Lihlooho tse ling tsa LC tsa ho rarolla mathata ha ho mohla li siiloeng ke nako, kaha ho na le litaba tsa ts'ebetso ea LC, joalo ka ha theknoloji ea lisebelisoa e ntlafala ha nako e ntse e feta.Ho na le litsela tse ngata tseo ka tsona mathata a ka hlahang tsamaisong ea LC mme a qetella a le boemong bo bobe ba tlhoro.
Ho bile monate ho ngola kholomo ena ea "LC Troubleshooting" le ho nahana ka lihlooho khoeli le khoeli, hobane lihlooho tse ling ha li tsoe mokhoeng oa ho etsa lipatlisiso.Ha re ntse re le lefapheng la lipatlisiso tsa chromatography lihlooho tse itseng kapa mehopolo e felloa ke nako kaha e nkeloa sebaka ke mehopolo e mecha le e betere, lebaleng la ho rarolla mathata, kaha sengoloa sa pele sa ho rarolla mathata se hlahile koranteng ena. 1983(1).Ka lilemo tse' maloa tse fetileng, ke tsepamisitse maikutlo a 'maloa a LC Troubleshooting sections on contemporary trending affecting liquid chromatography (LC) (mohlala, papiso e lekanyelitsoeng ea kutloisiso ea rona ea phello ea khatello ea ho boloka [2] New Advances) Tlhaloso ea rona ea liphetho tsa LC le mokhoa oa ho rarolla mathata ka khoeli ena ea 3 ea LC e qalileng letoto la lisebelisoa tsa ka khoeling ena ea December. 2021, e neng e tsepamisitse maikutlo ho tse ling tsa lihlooho tsa “bophelo le lefu” tsa LC ea ho rarolla mathata — elements that are great for any troubleshooter are important, ho sa tsotellehe lilemo tsa tsamaiso eo re e sebelisang.The core topic of this series is highly relevant to the LCGC’s famous “LC Troubleshooting Guide” chate ea lebota (4) leketlileng lilaboratoring tse ngata. litšobotsi.Hoa makatsa hore ebe chate ea lebota e thathamisa 44 lisosa tse fapaneng tse ka bakang sebopeho se futsanehileng sa tlhoro!Re ke ke ra nahana ka lintlha tsena kaofela ka botlalo sehloohong se le seng, kahoo tolokong ena ea pele ea sehlooho, ke tla tsepamisa maikutlo ho tse ling tseo ke li bonang hangata.Ke tšepa hore basebelisi ba LC ba bacha le ba baholo ba tla fumana malebela le likhopotso tse thusang tabeng ena ea bohlokoa.
Ke iphumana ke ntse ke tsoela pele ho araba lipotso tsa ho rarolla bothata ka "eng kapa eng e ka khoneha". Karabo ena e kanna ea utloahala e le bonolo ha ke nahana ka lintlha tseo ho leng thata ho li hlalosa, empa hangata ke fumana ho nepahetse. Ka lisosa tse ngata tse ka bang teng tsa boemo bo bobe ba maemo a holimo, ho bohlokoa ho ba le kelello e bulehileng ha re nahana hore na bothata e ka ba eng, le ho khona ho etelletsa pele lisosa tse ka bang teng ho qala ts'ebetso ea rona ea ho rarolla mathata, ho tsepamisa maikutlo ho tsona ke lintlha tsa bohlokoa haholo.
Bohato ba bohlokoa mosebetsing ofe kapa ofe oa ho rarolla mathata - empa eo ke nahanang hore ke ea tlaase - ke ho hlokomela hore ho na le bothata bo lokelang ho rarolloa.Ho lemoha hore bothata bo teng hangata ho bolela ho lemoha hore se etsahalang ka sesebelisoa se fapane le litebello tsa rona, tse bōpiloeng ke khopolo, tsebo ea matla, le phihlelo (5) . asymmetrical, boreleli, fluffy, etella pele, mohatla, joalo-joalo), empa hape le bophara.Litebello tsa rona bakeng sa sebopeho sa tlhōrō ea sebele li bonolo.Khopolo (6) e tšehetsa hantle tebello ea buka ea hore, maemong a mangata, litlhōrō tsa chromatographic li lokela ho ba le symmetrical le ho lumellana le sebōpeho sa kabo ea Gaussian, joalokaha ho bontšitsoe ho Figure ena, re tla tšohla taba ena e rarahaneng. sehlooho se reng nakong e tlang.Libopeho tse ling tsa tlhoro ho Setšoantšo sa 1 li bontša tse ling tsa menyetla e meng e neng e ka hlokomeloa-ka mantsoe a mang, litsela tse ling tseo lintho li ka senyehang ka tsona.Karolong e setseng ea karolo ena, re tla qeta nako re buisana ka mehlala e itseng e tobileng ea maemo a ka lebisang mefuteng ena ea sebōpeho.
Ka linako tse ling litlhōrō ha li hlokomeloe ho hang ka chromatogram moo ho lebelletsoeng hore li tlosoe litlhōrō, hangata le tsona li “fokola”).Lethathamo le lekhutšoane la mathata a ka bang teng le tharollo sehlopheng sena le ka fumanoa ho Lethathamo la I.
Joalokaha ho boletsoe ka holimo, potso ea hore na ho atolosoa ha tlhōrō e kae ho lokela ho mamelloa pele ho ela hloko le ho leka ho e lokisa ke taba e rarahaneng eo ke tla e tšohla sehloohong se tlang. Phihlelo ea ka ke hore ho atolosoa ha tlhōrō ea bohlokoa hangata ho tsamaisana le phetoho e kholo ea sebopeho sa tlhōrō, 'me peak tailing e tloaelehile ho feta pele ho tlhōrō kapa ho arohana.
E 'ngoe le e' ngoe ea litaba tsena e 'nile ea tšohloa ka ho qaqileng litabeng tse fetileng tsa Troubleshooting LC,' me babali ba thahasellang lihlooho tsena ba ka sheba lihlooho tsena tse fetileng bakeng sa tlhahisoleseding mabapi le lisosa le tharollo e ka 'nang ea e-ba teng litabeng tsena. Lintlha tse ling.
Peak mohatla, peak fronting, le petsoha kaofela li ka bakoa ke lik'hemik'hale kapa lintho tse etsahalang 'meleng,' me lethathamo la tharollo e ka bang teng mathateng ana le fapana haholo, ho itšetlehile ka hore na re sebetsana le bothata ba lik'hemik'hale kapa 'mele. ke tlhōrō e le 'ngoe feela kapa tse' maloa tse amehang, empa tse ling kaofela li shebahala li le ntle, sesosa se ka etsahala haholo ke lik'hemik'hale.
Lisosa tsa lik'hemik'hale tsa peak tailing li rarahane haholo hore li ka tšohloa ka bokhutšoanyane mona.'Mali ea thahasellang o fetisetsoa khatisong ea morao-rao ea "LC Troubleshooting" bakeng sa puisano e tebileng haholoanyane (10). Leha ho le joalo, ntho e bonolo ho leka ke ho fokotsa boima ba "analyte e kentsoeng" le ho bona hore na sebopeho sa tlhōrō se ntlafala. Analyte mass, kapa maemo a chromatographic a tlameha ho fetoloa e le hore libopeho tse ntle tsa tlhoro li ka fumanoa leha ho kentsoe bongata bo boholo.
Hape ho na le mabaka a mangata a 'mele a ka bakang mohatla o phahameng.Babali ba thahasellang puisano e qaqileng ea menyetla ba fetisetsoa khatisong e' ngoe ea morao-rao ea "LC Troubleshooting" (11) .E 'ngoe ea lisosa tse tloaelehileng tsa' mele tsa tlhōrō ea mohatla ke khokahanyo e mpe ka ntlha e 'ngoe pakeng tsa ente le detector (12) .Mohlala o feteletseng o bontšoa ho Figure 1d, nyeoe ena e ncha e hahiloeng libeke tse 'maloa tse fetileng. eo re neng re e-s'o e sebelise pele, 'me e kentse lupu e nyenyane ea ente ea molumo e nang le ferrule e entsoeng holim'a capillary ea tšepe e sa hloekang.Ka mor'a liteko tse ling tsa pele tsa ho rarolla mathata, re ile ra hlokomela hore botebo ba koung ea stator ea ente ea valve bo ne bo tebile haholo ho feta kamoo re neng re tloaetse kateng, ho fella ka molumo o moholo o shoeleng botlaaseng ba koung. felisa molumo o shoeleng ka tlase ho koung.
Litlhōrō tse kang tse bontšitsoeng ho Setšoantšo sa 1 li ka boela tsa bakoa ke mathata a 'mele kapa lik'hemik'hale.Sesosa se tloaelehileng sa' mele se lebisang pele ke hore karoloana ea bethe ha e tletse hantle, kapa hore likaroloana li hlophisitsoe bocha ha nako e ntse e ea. Joalo ka peak tailing e bakiloeng ke ketsahalo ena ea 'mele, tsela e molemo ka ho fetisisa ea ho lokisa sena ke ho nka sebaka sa kholumo le ho tsoela pele ho tsoela pele. "non-linear" ho boloka maemo. Tlas'a maemo a loketseng (linear), palo ea analyte e bolokiloeng ke mohato o emeng (ka hona, ntlha ea ho boloka) e amana ka ho toba le mahloriso a mohlahlobi kholumong.Chromatographically, sena se bolela hore ha boima ba analyte bo kentsoeng kholumong bo ntse bo eketseha, tlhōrō e fetoha e telele, empa boitšoaro bo sa tsitsang bo se bo pharaletse. litlhōrō ha li be telele feela empa li boetse li pharalla ha boima bo bongata bo kenngoa.Ho phaella moo, libopeho tse se nang moeli li khetholla sebōpeho sa litlhōrō tsa chromatographic, e leng se etsang hore ho be le likarolo tse etellang pele kapa tse tsamaeang ka morao.Joaloka ka boima bo bongata bo bakang peak mohatla (10), tlhōrō e etellang pele e bakoang ke ho boloka e sa tsitsang e ka boela ea fumanoa ka ho fokotsa ente e entsoeng ka mokhoa o etsang hore sebopeho sa analyte se ntlafatse. ka pele, kapa maemo a chromatographic a tlameha ho fetoloa ho fokotsa boitšoaro bona.
Ka linako tse ling re bona se bonahalang eka ke tlhōrō ea "ho arohana", joalokaha ho bontšitsoe setšoantšong sa 1f. Mohato oa pele oa ho rarolla bothata bona ke ho fumana hore na sebopeho sa tlhōrō se bakoa ke ho kopana ha karolo e itseng (ke hore, ho ba teng ha metsoako e 'meli e fapaneng empa e haufi-ufi eluting). litlhōrō "tse arohaneng" li amana le 'mele Ts'ebetso ha e amane le kholomo ka boeona.Hangata, leseli la bohlokoa ka ho fetisisa la qeto ena ke hore na litlhōrō tsohle tsa chromatogram li bonts'a libopeho tse arohaneng, kapa e le' ngoe kapa tse peli feela.Haeba e le 'ngoe kapa tse peli feela, mohlomong ke taba ea kopanelo; haeba litlhōrō tsohle li arohane, mohlomong ke taba ea 'mele, mohlomong e amana le kholomo ka boeona.
Litlhōrō tse arohaneng tse amanang le thepa ea 'mele ea kholomo ka boeona hangata li bakoa ke ho kenngoa ha karolo e itseng kapa li-outlet frits, kapa ho hlophisoa bocha ha likaroloana ka har'a kholomo, ho lumella karolo ea selefouno hore e phalle ka potlako ho feta mohato oa selefouno libakeng tse itseng tsa ho thehoa ha kanale ea likholomo .libakeng tse ling (11) . leha ho le joalo, ka phihlelo ea ka, hangata sena ke nako e khutšoanyane ho e-na le tharollo ea nako e telele.Sena se atisa ho bolaea ka litšiea tsa morao-rao haeba likaroloana li kopana hape ka har'a kholomo.Nakong ena, ho molemo ho nkela kholomo sebaka ebe u tsoela pele.
Tlhōrō ea Setšoantšo sa 1g, hape e tsoang ketsahalong ea morao tjena ka laborateng ea ka, hangata e bontša hore letšoao le phahame hoo le fihlileng pheletsong e phahameng ea likarabo tsa likarabo. e susumetsoa haholo ke mehloli e fapaneng ea lerata, joalo ka leseli le lahlehang le "lefifi la hona joale", ho etsa hore lets'oao le be le "fuzzy" haholo ka chebahalo ebile le ikemetse ka mahloriso a analyte. Ha sena se etsahala, hangata bothata bo ka rarolloa habonolo ka ho fokotsa molumo oa ente ea analyte-ho fokotsa molumo oa ente, ho hlapolla sampuli, kapa ka bobeli.
Sekolong sa chromatography, re sebelisa lets'oao la detector (ke hore, y-axis ho chromatogram) e le sesupo sa mahloriso a analyte sampoleng. Kahoo ho bonahala ho sa utloahale ho bona chromatogram e nang le lets'oao le ka tlase ho zero, kaha tlhaloso e bonolo ke hore sena se bonts'a khatello e mpe ea analyte - eo ehlile ha e khonehe 'meleng. (mohlala, UV-vis).
Tabeng ena, tlhōrō e mpe e bolela feela hore limolek'hule tse tsoang ho kholomo li monya khanya e fokolang ho feta mohato oa selefouno hang-hang pele le ka mor'a tlhōrō.Sena se ka etsahala, ka mohlala, ha ho sebelisoa maqhubu a batlang a le tlaase (<230 nm) le li-additives tsa mohato oa selefouno tse monyang boholo ba leseli ka maqhubu ana a maqhubu. sebelisa litlhōrō tse mpe ho lokisa curve ea calibration le ho fumana lintlha tse nepahetseng tsa palo, kahoo ha ho na lebaka la bohlokoa la ho li qoba ka nako e le 'ngoe (mokhoa ona ka linako tse ling o bitsoa "tlhahlobo e sa tobang ea UV") (13). mokhahlelo e le hore Ba monye khanya e nyane ho feta bahlahlobisisi.
Litlhōrō tse mpe li ka boela tsa hlaha ha u sebelisa refractive index (RI) ha index ea refractive ea likarolo tse ling ntle le analyte ka sampuli, joalo ka solvent matrix, e fapane le index ea refractive ea mobile phase. Sena se boetse se etsahala ka ho lemoha ha UV-vis, empa phello ena e atisa ho fokotsoa le ho fumanoa ha RI. mohato.
Karolong ea boraro ka taba ea motheo ea LC ho rarolla mathata, ke ile ka buisana ka maemo ao ho 'ona tlhōrō ea sebopeho se fapaneng le sebopeho se lebeletsoeng kapa se tloaelehileng sa tlhōrō.Tharollo e atlehang ea mathata a joalo e qala ka tsebo ea libopeho tse lebeletsoeng tlhōrōng (ho itšetlehile ka khopolo kapa phihlelo ea pele ka mekhoa e teng), kahoo ho kheloha litebellong tsena ho hlakile.Mathata a sebopeho sa tlhōrō a na le lisosa tse ngata tse fapaneng tse ka 'nang tsa e-ba teng, tse etellang pele ka ho pharaletseng, joalo-joalo. mabaka ao ke a bonang hangata.Ho tseba lintlha tsena ho fana ka sebaka se setle sa ho qala ho rarolla mathata, empa ha ho nke menyetla eohle.Babali ba thahasellang lethathamo le tebileng haholoanyane la lisosa le tharollo ba ka bua ka chate ea lebota ea LCGC “LC Troubleshooting Guide”.
(4) LCGC "LC Troubleshooting Guide" chate ea lebota.https://www.chromatographyonline.com/view/troubleshooting-wallchart (2021).
(6) A. Felinger, Tlhahlobo ea Boitsebiso le Ts'ebetso ea Lipontšo ho Chromatography (Elsevier, New York, NY, 1998), leqepheng la 43-96.
(8) Wahab MF, Dasgupta PK, Kadjo AF le Armstrong DW, Anal.Chim.Journal.Rev. 907, 31–44 (2016).https://doi.org/10.1016/j.aca.2015.11.043.