Hongcheng Wang, Bo Xu
Setsi sa R&D sa Kopo
Selelekela
Ho ea ka polarities e lekanyelitsoeng ea mokhahlelo o emeng le oa mohala, chromatography ea metsi e ka aroloa ka karolo e tloaelehileng ea chromatography (NPC) le karolo e khutlisetsoang ea chromatography (RPC).Bakeng sa RPC, polarity ea mobile phase e matla ho feta ea stationary phase.RPC e se e sebelisoa haholo mekhoeng ea karohano ea chromatography ea mokelikeli ka lebaka la ts'ebetso ea eona e phahameng, tharollo e ntle le mokhoa o hlakileng oa ho boloka.Ka hona RPC e loketse karohano le tlhoekiso ea metsoako e sa tšoaneng ea polar kapa e seng polar, ho akarelletsa le alkaloids, lik'habohaedreite, mafura acid, steroids, nucleic acids, amino acid, peptide, liprotheine, joalo-joalo RPC, ka ho fetisisa e sebediswa e emeng mokhahlelo ke. silika gel matrix e kopantsoeng le lihlopha tse sa tšoaneng tse sebetsang, ho akarelletsa le C18, C8, C4, phenyl, cyano, amino, joalo-joalo Har'a lihlopha tsena tse sebetsang tse kopantsoeng, tse sebelisoang ka ho fetisisa ke C18.Ho hakanngoa hore ho feta 80% ea RPC e se e sebelisa C18 bonded phase.Kahoo kholomo ea C18 ea chromatography e se e le kholomo ea bokahohle bakeng sa laboratori e 'ngoe le e 'ngoe.
Le hoja kholumo ea C18 e ka sebelisoa mefuteng e mengata ea likopo, leha ho le joalo, bakeng sa mehlala e meng e nang le polar haholo kapa e nang le hydrophilic haholo, litšiea tse tloaelehileng tsa C18 li ka 'na tsa e-ba le mathata ha li sebelisoa ho hloekisa mehlala e joalo.Ka RPC, li-solvents tse sebelisoang hangata li ka laeloa ho ea ka polarity ea tsona: metsi
Setšoantšo sa 1. Setšoantšo sa moralo oa mekhahlelo e tlamahaneng holim'a gel ea silica ka mohala o tloaelehileng oa C18 (ka ho le letšehali) le kholomo ea C18AQ (ka ho le letona).
Ho rarolla mathata a boletsoeng ka holimo, baetsi ba thepa ea ho paka ka chromatographic ba entse lintlafatso tsa tekheniki.E 'ngoe ea lintlafatso tsena ke ho etsa liphetoho tse ling holim'a matrix a silica, tse kang ho kenngoa ha lihlopha tsa hydrophilic cyano (joalokaha ho bontšitsoe karolong e nepahetseng ea Setšoantšo sa 1), ho etsa hore bokaholimo ba gel ea silica bo be hydrophilic haholoanyane.Kahoo liketane tsa C18 holim'a silika li ne li ka atolosoa ka botlalo tlas'a maemo a nang le metsi a mangata haholo 'me ho putlama ha hydrophobic phase ho ka qojoa.Litšiea tsena tse fetotsoeng tsa C18 li bitsoa litšiea tsa C18 tse nang le metsi, e leng litšiea tsa C18AQ, tse etselitsoeng maemo a metsi a mangata haholo 'me li ka mamella 100% tsamaiso ea metsi.Litšiea tsa C18AQ li 'nile tsa sebelisoa ka ho pharaletseng ha ho arohana le ho hloekisa metsoako e matla ea polar, ho akarelletsa le li-acids, li-peptide, li-nucleoside le li-vithamine tse qhibilihang ka metsing.
Desalting ke e 'ngoe ea lits'ebetso tse tloaelehileng tsa likholomo tsa C18AQ molemong oa tlhoekiso ea lisampole, e tlosang likaroloana tsa letsoai kapa li-buffer ka har'a mokelikeli oa sampole ho thusa ts'ebeliso ea sampole lithutong tse latelang.Ka poso ena, Brilliant Blue FCF e nang le polarity e matla e sebelisitsoe e le sampole mme ea hloekisoa kholomong ea C18AQ.Sehlapolli sa sampole se ile sa nkeloa sebaka ke organic solvent ho tsoa ho buffer solution, ka hona ho nolofaletsa mouoane o latelang oa rotary hammoho le ho boloka lihlapolli le nako ea ts'ebetso.Ho feta moo, bohloeki ba sampole bo ile ba ntlafatsoa ka ho tlosa litšila tse ling tsa sampole.
Karolo ea Teko
Setšoantšo sa 2. Sebopeho sa lik'hemik'hale sa sampuli.
Brilliant Blue FCF e sebelisitsoe e le mohlala posong ena.Ho hloeka ha sampuli e tala e ne e le 86% 'me sebopeho sa lik'hemik'hale sa sampuli se bontšitsoe ho Setšoantšo sa 2. Ho lokisetsa tharollo ea sampuli, 300 mg powdery crude solid of Brilliant Blue FCF e ile ea qhibiliha ka tharollo ea buffer ea 1 M NaH2PO4 'me ea sisinyeha hantle hore e be. tharollo e hlakileng ka ho feletseng.Ka mor'a moo, tharollo ea mohlala e ne e kenngoa ka har'a kholomo ea flash ka ente.Setupo sa liteko tsa ho hloekisa flash se thathamisitsoe ho Lethathamo la 1.
| Seletsa | SepaBean™ Machine2 | |||
| Likhatriji | 12 g SepaFlash C18 RP flash cartridge (silika e chitja, 20 - 45 μm, 100 Å, Nomoro 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 | |||
| Mokhahlelo oa selefouno | Solvent A: Metsi Solvent B: Methanol | |||
| Sekhahla sa phallo | 30 ml ka motsotso | |||
| Ho kenya mohlala | 300 mg (Brilliant Blue FCF ka bohloeki ba 86%) | |||
| Gradient | Nako (CV) | Solvent B (%) | Nako (CV) | Solvent B (%) |
| 0 | 10 | 0 | 0 | |
| 10 | 10 | 10 | 0 | |
| 10.1 | 100 | 10.1 | 100 | |
| 17.5 | 100 | 17.5 | 100 | |
| 17.6 | 10 | 17.6 | 0 | |
| 22.6 | 10 | 22.6 | 0 | |
Liphetho le puisano
SepaFlash C18AQ RP flash cartridge e ile ea sebelisoa bakeng sa sampole desalting le tlhoekiso.Ho ne ho sebelisoa "step gradient" moo metsi a hloekileng a neng a sebelisoa e le mohato o tsamaeang qalong ea tlhahiso, 'me o sebetsa bakeng sa li-volumes tse 10 (CV).Joalokaha ho bontšitsoe setšoantšong sa 3, ha u sebelisa metsi a hloekileng e le mohato oa selefouno, mohlala o ne o bolokiloe ka ho feletseng holim'a cartridge ea flash.Ka mor'a moo, methanol karolong ea mohala e ile ea eketseha ka ho toba ho 100% 'me gradient e bolokoa bakeng sa 7.5 CV.Mohlala o ile oa hlakoloa ho tloha ho 11.5 ho isa ho 13.5 CV.Likarolong tse bokelletsoeng, tharollo ea sampole e ile ea nkeloa sebaka ho tloha ho tharollo ea buffer ea NaH2PO4 ho methanol.Ha ho bapisoa le tharollo e nang le metsi a mangata haholo, methanol e ne e le bonolo haholo ho tlosoa ke mouoane oa rotary mohatong o latelang, o thusang lipatlisiso tse latelang.
Setšoantšo sa 3. Chromatogram e khanyang ea sampuli ho cartridge ea C18AQ.
Ho bapisa boitšoaro ba ho boloka cartridge ea C18AQ le cartridge e tloaelehileng ea C18 bakeng sa lisampole tsa polarity e matla, ho ile ha etsoa tlhahlobo ea papiso e tšoanang.SepaFlash C18 RP flash cartridge e ile ea sebelisoa 'me chromatogram ea flash bakeng sa sampuli e bontšitsoe ho Setšoantšo sa 4. Bakeng sa li-cartridges tse tloaelehileng tsa C18, tekanyo e phahameng ka ho fetisisa e mamelloang ke metsi e ka bang 90%.Ka hona, tekanyo ea ho qala e behiloe ho 10% methanol metsing a 90%.Joalokaha ho bontšitsoe setšoantšong sa 4, 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, ts'ebetso ea sampole desalting kapa tlhoekiso e ke ke ea phethoa.
Setšoantšo sa 4. Chromatogram e khanyang ea sampuli ka cartridge e tloaelehileng ea C18.
Ha ho bapisoa le linear gradient, tšebeliso ea step gradient e na le melemo e latelang:
1. Ts'ebeliso ea solvent le nako ea ho sebetsa bakeng sa tlhoekiso ea sampole e fokotsehile.
2. Sehlahisoa se lebisitsoeng se hlahisa tlhōrō e hlabang, e fokotsang palo ea likaroloana tse bokelitsoeng 'me kahoo e nolofaletsa mouoane o latelang oa rotary hammoho le ho boloka nako.
3. Sehlahisoa se bokelitsoeng se ka methanol e bonolo ho fetoha mouoane, kahoo nako ea ho omisa e fokotsehile.
Qetellong, bakeng sa tlhoekiso ea sampole e nang le polar e matla kapa e nang le hydrophilic haholo, li-cartridge tsa SepaFlash C18AQ RP tse kopantsoeng le sistimi e itokisang ea chromatography ea SepaBean™ Machine e ka fana ka tharollo e potlakileng le e sebetsang hantle.
Mabapi le SepaFlash Bonded Series C18 RP flash cartridges
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.
logi (joalokaha ho bontšitsoe ho Lethathamo la 2).
Nako ea poso: Aug-27-2018