Rui Huang, Bo Xu
Setsi sa R&D sa Kopo
Selelekela
Peptide ke motsoako o entsoeng ka li-amino acid, tseo e 'ngoe le e 'ngoe e nang le litšobotsi tse ikhethang tsa' mele le lik'hemik'hale ka lebaka la mefuta e fapaneng le tatellano ea masalla a amino acid a etsang tatellano ea eona.Ka nts'etsopele ea motsoako o tiileng oa lik'hemik'hale, motsoako oa lik'hemik'hale oa li-peptide tse fapaneng tse sebetsang o tsoetse pele haholo.Leha ho le joalo, ka lebaka la sebopeho se rarahaneng sa peptide e fumanoeng ke motsoako o tiileng oa karolo, sehlahisoa sa ho qetela se lokela ho hloekisoa ka mekhoa e tšepahalang ea karohano.Mekhoa e sebelisoang hangata ea tlhoekiso ea li-peptide e kenyelletsa ion exchange chromatography (IEC) le reversed-phase high performance liquid chromatography (RP-HPLC), tse nang le bofokoli ba ho kenya sampole e tlase, theko e phahameng ea mecha ea phatlalatso ea karohano, lisebelisoa tse rarahaneng le tse theko e boima tsa karohano. joalo-joalo Bakeng sa tlhoekiso e potlakileng ea li-peptide tse nyane tsa molek'hule (MW <1 kDa), nyeoe e atlehileng ea kopo e ile ea hatisoa pele ke Santai Technologies, moo khatriji ea SepaFlash RP C18 e ileng ea sebelisoa bakeng sa tlhoekiso e potlakileng ea thymopentin (TP-5) le sehlahiswa sa shebiloeng se fihlelang ditlhoko se ile sa fumanwa.
Setšoantšo sa 1. Li-amino acid tse 20 tse tloaelehileng (tse hlahisitsoeng ho www.bachem.com).
Ho na le mefuta e 20 ea li-amino acid tse tloaelehileng sebopehong sa li-peptide.Li-amino acid tsena li ka aroloa ka lihlopha tse latelang ho ea ka polarity le acid-base thepa: non-polar (hydrophobic), polar (uncharged), acidic kapa motheo (joalokaha ho bontšitsoe setšoantšong sa 1).Ka tatellano ea peptide, haeba li-amino acid tse etsang tatellano li le polar (joalo ka ha li tšoailoe ka 'mala o pinki setšoantšong sa 1), joalo ka Cysteine, Glutamine, Asparagine, Serine, Threonine, Tyrosine, joalo-joalo joale peptide ena e kanna ea ba le matla a matla. polarity le ho qhibiliha haholo metsing.Nakong ea ts'ebetso ea tlhoekiso ea lisampole tsena tse matla tsa polar peptide ka reversed-phase chromatography, ho tla etsahala ntho e bitsoang hydrophobic phase collapse (sheba lengolo la kopo le hatisitsoeng pele ke Santai Technologies: Hydrophobic Phase Collapse, AQ Reversed Phase Chromatography Columns and Their Applications).Ha ho bapisoa le litšiea tse tloaelehileng tsa C18, litšiea tse ntlafetseng tsa C18AQ li loketse ka ho fetisisa bakeng sa tlhoekiso ea li-sampuli tse matla tsa polar kapa tsa hydrophilic.Ka poso ena, ho ile ha sebelisoa peptide e matla ea polar e le sampole mme ea hloekisoa ke kholomo ea C18AQ.Ka lebaka leo, sehlahisoa se lebeletsoeng se fihlelang litlhoko se ile sa fumanoa 'me se ka sebelisoa lipatlisisong tse latelang le nts'etsopele.
| Seletsa | SepaBean™mochini 2 | |||
| Likhatriji | 12 g SepaFlash C18 RP flash cartridge (silika e chitja, 20 - 45 μm, 100 Å, Nunori ea taelo:SW-5222-012-SP) | 12 g SepaFlash C18AQ RP khatriji ea flash (silika e chitja, 20 - 45 μm, 100 Å, Nomoro ea odara:SW-5222-012-SP(AQ)) | ||
| Wavelength | 254 nm, 220 nm | 214 nm | ||
| Mokhahlelo oa selefouno | Solvent A: Metsi Solvent B: Acetonitrile | |||
| Sekhahla sa phallo | 15 mL/min | 20 ml / min | ||
| Ho kenya mohlala | 30 mg | |||
| Gradient | Nako (CV) | Solvent B (%) | Nako (mets) | Solvent B (%) |
| 0 | 0 | 0 | 4 | |
| 1.0 | 0 | 1.0 | 4 | |
| 10.0 | 6 | 7.5 | 18 | |
| 12.5 | 6 | 13.0 | 18 | |
| 16.5 | 10 | 14.0 | 22 | |
| 19.0 | 41 | 15.5 | 22 | |
| 21.0 | 41 | 18.0 | 38 | |
| / | / | 20.0 | 38 | |
| 22.0 | 87 | |||
| 29.0 | 87 | |||
Liphetho le puisano
Ho bapisa ts'ebetso ea tlhoekiso bakeng sa sampole ea peptide ea polar pakeng tsa kholomo e tloaelehileng ea C18 le kholomo ea C18AQ, re sebelisitse kholumo e tloaelehileng ea C18 bakeng sa tlhoekiso ea khanya ea sampole e le qalo.Joalokaha ho bontšitsoe Setšoantšong sa 2, ka lebaka la ho oa ha hydrophobic phase ea liketane tsa C18 tse bakoang ke karo-karolelano e phahameng ea metsi, sampuli e ne e sa bolokehe ka har'a cartridge e tloaelehileng ea C18 'me e ile ea hlakoloa ka ho toba ke mohato oa selefouno.Ka lebaka leo, mohlala ha oa ka oa aroloa ka katleho le ho hloekisoa.
Setšoantšo sa 2. Chromatogram ea flash ea sampuli ka cartridge e tloaelehileng ea C18.
Ka mor'a moo, re sebelisitse kholumo ea C18AQ bakeng sa tlhoekiso ea flash ea sampole.Joalo ka ha ho bonts'itsoe ho Setšoantšo sa 3, peptide e ile ea bolokoa ka nepo kholomong mme ea tsoa.Sehlahisoa se lebisitsoeng se ne se arohane le litšila tse ka har'a sampole e tala 'me ea bokelloa.Kamora ho hloekisoa ha lyophilization le ho hlahlobisisoa ke HPLC, sehlahisoa se hloekisitsoeng se na le bohloeki ba 98.2% mme se ka sebelisoa hape bakeng sa mehato e latelang ea lipatlisiso le nts'etsopele.
Setšoantšo sa 3. Chromatogram e khanyang ea sampuli ho cartridge ea C18AQ.
Qetellong, SepaFlash C18AQ RP flash cartridge e kopantsoeng le flash chromatography system SepaBean™mochini o ne o ka fana ka tharollo e potlakileng le e sebetsang bakeng sa tlhoekiso ea lisampole tse matla tsa polar kapa tsa hydrophilic.
Ho na le letoto la li-cartridges tsa SepaFlash C18AQ RP tse nang le litlhaloso tse fapaneng ho tloha ho Santai Technology (joalokaha ho bontšitsoe ho Lethathamo la 2).
| Nomoro ea Ntho | Boholo ba Kholomo | Sekhahla sa Phallo (mL/mots) | Khatello (psi/bar) |
| SW-5222-004-SP(AQ) | 5.4 g | 5-15 | 400/27.5 |
| SW-5222-012-SP(AQ) | 20 g | 10-25 | 400/27.5 |
| SW-5222-025-SP(AQ) | 33 g | 10-25 | 400/27.5 |
| SW-5222-040-SP(AQ) | 48 g | 15-30 | 400/27.5 |
| SW-5222-080-SP(AQ) | 105 g | 25-50 | 350/24.0 |
| SW-5222-120-SP(AQ) | 155 g | 30-60 | 300/20.7 |
| SW-5222-220-SP(AQ) | 300 g | 40-80 | 300/20.7 |
| SW-5222-330-SP(AQ) | 420 g | 40-80 | 250/17.2 |
Letlapa la 2. SepaFlash C18AQ RP flash cartridges.Lisebelisoa tsa ho paka: Silika e nang le matla a phahameng a spherical C18 (AQ) -e kopantsoeng, 20 - 45 μm, 100 Å.
Bakeng sa tlhaiso-leseling e batsi mabapi le lintlha tse qaqileng tsa mochini oa SepaBean™, kapa lintlha tsa ho odara ka li-flash cartridges tsa SepaFlash, ka kopo etela sebaka sa rona sa Marang-rang.
Nako ea poso: Oct-12-2018