Primerjava HPLC tehnik z uporabo reagentov ionskih parov ter mešanih separacijskih režimov za določevanje farmacevtskih učinkovin

Kastelic, Domen (author), Pompe, Matevž (mentor)

Za ločevanje polarnih in ionskih spojin v okviru reverzno fazne tekočinske kromatografije visoke ločljivosti se pogosto uporablja kromatografija ionskih parov in kromatografija z mešanimi separacijskimi režimi. V okviru magistrskega dela sem primerjal primernost uporabe teh dveh pristopov za razvoj metode za določevanje vsebnosti 4-aminofenola. Preverjena in ovržena je bila možnost dosega retencije 4-aminofenola na nekaterih pogostih reverzno-faznih kolonah proizvajalca Kinetex: C18, C8, Luna C5, Phenyl Hexyl in F5. Preučil sem vpliv uporabe različnih reagentov ionskih parov (natrijev dodecilsulfat, natrijev heptilsulfonat, trifluoroocetna kislina in perklorat) na stanje kromatografske kolone. Vpliv na kolono sem preveril z injiciranjem testne raztopine (uracil, naftalen, acetofenon, toluen) pred uporabo reagentov ionskih parov, po uporabi le teh in po različnih postopkih spiranja, namenjenih regeneraciji kolone. Na teh različnih stopnjah sem spremljal kromatografske parametre vrhov ter sipanje njihovih vrednosti. Natrijev dodecilsulfat in natrijev heptilsulfonat zagotovita retencijo 4-aminofenola na izbrani C18 kromatografski koloni. Z uporabo trifluoroocetne kisline in perklorata to ni bilo možno. Stacionarna faza je močno modificirana po uporabi natrijevega dodecilsulfata, za regeneracijo je zato potrebno dolgotrajno spiranje (>48 ur) pri agresivnih pogojih (pufer z visoko ionsko močjo – 100 mM in pH 6,0). Ostali reagenti ionskih parov niso signifikantno trajno modificirali stacionarne faze. Kolona Sielc Primesep 100 je bila izbrana kot ustrezna stacionarna faza z mešanimi separacijskimi režimi. Na obeh razvitih metodah je bila izvedena osnovna validacija, iz njih pa sta bili še modificirani ter preverjeni metodi za določevanje sorodnih substanc analita. Zaradi problematike modifikacije stacionarne faze, regeneracije s spiranjem, posledično težko dosegljive ponovljivosti med kolonami in nasploh zahtevnejše izvedbe pri uporabi reagentov ionskih parov se je za boljšo alternativo pri določevanju 4-aminofenola kot ionskega analita izkazala tehnika uporabe stacionarne faze z mešanimi separacijskimi režimi.

• Language: Slovenian
• Keywords: kromatografija ionskih parov, kromatografija z mešanimi separacijskimi režimi, 4-aminofenol
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2020
• PID: 20.500.12556/RUL-115971
• COBISS.SI-ID: 13572611
• Publication date in RUL: 04.05.2020

KASTELIC, Domen, 2020, Primerjava HPLC tehnik z uporabo reagentov ionskih parov ter mešanih separacijskih režimov za določevanje farmacevtskih učinkovin [online]. Master’s thesis. [Accessed 24 March 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=115971

Secondary language

• Language: English
• Title: Comparison of ion-pairing and mixed-mode HPLC techiques for the determination of active pharmaceutical ingredients

Abstract

For the separation of polar compounds in reversed-phase high performance liquid chromatography ion-pair chromatography and mixed-mode chromatography are often used. In the thesis these two aproaches were compared for 4-aminophenol assay method development. Possibility of retention of 4-aminophenol on some common reversed-phase columns by Kinetex: C18, C8, Luna C5, Phenyl-Hexyl and F5 was inspected and proved to not be possible. The effect of different ion-pairing reagents (sodium dodecylsulfate, sodium heptylsulfonate, trifluoroacetic acid and perchlorate) use on column condition was also explored. Effect on the column was studied by injecting test solution (uracil, naphthalene, acetophenone, toluene) before the use of ion-pairing reagents, after use and after different column washing procedures aimed at regenerating the column. Peak chromatographic parameters and their value dispersion was monitored at these different stages. Sodium dodecylsulfate and sodium heptylsulfonate achieve retention of 4-aminophenol on chosen C18 chromatographic column. This was not possible by using trifluoroacetic acid or perchlorate. The stationary phase is strongly modified by sodium dodecylsulfate and makes long (>48 h) and aggressive (buffer with high ionic strength – 100 mM and pH 6,0) washing necessary for column regeneration. Other ion-pairing reagents did not significantly permanently modify the stationary phase. Sielc Primesep 100 was chosen as a suitable mixed-mode column. Basic method validation was performed on developed methods as was further method modification and suitability for related substances assay. Problems such as stationary phase modification, washing needed for regeneration, consequently bad inter-column reproducibility and more demanding method development are the reason mixed-mode columns are a better alternative to ion-pairing reagents in case of ion analyte – 4-aminophenol assay.

• Keywords: ion-pair chromatography, mixed-mode chromatography, 4-aminophenol