Ezinye izihloko zokuxazulula iingxaki ze-LC azizange ziphelelwe yisikhathi, njengoko kukho imiba kwi-LC practice, njengoko iteknoloji yesixhobo iphucula ngokuhamba kwexesha.Kukho iindlela ezininzi apho iingxaki ezinokuvela kwinkqubo ye-LC kwaye ziphele kwincopho embi kakhulu.
Bekumnandi ukubhala le kholamu "LC Troubleshooting" kunye nokucinga ngezihloko nyanga nganye, kuba ezinye izihloko azinakuze ziphume kwisitayile.Ngelixa kwinkalo yophando lwechromatography izihloko ezithile okanye iimbono ziye ziphelelwe lixesha njengoko zithathelwe indawo ziimbono ezintsha nezingcono, kwinkalo yokucombulula ingxaki, ukusukela oko inqaku lokuqala lokusombulula ingxaki lavela kule jenali (inqaku le-LC lisekho) I-1983 (1) . Kule minyaka imbalwa idlulileyo, ndigxininise amacandelo amaninzi e-LC Troubleshooting kwiindlela zangoku ezichaphazela i-chromatography yolwelo (LC) (umzekelo, uthelekiso olunxulumene nokuqonda kwethu umphumo woxinzelelo ekugcinweni [2] Ukuqhubela phambili okutsha) Ukutolikwa kwethu kweziphumo ze-LC kunye nendlela yokusombulula ingxaki nge-lc chromatography ye-liquid (LC) (umzekelo, uthelekiso olunxulumene nokuqonda kwethu umphumo woxinzelelo ekugcinweni [2] Ukuqhubela phambili okutsha) Ukutolikwa kwethu kweziphumo ze-LC kunye nendlela yokusombulula ingxaki kunye nolu luhlu lwangoku lwe-LC lwenyanga (i-3) yokufaka i-instrument ye-lc yanamhlanje. I-2021, egxile kwezinye zezihloko "zobomi nokufa" ze-LC zokusombulula ingxaki - izinto ezilungele nawuphi na umxazululi ziyimfuneko, kungakhathaliseki ubudala benkqubo esiyisebenzisayo.Isihloko esingundoqo solu chungechunge sibaluleke kakhulu kwi-LCGC edumileyo "I-LC Troubleshooting Guide" itshathi yodonga (4) ejingayo kwiilabhoratri ezininzi. iimpawu.Ngokumangalisayo, itshathi eludongeni idwelisa 44 oonobangela ezahlukeneyo ezinokubakho ukumila incopho embi!Asikwazi ukuqwalasela yonke le miba ngokweenkcukacha kwinqaku elinye, ngoko ke kweli nqaku lokuqala kwisihloko, ndiza kugxila kwezinye zezona ndizibona rhoqo.Ndiyathemba ukuba abasebenzisi abancinci nabadala be-LC baya kufumana iingcebiso eziluncedo kunye nezikhumbuzo kwesi sihloko sibalulekileyo.
Ndizifumana ndisiya ndiphendula imibuzo yokucombulula ingxaki ngokuthi “nantoni na inokwenzeka”. Le mpendulo inokubonakala ilula xa siqwalasela imigqaliselo ekunzima ukuyitolika, kodwa ndiyifumana ifanelekile. Ngezizathu ezininzi ezinokubakho zokuma kwencopho embi, kubalulekile ukugcina ingqondo evulekileyo xa ucinga ukuba inokuba yintoni ingxaki, kwaye ukwazi ukubeka phambili oonobangela abanokuthi baqalise ukusombulula iingxaki zethu, ukugxila kwezona nzame zibalulekileyo.
Inyathelo eliphambili kuwo nawuphi na umsebenzi wokucombulula ingxaki - kodwa enye endicinga ukuba iphantsi - kukuqonda ukuba kukho ingxaki efuna ukusonjululwa.Ukuqonda ukuba kukho ingxaki kudla ngokuthetha ukuqaphela ukuba okwenzekayo kwisixhobo kuyahluka kwizinto esizilindeleyo, ezibunjwa yithiyori, ulwazi olunobungqina, kunye namava (5) .I "incopho yokuma" ayibhekiseli kuphela kwincopho ye-refertrical apha, i-peak ye-refertrical ayibhekiseli kuphela apha. asymmetrical, smooth, fluffy, lead edge, tailing, etc.), kodwa kwakhona kububanzi.Okulindelekileyo kwimilo yencopho yokwenyani kulula.Ithiyori (6) ixhasa kakuhle ulindelo lwencwadi yokufunda ukuba, kwiimeko ezininzi, iincopho zechromatographic kufuneka zilingane kwaye zihambelane nokumila kosasazo lweGaussian, njengoko kubonisiwe kuMzobo, siza kuxoxa ngobubanzi obungaphezulu. isihloko kwinqaku elizayo.Ezinye iimilo zencopho kuMfanekiso 1 zibonisa ezinye zezinye izinto ezinokwenzeka ezinokubonwa-ngamanye amazwi, ezinye zeendlela ezinokuhamba kakubi.Kwiintsalela yesi savenge, siza kuchitha ixesha sixoxa ngemizekelo ethile yeemeko ezinokukhokelela kwezi ntlobo zemilo.
Ngamanye amaxesha iincopho aziqatshelwa kwaphela kwichromatogram apho kulindeleke ukuba zihlanjululwe.Itshathi yodonga engentla ibonisa ukuba ukungabikho kwencopho (ucinga ukuba isampuli ngokwenene iqulethe umhlalutyi ojoliswe kuyo ekugxininiseni okufuneka yenze impendulo ye-detector yanele ukuyibona ngaphezu kwengxolo) idla ngokuhambelana nomba othile wesixhobo okanye iimeko zesigaba esiphathwayo esingachanekanga (ukuba sijongwe nakanye). iincopho, ngokuqhelekileyo "zibuthathaka" .Uluhlu olufutshane lweengxaki ezinokuthi zibe khona kunye nezisombululo kolu didi zingafumaneka kwiTheyibhile yoku-I.
Njengoko kukhankanyiwe ngasentla, umbuzo wobungakanani bencopho yokwandisa kufuneka inyanyezelwe ngaphambi kokuba inikele ingqalelo kwaye izame ukuyilungisa sisihloko esintsonkothileyo endiya kuxoxa ngaso kwinqaku elizayo.Amava am kukuba ukwanda kwencopho ebalulekileyo kuhlala kukhatshwa lutshintsho olubalulekileyo kwimilo yencopho, kwaye umsila wencopho uxhaphake kakhulu kune-pre-peak okanye uqhekeko.Nangona kunjalo, iincopho ezibizwa ngokuba yi-symmetrical , amava am kukuba ukwandiswa kwencopho ebalulekileyo kuhlala kukhatshwa lutshintsho olubalulekileyo kwimilo yencopho, kwaye umsila wencopho uxhaphake kakhulu kune-pre-peak okanye ukwahlulahlula.
Nganye yale miba ixutyushwe ngokweenkcukacha kwimiba yangaphambili ye- Troubleshooting LC , kwaye abafundi abanomdla kwezi zihloko banokubhekisela kula manqaku angaphambili ukuze bafumane ulwazi malunga nezizathu ezisisiseko kunye nezisombululo ezinokubakho kule miba. Iinkcukacha ezingakumbi.
Incopho yomsila, i-peak fronting, kunye nokwahlula konke kunokubangelwa yimichiza okanye i-physical phenomena, kwaye uluhlu lwezisombululo ezinokuthi zibekho kwezi ngxaki zihluka ngokubanzi, kuxhomekeke ekubeni sijongene nengxaki yekhemikhali okanye yomzimba.Amaxesha amaninzi, ngokuthelekisa iincopho ezahlukeneyo kwichromatogram, unokufumana iimpawu ezibalulekileyo malunga nokuba yeyiphi i-culprit.Ukuba yonke into ebangela ukuba i-shapeki ibonakaliswe kwi-peaks ebonakalayo. kuphela enye okanye iincopho ezimbalwa ezichaphazelekayo, kodwa ezinye zikhangeleka zilungile, unobangela weyona khemikhali inokwenzeka.
Izizathu zeekhemikhali zomsila we-peak zinzima kakhulu ukuba zixoxe ngokufutshane apha.Umfundi onomdla ubhekiswa kumcimbi wamva nje "I-LC Troubleshooting" kwingxoxo enzulu ngakumbi (10). Nangona kunjalo, into elula ukuzama ukunciphisa ubunzima bohlalutyi olujojowe kwaye ubone ukuba i-peak shape iphucula.Ukuba kunjalo, ke oku kuyinkcazo efanelekileyo yokuba le ngxaki incinci, kufuneka ilayishwe kwi-mass encinci. Hlalutya ubunzima, okanye iimeko zechromatographic kufuneka zitshintshwe ukuze kufumaneke iimilo zencopho elungileyo nokuba zitofwe ngobunzima obukhulu.
Kukho nezizathu ezininzi ezinokuthi zibe nomzimba wencopho yomsila.Abafundi abanomdla kwingxoxo eneenkcukacha ezinokwenzeka zibhekiswa komnye umba wamva nje we "LC Troubleshooting" (11) .Enye yezona zinto ziqhelekileyo ezibangela ukunyanzeliswa komzimba kwincopho yomsila kukudityaniswa okungahambi kakuhle kwinqanaba phakathi kwe-injector kunye ne-detector (12) .Umzekelo ogqithiseleyo uboniswa kuMzobo 1d, le nkqubo ye-valve eyakhiweyo kwiiveki ezimbalwa ezidlulileyo. esingazange sisebenzise ngaphambili, kwaye sifake i-loop encinci ye-injection ye-volume kunye ne-ferrule eyayibunjwe kwi-capillary yensimbi engenasici.Emva kweemvavanyo zokuqala zokusombulula ingxaki, saqonda ukuba ubunzulu be-port kwi-injection valve stator yayinzulu kakhulu kunokuba sasiqhelene nayo, okubangelwa umthamo omkhulu ofileyo ezantsi kwe-port. ukususa umthamo ofileyo ezantsi kwezibuko.
I-Peak fronts efana naleyo iboniswe kwi-Figure 1e nayo inokubangelwa iingxaki zomzimba okanye zekhemikhali.Isizathu esiqhelekileyo somzimba wecala elihamba phambili kukuba i-particle bed yekholomu ayipakishwanga kakuhle, okanye ukuba iinqununu ziye zahlengahlengiswa ngokuhamba kwexesha.Njengokuba i-peak tailing ebangelwa yile nto yomzimba, indlela efanelekileyo yokulungisa le nto kukutshintsha ikholomu kwaye siqhubeke sihamba ngemvelaphi yeekhemikhali kwaye siqhubeke sihamba. Iimeko zokugcina "ezingenamgca".Ngaphantsi kweemeko ezifanelekileyo (umgca), inani le-analyte egcinwe yisigaba esimileyo (ngoko ke, i-retention factor) ihambelana ngokuhambelana nokugxininiswa kwe-analyte kwikholamu.I-Chromatographically, oku kuthetha ukuba njengoko ubuninzi be-analyte injected in the column ukwanda, i-peak iba yinde, kodwa i-reten-line yokuziphatha iyaphulwa xa i-reten-line iyancipha. iincopho azigcini nje ngokuba zinde kodwa zibe zibanzi njengoko ubunzima obuninzi bujovwe.Ukongezelela, iimilo ezingezizo ezichanekileyo zimisela imilo yeencopho zechromatographic, okubangela ukuba kubekho imiphetho ekhokelayo okanye elandelanayo.Njengoko kunye nomthwalo onzima obangela i-peak tailing (10), i-peak ehamba phambili ebangelwa ukugcinwa kwe-nonlinear nayo inokufunyaniswa ngokunciphisa i-injected ye-injection peaks ibangela ukuba i-injection ye-analyte iphuculwe i-modified indlela yokuhlalutya kufuneka iphuculwe. edge edge, okanye iimeko zechromatographic kufuneka zitshintshwe ukunciphisa le ndlela yokuziphatha.
Ngamanye amaxesha siqwalasela into ebonakala ngathi yincopho "yokwahlulwa", njengoko kubonisiwe kuMfanekiso 1f. Inyathelo lokuqala ekusombululeni le ngxaki kukuqinisekisa ukuba ubume bencopho bubangelwa yintsebenziswano ephakathi (oko kukuthi, ubukho bemixube emibini eyahlukileyo kodwa esondeleleneyo eluting) Incopho “yokwahlulwa” inxulumene nomzimba Ukwenziwa komsebenzi akunanto yakwenza nomqolo ngokwawo.Amaxesha amaninzi, eyona nto ibalulekileyo kwesi sigqibo kukuba zonke iincopho kwichromatogram zibonisa iimilo ezicazululiweyo, okanye enye okanye ezimbini. ukuba zonke iincochoyi zahlulwe, mhlawumbi ngumba wenyama, kusenokwenzeka ukuba unxulumene nomqolo ngokwawo.
Ukwahlula iinqununu ezihambelana neempawu ezibonakalayo zekholomu ngokwayo ngokuqhelekileyo ngenxa yokuvalelwa kwe-inlet okanye i-outlet frits, okanye ukulungiswa kwakhona kweengqungquthela kwikholamu, ukuvumela ukuba isigaba esihambahambayo sihambe ngokukhawuleza kunesigaba esihambayo kwiindawo ezithile zokwakheka komzila wekholomu .kweminye imimandla (11) . nangona kunjalo, kumava am, oku kudla ixesha elifutshane kunokuba isisombululo sexesha elide.Oku kudla ngokubulala kunye neentsika zanamhlanje ukuba iinqununu ziphinda zidibanise ngaphakathi kwikholomu.Kule ngongoma, kungcono ukutshintsha ikholamu kwaye uqhubeke.
Incopho kuMzobo we-1g, kwakhona ukusuka kumzekelo wamva nje kwilebhu yam, ngokuqhelekileyo ibonisa ukuba umqondiso uphezulu kangangokuba ufikelele ekupheleni okuphezulu koluhlu lokuphendula.Ukufumana i-optical absorbance detectors (i-UV-vis kule meko), xa i-concentration ye-analyte iphezulu kakhulu, i-analyte ithatha ininzi yokukhanya okudlulayo kwi-detector flow cell, ishiya i-photographer encane kakhulu kwi-light detector. iphenjelelwa kakhulu yimithombo eyahlukeneyo yengxolo, njengokukhanya okulahlekileyo kunye "nobumnyama bamanje", okwenza umqondiso "ufuze" kakhulu ngenkangeleko kwaye uzimele kugxininiso lwe-analyte. Xa oku kusenzeka, ingxaki inokusonjululwa ngokulula ngokunciphisa umthamo wenaliti ye-analyte-ukunciphisa umthamo we-injection, ukuhlambulula isampuli, okanye zombini.
Kwisikolo sechromatography, sisebenzisa isibonakaliso se-detector (okt, i-y-axis kwichromatogram) njengesalathisi sogxininiso lwe-analyte kwisampulu. Ngoko kubonakala kungaqhelekanga ukubona ichromatogram enomqondiso ongaphantsi kwe-zero, njengoko utoliko olulula kukuba oku kubonisa ukugxilwa kwe-analyte negative - leyo ngokuqinisekileyo ayinakwenzeka ngokomzimba. (umz., UV-vis).
Kule meko, incopho embi ithetha nje ukuba iimolekyuli eziphuma kwikholamu zifunxa ukukhanya okuncinci kunesigaba esiphathwayo ngokwaso ngokukhawuleza ngaphambi nangemva kwencopho.Oku kunokwenzeka, umzekelo, xa kusetyenziswa amaza okubona aphantsi kakhulu (<230 nm) kunye nezongezelelo zesigaba esihambayo ezifunxa uninzi lokukhanya kula maza obude.Izongezo ezinjalo zinokuba yi-mobile phase solvent components okanye i-buffer ye-solvent components ezifana ne-buffer-solvents ezifana ne-buffer-solvent. sebenzisa iincopho ezingalunganga ukulungiselela ijika lokulinganisa kwaye ufumane ulwazi oluchanekileyo lobungakanani, ngoko akukho sizathu sisisiseko sokuziphepha ngese (le ndlela ngamanye amaxesha ibizwa ngokuba "ubhaqo lwe-UV olungathanga ngqo") (13) .Nangona kunjalo, ukuba sifuna ngokwenene ukuphepha iincopho ezingalunganga ngokupheleleyo, kwimeko yobhaqo lokufunxa, esona sisombululo kukusebenzisa inqanaba lobhaqo olwahlukileyo kunoko lokubona i-waveleng ephathwayo, isigaba ukuze Bafunxa ukukhanya okungaphantsi kunezihlalutyi.
Incopho ezingezizo zinokuvela xa kusetyenziswa isalathiso se-refractive (RI) xa isalathiso se-refractive samacandelo ngaphandle kwe-analyte kwisampulu, njenge-solvent matrix, yahlukile kwisalathiso se-refractive se-mobile phase.Oku kuyenzeka kwakhona ngokubhaqwa kwe-UV-vis, kodwa esi siphumo sithande ukuthotywa ngokunxulumene nobhaqo lwe-RI. isigaba.
Kwicandelo lesithathu kwisihloko esisisiseko se-LC yokusombulula ingxaki, ndaxoxa ngeemeko apho ukumila kwencopho ephawulweyo kwahluka ukusuka kwincopho elindelekileyo okanye eqhelekileyo imilo.Ukusombulula ingxaki esebenzayo yeengxaki ezinjalo iqala ngolwazi lweemilo zencopho elindelekileyo (ngokusekwe kwithiyori okanye amava angaphambili kunye neendlela ezikhoyo), ngoko ke ukutenxa kwezi zinto zilindelweyo zicacile.Iingxaki zemilo yencopho zinezizathu ezininzi ezahlukeneyo ezinokuthi zibekho, ezikhokelela kububanzi obuthile, njl njl. izizathu endizibona rhoqo.Ukwazi ezi nkcukacha kubonelela ngendawo elungileyo yokuqalisa ingxaki, kodwa ayibambi konke okunokwenzeka.Abafundi abanomdla kuluhlu olunzulu lwezizathu kunye nezisombululo banokubhekisa kwi-LCGC “IsiKhokelo se-LC Troubleshooting” itshathi yodonga.
(4) I-LCGC "I-LC Troubleshooting Guide" itshathi yodonga.https://www.chromatographyonline.com/view/troubleshooting-wallchart (2021).
(6) A. Felinger, uHlalutyo lweDatha kunye nokuPhathwa kweSignali kwiChromatography (Elsevier, New York, NY, 1998), iphepha 43-96.
(8) Wahab MF, Dasgupta PK, Kadjo AF kunye Armstrong DW, Anal.Chim.Journal.Rev. 907, 31–44 (2016).https://doi.org/10.1016/j.aca.2015.11.043.