Liquid chromatography ke mokhoa o ka sehloohong oa ho lekola litaba tsa karolo e 'ngoe le e' ngoe le litšila ka thepa e tala, li-intermediate, litokisetso le thepa ea ho paka, empa lintho tse ngata ha li na mekhoa e tloaelehileng ea ho itšetleha ka tsona, ka hona ho ke ke ha qojoa ho hlahisa mekhoa e mecha. Nts'etsopele ea mekhoa ea mohato oa mokelikeli, kholomo ea chromatographic ke motheo oa mokelikeli oa chromatography, kahoo mokhoa oa ho khetha kholomo e nepahetseng ea chromatographic e bohlokoa. Sengoliloeng sena, sengoli se tla hlalosa mokhoa oa ho khetha kholomo ea chromatography ea mokelikeli likarolong tse tharo: mehopolo ka kakaretso, maikutlo le boholo ba ts'ebeliso.
A.Kakaretso ea mehopolo ea ho khetha litšiea tsa chromatography tsa metsi
1. Hlahloba litšobotsi tsa 'mele le lik'hemik'hale tsa mohlahlobi: joalo ka sebopeho sa lik'hemik'hale, ho qhibiliha, botsitso (joalo ka hore na ho bonolo ho oxidized / ho fokotseha / hydrolyzed), acidity le alkalinity, joalo-joalo, haholo-holo sebopeho sa lik'hemik'hale ke senotlolo. ntlha ea ho khetholla thepa, joalo ka sehlopha sa conjugated se na le ho monya ho matla ha ultraviolet le fluorescence e matla;
2. Etsa qeto ea sepheo sa tlhahlobo: hore na karohano e phahameng, katleho e phahameng ea litšiea, nako e khutšoanyane ea ho hlahloba, kutloisiso e phahameng, ho hanyetsa khatello e phahameng, bophelo ba nako e telele, litšenyehelo tse tlaase, joalo-joalo;
- Khetha kholomo e loketseng ea chromatographic: utloisisa sebopeho, sebopeho sa 'mele le lik'hemik'hale tsa chromatographic filler, joalo ka boholo ba karoloana, boholo ba pore, mamello ea mocheso, mamello ea pH, adsorption ea analyte, jj.
- Lintlha tsa ho khetha litšiea tsa chromatography tsa metsi
Khaolo ena e tla tšohla lintlha tse lokelang ho nahanoa ha ho khethoa kholomo ea chromatography ho latela pono ea thepa ea 'mele le ea lik'hemik'hale ea kholomo ea chromatography ka boeona. 2.1 Filler matrix
2.1.1 Silica gel matrix Matrix ea ho tlatsa boholo ba likholomo tsa mokelikeli oa chromatography ke gel ea silica. Mofuta ona oa ho tlatsa o na le bohloeki bo phahameng, theko e tlase, matla a phahameng a mochini, 'me ho bonolo ho fetola lihlopha (tse kang phenyl bonding, amino bonding, cyano bonding, joalo-joalo), empa boleng ba pH le mocheso oo li o mamellang li fokotsehile: Mefuta e mengata ea pH ea li-filler tsa silika tsa gel matrix ke 2 ho isa ho 8, empa mefuta ea pH ea mekhahlelo e kopantsoeng ka ho khetheha ea silika e ka ba bophara ba 1.5 ho isa ho 10, hape ho na le mekhahlelo e kopantsoeng ka ho khetheha ea silika e tsitsitseng pH e tlase. tse kang Agilent ZORBAX RRHD stablebond-C18, e tsitsitseng pH 1 ho 8; moeli oa mocheso o kaholimo oa silika gel matrix hangata ke 60 ℃, 'me litšiea tse ling tsa chromatography li ka mamella mocheso oa 40 ℃ ho pH e phahameng.
2.1.2 Li-filler tsa polymer matrix Polymer hangata ke polystyrene-divinylbenzene kapa polymethacrylate. Melemo ea bona ke hore ba khona ho mamella pH e pharaletseng - e ka sebelisoa ho tloha ho 1 ho isa ho 14, 'me e hanyetsana le mocheso o phahameng (o ka fihla ka holimo ho 80 ° C). Ha ho bapisoa le li-filler tsa C18 tse thehiloeng ho silica, mofuta ona oa ho tlatsa o na le hydrophobicity e matla, 'me polymer e kholo e sebetsa haholo ho arola lisampole tse kang liprotheine. Mefokolo ea eona ke hore katleho ea kholomo e tlase mme matla a mochini a fokola ho feta a li-fillers tse thehiloeng ho silika. 2.2 Sebopeho sa karoloana
Boholo ba li-filler tsa sejoale-joale tsa HPLC ke likaroloana tse chitja, empa ka linako tse ling ke likaroloana tse sa tloaelehang. Likaroloana tsa spherical li ka fana ka khatello e tlase ea litšiea, katleho ea kholomo e phahameng, botsitso le bophelo bo bolelele; ha u sebelisa mekhahlelo ea li-mobile-viscosity e phahameng (e kang phosphoric acid) kapa ha sampuli ea tharollo e le li-viscous, likaroloana tse sa tloaelehang li na le sebaka se seholoanyane se khethehileng, se loketseng haholoanyane ketso e feletseng ea mekhahlelo e 'meli,' me theko e batla e le tlaase. 2.3 Boholo ba likaroloana
E nyenyane ea boholo ba likaroloana, e phahamisa katleho ea litšiea 'me e phahamisa karohano, empa e mpe le ho feta ho hanyetsa khatello e phahameng. Kholomo e sebelisoang haholo ke kholomo ea boholo ba likaroloana tse 5 μm; haeba tlhokahalo ea karohano e phahame, ho ka khethoa ho tlatsa 1.5-3 μm, e leng ho loketseng ho rarolla bothata ba ho arohana ha matrix a mang a rarahaneng le likarolo tse ngata tsa likarolo. UPLC e ka sebelisa li-filler tsa 1.5 μm; 10 μm kapa li-filler tse kholoanyane tsa boholo ba likaroloana hangata li sebelisoa bakeng sa litšiea tse itokiselitseng kapa tsa boitokisetso. 2.4 Likahare tsa khabone
Likahare tsa carbon li bua ka karolo ea karolo e tlanngoeng holim'a gel ea silika, e amanang le sebaka se itseng sa bokaholimo le sekoaelo sa mohato o tlanngoeng. Likahare tsa carbon tse phahameng li fana ka matla a phahameng a litšiea le tharollo e phahameng, 'me hangata e sebelisoa bakeng sa mehlala e rarahaneng e hlokang karohano e phahameng, empa ka lebaka la nako e telele ea ho sebelisana pakeng tsa mekhahlelo e' meli, nako ea ho hlahloba e telele; likholomo tsa chromatographic tsa khabone e tlase li na le nako e khuts'oane ea tlhahlobo mme li ka bonts'a likhetho tse fapaneng, 'me hangata li sebelisoa bakeng sa disampole tse bonolo tse hlokang tlhahlobo e potlakileng le disampole tse hlokang maemo a phahameng a metsi. Ka kakaretso, lihlahisoa tsa carbon tsa C18 li tloha ho 7% ho ea ho 19%. 2.5 Boholo ba pore le sebaka se itseng sa holim'a metsi
HPLC adsorption media ke likaroloana tse porous, 'me litšebelisano tse ngata li etsahala ka har'a pores. Ka hona, limolek'hule li tlameha ho kena ka har'a li-pores hore li phatlalatsoe le ho aroloa.
Boholo ba pore le sebaka se itseng sa bokaholimo ke mehopolo e 'meli e tlatsanang. Boholo ba pore bo bolela sebaka se seholo se ikhethileng, 'me ka tsela e fapaneng. Sebaka se seholo se ikhethileng se ka eketsa tšebelisano pakeng tsa limolek'hule tsa sampuli le mekhahlelo e tlamahaneng, ho ntlafatsa ho boloka, ho eketsa lisebelisoa tsa sampuli le matla a likholomo, le karohano ea likarolo tse rarahaneng. Li-filler tse nang le porous ka botlalo ke tsa mofuta ona oa li-filler. Bakeng sa ba nang le litlhoko tse phahameng tsa karohano, ho kgothaletswa ho khetha li-fillers tse nang le sebaka se seholo se khethehileng; sebaka se senyenyane se ikhethileng se ka fokotsa khatello ea morao, ho ntlafatsa ts'ebetso ea litšiea, le ho fokotsa nako ea tekano, e loketseng tlhahlobo ea gradient. Li-filler tsa li-core-shell ke tsa mofuta ona oa li-filler. Motheong oa ho netefatsa karohano, ho khothaletsoa ho khetha li-filler tse nang le sebaka se senyenyane se ikhethileng bakeng sa ba nang le litlhoko tse phahameng tsa tlhahlobo. 2.6 Bophahamo ba pore le matla a mochini
Pore volume, eo hape e tsejoang e le "pore volume", e bolela boholo ba molumo o se nang letho ka karoloana ea yuniti. E ka bonahatsa hantle matla a mochine oa ho tlatsa. Matla a mochini a li-filler tse nang le molumo o moholo oa pore a fokola hanyane ho feta a li-filler tse nang le molumo o monyane oa pore. Li-filler tse nang le molumo oa pore o ka tlase ho kapa o lekanang le 1.5 mL/g hangata li sebelisoa bakeng sa karohano ea HPLC, athe li-filler tse nang le bophahamo ba pore e kholo ho feta 1.5 mL/g li sebelisoa haholo bakeng sa chromatography ea limolek'hule le chromatography ea khatello e tlase. 2.7 Sekhahla sa Capping
Capping e ka fokotsa litlhōrō tsa mohatla tse bakoang ke tšebelisano lipakeng tsa metsoako le lihlopha tsa silanol tse pepeneneng (tse kang maqhama a ionic pakeng tsa metsoako ea alkaline le lihlopha tsa silanol, matla a van der Waals le maqhama a haedrojene pakeng tsa metsoako ea acidic le lihlopha tsa silanol), kahoo e ntlafatsa katleho ea kholomo le sebopeho sa tlhoro. . Mekhahlelo e tlanngoeng e sa kengoang e tla hlahisa likhetho tse fapaneng tse amanang le mekhahlelo e tlanngoeng, haholo-holo lisampole tsa polar.
- Sebaka sa ts'ebeliso ea litšiea tse fapaneng tsa mokelikeli oa chromatography
Khaolo ena e tla hlalosa boholo ba ts'ebeliso ea mefuta e fapaneng ea likholomo tsa mokelikeli oa chromatography maemong a mang.
3.1 Kholomo ea chromatographic ea mokhahlelo o khutliselitsoeng morao oa C18
Kholomo ea C18 ke kholomo e sebelisoang haholo-holo e khutliselitsoeng morao, e ka kopanang le liteko tsa litaba le ho se hloeke ha lintho tse ngata tsa manyolo, 'me e sebetsa ho lintho tse polar tse mahareng, tse fokolang le tse seng polar. Mofuta le litlhaloso tsa kholomo ea C18 chromatographic li lokela ho khethoa ho latela litlhoko tse ikhethileng tsa karohano. Ka mohlala, bakeng sa lintho tse nang le litlhoko tse phahameng tsa karohano, litlhaloso tsa 5 μm * 4.6 mm * 250 mm hangata li sebelisoa; bakeng sa lintho tse nang le matrices a karohano a rarahaneng le polarity e tšoanang, 4 μm * 4.6 mm * 250 mm litlhaloso kapa boholo ba likaroloana tse nyane li ka sebelisoa. Ka mohlala, mongoli o sebelisitse kholomo ea 3 μm * 4.6 mm * 250 mm ho bona litšila tse peli tsa genotoxic ho celecoxib API. Karohano ea lintho tse peli e ka fihla ho 2.9, e ntle haholo. Ho phaella moo, tlas'a motheo oa ho netefatsa karohano, haeba ho hlokahala tlhahlobo e potlakileng, mohala o khutšoanyane oa 10 mm kapa 15 mm o atisa ho khethoa. Ka mohlala, ha mongoli a sebelisa LC-MS/MS ho lemoha litšila tsa genotoxic ho piperaquine phosphate API, ho ile ha sebelisoa kholomo ea 3 μm * 2.1 mm * 100 mm. Karohano pakeng tsa ho se hloeke le karolo e kholo e ne e le 2.0, 'me ho fumanoa ha sampuli ho ka phethoa ka metsotso ea 5. 3.2 Kholomo ea phenyl e fetotsoeng
Phenyl column e boetse e le mofuta oa karolo e khutliselitsoeng. Mofuta ona oa litšiea o na le khetho e matla bakeng sa metsoako e nkhang hamonate. Haeba karabelo ea metsoako e nkhang hamonate e lekantsoeng ke kholomo e tloaelehileng ea C18 e fokola, o ka nahana ho nkela kholomo ea phenyl sebaka. Ka mohlala, ha ke ne ke etsa celecoxib API, karolo e ka sehloohong ea karabelo e lekantsoeng ke kholomo ea phenyl ea moetsi e tšoanang le tlhaloso e tšoanang (kaofela 5 μm * 4.6 mm * 250 mm) e ne e le ka makhetlo a 7 ho feta a kholomo ea C18. 3.3 Kholomo ea mokhahlelo o tloaelehileng
E le tlatsetso e sebetsang ho kholumo e fetotsoeng-mohato, kholomo ea mekhahlelo e tloaelehileng e loketse metsoako ea polar haholo. Haeba tlhōrō e ntse e potlakile haholo ha u ntse u e-na le karolo e fetang 90% ea metsi karolong e khutliselitsoeng-mohato, esita le haufi le ho fetana le tlhoro ea solvent, u ka nahana ka ho fetola kholomo e tloaelehileng. Mofuta ona oa kholomo o kenyelletsa kholomo ea hilic, kholomo ea amino, kholomo ea cyano, joalo-joalo.
3.3.1 Kholomo ea Hilic Kholomo ea Hilic hangata e kenya lihlopha tsa hydrophilic ka har'a ketane ea alkyl e tlanngoeng ho matlafatsa karabelo ho lintho tsa polar. Mofuta ona oa kholomo o loketse tlhahlobo ea lintho tse tsoekere. Mongoli o sebelisitse mofuta ona oa kholomo ha a etsa litaba le lintho tse amanang le xylose le lihlahisoa tsa eona. Li-isomers tsa motsoako oa xylose le tsona li ka aroloa hantle;
3.3.2 Kholomo ea amino le kholomo ea cyano Kholomo ea amino le kholomo ea cyano li bua ka kenyelletso ea liphetoho tsa amino le cyano qetellong ea ketane ea alkyl e kentsoeng, ka ho latellana, ho ntlafatsa khetho ea lintho tse khethehileng: mohlala, kholomo ea amino ke khetho e ntle. bakeng sa karohano ea tsoekere, li-amino acid, metheo, le li-amide; kholomo ea cyano e na le khetho e ntle ha e arola lintho tsa sebopeho sa hydrogenated le unhydrogenated ka lebaka la boteng ba li-conjugated bond. Kholomo ea Amino le kholomo ea cyano hangata e ka fetoloa lipakeng tsa kholumo e tloaelehileng ea karolo le kholumo e ka morao, empa ho fetoha khafetsa ha ho khothalletsoe. 3.4 Kholomo ea Chiral
Chiral column, joalokaha lebitso le bontša, e loketse ho arohana le ho hlahloba metsoako ea chiral, haholo-holo lefapheng la meriana. Mofuta ona oa kholomo o ka nahanoa ha litšiea tse tloaelehileng tsa mekhahlelo e fapaneng le mehala e tloaelehileng e sa khone ho fihlela karohano ea li-isomers. Mohlala, mongoli o sebelisitse kholomo ea 5 μm * 4.6 mm * 250 mm chiral ho arola li-isomers tse peli tsa 1,2-diphenylethylenediamine: (1S, 2S) -1, 2-diphenylethylenediamine le (1R, 2R) -1, 2 -diphenylethylenediamine, mme karohano pakeng tsa tse peli e fihlile hoo e ka bang 2.0. Leha ho le joalo, litšiea li bitsa chelete e ngata ho feta mefuta e meng ea litšiea, hangata 1W+/piece. Haeba ho na le tlhoko ea litšiea tse joalo, yuniti e hloka ho etsa tekanyetso e lekaneng. 3.5 Kholomo ea phapanyetsano ea ion
Litšiea tsa phapanyetsano ea ion li loketse karohano le tlhahlobo ea li-ion tse qosoang, joalo ka li-ion, liprotheine, li-nucleic acid le lintho tse ling tsa tsoekere. Ho ea ka mofuta oa li-filler, li arotsoe ka litšiea tsa phapanyetsano ea cation, litšiea tsa phapanyetsano ea anion, le litšiea tse matla tsa phapanyetsano ea cation.
Litšiea tsa phapanyetsano ea cation li kenyelletsa litšiea tse thehiloeng ka calcium le tsa haedrojene, tse loketseng haholo tlhahlobo ea lintho tsa cationic joalo ka li-amino acid. Ka mohlala, mongoli o sebelisitse litšiea tse thehiloeng ho calcium ha a hlahloba calcium gluconate le calcium acetate ka tharollo ea flushing. Lintho ka bobeli li ne li e-na le likarabo tse matla ho λ = 210nm, 'me tekanyo ea ho arohana e fihlile ho 3.0; mongoli o sebelisitse litšiea tse thehiloeng ho hydrogen ha a hlahloba lintho tse amanang le tsoekere. Lintho tse 'maloa tse kholo tse amanang - maltose, maltotriose le fructose - li ne li e-na le kutloisiso e phahameng tlas'a li-detectors tse fapaneng, tse nang le moeli oa ho lemoha o le tlase ho 0.5 ppm le tekanyo ea karohano ea 2.0-2.5.
Litšiea tsa phapanyetsano ea anion li loketse haholo-holo bakeng sa tlhahlobo ea lintho tsa anionic tse kang li-organic acid le li-ion tsa halogen; litšiea tse matla tsa phapanyetsano ea cation li na le bokhoni bo phahameng ba phapanyetsano ea ion le khetho, 'me li loketse karohano le tlhahlobo ea disampole tse rarahaneng.
Tse ka holimo ke selelekela sa mefuta le mefuta ea ts'ebeliso ea likholomo tse 'maloa tse tloaelehileng tsa mokelikeli oa chromatography hammoho le boiphihlelo ba sengoli. Ho na le mefuta e meng e ikhethang ea likholomo tsa chromatographic lits'ebetsong tsa 'nete, joalo ka likholomo tse kholo tsa pore chromatographic, litšiea tse nyane tsa chromatographic, likholomo tsa chromatography tse amanang, likholomo tsa chromatographic tse ngata, li-ultra-high performance liquid chromatography columns (UHPLC), likholomo tsa supercritical fluid chromatography ( SFC), joalo-joalo Ba phetha karolo ea bohlokoa mafapheng a fapaneng. Mofuta o ikhethileng oa kholomo ea chromatographic e lokela ho khethoa ho latela sebopeho le thepa ea sampole, litlhoko tsa karohano le merero e meng.
Nako ea poso: Jun-14-2024