Ezinye izihloko zokusombulula iingxaki ze-LC aziphelelwa lixesha, njengoko kukho iingxaki kwindlela yokusebenza ye-LC, nangona iteknoloji yezixhobo iphucuka ngokuhamba kwexesha. Zininzi iindlela apho iingxaki zinokuvela kwinkqubo ye-LC zize ziphele zikwimeko embi ye-peak. Xa kuvela imiba enxulumene ne-peak shape, uluhlu olufutshane lwezizathu ezinokubangela ezi ziphumo lunceda ukwenza lula amava ethu okusombulula iingxaki.
Bekumnandi ukubhala le kholamu ethi “LC Troubleshooting” nokucinga ngezihloko inyanga nenyanga, kuba ezinye izihloko azikaze ziphelelwe lixesha. Ngelixa ndikwicandelo le-chromatography uphando ngezihloko ezithile okanye iingcinga ziyaphelelwa lixesha njengoko zithathelwa indawo ziingcinga ezintsha nezingcono, kwicandelo le-troubleshooting, ukusukela oko kwavela inqaku lokuqala le-troubleshooting kule jenali (i-LC Journal ngelo xesha) kuba ezinye izihloko zisabalulekile) ngo-1983 (1). Kwiminyaka embalwa edlulileyo, ndigxile kumacandelo amaninzi e-LC Troubleshooting kwiindlela zanamhlanje ezichaphazela i-chromatography yolwelo (LC) (umzekelo, uthelekiso oluhambelanayo lokuqonda kwethu impembelelo yoxinzelelo ekugcinweni [2] Inkqubela phambili entsha) Ukutolika kwethu iziphumo ze-LC kunye nendlela yokusombulula iingxaki ngezixhobo ze-LC zanamhlanje. Kwinxalenye yale nyanga, ndiyaqhubeka nochungechunge lwam (3), oluqale ngoDisemba 2021, olugxile kwezinye zezihloko "zobomi nokufa" ze-LC troubleshooting - izinto ezilungileyo kuyo nayiphi na i-troubleshooter zibalulekile, nokuba mdala kangakanani na inkqubo esiyisebenzisayo. Isihloko esiphambili sale ngcelele sibaluleke kakhulu kwi-LCGC edumileyo. Itshathi yodonga ethi “LC Troubleshooting Guide” (4) exhonywe kwiilabhoratri ezininzi. Kwinxalenye yesithathu yolu ngcelele, ndikhethe ukugxila kwimiba enxulumene nemilo yencochoyi okanye iimpawu zencochoyi. Okumangalisayo kukuba, itshathi yodonga idwelisa izizathu ezingama-44 ezahlukeneyo ezinokubangela ukuba incochoyi ibe mbi! Asinakuyiqwalasela yonke le micimbi ngokweenkcukacha kwinqaku elinye, ngoko ke kule nxalenye yokuqala yesihloko, ndiza kugxila kwezinye zezo ndizibona rhoqo. Ndiyathemba ukuba abasebenzisi beLC abancinci nabadala baya kufumana iingcebiso eziluncedo kunye nezikhumbuzo ngalo mba ubalulekileyo.
Ndizifumana ndiphendula imibuzo yokusombulula iingxaki ngokuthi “nantoni na inokwenzeka”. Le mpendulo ingabonakala ilula xa ucinga ngezinto ezibonwayo ezinzima ukuzitolika, kodwa ndiyifumana ifanelekile rhoqo. Ngenxa yezizathu ezininzi ezinokubangela ukuba ingxaki ibe mbi, kubalulekile ukugcina ingqondo evulekileyo xa ucinga ukuba ingxaki inokuba yintoni, kwaye ukwazi ukubeka phambili izizathu ezinokubakho ukuze siqalise imizamo yethu yokusombulula iingxaki, sigxile kwezona zinto zixhaphakileyo, eli nqaku libaluleke kakhulu.
Inyathelo elibalulekileyo kuyo nayiphi na indlela yokusombulula iingxaki — kodwa endicinga ukuba ayixatyiswanga — kukuqonda ukuba kukho ingxaki ekufuneka isonjululwe. Ukuqonda ukuba kukho ingxaki kudla ngokuthetha ukuqonda ukuba okwenzekayo kwisixhobo kwahlukile kwizinto esizilindeleyo, ezibunjwe yithiyori, ulwazi oluvela kwi-empirical, kunye namava (5). "I-peak shape" ekubhekiswa kuyo apha ayibhekiseli kuphela kwimilo ye-peak (ehambelanayo, engalinganiyo, egudileyo, ethambileyo, ekhokelayo, emphethweni, njl.njl.), kodwa nakububanzi. Izinto esizilindeleyo kwimilo ye-peak yokwenyani zilula. Ithiyori (6) ixhasa kakuhle ulindelo lwencwadi yokuba, kwiimeko ezininzi, ii-chromatographic peaks kufuneka zibe zi-symmetrical kwaye zihambelane ne-shape yosasazo lwe-Gaussian, njengoko kubonisiwe kuMfanekiso 1a. Oko sikulindeleyo kububanzi be-peak yingxaki enzima ngakumbi, kwaye siza kuxoxa ngesi sihloko kwinqaku elizayo. Ezinye ii-peak shape kuMfanekiso 1 zibonisa ezinye zezinto ezinokwenzeka ezinokubonwa—ngamanye amazwi, ezinye zeendlela izinto ezinokuhamba gwenxa ngazo. Kwinxalenye eseleyo yale nkqubo, siza kuchitha ixesha sixoxa ngemizekelo ethile yeemeko ezinokuthi kungakhokelela kwezi ntlobo zeemilo.
Ngamanye amaxesha iincopho azibonwa konke konke kwi-chromatogram apho kulindeleke ukuba zingabonwa khona. Itshathi yodonga engasentla ibonisa ukuba ukungabikho kwencopho (ukuba isampuli iqulethe i-target analyte kwi-concentration ekufuneka yenze impendulo ye-detector yanele ukuyibona ngaphezu kwengxolo) idla ngokunxulumene nengxaki yesixhobo okanye iimeko zesigaba esingesiso (ukuba zibonwe kwaphela). iincopho, zihlala "zibuthathaka kakhulu"). Uluhlu olufutshane lweengxaki ezinokubakho kunye nezisombululo kolu didi lunokufumaneka kwiTheyibhile I.
Njengoko kukhankanyiwe apha ngasentla, umbuzo wokuba kufuneka kunyanyezelwe kangakanani ukwandiswa kweencopho ngaphambi kokuba kunikelwe ingqalelo kwaye kuzanywe ukuyilungisa ngumxholo onzima endiya kuwuxoxa kwinqaku elizayo. Amava am kukuba ukwandiswa kweencopho ezibalulekileyo kudla ngokuhamba notshintsho olukhulu kwimo yeencopho, kwaye ukususwa kweencopho kuqheleke kakhulu kune-pre-peak okanye ukwahlukana. Nangona kunjalo, iincopho ezilinganayo ngokwazo nazo ziyandiswa, ezinokubangelwa zizizathu ezimbalwa ezahlukeneyo:
Ngamnye wale miba uxoxiwe ngokweenkcukacha kwiincwadi zangaphambili ze-Troubleshooting LC, kwaye abafundi abanomdla kwezi zihloko banokubhekisa kula manqaku angaphambili ukuze bafumane ulwazi ngezizathu eziphambili kunye nezisombululo ezinokubakho kwezi ngxaki. Iinkcukacha ezithe vetshe.
Ukushiyana kweencochoyi, ukukhokela iincochoyi, kunye nokuqhekeka kwazo zonke zinokubangelwa zizinto zekhemikhali okanye zendalo, kwaye uluhlu lwezisombululo ezinokubakho kwezi ngxaki luyahluka kakhulu, kuxhomekeke ekubeni sijongene nengxaki yekhemikhali okanye yendalo. Rhoqo, ngokuthelekisa iincochoyi ezahlukeneyo kwi-chromatogram, ungafumana izikhokelo ezibalulekileyo malunga nokuba ngubani obangela le ngxaki. Ukuba zonke iincochoyi kwi-chromatogram zibonisa iimilo ezifanayo, unobangela mhlawumbi awungowomzimba. Ukuba kuphela iincochoyi ezimbalwa ezichaphazelekayo, kodwa ezinye zibonakala zilungile, unobangela mhlawumbi yikhemikhali.
Iimbangela zeekhemikhali zokutsalwa kwe-peak tailing zinzima kakhulu ukuba zingaxoxwa ngokufutshane apha. Umfundi onomdla uthunyelwe kumcimbi wakutshanje we-"LC Troubleshooting" ukuze axoxe ngokunzulu (10). Nangona kunjalo, into elula ukuyizama kukunciphisa ubunzima be-analyte efakwe kwi-analyte aze abone ukuba imo ye-peak iyaphucuka na. Ukuba kunjalo, ke le yingcaciso elungileyo yokuba ingxaki "kukugqithiswa kobunzima". Kule meko, indlela kufuneka inqunyelwe ekufakeni i-analyte mass encinci, okanye iimeko ze-chromatographic mazitshintshwe ukuze kufumaneke iimilo ezilungileyo ze-peak nokuba kufakwe i-mass mass enkulu.
Kukwakho nezizathu ezininzi ezinokubakho zomzimba zokutsala umsila ophezulu. Abafundi abanomdla kwingxoxo eneenkcukacha malunga namathuba bathunyelwa kwenye inqaku lakutshanje le-"LC Troubleshooting" (11). Enye yezona zinto zixhaphakileyo zomzimba zokutsala umsila ophezulu kukuqhagamshelana okungekho semgangathweni kwindawo ephakathi kwe-injector kunye ne-detector (12). Umzekelo ogqithisileyo uboniswe kuMfanekiso 1d, ofunyenwe kwilebhu yam kwiiveki ezimbalwa ezidlulileyo. Kule meko, sakhe inkqubo ene-injection valve entsha esasingazange siyisebenzise ngaphambili, saza safaka i-volume injection loop encinci ene-ferrule eyayibunjwe kwi-capillary yentsimbi engagqwaliyo. Emva kovavanyo lokuqala lokusombulula iingxaki, saqonda ukuba ubunzulu be-port kwi-injection valve stator babunzulu kakhulu kunokuba besiqhele ukwenza, nto leyo eyabangela ukuba kubekho i-dead volume enkulu ezantsi kwe-port. Le ngxaki isonjululwa ngokulula ngokutshintsha i-injection loop ngomnye umbhobho, singayilungisa i-ferrule kwindawo efanelekileyo ukuze sisuse i-dead volume ezantsi kwe-port.
Iimpondo ezingaphambili ezifana nezo ziboniswe kuMfanekiso 1e nazo zinokubangelwa ziingxaki zomzimba okanye zeekhemikhali. Isizathu esiqhelekileyo somzimba somphetho ongaphambili kukuba ibhedi yamasuntswana ekholamu ayipakishwanga kakuhle, okanye ukuba amasuntswana aphinde ahlelwa ngokuhamba kwexesha. Njengokuba kunjalo ngomsila wencochoyi obangelwe yile nto ibonakalayo, eyona ndlela ilungileyo yokulungisa oku kukutshintsha ikholamu uze uqhubeke. Ngokwesiseko, iimilo zencochoyi ezingaphambili ezinemvelaphi yeekhemikhali zihlala zivela kwinto esiyibiza ngokuba ziimeko zokugcina "ezingezizo ezingqalileyo". Phantsi kweemeko ezifanelekileyo (ezingqalileyo), ubungakanani be-analyte egcinwe yi-stationary phase (yiyo loo nto, i-retention factor) buhambelana ngokuthe ngqo noxinzelelo lwe-analyte kwikholamu. Ngokwe-chromatographic, oku kuthetha ukuba njengoko ubunzima be-analyte obufakwe kwikholamu bukhula, incochoyi iba nde, kodwa ingabi banzi. Olu lwalamano luyaphuka xa ukuziphatha kokugcina kungabi kungqamane, kwaye iincochoyi aziba zinde kuphela kodwa zibanzi njengoko ubunzima obuninzi bufakwa. Ukongeza, iimilo ezingezizo ezingqamane zimisela imo yeencochoyi ze-chromatographic, nto leyo ebangela imiphetho ekhokelayo okanye elandela emva. Njengakwi-mass overload ebangela i-peak tailing (10), incochoyi ukukhokela okubangelwa kukugcinwa okungacwangciswanga kungachongwa ngokunciphisa ubunzima be-analyte efakwe kwi-injection. Ukuba imo ye-peak iyaphucuka, indlela kufuneka iguqulwe ukuze ingadluli umgangatho we-injection obangela umda okhokelayo, okanye iimeko ze-chromatographic kufuneka zitshintshwe ukuze kuncitshiswe olu hlobo lokuziphatha.
Ngamanye amaxesha sibona into ebonakala ngathi yincochoyi "eyahlulwe", njengoko kubonisiwe kuMfanekiso 1f. Inyathelo lokuqala ekusombululeni le ngxaki kukufumanisa ukuba ingaba imo yencochoyi ibangelwa kukuhlangana okungaphelelanga (oko kukuthi, ukubakho kweekhompawundi ezimbini ezahlukeneyo kodwa ezifihlakeleyo). Ukuba kukho ii-analytes ezimbini ezahlukeneyo ezisondelana, ngoko ke ngumbandela wokuphucula isisombululo sazo (umzekelo, ngokwandisa ukukhetha, ukugcinwa, okanye inani leeplate), kwaye iincochoyi "ezahlulwe" ezibonakalayo zinxulumene nokusebenza komzimba akunanto yakwenza nekholamu ngokwayo. Rhoqo, eyona nto ibalulekileyo kwesi sigqibo kukuba ingaba zonke iincochoyi kwi-chromatogram zibonisa iimilo ezahlulwe, okanye enye okanye ezimbini kuphela. Ukuba yinye okanye ezimbini kuphela, mhlawumbi yingxaki yokuhlangana; ukuba zonke iincochoyi zahlulwe, mhlawumbi yingxaki yomzimba, mhlawumbi inxulumene nekholamu ngokwayo.
Iincopho ezahluliweyo ezinxulumene neempawu zomzimba zekholamu ngokwayo zihlala zibangelwa ziifrithi zokungena ezivalekileyo okanye eziphumayo, okanye ukuhlelwa kwakhona kwee-particles kwikholamu, okuvumela isigaba esihambayo ukuba sihambe ngokukhawuleza kunesigaba esihambayo kwiindawo ezithile zokwakheka kwetshaneli yekholamu .kweminye imimandla (11). Iifrithi ezivalekileyo ngamanye amaxesha zinokususwa ngokuguqula ukuhamba kwekholamu; nangona kunjalo, kumava am, oku kudla ngokuba sisisombululo sexesha elifutshane kunokuba sisisombululo sexesha elide. Oku kudla ngokuba yingozi kwiikholamu zanamhlanje ukuba ii-particles ziyaphinda zihlangane kwikholamu. Kweli nqanaba, kungcono ukutshintsha ikholamu uze uqhubeke.
Incopho kuMfanekiso 1g, nayo evela kwisiganeko sakutshanje kwilebhu yam, idla ngokubonisa ukuba isignali iphezulu kakhulu kangangokuba ifikelele kwinqanaba eliphezulu loluhlu lwempendulo. Kwizixhobo zokufunxa ukukhanya (i-UV-vis kule meko), xa uxinzelelo lwe-analyte luphezulu kakhulu, i-analyte ifunxa uninzi lokukhanya oludlula kwiseli yokuhamba kwe-detector, ishiya ukukhanya okuncinci kakhulu ukuba kubonwe. Phantsi kwezi meko, uphawu lombane oluvela kwi-photodetector luchaphazeleka kakhulu yimithombo eyahlukeneyo yengxolo, njengokukhanya okulahlekileyo kunye "nombane omnyama", okwenza uphawu lube "lufifi" kakhulu ngembonakalo kwaye luzimele kuxinzelelo lwe-analyte. Xa oku kusenzeka, ingxaki inokusonjululwa ngokulula ngokunciphisa umthamo we-injection ye-analyte—ukunciphisa umthamo we-injection, ukunciphisa isampuli, okanye zombini.
Kwisikolo se-chromatography, sisebenzisa isignali ye-detector (oko kukuthi, i-y-axis kwi-chromatogram) njengesalathisi soxinzelelo lwe-analyte kwisampulu. Ngoko ke kubonakala kungaqhelekanga ukubona i-chromatogram enophawu olungaphantsi kwe-zero, njengoko ingcaciso elula kukuba oku kubonisa uxinzelelo lwe-analyte olungalunganga - nto leyo engenakwenzeka ngokwasemzimbeni. Kumava am, iincopho ezingalunganga zihlala zibonwa xa kusetyenziswa ii-optical absorbance detectors (umz., i-UV-vis).
Kule meko, i-negative peak ithetha nje ukuba iimolekyuli eziphuma kwikholamu zifunxa ukukhanya okuncinci kune-mobile phase ngokwazo ngaphambi nasemva kwe-peak. Oku kunokwenzeka, umzekelo, xa kusetyenziswa ii-wavelengths ezisezantsi zokufumanisa (<230 nm) kunye nezongezo ze-mobile phase ezifunxa uninzi lokukhanya kwezi wavelengths. Ezo zongezo zinokuba zii-mobile phase solvent components ezifana ne-methanol okanye ii-buffer components ezifana ne-acetate okanye i-formate. Umntu unokusebenzisa ii-negative peaks ukulungiselela i-calibration curve kunye nokufumana ulwazi oluchanekileyo lobungakanani, ngoko ke akukho sizathu sisisiseko sokuziphepha ngokwazo (le ndlela ngamanye amaxesha ibizwa ngokuba yi-"indirect UV detection") (13). Nangona kunjalo, ukuba sifuna ngokwenene ukuphepha ii-negative peaks ngokupheleleyo, kwimeko yokufumanisa ukufunxa, isisombululo esingcono kukusebenzisa i-definition wavelength eyahlukileyo ukuze i-analyte ifunxe okungaphezulu kune-mobile phase, okanye itshintshe ukwakheka kwe-mobile phase ukuze ifunxe ukukhanya okuncinci kune-analytes.
Iincopho ezingalunganga zinokuvela xa kusetyenziswa ukufunyanwa kwe-refractive index (RI) xa i-refractive index yezinto ezingezizo i-analyte kwisampulu, njenge-solvent matrix, yahlukile kwi-refractive index yesigaba esiphathwayo. Oku kuyenzeka nangokufunyanwa kwe-UV-vis, kodwa esi siphumo sidla ngokuncitshiswa xa kuthelekiswa nokufunyanwa kwe-RI. Kuzo zombini iimeko, iincopho ezingalunganga zinokuncitshiswa ngokunxulumanisa ngokusondeleyo ukwakheka kwe-sample matrix nokwesigaba esiphathwayo.
Kwinxalenye yesithathu kwisihloko esisisiseko sokusombulula iingxaki ze-LC, ndixoxe ngeemeko apho imo yencochoyi ebonwayo yahlukile kwimo yencochoyi elindelekileyo okanye eqhelekileyo. Ukusombulula iingxaki ezinjalo kuqala ngolwazi lweemo zencochoyi ezilindelekileyo (ngokusekwe kwithiyori okanye kumava angaphambili ngeendlela ezikhoyo), ngoko ke ukuphambuka kwezi zinto zilindelekileyo kuyabonakala. Iingxaki zeemo zencochoyi zinezizathu ezininzi ezahlukeneyo ezinokubakho (ezibanzi kakhulu, ezinamacala amaninzi, ezikhokela phambili, njl.njl.). Kule nxalenye, ndixoxa ngokweenkcukacha ngezinye zezizathu endizibona rhoqo. Ukwazi ezi nkcukacha kunika indawo elungileyo yokuqala ukusombulula iingxaki, kodwa akubonisi zonke izinto ezinokwenzeka. Abafundi abanomdla kuluhlu olunzulu lwezizathu kunye nezisombululo banokubhekisa kwitshathi yodonga ethi "LC Troubleshooting Guide" ye-LCGC.
(4) Itshathi yodonga ye-LCGC “LC Troubleshooting Guide”. https://www.chromatographyonline.com/view/troubleshooting-wallchart (2021).
(6) A. Felinger, Uhlalutyo lweDatha kunye nokuCwangciswa kweSignali kwiChromatography (Elsevier, eNew York, NY, 1998), iphe. 43-96.
(8) UWahab MF, uDasgupta PK, uKadjo AF kunye no-Armstrong DW, u-Anal.Chim.Journal.Rev. 907, 31–44 (2016).https://doi.org/10.1016/j.aca.2015.11.043.