Hongcheng Wang, Bo Xu
Isicelo se-R&D Center
Isingeniso
Ngokuvumelana ne-polarity ehlobene yesigaba esimile nesigaba esihambayo, i-chromatography ewuketshezi ingahlukaniswa ngesigaba se-chromatography esivamile (NPC) kanye nesigaba se-chromatography ehlehlisiwe (RPC).Ku-RPC, i-polarity yesigaba seselula inamandla kunaleyo yesigaba esimile.I-RPC isiphenduke esetshenziswa kakhulu ezindleleni zokuhlukanisa i-chromatography ewuketshezi ngenxa yokusebenza kahle kwayo okuphezulu, ukulungiswa okuhle kanye nendlela yokugcinwa ecacile.Ngakho-ke i-RPC ifanele ukuhlukaniswa nokuhlanzwa kwezinhlanganisela ezihlukahlukene ze-polar noma ezingezona ze-polar, okuhlanganisa ama-alkaloid, ama-carbohydrate, ama-fatty acids, ama-steroid, ama-nucleic acid, ama-amino acid, ama-peptides, amaprotheni, njll. Ku-RPC, isigaba esivamile esisetshenziswa kakhulu i-silica gel matrix ehlanganiswe namaqembu ahlukahlukene asebenzayo, okuhlanganisa i-C18, i-C8, i-C4, i-phenyl, i-cyano, i-amino, njll. Phakathi kwala maqembu asebenzayo ahlanganisiwe, elisetshenziswa kakhulu yi-C18.Kulinganiselwa ukuthi ngaphezu kwama-80% we-RPC manje asebenzisa isigaba esiboshiwe se-C18.Ngakho-ke ikholomu ye-C18 chromatography isiphenduke ikholomu yomhlaba wonke okufanele ibe nayo yonke ilabhorethri.
Nakuba ikholomu ye-C18 ingasetshenziswa kuhlelo olubanzi kakhulu lwezinhlelo zokusebenza, nokho, kwamanye amasampuli ane-polar kakhulu noma ane-hydrophilic kakhulu, amakholomu avamile e-C18 angase abe nezinkinga lapho esetshenziselwa ukuhlanza amasampula anjalo.Ku-RPC, i-elution solvents evame ukusetshenziswa ingalawulwa ngokuvumelana ne-polarity yabo: amanzi < methanol
Umfanekiso 1. Umdwebo oyisikimu wezigaba eziboshiwe endaweni yejeli ye-silica kukholamu evamile ye-C18 (kwesokunxele) kanye nekholomu ye-C18AQ (kwesokudla).
Ukubhekana nezinkinga ezishiwo ngenhla, abakhiqizi bezinto zokupakisha ze-chromatographic benze ukuthuthukiswa kobuchwepheshe.Okunye kwalokhu kuthuthukiswa ukwenza izinguquko ezithile ebusweni be-silica matrix, njengokwethulwa kwamaqembu e-hydrophilic cyano (njengoba kuboniswe engxenyeni efanele yoMfanekiso 1), ukuze kwenziwe ingaphezulu lejeli ye-silica libe ne-hydrophilic.Ngakho-ke amaketanga e-C18 endaweni ye-silica anganwetshwa ngokugcwele ngaphansi kwezimo ezimanzi kakhulu futhi ukugoqa kwesigaba se-hydrophobic kungagwenywa.Lawa makholomu e-C18 aguquliwe abizwa ngokuthi amakholomu e-C18 anamanzi, okuwukuthi amakholomu e-C18AQ, aklanyelwe izimo ze-elution enamandla kakhulu futhi akwazi ukubekezelela isistimu yamanzi engu-100%.Amakholomu e-C18AQ asetshenziswe kabanzi ekuhlukaniseni nasekuhlanzweni kwezinhlanganisela eziqinile ze-polar, kuhlanganise nama-acids ephilayo, ama-peptide, ama-nucleosides namavithamini ancibilikayo emanzini.
I-Desalting ingenye yezinhlelo zokusebenza ezivamile zamakholomu e-C18AQ ekuhlanzweni kwe-flash kumasampuli, okukhipha usawoti noma izingxenye ze-buffer ku-solvent yesampula ukuze kube lula ukusetshenziswa kwesampula ezifundweni ezilandelayo.Kulokhu okuthunyelwe, i-Brilliant Blue FCF ene-polarity eqinile isetshenziswe njengesampula futhi yahlanzwa kukholomu ye-C18AQ.I-solvent eyisampula yathathelwa indawo yi-solvent ephilayo evela kusixazululo se-buffer, ngaleyo ndlela kube lula ukuhwamuka okujikelezayo okulandelayo kanye nokonga izinyibilikisi nesikhathi sokusebenza.Ngaphezu kwalokho, ukuhlanzeka kwesampula kwathuthukiswa ngokususa ukungcola okuthile kusampula.
Isigaba Sokuhlola
Umfanekiso 2. Isakhiwo samakhemikhali sesampula.
I-Brilliant Blue FCF isetshenziswe njengesampula kulokhu okuthunyelwe.Ukuhlanzeka kwesampula eluhlaza bekungama-86% futhi ukwakheka kwamakhemikhali kwesampula kuboniswe kuMfanekiso 2. Ukulungiselela isisombululo sesampula, u-300 mg oyimpuphu ongahluziwe oqinile we-Brilliant Blue FCF uhlakazwe kusixazululo sebhafa esingu-1 M NaH2PO4 futhi sanyakaza kahle ukuze sibe isixazululo esicacile ngokuphelele.Isixazululo sesampula sabe sesijovwa kukholamu yefuleshi ngomjovo.Ukusethwa kokuhlolwa kokuhlanzwa kwe-flash kufakwe kuhlu kuThebula 1.
| Insimbi | Umshini we-SepaBean™2 | |||
| Amakhatriji | 12 g SepaFlash C18 RP flash cartridge (silica eyindilinga, 20 - 45 μm, 100 Å, Inombolo ye-oda: SW-5222-012-SP) | 12 g SepaFlash C18AQ RP flash cartridge (silica eyindilinga, 20 - 45 μm, 100 Å, inombolo ye-oda:SW-5222-012-SP(AQ)） | ||
| Ubude begagasi | 254 nm | |||
| Isigaba seselula | I-solvent A: Amanzi I-solvent B: I-Methanol | |||
| Izinga lokugeleza | 30 mL/min | |||
| Isampula iyalayisha | 300 mg (Brilliant Blue FCF nobumsulwa be-86%) | |||
| I-Gradient | Isikhathi (CV) | I-solvent B (%) | Isikhathi (CV) | I-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 | |
Imiphumela nengxoxo
I-flash cartridge ye-SepaFlash C18AQ RP yasetshenziselwa isampula yokukhipha usawoti kanye nokuhlanza.Kwasetshenziswa i-step gradient lapho amanzi ahlanzekile asetshenziswa njengesigaba esihambayo ekuqaleni kwe-elution futhi iqhutshwa ngamakholomu angu-10 (CV).Njengoba kuboniswe kuMfanekiso 3, lapho kusetshenziswa amanzi ahlanzekile njengesigaba esihambayo, isampula ligcinwe ngokuphelele ku-flash cartridge.Okulandelayo, i-methanol esigabeni samaselula yanyuswa ngokuqondile yaba ngu-100% futhi igradient yagcinwa ifinyelela ku-7.5 CV.Isampula ikhishwe ku-11.5 kuya ku-13.5 CV.Kumafrakshini aqoqiwe, isisombululo sesampula sashintshwa kusukela kusixazululo sebhafa se-NaH2PO4 kuya ku-methanol.Uma kuqhathaniswa nesisombululo esinamanzi amaningi, i-methanol bekulula kakhulu ukususwa ngokuhwamuka kwe-rotary esinyathelweni esilandelayo, esenza kube lula ucwaningo olulandelayo.
Umfanekiso 3. I-flash chromatogram yesampula ku-cartridge ye-C18AQ.
Ukuze uqhathanise ukuziphatha kokugcinwa kwe-C18AQ cartridge kanye ne-C18 cartridge evamile yamasampuli e-polarity eqinile, ukuhlolwa kokuqhathanisa okufanayo kwenziwa.Kusetshenziswe i-flash cartridge ye-SepaFlash C18 RP futhi i-flash chromatogram yesampula yaboniswa kuMfanekiso 4. Kumakhathriji avamile e-C18, isilinganiso esiphezulu sesigaba samanzi esibekezeleleke sicishe sibe ngu-90%.Ngakho-ke i-gradient yokuqala yayibekwe ku-10% methanol emanzini angama-90%.Njengoba kuboniswe ku-Figure 4, ngenxa yokuwa kwesigaba se-hydrophobic samaketanga e-C18 okubangelwa isilinganiso esiphezulu se-aqueous, isampula ayizange igcinwe ku-cartridge evamile ye-C18 futhi yakhishwa ngokuqondile isigaba seselula.Ngenxa yalokho, ukusebenza kwesampula ye-desalting noma ukuhlanzwa akukwazi ukuqedwa.
Umfanekiso 4. I-flash chromatogram yesampula ku-cartridge evamile ye-C18.
Uma kuqhathaniswa ne-gradient yomugqa, ukusetshenziswa kwe-gradient yesinyathelo kunezinzuzo ezilandelayo:
1. Ukusetshenziswa kwe-solvent kanye nesikhathi sokusebenza sokuhlanza isampula sincishisiwe.
2. Umkhiqizo ohlosiwe uyaphunyuka ngokuphakama okucijile, okunciphisa umthamo wamafrakshini aqoqiwe futhi ngaleyo ndlela kusize ukuhwamuka okulandelayo okujikelezayo kanye nokonga isikhathi.
3. Umkhiqizo oqoqiwe uku-methanol okulula ukuhwamuka, ngaleyo ndlela isikhathi sokumisa siyancishiswa.
Sengiphetha, ukuze kuhlanzwe isampula eline-polar kakhulu noma eline-hydrophilic kakhulu, ama-flash cartridges e-SepaFlash C18AQ RP ahlanganiswe nohlelo olulungiselelwayo lwe-flash chromatography i-SepaBean™ Machine inganikeza isisombululo esisheshayo nesisebenzayo.
Mayelana ne-SepaFlash Bonded Series C18 RP flash cartridges
Kukhona uchungechunge lwama-flash cartridges e-SepaFlash C18AQ RP anemininingwane ehlukene evela ku-Santa Technology (njengoba kukhonjisiwe kuThebula 2).
| Inombolo Yento | Usayizi Wekholomu | Isilinganiso sokugeleza (mL/min) | Ubukhulu.Ingcindezi (i-psi/ibha) |
| I-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) | awu 33g | 10-25 | 400/27.5 |
| I-SW-5222-040-SP(AQ) | awu 48g | 15-30 | 400/27.5 |
| I-SW-5222-080-SP(AQ) | 105 g | 25-50 | 350/24.0 |
| I-SW-5222-120-SP(AQ) | awu 155g | 30-60 | 300/20.7 |
| I-SW-5222-220-SP(AQ) | 300 g | 40-80 | 300/20.7 |
| I-SW-5222-330-SP(AQ) | awu 420g | 40-80 | 250/17.2 |
Ithebula 2. I-SepaFlash C18AQ RP flash cartridges.
Izinto zokupakisha: I-silica eyisiyingi esebenza kahle kakhulu i-C18(AQ)-eboshiwe, 20 - 45 μm, 100 Å.
ilogi (njengoba kukhonjisiwe kuThebula 2).
Isikhathi sokuthumela: Aug-27-2018