resulted in adequate resolution between all five compounds, however,
the broad and diffuse peak shapes were still unacceptable.
Gradual introduction of methylene chloride on the expense of both
ethyl acetate and n-hexane resulted in a significant improvement
in peak shape without affecting resolution and this effect was more
pronounced by decreasing the ethyl acetate content of this quaternary
mixture. Indeed, the total replacement of ethyl acetate with
methylene chloride while maintaining the other 2 components
provided the best and the simplest developing system in terms
of resolution, peak shape and symmetry. Finally, the developing
system was adjusted by using [methylene chloride:n-hexane:
methanol] in the ratio [8.8:0.3:0.9, by volume] and the spots
of ALF, TER, PRZ, DOX and FIN were observed at Rf values equivalent
to 0.26 ± 0.02, 0.36 ± 0.02, 0.45 ± 0.02, 0.59 ± 0.02 and
0.69 ± 0.02, respectively. The chamber was saturated with the
mobile phase at room temperature for 15 min prior to development.
The optimized procedure resulted in well-defined spots with
reproducible Rf values.