Rui Huang, Bo Xu
Taupānga R&D Center
Kupu Whakataki
Ko te peptide he puhui i tito mai i nga waikawa amino, he mea motuhake to ia ahuatanga o te tinana me te matū na runga i nga momo rereke me te raupapa o nga toenga waikawa amino e noho ana i tona raupapa.Na te whanaketanga o te whakahiato matū o te wahanga totoka, kua nui te ahunga whakamua o te whakahiato matū o nga momo peptides kaha.Heoi, na te uaua o te hanganga o te peptide i puta mai i te whakahiatotanga o te wahanga totoka, me purea te hua whakamutunga ma nga tikanga wehewehe pono.Ko nga tikanga purenga e whakamahia nuitia ana mo nga peptides ko te chromatography whakawhiti katote (IEC) me te reversed-phase high performance liquid chromatography (RP-HPLC), kei a raatau nga ngoikoretanga o te iti o te kaha o te utaina tauira, te utu nui o te whakamaarama wehe, te uaua me te utu taputapu wehe, mo te purenga tere o nga peptides ngota iti (MW <1 kDa), he keehi tono angitu i whakaputaina i mua e Santai Technologies, i whakamahia he kariri SepaFlash RP C18 mo te purenga tere o te thymopentin (TP-5) me te i tutuki nga hua e tutuki ana i nga whakaritenga.
Whakaahua 1. 20 nga waikawa amino noa (he mea whakaputa mai i www.bachem.com).
E 20 nga momo waikawa amino e kitea ana i roto i te hanganga o nga peptides.Ka taea te wehewehea enei waikawa amino ki nga roopu e whai ake nei i runga i to ratou polarity me te waikawa-base: kore-polar (hydrophobic), polar (kaore he utu), waikawa, taketake ranei (penei i te Whakaaturanga 1).I roto i te raupapa peptide, mena he polar te nuinga o nga waikawa amino kei roto i te raupapa (e tohuhia ana ki te tae mawhero i te Whakaahua 1), penei i te Cysteine, Glutamine, Asparagine, Serine, Threonine, Tyrosine, me etahi atu ka kaha pea tenei peptide. polarity me te tino memeha ki te wai.I te wa o te purenga mo enei tauira peptide polar kaha na roto i te chromatography-waa-whakamuri, ka puta he ahuatanga e kiia ana ko te tiango o te wahanga hydrophobic (tirohia te tuhipoka tono i whakaputaina i mua e Santai Technologies: Hydrophobic Phase Collapse, AQ Reversed Phase Chromatography Columns me a raatau tono).Ka whakatauritea ki nga pou C18 auau, ko nga pou C18AQ kua pai ake he tino pai mo te purenga o nga tauira polar kaha, hydrophilic ranei.I tenei pou, he peptide polar kaha i whakamahia hei tauira ka purea e te pou C18AQ.Ko te mutunga mai, ko te hua e tutuki ana ki nga whakaritenga i whiwhi, ka taea te whakamahi i roto i nga rangahau me nga whanaketanga e whai ake nei.
| Taputapu | SepaBean™mihini 2 | |||
| Kaata | 12 g SepaFlash C18 RP kariri kohiko (silica porowhita, 20 - 45 μm, 100 Å, Whakataua te utu:SW-5222-012-SP) | 12 g SepaFlash C18AQ RP kariri kohiko (silica porohita, 20 - 45 μm, 100 Å, Tau ota:SW-5222-012-SP(AQ)) | ||
| Te roangaru | 254 nm, 220 nm | 214 nm | ||
| Waahanga pūkoro | Wairewa A: Wai Wairewa B: Acetonitrile | |||
| Rere rere | 15 mL/min | 20 mL/min | ||
| Uta tauira | 30 mg | |||
| Rōnaki | Wā (CV) | Wairewa B (%) | Wā (min) | Wairewa 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 | |||
Nga Hua me te Matapaki
Hei whakataurite i nga mahi purenga mo te tauira peptide polar i waenga i te pou C18 me te pou C18AQ, i whakamahia e matou he pou C18 mo te purenga uira o te tauira hei timatanga.E whakaatu ana i te Whakaahua 2, na te hinganga o te wahanga hydrophobic o nga mekameka C18 na te nui o te owehenga wai, kare i mau tonu te tauira ki runga i te kaata C18 o ia ra, katahi ka peia atu e te wahanga pūkoro.Ko te mutunga mai, kaore i tino tika te wehe me te purea o te tauira.
Whakaahua 2. Te chromatogram rama o te tauira i runga i te kariri C18.
I muri mai, i whakamahia e matou he pou C18AQ mo te purenga uira o te tauira.Ka rite ki te Whakaaturanga 3, ka mau tonu te peptide ki runga i te pou, katahi ka peia ki waho.I wehea te hua i whäia mai i ngä poke o te tauira mata ka kohia.Whai muri i te lyophilization, ka tātarihia e te HPLC, he 98.2% te ma o te hua kua purea, ka taea te whakamahi mo te rangahau me te whanaketanga o muri.
Whakaahua 3. Te chromatogram rama o te tauira i runga i te kariri C18AQ.
Hei mutunga, ka honoa te kariri kohiko SepaFlash C18AQ RP me te punaha chromatography flash SepaBean™Ka taea e te miihini te tuku otinga tere me te whai hua mo te purenga o nga tauira polar kaha, hydrophilic ranei.
He raupapa o nga kariri rama SepaFlash C18AQ RP me nga korero rereke mai i te Hangarau Santai (e whakaatuhia ana i te Ripanga 2).
| Tau Tuemi | Rahi Tīwae | Rere Rere (mL/mene) | Max.Pēhanga (psi/pa) |
| 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 |
Ripanga 2. SepaFlash C18AQ RP flash cartridges.Ko nga rauemi whakakakahu: He porowhita C18(AQ)-te tino pai, he silica here, 20 - 45 μm, 100 Å.
Mo etahi atu korero mo nga korero taipitopito o te miihini SepaBean™, me nga korero ota mo nga kaata rama raupapa SepaFlash, tirohia to maatau paetukutuku.
Te wa tuku: Oketopa-12-2018