CN106565732B - A method of using rosin based high molecular separating common camptotheca fruit alkali and 10-hydroxycamptothecine - Google Patents
A method of using rosin based high molecular separating common camptotheca fruit alkali and 10-hydroxycamptothecine Download PDFInfo
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- CN106565732B CN106565732B CN201611001162.2A CN201611001162A CN106565732B CN 106565732 B CN106565732 B CN 106565732B CN 201611001162 A CN201611001162 A CN 201611001162A CN 106565732 B CN106565732 B CN 106565732B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2/00—Processes of polymerisation
- C08F2/12—Polymerisation in non-solvents
- C08F2/16—Aqueous medium
- C08F2/20—Aqueous medium with the aid of macromolecular dispersing agents
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- C—CHEMISTRY; METALLURGY
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- C08F289/00—Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds not provided for in groups C08F251/00 - C08F287/00
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Abstract
The present invention discloses a kind of method for efficiently separating camptothecine and 10-hydroxycamptothecine using rosin based high molecular chromatographic column, with α-methacrylic acid (or methyl methacrylate) for monomer, maleic rosin acrylic acid glycol ester is crosslinking agent, prepares rosinyl polymer microsphere using micro suspension free radical polymerisation process；Microballoon is spherical porous material, and particle diameter distribution is 3-10 μm, average pore size 10-15nm, specific surface area 90-120m2/g, acid value 50-150mgKOH/g.Rosinyl polymer microsphere packing column machine wet method dress post is prepared into chromatographic column；By carrying out HPLC separation to camptothecine and 10-hydroxycamptothecine, Detection wavelength: 230-290nm, 30 ± 10 DEG C of temperature, flow velocity 0.3-1.0mL/min, the separating degree of camptothecine and 10-hydroxycamptothecine is 1.80-2.15.For the present invention to camptothecine and 10-hydroxycamptothecine separating degree with higher, selectivity is high, and this method high sensitivity, easy to operate, rapidly and efficiently, will not cause secondary pollution to drug, health care product, food.
Description
Technical field
The invention belongs to high performance liquid chromatography field, especially a kind of application rosin based high molecular chromatographic column is efficiently separated
The method of camptothecine and 10-hydroxycamptothecine.
Background technique
Camptothecine, chemical structural formula:
Camptothecine is indoles alkaloid contained in camplotheca acuminata, has significant anticancer activity, is the third of plant resources
Big natural anti-cancer drugs, camptothecine have a better effect intestines and stomach and head-neck carcinoma etc., by inhibiting topoisomerase I to play
Cytotoxicity；Structure is similar with 10-hydroxycamptothecine, but pharmacological action is different.
10-hydroxycamptothecine, chemical structural formula:
10-hydroxycamptothecine is the indoles alkaloid that content is less in camplotheca acuminata, can also be synthesized by camptothecine.It is not
Only the alkaloids more isolated than camplotheca acuminata have higher pharmacological activity, and generate less toxicity, structure and camptothecin
Seemingly, but pharmacological action is higher than camptothecine.
Liquid-phase chromatographic analysis refers to that mobile phase is that the chromatographic technique of liquid is being passed through by improving the granularity and column pressure of filler
The plate theory of gas-chromatography is introduced on the basis of the liquid column chromatography of allusion quotation, technically uses high pressure pump, efficiently
Stationary phase and highly sensitive detector, realize that analysis speed is fast, separative efficiency is high and operation automation, this chromatographic technique
Referred to as high performance liquid chromatography (High performance liquid chromatography, abbreviation HPLC).HPLC has
Following characteristics: (1) analysis speed is fast-usual analyzes a sample in 15~30min, some samples are even in 5min
It completes；(2) selectivity it is high-can the substance to otherness very little separation analysis is carried out to such as chiral material；(3) detection sensitivity
Height-UV detector is up to 0.01ng, and fluorescence and electrochemical detector are up to 0.1pg；Pillar Reusability, with a chromatography
Column separates different compounds；Sample size is few, is easy recycling；Sample is not destroyed after chromatographic column, can be collected single
It is prepared by component.
Currently, we find the following document about the method report that camptothecine and 10-hydroxycamptothecine separate:
1. the HPLC of camptothecine and 10-hydroxycamptothecine analyzes drug point in Liu Wenzhe, Qin Haiyan, Suo Zhirong Common Camptotheca Fruit
Analyse magazine .2005,25 (2): 168-170；It is mentioned that the liquid phase chromatogram condition of camptothecine and 10-hydroxycamptothecine, is used
Hypersil ODS chromatographic column (250mm × 4.0mm) carries out gradient elution, flow velocity 1.0mL/ by mobile phase of first alcohol and water
Min, Detection wavelength 254nm, 25 DEG C of column temperature.
2. Shi Weiguo, Zu YuanGang, 10-hydroxycamptothecine and vincoside-lactam is ground in Yang Lei's polyamide separation and purification Common Camptotheca Fruit
Study carefully CHINA JOURNAL OF CHINESE MATERIA MEDICA .2008,33 (21): 2486-2489；It is mentioned that the detection side of 10-hydroxycamptothecine and vincoside-lactam
Method, using C18Chromatographic column (250mm × 4.6mm), using acetonitrile and water as mobile phase, flow velocity 1.0mL/min, Detection wavelength 254nm,
35 DEG C of column temperature.
3. application number: 200510024487.8, denomination of invention: a kind of production method of 10-hydroxycamptothecine, it is mentioned that
The separation of camptothecine and 10-hydroxycamptothecine, use high pressure liquid phase prepare column (filler for little particle spherical silica gel, partial size 10
~16 μm) sample introduction upper prop under conditions of 10~30 DEG C, flow velocity is 250~350mL/min, sets ultraviolet detection wavelength 254nm.
About rosin based high molecular chromatographic column separating common camptotheca fruit alkali and 10-hydroxycamptothecine is applied, up to the present do not appear in the newspapers
Road.
Summary of the invention
The purpose of the present invention is to provide a kind of application rosin based high molecular chromatographic columns to efficiently separate camptothecine and 10- hydroxyl
The method of camptothecine, it is this method high sensitivity, easy to operate, rapidly and efficiently, drug, health care product, food will not be caused secondary
Pollution.
To achieve the above object, the present invention the following technical schemes are provided:
A method of camptothecine and 10-hydroxycamptothecine being efficiently separated using rosin based high molecular chromatographic column, by rosin
Rosin based high molecular chromatographic column is prepared with packing column machine wet method dress post as fixed phase stuffing in based high molecular microballoon；By abietyl
Macromolecule chromatographic column accesses liquid chromatograph, and the flow rate of mobile phase that liquid chromatograph is arranged is 0.3-1.0mL/min, Detection wavelength
230-290nm, 30 ± 10 DEG C of column oven；Starting sampling valve makes mobile phase by the mixed solution of camptothecine and 10-hydroxycamptothecine
It brings into rosin based high molecular chromatographic column, realizes the separation of camptothecine and 10-hydroxycamptothecine.
Rosinyl polymer microsphere separates the mechanism of two kinds of substances: in camptothecine and 10-hydroxycamptothecine molecule, itself band
There are C=O ,-OH group, can interact with-the COOH in rosinyl polymer microsphere, form stable associated matter, camplotheca acuminata
Alkali and 10-hydroxycamptothecine can dissociate H in mobile phase+, its active force between stationary phase can be weakened, therefore can be eluted
Get off.But the hydroxyl more than camptothecine of No. 10 positions in 10-hydroxycamptothecine molecule, it is different from stationary phase active force, it is flowing
The H dissociateed in dynamic phase+More, weaker with the active force of stationary phase, so during the separation process, 10-hydroxycamptothecine can be first
Appearance.
As the further improvement of technical solution, above-described rosinyl polymer microsphere particle diameter distribution is 3-10 μm,
Average pore size is 10-15nm, specific surface area 90-120m2/ g, acid value 50-150mgKOH/g.
As the further improvement of technical solution, above-described packing column machine wet method dress post is with constant pressure pump by abietyl height
Molecule microballoon is filled in sky chromatographic column, loads 120-180min under 3000psi pressure, continues to increase pressure to 3500psi
10-60min is loaded, after column flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto column cap, abietyl can be prepared
Macromolecule chromatographic column.
As the further improvement of technical solution, above-described rosinyl polymer microsphere the preparation method comprises the following steps: with α-
Methacrylic acid or methyl methacrylate are monomer, and maleic rosin acrylic acid glycol ester is crosslinking agent, certainly using micro suspension
Rosinyl polymer microsphere is prepared by base polymerization.
As the further improvement of technical solution, pine micro suspension free radical polymerisation process described above are as follows: by ionized water, ten
Sodium dialkyl sulfate, the water phase of polyvinyl alcohol composition and α-methacrylic acid or methyl methacrylate, crosslinking agent maleic rosin
The oil that acrylic acid glycol ester, solvent ethyl acetate, pore-foaming agent isooctane, initiator azodiisobutyronitrile form mixes, and uses
Dispersion machine is dispersed, and it is 3000-5000rpm, jitter time 5-20min that revolving speed, which is arranged, in dispersion machine, then carries out program liter
The rosinyl polymer microsphere can be obtained in temperature reaction.α-methacrylic acid or methyl methacrylate are present invention separation
The function monomer of camptothecine and 10-hydroxycamptothecine.
As the further improvement of technical solution, above-described oil is mutually and the mass ratio of water phase is 1:3-6.
As the further improvement of technical solution, routine described above temperature reaction are as follows: 70 DEG C of reaction 10- of temperature programming
The rosinyl polymer microsphere can be obtained in 80min, 80 DEG C of reactions 20-100min, 95 DEG C of reaction 30-60min.
As the further improvement of technical solution, deionized water, lauryl sodium sulfate, poly- second in above-described water phase
The mass ratio of enol is 100:0.1-0.5:1-3.
As the further improvement of technical solution, α-methacrylic acid or methyl methacrylate in above-described oil phase
Ester, crosslinking agent, solvent, pore-foaming agent, initiator quality ratio are 0.5-3:3-12.:50-100:1-5:0.1-2.
Compared with prior art, the invention has the benefit that
1. the rosin based high molecular chromatographic column that the present invention prepares, fixed phase stuffing dilation is small, large specific surface area, can
It for extracting the effective component in plant, and can use in organic solvent, the network knot of polymer will not be destroyed because of expansion
Structure prepares rosin based high molecular chromatographic column by filler of rosinyl polymer microsphere, because being rich in pore structure not of uniform size, has
There is not the phenomenon that be collapsed under higher flow velocity and pressure in the advantages that permeability is good, back pressure is low, efficient and high-throughput, and
And stability is good, reusable, after long-time use, the filler in chromatographic column is still without destruction, dissolution.
2. chromatographic column used in the present invention has preferable separating effect, best result to camptothecine and 10-hydroxycamptothecine
From degree up to 2.15 or more, with existing commodity column C18Column is compared, C18The separating degree of column is 1.74, and separating effect can be improved
24% or more.
3. rosinyl polymer microsphere is cheap and easy to get using the derivative of product of natural product rosin as raw material in the present invention, mechanical
Intensity is high, safe and non-toxic, can be used for the separation of food-grade.
4. the present invention prepares rosin based high molecular chromatographic column using wet method dress post, column effect is high, and crushing resistance is good.
5. HPLC high sensitivity of the invention, easy to operate, rapidly and efficiently.
Compared with prior art, the invention has the benefit that
1. in Detection wavelength: 230-290nm, mobile phase be 0.1% acetic acid methanol, 30 DEG C of temperature, flow velocity 0.5mL/min's
Under the conditions of, the separating degree ratio C of the invention patent18The separating degree of column is big.
2. the present invention applies rosin based high molecular chromatographic column separating common camptotheca fruit alkali and 10-hydroxycamptothecine, mobile phase is single, no
It needs to add buffer solution, is conducive to the preparation of future drugs, reduces the pollution again of drug.
3. the cost and C of rosin based high molecular chromatographic column fixed phase of the present invention18The stationary phase cost of column compares, the present invention
Stationary phase cost is relatively low.
Detailed description of the invention
Fig. 1 is C18Analysis chart of the column to camptothecine and 10-hydroxycamptothecine mixed solution；
Fig. 2 is analysis chart of the embodiment of the present invention 4 to camptothecine and 10-hydroxycamptothecine mixed solution；
Fig. 3 is analysis chart of the embodiment of the present invention 5 to camptothecine and 10-hydroxycamptothecine mixed solution；
Fig. 4 is analysis chart of the embodiment of the present invention 6 to camptothecine and 10-hydroxycamptothecine mixed solution；
Fig. 5 is analysis chart of the embodiment of the present invention 7 to camptothecine and 10-hydroxycamptothecine mixed solution
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to
The range that embodiment indicates.
The preparation of rosinyl polymer microsphere:
Embodiment 1:
200g deionized water, 0.4g lauryl sodium sulfate, 4.0g polyvinyl alcohol (ion are added into the beaker of 250mL
Water, lauryl sodium sulfate, polyvinyl alcohol mass ratio be 100:0.2:2), being heated to 95 DEG C makes polyvinyl alcohol and dodecyl
Sodium sulphate is completely dissolved, and obtains water phase, and being transferred in water-bath makes temperature be down to 55 DEG C of constant temperature.
3.00g maleic rosin acrylic acid glycol ester is weighed, is dissolved in 50.0g ethyl acetate, using ultrasonic wave dissolution solution,
After crosslinking agent is completely dissolved, 0.50g α-methacrylic acid, 1.00g isooctane, 0.10g azodiisobutyronitrile are sequentially added
(function monomer, crosslinking agent, solvent, pore-foaming agent, initiator quality ratio are 0.5:3:50:1:0.1), sonic oscillation 10min, dispersion
Uniformly, oily phase is made.
The oil prepared is added in water phase (oil is mutually and the mass ratio of water phase is 1:3.75), is divided with dispersion machine
It dissipates, dispersing speed 3000rpm, jitter time 5min obtain lotion, and after the completion of dispersion, lotion is moved to tri- mouthfuls of 500mL
In flask, heat up polymerization under the mixing speed of 100rad/min, constant temperature 10min when temperature rises to 70 DEG C, perseverance when rising to 80 DEG C
Warm 20min, constant temperature 30min when rising to 95 DEG C.
After reaction, product is sieved, the sieve pore of sieve is 100 mesh, and the polymer after screening is through 5000rad/min's
Revolving speed is centrifuged 5min, and polymer is transferred in 500mL beaker and is washed with distilled water for several times to remove extra dispersing agent, will be produced
Object successively uses ethyl acetate, ethyl alcohol, methanol solution Soxhlet extraction.The acetic acid second in polymer is finally removed with steam distillation
Ester, ethyl alcohol, methanol, obtain rosinyl polymer microsphere.
Through testing and analyzing, the rosinyl polymer microsphere particle diameter distribution that the present embodiment obtains is 3-10 μm, and average pore size is
10-15nm, specific surface area 90-120m2/ g, acid value 50-150mgKOH/g.
Embodiment 2:
Into the beaker of 500mL be added 400g deionized water, 2.00g lauryl sodium sulfate, 12.00g polyvinyl alcohol (from
Sub- water, lauryl sodium sulfate, polyvinyl alcohol mass ratio be 100:0.5:3), being heated to 95 DEG C makes polyvinyl alcohol and dodecane
Base sodium sulphate is completely dissolved, and obtains water phase, and being transferred in water-bath makes temperature be down to 55 DEG C of constant temperature.
12.00g maleic rosin acrylic acid glycol ester is weighed, is dissolved in 100.00g ethyl acetate, uses ultrasonic wave dissolution
Solution, after crosslinking agent is completely dissolved, it is different to sequentially add 3.00g methyl methacrylate, 5.00g isooctane, 2.00g azo two
Butyronitrile (function monomer, crosslinking agent, solvent, pore-foaming agent, initiator quality ratio are 3:12:100:5:2), sonic oscillation 10min, point
It dissipates uniformly, oil phase is made.
The oil prepared is added in water phase (oil is mutually and the mass ratio of water phase is 1:3.4), is dispersed with dispersion machine,
Dispersing speed is 4000rpm, and jitter time 10min obtains lotion, and after the completion of dispersion, lotion is moved to tri- mouthfuls of 1000mL
In flask, heat up polymerization under the mixing speed of 120rad/min, constant temperature 50min when temperature rises to 70 DEG C, perseverance when rising to 80 DEG C
Warm 60min, constant temperature 60min when rising to 95 DEG C.
After reaction, product is sieved, the sieve pore of sieve is 100 mesh, and the polymer after screening is through 5000rad/min's
Revolving speed is centrifuged 10min, and polymer is transferred in 500mL beaker and is washed with distilled water for several times to remove extra dispersing agent, will
Product successively uses ethyl acetate, ethyl alcohol, methanol solution Soxhlet extraction.The acetic acid in polymer is finally removed with steam distillation
Ethyl ester, ethyl alcohol, methanol, obtain rosinyl polymer microsphere.
Through testing and analyzing, the rosinyl polymer microsphere particle diameter distribution that the present embodiment obtains is 3-10 μm, and average pore size is
10-15nm, specific surface area 90-120m2/ g, acid value 50-150mgKOH/g.
Embodiment 3:
300g deionized water, 3.0g polyvinyl alcohol, 0.30g lauryl sodium sulfate (ion are added into the beaker of 400mL
Water, lauryl sodium sulfate, polyvinyl alcohol mass ratio be 100:0.1:1), being heated to 95 DEG C makes polyvinyl alcohol and dodecyl
Sodium sulphate is completely dissolved, and obtains water phase, and being transferred in water-bath makes temperature be down to 55 DEG C of constant temperature.
3.25g maleic rosin acrylic acid glycol ester is weighed, is dissolved in 80.0g ethyl acetate, using ultrasonic wave dissolution solution,
After crosslinking agent is completely dissolved, 1.55g α-methacrylic acid, 3.0g isooctane, 1.0g azodiisobutyronitrile (function are sequentially added
Energy monomer, crosslinking agent, solvent, pore-foaming agent, initiator quality ratio are 1.55:3.25:80:3:1), sonic oscillation 10min, dispersion
Uniformly, oily phase is made.
The oil prepared is added in water phase (oil is mutually and the mass ratio of water phase is 1:3.42), is divided with dispersion machine
It dissipates, dispersing speed 5000rpm, jitter time 20min obtain lotion, and after the completion of dispersion, lotion is moved to 500mL tri-
In mouth flask, heat up polymerization under the mixing speed of 100rad/min, constant temperature 80min when temperature rises to 70 DEG C, when rising to 80 DEG C
Constant temperature 100min, constant temperature 45min when rising to 95 DEG C.
After reaction, product is sieved, the sieve pore of sieve is 100 mesh, and the polymer after screening is through 5000rad/min's
Revolving speed is centrifuged 5min, and polymer is transferred in 500mL beaker and is washed with distilled water for several times to remove extra dispersing agent, will be produced
Object successively uses ethyl acetate, ethyl alcohol, methanol solution Soxhlet extraction.The acetic acid second in polymer is finally removed with steam distillation
Ester, ethyl alcohol, methanol, obtain rosinyl polymer microsphere.
Through testing and analyzing, the rosinyl polymer microsphere particle diameter distribution that the present embodiment obtains is 3-10 μm, and average pore size is
10-15nm, specific surface area 90-120m2/ g, acid value 50-150mgKOH/g.
The method for efficiently separating camptothecine and 10-hydroxycamptothecine using rosin based high molecular chromatographic column
Embodiment 4:
The resulting rosinyl polymer microsphere of embodiment 2 is prepared into chromatographic column using wet method dress post, specifically: packing column machine is wet
Method dress column is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, loads 140min under 3000psi pressure,
Continue increase pressure and load 30min to 3500psi, after column flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto column cap,
Rosin based high molecular chromatographic column can be prepared, being rinsed with 0.10% acetic acid methanol solution can sample introduction to baseline balance.
A method of camptothecine and 10-hydroxycamptothecine being efficiently separated using rosin based high molecular chromatographic column, according to such as
Lower step carries out:
(1) it prepares sample solution: taking appropriate camptothecine, 10-hydroxycamptothecine, dissolved with methanol, be configured to every 1L containing 2.5
×10-4The camplotheca acuminata of mol and 10-hydroxycamptothecine mixed solution, sample introduction；
(2) setup parameter: rosin based high molecular chromatographic column is accessed into liquid chromatograph, the mobile phase of liquid chromatograph is set
Flow velocity is 0.5mL/min, Detection wavelength 230nm, 30 DEG C of column oven；
(3) separate: starting sampling valve brings methanol in rosin based high molecular chromatographic column sample into, and sample volume is 20 μ L,
Realize the separation of camptothecine and 10-hydroxycamptothecine, as shown in Fig. 2, in retention time, when 24.68min acquired results occurs
10-hydroxycamptothecine peak occurs camptothecine peak, separating degree 2.15 in retention time 33.61min.
Embodiment 5:
The resulting rosinyl polymer microsphere of embodiment 1 is prepared into chromatographic column using wet method dress post, specifically: packing column machine is wet
Method dress column is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, loads 120min under 3000psi pressure,
Continue increase pressure and load 10min to 3500psi, after column flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto column cap,
Rosin based high molecular chromatographic column can be prepared, being rinsed with methanol can sample introduction to baseline balance.
A method of camptothecine and 10-hydroxycamptothecine being efficiently separated using rosin based high molecular chromatographic column, according to such as
Lower step carries out:
(1) it prepares sample solution: taking appropriate camptothecine, 10-hydroxycamptothecine, dissolved with methanol, be configured to every 1L containing 2.5
×10-4The camptothecine and 10-hydroxycamptothecine mixed solution of mol, sample introduction；
(2) setup parameter: rosin based high molecular chromatographic column is accessed into liquid chromatograph, the mobile phase of liquid chromatograph is set
Flow velocity is 0.3mL/min, Detection wavelength 254nm, 25 DEG C of column oven；
(3) separate: starting sampling valve brings methanol in rosin based high molecular chromatographic column sample into, and sample volume is 20 μ L,
Realize the separation of camptothecine and 10-hydroxycamptothecine, as shown in figure 3, in retention time, when 30.69min acquired results occurs
10-hydroxycamptothecine peak occurs camptothecine peak, separating degree 1.93 in retention time 39.38min.
Embodiment 6:
The resulting rosinyl polymer microsphere of embodiment 3 is prepared into chromatographic column using wet method dress post, specifically: packing column machine is wet
Method dress column is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, loads 160min under 3000psi pressure,
Continue increase pressure and load 50min to 3500psi, after column flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto column cap,
Rosin based high molecular chromatographic column can be prepared, being rinsed with methanol solution can sample introduction to baseline balance.
A method of camplotheca acuminata and 10-hydroxycamptothecine being efficiently separated using rosin based high molecular chromatographic column, according to as follows
Step carries out:
(1) it prepares sample solution: taking appropriate camptothecine, 10-hydroxycamptothecine, dissolved with methanol, be configured to every 1L containing 2.5
×10-4The camptothecine and 10-hydroxycamptothecine mixed solution of mol, sample introduction；
(2) setup parameter: rosin based high molecular chromatographic column is accessed into liquid chromatograph, the mobile phase of liquid chromatograph is set
Flow velocity is 0.8mL/min, Detection wavelength 290nm, 35 DEG C of column oven；
(3) separate: starting sampling valve brings methanol in rosin based high molecular chromatographic column sample into, and sample volume is 20 μ L,
Realize the separation of camptothecine and 10-hydroxycamptothecine, as shown in figure 4, in retention time, when 28.55min acquired results occurs
10-hydroxycamptothecine peak occurs camptothecine peak, separating degree 1.99 in retention time 36.61min.
Embodiment 7:
The resulting rosinyl polymer microsphere of embodiment 2 is prepared into chromatographic column using wet method dress post, specifically: packing column machine is wet
Method dress column is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, loads 180min under 3000psi pressure,
Continue increase pressure and load 60min to 3500psi, after column flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto column cap,
Rosin based high molecular chromatographic column can be prepared, being rinsed with 0.10% acetic acid methanol solution can sample introduction to baseline balance.
A method of camptothecine and 10-hydroxycamptothecine being efficiently separated using rosin based high molecular chromatographic column, according to such as
Lower step carries out:
(1) it prepares sample solution: taking appropriate camptothecine, 10-hydroxycamptothecine, dissolved with methanol, be configured to every 1L containing 2.5
×10-4The camptothecine and 10-hydroxycamptothecine mixed solution of mol, sample introduction；
(2) setup parameter: rosin based high molecular chromatographic column is accessed into liquid chromatograph, the mobile phase of liquid chromatograph is set
Flow velocity is 1.0mL/min, Detection wavelength 254nm, 30 DEG C of column oven；
(3) separate: starting sampling valve brings methanol in rosin based high molecular chromatographic column sample into, and sample volume is 20 μ L,
Realize the separation of camptothecine and 10-hydroxycamptothecine, acquired results are as shown in figure 5, there is 10- in retention time 26.96min
Hydroxycamptothecin peak occurs camptothecine peak, separating degree 2.10 in retention time 37.27min.
Claims (8)
1. a kind of method for efficiently separating camptothecine and 10-hydroxycamptothecine using rosin based high molecular chromatographic column, feature exist
In: rosin based high molecular chromatography is prepared using rosinyl polymer microsphere as fixed phase stuffing with packing column machine wet method dress post
Column；Rosin based high molecular chromatographic column is accessed into liquid chromatograph, the flow rate of mobile phase that liquid chromatograph is arranged is 0.3-1.0mL/
Min, Detection wavelength 230-290nm, 30 ± 10 DEG C of column oven；Starting sampling valve makes mobile phase by camptothecine and 10- hydroxy-camptothecin
The mixed solution of alkali is brought into rosin based high molecular chromatographic column, realizes the separation of camptothecine and 10-hydroxycamptothecine；
The rosinyl polymer microsphere the preparation method comprises the following steps: using α-methacrylic acid or methyl methacrylate as monomer,
Maleic rosin acrylic acid glycol ester is crosslinking agent, prepares rosinyl polymer microsphere using micro suspension free radical polymerisation process.
2. a kind of application rosin based high molecular chromatographic column according to claim 1 efficiently separates camptothecine and the happiness of 10- hydroxyl
The method for setting alkali, it is characterised in that: the rosinyl polymer microsphere particle diameter distribution is 3-10 μm, average pore size 10-
15nm, specific surface area 90-120m2/ g, acid value 50-150mgKOH/g.
3. a kind of application rosin based high molecular chromatographic column according to claim 1 or 2 efficiently separates camptothecine and 10- hydroxyl
The method of camptothecine, it is characterised in that: the packing column machine wet method dress post is to be filled rosinyl polymer microsphere with constant pressure pump
In empty chromatographic column, load 120-180min under 3000psi pressure, continue increase pressure to 3500psi load 10-60min,
After column flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto column cap, rosin based high molecular chromatographic column can be prepared.
4. a kind of application rosin based high molecular chromatographic column according to claim 1 or 2 efficiently separates camptothecine and 10- hydroxyl
The method of camptothecine, it is characterised in that: the micro suspension free radical polymerisation process are as follows: by deionized water, lauryl sodium sulfate,
The water phase and α-methacrylic acid or methyl methacrylate, crosslinking agent maleic rosin acrylic acid ethylene glycol of polyvinyl alcohol composition
The oil that ester, solvent ethyl acetate, pore-foaming agent isooctane, initiator azodiisobutyronitrile form mixes, and is divided with dispersion machine
It dissipates, it is 3000-5000rpm, jitter time 5-20min that revolving speed, which is arranged, in dispersion machine, and then carrying out temperature-programmed reaction can obtain
To the rosinyl polymer microsphere.
5. a kind of application rosin based high molecular chromatographic column according to claim 4 efficiently separates camptothecine and the happiness of 10- hydroxyl
The method for setting alkali, it is characterised in that: the oil is mutually and the mass ratio of water phase is 1:3-6.
6. a kind of application rosin based high molecular chromatographic column according to claim 4 efficiently separates camptothecine and the happiness of 10- hydroxyl
The method for setting alkali, it is characterised in that: the temperature-programmed reaction are as follows: 70 DEG C of reaction 10-80min of temperature programming, 80 DEG C of reactions
20-100min, 95 DEG C of reaction 30-60min, can be obtained the rosinyl polymer microsphere.
7. a kind of application rosin based high molecular chromatographic column according to claim 4 efficiently separates camptothecine and the happiness of 10- hydroxyl
The method for setting alkali, it is characterised in that: deionized water in the water phase, lauryl sodium sulfate, polyvinyl alcohol mass ratio be
100:0.1-0.5:1-3.
8. efficiently separating camptothecine and 10- according to a kind of any application rosin based high molecular chromatographic column of claim 5-7
The method of hydroxycamptothecin, it is characterised in that: α-methacrylic acid or methyl methacrylate in the oily phase, crosslinking agent,
Solvent, pore-foaming agent, initiator quality ratio are 0.5-3:3-12:50-100:1-5:0.1-2.
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