Hongcheng wang, bo xu
Isikhungo se-R & D Center
Ukuqalisa
Ngokwezinhlangano ezihlobene zesigaba se-station kanye nesigaba seselula, i-chromatography e-liquid ingahlukaniswa i-chromatography yesigaba esijwayelekile (NPC) ne-Chromatography yesigaba (RPC). Nge-RPC, ubukhosi besigaba seselula bunamandla kunalokho kwesigaba sokuma. I-RPC isiphenduke isetshenziswa kabanzi kuzindlela zokwehlukanisa kwe-chromatography ngenxa yokusebenza kwayo okuphezulu, ukulungiswa okuhle kanye nendlela yokugcina ecacile. Ngakho-ke i-RPC ilungele ukuhlukaniswa kanye nokuhlanjululwa kwezindawo ezahlukahlukene ze-polar noma ama-non-polar, ama-carbohydrate, amaprotheni, ama-matrix asetshenziswa kakhulu yi-silica gel matrix ehlanganiswe namaqembu ahlukahlukene asebenzayo, kufaka phakathi i-C18, C8, C4, UPhenyl, uCyano, i-Amino, njll. Phakathi kwalawa maqembu asebenza ngezibopho, asetshenziswa kakhulu yi-C18. Kulinganiselwa ukuthi ngaphezu kwama-80% we-RPC manje asebenzisa isigaba se-C18 Bonded. Ngakho-ke ikholomu ye-Chromaygraphy seliphenduke ikholomu okumele ibe ne-Universal lonke ilabhorethri.
Yize ikholomu ye-C18 ingasetshenziswa ezinhlobonhlobo zezicelo, noma kunjalo, kumasampula athile anama-polar noma ama-hydrophilic amaningi, amakholomu ajwayelekile we-C18 angaba nezinkinga lapho esetshenziselwa ukuhlanza amasampula anjalo. Ku-RPC, izixazululo ezivame ukusetshenziswa ezisetshenziswayo zingayalwa ngokuya nge-polarity yazo: amanzi <methanol <acetonitrile <ethanol <tetrahydroforan <isopropanol. Ukuqinisekisa ukugcinwa okuhle kwikholamu la masampula (i-polar eqinile noma i-hydrophilic ephezulu), ingxenye ephezulu yohlelo lwamanzi oludingekayo oludingekayo ukuze lusetshenziswe njengesigaba seselula. Kodwa-ke, lapho usebenzisa uhlelo lwamanzi ahlanzekile (kufaka phakathi amanzi ahlanzekile noma ikhambi likasawoti emsulwa) njengesigaba se-carbon esigabeni sekholamu esiteshini se-C18 kuvame ukugwema amanzi futhi kungagcinwa. Le nto ibizwa ngokuthi "isigaba se-hydrophobic Collapse" (njengoba kukhonjisiwe engxenyeni yesobunxele yesibalo 1). Yize lesi simo siphindisela emuva lapho ikholamu igezwa nge-sol sol solvents enjenge-methanol noma i-acetonitritle, isengadala ukulimala kwikholamu. Ngakho-ke, kuyadingeka ukuvikela lesi simo ukuthi kungenzeki.
Isibalo
Ukubhekana nezinkinga ezishiwo ngenhla, abakhiqizi be-Chromatographic Booking Actives benze ukuthuthuka kwezobuchwepheshe. Enye yalezi zinto zokuthuthuka yenza ukuguqulwa ebusweni be-matrix ye-silica, njengokuthulwa kwamaqembu we-hydrophilic Cyano (njengoba kukhonjisiwe engxenyeni efanele ye-Figue 1), ukwenza ingaphezulu le-silica gel ye-hydrophilic ngaphezulu. Ngakho-ke amaketanga e-C18 eLilica Surface anganwetshwa ngokuphelele ngaphansi kwezimo ezinamanzi amaningi kanye nokuwohloka kwesigaba se-hydrophobic kungagwenywa. Lawa makholomu we-C18 aguquliwe abizwa ngokuthi amakholomu we-Aquive C18, okungukuthi amakholomu we-C18AQ, enzelwe izimo ezinamanzi kakhulu futhi angabekezelela uhlelo olungamanzi alwayo. Amakholomu we-C18AQ asetshenziswe kabanzi ekuhlukanisweni nasekuhlanzeni kwamakhompiyutha aqinile we-polar, kufaka phakathi ama-organic acid, ama-peptides, ama-nucleosides namavithamini ancibilikayo amanzi.
I-Dealting kungenye yezicelo ezijwayelekile zekholomu ye-C18AQ ekuhlanzeni kwe-Flash kwamasampula, okususa izingxenye zikasawoti noma ama-buffer ku-solvent yesampula ukwenza isicelo sesampula ezifundweni ezilandelayo. Kulokhu okuthunyelwe, i-FCF eluhlaza okwesibhakabhaka ene-polarity eqinile yasetshenziswa njengesampula futhi ihlanjululwe kukholamu ye-C18AQ. I-solvent yesampula yathathelwa indawo yi-organic solvent kusuka kusisombululo se-buffer, ngaleyo ndlela yenza lula ukuhwamuka okulandelayo okujikelezayo kanye nokusindisa izixazululo nokusebenza kwesikhathi. Ngaphezu kwalokho, ubumsulwa besampula bathuthukiswa ngokususa ukungcola okuthile kusampula.
Isigaba Sokuhlola
Umdwebo 2. Isakhiwo samakhemikhali sesampula.
I-FCF eluhlaza okwesibhakabhaka yayisetshenziswa njengesampula kulokhu okuthunyelwe. Ubumsulwa besampula eluhlaza lalingama-86% futhi isakhiwo samakhemikhali sesampula saboniswa kuMfanekiso 2. Ukulungiselela isixazululo sesampula, i-300 mg powdery crude solifle ye-1 m nah2po4 buffer solution futhi yathuthumela ukuba yikhambi elicacile ngokuphelele. Isixazululo sesampula sabe sesifakwa kwikholomu ye-flash ngumjovo. Ukusethwa kokuhlola kokuhlanzwa kwe-Flash kufakwe kuhlu etafuleni 1.
| Into yokwenza utho | Umshini weSepabean ™2 | |||
| Ama-cartridges | I-12 g sepaflash C18 RP Flash Carthridge (Spherical Silica, 20 - 45 μm, Order Inombolo: SW-5222-012-SP) | I-12 g sepaflash C18AQ RP Flash Carthridge (spherical Silica, 20 - 45 μm, Inombolo ye-Order: SW-5222-012-SP (AQ)Isihlehlukene | ||
| Umbuzi | 254 nm | |||
| Isigaba | Solvent a: amanzi Solvent b: methanol | |||
| Izinga Lokugeleza | I-30 ml / min | |||
| Ukulayisha isampula | I-300 mg (Blue Blue FCF enobumsulwa be-86%) | |||
| I-gradient | Isikhathi (CV) | Solvent b (%) | Isikhathi (CV) | Solvent b (%) |
| 0 | 10 | 0 | 0 | |
| 10 | 10 | 10 | 0 | |
| I-10.1 | 100 | I-10.1 | 100 | |
| 17.5 | 100 | 17.5 | 100 | |
| 17.6 | 10 | 17.6 | 0 | |
| 22.6 | 10 | 22.6 | 0 | |
Imiphumela nengxoxo
Kwakusetshenziselwa i-Sepaflash C18AQ RP Flash cartridge ukuze isampula isampula nokuhlanzwa. Isinyathelo se-gradient sasetshenziswa lapho amanzi ahlanzekile asetshenziswa khona njengesigaba se-mobile ekuqaleni kokuqedwa futhi agijime amavolumu ayi-10 wekholamu (CV). Njengoba kukhonjisiwe kuMdwebo 3, lapho usebenzisa amanzi ahlanzekile njengesigaba seselula, isampula lagcinwa ngokuphelele ku-Flash Carthridge. Okulandelayo, i-methanol esigabeni eselula yanda ngokuqondile yaya ku-100% kanti i-gradient yagcinwa nge-7.5 CV. Isampula yavuswa kusuka ngo-11,5 kuye ku-13.5 CV. Ezingxekeni eziqoqiwe, ikhambi lesampula lathathelwa indawo yisixazululo se-NAH2PO4 Buffer ku-Methanol. Ukuqhathanisa isisombululo esinamanzi kakhulu, i-methanol yakulula kakhulu ukususwa ngokuhwamuka okujikelezayo esiteji esilandelayo, esisiza lolu cwaningo olulandelayo.
Umdwebo 3. I-flash chromatograph yesampula ku-C18AQ cartridge.
Ukuqhathanisa ukusebenza kokugcinwa kwe-C18AQ cartridge kanye ne-C18 Cartridge ye-C18 yamasampula yenhlekelele eqinile, kwenziwa ukuhlolwa kokuqhathanisa okufanayo. Kwasetshenziswa i-sepaflash C18 RP ye-Flash cartridge futhi i-flash chromatograph yesampula yaboniswa kuMdwebo 4. Kuma-cartridge ajwayelekile we-C18, isilinganiso esiphakeme kunazo zonke esibekelwe samanzi sokubekezela singaba ngu-90%. Ngakho-ke i-gradient yokuqala isethwe ku-10% Methanol emanzini angama-90%. Njengoba kukhonjisiwe kuMdwebo 4, ngenxa yokuwohloka kwesigaba se-hydrophobic kwamaketanga e-C18 adalwa yisilinganiso esiphakeme samanzi, isampula lagcinwa kancane ku-Cartridge evamile ye-C18 futhi lavunyelwa ngokuqondile yisigaba seselula. Ngenxa yalokhu, ukusebenza kwesampula ukuncipha noma ukuhlanzwa akunakuqedwa.
Umdwebo 4. I-flash chromatograph yesampula ku-C18 Cartridge.
Ukuqhathanisa i-gradient eqondile, ukusetshenziswa kwe-step gradient kunezinzuzo ezilandelayo:
1. Ukusetshenziswa kwe-solvent kanye nesikhathi sokuqalisa kokuhlanzwa kwesampula kuyancishiswa.
2
I-3. Umkhiqizo oqoqiwe use-methanol okulula ukuhanjiswa, ngakho-ke isikhathi sokusoma sincishisiwe.
Ekuphetheni, ngokuhlanjululwa kwesampula enamandla amakhulu noma ama-hydrophilic ahlanganisiwe, ama-sepaflash C18AQ RP Flash Cartridges ahlanganisa nesixazululo sokulungiselela i-Chromatfophy System Sepabean ™ Umshini osheshayo.
Mayelana nochungechunge lweSepaflash Bonded Cardgeges C98 RP
Kunochungechunge lwama-cartridgeges we-sepaflash C18AQ RP Flash anokucaciswa okuhlukile kubuchwepheshe beSantai (njengoba kukhonjisiwe kuThebula 2).
| Inombolo yento | Usayizi wekholomu | Izinga Lokugeleza (ml / min) | Max.prestrure (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 |
Ithebula 2. Sepaflash C18AQ RP Flash Cartridges.
Izinto zokupakisha: I-Spherical C18 esebenza kahle kakhulu
logy (njengoba kukhonjisiwe kuThebula 2).
Isikhathi sePosi: Aug-27-2018