Amazi ya chromatografiya nuburyo bwingenzi bwo gupima ibiri muri buri kintu hamwe n’umwanda mu bikoresho fatizo, abahuza, imyiteguro n’ibikoresho byo gupakira, ariko ibintu byinshi ntabwo bifite uburyo busanzwe bwo gushingiraho, bityo rero byanze bikunze bitezimbere uburyo bushya. Mugutezimbere uburyo bwicyiciro cyamazi, inkingi ya chromatografiya niyo nkingi ya chromatografiya yamazi, kuburyo rero bwo guhitamo inkingi ikwiye ya chromatografique ni ngombwa. Muri iyi ngingo, umwanditsi azasobanura uburyo bwo guhitamo inkingi ya chromatografiya yibice bitatu: ibitekerezo rusange, ibitekerezo hamwe nurwego rushyirwa mubikorwa.
A.Ibitekerezo rusange byo guhitamo inkingi ya chromatografiya
1. Suzuma imiterere yumubiri na chimique ya analyte: nkimiterere yimiti, solubilité, stabilite (nko kumenya niba byoroshye okiside / kugabanuka / hydrolyzed), acide na alkaline, nibindi, cyane cyane imiterere yimiti nurufunguzo. ikintu cyo kumenya imiterere, nkitsinda ryahujwe rifite imbaraga za ultraviolet zinjira cyane na fluorescence ikomeye;
2. Kugena intego yo gusesengura: niba gutandukana cyane, gukora inkingi ndende, igihe gito cyo gusesengura, sensibilité nyinshi, kurwanya umuvuduko ukabije, ubuzima bwinkingi ndende, igiciro gito, nibindi birakenewe;
- Hitamo inkingi ikwiye ya chromatografiya: sobanukirwa nibigize, imiterere yumubiri nubumashini byuzuza chromatografique, nkubunini bwibice, ingano ya pore, kwihanganira ubushyuhe, kwihanganira pH, adsorption ya analyte, nibindi.
- Ibitekerezo byo guhitamo amazi ya chromatografiya
Iki gice kizaganira kubintu bigomba kwitabwaho muguhitamo inkingi ya chromatografi uhereye kumiterere yumubiri na chimique ya chromatografi ubwayo. 2.1 Matrix yuzuza
2.1.1 Matrix ya Silica gel Matrix yuzuza inkingi nyinshi za chromatografiya ni silika gel. Ubu bwoko bwuzuza bufite isuku nyinshi, igiciro gito, imbaraga za mashini nyinshi, kandi biroroshye guhindura amatsinda (nka fenyl ihuza, guhuza amino, guhuza cyano, nibindi), ariko agaciro ka pH nubushyuhe bwihanganira ni bike: the pH urwego rwinshi rwa silika gel matrix yuzuza ni 2 kugeza 8, ariko pH urwego rwibintu byahinduwe byumwihariko silika gel ihujwe ibyiciro birashobora kuba binini nka 1.5 kugeza 10, kandi hariho na silika gel yahinduwe byumwihariko ibyiciro bihamye kuri pH nkeya, nka Agilent ZORBAX RRHD stabilbond-C18, ihagaze kuri pH 1 kugeza 8; ubushyuhe bwo hejuru ntarengwa ya materique ya silika gel ni 60 ℃, kandi inkingi zimwe za chromatografi zirashobora kwihanganira ubushyuhe bwa 40 ℃ kuri pH ndende.
2.1.2. Ibyiza byabo nuko bashobora kwihanganira intera nini ya pH - irashobora gukoreshwa murwego rwa 1 kugeza 14, kandi irwanya ubushyuhe bwinshi (irashobora kugera hejuru ya 80 ° C). Ugereranije na silika ishingiye kuri C18 yuzuza, ubu bwoko bwuzuza bufite hydrophobicity ikomeye, kandi polymer ya macroporome ifite akamaro kanini mugutandukanya ingero nka proteyine. Ibibi byayo ni uko inkingi ikora neza kandi imbaraga za mashini zikaba nke cyane kuruta izuzuye silika. 2.2 Imiterere
Ibyinshi muri HPLC byuzuza ni uduce duto, ariko rimwe na rimwe ni ibice bidasanzwe. Ibice bya serefegitura birashobora gutanga inkingi yo hasi, inkingi yo hejuru ikora neza, ituze nubuzima burebure; mugihe ukoresheje ibyiciro bigendanwa cyane (nka acide fosifori) cyangwa mugihe igisubizo cyicyitegererezo ari viscous, uduce duto duto dufite ubuso bunini bwihariye, ibyo bikaba bifasha cyane ibikorwa byuzuye byibyiciro byombi, kandi igiciro kiri hasi. Ingano y'ibice
Ingano ntoya, niko inkingi ikora neza kandi niko gutandukana, ariko niko birwanya umuvuduko mwinshi. Inkingi ikoreshwa cyane ni 5 μm ingano yubunini bwinkingi; niba gutandukana bisabwa ari hejuru, 1.5-3 μm yuzuza irashobora gutoranywa, bifasha mugukemura ikibazo cyo gutandukana kwa matrike igoye hamwe nibice byinshi. UPLC irashobora gukoresha 1.5 μm yuzuza; 10 mm cyangwa ingano nini yuzuye yuzuza akenshi ikoreshwa mugice cya kabiri gitegura cyangwa gitegura inkingi. 2.4 Ibirimo
Ibirimo bya karubone bivuga igipimo cyicyiciro cyahujwe hejuru ya silika gel, ifitanye isano nubuso bwihariye hamwe no gukwirakwiza icyiciro. Ibirimo byinshi bya karubone bitanga inkingi ndende nubushobozi buhanitse, kandi akenshi bikoreshwa kuburugero rugoye rusaba gutandukana cyane, ariko kubera igihe kirekire cyo gukorana hagati yibyiciro byombi, igihe cyo gusesengura ni kirekire; karubone nkeya ya chromatografi yinkingi ifite igihe gito cyo gusesengura kandi irashobora kwerekana amahitamo atandukanye, kandi akenshi ikoreshwa kuburugero rworoshye rusaba isesengura ryihuse hamwe nicyitegererezo gisaba ibihe byamazi yo hejuru. Muri rusange, karubone ya C18 iri hagati ya 7% na 19%. 2.5 Ingano nini nubuso bwihariye
HPLC itangazamakuru rya adsorption ni ibice byoroshye, kandi imikoranire myinshi ibera mumyenge. Kubwibyo, molekile zigomba kwinjira mu byobo kugirango zamamazwe kandi zitandukanye.
Ingano nini nubuso bwihariye nibintu bibiri byuzuzanya. Ingano ntoya isobanura ubuso bunini bwihariye, naho ubundi. Ubuso bunini bwihariye bushobora kongera imikoranire hagati ya molekile ntangarugero nicyiciro cyahujwe, kongera imbaraga zo kugumana, kongera urugero rwo gupakira hamwe nubushobozi bwinkingi, no gutandukanya ibice bigoye. Byuzuye byuzuye byuzuye mubwoko bwuzuye. Kubafite ibisabwa byo gutandukana cyane, birasabwa guhitamo ibyuzura bifite ubuso bunini bwihariye; agace gato kihariye gashobora kugabanya umuvuduko winyuma, kunoza imikorere yinkingi, no kugabanya igihe cyo kuringaniza, kibereye gusesengura buhoro buhoro. Core-shell yuzuza ni ubu bwoko bwuzuza. Hashingiwe ku kwemeza gutandukana, birasabwa guhitamo ibyuzuza bifite ubuso buto bwihariye kubantu bafite isesengura ryiza risabwa. 2.6 Ingano nini nimbaraga za mashini
Ingano ya pore, izwi kandi nka "pore volume", yerekeza ku bunini bwubunini bwubusa kuri buri gice. Irashobora kwerekana neza imbaraga za mashini zuzuza. Imbaraga zumukanishi zuzuza nubunini bunini bwa pore nintege nke ugereranije niyuzuza hamwe nubunini buto. Abuzuza amajwi ya pore munsi cyangwa angana na 1.5 mL / g bakoreshwa cyane mugutandukanya HPLC, mugihe abuzuza bafite pore irenga 1.5 mL / g bikoreshwa cyane cyane mukwirukana molekuline ya chromatografiya hamwe na chromatografiya yumuvuduko muke. Igipimo cyo gufata
Gufata birashobora kugabanya impinga yumurizo iterwa nubusabane hagati yimvange hamwe nitsinda rya silanol ryerekanwe (nko guhuza ionic guhuza ibibyimba bya alkaline hamwe nitsinda rya silanol, imbaraga za der der Waals hamwe na hydrogène ihuza aside aside hamwe nitsinda rya silanol), bityo bikazamura imikorere yinkingi nuburyo bwo hejuru . Ibice bidafunze ibyiciro bizatanga amahitamo atandukanye ugereranije nibice bifatanye, cyane cyane kuburugero.
- Ingano yimikorere ya chromatografiya itandukanye
Iki gice kizasobanura urwego rwimikorere yubwoko butandukanye bwamazi ya chromatografiya yinkingi zinyuze mubibazo bimwe.
3.1 Guhindura-icyiciro C18 inkingi ya chromatografiya
Inkingi ya C18 niyo ikoreshwa cyane ihinduranya -cyiciro-cyiciro, gishobora guhura nibirimo hamwe n ibizamini byanduye byibintu byinshi kama, kandi birakoreshwa mubintu bito-bito, byoroheje cyane kandi bidafite inkingi. Ubwoko nibisobanuro bya C18 chromatografi yinkingi bigomba guhitamo ukurikije ibisabwa byihariye byo gutandukana. Kurugero, kubintu bifite ibisabwa byo gutandukana cyane, 5 μm * 4,6 mm * 250 mm ibisobanuro bikunze gukoreshwa; kubintu bifite matrices yo gutandukanya bigoye hamwe na polarite isa, 4 μ m * 4,6 mm * 250 mm ibisobanuro cyangwa ingano ntoya irashobora gukoreshwa. Kurugero, umwanditsi yakoresheje inkingi ya 3 mm * 4,6 mm * 250 mm kugirango amenye umwanda wa genotoxique muri celecoxib API. Gutandukanya ibintu byombi birashobora kugera kuri 2.9, nibyiza. Mubyongeyeho, hashingiwe ku kwemeza gutandukana, niba bisabwa gusesengura byihuse, inkingi ngufi ya mm 10 cyangwa 15 mm iratoranywa. Kurugero, mugihe umwanditsi yakoresheje LC-MS / MS kugirango amenye umwanda wa genotoxique muri piperaquine fosifate API, hakoreshejwe inkingi ya 3 μm * 2,1 mm * 100 mm. Gutandukanya umwanda nibice byingenzi byari 2.0, kandi gutahura icyitegererezo birashobora kurangira muminota 5. 3.2 Inkingi ihinduye-fenil inkingi
Inkingi ya fenyl nayo ni ubwoko bwisubiraho -cyiciro. Ubu bwoko bwinkingi bufite guhitamo gukomeye kubintu byiza. Niba igisubizo cyibintu bya aromatic byapimwe ninkingi isanzwe ya C18 ifite intege nke, urashobora gutekereza gusimbuza inkingi ya fenyl. Kurugero, mugihe narimo nkora celecoxib API, igisubizo cyingenzi cyibipimo byapimwe ninkingi ya fenyl yumushinga umwe kandi ibisobanuro bimwe (byose 5 μm * 4,6 mm * 250 mm) byikubye inshuro 7 iby'inkingi ya C18. 3.3 Inkingi isanzwe
Nka nyongera yingirakamaro kuri rezo-fase inkingi, ibisanzwe-fonctionnement inkingi irakwiriye kubintu byinshi bya polar. Niba impinga ikirihuta cyane mugihe uteruye hamwe na 90% yicyiciro cyamazi mugice cyahinduwe -cyiciro, ndetse no hafi no guhuzagurika hamwe nimpinga ya solve, urashobora gutekereza gusimbuza inkingi isanzwe. Ubu bwoko bwinkingi burimo inkingi ya hilic, inkingi ya amino, inkingi ya cyano, nibindi.
3.3.1 Inkingi ya Hilic Inkingi ya Hilic isanzwe ikubiyemo amatsinda ya hydrophilique mumurongo wa alkyl uhujwe kugirango wongere igisubizo kubintu bya polar. Ubu bwoko bwinkingi burakwiriye gusesengura ibintu byisukari. Umwanditsi yakoresheje ubu bwoko bwinkingi mugihe akora ibirimo nibintu bifitanye isano na xylose nibiyikomokaho. Isomers yinkomoko ya xylose nayo irashobora gutandukana neza;
3.3. yo gutandukanya isukari, aside amine, shingiro, na amide; inkingi ya cyano ifite uburyo bwiza bwo guhitamo mugihe itandukanya hydrogenated na hydrogenated structure ibintu bisa bitewe nuko hariho imiyoboro ihujwe. Inkingi ya Amino na cyano inkingi irashobora guhindurwa hagati yicyiciro gisanzwe nicyiciro cyinyuma, ariko guhinduranya kenshi ntabwo byemewe. 3.4 Inkingi ya Chiral
Inkingi ya Chiral, nkuko izina ribigaragaza, irakwiriye gutandukanya no gusesengura ibice bya chiral, cyane cyane mubijyanye na farumasi. Ubu bwoko bwinkingi burashobora gusuzumwa mugihe gisanzwe gisubiza inyuma nibisanzwe icyiciro cyinkingi ntigishobora kugera kubitandukanya na isomers. Kurugero, umwanditsi yakoresheje 5 μm * 4,6 mm * 250 mm ya chiral inkingi kugirango atandukane isomers ebyiri za 1,2-diphenylethylenediamine: (1S, 2S) -1, 2-diphenylethylenediamine na (1R, 2R) -1, 2 -diphenylethylenediamine, no gutandukana byombi byageze kuri 2.0. Nyamara, chiral inkingi zihenze kuruta ubundi bwoko bwinkingi, mubisanzwe 1W + / igice. Niba hakenewe inkingi nkizo, igice gikeneye gukora bije ihagije. 3.5 Inkingi yo guhana
Inkingi ya Ion ikwiranye no gutandukanya no gusesengura ion zashizwemo, nka ion, proteyine, acide nucleic, hamwe nibintu bimwe byisukari. Ukurikije ubwoko bwuzuza, bagabanijwemo cation yo guhanahana inkingi, anion yo guhana inkingi, hamwe nimbaraga zikomeye zo guhana.
Inkingi zo guhanahana inkingi zirimo calcium ishingiye kuri calcium na hydrogène ishingiye ku nkingi, zikwiriye cyane cyane gusesengura ibintu bya cationic nka acide amine. Kurugero, umwanditsi yakoresheje calcium ishingiye kuri calcium mugihe asesenguye calcium gluconate na calcium acetate mugisubizo cyoroshye. Ibintu byombi byari bifite ibisubizo bikomeye kuri λ = 210nm, kandi impamyabumenyi yo gutandukana yageze kuri 3.0; umwanditsi yakoresheje hydrogène ishingiye ku nkingi iyo asesenguye ibintu bifitanye isano na glucose. Ibintu byinshi byingenzi bifitanye isano - maltose, maltotriose na fructose - byari bifite sensibilité nyinshi munsi ya disiketi zitandukanye, aho igipimo cyo gutahura kiri munsi ya 0.5 ppm na dogere ya 2.0-2.5.
Inkingi zo guhanahana Anion zirakwiriye cyane cyane gusesengura ibintu bya anionic nka acide organic na ion ya halogene; inkingi zikomeye zo guhana inkingi zifite ubushobozi bwo guhanahana ion no guhitamo, kandi birakwiriye gutandukana no gusesengura ingero zitoroshye.
Ibyavuzwe haruguru ni intangiriro yubwoko hamwe nibisabwa murwego rwibisanzwe byamazi ya chromatografiya hamwe nubunararibonye bwumwanditsi. Hariho ubundi bwoko bwihariye bwa chromatografiya mubikorwa bifatika, nkibinini binini bya chromatografiya, inkingi ntoya ya chromatografiya, inkingi ya chromatografiya, inkingi za chromatografi, multimode chromatografiya, ultra-high performance fluid chromatography columns (UHPLC), super superritical fluid chromatography columns (UHPLC) SFC), nibindi bigira uruhare runini mubice bitandukanye. Ubwoko bwihariye bwa chromatografi yinkingi bugomba gutoranywa ukurikije imiterere nimiterere yicyitegererezo, ibisabwa byo gutandukana nibindi bigamije.
Igihe cyo kohereza: Jun-14-2024