Hongcheng Wang, Bo Xu
Porogaramu R&D Ikigo
Intangiriro
Ukurikije polarite igereranije yicyiciro gihagaze nicyiciro kigendanwa, chromatografiya yamazi irashobora kugabanywa mubice bisanzwe bya chromatografiya (NPC) hanyuma bigahindura icyiciro cya chromatografiya (RPC).Kuri RPC, polarite yicyiciro kigendanwa irakomeye kuruta iyicyiciro gihagaze.RPC yahindutse imwe ikoreshwa muburyo butandukanye bwo gutandukanya chromatografiya bitewe nuburyo bukora neza, gukemura neza hamwe nuburyo bwo kubika neza.Kubwibyo RPC ikwiranye no gutandukanya no kweza ibice bitandukanye bya polar cyangwa bitari polar, harimo alkaloide, karubone, aside irike, steroid, acide nucleic, aside amine, peptide, proteyine, nibindi. Muri RPC, icyiciro gikunze gukoreshwa ni materique ya silika ihujwe nitsinda ritandukanye rikora, harimo C18, C8, C4, fenyl, cyano, amino, nibindi. Muri aya matsinda akora, ahuza cyane ni C18.Bigereranijwe ko hejuru ya 80% ya RPC ubu ikoresha icyiciro cya C18.Kubwibyo C18 chromatografiya inkingi yabaye ngombwa-kugira inkingi rusange kuri buri laboratoire.
Nubwo inkingi ya C18 ishobora gukoreshwa muburyo bwagutse bwa porogaramu, ariko, kuburugero rumwe rufite polar cyane cyangwa hydrophilique, inkingi zisanzwe za C18 zishobora kugira ibibazo mugihe zikoreshwa mugusukura izo ngero.Muri RPC, ibisanzwe bikoreshwa mumashanyarazi birashobora gutumizwa ukurikije polarite yabo: amazi
Igicapo 1. Igishushanyo mbonera cyibice bihujwe hejuru ya silika gel muburyo busanzwe bwa C18 (ibumoso) na C18AQ inkingi (iburyo).
Kugira ngo ukemure ibibazo byavuzwe haruguru, abakora ibikoresho byo gupakira chromatografi bakoze ibintu bya tekinike.Kimwe muri ibyo kunonosora ni uguhindura bimwe hejuru ya matrise ya silika, nko kwinjiza amatsinda ya hydrophilique cyano (nkuko bigaragara mugice cyiburyo cyishusho 1), kugirango ubuso bwa silika gel bube hydrophilique.Rero iminyururu ya C18 hejuru ya silika irashobora kwagurwa byuzuye mugihe cyamazi menshi kandi hydrophobique yo gusenyuka irashobora kwirindwa.Izi nkingi zahinduwe C18 zitwa inkingi zamazi C18, arizo C18AQ inkingi, zagenewe uburyo bwo gukuraho amazi menshi kandi zishobora kwihanganira sisitemu y'amazi 100%.Inkingi za C18AQ zakoreshejwe cyane mugutandukanya no kweza ibice bikomeye bya polar, harimo acide organic, peptide, nucleoside na vitamine zishonga mumazi.
Kurandura ni kimwe mubisanzwe byifashishwa byinkingi za C18AQ mugutunganya flash kuburugero, bivanaho umunyu cyangwa buffer mubice byicyitegererezo kugirango byorohereze ikoreshwa ryicyitegererezo mubushakashatsi bwakurikiyeho.Muri iyi nyandiko, Brilliant Blue FCF ifite polarite ikomeye yakoreshejwe nkicyitegererezo kandi cyezwa kumurongo wa C18AQ.Icyitegererezo cya solvent cyasimbujwe nimbaraga ziva mubisubizo bya buffer, bityo byoroshya guhinduka gukurikira kimwe no kuzigama imashanyarazi nigihe cyo gukora.Byongeye kandi, ubuziranenge bwicyitegererezo bwatejwe imbere mukuraho umwanda mubi.
Igice cy'Ubushakashatsi
Igishushanyo 2. Imiterere yimiti yicyitegererezo.
Brilliant Blue FCF yakoreshejwe nkicyitegererezo muriyi nyandiko.Ubuziranenge bwicyitegererezo kibisi bwari 86% kandi imiterere yimiti yicyitegererezo yerekanwe ku gishushanyo cya 2. Gutegura igisubizo cyicyitegererezo, 300 mg ya powdery yimbuto ikomeye ya Brilliant Blue FCF yashonga mumashanyarazi ya 1 M NaH2PO4 hanyuma iranyeganyega neza kugirango ibe igisubizo cyumvikana rwose.Igisubizo cyicyitegererezo noneho cyatewe muri flash inkingi nuwatewe inshinge.Ubushakashatsi bwakorewe flash yoza kurutonde murutonde 1.
| Igikoresho | SepaBean ™ Imashini2 | |||
| Cartridges | 12 g SepaFlash C18 RP flash cartridge (silika spherical, 20 - 45 μ mm, 100 Å, nimero yatanzwe: SW-5222-012-SP) | 12 g SepaFlash C18AQ RP flash cartridge (silika spherical, 20 - 45 μ mm, 100 Å, nimero ya ordre ： SW-5222-012-SP (AQ)） | ||
| Uburebure | 254 nm | |||
| Icyiciro cya mobile | Umuti A ： Amazi Umuti B ： Methanol | |||
| Igipimo cyo gutemba | 30 mL / min | |||
| Icyitegererezo | 300 mg (Brilliant Blue FCF ifite ubuziranenge bwa 86%) | |||
| Gradient | Igihe (CV) | Umuti B (%) | Igihe (CV) | Umuti 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 | |
Ibisubizo n'ibiganiro
SepaFlash C18AQ RP flash cartridge yakoreshejwe muburyo bwo gusiba no kweza.Intambwe ya gradient yakoreshejwe aho amazi meza yakoreshejwe nkicyiciro kigendanwa mugitangira cyo gukuraho no gukora kumubumbe winkingi 10 (CV).Nkuko bigaragara ku gishushanyo cya 3, mugihe ukoresheje amazi meza nkicyiciro kigendanwa, icyitegererezo cyagumishijwe rwose kuri flash cartridge.Ibikurikira, methanol mugice cya mobile yongerewe kuburyo bugaragara kugeza 100% kandi gradient yagumishijwe kuri 7.5 CV.Icyitegererezo cyakuwe kuri 11.5 kugeza 13.5 CV.Mu bice byakusanyirijwe hamwe, igisubizo cyicyitegererezo cyasimbuwe kuva NaH2PO4 buffer igisubizo kuri methanol.Ugereranije nigisubizo cyamazi menshi, methanol yari yoroshye cyane kuvanwaho no guhinduranya ibizunguruka mu ntambwe ikurikiraho, byorohereza ubushakashatsi bukurikira.
Igishushanyo 3. Flash chromatogramu yicyitegererezo kuri karitsiye ya C18AQ.
Kugereranya imyitwarire yo kugumana amakarito ya C18AQ hamwe na karitsiye ya C18 isanzwe kuburugero rwa polarite ikomeye, hakozwe ikigereranyo cyo kugereranya.Ikarita ya SepaFlash C18 RP yakoreshejwe kandi flash chromatogramu yicyitegererezo yerekanwe kumashusho ya 4. Kuri karitsiye ya C18 isanzwe, igipimo cyamazi cyihanganirwa cyane ni 90%.Kubwibyo gutangira gradient yashyizwe kuri 10% methanol mumazi 90%.Nkuko bigaragara ku gishushanyo cya 4, kubera hydrophobique yo gusenyuka kwiminyururu ya C18 yatewe n’igipimo kinini cy’amazi, icyitegererezo nticyagumishijwe cyane kuri karitsiye ya C18 isanzwe kandi cyatoranijwe mu cyiciro cya mobile.Nkigisubizo, ibikorwa byicyitegererezo cyo gusiba cyangwa kwezwa ntibishobora kurangira.
Igicapo 4. Flash chromatogramu yicyitegererezo kuri karitsiye ya C18 isanzwe.
Ugereranije n'umurongo ugereranya, ikoreshwa ryintambwe ya gradient ifite ibyiza bikurikira:
1. Gukoresha umusemburo no gukoresha igihe cyo kweza bigabanutse.
2. Igicuruzwa cyerekanwe kijya hejuru cyane, kigabanya ingano yibice byakusanyirijwe hamwe bityo bikorohereza guhinduranya bikurikira kimwe no gutakaza umwanya.
3. Ibicuruzwa byakusanyirijwe muri methanol byoroshye guhumeka, bityo igihe cyo kumisha kikagabanuka.
Mu gusoza, kugirango isukure ryicyitegererezo gifite polar cyangwa hydrophilique cyane, SepaFlash C18AQ RP flash cartridges ihujwe na sisitemu yo gutegura flash chromatografiya SepaBean ™ Imashini ishobora gutanga igisubizo cyihuse kandi cyiza.
Ibyerekeranye na SepaFlash Byahujwe Urukurikirane C18 RP flash cartridges
Hano hari urukurikirane rwa SepaFlash C18AQ RP flash cartridges hamwe nibisobanuro bitandukanye bivuye muri tekinoroji ya Santai (nkuko bigaragara mu mbonerahamwe ya 2).
| Umubare w'ingingo | Ingano yinkingi | Igipimo cy'Uruzi (mL / min) | Icyiciro (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 |
Imbonerahamwe 2. SepaFlash C18AQ RP flash cartridges.
Ibikoresho byo gupakira: Ubushobozi buhanitse bwa C18 (AQ)-silika ihambiriye, 20 - 45 μ mm, 100 Å.
logy (nkuko bigaragara mu mbonerahamwe ya 2).
Igihe cyo kohereza: Kanama-27-2018