Hongcheng Wang, Bo Xu
Ntchito R&D Center
Mawu Oyamba
Malinga ndi ma polarities a stationary phase and mobile phase, liquid chromatography ikhoza kugawidwa mu normal phase chromatography (NPC) ndi reversed phase chromatography (RPC).Kwa RPC, polarity ya gawo la mafoni ndi yamphamvu kuposa gawo loyima.RPC yakhala yomwe imagwiritsidwa ntchito kwambiri pamachitidwe olekanitsa amadzimadzi a chromatography chifukwa chakuchita bwino kwambiri, kusamvana bwino komanso kusungitsa bwino.Choncho RPC ndi oyenera kulekana ndi kuyeretsa zosiyanasiyana polar kapena sanali polar mankhwala, kuphatikizapo alkaloids, chakudya, mafuta zidulo, steroids, nucleic zidulo, amino zidulo, peptides, mapuloteni, etc. silika gel matrix yomwe imagwirizanitsidwa ndi magulu osiyanasiyana ogwira ntchito, kuphatikizapo C18, C8, C4, phenyl, cyano, amino, ndi zina zotero. Pakati pa magulu ogwira ntchito ogwirizanawa, omwe amagwiritsidwa ntchito kwambiri ndi C18.Akuti oposa 80% a RPC tsopano akugwiritsa ntchito gawo la C18.Chifukwa chake gawo la C18 chromatography lakhala gawo loyenera kukhala nalo pa labotale iliyonse.
Ngakhale ndime ya C18 ingagwiritsidwe ntchito pazinthu zambiri, komabe, kwa zitsanzo zina zomwe zimakhala za polar kapena za hydrophilic kwambiri, mizati ya C18 yokhazikika ikhoza kukhala ndi mavuto pamene ikugwiritsidwa ntchito kuyeretsa zitsanzo zoterezi.Mu RPC, zosungunulira zomwe zimagwiritsidwa ntchito kawirikawiri zimatha kulamulidwa malinga ndi polarity yawo: madzi
Chithunzi 1. Chithunzi chojambula cha magawo omangika pamwamba pa silika gel osakaniza pa C18 column (kumanzere) ndi C18AQ column (kumanja).
Kuti athane ndi mavuto omwe tawatchulawa, opanga zida zonyamula ma chromatographic apanga luso laukadaulo.Chimodzi mwazosinthazi ndikupanga zosintha zina pamwamba pa silika matrix, monga kuyambitsa magulu a hydrophilic cyano (monga momwe tawonetsera m'gawo loyenera la Chithunzi 1), kuti pamwamba pa silika gel ikhale ya hydrophilic.Choncho maunyolo a C18 pamtunda wa silika akhoza kufalikira mokwanira pansi pa mikhalidwe yamadzi kwambiri ndipo kugwa kwa gawo la hydrophobic kungapewedwe.Mizati ya C18 yosinthidwayi imatchedwa mizati yamadzi ya C18, yomwe ndi C18AQ mizati, yomwe imapangidwira mikhalidwe yamadzimadzi kwambiri ndipo imatha kulekerera 100% yamadzimadzi.Mizati ya C18AQ yakhala ikugwiritsidwa ntchito kwambiri pakulekanitsa ndi kuyeretsa mankhwala amphamvu a polar, kuphatikizapo organic acid, peptides, nucleosides ndi mavitamini osungunuka m'madzi.
Desalting ndi imodzi mwazinthu zomwe zimagwiritsidwa ntchito pamizere ya C18AQ poyeretsa kung'anima kwa zitsanzo, zomwe zimachotsa mchere kapena zida zosungunulira muzosungunulira zachitsanzo kuti zithandizire kugwiritsa ntchito zitsanzo m'maphunziro otsatirawa.Mu positi iyi, Brilliant Blue FCF yokhala ndi polarity yolimba idagwiritsidwa ntchito ngati chitsanzo ndikuyeretsedwa pagawo la C18AQ.Chosungunulira chachitsanzocho chinasinthidwa ndi organic solvent kuchokera ku buffer solution, motero kumathandizira kusungunuka kozungulira kotsatira komanso kupulumutsa zosungunulira ndi nthawi yogwiritsira ntchito.Komanso, chiyero cha chitsanzocho chinawongoleredwa pochotsa zonyansa zina mu chitsanzocho.
Gawo Loyesera
Chithunzi 2. Mapangidwe a mankhwala a chitsanzo.
Brilliant Blue FCF idagwiritsidwa ntchito ngati chitsanzo mu positi iyi.Chiyero cha chitsanzo chaiwisi chinali 86% ndipo mawonekedwe a mankhwala a chitsanzo akuwonetsedwa mu Chithunzi 2. Kukonzekera yankho lachitsanzo, 300 mg powdery crude solid of Brilliant Blue FCF inasungunuka mu 1 M NaH2PO4 buffer solution ndipo inagwedezeka bwino kuti ikhale yankho lomveka bwino.Yankho lachitsanzo lidalowetsedwa mugawo la flash ndi jekeseni.Kukonzekera koyesera kwa kuyeretsedwa kwa flash kwalembedwa mu Table 1.
| Chida | Makina a SepaBean™2 | |||
| Makatiriji | 12 g SepaFlash C18 RP flash cartridge (silica yozungulira, 20 - 45 μm, 100 Å, Nambala ya Order: SW-5222-012-SP) | 12 g SepaFlash C18AQ RP flash cartridge (silica yozungulira, 20 - 45 μm, 100 Å, Nambala ya Order: SW-5222-012-SP(AQ))） | ||
| Wavelength | 254 nm | |||
| Gawo la mafoni | Zosungunulira A: Madzi Zosungunulira B: Methanol | |||
| Mtengo woyenda | 30 ml / mphindi | |||
| Kutsegula kwachitsanzo | 300 mg (Brilliant Blue FCF ndi chiyero cha 86%) | |||
| Gradient | Nthawi (CV) | Zosungunulira B (%) | Nthawi (CV) | Zosungunulira 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 | |
Zotsatira ndi zokambirana
Katiriji ya SepaFlash C18AQ RP idagwiritsidwa ntchito pochotsa mchere komanso kuyeretsa.Step gradient idagwiritsidwa ntchito pomwe madzi oyera adagwiritsidwa ntchito ngati gawo loyambira poyambira ndikuyendetsa ma voliyumu 10 (CV).Monga momwe tawonetsera pa Chithunzi 3, pogwiritsira ntchito madzi oyera monga gawo la mafoni, chitsanzocho chinasungidwa kwathunthu pa cartridge ya flash.Kenako, methanol mu gawo la mafoni adawonjezedwa mwachindunji mpaka 100% ndipo gradient idasungidwa 7.5 CV.Chitsanzocho chinachotsedwa pa 11.5 mpaka 13.5 CV.M'zigawo zomwe zasonkhanitsidwa, yankho lachitsanzo linasinthidwa kuchoka ku NaH2PO4 buffer solution kupita ku methanol.Poyerekeza ndi njira yamadzimadzi kwambiri, methanol inali yosavuta kuchotsedwa ndi mpweya wozungulira mu sitepe yotsatira, yomwe imathandizira kafukufuku wotsatira.
Chithunzi 3. Chromatogram yowala ya chitsanzo pa cartridge ya C18AQ.
Poyerekeza machitidwe osungira katiriji a C18AQ ndi cartridge ya C18 yanthawi zonse ya zitsanzo za polarity amphamvu, kuyesa kofananitsa kofananira kunachitika.SepaFlash C18 RP flash cartridge inagwiritsidwa ntchito ndipo flash chromatogram ya chitsanzo inawonetsedwa mu Chithunzi 4. Kwa ma cartridges okhazikika a C18, chiwerengero chapamwamba kwambiri chololedwa ndi gawo la madzi ndi pafupifupi 90%.Chifukwa chake gradient yoyambira idayikidwa pa 10% methanol m'madzi 90%.Monga momwe tawonetsera pa Chithunzi 4, chifukwa cha kugwa kwa hydrophobic gawo la maunyolo a C18 chifukwa cha kuchuluka kwa madzi amadzimadzi, chitsanzocho sichinasungidwe pa cartridge ya C18 yokhazikika ndipo chinatulutsidwa mwachindunji ndi gawo la mafoni.Zotsatira zake, ntchito ya zitsanzo za desalting kapena kuyeretsa sikungatheke.
Chithunzi 4. Chromatogram yowala ya chitsanzo pa cartridge ya C18 yokhazikika.
Poyerekeza ndi mzere wa mzere, kugwiritsa ntchito step gradient kuli ndi zabwino izi:
1. Kugwiritsa ntchito zosungunulira ndi nthawi yoyendetsera kuyeretsa kwachitsanzo kumachepetsedwa.
2. The chandamale mankhwala elutes mu lakuthwa pachimake, amene amachepetsa voliyumu wa anasonkhanitsa tizigawo ting'onoting'ono motero facilitates zotsatirazi rotary evaporation komanso kupulumutsa nthawi.
3. Zomwe zasonkhanitsidwa zili mu methanol zomwe zimakhala zosavuta kusungunuka, motero nthawi yowuma imachepetsedwa.
Pomaliza, pofuna kuyeretsa chitsanzo chomwe chili ndi polar kwambiri kapena chopanda madzi kwambiri, makatiriji a SepaFlash C18AQ RP ophatikizika ndi makina okonzekera a chromatography a SepaBean™ Machine atha kupereka yankho lachangu komanso lothandiza.
Za SepaFlash Bonded Series C18 RP flash cartridges
Pali mndandanda wa makatiriji a SepaFlash C18AQ RP omwe ali ndi mawonekedwe osiyanasiyana ochokera ku Santai Technology (monga momwe tawonetsera mu Table 2).
| Nambala Yachinthu | Kukula kwa Mzere | Mtengo Woyenda (mL/mphindi) | Max.Pressure (psi/bar) |
| SW-5222-004-SP(AQ) | 5.4g pa | 5-15 | 400/27.5 |
| SW-5222-012-SP(AQ) | 20 g pa | 10-25 | 400/27.5 |
| SW-5222-025-SP(AQ) | 33 g pa | 10-25 | 400/27.5 |
| SW-5222-040-SP(AQ) | 48g pa | 15-30 | 400/27.5 |
| SW-5222-080-SP(AQ) | 105 g pa | 25-50 | 350/24.0 |
| SW-5222-120-SP(AQ) | 155g pa | 30-60 | 300/20.7 |
| SW-5222-220-SP(AQ) | 300 g pa | 40-80 | 300/20.7 |
| SW-5222-330-SP(AQ) | 420 g pa | 40-80 | 250/17.2 |
Table 2. SepaFlash C18AQ RP flash cartridges.
Zida zonyamula: Silika yolimba kwambiri yozungulira C18(AQ), 20 - 45 μm, 100 Å.
logi (monga momwe tawonetsera mu Table 2).
Nthawi yotumiza: Aug-27-2018