Rui Huang, Bo Xu
Porogaramu R&D Ikigo
Intangiriro
Peptide ni uruvange rugizwe na acide amine, buri kimwe gifite imiterere yihariye yumubiri nubumara bitewe nubwoko butandukanye hamwe nibisigisigi bya aside amine bigize urukurikirane.Hamwe niterambere rya synthesis ikomeye ya chimique, synthesis ya chimique ya peptide itandukanye ikora yateye imbere cyane.Ariko, kubera ibice bigoye bya peptide yabonetse hakoreshejwe synthesis ikomeye, ibicuruzwa byanyuma bigomba kwezwa nuburyo bwizewe bwo gutandukana.Uburyo bukoreshwa muburyo bwo kweza peptide burimo ion guhana chromatografiya (IEC) hamwe na fonctionnement ya fonctionnement yo hejuru ya chromatografiya (RP-HPLC), ifite ibibi byubushobozi buke bwo gupakira ibintu, igiciro kinini cyibitangazamakuru bitandukanya, ibikoresho bitandukanya kandi bihenze, Ibindi kugirango bisukure byihuse peptide ntoya (MW <1 kDa), urubanza rwatsinzwe rwatangajwe mbere na Santai Technologies, aho amakarito ya SepaFlash RP C18 yakoreshejwe mugusukura byihuse thymopentine (TP-5) na intego yibicuruzwa byujuje ibisabwa byabonetse.
Igicapo 1. 20 acide aminide isanzwe (yakuwe kuri www.bachem.com).
Hariho ubwoko 20 bwa aside amine ikunze kugaragara mubigize peptide.Aminide acide irashobora kugabanywamo amatsinda akurikira ukurikije polarite hamwe numutungo-fatizo wa aside: utari polar (hydrophobique), polar (utarishye), acide cyangwa shingiro (nkuko bigaragara ku gishushanyo 1).Muburyo bwa peptide, niba aside amine igizwe nuruhererekane ahanini ni polar (nkuko bigaragara mumabara yijimye ku gishushanyo 1), nka Cysteine, Glutamine, Asparagine, Serine, Threonine, Tyrosine, nibindi noneho iyi peptide irashobora kugira imbaraga zikomeye polarite kandi ube ushonga cyane mumazi.Mugihe cyo kweza kuri ziriya ngero zikomeye za polar peptide ukoresheje chromatografiya ihindagurika, ikintu cyitwa hydrophobic phase collapse kizabaho (reba inyandiko isaba mbere yatangajwe na Santai Technologies: Hydrophobic Phase Collapse, AQ Reversed Phase Chromatography Columns and Applications).Ugereranije ninkingi zisanzwe za C18, inkingi za C18AQ zinoze zirakwiriye cyane mugusukura inkingi zikomeye cyangwa hydrophilique.Muri iyi nyandiko, peptide ikomeye ya polar yakoreshejwe nkicyitegererezo kandi isukurwa ninkingi ya C18AQ.Nkigisubizo, ibicuruzwa bigenewe kubahiriza ibisabwa byabonetse kandi birashobora gukoreshwa mubushakashatsi niterambere bikurikira.
| Igikoresho | SepaBean™imashini 2 | |||
| Cartridges | 12 g SepaFlash C18 RP flash cartridge (silika spherical, 20 - 45 mm, 100 Å, Tegeka umubikira ： 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, 220 nm | 214 nm | ||
| Icyiciro cya mobile | Umuti A: Amazi Umuti B: Acetonitrile | |||
| Igipimo cyo gutemba | 15 mL / min | 20 mL / min | ||
| Icyitegererezo | 30 mg | |||
| Gradient | Igihe (CV) | Umuti B (%) | Igihe (min) | Umuti B (%) |
| 0 | 0 | 0 | 4 | |
| 1.0 | 0 | 1.0 | 4 | |
| 10.0 | 6 | 7.5 | 18 | |
| 12.5 | 6 | 13.0 | 18 | |
| 16.5 | 10 | 14.0 | 22 | |
| 19.0 | 41 | 15.5 | 22 | |
| 21.0 | 41 | 18.0 | 38 | |
| / | / | 20.0 | 38 | |
| 22.0 | 87 | |||
| 29.0 | 87 | |||
Ibisubizo n'ibiganiro
Kugereranya imikorere yo kweza icyitegererezo cya polar peptide hagati yinkingi isanzwe ya C18 ninkingi ya C18AQ, twakoresheje inkingi isanzwe ya C18 kugirango flash yeze yicyitegererezo nkintangiriro.Nkuko bigaragara ku gishushanyo cya 2, kubera icyiciro cya hydrophobique cyasenyutse cyumunyururu wa C18 cyatewe n’igipimo kinini cy’amazi, icyitegererezo nticyagumishijwe kuri karitsiye isanzwe ya C18 kandi cyatoranijwe mu cyiciro cya mobile.Nkigisubizo, icyitegererezo nticyatandukanijwe neza kandi cyezwa.
Igishushanyo 2. Flash chromatogramu yicyitegererezo kuri karitsiye ya C18 isanzwe.
Ibikurikira, twakoresheje inkingi ya C18AQ kugirango flash yeze ya sample.Nkuko bigaragara ku gishushanyo cya 3, peptide yagumishijwe neza ku nkingi hanyuma irasohoka.Igicuruzwa cyagenewe cyatandukanijwe n’umwanda uri mu cyitegererezo kibisi kandi cyegeranijwe.Nyuma ya lyophilisation hanyuma igasesengurwa na HPLC, ibicuruzwa bisukuye bifite ubuziranenge bwa 98.2% kandi birashobora gukoreshwa mubushakashatsi bukurikira niterambere.
Igishushanyo 3. Flash chromatogramu yicyitegererezo kuri karitsiye ya C18AQ.
Mugusoza, SepaFlash C18AQ RP flash cartridge ihujwe na flash ya chromatografiya ya SepaBean™imashini irashobora gutanga igisubizo cyihuse kandi cyiza mugusukura polar ikomeye cyangwa hydrophilique.
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.
Kubindi bisobanuro birambuye kubisobanuro birambuye bya mashini ya SepaBean ™, cyangwa amakuru yo gutumiza kuri seriveri ya flash cartridges ya SepaFlash, nyamuneka sura urubuga.
Igihe cyo kohereza: Ukwakira-12-2018