Liquid chromatography ndiye njira yayikulu yoyesera zomwe zili mugawo lililonse ndi zonyansa muzinthu zopangira, zapakatikati, zokonzekera ndi zonyamula, koma zinthu zambiri zilibe njira zokhazikika zodalira, chifukwa chake ndikofunikira kupanga njira zatsopano. Pakupangidwa kwa njira zamadzimadzi, gawo la chromatographic ndiye phata la chromatography yamadzi, kotero momwe mungasankhire gawo loyenera la chromatographic ndikofunikira. M'nkhaniyi, wolemba afotokoza momwe angasankhire gawo la chromatography yamadzimadzi kuchokera kuzinthu zitatu: malingaliro onse, malingaliro ndi kuchuluka kwa ntchito.
A. Malingaliro onse pakusankha mizati ya chromatography yamadzi
1. Ganizirani zakuthupi ndi mankhwala a analyte: monga mapangidwe a mankhwala, kusungunuka, kukhazikika (monga ngati n'kosavuta kukhala oxidized / kuchepetsa / hydrolyzed), acidity ndi alkalinity, etc., makamaka mapangidwe a mankhwala ndi chinsinsi. chinthu kudziwa katundu, monga gulu conjugated ali amphamvu ultraviolet mayamwidwe ndi fluorescence wamphamvu;
2. Dziwani cholinga cha kusanthula: ngati kupatukana kwakukulu, kugwira ntchito kwapamwamba kwambiri, nthawi yochepa yowunikira, kukhudzidwa kwakukulu, kukana kupanikizika, moyo wautali wautali, mtengo wotsika, ndi zina zotero;
- Sankhani ndime yoyenera ya chromatographic: kumvetsetsa kapangidwe kake, thupi ndi mankhwala a chromatographic filler, monga kukula kwa tinthu, kukula kwa pore, kulolerana kwa kutentha, kulolerana kwa pH, kutsatsa kwa analyte, ndi zina zambiri.
- Zoganizira posankha mizati ya liquid chromatography
Mutuwu ufotokoza zinthu zomwe ziyenera kuganiziridwa posankha gawo la chromatography kuchokera momwe zimakhalira komanso momwe zimapangidwira pagawo la chromatography palokha. 2.1 Filler matrix
2.1.1 Matrix a silika gelisi Matrix odzaza madzi ambiri amadzimadzi a chromatography ndi silika gel. Zodzaza zamtunduwu zimakhala ndi chiyero chambiri, mtengo wotsika, mphamvu zamakina apamwamba, ndipo ndizosavuta kusintha magulu (monga phenyl bonding, amino bonding, cyano bonding, etc.), koma mtengo wa pH ndi kutentha komwe amalekerera ndizochepa: the Mitundu ya pH yamafuta ambiri a silika gel matrix ndi 2 mpaka 8, koma mtundu wa pH wa magawo omangika mwapadera a silica gel ophatikizika amatha kukhala motalikirana ndi 1.5 mpaka 10, ndipo palinso magawo omangika mwapadera a silika omwe amakhala okhazikika pa pH yotsika, monga Agilent ZORBAX RRHD stablebond-C18, yomwe imakhala yokhazikika pa pH 1 mpaka 8; Kutentha kwapamwamba kwa silika gel matrix nthawi zambiri kumakhala 60 ℃, ndipo mizati ina ya chromatography imatha kupirira kutentha kwa 40 ℃ pa pH yayikulu.
2.1.2 Ma polymer matrix Polymer fillers nthawi zambiri amakhala polystyrene-divinylbenzene kapena polymethacrylate. Ubwino wawo ndikuti amatha kulekerera pH yochuluka - angagwiritsidwe ntchito pamtundu wa 1 mpaka 14, ndipo amatha kupirira kutentha kwakukulu (kutha kufika pamwamba pa 80 ° C). Poyerekeza ndi zodzaza silika zochokera ku C18, zodzaza zamtunduwu zimakhala ndi hydrophobicity yamphamvu, ndipo ma macroporous polima ndiwothandiza kwambiri pakulekanitsa zitsanzo monga mapuloteni. Zoyipa zake ndikuti magwiridwe antchito a mzati ndi otsika ndipo mphamvu zamakina ndi zofooka kuposa za silika-based fillers. 2.2 Maonekedwe a tinthu
Zambiri zamakono za HPLC ndi tinthu tating'onoting'ono tozungulira, koma nthawi zina timakhala tinthu tating'onoting'ono. Tinthu tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'onoting'ono, magwiridwe antchito apamwamba, kukhazikika komanso moyo wautali; mukamagwiritsa ntchito magawo a mafoni apamwamba kwambiri (monga phosphoric acid) kapena pamene njira yothetsera vutoli imakhala yowoneka bwino, tinthu tating'onoting'ono timakhala ndi malo akuluakulu enieni, omwe amathandiza kwambiri pazochitika zonse ziwiri, ndipo mtengo wake ndi wotsika kwambiri. 2.3 Kukula kwa tinthu
Zing'onozing'ono za tinthu tating'onoting'ono, ndizomwe zimapangidwira bwino komanso zimakhala zolekana, koma zimakhala zovuta kwambiri kukana kupanikizika. Mzere womwe umagwiritsidwa ntchito kwambiri ndi 5 μm kukula kwa tinthu; ngati kufunikira kolekanitsa kuli kwakukulu, 1.5-3 μm filler ikhoza kusankhidwa, yomwe imathandizira kuthetsa vuto la kulekana kwa matrix ena ovuta komanso zitsanzo zamagulu ambiri. UPLC imatha kugwiritsa ntchito 1.5 μm fillers; 10 μm kapena zokulirapo za kukula kwa tinthu nthawi zambiri zimagwiritsidwa ntchito popanga mizati yokonzekera kapena yokonzekera. 2.4 Zomwe zili ndi Carbon
Zomwe zili ndi mpweya zimatanthawuza kuchuluka kwa gawo lomangika pamwamba pa gelisi ya silika, yomwe imagwirizana ndi malo enieni komanso kufalikira kwa gawo. Mpweya wambiri wa carbon umapereka mphamvu yamtundu wapamwamba komanso kusamvana kwakukulu, ndipo nthawi zambiri amagwiritsidwa ntchito pa zitsanzo zovuta zomwe zimafuna kupatukana kwakukulu, koma chifukwa cha nthawi yayitali yolumikizana pakati pa magawo awiriwa, nthawi yowunikira ndi yaitali; mizati ya carbon low chromatographic columns imakhala ndi nthawi yofupikitsa yowunikira ndipo imatha kusonyeza zosankha zosiyanasiyana, ndipo nthawi zambiri zimagwiritsidwa ntchito pa zitsanzo zosavuta zomwe zimafuna kufufuza mofulumira ndi zitsanzo zomwe zimafuna mikhalidwe yambiri yamadzi. Nthawi zambiri, mpweya wa C18 umachokera ku 7% mpaka 19%. 2.5 Pore kukula ndi malo enieni
HPLC adsorption media ndi tinthu tating'onoting'ono, ndipo kuyanjana kwambiri kumachitika mu pores. Choncho, mamolekyu ayenera kulowa pores kuti adsorbed ndi kupatukana.
Kukula kwa pore ndi malo enieni ndi malingaliro awiri ogwirizana. Kukula kwa pore kumatanthauza malo akuluakulu enieni, ndipo mosiyana. Dera lalikulu lapadera likhoza kuonjezera mgwirizano pakati pa mamolekyu a zitsanzo ndi magawo omangika, kupititsa patsogolo kusungirako, kuonjezera kutsitsa kwachitsanzo ndi mphamvu ya mzere, ndi kulekanitsa kwa zigawo zovuta. Odzaza ndi porous fillers ali amtundu uwu wa fillers. Kwa iwo omwe ali ndi zofunikira zolekanitsa kwambiri, tikulimbikitsidwa kusankha zodzaza ndi malo akuluakulu enieni; malo ang'onoang'ono apadera amatha kuchepetsa kupsinjika kwa msana, kuwongolera magwiridwe antchito a mzati, ndikuchepetsa nthawi yofananira, yomwe ili yoyenera kusanthula gradient. Zodzaza zipolopolo zamtundu wamtundu uwu. Pamaziko owonetsetsa kulekana, tikulimbikitsidwa kusankha zodzaza ndi malo ang'onoang'ono apadera kwa iwo omwe ali ndi zofunikira zowunikira kwambiri. 2.6 Pore voliyumu ndi mphamvu zamakina
Voliyumu ya pore, yomwe imadziwikanso kuti "pore volume", imatanthawuza kukula kwa void void pagawo lililonse. Ikhoza kuwonetsa mphamvu zamakina a filler. Mphamvu zamakina zamafila okhala ndi voliyumu yayikulu ya pore ndizochepa pang'ono kuposa zodzaza ndi pore yaying'ono. Zodzaza ndi pore voliyumu yochepera kapena yofanana ndi 1.5 mL/g imagwiritsidwa ntchito kwambiri pakulekanitsa kwa HPLC, pomwe zodzaza ndi pore voliyumu yoposa 1.5 mL/g zimagwiritsidwa ntchito makamaka pakupatula ma chromatography ndi chromatography yotsika. 2.7 Mtengo wokwanira
Kuwombera kumatha kuchepetsa nsonga zam'mphepete zomwe zimayambitsidwa ndi kuyanjana pakati pamagulu ndi magulu a silanol owonekera (monga kugwirizana kwa ionic pakati pa mankhwala amchere ndi magulu a silanol, mphamvu za van der Waals ndi zomangira za haidrojeni pakati pamagulu a acidic ndi magulu a silanol), potero kumapangitsa kuti mizere ikhale yabwino komanso mawonekedwe apamwamba. . Magawo omangika osaphatikizidwa adzapanga zosankha zosiyanasiyana zokhudzana ndi magawo omangika, makamaka zitsanzo za polar.
- Kuchuluka kwa ntchito zamitundu yosiyanasiyana yamadzimadzi chromatography
Mutuwu ufotokoza kuchuluka kwa kagwiritsidwe ntchito kamitundu yosiyanasiyana yamadzi amadzimadzi a chromatography nthawi zina.
3.1 Gawo losinthidwa la C18 chromatographic column
Gawo la C18 ndilo gawo lomwe limagwiritsidwa ntchito kwambiri, lomwe limakwaniritsa zomwe zili ndi zoyezetsa zazinthu zambiri zamoyo, ndipo limagwiritsidwa ntchito kuzinthu zapakatikati, zofooka za polar komanso zopanda polar. Mtundu ndi mafotokozedwe a C18 chromatographic column ayenera kusankhidwa malinga ndi zofunikira zopatukana. Mwachitsanzo, pazinthu zomwe zili ndi zofunikira zolekanitsa, 5 μm * 4.6 mm * 250 mm nthawi zambiri amagwiritsidwa ntchito; kwa zinthu zomwe zimakhala ndi matrices ovuta olekanitsa ndi polarity ofanana, 4 μm * 4.6 mm * 250 mm specifications kapena tinthu tating'onoting'ono tingagwiritsidwe ntchito. Mwachitsanzo, wolembayo adagwiritsa ntchito gawo la 3 μm * 4.6 mm * 250 mm kuti azindikire zonyansa ziwiri za genotoxic mu celecoxib API. Kupatukana kwa zinthu ziwirizi kumatha kufika 2.9, zomwe ndi zabwino kwambiri. Kuonjezera apo, pansi pa chidziwitso cha kuonetsetsa kulekanitsa, ngati kusanthula mofulumira kumafunika, mzere waufupi wa 10 mm kapena 15 mm nthawi zambiri umasankhidwa. Mwachitsanzo, pamene wolemba anagwiritsa ntchito LC-MS/MS kuti azindikire kusayera kwa genotoxic mu piperaquine phosphate API, 3 μm * 2.1 mm * 100 mm column inagwiritsidwa ntchito. Kusiyanitsa pakati pa zonyansa ndi chigawo chachikulu chinali 2.0, ndipo kudziwika kwa chitsanzo kumatha kutha mu mphindi 5. 3.2 Reverse-phase phenyl column
Phenyl column ndi mtundu wa reverse-phase column. Mzati wamtunduwu uli ndi kusankha kwamphamvu kwamafuta onunkhira. Ngati kuyankha kwamafuta onunkhira omwe amayezedwa ndi gawo la C18 kuli kofooka, mutha kuganiziranso kusintha ndime ya phenyl. Mwachitsanzo, pamene ndinali kupanga celecoxib API, chigawo chachikulu kuyankha kuyezedwa ndi phenyl ndime ya wopanga yemweyo ndi mfundo zofanana (zonse 5 μm * 4.6 mm * 250 mm) anali pafupifupi 7 nthawi ya C18 ndime. 3.3 Mzere wanthawi zonse
Monga chowonjezera chothandizira pagawo losinthika, gawo lokhazikika ndiloyenera kuphatikizira kwambiri polar. Ngati pachimake akadali mofulumira kwambiri pamene eluting ndi oposa 90% amadzimadzi gawo mu n'zosiyana gawo ndime, ndipo ngakhale pafupi ndi overlops ndi nsonga zosungunulira, mukhoza kuganizira m'malo yachibadwa gawo ndime. Mzere wamtunduwu umaphatikizapo gawo la hilic, amino column, cyano column, ndi zina zotero.
3.3.1 Mzere wa Hilic Mzere wa Hilic nthawi zambiri umayika magulu a hydrophilic mu unyolo wa alkyl womangika kuti athandizire kuyankha ku zinthu za polar. Mzati wamtunduwu ndi woyenera kusanthula zinthu za shuga. Wolembayo adagwiritsa ntchito gawo lamtunduwu popanga zomwe zili ndi zinthu zokhudzana ndi xylose ndi zotuluka zake. Ma isomers a chotengera cha xylose amathanso kulekanitsidwa bwino;
3.3.2 Amino ndime ndi cyano ndime Amino ndi cyano ndime amatanthauza kumayambiriro amino ndi cyano zosintha kumapeto kwa unyolo womangidwa alkyl, motero, kusintha selectivity kwa zinthu zapadera: mwachitsanzo, amino ndime ndi chisankho chabwino. kwa kulekanitsa shuga, amino zidulo, maziko, ndi amides; cyano column ali bwino kusankha pamene kulekanitsa hydrogenated ndi unhydrogenated structural zinthu zofanana chifukwa kukhalapo kwa conjugated zomangira. Mzere wa amino ndi cyano nthawi zambiri umatha kusinthidwa pakati pa gawo lokhazikika ndi gawo losinthira, koma kusintha pafupipafupi sikuvomerezeka. 3.4 Chiral column
Chiral column, monga momwe dzinalo likusonyezera, ndiloyenera kulekanitsa ndi kusanthula mankhwala a chiral, makamaka pankhani ya mankhwala. Mzere wamtunduwu ukhoza kuganiziridwa pamene magawo ochiritsira ochiritsira ndi magawo abwinobwino sangathe kukwaniritsa kulekana kwa ma isomers. Mwachitsanzo, wolemba adagwiritsa ntchito 5 μm * 4.6 mm * 250 mm chiral column kuti alekanitse ma isomers awiri a 1,2-diphenylethylenediamine: (1S, 2S) -1, 2-diphenylethylenediamine ndi (1R, 2R) -1, 2 -diphenylethylenediamine, ndipo kulekana pakati pa awiriwa kunafika pafupifupi 2.0. Komabe, zipilala zachiral ndizokwera mtengo kuposa mitundu ina ya mizati, nthawi zambiri 1W +/chidutswa. Ngati pakufunika mizati yotere, gawolo liyenera kupanga bajeti yokwanira. 3.5 Mzere wosinthanitsa ion
Mizati yosinthira ion ndiyoyenera kupatukana ndi kusanthula ma ayoni omwe amaperekedwa, monga ayoni, mapuloteni, ma nucleic acid, ndi zinthu zina za shuga. Malingana ndi mtundu wa zodzaza, iwo amagawidwa m'mizere yosinthira ma cation, mizati yosinthanitsa ya anion, ndi mizati yamphamvu yosinthira ma cation.
Mizati yosinthira ma cation imaphatikizapo mizati yochokera ku calcium ndi haidrojeni, yomwe ili yoyenera kuwunikira zinthu za cationic monga ma amino acid. Mwachitsanzo, wolembayo adagwiritsa ntchito mizati yochokera ku calcium posanthula calcium gluconate ndi calcium acetate mu njira yosinthira. Zinthu zonsezi zinali ndi mayankho amphamvu pa λ = 210nm, ndipo digiri yolekanitsa idafika 3.0; wolemba adagwiritsa ntchito mizati yochokera ku haidrojeni posanthula zinthu zokhudzana ndi shuga. Zinthu zingapo zazikulu zokhudzana - maltose, maltotriose ndi fructose - zinali ndi chidwi chachikulu pansi pa zowunikira zosiyana, ndi malire ozindikira otsika ngati 0.5 ppm ndi digiri yopatukana ya 2.0-2.5.
Mizati yosinthira ya Anion ndiyoyenera makamaka kuwunika zinthu za anionic monga ma organic acid ndi ayoni a halogen; mizati yamphamvu yosinthira ma cation imakhala ndi mphamvu zapamwamba zosinthira ma ion ndi kusankha, ndipo ndizoyenera kupatukana ndi kusanthula zitsanzo zovuta.
Zomwe zili pamwambazi ndi chiyambi chabe cha mitundu ndi magwiritsidwe ntchito a magawo angapo amadzimadzi amadzimadzi a chromatography kuphatikiza ndi zomwe wolembayo adakumana nazo. Palinso mitundu ina yapadera ya mizati ya chromatographic pamapulogalamu enieni, monga mizati ya pore-pore chromatographic, mizati ya pore yaing'ono, mizati ya chromatography yolumikizana, mizati ya chromatographic multimode, mizati yapamwamba kwambiri yamadzimadzi amadzimadzi amadzimadzi (UHPLC), mizati yapamwamba kwambiri yamadzimadzi (UHPLC). SFC), etc. Amagwira ntchito yofunikira m'magawo osiyanasiyana. Mtundu wapadera wa mzere wa chromatographic uyenera kusankhidwa molingana ndi kapangidwe kake ndi katundu wa zitsanzo, zofunikira zolekanitsa ndi zolinga zina.
Nthawi yotumiza: Jun-14-2024