Hongcheng Wang, Bo Xu
Taupānga R&D Center
Kupu Whakataki
E ai ki nga polarities o te wahanga tu me te wahanga pūkoro, ka taea te wehea te chromatography wai ki te chromatography wahanga noa (NPC) me te chromatography wahanga whakamuri (RPC).Mo te RPC, ko te polarity o te wahanga waea he kaha ake i tera o te waahanga tuuturu.Ko te RPC te mea tino whakamahia i roto i nga momo wehenga chromatography wai na te kaha o te kaha, te pai o te whakatau me te maamaa o te pupuri.Na reira he pai te RPC mo te wehewehe me te purenga o nga momo momo polar, kore-polar ranei, tae atu ki nga pāhare, warowaihā, waikawa ngako, steroids, waikawa nucleic, waikawa amino, peptides, pūmua, me etahi atu. te matrix gel silica e hono ana ki nga momo roopu mahi, tae atu ki te C18, C8, C4, phenyl, cyano, amino, me etahi atu.E kiia ana, neke atu i te 80% o te RPC kei te whakamahi i te wahanga here C18.No reira kua noho te pou chromatography C18 hei pou mo te ao katoa mo ia taiwhanga.
Ahakoa ka taea te whakamahi i te pou C18 i roto i te whānuitanga o nga momo tono, engari, mo etahi tauira he tino polar, he tino hydrophilic ranei, ka raru pea nga pou C18 i te wa e whakamahia ana hei pure i aua tauira.I roto i te RPC, ka taea te tono nga whakarewa elution e whakamahia nuitia ana i runga i to ratou polarity: wai < methanol < acetonitrile < ethanol < tetrahydrofuran < isopropanol.Hei whakapumau i te pai o te pupuri i runga i te pou mo enei tauira (polar kaha, tino hydrophilic ranei), he nui te waahanga o te punaha wai hei whakamahi hei waahanga pūkoro.Heoi, i te wa e whakamahi ana i te punaha wai parakore (tae atu ki te wai parakore, te wairewa tote parakore ranei) hei waahanga pūkoro, ko te mekameka waro roa i runga i te wahanga tuutuu o te pou C18 ka karo i te wai me te uru ki a raua ano, ka hua ake te heke o te waa. kaha pupuri o te pou, ara kore pupuri ranei.Ko tenei ahuatanga e kiia ana ko te "tiango wahanga hydrophobic" (e whakaatuhia ana i te taha maui o te Whakaahua 1).Ahakoa ka taea te huri i tenei ahuatanga i te wa e horoi ana te pou ki nga whakarewa pararopi penei i te methanol me te acetonitrile, ka raru tonu te pou.No reira, he mea tika kia kore e puta tenei ahuatanga.
Whakaatu 1. Te hoahoa hoahoa o nga wahanga here i runga i te mata o te silica gel i te pou C18 (taha maui) me te pou C18AQ (matau).
Hei whakatika i nga raru kua whakahuahia ake nei, kua mahia e nga kaihanga taputapu whakakakahu chromatographic nga whakapainga hangarau.Ko tetahi o enei whakapainga kei te hanga i etahi whakarereketanga i runga i te mata o te matrix silica, penei i te whakaurunga o nga roopu cyano hydrophilic (e whakaatuhia ana i te taha matau o te Whakaahua 1), kia nui ake te hydrophilic o te mata o te silica gel.Koia nga mekameka C18 i runga i te mata silica ka taea te whakaroa i raro i nga tikanga tino wai, ka taea te karo i te tiango o te wahanga hydrophobic.Ko enei pou C18 kua whakarereketia e kiia ana ko nga pou C18 wai, ara ko nga pou C18AQ, he mea hangaia mo nga ahuatanga o te wai rere, ka taea te aro ki te 100% te punaha wai.Ko nga pou C18AQ kua whakamahia nuitia i roto i te wehenga me te purenga o nga puhui polar kaha, tae atu ki nga waikawa waro, peptides, nucleosides me nga huaora wairewa-wai.
Ko te whakakorenga tetahi o nga tono angamaheni o nga pou C18AQ i roto i te purenga uira mo nga tauira, e tango ana i te tote, i nga waahanga parepare ranei i roto i te tauira whakarewa hei whakahaere i te whakamahinga o te tauira i roto i nga rangahau o muri mai.I tenei pou, ko te Brilliant Blue FCF me te polarity kaha i whakamahia hei tauira me te purea i runga i te pou C18AQ.I whakakapihia te tauira whakarewa ki te whakarewa pararopi mai i te otinga buffer, na reira ka pai te whakaetonga hurihuri e whai ake nei me te whakaora i nga whakarewa me te wa mahi.I tua atu, i pai ake te ma o te tauira ma te tango i etahi poke i roto i te tauira.
Wāhanga Whakamātau
Whakaatu 2. Te hanganga matū o te tauira.
I whakamahia te Brilliant Blue FCF hei tauira i tenei pou.Ko te ma o te tauira mata ko te 86% me te hanganga matū o te tauira i whakaatuhia i te Whakaahua 2. Hei whakarite i te tauira otinga, 300 mg paura masivesivá totoka o Brilliant Blue FCF i rewa ki te 1 M NaH2PO4 otinga parepare me te wiri pai kia riro. he otinga tino marama.Ka werohia te tauira otinga ki roto i te pou hiko e te injector.Ko te tatūnga whakamātautau o te purenga uira kua whakarārangihia ki te Ripanga 1.
| Taputapu | Miihini SepaBean™2 | |||
| Kaata | 12 g SepaFlash C18 RP kariri kohiko (silica porowhita, 20 - 45 μm, 100 Å, Tau ota: 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 | |||
| Waahanga pūkoro | Wairewa A: Wai Wairewa B:Mewaro | |||
| Rere rere | 30 mL/min | |||
| Uta tauira | 300 mg (Brilliant Blue FCF me te ma o 86%) | |||
| Rōnaki | Wā (CV) | Wairewa B (%) | Wā (CV) | Wairewa 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 | |
Nga Hua me te Matapaki
I whakamahia he kariri kohiko SepaFlash C18AQ RP mo te tauira whakaheke me te purenga.I whakamahia te rōnaki hikoi i whakamahia ai te wai parakore hei wahanga pūkoro i te tiimatanga o te whakamaarama me te rere mo te 10 nga pukapuka pou (CV).Ka rite ki te Whakaaturanga 3, i te wa e whakamahi ana i te wai parakore hei waahanga pūkoro, ka mau tonu te tauira ki runga i te kariri uira.I muri mai, ko te methanol i roto i te waahanga pūkoro i piki tika ki te 100% ka mau tonu te rōnaki mo te 7.5 CV.I whakahekehia te tauira mai i te 11.5 ki te 13.5 CV.I roto i nga hautau i kohia, i whakakapihia te tauira otinga mai i te otinga parepare NaH2PO4 ki te methanol.Ki te whakatairite ki te wairewa tino wai, he ngawari ake te tango i te methanol ma te whakaetonga hurihuri i te taahiraa o muri mai, e whakahaere ana i nga rangahau e whai ake nei.
Whakaahua 3. Te chromatogram rama o te tauira i runga i te kariri C18AQ.
Hei whakataurite i te whanonga pupuri o te kariri C18AQ me te kariri C18 auau mo nga tauira polarity kaha, i mahia he whakamatautau whakatairite whakarara.I whakamahia he kariri kohiko SepaFlash C18 RP me te chromatogram rama mo te tauira i whakaatuhia i te Whakaahua 4. Mo nga kaata C18 auau, ko te 90% te tauwehenga wahanga wai e whakaaetia ana.Na reira i whakaritea te rōnaki tīmatanga ki te 10% methanol i te 90% wai.E whakaatu ana i te Whakaahua 4, 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, a, i tukuna tikatia e te wahanga pūkoro.Ko te mutunga mai, kare e taea te mahi o te tauira whakaheke tote, purenga ranei.
Whakaatu 4. Te chromatogram rama o te tauira i runga i te kariri C18.
Ki te whakataurite ki te rōnaki rārangi, ko te whakamahi i te rōnaki takahanga e whai ake nei nga painga:
1. Ka whakaitihia te whakamahi whakarewa me te wa oma mo te purenga tauira.
2. Ka heke te hua i whäia ki te tihi koi, ka whakaitihia te rärangi o ngä hautau kua kohia, ä, ka pai ake te whakaetonga hurihuri e whai ake nei me te whakaora i te wa.
3. Ko te hua kua kohia kei roto i te methanol he ngawari ki te whakaeto, na reira ka iti te wa whakamaroke.
Hei whakamutunga, mo te purenga o te tauira he tino polar, he tino hydrophilic ranei, SepaFlash C18AQ RP flash cartridges e hono ana ki te punaha chromatography flash preparative SepaBean™ Machine ka taea e te Miihini tere me te pai te tuku.
Mo te SepaFlash Bonded Series C18 RP flash cartridges
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.
logy (penei i te Ripanga 2).
Te wa tuku: Akuhata-27-2018