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.
Kube mnandi ukubhala le kholamu ethi "LC Troubleshooting" kwaye ucinga ngezihloko kwinyanga nganye, kuba ezinye izihloko azize ziphume kwisitayile.Ngelixa kwinkalo yophando lwechromatography izihloko ezithile okanye iimbono ziphelelwa lixesha njengoko zithathelwe indawo ziimbono ezintsha nezingcono, kwinkalo yokucombulula ingxaki, ekubeni inqaku lokuqala lokusombulula ingxaki livele kule jenali (i-LC ver8 izihloko ezidlulileyo zisekho iminyaka eyi-9 edlulileyo). , Ndigxininise amacandelo amaninzi e-LC Troubleshooting kwiintsingiselo zangoku ezichaphazela i-chromatography yolwelo (LC) (umzekelo, uthelekiso olunxulumeneyo lokuqonda kwethu ifuthe loxinzelelo ekugcinweni [2] Ukuqhubela phambili okutsha) Ukutolikwa kwethu kweziphumo zeLC kunye nendlela yokusombulula ingxaki ngezixhobo zanamhlanje zeLC.Kule nyanga yenyanga, uthelekiso oluhambelanayo lokuqonda kwethu isiphumo soxinzelelo ekugcinweni [2] Ukuqhubela phambili okutsha) Ukutolikwa kwethu kweziphumo zeLC kunye nendlela yokusombulula ingxaki ngezixhobo zeLC zanamhlanje.Kule nyanga yenyanga, uthelekiso olunxulumeneyo lokuqonda kwethu impembelelo yoxinzelelo ekugcinweni [2] Intuthuko entsha s of LC yokulungisa ingxaki - izinto ezilungele nawuphi na umlungisi zibalulekile, kungakhathaliseki ubudala benkqubo esiyisebenzisayo.Isihloko esingundoqo solu chungechunge sibaluleke kakhulu kwitshathi yodonga edumileyo ye-LCGC ye-"LC Troubleshooting Guide" (4) ejinga kwiilabhoratri ezininzi. Kwinxalenye yesithathu yolu ngcelele, ndakhetha ukugxila kwimiba enxulumene nencopho yeempawu ze-4 ezinokuthi zibekho kwitshathi yodonga okanye i-peak-shooting. t qwalasela yonke le miba ngokweenkcukacha kwinqaku elinye, ngoko ke kweli nqaku lokuqala kwisihloko, ndiya kugxininisa kwezinye endizibona 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 yokumilisela" ayibhekiseli kuphela apha, i-shapeal ye-fluesymmetric, i-peak, i-shapeal, i-supeal, i-filosymmetric, ayibhekiseli kuphela apha. i-ffy, i-edge ehamba phambili, umsila, njl.), kodwa kunye nobubanzi.Izinto esizilindeleyo kwimilo yencopho yokwenyani zilula.Ithiyori (6) ixhasa kakuhle ulindelo lwencwadi yokufunda ukuba, kwiimeko ezininzi, iincopho zechromatographic kufuneka zilingane kwaye zihambelane nemilo yokusasazwa kweGaussian, njengoko kubonisiwe kuMfanekiso 1a. Yintoni esiyilindeleyo kwinqaku lesihloko kwinqaku elinzima ngakumbi, kwaye silindele kwincopho yobubanzi bexesha elizayo. Uluhlu loku-1 lubonisa ezinye izinto ezinokwenzeka ezinokuthi ziqwalaselwe-ngamanye amazwi, ezinye zeendlela ezinokuhamba kakubi.Kwintsalela 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-chromato ibonakale ifanayo okanye i-peaks ebonakalayo ayikho enye i-peaks ebonakalayo. iincopho ziyachaphazeleka, kodwa ezinye zikhangeleka zilungile, unobangela unokuba yimichiza.
Izizathu zeekhemikhali zomsila we-peak zinzima kakhulu ukuba zixubushe ngokufutshane apha.Umfundi onomdla ubhekiswa kwinqaku lamva nje elithi "LC Troubleshooting" kwingxoxo enzulu ngakumbi (10) .Nangona kunjalo, into elula ukuzama ukunciphisa ubunzima bomhlalutyi ojojowe kwaye ubone ukuba i-peak shape iphucula.Ukuba kunjalo, ke oku kuyinkcazo efanelekileyo yokuba le ngxaki incinci, i-analyse incinci, kufuneka ilayishwe kwi-massess encinci. okanye iimeko zechromatographic kufuneka zitshintshwe ukuze iimilo zencopho ezilungileyo zifumaneke 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 isetyenzisiweyo kwi-valve entsha, safumana i-valve entsha kwiiveki ezimbalwa ezidlulileyo. ngaphambili, kwaye ifake i-loop encinci yesitofu somthamo kunye ne-ferrule eyayibunjwe kwi-capillary yensimbi engenasici.Emva kweminye imifuniselo yokuqala yokulungisa ingxaki, saqonda ukuba ubunzulu bezibuko kwi-stator ye-injection ye-injection yayinzulu kakhulu kunokuba sasiqhelene nayo, kubangele umthamo omkhulu ofileyo emazantsi e-port. .
I-Peak fronts efana naleyo iboniswe kwi-Figure 1e ingabangelwa iingxaki zomzimba okanye zekhemikhali.Isizathu esiqhelekileyo somzimba wecala elikhokelayo kukuba i-particle bed yekholomu ayipakishwanga kakuhle, okanye ukuba amaqhekeza aphinde alungelelanise ixesha elide.Njengokuba i-peak tailing ebangelwa yile nto yomzimba, indlela engcono yokulungisa oku kukutshintsha ikholomu kwaye siqhubeke sihamba nge-edge-pementally, i-edge-pementally call, i-edge, i-edge-pemental, i-edge-pemental, i-edge, i-edge, i-edge-pemental, i-edge, i-edge, i-edge, i-edge, i-edge-pemental. tion iimeko.Ngaphantsi kweemeko ezifanelekileyo (umgca), inani le-analyte egcinwe yisigaba esimileyo (kungoko, i-retention factor) ihambelana ngokuhambelana noxinaniso lwe-analyte kwikholamu.Chromatographically, oku kuthetha ukuba njengoko ubuninzi be-analyte efakwe kwikholamu inyuka, i-peak iba yinde, kodwa ingabi banzi. Ukongezwa, iimilo ezingezizo ezichanekileyo zinquma ubume be-chromatographic peaks, okubangelwa ukukhokela okanye ukulandelelana kwemiphetho.Njengoko kunye nomthwalo onzima obangela ukuba i-peak tailing (10), i-peak ehamba phambili ebangelwa ukugcinwa kwe-nonlinear nayo ingafunyaniswa ngokunciphisa ubunzima be-analyte injected.Ukuba i-peak shape iphucula, indlela kufuneka iguqulwe umgangatho obangela ukuba i-injection iguqulwe i-injection okanye i-injection ye-injection ukuba ingagqithisi i-ematoch ukuziphatha.
Ngamanye amaxesha siqwalasela into ebonakala ngathi yincopho “yokwahlulwa”, njengoko kubonisiwe kuMfanekiso 1f. Inyathelo lokuqala ekusombululeni le ngxaki kukuqinisekisa ukuba ubume bencopho bubangelwa kukudityaniswa kwenxalenye (oko kukuthi, ubukho bemihlanganisela emibini eyahlukileyo kodwa esondeleleneyo eluting). Ukuba ngenene kukho abahlalutyi ababini abahlukeneyo abasondelelana ngokusondeleyo, ngoko ke ngumbandela wokuphucula ngokucombulula ipleyiti okanye ipleyiti eyonyuswayo (i-applitivity) okanye i-applitivity eyonyukayo, i-applitivity okanye i-platerent. iincopho zinxulumene nomzimba Ukusebenza akunanto yakwenza nomqolo ngokwawo.Amaxesha amaninzi, eyona nto ibalulekileyo kwesi sigqibo kukuba zonke iincopho kwichromatogram zibonisa iimilo ezahlulahlulwe, okanye enye okanye ezimbini.Ukuba yinto enye okanye ezimbini, mhlawumbi ngumba wokusebenzisana;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.Kuma-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-detector ye-photographer encinci kakhulu kwi-bend detector. Imithombo eyahlukeneyo yengxolo, efana nokukhanya okulahlekileyo kunye "nobumnyama bangoku", 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 i-Scialcy Syrical (okt, i-B-AXIS kwi-chromatom) njengeyona nto iphambili kuyo i-zero 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 ngokwentelekiso (<230 nm) kunye nezongezo zesigaba esiphathwayo ezifunxa uninzi lokukhanya kwezi zavelengths.Izongezo ezinje zinokuba yi-mobile phase solvent components okanye i-negative phase solvent njenge-peak-metate components ezifana ne-peak-meace components. ukulungisa ijika lokulinganisa kunye nokufumana 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, isisombululo esona sisombululo kukusebenzisa ubhaqo olwahlukileyo lwe-wavelength, ukuze lufunxe ukubunjwa kwe-wavelength ephathwayo ukuze bafunxe ukubunjwa kwe-wavelength eshukumayo ukuze bafunxe inqanaba elisezantsi lokukhanya, ukufunxa ukwakheka kwe-mobile ukufunxa ngaphezulu kokuhlalutya kwenqanaba leselula tes.
Iincopho ezingezizo zinokuvela xa kusetyenziswa ubhaqo lwe-refractive index (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.
Kwicandelo lesithathu kwisihloko esisisiseko se-LC yokusombulula ingxaki, ndixoxe ngeemeko apho ukumila kwencopho ephawulweyo kuyahluka kwincopho elindelekileyo okanye eqhelekileyo. Ukusombulula iingxaki ezisebenzayo zeengxaki ezinjalo ziqala ngolwazi lweemilo zencopho elindelekileyo (ngokusekwe kwithiyori okanye amava angaphambili kunye neendlela esele zikho), ngoko ke ukutenxa kwezi zinto zilindelweyo kucacile. Iingxaki zemilo yencopho zinezizathu ezininzi ezahlukeneyo ezinokuthi zibekho, ezikhokelela kakhulu kwizizathu ezinokwenzeka, ezikhokelela kuluhlu olubanzi, njl. rhoqo.Ukwazi ezi nkcukacha kubonelela ngendawo elungileyo yokuqalisa ingxaki, kodwa ayibambi konke okunokwenzeka.Abafundi abanomdla kuluhlu olunzulu lwezizathu kunye nezisombululo banokubhekisa kwi-LCGC “LC Troubleshooting Guide” 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.