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.
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