CN109651387A - A kind of hud typed SiO of application2The method that@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-hydroxycamptothecine - Google Patents
A kind of hud typed SiO of application2The method that@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-hydroxycamptothecine Download PDFInfo
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- 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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Abstract
The present invention discloses a kind of hud typed SiO of application2The method that@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-hydroxycamptothecine, using methacrylic acid or methyl methacrylate as monomer, maleopimaric acid acrylic acid glycol ester or rosin acrylic acid acrylic acid glycol ester are crosslinking agent, it is coated to Silica Surface after being sufficiently mixed with radical initiator, so that it is carried out Raolical polymerizable and obtains hud typed SiO2@rosinyl polymer microsphere, microspherulite diameter are distributed as 2-50 μm, pore-size distribution 0.5-15nm, specific surface area 150-350m2/g.By hud typed SiO2@rosinyl polymer microsphere prepares chromatographic column with wet method dress post, HPLC separation is carried out to camptothecine and 10-hydroxycamptothecine using chromatographic column obtained, Detection wavelength: 254nm, 30 ± 10 DEG C of temperature, the separating degree of flow velocity 0.3-1.0mL/min, camptothecine and 10-hydroxycamptothecine is 2.45-3.47.The present invention is to camptothecine and 10-hydroxycamptothecine separating degree with higher and selectivity, and this method high sensitivity, easy to operate, rapidly and efficiently, environmental-friendly.
Description
Technical field
The invention belongs to high performance liquid chromatography fields, and in particular to a kind of hud typed SiO of application2@rosin based high molecular
The method that chromatographic column efficiently separates camptothecine and 10-hydroxycamptothecine.
Background technique
Camptothecine, chemical structural formula are as follows:
Camptothecine is indoles alkaloid contained in camplotheca acuminata, has significant anticancer activity, is the third-largest day of plant resources
Right anticancer drug, camptothecine have a better effect intestines and stomach and head-neck carcinoma etc., by inhibiting topoisomerase I to play cell
Toxicity；Structure is similar with 10-hydroxycamptothecine, but pharmacological action is different.
10-hydroxycamptothecine, chemical structural formula are as follows:
10-hydroxycamptothecine is the indoles alkaloid that content is less in camplotheca acuminata, can also be synthesized by camptothecine.It not only compares
The isolated alkaloids of camplotheca acuminata have higher pharmacological activity, and generate less toxicity, structure and camptothecin seemingly, but
Pharmacological action be higher than camptothecine, it is therefore necessary in Common Camptotheca Fruit camptothecine and 10-hydroxycamptothecine separate.
Liquid-phase chromatographic analysis refers to that mobile phase is the chromatographic technique of liquid.By improving the granularity and column pressure of filler, passing through
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 tool
There is the advantage that high separation efficiency, selectivity height, high sensitivity, analysis speed are fast and have a wide range of application.
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 C18 chromatographic 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 250-350mL/min, setting ultraviolet detection wavelength 254nm.
The above method be there is a problem that not ideal enough to the separating effect of camptothecine and 10-hydroxycamptothecine.
4. application number: 201611001162.2, denomination of invention: a kind of application rosin based high molecular separating common camptotheca fruit alkali and 10-
The method of hydroxycamptothecin, with α-methacrylic acid (or methyl methacrylate) for monomer, maleic rosin acrylic acid ethylene glycol
Ester is crosslinking agent, prepares rosinyl polymer microsphere using micro suspension free radical polymerisation process, rosinyl polymer microsphere is used
Packing column machine wet method dress post prepares 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.However, the preparation process of its fixed phase stuffing (rosinyl polymer microsphere) it is cumbersome and its preparation abietyl high score
The theoretical cam curve of sub- chromatographic column is lower, lower to the separating degree of 10-hydroxycamptothecine and camptothecine, it is therefore necessary to explore one
The method of kind highly efficient separating common camptotheca fruit alkali and 10-hydroxycamptothecine.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of hud typed SiO of application2@rosin based high molecular chromatography
The method that pillar height imitates separating common camptotheca fruit alkali and 10-hydroxycamptothecine, this method are with higher to camptothecine and 10-hydroxycamptothecine
Separating degree and selectivity, and this method high sensitivity, it is easy to operate, it is rapidly and efficiently, environmental-friendly.
Technical scheme is as follows:
A kind of hud typed SiO of application2@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-hydroxycamptothecine
Method, by hud typed SiO2@rosinyl polymer microsphere is prepared hud typed as fixed phase stuffing using wet method dress post
SiO2@rosin based high molecular chromatographic column；By hud typed SiO2@rosin based high molecular chromatographic column accesses liquid chromatograph, and liquid phase is arranged
Chromatographic flow rate of mobile phase be 0.3-1.0mL/min, Detection wavelength 254nm, 30 ± 10 DEG C of column oven；Starting sampling valve makes to flow
It is dynamic mutually to bring the mixed solution of camptothecine and 10-hydroxycamptothecine into hud typed SiO2In@rosin based high molecular chromatographic column, realize
The separation of camptothecine and 10-hydroxycamptothecine.
Hud typed SiO2@rosinyl polymer microsphere separates the mechanism of two kinds of substances: the chromatographic column that the present invention uses is consolidated
Determine the hud typed SiO of phase filling2@rosinyl polymer microsphere had both maintained silicon dioxide microsphere uniform particle diameter, and sphericity is good
Feature, while it is regular to have taken into account rosinyl polymer microsphere pore structure again, surface functional group advantage abundant, so with the microballoon
The hud typed SiO being prepared as stationary phase2@rosin based high molecular chromatographic column has higher than rosin based high molecular chromatographic column
Theoretical cam curve also significantly improves the separating degree of 10-hydroxycamptothecine and camptothecine.
As a preferred option of the technical scheme, the hud typed SiO2@rosinyl polymer microsphere particle diameter distribution is 2-50 μm,
Pore-size distribution 0.5-15nm, specific surface area 150-350m2/g。
As a preferred option of the technical scheme, the wet method dress post is with constant pressure pump by hud typed SiO2@rosin based high molecular
Microballoon is filled in sky chromatographic column, and column 30-120min is filled under the pressure of 3000-6000psi, after column flattens weighing apparatus, by chromatography
Column unloads and loads onto column cap to get hud typed SiO is arrived2@rosin based high molecular chromatographic column.
As a preferred option of the technical scheme, the hud typed SiO2@rosinyl polymer microsphere the preparation method comprises the following steps: with first
Base acrylic or methacrylic acid methyl esters is monomer, maleopimaric acid acrylic acid glycol ester or rosin acrylic acid acrylic acid second two
Alcohol ester is crosslinking agent, after being sufficiently mixed by a certain percentage with radical initiator, is uniformly coated to Silica Surface, is subsequently placed in water
Raolical polymerizable 6-10h is carried out in phase, Soxhlet extraction then is carried out to product with dehydrated alcohol, then be dried in vacuo
Hud typed SiO is made2@rosinyl polymer microsphere.
As a preferred option of the technical scheme, the mass ratio of the monomer, crosslinking agent and radical initiator is 1-50:1-100:
0.1-5。
As a preferred option of the technical scheme, the mass ratio 1:1-100 of the crosslinking agent and monomer and silica gel.
As a preferred option of the technical scheme, the water phase is the lauryl sodium sulfate aqueous solution that mass fraction is 1-15%.
As a preferred option of the technical scheme, the radical initiator is that organic peroxide initiator or azo draw
Send out agent；As the further preferred of technical solution, the radical initiator is dibenzoyl peroxide, azodiisobutyronitrile
Or azobisisoheptonitrile.
Compared with prior art, the invention has the benefit that
1. the hud typed SiO of the fixed phase stuffing for the chromatographic column that the present invention uses2@rosinyl polymer microsphere regular shape,
Particle diameter distribution is uniform, and pore structure is abundant and regular, and mechanical strength is big, and solvent tolerant degree is high, under higher flow velocity and pressure, does not have
The phenomenon that occurring be collapsed, and stability is good, and reusable, after long-time use, the filler in chromatographic column is still without broken
Bad, dissolution.
2. the hud typed SiO of the fixed phase stuffing for the chromatographic column that the present invention uses2@rosinyl polymer microsphere improves single
The characteristics of rosin-based polymer microspheres are to water sensitive, when mobile phase is the aqueous solution of different proportion, with hud typed SiO2@rosin
The chromatographic column that based high molecular microballoon is filled as stationary phase is still able to maintain good separating property.Further, since hud typed
SiO2@rosinyl polymer microsphere is relative to silica gel microball, rosin based polyalcohol of the surface bond with specific functional groups,
Therefore of the invention with hud typed SiO2The selectivity for the chromatographic column that@rosinyl polymer microsphere is filled as stationary phase is higher than again
The chromatographic column of single silicon dioxide microsphere filling.
3. the hud typed SiO of the fixed phase stuffing for the chromatographic column that the present invention uses2@rosinyl polymer microsphere both maintains two
Silicon oxide microsphere uniform particle diameter, the good feature of sphericity, while it is regular to have taken into account rosinyl polymer microsphere pore structure again, table
Face functional group advantage abundant, so the hud typed SiO being prepared using the microballoon as stationary phase2@rosin based high molecular color
Composing column has the theoretical cam curve for being higher than rosin based high molecular chromatographic column, and theoretical cam curve is increased to 10000 left sides by 6000 or so
It is right.
4. the present invention use with hud typed SiO2Chromatographic column of the@rosinyl polymer microsphere as fixed phase stuffing is right
Camptothecine and 10-hydroxycamptothecine have preferable separating effect, and separating degree is up to 3.47.Point of more existing commodity column C18 column
From degree 1.74,99.42% is improved, compared with the separating degree of the chromatographic column using rosinyl polymer microsphere as fixed phase stuffing
2.15, improve 61.40%.
5. the hud typed SiO of the fixed phase stuffing for the chromatographic column that the present invention uses2The preparation process of@rosinyl polymer microsphere
Simply, cost is relatively low, with good application prospect.
6. of the invention using hud typed SiO2@rosin based high molecular chromatographic column efficiently separates camptothecine and 10- hydroxyl is liked
The method high sensitivity of alkali is set, it is easy to operate, it is rapidly and efficiently, environmental-friendly.
Detailed description of the invention
Fig. 1 is analysis chart of the comparative example 1 of the present invention to camptothecine and 10-hydroxycamptothecine mixed solution；
Fig. 2 is analysis chart of the comparative example 2 of the present invention to camptothecine and 10-hydroxycamptothecine mixed liquor；
Fig. 3 is analysis chart of the Application Example 1 of the present invention to camptothecine and 10-hydroxycamptothecine mixed solution；
Fig. 4 is analysis chart of the Application Example 2 of the present invention to camptothecine and 10-hydroxycamptothecine mixed solution；
Fig. 5 is analysis chart of the Application Example 3 of the present invention to camptothecine and 10-hydroxycamptothecine mixed solution；
Fig. 6 is analysis chart of the Application Example 4 of the present invention to camptothecine and 10-hydroxycamptothecine mixed solution；
Fig. 7 is analysis chart of the Application Example 5 of the present invention to Common Camptotheca Fruit extracting solution；
Fig. 8 is detection figure of the Application Example 5 of the present invention to Common Camptotheca Fruit extracting solution.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is described in more detail, but is not limited to protection scope of the present invention.
Hud typed SiO2The preparation of@rosinyl polymer microsphere
Prepare embodiment 1
By 2.0g methacrylic acid, 2.0g maleopimaric acid acrylic acid glycol ester and 0.2g azodiisobutyronitrile are sufficiently mixed
After conjunction, it is uniformly coated to 400g Silica Surface, is subsequently placed in the lauryl sodium sulfate aqueous solution that 1000mL mass fraction is 1%
Middle progress Raolical polymerizable 8h, then carries out Soxhlet extraction to product with dehydrated alcohol, then be dried in vacuo, can be prepared by core
Shell mould SiO2@rosinyl polymer microsphere.
Through testing and analyzing, the obtained hud typed SiO of the present embodiment2The centralized particle diameter of@rosinyl polymer microsphere
In 2-10 μ m, average pore size 0.50nm, specific surface area 350m2/g。
Prepare embodiment 2
By 7.5g methacrylic acid, 1.5g rosin acrylic acid acrylic acid glycol ester and 2.25g azobisisoheptonitrile are abundant
After mixing, it is uniformly coated to 225g Silica Surface, it is water-soluble to be subsequently placed in the lauryl sodium sulfate that 100mL mass fraction is 5%
Raolical polymerizable 9h is carried out in liquid, Soxhlet extraction then is carried out to product with dehydrated alcohol, then be dried in vacuo, can be prepared by
Hud typed SiO2@rosinyl polymer microsphere.
Through testing and analyzing, the obtained hud typed SiO of the present embodiment2The centralized particle diameter of@rosinyl polymer microsphere
Near 12-18 μm, average pore size 3.79nm, specific surface area 247m2/g。
Prepare embodiment 3
By 0.5g methyl methacrylate, 10g maleopimaric acid acrylic acid glycol ester and 14mg dibenzoyl peroxide
After being sufficiently mixed, it is uniformly coated to 10.5g Silica Surface, is subsequently placed in the dodecyl sulphate that 100mL mass fraction is 15%
Raolical polymerizable 10h is carried out in sodium water solution, Soxhlet extraction then is carried out to product with dehydrated alcohol, then be dried in vacuo,
It can be prepared by hud typed SiO2@rosinyl polymer microsphere.
Through testing and analyzing, the obtained hud typed SiO of the present embodiment2The centralized particle diameter of@rosinyl polymer microsphere
Near 25-30 μm, average pore size 6.01nm, specific surface area 312m2/g。
Prepare embodiment 4
By 25g methacrylic acid, 35g rosin acrylic acid acrylic acid glycol ester is sufficiently mixed with 3g azobisisoheptonitrile
Afterwards, it is uniformly coated to 3000g Silica Surface, is subsequently placed in the lauryl sodium sulfate aqueous solution that 100mL mass fraction is 5%
Raolical polymerizable 6h is carried out, Soxhlet extraction then is carried out to product with dehydrated alcohol, then be dried in vacuo, can be prepared by nucleocapsid
Type SiO2@rosinyl polymer microsphere.
Through testing and analyzing, the obtained hud typed SiO of the present embodiment2The centralized particle diameter of@rosinyl polymer microsphere
Near 32-40 μm, average pore size 12.79nm, specific surface area 187m2/g。
Prepare embodiment 5
By 50g methyl methacrylate, 100g maleopimaric acid acrylic acid glycol ester is filled with 5g dibenzoyl peroxide
After dividing mixing, it is uniformly coated to 11250g Silica Surface, is subsequently placed in the lauryl sodium sulfate that 100mL mass fraction is 15%
Raolical polymerizable 7h is carried out in aqueous solution, Soxhlet extraction then is carried out to product with dehydrated alcohol, then be dried in vacuo
Hud typed SiO is made2@rosinyl polymer microsphere.
Through testing and analyzing, the obtained hud typed SiO of the present embodiment2The centralized particle diameter of@rosinyl polymer microsphere
Near 42-50 μm, average pore size 15.00nm, specific surface area 150m2/g。
Using hud typed SiO2The method that@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-hydroxycamptothecine
The method of comparative example 1 application C18 post separation camptothecine and 10-hydroxycamptothecine
Commodity column C18 column, to be commercially available on the market.
A method of using C18 post separation camptothecine and 10-hydroxycamptothecine, carry out in accordance with the following steps:
(1) it prepares sample solution: taking appropriate camptothecine, 10-hydroxycamptothecine, dissolved with methanol, be configured to every 1L and contain
2.5×10-4The camptothecine and 10-hydroxycamptothecine mixed solution of mol, sample introduction；
(2) setup parameter: accessing liquid chromatograph for C18 column, and the flow rate of mobile phase that liquid chromatograph is arranged is 0.5mL/
Min, Detection wavelength 254nm, 30 DEG C of column oven；
(3) separate: starting sampling valve bring into methanol sample in C18 column, sample volume be 20 μ L, realize camptothecine and
The separation of 10- hydroxycamptothecin, acquired results are as shown in Figure 1, there is 10-hydroxycamptothecine in retention time 30.11min
Peak occurs camptothecine peak, separating degree 1.74 in retention time 39.83min.
The method of comparative example 2 application rosin based high molecular chromatographic column separating common camptotheca fruit alkali and 10-hydroxycamptothecine
Rosin based high molecular chromatographic column, according to application No. is the methods that 201611001162.2 application for a patent for invention is announced
It is prepared.
A method of using rosin based high molecular chromatographic column separating common camptotheca fruit alkali and 10-hydroxycamptothecine, according to following step
It is rapid to carry out:
(1) it prepares sample solution: taking appropriate camptothecine, 10-hydroxycamptothecine, dissolved with methanol, be configured to every 1L and contain
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 0.5mL/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 Fig. 2, there is 10- in retention time 24.68min
Hydroxycamptothecin peak occurs camptothecine peak, separating degree 2.15 in retention time 33.61min.
Application Example 1
The resulting hud typed SiO of embodiment 2 will be prepared2@rosinyl polymer microsphere prepares chromatographic column using wet method dress post,
Specifically:
Wet method dress post is with constant pressure pump by hud typed SiO2@rosinyl polymer microsphere is filled in sky chromatographic column,
30min is loaded under 3000psi pressure, after column flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto column cap, can be prepared
Obtain hud typed SiO2@rosin based high molecular chromatographic column, being rinsed with methanol solution can sample introduction to baseline balance.
A kind of hud typed SiO of application2@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-hydroxycamptothecine
Method carries out in accordance with the following steps:
(1) it prepares sample solution: taking appropriate camptothecine, 10-hydroxycamptothecine, dissolved with methanol, be configured to every 1L and contain
2.5×10-4The camplotheca acuminata of mol and 10-hydroxycamptothecine mixed solution, sample introduction；
(2) setup parameter: by hud typed SiO2@rosin based high molecular chromatographic column accesses liquid chromatograph, and liquid phase color is arranged
The flow rate of mobile phase of spectrometer be 0.3mL/min, Detection wavelength 254nm, 25 DEG C of column oven；
(3) separate: starting sampling valve makes methanol bring sample into hud typed SiO2In@rosin based high molecular chromatographic column, into
Sample amount is 20 μ L, realizes the separation of camptothecine and 10-hydroxycamptothecine, acquired results are as shown in figure 3, in retention time
Occur 10-hydroxycamptothecine peak when 12.08min, occurs camptothecine peak, separating degree 2.45 in retention time 14.61min.
Application Example 2
The resulting hud typed SiO of embodiment 1 will be prepared2@rosinyl polymer microsphere prepares chromatographic column using wet method dress post,
Specifically: wet method dress post is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, is filled under 4500psi pressure
60min is filled out, after column flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto column cap, hud typed SiO can be prepared2@
Rosin based high molecular chromatographic column, being rinsed with methanol can sample introduction to baseline balance.
A kind of hud typed SiO of application2@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-hydroxycamptothecine
Method carries out in accordance with the following steps:
(1) it prepares sample solution: taking appropriate camptothecine, 10-hydroxycamptothecine, dissolved with methanol, be configured to every 1L and contain
2.5×10-4The camptothecine and 10-hydroxycamptothecine mixed solution of mol, sample introduction；
(2) setup parameter: by hud typed SiO2@rosin based high molecular chromatographic column accesses liquid chromatograph, and liquid phase color is arranged
The flow rate of mobile phase of spectrometer be 0.5mL/min, Detection wavelength 254nm, 30 DEG C of column oven；
(3) separate: starting sampling valve makes methanol bring sample into hud typed SiO2In@rosin based high molecular chromatographic column, into
Sample amount is 20 μ L, realizes the separation of camptothecine and 10-hydroxycamptothecine, acquired results are as shown in figure 4, in retention time
Occur 10-hydroxycamptothecine peak when 11.49min, occurs camptothecine peak, separating degree 2.73 in retention time 13.38min.
Application Example 3
The resulting hud typed SiO of embodiment 1 will be prepared2@rosinyl polymer microsphere prepares chromatographic column using wet method dress post,
Specifically: wet method dress post is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, is filled under 5500psi pressure
100min is filled out, after column flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto column cap, can be prepared hud typed
SiO2 rosin based high molecular chromatographic column, being rinsed with methanol-water (80:20, V/V) can sample introduction to baseline balance.
A kind of hud typed SiO of application2@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-hydroxycamptothecine
Method carries out in accordance with the following steps:
(1) it prepares sample solution: taking appropriate camptothecine, 10-hydroxycamptothecine, dissolved with methanol, be configured to every 1L and contain
2.5×10-4The camptothecine and 10-hydroxycamptothecine mixed solution of mol, sample introduction；
(2) setup parameter: by hud typed SiO2@rosin based high molecular chromatographic column accesses liquid chromatograph, and liquid phase color is arranged
The flow rate of mobile phase of spectrometer be 0.7mL/min, Detection wavelength 254nm, 35 DEG C of column oven；
(3) separate: starting sampling valve makes methanol bring sample into hud typed SiO2In@rosin based high molecular chromatographic column, into
Sample amount is 20 μ L, realizes the separation of camptothecine and 10-hydroxycamptothecine, acquired results are as shown in figure 5, in retention time
Occur 10-hydroxycamptothecine peak when 16.69min, occurs camptothecine peak, separating degree 3.47 in retention time 20.38min.
Application Example 4
The resulting hud typed SiO of embodiment 3 will be prepared2@rosinyl polymer microsphere prepares chromatographic column using wet method dress post,
Specifically: wet method dress post is that rosinyl polymer microsphere is filled in sky chromatographic column with constant pressure pump, is filled under 6000psi pressure
120min is filled out, after column flattens weighing apparatus, chromatographic column is removed from packing column machine, loads onto column cap, can be prepared hud typed
SiO2@rosin based high molecular chromatographic column, being rinsed with methanol can sample introduction to baseline balance.
A kind of hud typed SiO of application2@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-hydroxycamptothecine
Method carries out in accordance with the following steps:
(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: by hud typed SiO2@rosin based high molecular chromatographic column accesses liquid chromatograph, and liquid phase color is arranged
The flow rate of mobile phase of spectrometer be 1.0mL/min, Detection wavelength 254nm, 40 DEG C of column oven；
(3) separate: starting sampling valve makes methanol bring sample into hud typed SiO2In@rosin based high molecular chromatographic column, into
Sample amount is 20 μ L, realizes the separation of camptothecine and 10-hydroxycamptothecine, acquired results are as shown in fig. 6, in retention time
Occur 10-hydroxycamptothecine peak when 11.20min, occurs camptothecine peak, separating degree 3.11 in retention time 13.58min.
Application Example 5
(1) the thick of camptothecine mentions: weighing the dry Common Camptotheca Fruit powder of 50g, is put into the round-bottomed flask containing 300mL ethyl alcohol and adds
Hot continuous extraction 3h, is concentrated to give camptothecine medicinal extract to extracting solution after stopping heating.
(2) essence of camptothecine mentions: weighing 0.1g camptothecine medicinal extract, is dissolved in preparation in 10mL methanol and obtains sample solution.It will
The hud typed SiO that embodiment 3 obtains2Hud typed SiO of the@rosinyl polymer microsphere as fixed phase stuffing2@abietyl high score
Sub- chromatographic column accesses liquid chromatograph, and the flow rate of mobile phase that liquid chromatograph is arranged is 0.3mL/min, Detection wavelength 254nm, column
25 DEG C of incubator；Starting sampling valve makes methanol bring sample into hud typed SiO2In@rosin based high molecular chromatographic column, sample volume is
20 μ L, realize the purifying of camptothecine, and acquired results are as shown in Figure 7.Detected with C18 column, testing conditions: Detection wavelength is
254nm, 25 DEG C of column temperature, testing result is as shown in figure 8, camptothecine purity is 92.0%.
Claims (9)
1. a kind of hud typed SiO of application2@rosin based high molecular chromatographic column efficiently separates the side of camptothecine and 10-hydroxycamptothecine
Method, it is characterised in that: by hud typed SiO2@rosinyl polymer microsphere is prepared into as fixed phase stuffing using wet method dress post
To hud typed SiO2@rosin based high molecular chromatographic column；By hud typed SiO2@rosin based high molecular chromatographic column accesses liquid chromatograph,
Be arranged liquid chromatograph flow rate of mobile phase be 0.3-1.0mL/min, Detection wavelength 254nm, 30 ± 10 DEG C of column oven；Start into
Sample valve makes mobile phase bring the mixed solution of camptothecine and 10-hydroxycamptothecine into hud typed SiO2@rosin based high molecular chromatography
In column, the separation of camptothecine and 10-hydroxycamptothecine is realized.
2. the hud typed SiO of application according to claim 12@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-
The method of hydroxycamptothecin, it is characterised in that: the hud typed SiO2@rosinyl polymer microsphere particle diameter distribution is 2-50 μ
M, pore-size distribution 0.5-15nm, specific surface area 150-350m2/g。
3. the hud typed SiO of application according to claim 1 or 22@rosin based high molecular chromatographic column efficiently separate camptothecine with
The method of 10-hydroxycamptothecine, it is characterised in that: the wet method dress post is with constant pressure pump by hud typed SiO2@abietyl is high
Molecule microballoon is filled in sky chromatographic column, and column 30-120min is filled under the pressure of 3000-6000psi, will after column flattens weighing apparatus
Chromatographic column unloads and loads onto column cap to get hud typed SiO is arrived2@rosin based high molecular chromatographic column.
4. the hud typed SiO of application according to claim 1 or 22@rosin based high molecular chromatographic column efficiently separate camptothecine with
The method of 10-hydroxycamptothecine, it is characterised in that: the hud typed SiO2@rosinyl polymer microsphere the preparation method comprises the following steps:
Using methacrylic acid or methyl methacrylate as monomer, maleopimaric acid acrylic acid glycol ester or rosin acrylic acid acrylic acid
Glycol ester is crosslinking agent, after being sufficiently mixed by a certain percentage with radical initiator, is uniformly coated to Silica Surface, then sets
Raolical polymerizable 6-10h is carried out in water phase, Soxhlet extraction then is carried out to product with dehydrated alcohol, then be dried in vacuo,
It can be prepared by hud typed SiO2@rosinyl polymer microsphere.
5. the hud typed SiO of application according to claim 42@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-
The method of hydroxycamptothecin, it is characterised in that: the mass ratio of the monomer, crosslinking agent and radical initiator is 1-50:1-
100:0.1-5。
6. the hud typed SiO of application according to claim 42@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-
The method of hydroxycamptothecin, it is characterised in that: the mass ratio 1:1-100 of the crosslinking agent and monomer and silica gel.
7. the hud typed SiO of application according to claim 42@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-
The method of hydroxycamptothecin, it is characterised in that: the water phase is the lauryl sodium sulfate aqueous solution that mass fraction is 1-15%.
8. any described using hud typed SiO according to claim 4-72@rosin based high molecular chromatographic column efficiently separates camptothecine
With the method for 10-hydroxycamptothecine, it is characterised in that: the radical initiator is organic peroxide initiator or idol
Nitrogen class initiator.
9. the hud typed SiO of application according to claim 82@rosin based high molecular chromatographic column efficiently separates camptothecine and 10-
The method of hydroxycamptothecin, it is characterised in that: the radical initiator be dibenzoyl peroxide, azodiisobutyronitrile or
Azobisisoheptonitrile.
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