Hongcheng Wang, Bo Xu
Daim ntawv thov R&D Center
Taw qhia
Raws li cov txheeb ze polarities ntawm qhov chaw nres tsheb thiab theem mobile, kua chromatography tuaj yeem muab faib ua theem ib txwm muaj chromatography (NPC) thiab thim rov qab theem chromatography (RPC).Rau RPC, qhov polarity ntawm lub xov tooj ntawm tes muaj zog dua li ntawm theem nyob ruaj khov.RPC tau dhau los ua cov khoom siv dav tshaj plaws hauv cov kua chromatography sib cais hom vim nws cov kev ua haujlwm siab, kev daws teeb meem zoo thiab kev ceev ceev ceev.Yog li ntawd RPC yog tsim rau kev sib cais thiab purification ntawm ntau yam polar los yog tsis-polar tebchaw, xws li alkaloids, carbohydrates, fatty acids, steroids, nucleic acids, amino acids, peptides, proteins, thiab lwm yam. Nyob rau hauv RPC, feem ntau siv nyob rau hauv cov theem nyob ruaj khov yog silica gel matrix uas yog sib koom nrog ntau pawg ua haujlwm, suav nrog C18, C8, C4, phenyl, cyano, amino, thiab lwm yam.Nws tau kwv yees tias ntau dua 80% ntawm RPC tam sim no siv C18 sib koom ua ke.Yog li C18 chromatography kem tau dhau los ua ib qho yuav tsum muaj universal kem rau txhua chav kuaj.
Txawm hais tias C18 kem tuaj yeem siv tau ntau yam ntawm cov ntawv thov, txawm li cas los xij, rau qee cov qauv uas yog polar heev lossis hydrophilic, cov kab C18 tsis tu ncua yuav muaj teeb meem thaum siv los ntxuav cov qauv no.Hauv RPC, feem ntau siv cov kuab tshuaj elution tuaj yeem raug txiav txim raws li lawv cov polarity: dej < methanol < acetonitrile < ethanol < tetrahydrofuran < isopropanol .Txhawm rau kom muaj kev tuav pov hwm zoo nyob rau hauv kem rau cov qauv no (muaj zog polar lossis hydrophilic), qhov feem pua ntawm cov dej hauv dej yog qhov tsim nyog yuav tsum tau siv los ua lub sijhawm txawb.Txawm li cas los xij, thaum siv cov dej ntshiab huv (xws li cov dej ntshiab los yog cov ntsev ntsev ntshiab) raws li lub sij hawm txawb, cov pa roj carbon monoxide ntev nyob rau theem nyob ruaj ruaj ntawm C18 kem nyhav kom tsis txhob muaj dej thiab sib tov sib xyaw, ua rau muaj kev txo qis hauv lub sijhawm. tuav lub peev xwm ntawm kab los yog txawm tsis muaj tuav.Qhov tshwm sim no hu ua "hydrophobic theem vau" (raws li qhia nyob rau sab laug ntawm daim duab 1).Txawm hais tias qhov xwm txheej no yog thim rov qab thaum lub kem ntxuav nrog cov kuab tshuaj organic xws li methanol lossis acetonitrile, nws tseem tuaj yeem ua rau lub cev puas tsuaj.Yog li ntawd, nws yog ib qho tsim nyog kom tsis txhob muaj qhov xwm txheej no tshwm sim.
Daim duab 1. Daim duab schematic ntawm cov theem sib koom ua ke ntawm qhov chaw ntawm silica gel nyob rau hauv ib txwm C18 kem (sab laug) thiab C18AQ kem (txoj cai).
Txhawm rau daws cov teeb meem saum toj no, cov khoom siv chromatographic packing manufacturers tau txhim kho kev txhim kho.Ib qho ntawm cov kev txhim kho no yog ua qee qhov kev hloov kho ntawm qhov chaw ntawm silica matrix, xws li kev taw qhia ntawm hydrophilic cyano pawg (raws li qhia nyob rau hauv txoj cai ntawm daim duab 1), ua rau lub nto ntawm silica gel ntau hydrophilic.Yog li C18 chains ntawm silica nto tuaj yeem txuas ntxiv nyob rau hauv cov dej muaj dej ntau thiab cov theem hydrophobic yuav raug zam.Cov kab C18 hloov kho no hu ua aqueous C18 kab, uas yog C18AQ kab, uas yog tsim los rau cov dej elution heev thiab tuaj yeem zam tau 100% aqueous system.C18AQ txhua kab tau siv dav hauv kev sib cais thiab kev ua kom huv ntawm cov polar muaj zog, nrog rau cov organic acids, peptides, nucleosides thiab dej-soluble vitamins.
Desalting yog ib qho ntawm cov kev siv ntawm C18AQ txhua kab hauv flash purification rau cov qauv, uas tshem tawm cov ntsev los yog cov khoom tsis nyob hauv cov qauv hnyav los pab txhawb kev siv cov qauv hauv cov kev tshawb fawb tom ntej.Hauv cov ntawv tshaj tawm no, Brilliant Blue FCF nrog cov polarity muaj zog tau siv los ua cov qauv thiab ua kom huv ntawm C18AQ kem.Cov qauv hnyav tau hloov los ntawm cov kuab tshuaj organic los ntawm cov tshuaj tsis haum, yog li ua kom yooj yim rau cov evaporation nram qab no nrog rau kev txuag cov kuab tshuaj thiab lub sijhawm ua haujlwm.Tsis tas li ntawd, qhov purity ntawm tus qauv tau txhim kho los ntawm kev tshem tawm qee qhov impurities hauv cov qauv.
Ntu kev sim
Daim duab 2. Cov qauv tshuaj ntawm cov qauv.
Lub Brilliant Blue FCF tau siv los ua tus qauv hauv tsab ntawv no.Lub purity ntawm cov qauv raw yog 86% thiab cov qauv tshuaj ntawm cov qauv tau pom nyob rau hauv daim duab 2. Txhawm rau npaj cov qauv kev daws teeb meem, 300 mg hmoov crude khoom ntawm Brilliant Blue FCF yog yaj nyob rau hauv 1 M NaH2PO4 buffer tov thiab co zoo los ua. ib tug meej meej tov.Cov qauv kev daws teeb meem yog tom qab txhaj rau hauv kem flash los ntawm ib qho injector.Kev sim teeb tsa ntawm lub flash purification yog teev nyob rau hauv Table 1.
| Ntsuas | SepaBean ™ tshuab2 | |||
| Cartridges | 12 g SepaFlash C18 RP flash cartridge (spherical silica, 20 - 45 μm, 100 Å, Order naj npawb: SW-5222-012-SP) | 12 g SepaFlash C18AQ RP flash cartridge (spherical silica, 20 - 45 μm, 100 Å, Order naj npawb: SW-5222-012-SP(AQ))) | ||
| Lub wavelength | 254nm ua | |||
| Mobile theem | Solvent A: Dej Cov kuab tshuaj B: Methanol | |||
| Tus nqi ntws | 30 ml / min | |||
| Sample loading | 300 mg (Brilliant Blue FCF nrog purity ntawm 86%) | |||
| Gradient | Lub sijhawm (CV) | Cov kuab tshuaj B (%) | Lub sijhawm (CV) | Cov kuab tshuaj B (%) |
| 0 | 10 | 0 | 0 | |
| 10 | 10 | 10 | 0 | |
| 10.1 | 100 | 10.1 | 100 | |
| 17.5 Nws | 100 | 17.5 Nws | 100 | |
| 17.6 ib | 10 | 17.6 ib | 0 | |
| 22.6 ib | 10 | 22.6 ib | 0 | |
Cov txiaj ntsig thiab kev sib tham
Ib tug SepaFlash C18AQ RP flash cartridge tau siv rau cov qauv desalting thiab purification.Cov kauj ruam gradient tau siv nyob rau hauv uas cov dej ntshiab tau siv los ua lub sijhawm txawb thaum pib elution thiab khiav rau 10 kem ntim (CV).Raws li pom nyob rau hauv daim duab 3, thaum siv cov dej ntshiab raws li lub sij hawm mobile, cov qauv tau khaws cia tag nrho ntawm lub flash cartridge.Tom ntej no, cov methanol nyob rau hauv lub mobile theem tau ncaj qha nce mus rau 100% thiab cov gradient tau khaws cia rau 7.5 CV.Cov qauv tau raug tshem tawm ntawm 11.5 txog 13.5 CV.Hauv cov seem uas tau sau tseg, cov qauv tshuaj tau hloov los ntawm NaH2PO4 tsis muaj tshuaj rau methanol.Muab piv nrog cov tshuaj aqueous heev, methanol tau yooj yim dua kom tshem tawm los ntawm rotary evaporation hauv cov kauj ruam tom ntej, uas ua rau kev tshawb fawb hauv qab no.
Daim duab 3. Lub teeb chromatogram ntawm tus qauv ntawm C18AQ cartridge.
Txhawm rau sib piv tus cwj pwm tuav ntawm C18AQ cartridge thiab C18 cartridge tsis tu ncua rau cov qauv ntawm cov polarity muaj zog, qhov kev sib piv sib piv tau ua tiav.Ib tug SepaFlash C18 RP flash cartridge tau siv thiab flash chromatogram rau cov qauv tau pom nyob rau hauv daim duab 4. Rau li niaj zaus C18 cartridges, qhov siab tshaj aqueous theem piv yog hais txog 90%.Yog li qhov pib gradient tau teem rau ntawm 10% methanol hauv 90% dej.Raws li pom nyob rau hauv daim duab 4, vim lub hydrophobic theem vau ntawm lub C18 chains tshwm sim los ntawm high aqueous ratio, tus qauv twb NW nyuam qhuav khaws cia nyob rau hauv ib txwm C18 cartridge thiab tau ncaj qha eluted tawm los ntawm lub mobile theem.Yog li ntawd, kev ua haujlwm ntawm cov qauv desalting lossis purification tsis tuaj yeem ua tiav.
Daim duab 4. Lub teeb chromatogram ntawm cov qauv ntawm lub cartridge C18 tsis tu ncua.
Sib piv nrog cov linear gradient, kev siv cov kauj ruam gradient muaj qhov zoo hauv qab no:
1. Kev siv cov kuab tshuaj thiab lub sijhawm ua haujlwm rau cov qauv purification raug txo.
2. Lub hom phiaj cov khoom elutes nyob rau hauv ib tug ntse ncov, uas yuav txo tau lub ntim ntawm sau fractions thiab yog li pab txhawb cov nram qab no rotary evaporation thiab txuag lub sij hawm.
3. Cov khoom ntim khoom yog nyob rau hauv methanol uas yooj yim rau evaporated, yog li lub sij hawm ziab yog txo.
Hauv kev xaus, rau kev ua kom huv ntawm cov qauv uas muaj zog polar los yog hydrophilic heev, SepaFlash C18AQ RP flash cartridges ua ke nrog kev npaj flash chromatography system SepaBean ™ Tshuab tuaj yeem muab kev daws teeb meem sai thiab muaj txiaj ntsig.
Hais txog SepaFlash Bonded Series C18 RP flash cartridges
Muaj ib tug series ntawm SepaFlash C18AQ RP flash cartridges nrog txawv specifications ntawm Santai Technology (raws li qhia nyob rau hauv Table 2).
| Yam khoom naj npawb | Kem Loj | Tus nqi ntws (ml / min) | Max. Siab (psi/bar) |
| SW-5222-004-SP(AQ) | 5,4g e | 5-15 | 400/27.5 |
| SW-5222-012-SP(AQ) | 20 g ib | 10-25 | 400/27.5 |
| SW-5222-025-SP(AQ) | 33g ua | 10-25 | 400/27.5 |
| SW-5222-040-SP (AQ) | 48g ua | 15-30 | 400/27.5 |
| SW-5222-080-SP(AQ) | 10 5g | 25-50 | 350/24.0 Nws |
| SW-5222-120-SP(AQ) | 15 5g | 30-60 | 300/20.7 |
| SW-5222-220-SP(AQ) | 300 g | 40-80 : kuv | 300/20.7 |
| SW-5222-330-SP(AQ) | 420g ua | 40-80 : kuv | 250/17.2 |
Table 2. SepaFlash C18AQ RP flash cartridges.
Cov ntaub ntawv ntim: High-efficiency spherical C18 (AQ)-bonded silica, 20 - 45 μm, 100 Å.
logy (raws li qhia hauv Table 2).
Post lub sij hawm: Aug-27-2018