The effect of mobile phase water content on the separation mechanism of small polar molecules.

U okviru ovog rada proučavano je hromatografsko ponašanje serije Ru(II) kompleksa i odabranih anestetika u uslovima hromatografije hidrofilnih interakcija (Hydrophilic Interaction Liquid Chromatography, HILIC). Sistematski je ispitivan uticaj sastava mobilne faze, tačnije sadržaja vode u mobilnoj fazi, na retenciju ovih jedinjenja u različitim hromatografskim sistemima primenom visokoefikasne tankoslojne hromatografije (High Performance Thin-Layer Chromatography, HPTLC) kao brze i efikasne hromatografske tehnike, koja, s obzirom na minimalnu upotrebu rastvarača i potrebu za derivatizacijom, spada u metode zelene analitičke hemije. Za hromatografsko ispitivanje izabrani su sorbenti različite polarnosti i adsorpcionih karakteristika, a kao mobilna faza izabrane su binarne smeše vode i organskih rastvarača različitog sastava, od čiste vode do čistog organskog rastvarača. Utvrđeno je da povećanje sadržaja vode u mobilnoj fazi dovodi do promene mehanizma odvajanja, odnosno obrtanja retencionog redosleda ispitivanih jedinjenja i pretpostavljeno da se sa povećanjem sadržaja vode u mobilnoj fazi mehanizam odvajanja menja od adsorpcije do particije, a retencione krive imaju karakterističan U oblik. Prelaz između adsorpcionog i particionog mehanizma je kontinualan i zavisi od hemijske prirode odvajanih supstanci, stacionarne faze kao i organske komponente mobilne faze. Silika-gel se može smatrati najpogodnijim stacionarnom fazom za odvajanje i sistematsko praćenje hromatografskog ponašanja ispitivanih jedinjenja u HILIC uslovima, dok se acetonitril pokazao kao najpodesniji rastvarač. Dobijeni rezultati doprinose razumevanju dominantnog mehanizma odvajanja, odnosno vrste i jačina interakcija koje se ostvaruju između odvajanih supstanci i stacionarne i mobilne faze, a mogu se upotrebiti i za određivanje važnih fizičko-hemijskih karakteristika analita, naročito lipofilnosti. Takođe, predloženi su optimalni sistemi za brzu i efikasnu analizu polarnih supstanci što je od velikog praktičnog značaja za analitiku lekova, hrane i uzoraka iz životne sredine. Razvijena je i validovana HPTLC metoda za kvantitativno određivanje amigdalina iz različitih uzoraka. Takođe, predložena je i validovana HPTLC metoda za kvantitativno određivanje deset najčešće korišćenih sintetičkih boja za životne namirnice.In this study, chromatographic behavior of the Ru(II) complex, and selected anesthetics, was studied under conditions of hydrophilic interaction chromatography (Hydrophilic Interaction Liquid Chromatography, HILIC). The effect of mobile phase composition, precisely, water content in the mobile phase used, on the retention of these compounds in different chromatographic systems was systematically examined using High Performance Thin-Layer Chromatography (HPTLC) as a fast and efficient chromatographic technique which, given the minimal use solvent and the need for derivatisation, belongs to the methods of green analytical chemistry. For the chromatographic investigations the sorbents of different polarity and adsorption characteristics were selected, and mixtures of water and organic solvents of various compositions, from pure water to pure organic solvent, were selected as the mobile phase. It was established that increasing the amount of water in the mobile phase leads to a conversion of the separation mechanism, and assumed that as the water content increases in the mobile phase, the separation mechanism changes from adsorption to the partition, and the retention curves have a characteristic U shape. The conversion between the adsorption and partition mechanisms is most likely continuous and depends on chemical nature of separated substances, stationary phase as well as on organic component of mobile phase. Silica-gel can be considered as the most suitable stationary phase for the systematic investigation of the chromatographic behavior of the test compounds in HILIC conditions, whereas acetonitrile has been shown to be the most suitable solvent. Two new HPTLC methods, for the quantitative determination of the ten most commonly used synthetic food colors and amygdalin from different samples, were developed and proposed. The results obtained contribute to the understanding of the dominant separation mechanism, the type and intensity of the interactions that occur between separated substances and the stationary and mobile phases, and can also be used to determine important physical and chemical characteristics of the compaunds investigated, especially lipophilicity. In addition, the optimal systems for rapid and efficient analysis of polar substances have been proposed, which is of great practical importance for the analysis of drugs, food and environmental samples