitrile, chloroform, ethyl acetate, in acetone
One or more, preferably acetonitrile.The present invention is by comparing feed grade canthaxanthin (purity 10%) in different organic examinations
Dissolution degree in agent, to achieve the purpose that the extraction canthaxanthin crude product as much as possible from feed addictive.Extract sample
Analysis chromatogram is shown in Fig. 2.The extraction efficiency of each Extraction solvent is shown in Table 1.
The present invention has inquired into different aglucons, granularity and the aperture of canthaxanthin extractant, chromatographic stuffing by control variate method
And the selection of mobile phase isolates and purifies the influence of effect to canthaxanthin.The result shows that different extractants is for canthaxanthin
Extraction efficiency is different, and gap is larger.The wherein opposite highest of efficiency that acetonitrile is extracted in feed addictive.
Influence of the different aglucons of chromatographic stuffing to canthaxanthin separating effect is also different: phenyl, pentafluorophenyl group, phenyl-pentabromide
Tetra- kinds of base, C18 aglucons can arrive preferable purification effect, and sample purity reaches 96.83%-99.50% after purification, purify yield
For 80%-90%: wherein C18 aglucon is better than other two kinds of filler aglucons for the purification effect of canthaxanthin, and has statistics
Difference in meaning.The granularity of chromatographic stuffing and aperture also have an impact to separation.Experiment discovery, the granularity of filler are got over
Small, separating degree is higher, and yield is also higher.For this purpose, suppress in standby chromatography middle, we have selected granularity for 15 μm of filler.And
The aperture of filler does not have larger impact to the purity and yield of canthaxanthin, but has apparent influence to single sample volume.Its mesoporous
Diameter isFiller applied sample amount highest.
The selection of mobile phase is affected to the present invention.In methanol, acetonitrile, the organic reagents such as tetrahydrofuran, ethyl acetate
Two-by-two proportion or methanol, water, acetonitrile three match two-by-two in find, different reagent separating effects have significance difference away from.Research
It was found that the mobile phase is methanol-acetonitrile, methanol-tetrahydrofuran, acetonitrile-tetrahydrofuran, separating effect is preferable, preferably second
Nitrile-tetrahydrofuran (35-65) purifies yield highest.
The fraction collection time of the invention is higher on purity and yield influence, maintains high purity, will greatly reduce sample
The rate of recovery of product.Therefore, according to having there is experience, under the conditions of maintaining the rate of recovery 90%, the purity of sample is improved as far as possible, therefore
Selecting elution volume for 1.5-2 times is optimum condition.
The chromatographic purification method of high-purity canthaxanthin be using from the sample extracted in chicken feed addictive purpose product and
Other impurities relative hydrophobicity and selective difference in specific stationary phase and mobile phase are purified.The method of the present invention
Simple possible, study sample applied sample amount is big, after purification sample purity height and high income, and the at low cost and process stabilizing of purifying is reliable,
Suitable for preparation of industrialization high-purity canthaxanthin.
The method of the present invention is suitable for middle pressure preparative scale chromatography.
The experimental result of the above method can be refering to Fig. 2-Fig. 7, as shown in Fig. 2, peak1 is that we will be from system in figure
The canthaxanthin sterling isolated and purified in standby chromatography；As shown in figure 3, peak1 i.e. our collected eluents containing canthaxanthin in figure；
As shown in figure 4, during preparation altogether on sample three times, due to multiple loading, baseline is uneven, but has no effect on the collection of canthaxanthin；Such as
Shown in Fig. 5, peak is canthaxanthin sterling, and purity is up to 99.5%；As shown in fig. 6, the substance that nucleocytoplasmic ratio is 565.4002 is
Our Chinese blister beetles to be detected, other fragment peaks do not refer to；As shown in fig. 7, from figure for the integral of hydrogen and with text
The hydrogen spectrogram comparison of canthaxanthin is offered it can be seen that this substance is canthaxanthin sterling prepared by the present invention.
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, combined with specific embodiments below into
One step illustrates technical solution of the present invention.But the present invention is not limited to listed embodiments, should also be included in the present invention and be wanted
Other any well known changes in the interest field asked.
" one embodiment " or " embodiment " referred to herein, which refers to, may be included at least one implementation of the invention
A particular feature, structure, or characteristic." in one embodiment " that different places occur in the present specification not refers both to same
A embodiment, nor the individual or selective embodiment mutually exclusive with other embodiments.
Term " reverse phase silica gel " is the silica material that surface is coated with hydrophobic matrix, and wherein hydrophobic matrix can be alkane
Hydrocarbon；" coating " functional group i.e. different using the Si-OH group bonding of Silica Surface, becomes suitable for different clastotypes
Chromatography fill out；Can be generally chemically modified by three kinds of approach to silica gel to prepare silica gel bonded phase: coating, entirety are repaired
The chemical modification method of decorations method and surface Si-OH.In the present invention, term " reverse phase silica gel " and term " advanced silica-gel carrier " contain
Justice is identical, and the two can be interchanged；Term " preparation chromatography " is interpreted as preparing net product at industrial scale.
1. test material and instrument
Acetonitrile (CH3CN, prepare chromatographic grade) etc. organic solvents purchased from upper starfish can high purity reagent Co., Ltd, preparative color
Spectrum column (15mm × 460mm), filler and preparative efficient liquid phase instrument are all from the limited public affairs of Suzhou Correspondent chromatographic separation and purification
Department.
2. sample purity detection method:
It is detected using sample of the reversed phase chromatography method to study and purified pool, pillar uses ODS-C18-5 μm of color
It composes column (4.6mm × 250mm).Flow velocity is 1mL/min, and column temperature is 25 DEG C, Detection wavelength 470nm.Isocratic elution 20min.It protects
Staying the time in the main peak of 12-13min is purpose product.Purity, which calculates, uses area normalization method.
The extracting method of canthaxanthin in feed addictive is implemented in preparation.
Suitable chicken feed addictive 100g is weighed, quarter is divided into, is separately added into acetonitrile, acetone, ethyl acetate, trichlorine
Each 200mL of methane after stirring 60min under magnetic stirring apparatus, crosses 0.45 μm of filter membrane, collects filtrate, obtain canthaxanthin extracting solution.
Embodiment 1
Study influence of the different extractants to the extraction efficiency of canthaxanthin in feed addictive
Experimental method: the study sample is as above, is 10% feed grade canthaxanthin.Chromatographic purification method uses
(C18-15μm-) carried out on middle pressure preparative chromatographic column (15mm × 460mm), filling pressure is 3MPa；Elution stream
Dynamic is mutually methanol；Elution flow rate is 18mL/min；Detection wavelength is 470nm.
Implementation steps:
(1) feed addictive is extracted using extracting solution；
(2) the canthaxanthin study sample loading pillar for extracting 10mL is then steady to baseline using equilibrium liquid balance pillar
It is fixed.
(3) it uses mobile phase for methanol, is eluted, monitor wavelength 470nm.
(4) acquisition time is 12-13min；
Influence of 1 different solvents of table to purity and yield
From table 1 it follows that there is no too many differences in the canthaxanthin purity of four kinds of solvent extractions, but yield difference is huge
Greatly.Extraction solvent, yield highest are done with acetonitrile.Therefore acetonitrile is selected to extract the Chinese blister beetle in feed addictive as Extraction solvent
It is yellow.
Embodiment 2
Study influence of the chromatographic stuffing different grain size to the separating effect of canthaxanthin in feed addictive
Experimental method: the study sample is as above, is 10% feed grade canthaxanthin.Chromatographic purification method is pressed in using
It is carried out on preparative scale chromatography column (15mm × 460mm), filling pressure is 3MPa；Elution is methanol with mobile phase；Elution flow rate is
18mL/min；Detection wavelength is 470nm.
Implementation steps:
(1) feed addictive is extracted using acetonitrile；
(2) the 10mL canthaxanthin study sample extracted is loaded to middle pressure glass column, pillar is then balanced using equilibrium liquid
To baseline stability.It is loaded respectively in middle pressure glass column:
C18-15μm-C18-20μm-C18-20μm-
(3) it uses mobile phase for methanol, is eluted, monitor wavelength 470nm.
(4) acquisition time is 12-13min；
Test result is shown in Table 2
Influence of the different filler particle sizes of table 2 to purity and yield
The experimental results showed that filler particle size is smaller, sample purity and yield are higher after purification.As can be seen from the table,
The filler that 15 μm of granularity is best for the purification effect of canthaxanthin, and sample purity is higher after purification for performance, is 99.31%, and
Yield is purified also with respect to highest.Therefore the filler that preferred size is 15 μm uses filler as canthaxanthin purifying.
Embodiment 3
Study the influence that isolates and purifies of the chromatographic stuffing different pore size to canthaxanthin in feed addictive
Experimental method: the study sample is the canthaxanthin study solution that acetonitrile extracts: chromatographic purification method uses silicon
Glue filler is identical, granularity is identical (C18-15 μm) but aperture is different Filler making
It is carried out on standby type chromatographic column (15mm × 460mm), filling pressure is 3MPa；Elution mobile phase first methanol, elution flow rate are
18mL/min；Detection wavelength is 470nm
Implementation steps:
(1) feed addictive is extracted using acetonitrile；
(2) the 10mL canthaxanthin study sample extracted is loaded to middle pressure glass column, pillar is then balanced using equilibrium liquid
To baseline stability.It is loaded respectively in middle pressure glass column:
C18-15μm-C18-15μm-C18-15μm-C18-15μm-
(3) it uses mobile phase for methanol, is eluted, monitor wavelength 470nm.
(4) acquisition time is 12-13min；
Test result is shown in Table 3
Influence of the different filler apertures of table 3 to purity and yield
The experimental results showed that filler aperture is smaller, single applied sample amount is bigger.As can be seen from the table, four kinds of apertures
Filler is more close for the purification effect of canthaxanthin, but aperture isFiller its single applied sample amount it is higher, this leads to list
Secondary resulting canthaxanthin sterling amount is also more.Under the conditions of preparing the canthaxanthin sterling of equivalent, the reagent cost of consumption is minimum.
Therefore preferably aperture isFiller as canthaxanthin purifying use filler.
Embodiment 4
Influence of the based filler to separating effect is matched in research
Experimental method: the study sample is as above, is 10% feed grade canthaxanthin.Chromatographic purification method is pressed in using
It is carried out on preparative scale chromatography column (15mm × 460mm), filling pressure is 3MPa；Elution is methanol with mobile phase；Elution flow rate is
18mL/min；Detection wavelength is 470nm.
Implementation steps:
(1) feed addictive is extracted using acetonitrile；
(2) the 10mL canthaxanthin study sample extracted is loaded to middle pressure glass column, pillar is then balanced using equilibrium liquid
To baseline stability.It is loaded respectively in middle pressure glass column:
C4-15μm-C8-15μm-C18-15μm-- 15 μm of phenyl-15 μm of pentafluorophenyl group-15 μm of penta-bromophenyl-- C18-15 μm of pentafluorophenyl group-- C18-15 μm of penta-bromophenyl-Phenyl-pentabromide
- C8-15 μm of base-
(3) it uses mobile phase for methanol, is eluted, monitor wavelength 470nm.
(4) acquisition time is 12-13min；
Test result is shown in Table 4
Influence of the different fillers of table 4 to purity and yield
Experimental result: as can be seen from the table ,-C18-5 μm of pentafluorophenyl group-Purifying of the mixed fillers for canthaxanthin
Effect is best, and sample purity is higher after purification for performance, reaches 99.16%, and purifies yield also with respect to highest.Therefore preferably five
- C18-5 μm of fluorophenyl-Filler uses filler as canthaxanthin purifying.
Embodiment 5
Study the influence that isolates and purifies of the different eluant, eluents to canthaxanthin in feed addictive
Experimental method: the study sample is as above, is 10% feed grade canthaxanthin.Chromatographic purification method is pressed in using
It is carried out on preparative scale chromatography column (15mm × 460mm), filling pressure is 3MPa；Elution flow rate is 18mL/min；Detection wavelength is
470nm。
Implementation steps:
(1) feed addictive is extracted using acetonitrile；
(2) the 10mL canthaxanthin study sample extracted is loaded to middle pressure glass column, pillar is then balanced using equilibrium liquid
To baseline stability.Filling in middle pressure glass column: C18-15 μm-
(3) mobile phase is used to be eluted, and monitors wavelength 470nm.
Mobile phase is respectively as follows: methanol: acetonitrile, methanol: tetrahydrofuran, tetrahydrofuran: acetonitrile, acetonitrile: water, ethyl acetate:
Acetonitrile
(4) acquisition time is 12-13min；
Test result is shown in Table 5
The influence of the different elution flowing relative purities and yield of table 5
Experimental result: as can be seen from the table, the selection of mobile phase is affected to the purifying of canthaxanthin and yield.In first
Alcohol, acetonitrile, the organic reagents such as tetrahydrofuran, ethyl acetate two two match or methanol, water, send out in the proportion of acetonitrile threes two
It is existing, different reagent separating effects have significance difference away from.The study found that the mobile phase be methanol-acetonitrile, methanol-tetrahydrofuran,
Acetonitrile-tetrahydrofuran, separating effect is preferable, preferably methanol-acetonitrile (50:50), purifies yield highest.
Embodiment 6
The research present invention is compared with related Chinese patent is in main technologic parameters and purification effect, and see Table 6 for details
In conclusion a kind of solvent pairs combination medium pressure liquid chromatography of the present invention divides the side of purification of high-purity canthaxanthin
Method prepares purity up to 99% or more canthaxanthin sterling using medium pressure liquid chromatography.The organic reagent that this invention uses, such as second
Nitrile etc. can be recycled using technological means, not will cause environmental pollution and reagent waste.Followed by, chromatographic purification does not need
Using photo-thermal, the oxygen containing condition for being easily denaturalized canthaxanthin, environment is mild, easy to operate.The canthaxanthin that chromatography is isolated is pure
Degree greatly increases, and is increased to 99% or more by 2.5%, 10% common in the market equal size, is more applicable for as reference substance
Matter, as food, the reference substance of pharmaceuticals industry canthaxanthin analyte.Crude samples concentration can be quickly increased to by this method by 10%
99% or more, and after purification sample yield 95% or more, have it is stronger can amplification, be suitable as industrialized purification Chinese blister beetle
Yellow chromatographic purification method.
It should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to preferable
Embodiment describes the invention in detail, those skilled in the art should understand that, it can be to technology of the invention
Scheme is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered in this hair
In bright scope of the claims.
Claims (10)
1. a kind of solvent pairs combination medium pressure liquid chromatography divides the method for purification of high-purity canthaxanthin, which is characterized in that comprising steps of
(1) canthaxanthin is extracted from raw material using single solvent；
(2) middle pressure glass column is loaded to be chromatographed；
(3) double base solvent isocratic elution is utilized；
(4) subject component is collected up to sterling.
2. the side that 1. a kind of solvent pairs combination medium pressure liquid chromatography according to claim 1 divides purification of high-purity canthaxanthin
Method, it is characterised in that: extractant is acetonitrile, chloroform, ethyl acetate, any one or more in acetone in step (1).
3. a kind of solvent pairs combination medium pressure liquid chromatography according to claim 1 divides the method for purification of high-purity canthaxanthin,
It is characterized by: in step (2) chromatographic stuffing aglucon be phenyl, pentafluorophenyl group, penta-bromophenyl or C18 in any one or
Two kinds of person combinations.
4. a kind of solvent pairs combination medium pressure liquid chromatography according to claim 3 divides the method for purification of high-purity canthaxanthin,
It is characterized by: the aglucon of chromatographic stuffing is C18 and two kinds of pentafluorophenyl group combinations in step (2), the C18 and pentafluorophenyl group
Ratio is 2:8.
5. a kind of solvent pairs combination medium pressure liquid chromatography according to claim 1 divides the method for purification of high-purity canthaxanthin,
It is characterized by: the average particle size of chromatographic stuffing is 15-35 μm in step (2), the aperture of the chromatographic stuffing isOr
6. a kind of solvent pairs combination medium pressure liquid chromatography according to claim 5 divides the method for purification of high-purity canthaxanthin,
It is characterized by: the average particle size of chromatographic stuffing is 15 μm in step (2), the aperture of the chromatographic stuffing isSilica gel
Matrix fill.
7. a kind of solvent pairs combination medium pressure liquid chromatography according to claim 1 divides the method for purification of high-purity canthaxanthin,
It is characterized by: the A phase of double base solvent described in step (3) and B phase are methanol, acetonitrile, tetrahydrofuran, appointing in ethyl acetate
Two kinds of meaning.
8. a kind of solvent pairs combination medium pressure liquid chromatography according to claim 7 divides the method for purification of high-purity canthaxanthin,
It is characterized by: the A phase of double base solvent described in step (3) is acetonitrile and tetrahydrofuran, mixed proportion 35:65 with B phase.
9. a kind of solvent pairs combination medium pressure liquid chromatography according to claim 1 divides the method for purification of high-purity canthaxanthin,
It is characterized by: collecting the fraction period in step (4) is 1~5 times of column volume of elution.
10. a kind of solvent pairs combination medium pressure liquid chromatography according to claim 9 divides the method for purification of high-purity canthaxanthin,
It is characterized by: collecting the fraction period in step (4) is 1.5 times~2.0 times column volumes of elution.
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| CN110749689A (en) * | 2019-11-07 | 2020-02-04 | 贵州省兽药饲料监察所(贵州省兽药残留监测中心) | Method for measuring content of cantharis yellow colorant in feed |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110749689A (en) * | 2019-11-07 | 2020-02-04 | 贵州省兽药饲料监察所(贵州省兽药残留监测中心) | Method for measuring content of cantharis yellow colorant in feed |
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