Amasu E-Chromatography Engqimbeni Encane Ekhemisi
I-Pendahuluan
I-Thin Layer Chromatography (TLC) iyindlela yokuhlaziya evame kakhulu esetshenziswa embonini yezemithi. Le ndlela yaziwa ngokulula kwayo, isivinini, kanye nokungabizi kakhulu, kanye nekhono layo lokunikeza ulwazi olubalulekile mayelana nokwakheka kwesampula. Emalabhorethri ezemithi, i-TLC ivame ukusetshenziswa ocwaningweni nasekuthuthukisweni kwemithi, ekuhlolweni kwezinto zokusetshenziswa, kanye nasekulawulweni kwekhwalithi yomkhiqizo oqediwe. Nakuba izinsimbi zokuhlaziya zesimanje ezifana ne-HPLC kanye ne-LC-MS zisetshenziswa kakhulu, i-TLC isalokhu ibalulekile ngenxa yokuthi kulula ukuyisebenzisa kanye nekhono layo njengendlela yokuhlola yokuqala.
Izimiso Eziyisisekelo ze-Chromatography Yezingqimba Ezincane
Isimiso se-TLC sisekelwe ekuhlukanisweni kwezingxenye zengxube ngokusekelwe ekwehlukeni kwazo ekuhlanganiseni kwezigaba ezimbili: isigaba esimile kanye nesigaba esihambayo. Isigaba esimile ngokuvamile siwungqimba oluncane lwe-adsorbent njenge-silica gel, i-alumina, noma i-cellulose embozwe engilazini, i-aluminium, noma ipuleti lepulasitiki. Isigaba esihambayo, okwamanje, siyingxube ye-solvent noma ye-solvent ehamba ngesigaba esimile ngenxa yesenzo se-capillary.
Uma isampula ibonwa phansi kwepuleti futhi ipuleti lifakwa esitsheni esisathuthuka esiqukethe isigaba esihambayo, isinyibilikisi sizohamba siye phezulu. Izingxenye ezincibilika kakhulu esigabeni esihambayo futhi ezisebenzisana kancane nesigaba esimiyo zizohamba ziye phambili, kuyilapho izingxenye ezisebenzisana kakhulu nesigaba esimiyo zizogcinwa futhi zingasheshi ukuhamba. Umphumela uba ukuhlukaniswa ngesimo samabala ezindaweni ezahlukene.
Izingxenye Eziyinhloko ku-TLC
Ekwenzeni kwezokwelapha, impumelelo ye-TLC incike kakhulu ekukhetheni kanye nekhwalithi yezingxenye zayo eziyinhloko:
1. Ipuleti le-TLC
Amapuleti avame ukumbozwa nge-polar silica gel (SiO₂). I-silica gel ifanelekela ukuhlukanisa izinhlobo eziningi zama-compound emithi, ikakhulukazi lawo ane-polarities ehlukene. Amapuleti e-reversed-phase, njenge-C18, ayatholakala futhi kuma-compound amaningi angewona ama-polar noma ama-compound athile okunzima ukuwahlukanisa esigabeni esijwayelekile.
2. Isigaba esiphathwayo (eluent)
I-eluent ingaba yi-solvent eyodwa noma ingxube. Ukukhetha kuncike ezicini zamakhemikhali ze-analyte, ikakhulukazi i-polarity yayo. Izingxube ezivamile zifaka i-chloroform–methanol, i-ethyl acetate–hexane, noma i-toluene–acetate. Isilinganiso se-solvent sivame ukulungiswa ukuze kufezwe ukuhlukaniswa okubukhali.
3. Umkhumbi wokuthuthukiswa
Isitsha kumele sivalwe kahle ukuze igumbi ligcwale umusi we-solvent. Lokhu kugcwala kusiza ekukhiqizeni intuthuko eqhubekayo kanye nezindawo ezingcono.
4. Amasampula kanye nezindinganiso zokuqhathanisa
Ekuhlaziyweni kwemithi, amasampula avame ukuqhathaniswa nezindinganiso ukuqinisekisa ubunjalo noma ubumsulwa. Ukubona indinganiso kanye nesampula epuletini elifanayo kwandisa ukuthembeka kokuchazwa.
5. Ukutholwa kwamabala
Amabala angabonakala ngokubonakalayo uma enombala, noma kusetshenziswa ukukhanya kwe-UV (254 nm noma 366 nm). Ngaphandle kwalokho, ama-reagents okufafaza njenge-ninhydrin (yama-amino acid), i-Dragendorff (yama-alkaloid), i-anisaldehyde sulfate (yama-metabolites ahlukahlukene), noma i-iodine angasetshenziswa.
Izigaba Zokusetshenziswa kwe-KLT
Inqubo ye-TLC ekhemisi ngokuvamile ihlanganisa izinyathelo ezibalulekile ezilandelayo:
1. Ukulungiselela ipuleti
Uma usebenzisa amapuleti alungele ukusetshenziswa, lesi sinyathelo silula. Kodwa-ke, kwezinye izimo, amapuleti angadinga ukuvuselelwa ngokushisa (isb., ku-100–120°C) ukuze kususwe umswakama ongathinta ukuhlukana.
2. Ukubona isampula
Isampula ibonwa kusetshenziswa i-capillary noma i-micropipette emgqeni wokuqala, ngebanga elithile phakathi kwamabala. Amabala kufanele abe mancane futhi abe makhulu ukuvimbela indawo ukuthi ingasabalali.
3. Ukuthuthukiswa kwe-Chromatogram
Ipuleti lifakwa ekamelweni elikhulayo elinezinga le-solvent ngaphansi komugqa oqondile. I-solvent ivunyelwe ukuthi ikhuphukele ezingeni elifunekayo, bese ipuleti lisuswa bese lomiswa.
4. Ukubona ngeso lengqondo kanye nokuhlonza izindawo
Ngemva kokoma, amabala abonakala nge-UV noma ama-reagents okufutha. Izikhundla zamabala ziyalinganiswa ukuze kubalwe inani le-Rf (Retardation factor):
\[
R_f = \frac{\text{spot travel distance}}{\text{solvent front travel distance}}
\]
Inani le-Rf lingasetshenziswa njengepharamitha yokuhlonza, ikakhulukazi uma liqhathaniswa nezindinganiso futhi lenziwa ngaphansi kwezimo ezifanayo zokuhlaziya.
Izicelo ze-TLC ku-Pharmacy
I-TLC inezinhlobo ezahlukene zezicelo ezibaluleke kakhulu emhlabeni wezokwelapha, okuhlanganisa:
1. Ukuhlonza izinto zokusetshenziswa kwezidakamizwa
I-TLC ivame ukusetshenziswa ukuqinisekisa ukuthi izinto zokusetshenziswa ezitholakele ziyahlangabezana nezimfuneko. Isibonelo, ukuhlonza inhlanganisela ethile esebenzayo kungenziwa ngokuqhathanisa i-Rf yesampula nendinganiso esemthethweni.
2. Ukuhlolwa kobumsulwa kanye nokutholwa kokungcola
Ekulawuleni ikhwalithi, i-TLC ingabonisa ukuba khona kokungcola noma imikhiqizo yokuwohloka. Amachashaza engeziwe ngale kwendawo eyinhloko abonisa ukungcola okungenzeka.
3. Ukuqapha inqubo yokwenziwa
Ocwaningweni nasekuthuthukisweni kwezidakamizwa, i-TLC isetshenziswa ukuqapha ukusabela kwamakhemikhali: ukuthi ngabe ama-reactants asekhona yini, ukuthi imikhiqizo yakhekile yini, nokuthi ukusabela kufinyelela nini eqophelweni laso elihle.
4. Ukuhlaziywa kwemithi yezitshalo kanye namakhambi
I-TLC isetshenziswa kabanzi ekuhlaziyeni izithasiselo zezitshalo zokwelapha. Amanye ama-pharmacopoeias aphinde abhale izindlela ze-TLC zokuhlonza ama-marker compounds emikhiqizweni yamakhambi.
5. Ukuhlolwa okulula kokuqina
Ezifundweni zokuqala zokuzinza, i-TLC ingasiza ekutholeni ukwakheka kwemikhiqizo yokuwohloka ngenxa yokushisa, ukukhanya, noma umswakama.
Izinzuzo kanye nokulinganiselwa kwe-TLC
Ukuvelela
– Kuyashesha futhi kuyonga uma kuqhathaniswa nezindlela eziyinkimbinkimbi zokusebenzisa amathuluzi.
– Kulula ukukwenza futhi akudingi imishini ebizayo.
– Iguquguquka, ngoba ingahlukanisa izinhlobo eziningi zama-compounds ngokushintshashintsha kwezigaba ze-eluents kanye nezigaba ezimile.
- Ukuhlola okusebenzayo, okufanelekela izigaba zokuqala zokuhlaziya noma zocwaningo.
Ukulinganiselwa
– Ukuphindaphindeka kwesinye isikhathi kuba kubi uma izimo zingalawulwa ngokuqinile (ukugcwala kwemikhumbi, umswakama, ikhwalithi yesinyibilikisi).
– Ukulinganisa kunqunyelwe, yize kungathuthukiswa nge-densitometry (TLC Scanner).
– Ukuzwela ngokuvamile kuphansi kune-HPLC noma i-LC-MS.
Intuthuko Yanamuhla: HPTLC
Njengokuthuthukiswa kwe-TLC, i-High-Performance Thin-Layer Chromatography (HPTLC) iyaziwa. I-HPTLC isebenzisa amapuleti anezinhlayiya ezincane kanye nesendlalelo esifanayo, okuholela ekuhlukanisweni okubukhali nokuzwela kakhulu okungahlaziywa ngokwezibalo kusetshenziswa ama-scan e-densitometric. Embonini yemithi, i-HPTLC inikeza ibhalansi phakathi kokulula kwe-TLC kanye nokunemba kwezindlela zesimanje zokusebenzisa amathuluzi.
Isiphetho
I-Thin Layer Chromatography (TLC) isalokhu iyindlela ebalulekile yokuhlaziya embonini yezemithi ngenxa yobulula bayo, isivinini, kanye nokuguquguquka. I-TLC iwusizo ekuhlonzeni, ekuhlolweni kobumsulwa, ekuqapheni ukwenziwa, kanye nasekuhlaziyweni komkhiqizo wemvelo. Naphezu kwemikhawulo ekulinganisweni nasekuzweleni, i-TLC isalokhu iyisinqumo esiyinhloko sokuhlola nokuhlola okuvamile. Ngokuthuthuka kwe-HPTLC, amakhono e-TLC akhuphukile, okwenza ukuthi ihlangabezane nezidingo zokuhlaziywa kwezemithi okuqinile nokujwayelekile.
Uma ufisa, ngingangeza izibonelo zesifundo secala (isb., ukuhlonza i-paracetamol, i-caffeine, noma ama-marker compounds ku-turmeric) kanye nezincomo zezigaba zokuhamba kanye nezindlela zokubona ezivame ukusetshenziswa elabhorethri.
