Ingingo zimwe na zimwe zo gukemura ibibazo bya LC ntizijya zishaje, kuko hari ibibazo mu mikorere ya LC, ndetse n'uko ikoranabuhanga ry'ibikoresho rigenda ritera imbere uko igihe kigenda gihita. Hari uburyo bwinshi ibibazo bishobora kuvuka muri sisitemu ya LC bikagera mu mwanya mubi. Iyo havutse ibibazo bijyanye n'imiterere y'umwanya, urutonde rugufi rw'impamvu zishobora gutera ibi bisubizo bifasha koroshya uburyo dukemura ibibazo.
Byari bishimishije kwandika iyi nkingi "LC Errorbing" no gutekereza ku ngingo buri kwezi, kuko zimwe mu ngingo zitajya zihinduka. Mu gihe ndi mu rwego rwa chromatography, ubushakashatsi ku ngingo zimwe na zimwe cyangwa ibitekerezo bimwe na bimwe birasaza kuko bisimbura ibitekerezo bishya kandi byiza, mu rwego rwo gukemura ibibazo, kuva aho inyandiko ya mbere yo gukemura ibibazo yasohotse muri iki kinyamakuru (LC Journal icyo gihe) kubera ko ingingo zimwe na zimwe zigifite akamaro) mu 1983 (1). Mu myaka mike ishize, nibanze ku bice byinshi bya LC Errorbing ku miterere ya none igira ingaruka kuri chromatography y'amazi (LC) (urugero, kugereranya uko twumva ingaruka z'igitutu ku kugumana [2] Iterambere rishya) Gusobanura kwacu ibyavuye muri LC n'uburyo bwo gukemura ibibazo hakoreshejwe ibikoresho bigezweho bya LC. Mu gice cy'uku kwezi, ndakomeza urukurikirane rwanjye (3), rwatangiye mu Kuboza 2021, rwibanze ku ngingo zimwe na zimwe "ubuzima n'urupfu" zo gukemura ibibazo bya LC - ibintu byiza ku muntu wese ukemura ibibazo ni ingenzi, uko sisitemu dukoresha yaba imeze kose. Ingingo nyamukuru y'uru rukurikirane ifitanye isano cyane n'icyamamare cya LCGC Imbonerahamwe y'urukuta rwa "LC Error Guide" (4) iri muri laboratwari nyinshi. Ku gice cya gatatu cy'uru rukurikirane, nahisemo kwibanda ku bibazo bijyanye n'imiterere y'agasongero cyangwa imiterere y'agasongero. Igitangaje ni uko imbonerahamwe y'urukuta igaragaza impamvu 44 zitandukanye zishobora gutera imiterere mibi y'agasongero! Ntidushobora gusuzuma ibi bibazo byose mu buryo burambuye mu ngingo imwe, bityo muri iki gice cya mbere kuri iyi ngingo, ndibanda kuri bimwe mu byo mbona kenshi. Nizeye ko abakiri bato n'abakuze bakoresha LC bazabona inama n'ibyibutsa by'ingirakamaro kuri iyi ngingo y'ingenzi.
Nsanga nkomeza gusubiza ibibazo byo gukemura ibibazo nkoresheje "ikintu cyose kirashoboka". Iki gisubizo gishobora gusa n'icyoroshye iyo utekereje ku byo wabonye bigoye gusobanura, ariko nsanga akenshi ari byiza. Kubera impamvu nyinshi zishobora gutuma ikibazo kirushaho kuba kibi, ni ngombwa kugira ibitekerezo bifunguye iyo utekereje ku kibazo gishobora kuba ari cyo, no kubasha gushyira imbere impamvu zishobora kubaho kugira ngo utangire ibikorwa byacu byo gukemura ibibazo, twibanda ku bintu bikunze kubaho, iyi ngingo ni ingenzi cyane. birashoboka.
Intambwe y'ingenzi mu myitozo iyo ari yo yose yo gukemura ibibazo - ariko nkeka ko idahabwa agaciro gahagije - ni ukumenya ko hari ikibazo kigomba gukemurwa. Kwemera ko hari ikibazo akenshi bivuze kumenya ko ibibera ku gikoresho bitandukanye n'ibyo twiteze, bigizwe n'inyigisho, ubumenyi bw'ubuhanga, n'uburambe (5). "Ishusho y'agahebuzo" ivugwa hano mu by'ukuri ntiyerekeza gusa ku ishusho y'agahebuzo (ingana, idangana, yoroshye, yoroshye, igana imbere, inyuma, n'ibindi), ahubwo no ku bugari. Ibyo twiteze ku ishusho nyayo y'agahebuzo biroroshye. Inyigisho (6) ishyigikira neza ibyo igitabo cyiteze ko, mu bihe byinshi, imisozi ya chromatographic igomba kuba ingana kandi ijyanye n'imiterere y'ikwirakwizwa rya Gaussian, nk'uko bigaragara ku Ishusho ya 1a. Icyo twiteze ku bugari bw'agahebuzo ni ikibazo kigoye cyane, kandi tuzaganira kuri iyi ngingo mu nkuru itaha. Izindi shusho z'agahebuzo ziri ku Ishusho ya 1 zigaragaza zimwe mu zindi ngero zishobora kugaragara - mu yandi magambo, zimwe mu nzira ibintu bishobora kugenda nabi. Mu gice gisigaye cy'iki gice, tuzamara igihe tuganira ku ngero zimwe na zimwe z'imimerere bishobora gutuma habaho ubwo bwoko bw'imiterere.
Hari igihe imisozi miremire itagaragara na gato muri chromatogram aho byitezwe ko itazagaragara. Imbonerahamwe iri hejuru yerekana ko kutagira umusozi munini (dufashe ko icyitegererezo kirimo icyerekezo cy’isesengura ku rugero rugomba gutuma igisubizo cy’icyuma gipima ubushyuhe gihagije kugira ngo kibone hejuru y’urusaku) akenshi bifitanye isano n’ikibazo cy’ibikoresho cyangwa imiterere itari yo y’urwego rw’itsinda (niba byagaragaye na gato). imisozi miremire, akenshi "ifite intege nke cyane"). Urutonde rugufi rw’ibibazo n’ibisubizo bishobora kubaho muri iki cyiciro ruboneka mu mbonerahamwe ya mbere.
Nkuko byavuzwe haruguru, ikibazo cy'ingano yo kwagura uburebure bw'umurambararo kigomba kwihanganirwa mbere yo kwita ku ngingo no kugerageza kuyikosora ni ingingo igoye nzaganiraho mu nkuru itaha. Ubunararibonye bwanjye ni uko kwagura uburebure bw'umurambararo mu buryo bukomeye akenshi biherekezwa n'impinduka zikomeye mu miterere y'uburebure bw'umurambararo, kandi gucika kw'umurambararo mu gice cy'umurambararo bikunze kugaragara kuruta gucikamo kabiri cyangwa gucikamo kabiri. Ariko, uburebure bw'umurambararo busanzwe nabwo buragabanuka, bishobora guterwa n'impamvu zitandukanye:
Buri kibazo muri ibi cyaganiriweho birambuye mu nomero zabanje za Troubleshooting LC, kandi abasomyi bashishikajwe n'izi ngingo bashobora kureba izi ngingo zabanje kugira ngo bamenye impamvu nyamukuru n'ibisubizo bishoboka kuri ibi bibazo. Ibisobanuro birambuye.
Kugabanuka kw'isonga, kuzamuka kw'isonga, no gucikamo ibice byose bishobora guterwa n'ibintu bya shimi cyangwa imiterere, kandi urutonde rw'ibisubizo bishobora gutangwa kuri ibi bibazo rutandukanye cyane, bitewe n'uko tuba duhanganye n'ikibazo cya shimi cyangwa imiterere. Akenshi, ugereranije imiterere itandukanye muri chromatogram, ushobora kubona ibimenyetso by'ingenzi ku mpamvu yabyo. Niba imiterere yose muri chromatogram igaragaza imiterere isa, impamvu ishobora kuba atari imiterere ifatika. Niba imiterere imwe cyangwa mike gusa yagizweho ingaruka, ariko indi yose isa neza, impamvu ishobora kuba imiterere ishingiye kuri shimi.
Impamvu zitera ubwinshi bw'ibice bitera ubwinshi bw'ibice binini ni ingorabahizi cyane ku buryo tutazivugaho muri make hano. Umusomyi ushishikajwe yohererejwe ku kinyamakuru giherutse gutangazwa cyitwa "LC Troubleshooting" kugira ngo tuganireho byimbitse (10). Ariko, ikintu cyoroshye kugerageza ni ukugabanya uburemere bw'igipimo cy'ubuhanga mu gupima no kureba niba imiterere y'igipimo irushaho kuba myiza. Niba ari uko bimeze, iki ni ikimenyetso cyiza cy'uko ikibazo ari "ubwinshi bw'ibice binini". Muri iki gihe, uburyo bugomba kugarukira ku gutera ubwinshi bw'ibice binini by'ubuhanga mu gupima, cyangwa se imiterere ya chromatographic igomba guhinduka kugira ngo imiterere myiza y'igipimo ishobore kuboneka nubwo haba hari ubwinshi bunini bw'ibice binini.
Hariho kandi impamvu nyinshi zishobora gutuma habaho gucika intege mu buryo bugaragara. Abasomyi bashishikajwe no kuganira ku buryo burambuye ku byashoboka bohererezwa ikindi kinyamakuru giherutse kuvuga "LC Troubleshooting" (11). Imwe mu mpamvu zikunze kugaragara zo gucika intege mu buryo bugaragara ni uguhuza nabi hagati y'icyuma gitera injection na detector (12). Urugero rukabije rugaragara ku Ishusho ya 1d, yabonetse muri laboratwari yanjye mu byumweru bike bishize. Muri uru rubanza, twubatse sisitemu ifite valve nshya yo gutera injection tutari twakoresheje mbere, maze dushyiraho agace gato k'icyuma gitera injection hamwe na ferrule yari yabumbwe ku gasanduku k'icyuma kitagira umugese. Nyuma y'igerageza rya mbere ryo gukemura ibibazo, twabonye ko ubujyakuzimu bw'aho hantu muri stator ya valve yo gutera injection bwari burebure cyane kuruta uko twari tumenyereye, bigatuma habaho fluid nini hasi y'aho hantu. Iki kibazo gikemurwa byoroshye dusimbuza agace k'icyuma gitera injection n'undi muyoboro, dushobora guhindura ferrule mu mwanya ukwiye kugira ngo dukureho fluid iri hasi y'aho hantu.
Impande zo hejuru nk'izigaragara ku Ishusho ya 1e zishobora guterwa n'ibibazo by'umubiri cyangwa ibya shimi. Impamvu isanzwe y'inkombe y'imbere ni uko agace k'inkingi kadapakiye neza, cyangwa ko uduce twahindutse uko igihe kigenda gihita. Kimwe no kugabanuka kw'inkombe bitewe n'iki kintu gifatika, uburyo bwiza bwo kubikemura ni ugusimbuza inkingi no gukomeza. Mu by'ukuri, imiterere y'inkombe y'imbere ifite inkomoko ya shimi ikunze kuva ku byo twita "ibintu bidahuza". Mu bihe byiza (by'umurongo), ingano ya analyte igumana n'igice gihoraho (bityo, ikintu cyo kugumana) ifitanye isano ku murongo n'ubwinshi bw'alyte mu nkingi. Mu buryo bwa chromatographic, ibi bivuze ko uko uburemere bwa analyte bushyirwa mu nkingi bwiyongera, impera iba ndende, ariko ntibe nini. Iyi sano iracika iyo imyitwarire yo kugumana idahuje umurongo, kandi imisozi ntirushaho kuba miremire gusa ahubwo inagutse uko uburemere bwinshi bushyirwamo. Byongeye kandi, imiterere idahuje umurongo igena imiterere y'inkombe z'inyuma, bigatuma impande ziyobora cyangwa zikurikira. Kimwe no ku kwiyongera k'umubyimba bitera kwiyongera kw'inkombe (10), impera Kuyobora guterwa no kugumana kw'imitsi mu buryo butari umurongo bishobora kandi gupimwa hakoreshejwe kugabanya uburemere bw'imashini zikoresha uburyo bwa analyte. Iyo imiterere y'imashini iteye imbere, uburyo bugomba guhindurwa kugira ngo butarenga ubwiza bw'imashini zikoresha uburyo bwa edge, cyangwa se imiterere ya chromatographic igomba guhinduka kugira ngo iyi myitwarire igabanye.
Hari igihe tubona ikintu gisa n'aho ari agasongero "gacitse", nk'uko bigaragara ku Ishusho ya 1f. Intambwe ya mbere mu gukemura iki kibazo ni ukumenya niba imiterere y'agasongero iterwa no guhuza igice (ni ukuvuga kuba hari ibintu bibiri bitandukanye ariko bitandukanya cyane). Niba koko hari isesengura ribiri ritandukanye rihurira hafi, ni ngombwa kunoza ubushobozi bwaryo (urugero, binyuze mu kongera guhitamo, kugumana, cyangwa umubare w'amasahani), kandi impuzandengo zigaragara "zitandukanya" zifitanye isano n'imikorere y'umubiri ntaho zihuriye n'inkingi ubwayo. Akenshi, ikintu cy'ingenzi kuri iki cyemezo ni ukumenya niba impuzandengo zose muri chromatogram zigaragaza imiterere itandukanya, cyangwa imwe cyangwa ebyiri gusa. Niba ari imwe cyangwa ebyiri gusa, birashoboka ko ari ikibazo cyo guhuza; niba impuzandengo zose zitandukaniye, birashoboka ko ari ikibazo cy'umubiri, birashoboka cyane ko gifitanye isano n'inkingi ubwayo.
Imisozo igabanyijemo ibice ijyanye n'imiterere y'inkingi ubwayo akenshi iterwa n'imirongo ifunganye cyangwa isohoka, cyangwa kuvugurura ibice biri mu nkingi, bigatuma igice cyo kugenda gitembera vuba kurusha igice cyo kugenda mu bice bimwe na bimwe by'imiyoboro y'inkingi. mu tundi turere (11). Igice cyo kuziba igice gishobora gukurwaho no gusubiza inyuma inzira inyura mu nkingi; icyakora, mu bunararibonye bwanjye, ubusanzwe iki ni igisubizo cy'igihe gito aho kuba igisubizo cy'igihe kirekire. Ibi akenshi biba bibi iyo ibice bigezweho bihuye mu nkingi. Kuri iyi ngingo, ni byiza gusimbuza inkingi no gukomeza.
Isonga riri ku Ishusho ya 1g, naryo riherutse kuba muri laboratwari yanjye, rikunze kwerekana ko ikimenyetso kiri hejuru cyane ku buryo kigeze ku rwego rwo hejuru rw'igisubizo. Ku bikoresho bipima urumuri (UV-vis muri uru rubanza), iyo igipimo cy'urumuri cya analyte kiri hejuru cyane, analyte yinjiza urumuri rwinshi runyura mu kazu k'urumuri, bigatuma urumuri ruke cyane ruboneka. Muri ibi bihe, ikimenyetso cy'amashanyarazi kiva kuri photodetector kigirwaho ingaruka zikomeye n'amasoko atandukanye y'urusaku, nk'urumuri rudasobanutse n' "umuyoboro w'amazi wijimye", bigatuma ikimenyetso "kidasobanutse" mu buryo busa kandi kidafite igipimo cy'urumuri cya analyte. Iyo ibi bibaye, ikibazo gishobora gukemurwa byoroshye binyuze mu kugabanya ingano y'urumuri rwa analyte - kugabanya ingano y'urumuri, kugabanya icyitegererezo, cyangwa byombi.
Mu ishuri rya chromatography, dukoresha ikimenyetso cy’isuzuma (ni ukuvuga umurongo wa y muri chromatogram) nk'ikimenyetso cy’icyitegererezo cy’isesengura mu rugero. Bityo rero bisa nkaho bitangaje kubona chromatogram ifite ikimenyetso kiri munsi ya zeru, kuko ibisobanuro byoroshye ari uko ibi bigaragaza icyitegererezo cy’isesengura kibi - birumvikana ko bidashoboka ku mubiri. Mu bunararibonye bwanjye, uburebure bubi bukunze kugaragara iyo hakoreshejwe ibikoresho by’isuzuma bikoresha optique absorbance (urugero, UV-vis).
Muri iki gihe, uburebure bwa negative busobanura gusa ko molekile zisohoka mu nkingi zifata urumuri ruto ugereranije n’urwego rwa mobile ubwarwo mbere na nyuma y’urwego. Ibi bishobora kubaho, urugero, iyo hakoreshejwe uburebure bwa detection ndende (<230 nm) n’inyongera za mobile phase zifata urumuri rwinshi kuri ubu burebure bwa wavelength. Inyongera nk’izo zishobora kuba ibice bya mobile phase solvent nka methanol cyangwa ibice bya buffer nka acetate cyangwa formate. Umuntu ashobora gukoresha uburebure bwa negative kugira ngo ategure calibration curve no kubona amakuru nyayo y’imibare, bityo nta mpamvu y’ibanze yo kubyirinda (ubu buryo rimwe na rimwe bwitwa "indirect UV detection") (13). Ariko, niba koko dushaka kwirinda uburebure bwa negative burundu, mu gihe cyo kubona absorbance, igisubizo cyiza ni ugukoresha uburebure bwa detection butandukanye kugira ngo analyte yinjire byinshi kurusha mobile phase, cyangwa guhindura imiterere ya mobile phase kugira ngo yinjire urumuri ruto ugereranyije na analyte.
Imisozo mibi ishobora kandi kugaragara iyo hakoreshejwe uburyo bwo gupima imiterere y’urumuri (RI) iyo imiterere y’urumuri rw’ibice bitari isesengura mu rugero, nka matrix ya solvent, itandukanye n’imiterere y’urumuri rw’urumuri rw’itsinda rigendanwa. Ibi kandi bibaho no mu gupima UV-vis, ariko iyi ngaruka ikunda kugabanuka ugereranyije no gupima RI. Muri ibyo bihe byombi, imiterere mibi ishobora kugabanuka binyuze mu guhuza neza imiterere y’urumuri rw’icyitegererezo n’iy’urumuri rugendanwa.
Mu gice cya gatatu ku ngingo y'ibanze yo gukemura ibibazo bya LC, navuze ku bihe aho imiterere y'agasongero kabonetse itandukanye n'imiterere y'agasongero iteganijwe cyangwa isanzwe. Gukemura ibibazo nk'ibi bitangirana n'ubumenyi bw'imiterere y'agasongero iteganijwe (ishingiye ku nyigisho cyangwa ubunararibonye bwabanje ku buryo busanzwe), bityo gutandukana n'ibyo byitezwe biragaragara. Ibibazo by'imiterere y'agasongero bifite impamvu nyinshi zitandukanye zishobora gutera (zinini cyane, zihindagurika, ziyobora, nibindi). Muri iki gice, ndavuga ku buryo burambuye zimwe mu mpamvu mbona kenshi. Kumenya ibi bisobanuro bitanga ahantu heza ho gutangirira gukemura ibibazo, ariko ntibigaragaza uburyo bwose bushoboka. Abasomyi bashishikajwe n'urutonde rw'impamvu n'ibisubizo byimbitse bashobora kureba imbonerahamwe y'urukuta ya LCGC "LC Troubleshooting Guide".
(4) Imbonerahamwe y'urukuta ya LCGC "LC Error Guide". https://www.chromatographyonline.com/view/troubleshooting-wallchart (2021).
(6) A. Felinger, Isesengura ry'amakuru no gutunganya ibimenyetso muri chromatography (Elsevier, New York, NY, 1998), pp. 43-96.
(8) Wahab MF, Dasgupta PK, Kadjo AF na Armstrong DW, Anal.Chim.Journal.Rev. 907, 31–44 (2016). https://doi.org/10.1016/j.aca.2015.11.043.