Under a RP-LC separation, the more polar compounds elutefirst. However, since the chromatographic determination of thecolorants is performed, normally, at pH around 7, the acidicand alkaline groups present in the molecules can change the
elution sequence [37]. Thus, under the conditions of the current
experiments, colorants are carried along with the organic
solvent (mixture of methanol:acetonitrile). The addition of acetonitrile
was found to improve significantly the asymmetry of
the peaks, which has also been noticed when used in methanol
[38]. However, the addition of an inorganic electrolyte to the
mobile phase, as a modifier, is necessary in order to improve
the separation of ionizable species and to obtain the separation
in reasonably short analysis time. Ammonium acetate buffer
has been used as a modifier for the purification and separation
of seven azo colorants by RP-HPLC and preparative liquid
chromatography [39]. It was also used (at a pH 5) for the separation
and determination of two (E 102 and E 110) [38] or
five azo food dyes (E 102, E 104, E 110, E 122 and E 124)
in soft drinks by RP-HPLC [23]. A gradient elution prepared
by a mixture of sodium acetate buffer (0.1 M and pH 7) with
acetonitrile was used for the successful separation of 14 synthetic
food colorants in standard solutions [22]. It was also used
for the successful determination of eight azo-colorants in foodstuff
by liquid chromatography–electrospray-mass spectrometry
(LC–ESI-MS)