Ezinye izihloko zokuxazulula izinkinga ze-LC azikaze ziphelelwe yisikhathi, njengoba kunezinkinga ekusebenzeni kwe-LC, noma ngabe ubuchwepheshe bezinsimbi buthuthuka ngokuhamba kwesikhathi. Kunezindlela eziningi lapho izinkinga zingavela khona ohlelweni lwe-LC futhi zigcine zisesimweni esibi se-peak. Lapho kuvela izinkinga ezihlobene ne-peak, uhlu olufushane lwezimbangela ezingaba khona zale miphumela lusiza ukwenza lula ulwazi lwethu lokuxazulula izinkinga.
Bekumnandi ukubhala le kholomu ethi “LC Troubleshooting” nokucabanga ngezihloko njalo ngenyanga, ngoba ezinye izihloko azikaze ziphelelwe yisikhathi. Ngesikhathi ngisemkhakheni we-chromatography ucwaningo lwezihloko noma imibono ethile iyaphelelwa yisikhathi njengoba ithathelwa indawo yimibono emisha nengcono, emkhakheni we-troubleshooting, selokhu kwavela isihloko sokuqala sokuxazulula izinkinga kulo magazini (i-LC Journal ngaleso sikhathi) njengoba ezinye izihloko zisabalulekile) ngo-1983(1). Eminyakeni embalwa edlule, ngigxile ezigabeni eziningana ze-LC Troubleshooting ezindleleni zesimanje ezithinta i-liquid chromatography (LC) (isibonelo, ukuqhathanisa okuhlobene kokuqonda kwethu ngomphumela wokucindezela ekugcinweni [2] Intuthuko Entsha) Ukuhumusha kwethu imiphumela ye-LC kanye nendlela yokuxazulula izinkinga ngamathuluzi e-LC yesimanje. Esigabeni sale nyanga, ngiyaqhubeka nochungechunge lwami (3), olwaqala ngoDisemba 2021, olwalugxile kwezinye zezihloko “zokuphila nokufa” zokuxazulula izinkinga ze-LC - izinto ezinhle kunoma yimuphi umxazululi wezinkinga zibalulekile, kungakhathaliseki ukuthi uhlelo esilusebenzisayo lungakanani. Isihloko esiyinhloko salolu chungechunge sibaluleke kakhulu kuhlelo oludumile lwe-LCGC Ishadi lodonga elithi “LC Troubleshooting Guide” (4) elilenga ezindaweni eziningi zokucwaninga. Engxenyeni yesithathu yalolu chungechunge, ngikhethe ukugxila ezindabeni ezihlobene nesimo se-peak noma izici ze-peak. Ngokumangalisayo, ishadi lodonga libala izimbangela ezingu-44 ezahlukene ezingaba khona zesimo se-peak esibi! Asikwazi ukucabangela zonke lezi zinkinga ngokuningiliziwe esihlokweni esisodwa, ngakho kulesi sigaba sokuqala ngesihloko, ngizogxila kwezinye zalezo engivame ukuzibona. Ngithemba ukuthi abasebenzisi be-LC abasebasha nabadala bazothola amathiphu nezikhumbuzo eziwusizo ngalesi sihloko esibalulekile.
Ngizithola ngiphendula imibuzo yokuxazulula izinkinga ngokuthi “noma yini ingenzeka”. Le mpendulo ingase ibonakale ilula uma ucabanga ngokubona okunzima ukukuchaza, kodwa ngikuthola kufaneleka njalo. Njengoba kunezimbangela eziningi ezingaba khona zokuma okungekuhle kwe-peak, kubalulekile ukugcina ingqondo evulekile lapho ucabanga ukuthi inkinga ingaba yini, futhi ukwazi ukubeka phambili izimbangela ezingaba khona ukuze uqale imizamo yethu yokuxazulula izinkinga, ugxile kulezo zindlela ezivame kakhulu, leli phuzu libaluleke kakhulu. kungenzeka.
Isinyathelo esibalulekile kunoma yikuphi ukuvivinya ukuxazulula izinkinga — kodwa engicabanga ukuthi akunakwa kakhulu — ukuqaphela ukuthi kunenkinga okudingeka ixazululwe. Ukuqaphela ukuthi kunenkinga kuvame ukusho ukuqaphela ukuthi okwenzeka kuthuluzi kuhlukile kokulindelwe kwethu, okubunjwe yithiyori, ulwazi olubonakalayo, kanye nokuhlangenwe nakho (5). "Isimo se-peak" okukhulunywa ngaso lapha empeleni asibhekiseli kuphela esimweni se-peak (esilinganayo, esingalingani, esibushelelezi, esithambile, esiholayo, umsila, njll.), kodwa futhi nasebubanzini. Okulindelwe kwethu ngesimo se-peak sangempela kulula. Ithiyori (6) isekela kahle ukulindela kwencwadi ukuthi, ezimweni eziningi, iziqongo ze-chromatographic kufanele zibe zi-symmetrical futhi zivumelane nesimo sokusatshalaliswa kwe-Gaussian, njengoba kuboniswe kuMfanekiso 1a. Lokho esikulindele kububanzi be-peak kuyinkinga eyinkimbinkimbi kakhulu, futhi sizoxoxa ngalesi sihloko esihlokweni esizayo. Ezinye izimo ze-peak kuMfanekiso 1 zibonisa ezinye zezinye izinto ezingase zibonwe—ngamanye amazwi, ezinye zezindlela izinto ezingase zihambe kabi ngazo. Esigabeni esisele salesi sigaba, sizochitha isikhathi sixoxa ngezibonelo ezithile zezimo kungaholela kulezi zinhlobo zezimo.
Ngezinye izikhathi iziqongo azibonwa nhlobo ku-chromatogram lapho kulindeleke ukuthi zingabonakali khona. Ishadi lodonga elingenhla libonisa ukuthi ukungabikho kwesiqongo (uma sicabanga ukuthi isampula empeleni iqukethe i-target analyte ekugxilweni okufanele yenze impendulo yomtholi yanele ukuyibona ngaphezu komsindo) kuvame ukuhlotshaniswa nenkinga ethile yethuluzi noma izimo zesigaba esingesihle (uma zibonwa nhlobo). iziqongo, ngokuvamile "zibuthakathaka kakhulu"). Uhlu olufushane lwezinkinga ezingaba khona kanye nezixazululo kulesi sigaba lungatholakala kuThebula I.
Njengoba kushiwo ngenhla, umbuzo wokuthi kufanele kubekezelelwe ukwanda kwezinga eliphezulu ngaphambi kokunaka nokuzama ukulungisa kuyisihloko esiyinkimbinkimbi engizoxoxa ngaso esihlokweni esizayo. Okuhlangenwe nakho kwami ​​​​ukuthi ukwanda kwezinga eliphezulu okukhulu kuvame ukuhambisana noshintsho olukhulu esimweni sezinga eliphezulu, futhi umsila wezinga eliphezulu uvame kakhulu kunokwandulela ukuphakama noma ukuhlukana. Kodwa-ke, iziqongo ezilinganiselwe nazo ziyanwetshwa, okungabangelwa yizizathu ezimbalwa ezahlukene:
Ngayinye yalezi zinkinga ixoxwe ngokuningiliziwe ezindabeni zangaphambilini ze-Troubleshooting LC, futhi abafundi abanentshisekelo kulezi zihloko bangabheka lezi zihloko zangaphambilini ukuthola ulwazi ngezimbangela eziyinhloko kanye nezixazululo ezingaba khona zalezi zinkinga. Imininingwane eyengeziwe.
Ukushiyana kweziqongo, ukuhola phambili kweziqongo, kanye nokuhlukana konke kungabangelwa yizinto zamakhemikhali noma zomzimba, futhi uhlu lwezixazululo ezingaba khona zalezi zinkinga luyahlukahluka kakhulu, kuye ngokuthi sibhekene nenkinga yamakhemikhali noma yomzimba. Ngokuvamile, ngokuqhathanisa iziqongo ezahlukene ku-chromatogram, ungathola izinkomba ezibalulekile mayelana nokuthi iyiphi imbangela. Uma zonke iziqongo ku-chromatogram zibonisa izimo ezifanayo, imbangela cishe ayiyona ebonakalayo. Uma iziqongo ezimbalwa kuphela ezithintekile, kodwa ezinye zibukeka zilungile, imbangela cishe iyikhemikhali.
Izimbangela zamakhemikhali zokuncipha kwesisindo sezinsika ziyinkimbinkimbi kakhulu ukuba zingaxoxwa kafushane lapha. Umfundi onentshisekelo ubhekiselwe kumagazini wakamuva we-"LC Troubleshooting" ukuze axoxe kabanzi (10). Kodwa-ke, into elula ukuyizama ukunciphisa isisindo se-analyte ejojiwe bese ubona ukuthi isimo se-peak siyathuthuka yini. Uma kunjalo, khona-ke lokhu kuyinkomba enhle yokuthi inkinga "ukulayisha ngokweqile kwesisindo". Kulesi simo, indlela kumele ikhawulelwe ekujoveni izinsika ezincane ze-analyte, noma izimo ze-chromatographic kumele zishintshwe ukuze kutholakale izimo ezinhle zezinsika ngisho nangokufakwa kwezinsika ezinkulu.
Kukhona futhi izizathu eziningi ezibonakalayo ezingaba khona zokudonsa i-peak tail. Abafundi abanesifiso sokuxoxa ngokuningiliziwe ngamathuba babhekiselwa kwenye inkinga yakamuva ethi “LC Troubleshooting” (11). Enye yezimbangela ezivame kakhulu zomzimba zokudonsa i-peak tail ukuxhumana okungekuhle endaweni ephakathi kwe-injector kanye ne-detector (12). Isibonelo esibi kakhulu siboniswe ku-Figure 1d, esitholwe elabhorethri yami emasontweni ambalwa edlule. Kulesi simo, sakha uhlelo nge-valve entsha yokujova esasingakaze siyisebenzise ngaphambili, futhi safaka i-loop encane yokujova ngevolumu ene-ferrule eyayibunjwe kwi-capillary yensimbi engagqwali. Ngemva kokuhlola kokuqala kokuxazulula izinkinga, saqaphela ukuthi ukujula kwe-port ku-stator ye-injection valve kwakujule kakhulu kunalokho esasikujwayele, okwaholela ku-volume enkulu efile phansi kwe-port. Le nkinga ixazululwa kalula ngokufaka i-injection loop ngelinye ithubhu, singalungisa i-ferrule endaweni efanele ukuze sisuse i-volume efile phansi kwe-port.
Izimpondo ezingaphambili njengalezo eziboniswe kuMfanekiso 1e nazo zingabangelwa izinkinga zomzimba noma zamakhemikhali. Imbangela evamile yomzimba yomphetho oholayo ukuthi umbhede wezinhlayiya zekholomu awupakishwanga kahle, noma ukuthi izinhlayiya sezihleleke kabusha ngokuhamba kwesikhathi. Njengokunamathela okuphezulu okubangelwa yilesi simo somzimba, indlela engcono kakhulu yokulungisa lokhu ukufaka ikholomu esikhundleni sekholomu bese uqhubeka. Ngokuyisisekelo, izimo zezimpondo ezingaphambili zomphetho oholayo ezinemvelaphi yamakhemikhali zivame ukuvela kulokho esikubiza ngokuthi izimo zokugcina "ezingezona eziqondile". Ngaphansi kwezimo ezifanele (eziqondile), inani le-analyte eligcinwe yisigaba esimile (ngakho-ke, isici sokugcina) lihlobene ngokuqondile nokuhlushwa kwe-analyte kukholamu. Ngokwe-chromatographic, lokhu kusho ukuthi njengoba isisindo se-analyte esifakwe kukholamu sikhula, isiqongo siba side, kodwa singabi sikhulu. Lobu budlelwano buyaphuka lapho ukuziphatha kokugcina kungabi okuqondile, futhi iziqongo azigcini nje ngokuba zinde kodwa futhi zibanzi njengoba kufakwa isisindo esiningi. Ngaphezu kwalokho, izimo ezingezona eziqondile zinquma ukuma kweziqongo ze-chromatographic, okuholela emaphethelweni aholayo noma alandelanayo. Njengokunqwabelana kwesisindo okubangela ukudonsa okuphezulu (10), isiqongo ukuhola okubangelwa ukugcinwa okungeyona i-linear kungatholakala nangokunciphisa isisindo se-analyte esijovwe. Uma isimo se-peak sithuthuka, indlela kumele iguqulwe ukuze ingadluli ikhwalithi yomjovo ebangela umkhawulo oholayo, noma izimo ze-chromatographic kumele zishintshwe ukuze kuncishiswe lokhu kuziphatha.
Ngezinye izikhathi sibona lokho okubonakala sengathi yi-"split" peak, njengoba kuboniswe ku-Figure 1f. Isinyathelo sokuqala ekuxazululeni le nkinga ukunquma ukuthi ngabe ukuma kwe-peak kungenxa yokuhlangana okungaphelele (okungukuthi, ukuba khona kwamakhemikhali amabili ahlukile kodwa acishe afane). Uma empeleni kukhona abahlaziyi ababili abahlukene abacishe bafane, khona-ke kuyindaba yokuthuthukisa ukuxazululwa kwabo (isibonelo, ngokwandisa ukukhetha, ukugcinwa, noma inani lamapuleti), futhi iziqongo "eziqhekekile" ezibonakalayo zihlobene nokusebenza ngokomzimba akunalutho oluhlobene nekholomu uqobo. Ngokuvamile, inkomba ebaluleke kakhulu kulesi sinqumo ukuthi ngabe zonke iziqongo ku-chromatogram zibonisa ukuma okuhlukanisiwe, noma eyodwa noma ezimbili nje. Uma kuyinye noma ezimbili nje, mhlawumbe kuyinkinga yokuhlangana; uma zonke iziqongo zihlukanisiwe, mhlawumbe kuyinkinga ebonakalayo, cishe ehlobene nekholomu uqobo.
Iziqongo ezihlukanisiwe ezihlobene nezakhiwo zomzimba zekholomu ngokwayo zivame ukubangelwa yizingqimba zokungena noma zokuphuma ezivalekile kancane, noma ukuhlelwa kabusha kwezinhlayiya kukholomu, okuvumela isigaba esihambayo ukuthi sigeleze ngokushesha kunesigaba esihambayo ezindaweni ezithile zokwakheka kwesiteshi sekholomu .kwezinye izifunda (11). Izingqimba ezivalekile kancane ngezinye izikhathi zingasuswa ngokuguqula ukugeleza kukholomu; noma kunjalo, ngokuhlangenwe nakho kwami, lokhu kuvame ukuba yisisombululo sesikhashana kunokuba yisixazululo sesikhathi eside. Lokhu kuvame ukuba yingozi ngamakholomu anamuhla uma izinhlayiya zihlangana kabusha kukholomu. Kuleli qophelo, kungcono ukufaka esikhundleni sekholomu bese uqhubeka.
Iphuzu eliphakeme kuMfanekiso 1g, futhi elivela esibonelweni samuva nje elabhorethri yami, livame ukukhombisa ukuthi isignali iphakeme kakhulu kangangokuthi isifinyelele esiphethweni esiphezulu sebanga lokuphendula. Kuma-optical absorbance detectors (UV-vis kulesi simo), lapho ukuhlushwa kwe-analyte kuphezulu kakhulu, i-analyte imunca iningi lokukhanya elidlula kuseli lokugeleza kwe-detector, okushiya ukukhanya okuncane kakhulu ukuthi kutholakale. Ngaphansi kwalezi zimo, isignali kagesi evela ku-photodetector ithonywa kakhulu yimithombo ehlukahlukene yomsindo, njengokukhanya okulahlekile kanye "nogesi omnyama", okwenza isignali "ibe yi-fuzzy" kakhulu ngokubukeka futhi izimele ekuhlushweni kwe-analyte. Uma lokhu kwenzeka, inkinga ingaxazululwa kalula ngokunciphisa ivolumu yokujova ye-analyte - ukunciphisa ivolumu yokujova, ukunciphisa isampula, noma kokubili.
Esikoleni se-chromatography, sisebenzisa isignali ye-detector (okungukuthi, i-y-axis ku-chromatogram) njengesibonakaliso sokuhlushwa kwe-analyte kusampula. Ngakho-ke kubonakala kungavamile ukubona i-chromatogram enesignali engaphansi kwezinga-zero, njengoba incazelo elula iwukuthi lokhu kubonisa ukuhlushwa kwe-analyte okungekuhle - okungenakwenzeka ngokomzimba. Ngokwami, iziqongo ezingezinhle zivame ukubonwa lapho kusetshenziswa ama-optical absorbance detectors (isb., i-UV-vis).
Kulesi simo, i-negative peak isho nje ukuthi ama-molecule aphuma kukholamu amunca ukukhanya okuncane kune-mobile phase uqobo ngaphambi nangemva kwe-peak. Lokhu kungenzeka, isibonelo, uma kusetshenziswa ama-wavelengths aphansi okuthola (<230 nm) kanye nezithasiselo ze-mobile phase ezimunca ukukhanya okuningi kula ma-wavelengths. Izithasiselo ezinjalo zingaba izingxenye ze-mobile phase solvent njenge-methanol noma izingxenye ze-buffer njenge-acetate noma i-formate. Umuntu angasebenzisa ama-negative peaks ukulungisa i-calibration curve futhi athole ulwazi olunembile lwe-quantitative, ngakho-ke asikho isizathu esiyisisekelo sokuzigwema ngokwazo (le ndlela ngezinye izikhathi ibizwa ngokuthi "ukutholwa kwe-UV okungaqondile") (13). Kodwa-ke, uma sifuna ngempela ukugwema ama-negative peaks ngokuphelele, esimweni sokutholwa kokumunca, ikhambi elingcono kakhulu ukusebenzisa i-wavelength ehlukile yokuthola ukuze i-analyte imunce okungaphezu kwe-mobile phase, noma ishintshe ukwakheka kwe-mobile phase ukuze imunce ukukhanya okuncane kune-analytes.
Iziqongo ezingezinhle zingavela futhi uma kusetshenziswa ukutholwa kwe-refractive index (RI) lapho i-refractive index yezingxenye ezingezona i-analyte kusampula, njenge-solvent matrix, ihlukile ku-refractive index yesigaba esiphathwayo. Lokhu kwenzeka futhi ngokutholwa kwe-UV-vis, kodwa lo mphumela uvame ukuncishiswa uma kuqhathaniswa nokutholwa kwe-RI. Kuzo zombili izimo, iziqongo ezingezinhle zingancishiswa ngokufanisa ukwakheka kwe-sample matrix nalokho kwesigaba esiphathwayo.
Engxenyeni yesithathu ngesihloko esiyisisekelo sokuxazulula izinkinga ze-LC, ngixoxe ngezimo lapho isimo se-peak esibonwe sihlukile esimweni se-peak esilindelekile noma esivamile. Ukuxazulula izinkinga eziphumelelayo zezinkinga ezinjalo kuqala ngolwazi lwezimo ze-peak ezilindelekile (ngokusekelwe kumbono noma kokuhlangenwe nakho kwangaphambilini ngezindlela ezikhona), ngakho-ke ukuphambuka kulezi zilindelo kusobala. Izinkinga zesimo se-peak zinezimbangela eziningi ezahlukene ezingaba khona (ezibanzi kakhulu, ezingemuva, eziholela phambili, njll.). Kulesi sigaba, ngixoxa ngokuningiliziwe ngezinye zezizathu engizibona njalo. Ukwazi le mininingwane kunikeza indawo enhle yokuqala ukuxazulula izinkinga, kodwa akubonisi wonke amathuba. Abafundi abanesifiso sokuthola uhlu olujulile lwezimbangela nezixazululo bangabhekisela eshadini lodonga le-LCGC elithi “LC Troubleshooting Guide”.
(4) Ishadi lodonga le-LCGC “LC Troubleshooting Guide” https://www.chromatographyonline.com/view/troubleshooting-wallchart (2021).
(6) A. Felinger, Ukuhlaziywa Kwedatha Nokucubungula Izimpawu ku-Chromatography (Elsevier, New York, NY, 1998), amakhasi 43-96.
(8) UWahab MF, uDasgupta PK, uKadjo AF no-Armstrong DW, u-Anal.Chim.Journal.Rev. 907, 31–44 (2016).https://doi.org/10.1016/j.aca.2015.11.043.