Hongcheng Wang, Bo Xu
Isicelo se-R&D Centre
Intshayelelo
Ngokuhambelana nemilinganiselo yesigaba esimileyo kunye nesigaba esishukumayo, ichromatography yolwelo inokohlulwa ibe yinqanaba lesiqhelo lechromatography (NPC) kunye nesigaba sechromatography ebuyiselwe umva (RPC).Kwi-RPC, i-polarity yesigaba esiphathwayo sinamandla kuneso sigaba sokumisa.I-RPC iye yaba yeyona isetyenziswa kakhulu kwiindlela zokwahlula ichromatography elulwelo ngenxa yokusebenza kwayo okuphezulu, isisombululo esihle kunye nendlela yokugcinwa ecacileyo.Ngoko ke i-RPC ilungele ukwahlula kunye nokuhlanjululwa kweekhompawundi ezahlukeneyo ze-polar okanye ezingekho-polar, ezibandakanya i-alkaloids, i-carbohydrates, i-fatty acids, i-steroids, i-nucleic acids, i-amino acids, i-peptides, iiprotheni, njl. Kwi-RPC, isigaba esiqhelekileyo esisetyenziswayo i-silica gel matrix edibeneyo kunye namaqela ahlukeneyo asebenzayo, kubandakanywa i-C18, i-C8, i-C4, i-phenyl, i-cyano, i-amino, njl. Phakathi kwala maqela asebenzayo adibeneyo, eyona isetyenziswa kakhulu yiC18.Kuqikelelwa ukuba ngaphezu kwe-80% ye-RPC ngoku isebenzisa isigaba se-C18 edibeneyo.Ke ngoko ikholamu yeC18 yechromatography iye yaba yintsika yendalo yonke kwilabhoratri nganye.
Nangona ikholamu ye-C18 ingasetyenziselwa kuluhlu olubanzi kakhulu lwezicelo, nangona kunjalo, kwezinye iisampuli eziphantsi kakhulu okanye i-hydrophilic kakhulu, iikholomu eziqhelekileyo ze-C18 zinokuba neengxaki xa zisetyenziselwa ukuhlambulula iisampulu ezinjalo.Kwi-RPC, i-solvents elution esetyenziswa ngokuqhelekileyo inokumiselwa ngokwe-polarity yabo: amanzi
Umzobo 1. Umzobo oqingqiweyo wezigaba ezidibeneyo kumphezulu we-silica gel kwikholamu eqhelekileyo ye-C18 (ngasekhohlo) kunye nekholamu ye-C18AQ (ekunene).
Ukujongana neengxaki ezikhankanywe ngasentla, abavelisi bezinto zokupakisha zechromatographic baye benza ukuphuculwa kobugcisa.Enye yale mpucuko yenza uhlengahlengiso oluthile kumphezulu we-silica matrix, njengokungeniswa kwamaqela e-hydrophilic cyano (njengoko kuboniswe kwindawo efanelekileyo yoMzobo 1), ukwenza umphezulu we-silica gel ube ne-hydrophilic.Ngaloo ndlela amaketanga e-C18 kwi-silica surface inokwandiswa ngokupheleleyo phantsi kweemeko ezinamanzi amaninzi kunye nokuwa kwesigaba se-hydrophobic kunokugwenywa.Ezi zikholamu ze-C18 ezilungisiweyo zibizwa ngokuba ziikholamu ze-C18 ezinamanzi, ezizezi iikholomu ze-C18AQ, ezilungiselelwe iimeko zokulunywa kwamanzi kakhulu kwaye zinokunyamezela i-100% yenkqubo yamanzi.Iikholomu ze-C18AQ zisetyenziswe ngokubanzi ekuhlukaneni kunye nokuhlanjululwa kwezinto eziqinileyo ze-polar, ezibandakanya i-organic acids, i-peptides, i-nucleosides kunye namavithamini anyibilikayo emanzini.
I-Desalting yenye yezicelo eziqhelekileyo zeekholamu ze-C18AQ kwi-flash yokucoca iisampulu, esusa ityuwa okanye i-buffer components kwi-solvent yesampuli ukuququzelela ukusetyenziswa kwesampuli kwizifundo ezilandelayo.Kule post, i-Brilliant Blue FCF ene-polarity enamandla isetyenziswe njengesampuli kwaye yahlanjululwa kwikholamu yeC18AQ.I-solvent yesampulu yatshintshwa sisinyibilikisi se-organic ukusuka kwisisombululo se-buffer, ngaloo ndlela iququzelela ukuphuphuma komphunga okulandelayo kunye nokugcina izinyibilikisi kunye nexesha lokusebenza.Ngaphezu koko, ukucoceka kwesampulu kwaphuculwa ngokususa ukungcola okuthile kwisampuli.
iCandelo loMfuniselo
Umzobo 2. Ukwakhiwa kweekhemikhali zesampuli.
I-Brilliant Blue FCF isetyenziswe njengesampulu kwesi sithuba.Ukucoceka kwesampulu eluhlaza kwakuyi-86% kunye nesakhiwo sekhemikhali sesampuli siboniswe kuMzobo 2. Ukulungiselela isisombululo sesampuli, i-300 mg powdery crude solid ye-Brilliant Blue FCF yachithwa kwi-1 M i-NaH2PO4 isisombululo se-buffer kwaye yaxubha kakuhle ukuba ibe. isisombululo esicacileyo ngokupheleleyo.Isisombululo sesampulu sathi safakwa kwikholamu yeflash nge-injector.Ukusetwa kovavanyo lokucocwa kweflash kudweliswe kwiThebhile yoku-1.
| Isixhobo | SepaBean™ Machine2 | |||
| Iikhatriji | 12 g SepaFlash C18 RP flash cartridge (i-silica engqukuva, 20 - 45 μm, 100 Å, inombolo ye-oda: SW-5222-012-SP) | 12 g SepaFlash C18AQ RP flash cartridge (silica spherical, 20 - 45 μm, 100 Å, inombolo Order: SW-5222-012-SP(AQ))） | ||
| Ubude bamaza | 254 nm | |||
| Isigaba esiphathwayo | Isinyibilikisi A: Amanzi Isinyibilikisi B: Methanol | |||
| Umgangatho wohambo | 30 mL/min | |||
| Ukulayisha isampulu | 300 mg (Brilliant Blue FCF kunye nokusulungeka kwe86%) | |||
| Umgangatho | Ixesha (CV) | Isinyibilikisi B (%) | Ixesha (CV) | Isinyibilikisi 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 | |
Iziphumo neengxoxo
I-SepaFlash ye-C18AQ RP ye-flash cartridge yayisetyenziselwa isampuli desalting kunye nokucoca.Inyathelo lokuthamba kwasetyenziswa apho amanzi acocekileyo ayesetyenziswa njengenqanaba elihambayo ekuqaleni koluhlaziyo kwaye aqhutywe kwiikholamu ezili-10 (CV).Njengoko kuboniswe kwi-Figure 3, xa usebenzisa amanzi acocekileyo njengesigaba seselula, isampuli igcinwe ngokupheleleyo kwi-flash cartridge.Okulandelayo, i-methanol kwisigaba esihambayo yandiswe ngokuthe ngqo ukuya kwi-100% kwaye i-gradient yagcinwa kwi-7.5 CV.Isampulu yahluthwa ukusuka kwi-11.5 ukuya kwi-13.5 CV.Kumaqhezu aqokelelweyo, isisombululo sesampuli satshintshwa kwisisombululo se-NaH2PO4 se-buffer kwi-methanol.Xa kuthelekiswa nesisombululo esinamanzi kakhulu, i-methanol yayilula kakhulu ukuba isuswe ngokunyuka kwe-rotary kwisinyathelo esilandelayo, esiququzelela uphando olulandelayo.
Umzobo 3. I-chromatogram ye-flash yesampuli kwi-cartridge ye-C18AQ.
Ukuthelekisa ukuziphatha kokugcinwa kwe-cartridge ye-C18AQ kunye ne-cartridge ye-C18 eqhelekileyo kwiisampuli ze-polarity eqinile, uvavanyo lokuthelekisa oluhambelanayo lwenziwa.I-cartridge ye-SepaFlash ye-C18 RP ye-flash isetyenzisiwe kwaye i-chromatogram ye-flash yesampuli iboniswe kwi-Figure 4. Kwii-cartridges eziqhelekileyo ze-C18, umlinganiselo ophezulu wokunyamezela wesigaba se-aqueous malunga ne-90%.Ngoko ke i-gradient yokuqala yamiselwa kwi-10% yemethanol kumanzi angama-90%.Njengoko kuboniswe kwi-Figure 4, ngenxa yokuwa kwesigaba se-hydrophobic yeentambo ze-C18 ezibangelwa umlinganiselo ophezulu we-aqueous ratio, isampuli ayizange igcinwe kwi-cartridge ye-C18 eqhelekileyo kwaye ikhutshwe ngokuthe ngqo kwisigaba seselula.Ngenxa yoko, ukusebenza kwesampulu ye-desalting okanye ukuhlanjululwa akukwazi ukugqitywa.
Umzobo 4. I-chromatogram ye-flash yesampuli kwi-cartridge ye-C18 eqhelekileyo.
Xa kuthelekiswa nomgangatho womgca, ukusetyenziswa kwenqanaba lokuthambeka kunezi nzuzo zilandelayo:
1. Ukusetyenziswa kwe-solvent kunye nexesha lokuqhuba ukuhlanjululwa kwesampuli liyancitshiswa.
2. Imveliso ekujoliswe kuyo elutes kwincopho ebukhali, nto leyo enciphisa umthamo amaqhezu eziqokelelweyo kwaye ngaloo ndlela kube lula ukuphuphuma rotary zilandelayo kwakunye ukonga ixesha.
3. Imveliso eqokelelweyo ikwi-methanol ekulula ukuyikhupha, ngoko ke ixesha lokumisa liyancipha.
Ukuqukumbela, ukucocwa kwesampulu ephola ngamandla okanye ene-hydrophilic kakhulu, iikhatriji ze-SepaFlash C18AQ RP ezidityaniswa nenkqubo elungiselelayo yechromatography ye-flash ye-SepaBean™ Machine inokubonelela ngesisombululo esikhawulezayo nesisebenzayo.
Malunga SepaFlash Bonded Series C18 RP flash cartridges
Kukho uluhlu lwee-cartridges ze-SepaFlash C18AQ RP ezineenkcazo ezahlukeneyo kwi-Santai Technology (njengoko kuboniswe kwiThebhile 2).
| Inombolo yento | Ubungakanani beKholam | Umgangatho wohambo (mL/min) | Uxinzelelo olukhulu (psi/bar) |
| SW-5222-004-SP(AQ) | 5.4 g | 5-15 | 400/27.5 |
| I-SW-5222-012-SP(AQ) | 20 g | 10-25 | 400/27.5 |
| I-SW-5222-025-SP(AQ) | 33 g | 10-25 | 400/27.5 |
| I-SW-5222-040-SP(AQ) | 48 g | 15-30 | 400/27.5 |
| I-SW-5222-080-SP(AQ) | 105 g | 25-50 | 350/24.0 |
| I-SW-5222-120-SP(AQ) | 155 g | 30-60 | 300/20.7 |
| I-SW-5222-220-SP(AQ) | 300 g | 40-80 | 300/20.7 |
| I-SW-5222-330-SP(AQ) | 420 g | 40-80 | 250/17.2 |
Itheyibhile 2. SepaFlash C18AQ RP flash cartridges.
Izinto zokupakisha: Ukusebenza okuphezulu kwe-spherical C18 (AQ) -i-silica edibeneyo, i-20 - 45 μm, 100 Å.
ilogi (njengoko kubonisiwe kwiThebhile 2).
Ixesha lokuposa: Aug-27-2018