Cov kua dej chromatography yog txoj hauv kev tseem ceeb rau kev ntsuam xyuas cov ntsiab lus ntawm txhua lub ntsiab lus thiab cov khoom tsis huv hauv cov khoom siv raw, cov khoom nruab nrab, kev npaj thiab cov khoom ntim, tab sis ntau yam khoom tsis muaj cov txheej txheem kev vam khom, yog li nws yog qhov tsim nyog los tsim txoj hauv kev tshiab. Hauv kev txhim kho cov txheej txheem ua kua theem, cov kab chromatographic kem yog qhov tseem ceeb ntawm cov kua chromatography, yog li yuav ua li cas xaiv cov kab chromatographic haum yog qhov tseem ceeb. Hauv tsab xov xwm no, tus sau yuav piav qhia yuav ua li cas xaiv cov kua chromatography kem los ntawm peb yam: tag nrho cov tswv yim, kev txiav txim siab thiab daim ntawv thov.
A. Tag nrho cov tswv yim rau xaiv cov kua chromatography kab
1. Ntsuas lub cev thiab cov khoom siv tshuaj lom neeg ntawm cov tshuaj ntsuam xyuas: xws li cov qauv tshuaj, solubility, stability (xws li seb nws puas yooj yim rau oxidized / txo / hydrolyzed), acidity thiab alkalinity, thiab lwm yam, tshwj xeeb tshaj yog cov qauv tshuaj yog tus yuam sij. qhov tseem ceeb hauv kev txiav txim siab cov khoom, xws li cov pab pawg sib txuas muaj zog ultraviolet absorption thiab muaj zog fluorescence;
2. Txiav txim siab lub hom phiaj ntawm kev tsom xam: seb puas muaj kev sib cais siab, siab kem efficiency, luv luv tsom xam lub sij hawm, siab rhiab heev, siab tsis kam, ntev kem lub neej, tsis tshua muaj nqi, thiab lwm yam yuav tsum tau;
- Xaiv cov kab chromatographic tsim nyog: nkag siab txog qhov muaj pes tsawg leeg, lub cev thiab tshuaj lom neeg ntawm chromatographic muab tub lim, xws li qhov loj me, qhov pore loj, qhov kub thiab txias, pH kam rau ua, adsorption ntawm cov tshuaj ntsuam xyuas, thiab lwm yam.
- Kev txiav txim siab rau xaiv cov kua chromatography kab
Tshooj lus no yuav tham txog cov xwm txheej uas yuav tsum tau txiav txim siab thaum xaiv kab chromatography los ntawm qhov kev xav ntawm lub cev thiab tshuaj lom neeg ntawm chromatography kem nws tus kheej. 2.1 Filler matrix
2.1.1 Silica gel matrix Cov muab tub lim matrix ntawm feem ntau cov kua chromatography kab yog silica gel. Hom muab tub lim no muaj purity siab, tsis tshua muaj nqi, siab mechanical zog, thiab yog ib qho yooj yim los hloov cov pab pawg (xws li phenyl bonding, amino bonding, cyano bonding, thiab lwm yam), tab sis tus nqi pH thiab kub ntau yam nws tolerates yog txwv: lub pH ntau ntawm silica gel matrix fillers feem ntau yog 2 txog 8, tab sis pH ntau yam tshwj xeeb hloov silica gel bonded theem tuaj yeem dav li 1.5 txog 10, thiab kuj tseem muaj cov txheej txheem tshwj xeeb hloov silica gel bonded theem uas ruaj khov ntawm pH tsawg, xws li Agilent ZORBAX RRHD stabilitybond-C18, uas ruaj khov ntawm pH 1 txog 8; Lub siab kub txwv ntawm silica gel matrix feem ntau yog 60 ℃, thiab qee cov kab chromatography tuaj yeem tiv taus qhov kub ntawm 40 ℃ ntawm pH siab.
2.1.2 Polymer matrix Polymer fillers feem ntau yog polystyrene-divinylbenzene lossis polymethacrylate. Lawv qhov zoo yog tias lawv tuaj yeem tiv taus ntau yam pH - lawv tuaj yeem siv rau hauv thaj tsam ntawm 1 txog 14, thiab lawv muaj peev xwm tiv taus qhov kub siab tshaj (tuaj yeem ncav cuag 80 ° C). Piv nrog rau silica-raws li C18 fillers, hom muab tub lim no muaj zog hydrophobicity, thiab cov macroporous polymer zoo heev hauv kev sib cais cov qauv xws li cov protein. Nws qhov tsis zoo yog qhov kev ua tau zoo ntawm cov kem qis dua thiab cov neeg kho tshuab lub zog tsis muaj zog dua li ntawm silica-based fillers. 2.2 Particle puab
Feem ntau cov niaj hnub HPLC fillers yog kheej kheej, tab sis qee zaum lawv yog cov khoom tsis sib xws. Spherical hais tuaj yeem muab cov kab qis qis, siab dua kem efficiency, stability thiab lub neej ntev; thaum siv high-viscosity mobile theem (xws li phosphoric acid) los yog thaum cov qauv tov yog nkig, irregular hais muaj ib tug loj cheeb tsam ntawm lub nto, uas yog ntau conducive rau tag nrho cov kev txiav txim ntawm ob theem, thiab tus nqi kuj tsawg. 2.3 Particle loj
Qhov me me ntawm qhov me me, qhov ua tau zoo ntawm cov kab ke ntau dua thiab qhov sib cais ntau dua, tab sis qhov phem dua qhov kev ua haujlwm siab. Feem ntau siv kem yog 5 μm particle loj kem; Yog hais tias qhov kev sib cais yuav tsum siab, 1.5-3 μm muab tub lim tuaj yeem xaiv tau, uas yog qhov tsim nyog rau kev daws teeb meem kev sib cais ntawm qee qhov kev sib xyaw ua ke thiab ntau cov qauv. UPLC tuaj yeem siv 1.5 μm muab tub lim; 10 μm los yog loj dua particle loj fillers feem ntau yog siv rau semi-preparative lossis preparative kab. 2.4 Cov ntsiab lus carbon
Cov ntsiab lus carbon yog hais txog qhov kev faib ua feem ntawm kev sib koom ua ke rau ntawm qhov chaw ntawm silica gel, uas muaj feem xyuam rau thaj chaw tshwj xeeb thiab cov txheej txheem sib koom ua ke. Cov ntsiab lus carbon siab muab cov kab ke siab siab thiab kev daws teeb meem siab, thiab feem ntau yog siv rau cov qauv nyuaj uas yuav tsum tau muaj kev sib cais siab, tab sis vim muaj kev cuam tshuam ntev ntawm ob theem, lub sij hawm tsom xam yog ntev; Cov ntsiab lus tsis tshua muaj cov ntsiab lus chromatographic kab muaj lub sijhawm tshawb xyuas luv dua thiab tuaj yeem pom cov kev xaiv sib txawv, thiab feem ntau yog siv rau cov qauv yooj yim uas yuav tsum tau soj ntsuam sai thiab cov qauv uas yuav tsum tau muaj cov txheej txheem aqueous siab. Feem ntau, cov ntsiab lus carbon ntawm C18 yog li ntawm 7% mus rau 19%. 2.5 Pore loj thiab thaj chaw tshwj xeeb
HPLC adsorption media yog cov khoom ntxeem tau, thiab kev sib cuam tshuam feem ntau tshwm sim hauv qhov hws. Yog li ntawd, molecules yuav tsum nkag mus rau hauv lub pores kom tau adsorbed thiab sib cais.
Pore loj thiab thaj chaw tshwj xeeb yog ob lub ntsiab lus ntxiv. Qhov pore me me txhais tau hais tias qhov loj ntawm qhov chaw, thiab rov ua dua. Ib cheeb tsam loj tshwj xeeb tuaj yeem ua rau muaj kev sib cuam tshuam ntawm cov qauv molecules thiab cov txheej txheem sib txuas, txhim khu kev tuav pov hwm, ua kom cov qauv thauj khoom thiab kem muaj peev xwm, thiab sib cais ntawm cov khoom sib txawv. Tag nrho cov ntxeem tau muab tso rau hauv hom fillers no. Rau cov neeg uas xav tau kev sib cais siab, nws raug nquahu kom xaiv cov fillers nrog thaj tsam loj; Cov cheeb tsam me me tuaj yeem txo qis rov qab siab, txhim kho kab ke kev ua haujlwm, thiab txo lub sijhawm sib npaug, uas yog tsim rau kev soj ntsuam gradient. Core-plhaub fillers belongs rau hom fillers. Ntawm qhov chaw ntawm kev ua kom muaj kev sib cais, nws raug nquahu kom xaiv cov fillers nrog me me tshwj xeeb thaj chaw rau cov neeg uas muaj kev tsom xam ua haujlwm siab. 2.6 Pore ntim thiab txhua yam muaj zog
Pore ntim, tseem hu ua "pore ntim", hais txog qhov loj ntawm qhov tsis muaj qhov ntim ntawm ib chav tsev particle. Nws tuaj yeem cuam tshuam zoo rau txhua yam zog ntawm qhov muab tub lim. Lub zog txhua yam ntawm fillers nrog loj pore ntim yog me ntsis tsis muaj zog dua li ntawm fillers nrog me me pore ntim. Fillers nrog pore ntim tsawg dua lossis sib npaug li 1.5 mL / g feem ntau yog siv rau kev sib cais ntawm HPLC, thaum cov khoom ntim nrog pore ntim ntau dua 1.5 mL / g feem ntau yog siv rau molecular exclusion chromatography thiab low-pressure chromatography. 2.7 Capping tus nqi
Capping tuaj yeem txo cov tailing peaks tshwm sim los ntawm kev sib cuam tshuam ntawm cov tebchaw thiab cov pab pawg silanol (xws li ionic bonding ntawm alkaline tebchaw thiab silanol pawg, van der Waals rog thiab hydrogen bonds ntawm acidic compounds thiab silanol pawg), yog li txhim kho kem efficiency thiab ncov zoo. . Uncapped bonded phases yuav tsim cov kev xaiv sib txawv ntawm cov txheej txheem capped bonded, tshwj xeeb tshaj yog rau cov qauv polar.
- Daim ntawv thov Scope ntawm cov kua chromatography sib txawv
Tshooj lus no yuav piav qhia txog daim ntawv thov ntawm ntau hom kua chromatography kab los ntawm qee kis.
3.1 Rov qab-theem C18 chromatographic kem
C18 kem yog cov feem ntau siv thim rov qab-theem kem, uas tuaj yeem ua tau raws li cov ntsiab lus thiab impurity xeem ntawm feem ntau cov organic tshuaj, thiab muaj feem xyuam rau nruab nrab-polar, weakly polar thiab non-polar tshuaj. Hom thiab specification ntawm C18 chromatographic kem yuav tsum raug xaiv raws li cov kev cai sib cais tshwj xeeb. Piv txwv li, rau cov khoom uas muaj kev sib cais siab, 5 μm * 4.6 mm * 250 mm specifications feem ntau yog siv; rau cov khoom uas muaj kev sib cais matrices thiab zoo sib xws polarity, 4 μm * 4.6 mm * 250 mm specifications los yog me me particle loj yuav siv tau. Piv txwv li, tus sau tau siv 3 μm * 4.6 mm * 250 mm kem txhawm rau txheeb xyuas ob lub genotoxic impurities hauv celecoxib API. Kev sib cais ntawm ob yam khoom tuaj yeem ncav cuag 2.9, uas yog qhov zoo heev. Tsis tas li ntawd, nyob rau hauv qhov kev pom zoo ntawm kev ua kom muaj kev sib cais, yog tias xav tau kev tshuaj xyuas sai, feem ntau xaiv ib kem luv ntawm 10 mm lossis 15 mm. Piv txwv li, thaum tus kws sau ntawv siv LC-MS / MS txhawm rau txhawm rau txhawm rau kuaj genotoxic impurity hauv piperaquine phosphate API, 3 μm * 2.1 mm * 100 mm kem tau siv. Kev sib cais ntawm cov impurity thiab cov ntsiab lus tseem ceeb yog 2.0, thiab kev kuaj xyuas cov qauv tuaj yeem ua tiav hauv 5 feeb. 3.2 Rov qab-theem phenyl kem
Phenyl kem kuj yog ib hom kab rov qab-theem. Hom kab no muaj zog xaiv rau cov tshuaj tsw qab. Yog tias cov lus teb ntawm cov tshuaj tsw qab uas ntsuas los ntawm kab C18 zoo tib yam tsis muaj zog, koj tuaj yeem xav txog kev hloov phenyl kem. Piv txwv li, thaum kuv tab tom ua celecoxib API, lub ntsiab lus teb tau ntsuas los ntawm phenyl kem ntawm tib lub chaw tsim khoom thiab cov lus qhia tib yam (tag nrho 5 μm * 4.6 mm * 250 mm) yog li 7 npaug ntawm C18 kem. 3.3 Ib txwm-phase kem
Raws li ib qho txiaj ntsig ntxiv rau cov kab rov qab-theem, kab ke ib txwm yog tsim rau cov tebchaw uas muaj zog heev. Yog hais tias lub ncov tseem ceev heev thaum eluting nrog ntau tshaj 90% aqueous theem nyob rau hauv lub reversed-theem kem, thiab txawm nyob ze thiab overlaps nrog cov hnyav ncov, koj muaj peev xwm txiav txim siab hloov lub qub-theem kem. Hom kab no suav nrog hilic kem, amino kem, cyano kem, thiab lwm yam.
3.3.1 Hilic kem Hilic kem feem ntau embeds hydrophilic pawg nyob rau hauv bonded alkyl saw los txhim kho cov lus teb rau polar tshuaj. Hom kab no haum rau kev tsom xam cov khoom qab zib. Tus sau tau siv hom kab ntawv no thaum ua cov ntsiab lus thiab lwm yam khoom ntawm xylose thiab nws cov derivatives. Cov isomers ntawm xylose derivative kuj tuaj yeem sib cais zoo;
3.3.2 Amino kem thiab cyano kem Amino kem thiab cyano kem xa mus rau cov kev taw qhia ntawm cov amino thiab cyano hloov kho nyob rau hauv qhov kawg ntawm cov bonded alkyl saw, feem, los txhim kho cov selectivity rau tshwj xeeb tshuaj: piv txwv li, amino kem yog ib tug zoo xaiv. rau kev sib cais ntawm cov suab thaj, amino acids, bases, thiab amides; cyano kem muaj kev xaiv zoo dua thaum sib cais hydrogenated thiab unhydrogenated cov qauv zoo sib xws vim muaj cov ntawv sib txuas. Amino kem thiab cyano kem feem ntau tuaj yeem hloov pauv ntawm cov kab ke ib txwm thiab cov kab rov qab, tab sis tsis pom zoo hloov pauv. 3.4 Chiral kab
Chiral kem, raws li lub npe qhia, yog tsim nyog rau kev sib cais thiab tsom xam ntawm chiral compounds, tshwj xeeb tshaj yog nyob rau hauv lub teb ntawm kws tshuaj. Hom kab no tuaj yeem txiav txim siab thaum cov qauv rov qab theem thiab cov kab ke ib txwm tsis tuaj yeem ua tiav kev sib cais ntawm isomers. Piv txwv li, tus sau tau siv 5 μm * 4.6 mm * 250 mm chiral kem cais ob isomers ntawm 1,2-diphenylethylenediamine: (1S, 2S)-1, 2-diphenylethylenediamine thiab (1R, 2R)-1, 2 -diphenylethylenediamine, thiab kev sib cais ntawm ob mus txog 2.0. Txawm li cas los xij, chiral kab yog kim dua lwm hom kab, feem ntau 1W + / thooj. Yog tias xav tau cov kab ntawv zoo li no, chav tsev yuav tsum tau ua kom muaj peev txheej txaus. 3.5 Ion pauv kab
Cov kab sib pauv ion yog tsim nyog rau kev sib cais thiab kev soj ntsuam ntawm cov ions them, xws li ions, proteins, nucleic acids, thiab qee cov khoom qab zib. Raws li hom muab tub lim, lawv tau muab faib ua cation txauv kab, anion pauv kab, thiab muaj zog cation txauv kab.
Cation txauv kab muaj xws li calcium-based thiab hydrogen-based kab, uas yog tsim nyog rau kev tsom xam cov cationic tshuaj xws li amino acids. Piv txwv li, tus kws sau ntawv tau siv cov kab ntawv calcium uas yog thaum txheeb xyuas calcium gluconate thiab calcium acetate hauv cov tshuaj yaug dej. Ob yam tshuaj muaj cov lus teb muaj zog ntawm λ = 210nm, thiab kev sib cais degree mus txog 3.0; tus kws sau ntawv siv cov kab ntawv hydrogen-based thaum txheeb xyuas cov tshuaj muaj txiaj ntsig ntawm cov piam thaj. Ob peb yam tseem ceeb - maltose, maltotriose thiab fructose - muaj rhiab heev nyob rau hauv cov khoom sib txawv, nrog rau kev kuaj pom tsawg li 0.5 ppm thiab qib sib cais ntawm 2.0-2.5.
Anion txauv kab yog tsim nyog rau kev tsom xam ntawm anionic tshuaj xws li organic acids thiab halogen ions; muaj zog cation txauv kab muaj ntau dua ion pauv peev xwm thiab kev xaiv, thiab tsim nyog rau kev sib cais thiab kev soj ntsuam ntawm cov qauv nyuaj.
Cov saum toj no tsuas yog ib qho kev taw qhia rau cov hom thiab daim ntawv thov ntau yam ntawm ntau hom kua chromatography kab nrog rau tus sau nws tus kheej kev paub. Muaj lwm yam tshwj xeeb ntawm chromatographic kab hauv kev siv tiag tiag, xws li cov pore chromatographic kab loj, me me-pore chromatographic kab, affinity chromatography kab, multimode chromatographic kab, ultra-high performance kua chromatography kab (UHPLC), supercritical fluid chromatography kab ( SFC), thiab lwm yam. Lawv ua lub luag haujlwm tseem ceeb hauv ntau qhov chaw. Yam tshwj xeeb ntawm chromatographic kem yuav tsum raug xaiv raws li cov qauv thiab cov khoom ntawm cov qauv, cov kev cai sib cais thiab lwm yam.
Post lub sij hawm: Jun-14-2024