The results obtained in these three tests, however, were unsuccessful.
The INS122, INS133, INS143 colorants did not have a
satisfactory chromatographic separation with the presence of double
peaks in the chromatograms. The INS143 colorant presented a
retention time of 41 minutes. Thus, it was necessary to make
changes in the composition of the mobile phase to improve these
results. The best results were achieved by use of the mobile phase
containing an acetonitrile–methanol (20:80) mixture and 1% (m/v)
ammonium acetate solution at pH 7.0 (adjusted with ammonium
hydroxide solution). Increasing the pH of the aqueous portion of
the mobile phase resulted in a poor resolution of the separation
of some colorants. A higher concentration of ammonium acetate
led to an increase of the retention time, probably due the enhanced
interaction of the analyte with the stationary phase.