Hongcheng Wang, Bo Xu
Ka Papahana R&D Center
Hoʻolauna
Wahi a nā polarities pili o ka pae kū a me ka pae kelepona, hiki ke hoʻokaʻawale ʻia ka chromatography wai i ka chromatography maʻamau (NPC) a me ka chromatography reversed phase (RPC).No RPC, ʻoi aku ka ikaika o ka polarity o ka pae kelepona ma mua o ka pae paʻa.Ua lilo ʻo RPC i mea hoʻohana nui loa i nā ʻano hoʻokaʻawale chromatography wai ma muli o kona kiʻekiʻe kiʻekiʻe, hoʻonā maikaʻi a me ka mīkini paʻa paʻa.No laila ua kūpono ʻo RPC no ka hoʻokaʻawale ʻana a me ka hoʻomaʻemaʻe ʻana i nā pūhui polar a i ʻole polar like ʻole, me nā alkaloids, carbohydrates, fatty acids, steroids, nucleic acids, amino acids, peptides, proteins, etc. ka silica gel matrix i hoʻopaʻa ʻia me nā hui hana like ʻole, me C18, C8, C4, phenyl, cyano, amino, etc.Ua manaʻo ʻia ʻoi aku ma mua o 80% o RPC i kēia manawa e hoʻohana nei i ka pae paʻa C18.No laila, ua lilo ke kolamu chromatography C18 i kolamu pono no kēlā me kēia keʻena hana.
ʻOiai hiki ke hoʻohana ʻia ke kolamu C18 ma kahi ākea loa o nā noi, akā naʻe, no kekahi mau laʻana he polar loa a hydrophilic nui paha, pilikia paha nā kolamu C18 maʻamau ke hoʻohana ʻia e hoʻomaʻemaʻe i ia mau laʻana.I loko o RPC, hiki ke kauoha ʻia nā mea hoʻoheheʻe elution maʻamau e like me ko lākou polarity: wai < methanol < acetonitrile < ethanol < tetrahydrofuran < isopropanol.No ka hōʻoia ʻana i ka mālama maikaʻi ʻana i ke kolamu no kēia mau laʻana (polar ikaika a i ʻole hydrophilic kiʻekiʻe), pono e hoʻohana ʻia ka ʻāpana kiʻekiʻe o ka ʻōnaehana wai e like me ke kaʻina kelepona.Eia nō naʻe, i ka hoʻohana ʻana i ka ʻōnaehana wai maʻemaʻe (me ka wai maʻemaʻe a i ʻole ka wai paʻakai maʻemaʻe) ma ke ʻano o ke kaʻa kaʻahele, ʻo ke kaula kalapona lōʻihi ma ka pae paʻa o ka kolamu C18 e pale aku i ka wai a hui pū kekahi me kekahi, e hopena i ka emi koke o ka. hiki ke hoʻopaʻa i ke kolamu a i ʻole paʻa ʻole.Kapa ʻia kēia ʻano he "hydrophobic phase collapse" (e like me ka mea i hōʻike ʻia ma ka ʻaoʻao hema o ke Kiʻi 1).ʻOiai hiki ke hoʻohuli ʻia kēia kūlana ke holoi ʻia ke kolamu me nā mea hoʻoheheʻe kūlohelohe e like me ka methanol a i ʻole acetonitrile, hiki nō ke hōʻeha i ke kolamu.No laila, pono e pale aku i kēia kūlana.
Kiʻi 1. ʻO ke kiʻikuhi kikoʻī o nā pae i hoʻopaʻa ʻia ma ka ʻili o ka silica gel ma ke kolamu C18 maʻamau (hema) a me ke kolamu C18AQ (akau).
No ka hoʻoponopono ʻana i nā pilikia i ʻōlelo ʻia ma luna, ua hana nā mea hana chromatographic packing material i nā hoʻomaikaʻi ʻenehana.ʻO kekahi o kēia mau hoʻomaikaʻi ke hana nei i kekahi mau hoʻololi ʻana ma luna o ka ʻili o ka silica matrix, e like me ka hoʻokomo ʻana o nā pūʻulu cyano hydrophilic (e like me ka hōʻike ʻana ma ka ʻaoʻao ʻākau o ke Kiʻi 1), e hoʻonui i ka hydrophilic i ka ʻili o ka silica gel.No laila hiki ke hoʻonui piha ʻia nā kaulahao C18 ma ka ʻili silica ma lalo o nā kūlana wai nui a hiki ke pale ʻia ka hāʻule ʻana o ka pae hydrophobic.Ua kapa ʻia kēia mau kolamu C18 i hoʻololi ʻia he kolamu wai C18, ʻo ia hoʻi nā kolamu C18AQ, i hoʻolālā ʻia no nā kūlana elution wai nui a hiki ke ʻae i ka ʻōnaehana aqueous 100%.Ua hoʻohana nui ʻia nā kolamu C18AQ i ka hoʻokaʻawale ʻana a me ka hoʻomaʻemaʻe ʻana i nā pūhui polar ikaika, me nā waikawa organik, peptides, nucleosides a me nā huaora hiki ke hoʻoheheʻe ʻia i ka wai.
ʻO ka desalting kekahi o nā noi maʻamau o nā kolamu C18AQ i ka hoʻomaʻemaʻe uila no nā laʻana, kahi e hoʻoneʻe ai i ka paʻakai a i ʻole nā mea pale i loko o ka hāpana hāpana e hoʻomaʻamaʻa i ka noi ʻana o ka hāpana i nā haʻawina ma hope.Ma kēia pou, ua hoʻohana ʻia ka Brilliant Blue FCF me ka polarity ikaika e like me ka laʻana a hoʻomaʻemaʻe ʻia ma ke kolamu C18AQ.Ua hoʻololi ʻia ka mea hoʻoheheʻe laʻana e ka mea hoʻoheheʻe organik mai ka hopena buffer, no laila e hoʻomaʻamaʻa i ka hoʻoheheʻe ʻana o ka rotary a me ka mālama ʻana i nā solvents a me ka manawa hana.Eia kekahi, ua hoʻomaikaʻi ʻia ka maʻemaʻe o ka hāpana ma ka wehe ʻana i kekahi mau haumia i loko o ka hāpana.
Māhele hoʻokolohua
Kiʻi 2. ʻO ke ʻano kemika o ka hāpana.
Ua hoʻohana ʻia ka Brilliant Blue FCF ma ke ʻano he laʻana ma kēia pou.ʻO ka maʻemaʻe o ka hāpana maka he 86% a ua hōʻike ʻia ke ʻano kemika o ka hāpana ma ke Kiʻi 2. No ka hoʻomākaukau ʻana i ka hāpana hāpana, ua hoʻoheheʻe ʻia ka 300 mg pauda paʻa paʻa o Brilliant Blue FCF i 1 M NaH2PO4 buffer solution a haʻalulu maikaʻi ʻia e lilo. he hopena maopopo loa.A laila, hoʻokomo ʻia ka hāpana hāpana i loko o ke kolamu uila e kahi injector.Hōʻike ʻia ka hoʻonohonoho hoʻokolohua o ka hoʻomaʻemaʻe uila ma ka Papa 1.
| Mea hana | Mīkini SepaBean™2 | |||
| Nā pahu pahu | 12 g SepaFlash C18 RP pahu pahu uila (spherical silica, 20 - 45 μm, 100 Å, Helu kauoha: SW-5222-012-SP) | 12 g SepaFlash C18AQ RP pahu pahu uila (spherical silica, 20 - 45 μm, 100 Å, Helu kauoha:SW-5222-012-SP(AQ)） | ||
| Loʻihi nalu | 254 nm | |||
| Māhele kelepona | Solvent A: Wai Mea hoʻoheheʻe B: Methanol | |||
| Kahe kahe | 30 mL/min | |||
| Hoʻouka laʻana | 300 mg (Brilliant Blue FCF me ka maʻemaʻe o 86%) | |||
| Gradient | Manawa (CV) | Mea hoʻoheheʻe B (%) | Manawa (CV) | Mea hoʻoheheʻe 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 | |
Nā hualoaʻa a me ke kūkākūkā
Ua hoʻohana ʻia kahi pahu pahu uila SepaFlash C18AQ RP no ka laʻana desalting a me ka hoʻomaʻemaʻe.Ua hoʻohana ʻia ka ʻanuʻu ʻanuʻu kahi i hoʻohana ʻia ai ka wai maʻemaʻe e like me ke kaʻina neʻe i ka hoʻomaka ʻana o ka elution a holo no 10 kolamu puke (CV).E like me ka mea i hōʻike ʻia ma ke Kiʻi 3, i ka wā e hoʻohana ai i ka wai maʻemaʻe e like me ka pae paʻa, ua paʻa loa ka hāpana ma ka pahu uila.Ma hope aʻe, ua hoʻonui pololei ʻia ka methanol i ka pae kelepona i 100% a mālama ʻia ka gradient no 7.5 CV.Ua hoʻokuʻu ʻia ka laʻana mai 11.5 a i 13.5 CV.I nā hakina i hōʻiliʻili ʻia, ua hoʻololi ʻia ka hāpana hāpana mai NaH2PO4 buffer solution i ka methanol.I ka hoʻohālikelike ʻana me ka wai wai nui, ʻoi aku ka maʻalahi o ka wehe ʻana i ka methanol e ka rotary evaporation i ka pae hope, e hoʻomaʻamaʻa i kēia noiʻi.
Kiʻi 3. ʻO ka chromatogram flash o ka hāpana ma kahi pahu pahu C18AQ.
No ka hoʻohālikelike ʻana i ka hana paʻa o ka pahu pahu C18AQ a me ka pahu pahu C18 maʻamau no nā laʻana o ka polarity ikaika, ua hana ʻia ka hoʻohālikelike hoʻohālikelike.Ua hoʻohana ʻia kahi pahu pahu uila SepaFlash C18 RP a ua hōʻike ʻia ka chromatogram flash no ka hāpana ma ke Kiʻi 4. No nā pahu pahu C18 maʻamau, ʻo ka nui o ka lākiō wai wai i ʻae ʻia ma kahi o 90%.No laila ua hoʻonoho ʻia ka gradient hoʻomaka ma 10% methanol i 90% wai.E like me ka mea i hōʻike ʻia ma ke Kiʻi 4, ma muli o ka hāʻule ʻana o ka pae hydrophobic o nā kaulahao C18 i hoʻokumu ʻia e ka ratio wai kiʻekiʻe, ua paʻa ʻole ka hāpana ma ka pahu pahu C18 maʻamau a ua hoʻokaʻawale pololei ʻia e ka pae kelepona.ʻO ka hopena, ʻaʻole hiki ke hoʻopau ʻia ka hana o ka hoʻoheheʻe ʻana a i ʻole ka hoʻomaʻemaʻe ʻana.
Kiʻi 4. ʻO ka chromatogram flash o ka hāpana ma kahi pahu pahu C18 maʻamau.
Ke hoʻohālikelike nei me ka linear gradient, ʻo ka hoʻohana ʻana i ka gradient step he mau mea maikaʻi aʻe:
1. Hoʻemi ʻia ka hoʻohana ʻana i ka solvent a me ka manawa holo no ka hoʻomaʻemaʻe laʻana.
2. Huli 'ia ka hua i ho'opa'a 'ia ma ka piko 'oi, e ho'emi ana i ka nui o nā hakina i ohi 'ia a pēlā e ho'oma'ama'a ai i ka ho'ohehe'e 'ana a'e a me ka mālama 'ana i ka manawa.
3. ʻO ka huahana i hōʻiliʻili ʻia i loko o ka methanol i maʻalahi ke hoʻoheheʻe ʻia, no laila ua hoʻemi ʻia ka manawa maloʻo.
I ka hopena, no ka hoʻomaʻemaʻe ʻana i ka laʻana i polar ikaika a i ʻole hydrophilic nui, SepaFlash C18AQ RP pahu pahu uila e hui pū ana me ka ʻōnaehana chromatography flash preparative SepaBean™ Machine hiki ke hāʻawi i kahi hopena wikiwiki a maikaʻi.
E pili ana i nā pahu pahu uila SepaFlash Bonded Series C18 RP
Aia kekahi pūʻulu o nā pahu uila SepaFlash C18AQ RP me nā kikoʻī like ʻole mai Santai Technology (e like me ka hōʻike ʻana ma ka Papa 2).
| Helu 'ikamu | Ka nui o ke kolamu | Kahe Kahe (mL/min) | Max.Pressure (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 |
Papa 2. SepaFlash C18AQ RP pahu pahu flash.
Mea hoʻopili: High-efficiency spherical C18(AQ)-hoʻopaʻa silica, 20 - 45 μm, 100 Å.
logy (e like me ka hoike ma ka Papa 2).
Ka manawa hoʻouna: ʻAukake-27-2018