I-chromatography ye-Liquid yindlela ephambili yokuvavanya umxholo wecandelo ngalinye kunye nokungcola kwizinto eziluhlaza, i-intermediates, amalungiselelo kunye nezinto zokupakisha, kodwa izinto ezininzi azikho iindlela eziqhelekileyo zokuxhomekeka, ngoko ke akunakuphepheka ukuphuhlisa iindlela ezintsha. Kuphuhliso lweendlela zesigaba solwelo, ikholamu yechromatographic ngundoqo wechromatography yolwelo, ngoko ke indlela yokukhetha ikholamu efanelekileyo yechromatographic ibalulekile. Kweli nqaku, umbhali uya kuchaza indlela yokukhetha ikholamu yechromatography elulwelo kwimiba emithathu: izimvo ngokubanzi, ukuqwalaselwa kunye nobubanzi besicelo.
A.Iingcamango zizonke zokukhetha ikholamu yechromatography engamanzi
1. Ukuvavanya iimpawu ezibonakalayo kunye neekhemikhali ze-analyte: ezifana nokwakheka kweekhemikhali, ukunyibilika, ukuzinza (njengokuthi kulula ukuba i-oxidized / iyancipha / i-hydrolyzed), i-acidity kunye ne-alkalinity, njl., ngakumbi i-chemical structure ingundoqo. into ekumiseleni iipropathi, ezifana neqela elidityanisiweyo line-ultraviolet eyomeleleyo kunye ne-fluorescence eyomeleleyo;
2. Qinisekisa injongo yokuhlalutya: ingaba ukuhlukana okuphezulu, ukusebenza kakuhle kwekholomu, ixesha elifutshane lokuhlalutya, uvakalelo oluphezulu, ukuxhatshazwa koxinzelelo oluphezulu, ubomi bekholomu ende, ixabiso eliphantsi, njl.
- Khetha ikholamu yechromatographic efanelekileyo: qonda ukubunjwa, iipropati ezibonakalayo kunye neekhemikhali ze-chromatographic filler, ezifana nobukhulu be-particle, ubukhulu be-pore, ukunyamezela kweqondo lokushisa, ukunyamezela kwe-pH, i-adsorption ye-analyte, njl.
- Iingqwalasela zokukhetha iikholamu zekhromatografi yolwelo
Esi sahluko siza kuxubusha izinto eziza kuqwalaselwa xa ukhetha ikholamu yechromatography ukusuka kumbono weempawu ezibonakalayo kunye neekhemikhali zekholamu yechromatography ngokwayo. 2.1 Imatriksi yokuzalisa
2.1.1 I-silica gel matrix I-matrix yokuzalisa uninzi lwezintlu zekhromatografi ezimanzi yijeli ye-silica. Olu hlobo lokuzalisa lunokucoceka okuphezulu, ixabiso eliphantsi, amandla amakhulu omatshini, kwaye kulula ukuguqula amaqela (afana ne-phenyl bonding, i-amino bonding, i-cyano bonding, njl. Uluhlu lwe-pH uninzi lwe-silica gel matrix fillers yi-2 ukuya kwi-8, kodwa uluhlu lwe-pH lwezigaba ezidityanisiweyo ze-silica ezidityanisiweyo zinokuba banzi nge-1.5 ukuya kwi-10, kwaye kukho izigaba ezidityaniswe ngokukodwa ze-silica gel ezizinzileyo kwi-pH ephantsi. njenge-Agilent ZORBAX RRHD stablebond-C18, ezinzileyo kwi-pH 1 ukuya kwi-8; Umda wobushushu obuphezulu be-silica gel matrix uqhele ukuba yi-60 ℃, kwaye ezinye iikholamu zechromatography zinokunyamezela ubushushu obungama-40 ℃ kwi-pH ephezulu.
2.1.2 Iipolymer matrix Izizalisi zePolymer ubukhulu becala ziyi-polystyrene-divinylbenzene okanye i-polymethacrylate. Iinzuzo zabo kukuba ziyakwazi ukunyamezela uluhlu olubanzi lwe-pH - zingasetyenziswa kwi-1 ukuya kwi-14, kwaye ziyakwazi ukumelana nokushisa okuphezulu (zinokufikelela ngaphezu kwe-80 ° C). Xa kuthelekiswa ne-silica-based C18 fillers, olu hlobo lokuzalisa lune-hydrophobicity enamandla, kwaye i-polymer ye-macroporous iphumelele kakhulu ekuhlukaniseni iisampuli ezifana neeprotheni. Ukungalungi kwayo kukuba ukusebenza kakuhle kwekholomu kuphantsi kwaye amandla omatshini abuthathaka kunokuba afakwe kwi-silica-based fillers. 2.2 Imilo yesuntswana
Uninzi lwangoku lwe-HPLC fillers ngamasuntswana angqukuva, kodwa ngamanye amaxesha ngamasuntswana angaqhelekanga. Iincinci zeSpherical zinokubonelela ngoxinzelelo lwekholamu ephantsi, ukusebenza kakuhle kwekholomu ephezulu, ukuzinza kunye nobomi obude; xa usebenzisa izigaba ezihamba phambili ze-viscosity (ezifana ne-phosphoric acid) okanye xa isisombululo sesampuli sibonakala, iinqununu ezingaqhelekanga zinendawo enkulu yendawo ethile, ehambelana kakhulu nesenzo esipheleleyo sezigaba ezimbini, kwaye ixabiso liphantsi. 2.3 Ubungakanani besuntswana
Ubuncinci besayizi ye-particle, iphezulu i-column esebenzayo kwaye iphakamileyo yokuhlukana, kodwa kubi kakhulu ukuxhatshazwa koxinzelelo oluphezulu. Ikholamu esetyenziswa ngokuqhelekileyo yikholomu ye-5 μm yobungakanani be-particle; ukuba imfuno yokwahlukana iphezulu, i-filler ye-1.5-3 μm inokukhethwa, efanelekileyo ekusombululeni ingxaki yokwahlukana kwe-matrix eyinkimbinkimbi kunye neesampuli zamacandelo amaninzi. I-UPLC ingasebenzisa i-1.5 μm fillers; I-10 μm okanye i-particle size size fillers isetyenziswe rhoqo kwiikholamu zokulungiselela isiqingatha okanye zokulungiselela. 2.4 Umxholo wekhabhoni
Umxholo wekhabhoni ubhekiselele kumlinganiselo wesigaba esibotshiweyo kumphezulu wejeli ye-silica, enxulumene nommandla othile ongaphezulu kunye nesigaba esidityanisiweyo. Umxholo wekhabhoni ophezulu unikeza umthamo ophezulu wekholomu kunye nesisombululo esiphezulu, kwaye sihlala sisetyenziselwa iisampuli ezinzima ezifuna ukuhlukana okuphezulu, kodwa ngenxa yexesha elide lokusebenzisana phakathi kwezigaba ezimbini, ixesha lokuhlalutya lide; i-carbon content chromatographic columns inexesha elifutshane lokuhlalutya kwaye lingabonisa izinto ezikhethiweyo ezahlukeneyo, kwaye zihlala zisetyenziselwa iisampuli ezilula ezifuna uhlalutyo olukhawulezayo kunye neesampuli ezifuna iimeko zesigaba esiphezulu samanzi. Ngokuqhelekileyo, umxholo wekhabhoni weC18 uvela kwi-7% ukuya kwi-19%. 2.5 Ubungakanani bepore kunye nommandla othile womphezulu
Imidiya ye-adsorption ye-HPLC ngamasuntswana angama-porous, kwaye uninzi lwentsebenziswano lwenzeka kwi-pores. Ke ngoko, iimolekyuli kufuneka zingene kwiipores ukuze zibhengezwe kwaye zihlulwe.
Ubungakanani bePore kunye nommandla othile ongaphezulu ziikhonsepthi ezimbini ezihambelanayo. Ubungakanani bemingxuma encinci buthetha indawo enkulu yendawo ethile, kwaye ngokuphambeneyo. Indawo enkulu yendawo ethile inokunyusa intsebenziswano phakathi kweeamolekyu zesampula kunye nezigaba ezidibeneyo, ukwandisa ukugcinwa, ukwandisa isampuli yokulayisha kunye nomthamo wekholomu, kunye nokuhlukana kwamacandelo anzima. Iifayili ezigcweleyo ezigcweleyo zolu hlobo lwezihluzi. Kulabo abaneemfuno eziphezulu zokwahlula, kucetyiswa ukuba ukhethe iifayili ezinommandla omkhulu ocacileyo; indawo encinci yendawo encinci inokunciphisa uxinzelelo lwangasemva, iphucule ukusebenza kakuhle kwekholomu, kunye nokunciphisa ixesha lokulinganisa, elilungele ukuhlalutya i-gradient. Izihluzi ze-core-shell zikolu hlobo lokuzalisa. Kwisiseko sokuqinisekisa ukuhlukana, kuyacetyiswa ukuba ukhethe iifayili ezinommandla omncinci ocacileyo kulabo abaneemfuno eziphezulu zokuhlalutya. 2.6 Umthamo wepore kunye namandla omatshini
Umthamo wepore, owaziwa ngokuba yi "pore volume", ubhekisa kubungakanani bomthamo ongenanto kwiyunithi yeyunithi. Ingabonakalisa kakuhle amandla omatshini wokugcwalisa. Amandla omatshini wezihluzi ezinomthamo omkhulu wepore ubuthathaka kancinci kunezahluli ezinomthamo omncinci wepore. Iifayili ezinomthamo we-pore ongaphantsi okanye olingana ne-1.5 mL / g zisetyenziselwa kakhulu ukwahlukana kwe-HPLC, ngelixa iifayili ezinomthamo we-pore omkhulu kune-1.5 mL / g zisetyenziselwa ikakhulu ukukhutshwa kwe-molecular chromatography kunye ne-chromatography yoxinzelelo oluphantsi. 2.7
I-Capping inokunciphisa iincopho ze-tailing ezibangelwa yintsebenziswano phakathi kweekhompawundi kunye namaqela e-silanol abonakalisiweyo (njengokubambisana kwe-ionic phakathi kweekhompawundi ze-alkaline kunye namaqela e-silanol, amandla e-van der Waals kunye neebhondi ze-hydrogen phakathi kweekhompawundi ze-acidic kunye namaqela e-silanol), ngaloo ndlela kuphuculwe ukusebenza kakuhle kwekholomu kunye nokuma okuphezulu. . Izigaba ezidityanisiweyo ezingafakwanga ziya kuvelisa ukhetho olwahlukileyo olunxulumene nezigaba ezidityanisiweyo, ngakumbi kwiisampulu zepolar.
- Umda wosetyenziso lwekholam ezahlukeneyo zekhromatografi yolwelo
Esi sahluko siza kuchaza umda wesicelo seentlobo ezahlukeneyo zekholamu yolwelo lwechromatography ngezinye iimeko.
3.1 Ikholamu yechromatographic yesigaba esibuyiselwe umva
Ikholomu ye-C18 iyona nto isetyenziswa ngokuqhelekileyo kwikholamu yesigaba esiguqulwayo, esinokuhlangabezana nomxholo kunye neemvavanyo zokungcola kwezinto ezininzi eziphilayo, kwaye zisebenza kwi-medium-polar, i-polar ebuthakathaka kunye ne-non-polar substances. Uhlobo kunye neenkcukacha zekholamu yechromatographic C18 kufuneka zikhethwe ngokweemfuno ezithile zokwahlula. Ngokomzekelo, kwizinto ezineemfuno eziphezulu zokuhlukana, i-5 μm * 4.6 mm * 250 mm imilinganiselo isetyenziswa rhoqo; kwizinto ezinematriki zokwahlula ezintsonkothileyo kunye ne-polarity efanayo, i-4 μm * 4.6 mm * 250 mm inkcazo okanye i-particle encinci ingasetyenziswa. Ngokomzekelo, umbhali wasebenzisa ikholomu ye-3 μm * 4.6 mm * 250 mm ukufumana ukungcola okubili kwe-genotoxic kwi-celecoxib API. Ukwahlulwa kwezinto ezimbini kunokufikelela kwi-2.9, eyona nto ibalaseleyo. Ukongezelela, phantsi kwesiseko sokuqinisekisa ukuhlukana, ukuba uhlalutyo olukhawulezayo lufunekayo, ikholamu emfutshane ye-10 mm okanye i-15 mm isoloko ikhethwa. Ngokomzekelo, xa umbhali esebenzisa i-LC-MS / MS ukufumanisa ukungcola kwe-genotoxic kwi-piperaquine phosphate API, ikholomu ye-3 μm * 2.1 mm * 100 mm isetyenziswe. Ukwahlula phakathi kokungcola kunye necandelo eliphambili laliyi-2.0, kwaye ukufunyanwa kwesampuli kunokugqitywa kwimizuzu ye-5. 3.2 Ikholamu yephenyl yesigaba esibuyiselwe umva
Ikholamu yePhenyl nayo luhlobo lwekholamu yesigaba esibuyiselwe umva. Olu hlobo lwekholamu lunokukhetha okunamandla kwiikhompawundi ezinuka kamnandi. Ukuba impendulo yeekhompawundi ezinuka kamnandi ezilinganiswe yikholamu eqhelekileyo yeC18 ibuthathaka, unokuqwalasela ukubuyisela ikholamu yephenyl. Ngokomzekelo, xa ndenza i-celecoxib API, impendulo yecandelo eliphambili elilinganiswa nekholamu ye-phenyl yomenzi ofanayo kunye neenkcukacha ezifanayo (zonke i-5 μm * 4.6 mm * 250 mm) malunga namaxesha angama-7 kwikholamu ye-C18. 3.3 Ikholam yesigaba esiqhelekileyo
Njengesongezelelo esisebenzayo kwikholamu yesigaba esiguqulwayo, ikholamu yesigaba esiqhelekileyo ifanelekile kwiikhompawundi ze-polar. Ukuba i-peak isakhawuleza kakhulu xa i-eluting ngaphezu kwe-90% yesigaba se-aqueous kwikholamu ye-reverse-phase column, kwaye isondele kwaye igqithe kunye nencopho ye-solvent, unokucinga ngokutshintshela ikholamu yesigaba esiqhelekileyo. Olu hlobo lwekholomu lubandakanya ikholamu ye-hilic, ikholamu ye-amino, ikholamu ye-cyano, njl.
3.3.1 Ikholamu ye-Hilic Ikholamu ye-Hilic idla ngokufaka amaqela e-hydrophilic kwikhonkco le-alkyl elidityanisiweyo ukuze kuphuculwe impendulo kwizinto ze-polar. Olu hlobo lwekholomu lufanelekile uhlalutyo lwezinto zeswekile. Umbhali wasebenzisa olu hlobo lwekholomu xa esenza umxholo kunye nezinto ezinxulumene nexylose kunye nezinto eziphuma kuzo. Ii-isomers ze-xylose derivative nazo zinokwahlulwa kakuhle;
3.3.2 Ikholomu ye-amino kunye nekholomu ye-cyano Ikholomu ye-amino kunye nekholomu ye-cyano ibhekisela ekuqalisweni kwe-amino kunye nokuguqulwa kwe-cyano ekupheleni kwekhonkco ye-alkyl eboshiweyo, ngokulandelanayo, ukuphucula ukukhethwa kwezinto ezikhethekileyo: umzekelo, ikholomu ye-amino lukhetho oluhle. ukwahlula iswekile, iiamino acids, iziseko kunye neeamide; ikholamu yecyano inokhetho olungcono xa kwahlula i-hydrogenated kunye ne-unhydrogenated yesakhiwo sezinto ezifanayo ngenxa yobukho be-conjugated bonds. Ikholomu ye-Amino kunye nekholomu ye-cyano inokutshintshwa rhoqo phakathi kwekholamu yesigaba esiqhelekileyo kunye nekholamu yesigaba esibuyisela umva, kodwa ukutshintsha rhoqo akukhuthazwa. 3.4 Ikholamu yeChiral
Ikholomu ye-Chiral, njengoko igama libonisa, ifanelekile ukuhlukana kunye nohlalutyo lwe-chiral compounds, ngokukodwa kwintsimi yamachiza. Olu hlobo lwekholomu lunokuthi luqwalaselwe xa iikholomu zesigaba esiqhelekileyo kunye nezigaba eziqhelekileyo azikwazi ukufezekisa ukuhlukana kwee-isomers. Umzekelo, umbhali usebenzise i-5 μm * 4.6 mm * 250 mm ikholamu ye-chiral ukwahlula isomers ezimbini ze-1,2-diphenylethylenediamine: (1S, 2S) -1, 2-diphenylethylenediamine kunye (1R, 2R) -1, 2 -diphenylethylenediamine, kwaye ukwahlukana phakathi kwezi zibini kufikelele malunga ne-2.0. Nangona kunjalo, iikholamu ze-chiral zibiza kakhulu kunezinye iindidi zekholamu, ngokuqhelekileyo i-1W +/piece. Ukuba kukho imfuneko yeekholomu ezinjalo, iyunithi kufuneka yenze uhlahlo lwabiwo-mali olwaneleyo. 3.5 Ikholamu yokutshintshiselana nge-ion
Iikholamu zokutshintshiselana kwe-Ion zifanelekile ukwahlula kunye nohlalutyo lwee-ion ezihlawulisiweyo, ezifana ne-ion, iiprotheni, i-nucleic acids, kunye nezinye izinto zeswekile. Ngokohlobo lokuzalisa, zahlulwe zibe yikholomu zotshintshiselwano lwe-cation, iikholomu zotshintshiselwano lwe-anion, kunye neekholamu zotshintshiselwano ezinamandla.
Iikholamu zokutshintshiselana nge-cation ziquka i-calcium-based based and hydrogen-based columns, eyona nto ifaneleke kakhulu uhlalutyo lwezinto ze-cationic ezifana ne-amino acids. Ngokomzekelo, umbhali wasebenzisa iikholomu ezisekelwe kwi-calcium xa ehlalutya i-calcium gluconate kunye ne-calcium acetate kwisisombululo sokugungqa. Zomibini izinto zineempendulo ezinamandla kwi-λ=210nm, kwaye idigri yokwahlukana yafikelela kwi-3.0; umbhali wasebenzisa iikholomu ezisekelwe kwi-hydrogen xa ehlalutya izinto ezinxulumene ne-glucose. Izinto ezininzi ezinxulumene nazo - i-maltose, i-maltotriose kunye ne-fructose - yayinovakalelo oluphezulu phantsi kwee-detectors ezihlukeneyo, kunye nomda wokufumanisa ophantsi kwe-0.5 ppm kunye ne-degree yokwahlukana kwe-2.0-2.5.
Iikholomu zotshintshiselwano ze-Anion zifanelekile kakhulu uhlalutyo lwezinto ze-anionic ezifana ne-organic acids kunye ne-halogen ions; iikholomu zokutshintshiselana nge-cation ezinamandla zinomthamo ophezulu wokutshintshiselana kwe-ion kunye nokukhetha, kwaye zifanelekile ukwahlukana kunye nohlalutyo lweesampuli ezinzima.
Oku ngasentla sisingeniso nje kwiindidi kunye noluhlu lwezicelo lwekholamu ezininzi zolwelo oluqhelekileyo lwechromatography edityaniswe namava ombhali. Kukho ezinye iindidi ezikhethekileyo zekholamu yechromatographic kwizicelo ezizizo, ezinje ngekholamu yechromatographic enepore enkulu, ikholamu yechromatographic encinci, affinity chromatography columns, multimode chromatographic columns, Ultra-high performance liquid chromatography columns (UHPLC), supercritical fluid chromatography columns (UHPLC) SFC), njl. Badlala indima ebalulekileyo kwiinkalo ezahlukeneyo. Uhlobo oluthile lwekholamu yechromatographic kufuneka ikhethwe ngokwesakhiwo kunye neempawu zesampuli, iimfuno zokuhlukana kunye nezinye iinjongo.
Ixesha lokuposa: Jun-14-2024