CN102898510A - Method for separating enramycin A from enramycin B - Google Patents
Method for separating enramycin A from enramycin B Download PDFInfo
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- CN102898510A CN102898510A CN201210433029XA CN201210433029A CN102898510A CN 102898510 A CN102898510 A CN 102898510A CN 201210433029X A CN201210433029X A CN 201210433029XA CN 201210433029 A CN201210433029 A CN 201210433029A CN 102898510 A CN102898510 A CN 102898510A
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- enramycin
- aqueous solution
- monobasic
- sodium phosphate
- methyl alcohol
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- NJCUSQKMYNTYOW-MWUYRYRWSA-N enramicina Chemical compound O.N1C(=O)NC(=O)C(C=2C=C(Cl)C(O)=C(Cl)C=2)NC(=O)C(CO)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(CC2N=C(N)NC2)NC(=O)C(CCCNC(N)=O)NC(=O)C(C(C)O)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C(C)O)NC(=O)N(CCCCN)C(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)/C=C/C=C/CCCCC(C)CC)C(C)OC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C)NC(=O)C1CC1CNC(N)=N1 NJCUSQKMYNTYOW-MWUYRYRWSA-N 0.000 title claims abstract description 181
- 108700041171 enramycin Proteins 0.000 title claims abstract description 181
- 229950003984 enramycin Drugs 0.000 title claims abstract description 181
- JPYWPHBUMZRLPO-DLYWSANHSA-N 5tq7z201b8 Chemical compound C([C@H]1C(=O)N[C@H](C)C(=O)N[C@H](C(=O)O[C@H](C)[C@@H](C(N[C@@H](C(=O)N[C@H](CCCN)C(=O)N[C@@H](C(=O)N[C@H](C(=O)N[C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCCNC(N)=O)C(=O)N[C@H](C[C@H]2NC(N)=NC2)C(=O)N[C@H](C(=O)N[C@H](CO)C(=O)N[C@H](C(=O)NCC(=O)N1)C=1C=C(Cl)C(O)=C(Cl)C=1)C=1C=CC(O)=CC=1)[C@H](C)O)C=1C=CC(O)=CC=1)C=1C=CC(O)=CC=1)[C@@H](C)O)C=1C=CC(O)=CC=1)=O)NC(=O)[C@H](CC(O)=O)NC(=O)\C=C/C=C/CCCCC(C)CC)C=1C=CC(O)=CC=1)[C@@H]1CN=C(N)N1 JPYWPHBUMZRLPO-DLYWSANHSA-N 0.000 title claims abstract description 139
- 238000000034 method Methods 0.000 title claims abstract description 44
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 246
- 239000007864 aqueous solution Substances 0.000 claims abstract description 97
- 238000012856 packing Methods 0.000 claims abstract description 82
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims abstract description 68
- 238000010828 elution Methods 0.000 claims abstract description 56
- 239000000203 mixture Substances 0.000 claims abstract description 45
- 238000011068 loading method Methods 0.000 claims abstract description 15
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims abstract description 14
- 229910019142 PO4 Inorganic materials 0.000 claims description 93
- 239000010452 phosphate Substances 0.000 claims description 93
- 239000001488 sodium phosphate Substances 0.000 claims description 56
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 56
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 56
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 38
- 229940045641 monobasic sodium phosphate Drugs 0.000 claims description 38
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 38
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 28
- 239000006166 lysate Substances 0.000 claims description 26
- 239000000377 silicon dioxide Substances 0.000 claims description 14
- 238000004587 chromatography analysis Methods 0.000 claims description 13
- 239000012535 impurity Substances 0.000 claims description 13
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 13
- 239000002245 particle Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 abstract description 2
- DFIWJEVKLWMZBI-UHFFFAOYSA-M sodium;dihydrogen phosphate;phosphoric acid Chemical compound [Na+].OP(O)(O)=O.OP(O)([O-])=O DFIWJEVKLWMZBI-UHFFFAOYSA-M 0.000 abstract 4
- 238000004090 dissolution Methods 0.000 abstract 1
- 238000010898 silica gel chromatography Methods 0.000 abstract 1
- 238000002791 soaking Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 27
- 238000004128 high performance liquid chromatography Methods 0.000 description 11
- 238000000926 separation method Methods 0.000 description 9
- 238000000605 extraction Methods 0.000 description 7
- 229920005989 resin Polymers 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 229920001429 chelating resin Polymers 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000193738 Bacillus anthracis Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000193155 Clostridium botulinum Species 0.000 description 1
- 241000193468 Clostridium perfringens Species 0.000 description 1
- 241000193449 Clostridium tetani Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 241000971005 Streptomyces fungicidicus Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a method for separating enramycin A from enramycin B. The method comprises the following steps of: 1, dissolving a mixture of the enramycin A and the enramycin B; 2, soaking an octadecyl reversed phase bonded silica gel chromatography filling in methanol, and performing column packing; 3, loading a dissolution solution in the step 1 onto a reversed phase chromatography column, and eluting by using a sodium dihydrogen phosphate-phosphoric acid aqueous solution in an amount which is 4 to 6 times of the volume of the filling; 4, eluting the reversed phase chromatography column which contains the enramycin A and the enramycin B and is obtained in the step 3 by using the sodium dihydrogen phosphate-phosphoric acid aqueous solution containing 32 to 36 percent of methanol; 5, increasing the content of the methanol in the sodium dihydrogen phosphate-phosphoric acid aqueous solution step by step until the content is 40 percent, and performing gradient elution; and 6, eluting the reversed phase chromatography column treated in the step 4 by using the sodium dihydrogen phosphate-phosphoric acid aqueous solution containing 40 to 50 percent of methanol. By the method, the enramycin A can be separated from the enramycin B effectively.
Description
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Technical fieldThe invention belongs to separation technology field, relate to a kind of method of using reversed-phase bonded silica thin layer chromatography separation and purification enramycin.
Background technologyEnramycin (enramycin) has another name called Enramycin, enramycin, enramycin, enramycin, is by actinomycetes (Streptomyces
Fungicidicus) fermentation and get, be the polypeptide antibiotics that unsaturated fatty acids is combined with tens seed amino acids, main ingredient has enramycin A and B, is the application of its hydrochloride form.Gram-positive microorganism there is remarkable bacteriostatic action, mainly hinders the synthetic of bacteria cell wall.Sensitive bacterial has staphylococcus aureus, staphylococcus epidermidis, Staphylococcus citreus, streptococcus pyogenes etc.And streptococcus pneumoniae, Bacillus subtilus, anthrax bacillus, clostridium tetani, Clostridium botulinum, clostridium perfringens are also responsive.White or yellowish white powder (crude product gray or dun powder have special smell).234～238 ℃ of decomposition are soluble in dilute hydrochloric acid, are slightly soluble in water, methyl alcohol, ethanol, are insoluble to acetone.
Enramycin has very strong activity to gram-positive microorganism, and is particularly very strong to harmful clostridium (Clostridium) restraint in the intestines.Be not easy behind the life-time service to develop immunity to drugs, because it has changed the bacterial flora in the enteron aisle, thus effective to utilizing of nutritive ingredient in the feed, can promote pig, chicken to increase weight and improve food conversion ratio.
For fear of because antibiotic abuse causes the Resistant strain amount reproduction or the residual quantity of medicine in livestock product increased, growth of animals or poultry and HUMAN HEALTH are caused direct harm, the antibiotic feed additive of using the livestock and poultry special use is extremely urgent.Because enramycin has good growth promotion and improves the effect of efficiency of feed utilization, enramycin is for oral administration extremely to be difficult for being absorbed, and drug main will excrete by ight soil.Therefore recommended as the microbiotic growth promoter by many countries in the world.Main source is the mycelium pre-mixture of Schering Plough company on the market.
The molecular formula of enramycin A is C107H138Cl2N26O31, and molecular weight is 2355; The molecular formula of enramycin B is C108H140Cl2N26O31, and molecular weight is 2369; Chemical formula as shown below, the difference of enramycin A and enramycin B are enramycin A position methyl on the R group position, and enramycin B is ethyl.For enramycin A and enramycin B are effectively studied and detect, need separation and purification to obtain purer monomeric compound and study in contrast.
Record comes the separation and Extraction Enramycin with macroporous adsorbent resin Amberlite XAD-4, Amberlite XAD-16 etc. among the Chinese invention patent application ZL200910032340.1 " a kind of enramycin producing strain and utilize the method for macroporous resin extraction ".But this inventive method can only be controlled at 60%～71% for the productive rate of enramycin in the mycelium, and the rate of recovery is lower, and too many for large-scale industrial production consume, content detection aspect also difficulty satisfies accuracy requirement.
Used macropore acidulous cation resin separation and Extraction enramycin in the Chinese invention patent application 201010213986.2 " using the method for macropore acidulous cation resin separation and Extraction enramycin ", use a large amount of strong acid and strong bases to process ion exchange resin in the purge process, cause the wastewater treatment difficulty to strengthen, and the solution of sample also uses basic solution to regulate, thereby causes the unstable of sample.The sample purity that obtains by above method simultaneously is total purity of two kinds of materials of AB.
Therefore need the fast separation and purification of a kind of method simple to operation to obtain enramycin A and enramycin B sterling.
Summary of the inventionThe purpose of this invention is to provide a kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, gets the mixture that contains enramycin A and enramycin B and dissolves, and lysate is the aqueous hydrochloric acid of 30% methyl alcohol, and pH is 3.0-4.0; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 10-50 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.01-0.05M SODIUM PHOSPHATE, MONOBASIC of the pH4.0-5.5 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 1-10mg mixture/mL packing volume, again with the SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4-6 times of packing volume-phosphate aqueous solution wash-out impurity, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 32%-36% methyl alcohol of 1-3 times of packing volume that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out wash-out, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 3-6 times of packing volume-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, the reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 40%-50% methyl alcohol of 3-8 times of packing volume that step 4 is processed carries out wash-out, collects to such an extent that contain the elutriant B of enramycin B.
The gross weight of enramycin A and enramycin B accounts for more than 50% of mixture total weight amount in the step 1 of the present invention, and the weight ratio of enramycin A and enramycin B is 1:0.5-1.5.
The aqueous hydrochloric acid consumption of 30% methyl alcohol of dissolving mixt is the 0.25-0.5mL/mg mixture in the step 1 of the present invention.
Type of elution in step 4 of the present invention and the step 6 is gradient elution or stepwise elution.Preferred type of elution is the stepwise elution that contains the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 of 35% methyl alcohol in the step 4.Preferred type of elution is the stepwise elution that contains the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 of 45% methyl alcohol in the step 6.
Flow rate of mobile phase of the present invention is 10-25 packing volume per hour, wherein preferred 15 packing volumes per hour.
SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution of the present invention is preferably 0.03M SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution of pH4.0.
Process through method of the present invention, effectively separated at enramycin A and enramycin B, be present in respectively among elutriant A and the elutriant B.Method of the present invention can cooperate with other existing separation methods, thus difference purifying enramycin A and enramycin B.
EmbodimentThe below is further expalined the part term among the present invention:
" mixture that contains enramycin A and enramycin B " described in the step 1 of the present invention can be the enramycin crude product that obtains with the prior art preliminary purification, such as Chinese patent ZL200910032340.1 " a kind of enramycin producing strain and utilize the method for macroporous resin extraction ", ZL201110344875.X " the extraction Isolation and purification method of enramycin ", the product that the disclosed methods such as ZL201010213986.2 " using the method for macropore acidulous cation resin separation and Extraction enramycin " obtain also can be commercially available satisfactory enramycin crude product.In particular cases, also can be artificial enramycin A and enramycin B sterling to be mixed in proportion to verify the method.
" aqueous hydrochloric acid " of the present invention namely carries out the adjusting of pH value with hydrochloric acid, pH is adjusted to claimed range.
The collocation method of " SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution " of the present invention is undertaken by the prior art in reference book, the document.
" flow rate of mobile phase " of the present invention when being loading and during wash-out sample and the flow velocity of SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, the flow velocity of stages can be inconsistent, regulates in scope as required.
" progressively improve methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandon elutriant " wherein 40% is capture range in the step 5 of the present invention, and the elutriant that abandons does not contain 40% elutriant.
The purpose of this invention is to provide a kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, gets the mixture that contains enramycin A and enramycin B and dissolves, and lysate is the aqueous hydrochloric acid of 30% methyl alcohol, and pH is 3-4; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 10-50 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.01-0.05M SODIUM PHOSPHATE, MONOBASIC of the pH4.0-5.5 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 1-10mg mixture/mL packing volume, again with the SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4-6 times of packing volume-phosphate aqueous solution wash-out impurity, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 32%-36% methyl alcohol of 1-3 times of packing volume that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out wash-out, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 3-6 times of packing volume-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, the reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 40%-50% methyl alcohol of 3-8 times of packing volume that step 4 is processed carries out wash-out, collects to such an extent that contain the elutriant B of enramycin B.
The gross weight of enramycin A and enramycin B accounts for more than 50% of mixture total weight amount in the step 1 of the present invention, and the weight ratio of enramycin A and enramycin B is 1:0.5-1.5.
The aqueous hydrochloric acid consumption of 30% methyl alcohol of dissolving mixt is the 0.25-0.5mL/mg mixture in the step 1 of the present invention.
Type of elution in step 4 of the present invention and the step 6 is gradient elution or stepwise elution.Preferred type of elution is the stepwise elution that contains the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 of 35% methyl alcohol in the step 4.Preferred type of elution is the stepwise elution that contains the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 of 45% methyl alcohol in the step 6.
Flow rate of mobile phase of the present invention is 10-25 packing volume per hour, wherein preferred 15 packing volumes per hour.
SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution of the present invention is preferably 0.03M SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution of pH4.0.
Enramycin A and enramycin B purity among employing HPLC detection elutriant A and the elutriant B account for the enramycin total content with enramycin A in the elutriant respectively and enramycin B accounts for the separating effect that the enramycin total content is weighed enramycin A and enramycin B.Liquid phase chromatogram condition: ODS C18(250mm*4.6mm, 5 μ), 35 ℃ of column temperatures, flow velocity 1ml/min detects wavelength 267nm, moving phase: 15% acetonitrile: 15% methyl alcohol: 70% water (SODIUM PHOSPHATE, MONOBASIC-phosphoric acid buffer pH=4.5).
Embodiment 1:A kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, getting the mixture 100mg that contains enramycin A and enramycin B dissolves, (gross weight of enramycin A and enramycin B accounts for 50% of mixture total weight amount, the weight ratio of enramycin A and enramycin B is 1:0.5), lysate is the aqueous hydrochloric acid of 30% methyl alcohol of 25mL, and pH is 3; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 10 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.01M SODIUM PHOSPHATE, MONOBASIC of the pH4.0 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 1mg mixture/mL packing volume, uses SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4 times of packing volumes-phosphate aqueous solution wash-out impurity again, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 32%-36% methyl alcohol of 1 times of packing volume that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out gradient elution, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 3 times of packing volumes-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 40%-50% methyl alcohol of 3 times of packing volumes that step 4 is processed carries out gradient elution (wherein the elutriant at 40% place is collected into elutriant B), collects to such an extent that contain the elutriant B of enramycin B.Flow rate of mobile phase is 15 packing volumes per hour.
Detect enramycin A and enramycin B purity through HPLC, enramycin A accounts for more than 98% of enramycin total content among the elutriant A, and enramycin B accounts for more than 98% of enramycin total content among the elutriant B.
Embodiment 2:A kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, getting the mixture 100mg that contains enramycin A and enramycin B dissolves, (gross weight of enramycin A and enramycin B accounts for 60% of mixture total weight amount, the weight ratio of enramycin A and enramycin B is 1:1.5), lysate is the aqueous hydrochloric acid of 30% methyl alcohol of 50mL, and pH is 4.0; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 50 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.05M SODIUM PHOSPHATE, MONOBASIC of the pH5.5 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 10mg mixture/mL packing volume, uses SODIUM PHOSPHATE, MONOBASIC in the step 2 of 6 times of packing volumes-phosphate aqueous solution wash-out impurity again, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 33%-35% methyl alcohol of 3 times of packing volumes that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out gradient elution, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 6 times of packing volumes-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, the reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 42%-48% methyl alcohol of 8 times of packing volumes that step 4 is processed carries out gradient elution, collects to such an extent that contain the elutriant B of enramycin B.Flow rate of mobile phase is 25 packing volumes per hour.
Detect enramycin A and enramycin B purity through HPLC, enramycin A accounts for more than 98% of enramycin total content among the elutriant A, and enramycin B accounts for more than 98% of enramycin total content among the elutriant B.
Embodiment 3:A kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, getting the mixture 100mg that contains enramycin A and enramycin B dissolves, (gross weight of enramycin A and enramycin B accounts for 70% of mixture total weight amount, the weight ratio of enramycin A and enramycin B is 1:1), lysate is the aqueous hydrochloric acid of 30% methyl alcohol of 30mL, and pH is 3.1; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 15 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.02M SODIUM PHOSPHATE, MONOBASIC of the pH4.5 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 5mg mixture/mL packing volume, uses SODIUM PHOSPHATE, MONOBASIC in the step 2 of 5 times of packing volumes-phosphate aqueous solution wash-out impurity again, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 32%-34% methyl alcohol of 1.5 times of packing volumes that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out gradient elution, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 5 times of packing volumes-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, the reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 44%-46% methyl alcohol of 4 times of packing volumes that step 4 is processed carries out gradient elution, collects to such an extent that contain the elutriant B of enramycin B.Flow rate of mobile phase is 20 packing volumes per hour.
Detect enramycin A and enramycin B purity through HPLC, enramycin A accounts for more than 98% of enramycin total content among the elutriant A, and enramycin B accounts for more than 98% of enramycin total content among the elutriant B.
Embodiment 4:A kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, getting the mixture 100mg that contains enramycin A and enramycin B dissolves, (gross weight of enramycin A and enramycin B accounts for 80% of mixture total weight amount, the weight ratio of enramycin A and enramycin B is 1:0.7), lysate is the aqueous hydrochloric acid of 30% methyl alcohol of 40mL, and pH is 3.2; Step 2, be after the octadecyl reversed-phase bonded silica chromatographic stuffing of 20 μ m soaks with methyl alcohol with particle diameter, has the dress post (wrongly write with the pH4.0 that contains 30% methyl alcohol again?) 0.03M SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution balance chromatography column after to make the reversed phase chromatography post for subsequent use; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 6mg mixture/mL packing volume, uses SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4.5 times of packing volumes-phosphate aqueous solution wash-out impurity again, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 36% methyl alcohol of 2.5 times of packing volumes that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out stepwise elution, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4 times of packing volumes-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, the reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 45% methyl alcohol of 5 times of packing volumes that step 4 is processed carries out stepwise elution, collects to such an extent that contain the elutriant B of enramycin B.Flow rate of mobile phase is 10 packing volumes per hour.
Detect enramycin A and enramycin B purity through HPLC, enramycin A accounts for more than 98% of enramycin total content among the elutriant A, and enramycin B accounts for more than 96% of enramycin total content among the elutriant B.
Embodiment 5:A kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, getting the mixture 100mg that contains enramycin A and enramycin B dissolves, (gross weight of enramycin A and enramycin B accounts for 90% of mixture total weight amount, the weight ratio of enramycin A and enramycin B is 1:1.2), lysate is the aqueous hydrochloric acid of 30% methyl alcohol of 35mL, and pH is 3.3; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 25 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.04M SODIUM PHOSPHATE, MONOBASIC of the pH4.3 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 3mg mixture/mL packing volume, uses SODIUM PHOSPHATE, MONOBASIC in the step 2 of 5.5 times of packing volumes-phosphate aqueous solution wash-out impurity again, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4 adopts the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 35% methyl alcohol of 2 times of packing volumes that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out stepwise elution, collects to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 5.5 times of packing volumes-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 40% methyl alcohol of 6 times of packing volumes that step 4 is processed carries out stepwise elution (wherein the elutriant at 40% place is collected into elutriant B), collects to such an extent that contain the elutriant B of enramycin B.Flow rate of mobile phase is 23 packing volumes per hour.
Detect enramycin A and enramycin B purity through HPLC, enramycin A accounts for more than 97% of enramycin total content among the elutriant A, and enramycin B accounts for more than 98% of enramycin total content among the elutriant B.
Embodiment 6:A kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, getting the mixture 100mg that contains enramycin A and enramycin B dissolves, (gross weight of enramycin A and enramycin B accounts for 100% of mixture total weight amount, the weight ratio of enramycin A and enramycin B is 1:1.3), lysate is the aqueous hydrochloric acid of 30% methyl alcohol of 45mL, and pH is 3.4; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 30 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.03M SODIUM PHOSPHATE, MONOBASIC of the pH5.3 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 8mg mixture/mL packing volume, uses SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4.8 times of packing volumes-phosphate aqueous solution wash-out impurity again, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 32% methyl alcohol of 2.2 times of packing volumes that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out stepwise elution, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4.5 times of packing volumes-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, the reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 50% methyl alcohol of 7 times of packing volumes that step 4 is processed carries out stepwise elution, collects to such an extent that contain the elutriant B of enramycin B.Flow rate of mobile phase is 19 packing volumes per hour.
Detect enramycin A and enramycin B purity through HPLC, enramycin A accounts for more than 97% of enramycin total content among the elutriant A, and enramycin B accounts for more than 98% of enramycin total content among the elutriant B.
Embodiment 7:A kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, getting the mixture 100mg that contains enramycin A and enramycin B dissolves, (gross weight of enramycin A and enramycin B accounts for 65% of mixture total weight amount, the weight ratio of enramycin A and enramycin B is 1:0.8), lysate is the aqueous hydrochloric acid of 30% methyl alcohol of 23mL, and pH is 3.5; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 35 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.03M SODIUM PHOSPHATE, MONOBASIC of the pH4.0 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 2mg mixture/mL packing volume, uses SODIUM PHOSPHATE, MONOBASIC in the step 2 of 5.3 times of packing volumes-phosphate aqueous solution wash-out impurity again, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 35% methyl alcohol of 1.2 times of packing volumes that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out stepwise elution, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 3.5 times of packing volumes-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, the reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 45% methyl alcohol of 3.5 times of packing volumes that step 4 is processed carries out stepwise elution, collects to such an extent that contain the elutriant B of enramycin B.Flow rate of mobile phase is 12 packing volumes per hour.
Detect enramycin A and enramycin B purity through HPLC, enramycin A accounts for more than 98% of enramycin total content among the elutriant A, and enramycin B accounts for more than 96% of enramycin total content among the elutriant B.
Embodiment 8:A kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, getting the mixture 100mg that contains enramycin A and enramycin B dissolves, (gross weight of enramycin A and enramycin B accounts for 55% of mixture total weight amount, the weight ratio of enramycin A and enramycin B is 1:0.6), lysate is the aqueous hydrochloric acid of 30% methyl alcohol of 28mL, and pH is 3.6; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 40 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.05M SODIUM PHOSPHATE, MONOBASIC of the pH5.2 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 4mg mixture/mL packing volume, uses SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4.3 times of packing volumes-phosphate aqueous solution wash-out impurity again, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 33%-36% methyl alcohol of 2.2 times of packing volumes that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out gradient elution, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 5.3 times of packing volumes-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, the reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 42% methyl alcohol of 4.5 times of packing volumes that step 4 is processed carries out stepwise elution, collects to such an extent that contain the elutriant B of enramycin B.Flow rate of mobile phase is 22 packing volumes per hour.
Detect enramycin A and enramycin B purity through HPLC, enramycin A accounts for more than 98% of enramycin total content among the elutriant A, and enramycin B accounts for more than 95% of enramycin total content among the elutriant B.
Embodiment 9:A kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, getting the mixture 100mg that contains enramycin A and enramycin B dissolves, (gross weight of enramycin A and enramycin B accounts for 75% of mixture total weight amount, the weight ratio of enramycin A and enramycin B is 1:1.1), lysate is the aqueous hydrochloric acid of 30% methyl alcohol of 38mL, and pH is 3.7; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 45 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.04M SODIUM PHOSPHATE, MONOBASIC of the pH4.8 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 7mg mixture/mL packing volume, uses SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4.7 times of packing volumes-phosphate aqueous solution wash-out impurity again, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 33% methyl alcohol of 1.8 times of packing volumes that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out stepwise elution, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 3.8 times of packing volumes-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, the reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 48% methyl alcohol of 5.5 times of packing volumes that step 4 is processed carries out stepwise elution, collects to such an extent that contain the elutriant B of enramycin B.Flow rate of mobile phase is 17 packing volumes per hour.
Detect enramycin A and enramycin B purity through HPLC, enramycin A accounts for more than 98% of enramycin total content among the elutriant A, and enramycin B accounts for more than 98% of enramycin total content among the elutriant B.
Embodiment 10:A kind of method of separating enramycin A and enramycin B, it comprises the steps: step 1, getting the mixture 100mg that contains enramycin A and enramycin B dissolves, (gross weight of enramycin A and enramycin B accounts for 85% of mixture total weight amount, the weight ratio of enramycin A and enramycin B is 1:1.4), lysate is the aqueous hydrochloric acid of 30% methyl alcohol of 43mL, and pH is 3.8; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 12 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.01M SODIUM PHOSPHATE, MONOBASIC of the pH5.4 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 9mg mixture/mL packing volume, uses SODIUM PHOSPHATE, MONOBASIC in the step 2 of 5.7 times of packing volumes-phosphate aqueous solution wash-out impurity again, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 34% methyl alcohol of 2.8 times of packing volumes that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out stepwise elution, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4.7 times of packing volumes-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 40%-45% methyl alcohol of 6.5 times of packing volumes that step 4 is processed carries out gradient elution (collecting the elutriant at 40% place), collects to such an extent that contain the elutriant B of enramycin B.Flow rate of mobile phase is 14 packing volumes per hour.
Detect enramycin A and enramycin B purity through HPLC, enramycin A accounts for more than 95% of enramycin total content among the elutriant A, and enramycin B accounts for more than 98% of enramycin total content among the elutriant B.
More than each embodiment illustrate and adopt method of the present invention can separate effectively rapidly enramycin A and enramycin B, thereby obtain purer enramycin A and enramycin B.
Claims (9)
1. method of separating enramycin A and enramycin B, it comprises the steps: step 1, gets the mixture that contains enramycin A and enramycin B and dissolves, and lysate is the aqueous hydrochloric acid of 30% methyl alcohol, and pH is 3.0-4.0; Step 2 is after the octadecyl reversed-phase bonded silica chromatographic stuffing of 10-50 μ m soaks with methyl alcohol with particle diameter, and the dress post is for subsequent use with making the reversed phase chromatography post behind the 0.01-0.05M SODIUM PHOSPHATE, MONOBASIC of the pH4.0-5.5 that contains 30% methyl alcohol-phosphate aqueous solution balance chromatography column again; Step 3, with lysate loading reversed phase chromatography post in the step 1, applied sample amount is 1-10mg mixture/mL packing volume, again with the SODIUM PHOSPHATE, MONOBASIC in the step 2 of 4-6 times of packing volume-phosphate aqueous solution wash-out impurity, forms the reversed phase chromatography post that contains enramycin A and enramycin B; Step 4, adopt the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 32%-36% methyl alcohol of 1-3 times of packing volume that the reversed phase chromatography post that contains enramycin A and enramycin B that step 3 obtains is carried out wash-out, collect to such an extent that contain the elutriant A of enramycin A; Step 5 adopts SODIUM PHOSPHATE, MONOBASIC in the step 2 of 3-6 times of packing volume-phosphate aqueous solution wash-out, improves progressively that methanol content to 40% carries out gradient elution in SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution, abandons elutriant; Step 6, the reversed phase chromatography post after adopting SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 that contains 40%-50% methyl alcohol of 3-8 times of packing volume that step 4 is processed carries out wash-out, collects to such an extent that contain the elutriant B of enramycin B.
2. a kind of method of separating enramycin A and enramycin B according to claim 1, it is characterized in that: the gross weight of enramycin A and enramycin B accounts for more than 50% of mixture total weight amount in the described step 1, and the weight ratio of enramycin A and enramycin B is 1:0.5-1.5.
3. a kind of method of separating enramycin A and enramycin B according to claim 1, it is characterized in that: the consumption of the aqueous hydrochloric acid of 30% methyl alcohol is the 0.25-0.5mL/mg mixture in the described step 1.
4. a kind of method of separating enramycin A and enramycin B according to claim 1, it is characterized in that: the type of elution in described step 4 and the step 6 is gradient elution or stepwise elution.
5. a kind of method of separating enramycin A and enramycin B according to claim 1 is characterized in that: the type of elution in the described step 4 is the stepwise elution that contains the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 of 35% methyl alcohol.
6. a kind of method of separating enramycin A and enramycin B according to claim 1 is characterized in that: the type of elution in the described step 6 is the stepwise elution that contains the SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution in the step 2 of 45% methyl alcohol.
7. a kind of method of separating enramycin A and enramycin B according to claim 1 is characterized in that: flow rate of mobile phase is 10-25 packing volume per hour.
8. a kind of method of separating enramycin A and enramycin B according to claim 1 is characterized in that: omnidistance flow velocity is 15 packing volumes per hour.
9. a kind of method of separating enramycin A and enramycin B according to claim 1, it is characterized in that: described SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution is 0.03M SODIUM PHOSPHATE, MONOBASIC-phosphate aqueous solution of pH4.0.
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| CN102898510B (en) | 2014-05-07 |
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