I-Liquid chromatography iyindlela eyinhloko yokuhlola okuqukethwe kwengxenye ngayinye kanye nokungcola ezintweni ezingavuthiwe, okuphakathi, amalungiselelo nezinto zokupakisha, kodwa izinto eziningi azinazo izindlela ezijwayelekile zokuthembela kuzo, ngakho-ke akunakugwenywa ukuthuthukisa izindlela ezintsha. Ekuthuthukisweni kwezindlela zesigaba soketshezi, ikholomu ye-chromatographic iwumgogodla we-chromatography ewuketshezi, ngakho-ke indlela yokukhetha ikholomu efanelekile yechromatographic ibalulekile. Kulesi sihloko, umbhali uzochaza indlela yokukhetha ikholomu ye-chromatography ewuketshezi ezicini ezintathu: imibono iyonke, ukucatshangelwa kanye nobubanzi bohlelo lokusebenza.
A.Imibono isiyonke yokukhetha amakholomu echromatography ewuketshezi
1. Hlola izici ezibonakalayo namakhemikhali ze-analyte: njengesakhiwo samakhemikhali, ukuncibilika, ukuzinza (njengokuthi kulula yini ukufakwa i-oxidized/ukunciphisa/i-hydrolyzed), i-acidity ne-alkalinity, njll., ikakhulukazi isakhiwo samakhemikhali siyisihluthulelo. isici ekunqumeni izakhiwo, njengeqembu elihlanganisiwe linokumunca okunamandla kwe-ultraviolet kanye ne-fluorescence eqinile;
2. Thola inhloso yokuhlaziya: ukuthi ngabe ukuhlukaniswa okuphezulu, ukusebenza kahle kwekholomu ephezulu, isikhathi sokuhlaziya esifushane, ukuzwela okukhulu, ukumelana nokucindezela okukhulu, ukuphila kwekholomu ende, izindleko eziphansi, njll.
- Khetha ikholomu efanelekile ye-chromatographic: qonda ukwakheka, izici zomzimba nezamakhemikhali zesigcwalisi se-chromatographic, njengosayizi wezinhlayiyana, usayizi wembotshana, ukubekezelela izinga lokushisa, ukubekezelelwa kwe-pH, i-adsorption ye-analyte, njll.
- Ukucatshangelwa kokukhetha amakholomu e-chromatography ewuketshezi
Lesi sahluko sizoxoxa ngezinto okufanele zicatshangelwe lapho kukhethwa ikholomu ye-chromatography ngokombono wezakhiwo zomzimba nezamakhemikhali zekholomu ye-chromatography ngokwayo. 2.1 I-matrix yokugcwalisa
2.1.1 I-matrix yejeli ye-silica I-matrix yokugcwalisa yamakholomu amaningi e-chromatography ewuketshezi ijeli ye-silica. Lolu hlobo lokugcwalisa lunokuhlanzeka okuphezulu, izindleko eziphansi, amandla aphezulu emishini, futhi kulula ukuwashintsha amaqembu (njenge-phenyl bonding, amino bonding, cyano bonding, njll.), kodwa inani le-pH kanye nobubanzi bezinga lokushisa elibubekezelela bulinganiselwe: i Uhlu lwe-pH lwezigcwali eziningi ze-silica gel matrix zingu-2 kuye ku-8, kodwa uhla lwe-pH lwezigaba eziboshwe ngokukhethekile ze-silica gel lungaba ububanzi obusukela ku-1.5 kuye ku-10, futhi kukhona nezigaba ezihlanganiswe ngokukhethekile ze-silica gel ezizinzile ku-pH ephansi, njenge-Agilent ZORBAX RRHD stablebond-C18, ezinzile ku-pH 1 kuya ku-8; umkhawulo wokushisa ongaphezulu we-silica gel matrix uvamise ukuba ngu-60 ℃, futhi amanye amakholomu echromatography akwazi ukubekezelela izinga lokushisa elingu-40 ℃ ku-pH ephezulu.
2.1.2 Izigcwalisi ze-polymer matrix Polymer ngokuvamile ziyi-polystyrene-divinylbenzene noma i-polymethacrylate. Izinzuzo zabo ukuthi ziyakwazi ukubekezelela ububanzi be-pH - zingasetshenziswa ku-1 kuya ku-14, futhi zikwazi ukumelana nokushisa okuphezulu (zingafinyelela ngaphezu kuka-80 ° C). Uma kuqhathaniswa nezihlungi ze-C18 ezisekelwe ku-silica, lolu hlobo lokugcwalisa lune-hydrophobicity eqinile, futhi i-polymer ye-macroporous iphumelela kakhulu ekuhlukaniseni amasampula afana namaprotheni. Ukungalungi kwayo ukuthi ukusebenza kahle kwekholomu kuphansi futhi amandla emishini abuthakathaka kunalawo okugcwalisa i-silica-based. 2.2 Isimo se-particle
Izigcwalisi eziningi zesimanje ze-HPLC ziyizinhlayiya eziyindilinga, kodwa kwesinye isikhathi ziyizinhlayiya ezingajwayelekile. Izinhlayiya eziyindilinga zinganikeza ukucindezela kwekholomu ephansi, ukusebenza kahle kwekholomu ephakeme, ukuzinza nokuphila isikhathi eside; lapho usebenzisa izigaba zeselula ze-viscosity ephezulu (njenge-phosphoric acid) noma uma isisombululo sesampula sibonakala, izinhlayiya ezingavamile zinendawo enkulu yendawo ethize, evumelana kakhulu nesenzo esigcwele sezigaba ezimbili, futhi intengo iphansi kakhulu. 2.3 Usayizi wezinhlayiyana
Ubukhulu bezinhlayiyana buncane, buphakamisa ukusebenza kahle kwekholomu futhi kuphakeme ukuhlukana, kodwa kubi nakakhulu ukumelana nokucindezela okukhulu. Ikholomu esetshenziswa kakhulu yikholomu yosayizi wezinhlayiyana ezingu-5 μm; uma isidingo sokuhlukaniswa siphezulu, isigcwalisi esingu-1.5-3 μm singakhethwa, esivumelana nokuxazulula inkinga yokuhlukaniswa kwe-matrix ethile eyinkimbinkimbi kanye namasampula ezingxenye eziningi. I-UPLC ingasebenzisa izigcwalisi ze-1.5 μm; 10 μm noma izigcwalisi zosayizi wezinhlayiyana ezinkulu zivame ukusetshenziselwa amakholomu okulungiselela kancane noma okulungiselela. 2.4 Okuqukethwe kwekhabhoni
Okuqukethwe kwekhabhoni kubhekisela engxenyeni yesigaba esiboshiwe endaweni yejeli ye-silica, ehlobene nendawo ethile engaphezulu kanye nokufakwa kwesigaba esiboshiwe. Okuqukethwe kwekhabhoni ephezulu kunikeza umthamo wekholomu ephezulu kanye nokulungiswa okuphezulu, futhi kuvame ukusetshenziselwa amasampula ayinkimbinkimbi adinga ukuhlukaniswa okuphezulu, kodwa ngenxa yesikhathi eside sokuxhumana phakathi kwezigaba ezimbili, isikhathi sokuhlaziya side; amakholomu e-chromatographic okuqukethwe kwekhabhoni ephansi anesikhathi esifushane sokuhlaziya futhi angabonisa ukukhetha okuhlukile, futhi avame ukusetshenziselwa amasampula alula adinga ukuhlaziya okusheshayo namasampuli adinga izimo zesigaba samanzi aphezulu. Ngokuvamile, okuqukethwe kwekhabhoni ye-C18 kusuka ku-7% kuya ku-19%. 2.5 Ubukhulu bembotshana kanye nendawo ethile
I-HPLC adsorption media iyizinhlayiya ezinama-porous, futhi ukusebenzisana okuningi kwenzeka ezimbotsheni. Ngakho-ke, ama-molecule kufanele angene ezimbotsheni ukuze adsorbed futhi ahlukaniswe.
Usayizi we-Pore kanye nendawo ethile yendawo yimibono emibili ehambisanayo. Ubukhulu bembotshana encane busho indawo enkulu ethile, futhi ngokuphambene nalokho. Indawo enkulu ethile ingakhuphula ukusebenzisana phakathi kwama-molecule esampula nezigaba eziboshiwe, ithuthukise ukugcinwa, ikhulise isampula yokulayisha kanye nomthamo wekholomu, nokuhlukaniswa kwezingxenye eziyinkimbinkimbi. Izigcwalisi ezinama-porous ngokugcwele zingezalolu hlobo lokugcwalisa. Kulabo abanezidingo eziphakeme zokuhlukanisa, kunconywa ukukhetha ama-fillers anendawo enkulu ethize; indawo encane ethize inganciphisa ukucindezela emuva, ithuthukise ukusebenza kahle kwekholomu, futhi inciphise isikhathi sokulinganisa, esifanele ukuhlaziya i-gradient. Izigcwalisi ze-Core-shell zingezalolu hlobo lokugcwalisa. Ngokwesisekelo sokuqinisekisa ukuhlukana, kunconywa ukukhetha ama-fillers anendawo encane ethize yalabo abanezidingo eziphezulu zokuhlaziya. 2.6 Ivolumu yembotshana namandla emishini
I-pore volume, eyaziwa nangokuthi "i-pore volume", ibhekisela kusayizi wevolumu engenalutho ngeyunithi ngayinye. Ingabonisa kahle amandla omshini we-filler. Amandla omshini wama-filler anevolumu enkulu ye-pore abuthakathaka kancane kunalawo okugcwalisa anevolumu encane yembobo. Izigcwalisi ezinevolumu yembotshana engaphansi noma elingana no-1.5 mL/g isetshenziswa kakhulu ekuhlukaniseni i-HPLC, kuyilapho izigcwalisi ezinevolumu yembotshana enkulu kuno-1.5 mL/g zisetshenziselwa ikakhulukazi i-chromatography yokukhishwa kwamangqamuzana kanye ne-chromatography enengcindezi ephansi. 2.7 Isilinganiso sokulinganisa
I-Capping inganciphisa iziqongo zomsila ezibangelwa ukusebenzisana phakathi kwezinhlanganisela namaqembu e-silanol aveziwe (okufana nokuhlangana kwe-ionic phakathi kwezinhlanganisela ze-alkaline namaqembu e-silanol, amandla e-van der Waals kanye nezibopho ze-hydrogen phakathi kwezinhlanganisela ze-asidi namaqembu e-silanol), ngaleyo ndlela kuthuthukiswe ukusebenza kahle kwekholomu nokuma okuphezulu. . Izigaba eziboshiwe ezingafakwanga zizokhiqiza ukukhetha okuhlukile okuhlobene nezigaba eziboshiwe, ikakhulukazi amasampula e-polar.
- Ububanzi bohlelo lokusebenza lwamakholomu ahlukene we-liquid chromatography
Lesi sahluko sizochaza ububanzi bohlelo lokusebenza lwezinhlobo ezahlukene zamakholomu e-chromatography ewuketshezi kwezinye izimo.
3.1 Ikholomu ye-chromatographic yesigaba esibuyiselwe emuva C18
Ikholomu ye-C18 iyikholomu yesigaba esihlanekezelwe esetshenziswa kakhulu, engahlangabezana nokuhlolwa kokuqukethwe nokungcola kwezinto eziningi eziphilayo, futhi isebenza ezintweni ezitholakala endaweni epholile emaphakathi, ebuthaka kalula nezingezona ezomhlaba. Uhlobo nokucaciswa kwekholomu ye-C18 chromatographic kufanele kukhethwe ngokuya ngezidingo ezithile zokuhlukaniswa. Isibonelo, ezintweni ezinezidingo zokuhlukaniswa okuphezulu, ukucaciswa okungu-5 μm*4.6 mm*250 mm kuvame ukusetshenziswa; ezintweni ezinama-matrices okuhlukanisa ayinkimbinkimbi kanye ne-polarity efanayo, 4 μm * 4.6 mm * 250 mm ukucaciswa noma osayizi bezinhlayiyana ezincane zingasetshenziswa. Isibonelo, umbhali usebenzise ikholomu engu-3 μm*4.6 mm*250 mm ukuze athole ukungcola okubili kwe-genotoxic ku-celecoxib API. Ukuhlukaniswa kwezinto ezimbili kungafinyelela ku-2.9, okuyinto enhle kakhulu. Ukwengeza, ngaphansi kwesisekelo sokuqinisekisa ukuhlukana, uma ukuhlaziya okusheshayo kuyadingeka, ikholomu emfushane ye-10 mm noma i-15 mm ivame ukukhethwa. Isibonelo, lapho umbhali esebenzisa i-LC-MS/MS ukuze athole ukungcola kwe-genotoxic ku-piperaquine phosphate API, kusetshenziswe ikholomu engu-3 μm*2.1 mm*100 mm. Ukuhlukaniswa phakathi kokungcola nengxenye eyinhloko kwakungu-2.0, futhi ukutholwa kwesampula kungaqedwa emaminithini angu-5. 3.2 Ikholomu ye-phenyl yesigaba esibuyiselwe emuva
Ikholomu ye-Phenyl futhi iwuhlobo lwekholomu yesigaba esibuyiselwe emuva. Lolu hlobo lwekholomu lunokukhethwa okuqinile kwezinhlanganisela ezinuka kamnandi. Uma impendulo yenhlanganisela yephunga elimnandi elinganiswa ngekholomu evamile ye-C18 ibuthakathaka, ungacabanga ukufaka esikhundleni sekholomu ye-phenyl. Isibonelo, ngenkathi ngenza i-celecoxib API, impendulo yengxenye eyinhloko ikalwa ngekholomu ye-phenyl yomkhiqizi ofanayo kanye nokucaciswa okufanayo (konke okungu-5 μm*4.6 mm*250 mm) kwakucishe kube izikhathi ezingu-7 kunekholomu ye-C18. 3.3 Ikholomu yesigaba esijwayelekile
Njengesengezo esisebenzayo kukholomu yesigaba esihlehlisiwe, ikholomu yesigaba esivamile ifanele izinhlanganisela ze-polar ephezulu. Uma isiqongo sisashesha kakhulu uma sikhuphuka ngesigaba samanzi esingaphezu kuka-90% kukholomu yesigaba esibuyiselwe emuva, futhi sisondele futhi sigqagqene nesiqongo se-solvent, ungacabanga ukufaka esikhundleni sekholomu yesigaba esivamile. Lolu hlobo lwekholomu luhlanganisa ikholomu ye-hilic, ikholomu ye-amino, ikholomu ye-cyano, njll.
3.3.1 Ikholomu ye-Hilic Ikholomu ye-Hilic ivamise ukushumeka amaqembu e-hydrophilic ochungechungeni lwe-alkyl eliboshiwe ukuze kuthuthukiswe ukusabela ezintweni ezipholile. Lolu hlobo lwekholomu lufanele ukuhlaziywa kwezinto ezishukela. Umbhali usebenzise lolu hlobo lwekholomu lapho enza okuqukethwe nezinto ezihlobene ze-xylose nokuphuma kwayo. Ama-isomers we-xylose derivative nawo angahlukaniswa kahle;
3.3.2 Ikholomu ye-amino nekholomu yecyano Ikholomu ye-amino nekholomu yecyano ibhekisela ekwethulweni kokuguqulwa kwe-amino necyano ekugcineni kweketango le-alkyl eliboshiwe, ngokulandelana, ukuze kuthuthukiswe ukukhetha kwezinto ezikhethekile: isibonelo, ikholomu ye-amino iyisinqumo esihle. ukuhlukaniswa koshukela, ama-amino acid, izisekelo, nama-amide; Ikholomu ye-cyano inokukhetha okungcono uma ihlukanisa izinto ezifanayo zesakhiwo se-hydrogenated ne-unhydrogenated ngenxa yokuba khona kwamabhondi ahlanganisiwe. Ikholomu ye-amino kanye nekholomu yecyano ngokuvamile ingashintshwa phakathi kwekholomu yesigaba esivamile nekholomu yesigaba sokuhlehla, kodwa ukushintsha njalo akunconyiwe. 3.4 Ikholomu ye-Chiral
Ikholomu yeChiral, njengoba igama libonisa, ifanele ukuhlukaniswa nokuhlaziywa kwezinhlanganisela ze-chiral, ikakhulukazi emkhakheni wezokwelapha. Lolu hlobo lwekholomu lungacatshangelwa lapho amakholomu esigaba esibuyela emuva esivamile engakwazi ukufinyelela ukuhlukaniswa kwama-isomer. Isibonelo, umbhali usebenzise ikholomu ye-chiral engu-5 μm*4.6 mm*250 mm ukuhlukanisa ama-isomer amabili we-1,2-diphenylethylenediamine: (1S, 2S) -1, 2-diphenylethylenediamine kanye (1R, 2R) -1, 2 -diphenylethylenediamine, futhi ukuhlukaniswa phakathi kokubili kwafinyelela cishe ku-2.0. Nokho, amakholomu e-chiral abiza kakhulu kunezinye izinhlobo zamakholomu, ngokuvamile angu-1W+/ucezu. Uma kunesidingo samakholomu anjalo, iyunithi idinga ukwenza isabelomali esanele. 3.5 Ikholomu yokushintsha i-ion
Amakholomu okushintshanisa ama-ion afanele ukuhlukaniswa nokuhlaziywa kwama-ion ashajiwe, njengama-ion, amaprotheni, ama-nucleic acid, nezinye izinto zikashukela. Ngokohlobo lwesigcwalisi, ahlukaniswe amakholomu okushintshaniswa kwe-cation, amakholomu okushintshanisa ama-anion, namakholomu okushintshaniswa kwe-cation aqinile.
Amakholomu okushintshanisa i-cation ahlanganisa amakholomu asekelwe ku-calcium kanye nasekelwe ku-hydrogen, afaneleka kakhulu ukuhlaziya izinto ze-cationic ezifana nama-amino acid. Isibonelo, umbhali wasebenzisa amakholomu asekelwe ku-calcium lapho ehlaziya i-calcium gluconate ne-calcium acetate kusixazululo sokuguquguquka. Zombili izinto zinezimpendulo eziqinile ku-λ=210nm, futhi idigri yokuhlukanisa yafinyelela ku-3.0; umbhali wasebenzisa amakholomu asekelwe ku-hydrogen lapho ehlaziya izinto ezihlobene ne-glucose. Izinto eziningana ezibalulekile ezihlobene - i-maltose, i-maltotriose ne-fructose - yayinokuzwela okuphezulu ngaphansi kwezitholi ezihlukanisayo, ezinomkhawulo wokutholwa ophansi njengo-0.5 ppm kanye nedigri yokuhlukanisa engu-2.0-2.5.
Amakholomu okushintshanisa ama-Anion afaneleka kakhulu ukuhlaziya izinto ze-anionic ezifana nama-acids ephilayo nama-halogen ions; amakholomu okushintshaniswa kwe-cation aqinile anomthamo wokushintshaniswa kwe-ion ephakeme nokukhetha, futhi afanele ukuhlukaniswa nokuhlaziywa kwamasampuli ayinkimbinkimbi.
Okungenhla kumane kuyisethulo sezinhlobo nobubanzi bokusetshenziswa kwamakholomu amaningana avamile we-chromatography ewuketshezi ahlanganiswe nolwazi lombhali. Kukhona ezinye izinhlobo ezikhethekile zamakholomu e-chromatographic ezinhlelweni zangempela, njengamakholomu e-chromatographic anezimbotshana ezinkulu, amakholomu e-chromatographic anezimbotshana ezincane, amakholomu e-affinity chromatography, amakholomu e-chromatographic amaningi, amakholomu e-chromatography esebenzayo ephezulu kakhulu (UHPLC), amakholomu e-supercritical fluid chromatography ( SFC), njll. Badlala indima ebalulekile emikhakheni ehlukene. Uhlobo oluthile lwekholomu ye-chromatographic kufanele lukhethwe ngokuya ngesakhiwo nezici zesampula, izimfuneko zokuhlukaniswa nezinye izinjongo.
Isikhathi sokuthumela: Jun-14-2024