Liquid chromatography ndiyo nzira huru yekuyedza zviri mukati mechikamu chega chega uye kusvibiswa kwezvinhu zvakabikwa, zvepakati, gadziriro uye zvekurongedza zvinhu, asi zvinhu zvakawanda hazvina nzira dzakajairwa dzekuvimba nazvo, saka hazvidzivisike kugadzira nzira nyowani. Mukuvandudza nzira dzechikamu chemvura, iyo chromatographic koramu ndiyo musimboti weiyo mvura chromatography, saka nzira yekusarudza yakakodzera chromatographic column yakakosha. Muchinyorwa chino, munyori anotsanangura nzira yekusarudza yemvura chromatography column kubva pamatatu maficha: yakazara mazano, kufunga uye chiyero chekushandisa.
A.Zvose mazano ekusarudza liquid chromatography columns
1. Ongorora chimiro chemuviri uye chemakemikari emu analyte: zvakadai semakemikari maitiro, solubility, kugadzikana (sekunge zviri nyore kuva oxidized / kuderedzwa / hydrolyzed), acidity uye alkalinity, nezvimwewo, kunyanya chimiro chemakemikari ndicho chinhu chinokosha. chinhu pakusarudza zvivakwa, senge boka rakasanganiswa rine simba rekunyura ultraviolet uye yakasimba fluorescence;
2. Sarudza chinangwa chekuongorora: kana kupatsanurwa kwepamusoro, kushanda kwekoramu yakakwirira, nguva pfupi yekuongorora, kunzwisiswa kwepamusoro, kupikisa kwepamusoro, hupenyu hurefu hwekoramu, mari shoma, nezvimwewo zvinodiwa;
- Sarudza yakakodzera chromatographic koramu: nzwisisa kuumbwa, kwemuviri uye kemikari zvimiro zvechromatographic filler, senge chidimbu saizi, saizi yepore, kushivirira kwekushisa, pH kushivirira, adsorption yeanalyte, nezvimwe.
- Mafungiro ekusarudza liquid chromatography columns
Chitsauko ichi chichakurukura zvinhu zvinofanirwa kutariswa pakusarudza koramu yekromatografia kubva pakuona kwechimiro chemuviri uye chemakemikari zvimiro zvekromatografia pachezvayo. 2.1 Filler matrix
2.1.1 Silica gel matrix Iyo yekuzadza matrix yemvura zhinji chromatography column isilica gel. Rudzi urwu rwekuzadza rune kuchena kwakakwira, mutengo wakaderera, simba rakawanda remuchina, uye iri nyore kushandura mapoka (senge phenyl bonding, amino bonding, cyano bonding, nezvimwewo), asi iyo pH kukosha uye tembiricha yainoshivirira ishoma: iyo pH huwandu hweakawanda silica gel matrix fillers ndeye 2 kusvika 8, asi iyo pH yemhando yakagadziridzwa silica gel yakasungirirwa zvikamu inogona kuhupamhi se1.5 kusvika gumi, uye ipapo. ariwo anonyanya kugadziriswa silica gel bonded phases iyo yakagadzikana pane yakaderera pH, yakadai seAgilent ZORBAX RRHD stablebond-C18, iyo yakagadzikana pH 1 kusvika ku8; Iyo yepamusoro tembiricha muganho we silica gel matrix inowanzo 60 ℃, uye mamwe makoramu echromatography anogona kushivirira tembiricha ye40 ℃ pakakwirira pH.
2.1.2 Polymer matrix Polymer mafirita akanyanya polystyrene-divinylbenzene kana polymethacrylate. Zvavakanakira ndezvekuti vanogona kushivirira yakafara pH siyana - inogona kushandiswa muhuwandu hwe1 kusvika 14, uye ivo vanonyanya kurwisa tembiricha yepamusoro (inogona kusvika pamusoro pe80 ° C). Kuenzaniswa ne silica-based C18 fillers, iyi mhando yekuzadza ine yakasimba hydrophobicity, uye iyo macroporous polymer inoshanda zvakanyanya mukuparadzanisa sampuli senge mapuroteni. Zvayakaipira ndezvekuti ikorumu inoshanda yakadzikira uye simba remuchina harina kusimba pane iro resilica-based fillers. 2.2 Chimiro chechikamu
Mazhinji emazuva ano HPLC mafirita akatenderedzwa zvimedu, asi dzimwe nguva hazvina kujairika zvimedu. Spherical particles inogona kupa yakaderera column kudzvinyirirwa, yakakwirira koramu kunyatsoshanda, kugadzikana uye hupenyu hurefu; paunoshandisa high-viscosity mobile phases (yakadai sephosphoric acid) kana apo mhinduro yemuenzaniso iine viscous, zvimedu zvisingaenzaniswi zvine nzvimbo yakakura yepamusoro, iyo inonyatsoita kuti iite zvizere zvezvikamu zviviri, uye mutengo wacho wakaderera. 2.3 Chikamu saizi
Iyo diki saizi yechikamu, iyo yakakwirira yekoramu inoshanda uye yakakwirira kupatsanurwa, asi yakaipisisa iyo yakanyanya kudzvinyirira kuramba. Koramu inonyanya kushandiswa ndeye 5 μm particle size column; kana kupatsanurwa kunodiwa kwakakwirira, 1.5-3 μm filler inogona kusarudzwa, iyo inobatsira kugadzirisa dambudziko rekuparadzanisa kwemamwe matrix akaoma uye akawanda-chikamu sampuli. UPLC inogona kushandisa 1.5 μm fillers; 10 μm kana yakakura particle size fillers anowanzo shandiswa semi-yekugadzirira kana kugadzirira makoramu. 2.4 Carbon content
Kabhoni yemukati inoreva chikamu chechikamu chakasungirirwa pamusoro pesilica gel, ine hukama nenzvimbo yakatarwa uye yakasungwa chikamu chekuvhara. High carbon content inopa huwandu hwekoramu uye kugadziriswa kwepamusoro, uye inowanzoshandiswa kune sampuli dzakaoma dzinoda kuparadzaniswa kwepamusoro, asi nekuda kwenguva yakareba yekubatana pakati pezvikamu zviviri, nguva yekuongorora yakareba; low carbon content chromatographic columns ine nguva pfupi yekuongorora uye inogona kuratidza zvakasiyana-siyana, uye inowanzoshandiswa kune sampuli dzakareruka dzinoda kukurumidza kuongororwa uye sampuli dzinoda mamiriro epamusoro emvura. Kazhinji, iyo kabhoni yeC18 inotangira kubva ku7% kusvika ku19%. 2.5 Pore saizi uye chaiyo nzvimbo
HPLC adsorption media ane porous particles, uye kupindirana kwakawanda kunoitika mumapores. Naizvozvo, mamorekuru anofanirwa kupinda mumapores kuti adsorbed uye aparadzaniswe.
Saizi yePore uye yakananga nzvimbo yepasi ipfungwa mbiri dzinopindirana. Diki pore size inoreva nzvimbo yakakura chaiyo, uye zvakasiyana. Nzvimbo hombe chaiyo yepamusoro inogona kuwedzera kudyidzana pakati pemamolekemu emuenzaniso uye zvikamu zvakasungwa, kuwedzera kuchengetwa, kuwedzera sampuli kurodha uye makoramu kugona, uye kupatsanurwa kwezvinhu zvakaomarara. Zvizere porous fillers ndezverudzi urwu rwekuzadza. Kune avo vane yakakwira yekuparadzanisa zvinodiwa, zvinokurudzirwa kusarudza mafirita ane hombe chaiyo nzvimbo; diki yakananga nzvimbo inogona kuderedza kumashure kumanikidzwa, kunatsiridza mbiru kushanda zvakanaka, uye kuderedza yakaenzana nguva, iyo inokodzera gradient kuongororwa. Core-shell fillers ndeyemhando iyi yemafirita. Pachimiro chekuona kupatsanurwa, zvinokurudzirwa kusarudza mafirita ane diki chaiyo nzvimbo kune avo vane yakakwira ongororo inoshanda zvinodiwa. 2.6 Pore vhoriyamu uye simba remuchina
Pore vhoriyamu, inozivikanwawo se "pore vhoriyamu", inoreva saizi yevhoriyamu isina chinhu pachikamu chechikamu. Inogona kunyatso kuratidza simba remagetsi rekuzadza. Iyo mechanic simba yemafirita ane hombe pore vhoriyamu ishoma pane iyo yemafirita ane diki pore vhoriyamu. Mafirita ane pore vhoriyamu isingasviki kana kuenzana ne1.5 mL/g anonyanya kushandiswa kupatsanurwa kweHPLC, nepo mafirita ane pore vhoriyamu yakakura kupfuura 1.5 mL/g anonyanya kushandiswa kumisa mamorekuru chromatography uye yakaderera-kudzvanywa chromatography. 2.7 Capping rate
Kuvharisa kunogona kuderedza nhongonya dzemuswe dzinokonzerwa nekudyidzana pakati pemakomponi uye akafumurwa mapoka e silanol (senge ionic bonding pakati pealkaline makomisheni nemapoka e silanol, van der Waals masimba uye hydrogen zvisungo pakati peasidhi makomisheni nemapoka e silanol), nekudaro kuvandudza mashandiro ekoramu uye chimiro chepamusoro. . Zvikamu zvakasungwa zvisina kuvharwa zvinogadzira zvakasiyana-siyana zvine chekuita neakavharwa akasungwa zvikamu, kunyanya kune polar samples.
- Chiyero chekushandisa cheakasiyana emvura chromatography makoramu
Ichi chitsauko chinotsanangura chiyero chekushandiswa kwemhando dzakasiyana dzemvura chromatography makoramu kuburikidza nezvimwe zviitiko.
3.1 Reverse-phase C18 chromatographic column
Iyo C18 column ndiyo inonyanya kushandiswa reverse-phase column, iyo inogona kusangana nezviri mukati uye kusachena bvunzo yezvakawanda organic zvinhu, uye inoshanda kune yepakati-polar, isina kusimba polar uye isiri-polar zvinhu. Mhando uye kutsanangurwa kweC18 chromatographic column inofanira kusarudzwa zvichienderana nezvinodiwa zvekuparadzanisa. Semuenzaniso, kune zvinhu zvine zvinodiwa zvekuparadzanisa zvakanyanya, 5 μm * 4.6 mm * 250 mm zvinyorwa zvinowanzoshandiswa; kune zvinhu zvine matrices akaoma ekuparadzanisa uye akafanana polarity, 4 μm * 4.6 mm * 250 mm tsanangudzo kana diki diki hukuru inogona kushandiswa. Semuenzaniso, munyori akashandisa 3 μm * 4.6 mm * 250 mm column kuti aone maviri genotoxic tsvina mu celecoxib API. Kuparadzaniswa kwezvinhu zviviri izvi kunogona kusvika 2.9, iyo yakanaka kwazvo. Mukuwedzera, pasi pechirevo chekuvimbisa kuparadzana, kana kuongororwa nokukurumidza kuchidikanwa, chikamu chiduku che 10 mm kana 15 mm chinowanzosarudzwa. Semuenzaniso, munyori paakashandisa LC-MS/MS kuona kusachena kwegenotoxic mupiperaquine phosphate API, 3 μm * 2.1 mm * 100 mm column yakashandiswa. Kuparadzaniswa pakati pekusachena uye chikamu chikuru chaiva 2.0, uye kuonekwa kwemuenzaniso kunogona kupedzwa mumaminitsi mashanu. 3.2 Reversed-phase phenyl column
Phenyl column iriwo mhando ye reversed-phase column. Rudzi urwu rwekoramu rune sarudzo yakasimba yezvinonhuhwirira. Kana mhinduro yemishonga inonhuhwirira yakayerwa neyakajairika C18 column isina simba, unogona kufunga kutsiva iyo phenyl column. Semuenzaniso, pandakanga ndichigadzira celecoxib API, mhinduro huru yechikamu yakayerwa nephenyl column yemugadziri mumwe chete uye kutsanangurwa kwakafanana (zvese 5 μm * 4.6 mm * 250 mm) yaive yakapetwa ka7 iyo yeC18 column. 3.3 Normal-phase column
Sekuwedzera kunoshanda kune yakadzoserwa-chikamu chikamu, yakajairika-chikamu chikamu chakakodzera kune yakanyanya polar makomisheni. Kana iyo peak ichiri kukurumidza kwazvo kana ichidonhedza neinodarika 90% chikamu chemvura mune yakadzoserwa-chikamu chikamu, uye kunyange padyo uye nekupindirana nepamusoro peinonyungudutsa, unogona kufunga kutsiva yakajairwa-chikamu chikamu. Mhando yekoramu iyi inosanganisira hilic column, amino column, cyano column, nezvimwewo.
3.3.1 Hilic column Hilic column inowanzopinza hydrophilic mapoka muakasungwa alkyl cheni kuti awedzere mhinduro kuzvinhu zvepolar. Rudzi urwu rwekoramu rwakakodzera kuongororwa kwezvinhu zvehuga. Munyori akashandisa rudzi urwu rwekoramu paaiita zvirimo uye zvine chekuita nezve xylose uye zvinobva pairi. Isomers ye xylose derivative inogonawo kuparadzaniswa zvakanaka;
3.3.2 Amino column uye cyano column Amino column uye cyano column inoreva kuiswa kweamino uye cyano kugadziriswa pamagumo eketani yakasungwa alkyl, maererano, kuvandudza kusarudzwa kwezvinhu zvakakosha: somuenzaniso, amino column isarudzo yakanaka. nokuda kwekuparadzaniswa kweshuga, amino acids, mabhesi, uye amides; cyano column ine zvirinani selectivity pakuparadzanisa hydrogenated uye unhydrogenated structural zvinhu zvakafanana nokuda kuvapo conjugated zvisungo. Amino column uye cyano column inogona kazhinji kuchinjwa pakati peyakajairwa chikamu chikamu uye reverse chikamu chikamu, asi kazhinji kuchinja hakukurudzirwe. 3.4 Chiral column
Chiral column, sezvinoratidzwa nezita, inokodzera kuparadzaniswa uye kuongororwa kwechiral compounds, kunyanya mumunda wemishonga. Rudzi urwu rwekoramu runogona kutariswa kana yakajairwa reverse chikamu uye yakajairika chikamu makoramu haigone kuwana kupatsanurwa kwemaisomers. Semuenzaniso, munyori akashandisa 5 μm * 4.6 mm * 250 mm chiral column kupatsanura maviri isomers e1,2-diphenylethylenediamine: (1S, 2S) -1, 2-diphenylethylenediamine uye (1R, 2R) -1, 2. -diphenylethylenediamine, uye kupatsanurwa pakati pezviviri kwakasvika anenge 2.0. Zvisinei, chiral columns inodhura kupfuura mamwe marudzi emakoramu, kazhinji 1W +/chidimbu. Kana pane kudiwa kwemakoramu akadaro, chikwata chinoda kuita bhajeti yakakwana. 3.5 Ion exchange column
Ion exchange columns inokodzera kupatsanurwa uye kuongororwa kweiyoni inochajiwa, senge ion, mapuroteni, nucleic acids, uye zvimwe zvinhu zveshuga. Zvinoenderana nerudzi rwekuzadza, ivo vakakamurwa kuita cation exchange column, anion exchange columns, uye yakasimba cation exchange column.
Cation exchange columns inosanganisira calcium-based uye hydrogen-based columns, iyo inonyanya kukodzera kuongororwa kwezvinhu cationic zvakadai seamino acids. Semuenzaniso, munyori akashandisa calcium-based columns paaiongorora calcium gluconate uye calcium acetate mukugadzirisa mhinduro. Zvinhu zviviri izvi zvine mhinduro dzakasimba pa λ = 210nm, uye dhigirii yekuparadzanisa yakasvika 3.0; munyori akashandisa hydrogen-based columns paaiongorora zvinhu zvine chokuita neglucose. Zvinhu zvinoverengeka zvakakura zvine hukama - maltose, maltotriose uye fructose - yaive nekunzwa kwakanyanya pasi pema detectors akasiyana, ane muganho wekuona wakaderera se0.5 ppm uye dhigirii yekuparadzanisa ye2.0-2.5.
Anion exchange columns inonyanya kukodzera kuongororwa kweanionic zvinhu zvakadai seorganic acids uye halogen ions; yakasimba cation exchange columns ine yakakwirira ion yekutsinhana simba uye selectivity, uye inokodzera kupatsanurwa uye kuongororwa kwemasampuli akaoma.
Izvo zviri pamusoro ingori sumo yemhando uye mashandisirwo emhando dzakawanda dzinozivikanwa mvura chromatography makoramu akasanganiswa neruzivo rwemunyori. Kune mamwe marudzi akakosha emakoramu echromatographic mumashandisirwo chaiwo, senge hombe-pore chromatographic makoramu, diki-pore chromatographic makoramu, affinity chromatography makoramu, multimode chromatographic columns, Ultra-yakakwirira performance liquid chromatography columns (UHPLC), supercritical fluid chromatography columns ( SFC), nezvimwewo Vanoita basa rakakosha munzvimbo dzakasiyana. Iyo chaiyo mhando yekromatographic column inofanirwa kusarudzwa zvinoenderana nechimiro uye zvimiro zvemuenzaniso, kupatsanurwa zvinodiwa uye zvimwe zvinangwa.
Nguva yekutumira: Jun-14-2024