CN108772042A - A kind of anthraquinone derivative bonded silica gel stationary phase, preparation method and ginsenoside detection application - Google Patents
A kind of anthraquinone derivative bonded silica gel stationary phase, preparation method and ginsenoside detection application Download PDFInfo
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Abstract
The invention discloses the detections of a kind of anthraquinone derivative bonded silica gel stationary phase, preparation method and ginsenoside to apply.It is a discovery of the invention that being bonded 1,2- dihydroxy anthraquinones, 2,6- dihydroxy anthraquinones or 1 in Silica Surface, 8- dihydroxy anthraquinones can significantly improve separating effect of the silica gel to ginsenoside RZ1 and RK1.1,2- dihydroxy anthraquinone, 2,6- dihydroxy anthraquinones, 1,4- dihydroxy anthraquinones or 1, although 8- dihydroxy anthraquinones are all dihydroxy-anthracene quinone structures, 1, the bonding of 4- dihydroxy anthraquinones can not enhance separating capacity of the silica gel to ginsenoside RZ1 and RK1, this may be related with hydroxy position.Thus it is possible to produce prepare 1,2- dihydroxy anthraquinones, 2,6- dihydroxy anthraquinones or 1, the exclusive analysis chromatographic column of the chromatographic columns of 8- dihydroxy anthraquinone bonded silica gel fillers as ginsenoside RZ1 and RK1, of low cost, application method is simple.
Description
Technical field
The invention belongs to analyze detection field, it is related to the separation of isomeric compound, and in particular to a kind of anthraquinone derivative
Bonded silica gel stationary phase, preparation method and ginsenoside detection application.
Background technology
Ginsenoside is the principle active component of the panax species such as rare medicinal herbs ginseng and American Ginseng, is had preferable anti-
The pharmacological activity such as tumour, anti-inflammatory, anti-oxidant and inhibition Apoptosis.
Ginsenoside broad categories, wherein also multipair or multigroup isomer.Some isomer chemical polarities
Closely similar, causing the separation of these isomers to be analyzed, there are larger difficulties.By taking ginsenoside RZ1, RK1 and RG5 as an example,
Three's isomer each other, from biological relations, C20 hydroxyls and the C21 hydrogen of ginsenoside RG3 occur elimination reaction and obtain people
Join saponin(e RK1, C20 hydroxyls and the C22 hydrogen of ginsenoside RG3 occur elimination reaction and obtains ginsenoside RG5 and RZ1, ginseng soap
Glycosides RK1 is double-bond positional isomerization with RG5, RZ1, and ginsenoside RG5 and RZ1 is double bond cis-trans isomerism.Wherein, ginsenoside RZ1
It is closely similar in the retention behavior of liquid chromatogram with ginsenoside RK1, it is extremely difficult to detach.With the earliest finders of ginsenoside RZ1
(bibliography for Sang Myung LEE：Ginsenosides from Heat Processed Ginseng,Chem
Pharm Bull, 2009), researcher uses two-dimensional liquid chromatography and just separates ginsenoside RZ1 and ginsenoside RK1.But
It is that, one skilled in the art will appreciate that two-dimensional liquid chromatography develops very immature, poor reproducibility, drug quality control cannot be met
The strict demand of system.Pharmacopoeia of each country is all very prudent to two-dimensional liquid chromatography, and only United States Pharmacopeia has recorded individually extremely at present
It is difficult to the kind detached.Chinese patent CN107505409A discloses a kind of side of separation ginsenoside RZ1 and ginsenoside RK1
Method, this method is using a kind of Chiral Mobile Phase additive process based on reversed-phase silica gel column chromatography, although separating degree is good, hand
Property mobile phase prepare relatively complicated, and must face with now matching, it is poor to flow phase stability.
Therefore, it is necessary to develop a kind of stationary phase, using common mobile phase can efficiently separate ginsenoside RZ1 with
RK1。
Invention content
It is an object of the invention to overcome the deficiencies in the prior art, the first purpose is to provide a kind of anthraquinone derivative bonded silica
Glue stationary phase, the second purpose are to provide the preparation method of the stationary phase, and third purpose is to provide the ginsenoside inspection of the stationary phase
Survey application.
The above-mentioned purpose of the present invention is achieved by following technical solution：
Technical solution one：
A kind of anthraquinone derivative bonded silica gel stationary phase is obtained in Silica Surface bonding 1,2- dihydroxy anthraquinones.
The preparation method of above-mentioned anthraquinone derivative bonded silica gel stationary phase, includes the following steps：
Step S1 first uses 3mol/L salt acid soak silica gel 12h, reheating reflux 10h to be then washed with water repeatedly to neutrality, most
It is washed 2 times with acetone afterwards, baking water removal activation 6h, is stored in spare in drier after cooling at 160 DEG C；
Step S2 takes the dry silica gel that 6.0g is activated, and 80mL dry toluenes are added, are added with stirring 4.0mL KH-560
With 3 drop Triethylamine catalysts, it is heated to reflux under nitrogen protection for 24 hours, it is cooling, for 24 hours with toluene extracting, acetone, methanol are used successively
It is washed with acetone, is dried in vacuo 8h at 80 DEG C, obtains coupling agent bonded silica gel GBS；
Step S3, under heating stirring, 1, the 2- dihydroxy anthraquinones for keeping 1.0g fully dry are dissolved into the anhydrous new steaming first of 80mL
In benzene, 4.0g GBS are subsequently added under stirring, 2 drop perchloric acid are heated to reflux for 24 hours under nitrogen protection, cooling, with methanol Soxhlet
Remaining 1,2- dihydroxy anthraquinones are extracted, until washing lotion is water white transparency, 8h is dried in vacuo at 80 DEG C, obtains 1,2- dihydroxy anthraquinones
Bonded silica gel stationary phase, kept dry after vacuum dried 10h.
Preferably, the silica gel is spherical silica gel, 5 μm of grain size, apertureSpecific surface area is 450m2/g。
A kind of exclusive chromatographic column for detaching ginsenoside RZ1 and RK1, stationary phase are above-mentioned anthraquinone derivative key
Close silica gel solid phase.
A kind of chromatographic process of separation ginsenoside RZ1 and RK1, chromatographic parameter are as follows：
Chromatographic column：Above-mentioned chromatographic column；
Mobile phase A phase：Water；
Mobile phase B phase：Acetonitrile；
Elution program：0-5min, 35%B；5-20min, 35% → 85%B.
Preferably, flow velocity 1.0mL/min.
Preferably, column temperature is 35 DEG C.
Preferably, Detection wavelength 203nm.
Technical solution two：
A kind of anthraquinone derivative bonded silica gel stationary phase is obtained in Silica Surface bonding 2,6- dihydroxy anthraquinones.
The preparation method of above-mentioned anthraquinone derivative bonded silica gel stationary phase, includes the following steps：
Step S1 first uses 3mol/L salt acid soak silica gel 12h, reheating reflux 10h to be then washed with water repeatedly to neutrality, most
It is washed 2 times with acetone afterwards, baking water removal activation 6h, is stored in spare in drier after cooling at 160 DEG C；
Step S2 takes the dry silica gel that 6.0g is activated, and 80mL dry toluenes are added, are added with stirring 4.0mL KH-560
With 3 drop Triethylamine catalysts, it is heated to reflux under nitrogen protection for 24 hours, it is cooling, for 24 hours with toluene extracting, acetone, methanol are used successively
It is washed with acetone, is dried in vacuo 8h at 80 DEG C, obtains coupling agent bonded silica gel GBS；
Step S3, under heating stirring, 2, the 6- dihydroxy anthraquinones for keeping 1.0g fully dry are dissolved into the anhydrous new steaming first of 80mL
In benzene, 4.0g GBS are subsequently added under stirring, 2 drop perchloric acid are heated to reflux for 24 hours under nitrogen protection, cooling, with methanol Soxhlet
Remaining 2,6- dihydroxy anthraquinones are extracted, until washing lotion is water white transparency, 8h is dried in vacuo at 80 DEG C, obtains 2,6- dihydroxy anthraquinones
Bonded silica gel stationary phase, kept dry after vacuum dried 10h.
Preferably, the silica gel is spherical silica gel, 5 μm of grain size, apertureSpecific surface area is 450m2/g。
A kind of exclusive chromatographic column for detaching ginsenoside RZ1 and RK1, stationary phase are above-mentioned anthraquinone derivative key
Close silica gel solid phase.
A kind of chromatographic process of separation ginsenoside RZ1 and RK1, chromatographic parameter are as follows：
Chromatographic column：Above-mentioned chromatographic column；
Mobile phase A phase：Water；
Mobile phase B phase：Acetonitrile；
Elution program：0-5min, 35%B；5-20min, 35% → 85%B.
Preferably, flow velocity 1.0mL/min.
Preferably, column temperature is 35 DEG C.
Preferably, Detection wavelength 203nm.
Technical solution three：
A kind of anthraquinone derivative bonded silica gel stationary phase is obtained in Silica Surface bonding 1,8- dihydroxy anthraquinones.
The preparation method of above-mentioned anthraquinone derivative bonded silica gel stationary phase, includes the following steps：
Step S1 first uses 3mol/L salt acid soak silica gel 12h, reheating reflux 10h to be then washed with water repeatedly to neutrality, most
It is washed 2 times with acetone afterwards, baking water removal activation 6h, is stored in spare in drier after cooling at 160 DEG C；
Step S2 takes the dry silica gel that 6.0g is activated, and 80mL dry toluenes are added, are added with stirring 4.0mL KH-560
With 3 drop Triethylamine catalysts, it is heated to reflux under nitrogen protection for 24 hours, it is cooling, for 24 hours with toluene extracting, acetone, methanol are used successively
It is washed with acetone, is dried in vacuo 8h at 80 DEG C, obtains coupling agent bonded silica gel GBS；
Step S3, under heating stirring, 1, the 8- dihydroxy anthraquinones for keeping 1.0g fully dry are dissolved into the anhydrous new steaming first of 80mL
In benzene, 4.0g GBS are subsequently added under stirring, 2 drop perchloric acid are heated to reflux for 24 hours under nitrogen protection, cooling, with methanol Soxhlet
Remaining 1,8- dihydroxy anthraquinones are extracted, until washing lotion is water white transparency, 8h is dried in vacuo at 80 DEG C, obtains 1,8- dihydroxy anthraquinones
Bonded silica gel stationary phase, kept dry after vacuum dried 10h.
Preferably, the silica gel is spherical silica gel, 5 μm of grain size, apertureSpecific surface area is 450m2/g。
A kind of exclusive chromatographic column for detaching ginsenoside RZ1 and RK1, stationary phase are above-mentioned anthraquinone derivative key
Close silica gel solid phase.
A kind of chromatographic process of separation ginsenoside RZ1 and RK1, chromatographic parameter are as follows：
Chromatographic column：Above-mentioned chromatographic column；
Mobile phase A phase：Water；
Mobile phase B phase：Acetonitrile；
Elution program：0-5min, 35%B；5-20min, 35% → 85%B.
Preferably, flow velocity 1.0mL/min.
Preferably, column temperature is 35 DEG C.
Preferably, Detection wavelength 203nm.
Advantageous effect：
It is a discovery of the invention that being bonded 1,2- dihydroxy anthraquinones, 2,6- dihydroxy anthraquinones or 1,8- dihydroxy-anthracenes in Silica Surface
Quinone can significantly improve separating effect of the silica gel to ginsenoside RZ1 and RK1.1,2- dihydroxy anthraquinones, 2,6- dihydroxy anthraquinones,
Isosorbide-5-Nitrae-dihydroxy anthraquinone or 1, although 8- dihydroxy anthraquinones are all dihydroxy-anthracene quinone structures, the bonding of Isosorbide-5-Nitrae-dihydroxy anthraquinone
Separating capacity of the silica gel to ginsenoside RZ1 and RK1 can not be enhanced, this may be related with hydroxy position.Thus it is possible to produce
The chromatographic column of 1,2- dihydroxy anthraquinones, 2,6- dihydroxy anthraquinones or 1,8- dihydroxy anthraquinone bonded silica gel fillers is prepared as ginseng
The exclusive analysis chromatographic column of saponin(e RZ1 and RK1, of low cost, application method is simple.
Description of the drawings
Fig. 1 is the chemical structural formula of anthraquinone derivative A~D；
Fig. 2 is separation chromatogram of the anthraquinone derivative A bonded silica gel stationary phases to ginsenoside RZ1 and RK1；
Fig. 3 is separation chromatogram of the anthraquinone derivative B bonded silica gel stationary phases to ginsenoside RZ1 and RK1；
Fig. 4 is separation chromatogram of the anthraquinone derivative C bonded silica gel stationary phases to ginsenoside RZ1 and RK1；
Fig. 5 is separation chromatogram of the anthraquinone derivative D bonded silica gel stationary phases to ginsenoside RZ1 and RK1；
Fig. 6 is separation chromatogram of the nonbonding silica gel solid phase to ginsenoside RZ1 and RK1.
Specific implementation mode
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but the guarantor of the present invention is not limited with this
Protect range.
Embodiment 1：The preparation of anthraquinone derivative bonded silica gel stationary phase and dress column
One, the chemical structural formula of anthraquinone derivative A~D is as shown in Figure 1, respectively 1,2- dihydroxy anthraquinones, 2,6- dihydroxies
Base anthraquinone, 1,4- dihydroxy anthraquinones, 1,8- dihydroxy anthraquinones.
Two, the preparation process of anthraquinone derivative A~D bonded silica gel stationary phases is as follows：
Step S1 first uses 3mol/L salt acid soaks silica gel (spherical silica gel, 5 μm of grain size, apertureSpecific surface area is
450m2/ g, purchased from match branch skill) 12h, reheating flows back 10h, is then washed with water to neutrality, finally washed 2 times with acetone repeatedly,
Baking water removal activation 6h, is stored in spare in drier after cooling at 160 DEG C；
Step S2 takes the dry silica gel that 6.0g is activated, and 80mL dry toluenes are added, are added with stirring 4.0mL KH-560
With 3 drop Triethylamine catalysts, it is heated to reflux under nitrogen protection for 24 hours, it is cooling, for 24 hours with toluene extracting, acetone, methanol are used successively
It is washed with acetone, is dried in vacuo 8h at 80 DEG C, obtains coupling agent bonded silica gel (GBS)；
Step S3, under heating stirring, it is anhydrous new that anthraquinone derivative A, B, C or D for keeping 1.0g fully dry are dissolved into 80mL
It steams in toluene, 4.0g GBS is subsequently added under stirring, 2 drop perchloric acid are heated to reflux for 24 hours under nitrogen protection, cooling, use methanol
Anthraquinone derivative A, B, C or D of Soxhlet extraction remnants is dried in vacuo 8h at 80 DEG C, obtains anthraquinone and spread out until washing lotion is water white transparency
Biological A~D bonded silica gel stationary phases, kept dry after vacuum dried 10h.
Three, the filling of anthraquinone derivative A~D bonded silica gels chromatographic column
Make homogenate agent with isopropanol, by anthraquinone derivative A~D bonded silica gels filling stainless steel chromatographic column (4.6mm i.d.
× 150mm) in be used as stationary phase, rinsed repeatedly with water and methanol, it is spare.
Embodiment 2：Separating effect of the anthraquinone derivative A bonded silica gel stationary phases to ginsenoside RZ1 and RK1
Ginsenoside RZ1 and ginsenoside RK1 reference substance purity are not less than 98%.
The preparation of mixed reference substance solution：Precision weighs ginsenoside RZ1 respectively and ginsenoside RK1 reference substances are each
10mg is set in 20ml measuring bottles, with acetonitrile dissolving and constant volume, is shaken up, is obtained mixed reference substance solution.
HPLC chromatogram parameter：
Chromatograph：2695 type high performance liquid chromatographs of Waters；
Chromatographic column：Anthraquinone derivative A bonded silica gels chromatographic column (150 × 4.6mm, preparation method are shown in embodiment 1)；
Mobile phase A phase：Water；
Mobile phase B phase：Acetonitrile；
Elution program：0-5min, 35%B；5-20min, 35% → 85%B；
Flow velocity：1.0mL/min；
Column temperature：35℃；
Detection wavelength：203nm.
Precision measures 10 μ L mixed reference substance solutions and injects liquid chromatograph, records chromatogram, as shown in Figure 2.
As it is clear from fig. 2 that ginsenoside RZ1 and RK1 separating degrees are good, reach baseline separation.
Embodiment 3：Separating effect of the anthraquinone derivative B bonded silica gel stationary phases to ginsenoside RZ1 and RK1
Ginsenoside RZ1 and ginsenoside RK1 reference substance purity are not less than 98%.
The preparation of mixed reference substance solution：Precision weighs ginsenoside RZ1 respectively and ginsenoside RK1 reference substances are each
10mg is set in 20ml measuring bottles, with acetonitrile dissolving and constant volume, is shaken up, is obtained mixed reference substance solution.
HPLC chromatogram parameter：
Chromatograph：2695 type high performance liquid chromatographs of Waters；
Chromatographic column：Anthraquinone derivative B bonded silica gels chromatographic column (150 × 4.6mm, preparation method are shown in embodiment 1)；
Mobile phase A phase：Water；
Mobile phase B phase：Acetonitrile；
Elution program：0-5min, 35%B；5-20min, 35% → 85%B；
Flow velocity：1.0mL/min；
Column temperature：35℃；
Detection wavelength：203nm.
Precision measures 10 μ L mixed reference substance solutions and injects liquid chromatograph, records chromatogram, as shown in Figure 3.
It can be seen from figure 3 that ginsenoside RZ1 and RK1 separating degrees are good, reach baseline separation.
Embodiment 4：Separating effect of the anthraquinone derivative C bonded silica gel stationary phases to ginsenoside RZ1 and RK1
Ginsenoside RZ1 and ginsenoside RK1 reference substance purity are not less than 98%.
The preparation of mixed reference substance solution：Precision weighs ginsenoside RZ1 respectively and ginsenoside RK1 reference substances are each
10mg is set in 20ml measuring bottles, with acetonitrile dissolving and constant volume, is shaken up, is obtained mixed reference substance solution.
HPLC chromatogram parameter：
Chromatograph：2695 type high performance liquid chromatographs of Waters；
Chromatographic column：Anthraquinone derivative C bonded silica gels chromatographic column (150 × 4.6mm, preparation method are shown in embodiment 1)；
Mobile phase A phase：Water；
Mobile phase B phase：Acetonitrile；
Elution program：0-5min, 35%B；5-20min, 35% → 85%B；
Flow velocity：1.0mL/min；
Column temperature：35℃；
Detection wavelength：203nm.
Precision measures 10 μ L mixed reference substance solutions and injects liquid chromatograph, records chromatogram, as shown in Figure 4.
As seen from Figure 4, ginsenoside RZ1 and RK1 co-elutes, can not detach.
Embodiment 5：Separating effect of the anthraquinone derivative D bonded silica gel stationary phases to ginsenoside RZ1 and RK1
Ginsenoside RZ1 and ginsenoside RK1 reference substance purity are not less than 98%.
The preparation of mixed reference substance solution：Precision weighs ginsenoside RZ1 respectively and ginsenoside RK1 reference substances are each
10mg is set in 20ml measuring bottles, with acetonitrile dissolving and constant volume, is shaken up, is obtained mixed reference substance solution.
HPLC chromatogram parameter：
Chromatograph：2695 type high performance liquid chromatographs of Waters；
Chromatographic column：Anthraquinone derivative D bonded silica gels chromatographic column (150 × 4.6mm, preparation method are shown in embodiment 1)；
Mobile phase A phase：Water；
Mobile phase B phase：Acetonitrile；
Elution program：0-5min, 35%B；5-20min, 35% → 85%B；
Flow velocity：1.0mL/min；
Column temperature：35℃；
Detection wavelength：203nm.
Precision measures 10 μ L mixed reference substance solutions and injects liquid chromatograph, records chromatogram, as shown in Figure 5.
From figure 5 it can be seen that ginsenoside RZ1 and RK1 separating degrees are good, reach baseline separation.
Embodiment 6：Separating effect of the nonbonding silica gel solid phase to ginsenoside RZ1 and RK1
Ginsenoside RZ1 and ginsenoside RK1 reference substance purity are not less than 98%.
The preparation of mixed reference substance solution：Precision weighs ginsenoside RZ1 respectively and ginsenoside RK1 reference substances are each
10mg is set in 20ml measuring bottles, with acetonitrile dissolving and constant volume, is shaken up, is obtained mixed reference substance solution.
HPLC chromatogram parameter：
Chromatograph：2695 type high performance liquid chromatographs of Waters；
Chromatographic column：(150 × 4.6mm directly uses spherical silica gel to be filled according to 1 method of embodiment to nonbonding silica gel chromatographic column
Column)；
Mobile phase A phase：Water；
Mobile phase B phase：Acetonitrile；
Elution program：0-5min, 35%B；5-20min, 35% → 85%B；
Flow velocity：1.0mL/min；
Column temperature：35℃；
Detection wavelength：203nm.
Precision measures 10 μ L mixed reference substance solutions and injects liquid chromatograph, records chromatogram, as shown in Figure 6.
As seen from Figure 6, ginsenoside RZ1 and RK1 co-elutes, can not detach.
Above-described embodiment shows that silica gel can be significantly improved to ginseng soap in Silica Surface bonding anthraquinone derivative A, B or D
The separating effect of glycosides RZ1 and RK1.Although anthraquinone derivative A~D is dihydroxy-anthracene quinone structure, the key of anthraquinone derivative C
Merging cannot enhance separating capacity of the silica gel to ginsenoside RZ1 and RK1, this may be related with hydroxy position.Therefore, Ke Yisheng
Production prepares exclusive analysis chromatography of the chromatographic column as ginsenoside RZ1 and RK1 of anthraquinone derivative A, B or D bonded silica gel filler
Column, of low cost, application method is simple.
The effect of above-described embodiment is specifically to introduce the essentiality content of the present invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
Claims (8)
1. a kind of anthraquinone derivative bonded silica gel stationary phase, it is characterised in that：It is obtained in Silica Surface bonding 1,2- dihydroxy anthraquinones
It arrives.
2. the preparation method of anthraquinone derivative bonded silica gel stationary phase described in claim 1, which is characterized in that including walking as follows
Suddenly：
Step S1 first uses 3mol/L salt acid soak silica gel 12h, reheating reflux 10h to be then washed with water repeatedly to neutrality, finally used
Acetone is washed 2 times, and baking water removal activation 6h, is stored in spare in drier after cooling at 160 DEG C；
Step S2 takes the dry silica gel that 6.0g is activated, and 80mL dry toluenes are added, and is added with stirring the drops of 4.0mLKH-560 and 3
Triethylamine catalyst is heated to reflux for 24 hours under nitrogen protection, cooling, for 24 hours with toluene extracting, uses acetone, methanol and acetone successively
It washs, is dried in vacuo 8h at 80 DEG C, obtains coupling agent bonded silica gel GBS；
Step S3, under heating stirring, 1, the 2- dihydroxy anthraquinones for keeping 1.0g fully dry are dissolved into the anhydrous new steaming toluene of 80mL,
4.0g GBS are subsequently added under stirring, 2 drop perchloric acid are heated to reflux for 24 hours under nitrogen protection, cooling, with methanol Soxhlet extraction
Remaining 1,2- dihydroxy anthraquinones are dried in vacuo 8h until washing lotion is water white transparency at 80 DEG C, obtain the bonding of 1,2- dihydroxy anthraquinones
Silica gel solid phase, kept dry after vacuum dried 10h.
3. preparation method according to claim 2, it is characterised in that：The silica gel is spherical silica gel, 5 μm of grain size, apertureSpecific surface area is 450m2/g。
4. a kind of exclusive chromatographic column for detaching ginsenoside RZ1 and RK1, it is characterised in that：Its stationary phase is claim 1
The anthraquinone derivative bonded silica gel stationary phase.
5. a kind of chromatographic process of separation ginsenoside RZ1 and RK1, which is characterized in that chromatographic parameter is as follows：
Chromatographic column：Chromatographic column described in claim 4；
Mobile phase A phase：Water；
Mobile phase B phase：Acetonitrile；
Elution program：0-5min, 35%B；5-20min, 35% → 85%B.
6. chromatographic process according to claim 5, it is characterised in that：Flow velocity is 1.0mL/min.
7. chromatographic process according to claim 5, it is characterised in that：Column temperature is 35 DEG C.
8. chromatographic process according to claim 5, it is characterised in that：Detection wavelength is 203nm.
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