Optimization of separation conditions
Vitamin separation was firstly optimized using CZE. For this purpose, a test mixture containing the eight vitamin standards (at
100 mg mL1 ) described in the Chemicals section was used. Most CE studies related to vitamin separation have been mainly performed using borate buffers at different concentrations (20e50 mM) and at pH values comprised between 8.0 and 9.5 (in order to assure that analytes are in their ionic form).For this purpose, different borate contents (ranged from 10 to 60 mM) at a pH of 8.5 were first tried. The results obtained are shown in Fig. 2. As it can be observed, in general, analysis time increased when the borate content increased, which could be explained in terms of an ionic strength effect, giving as a result an EOF decrease. The migration order of vitamins was similar to that found in literature . Thus, vitamins B2, B3 (nicotinic acid), B5, B6 and C were all were negatively charged at pH 8.5, and their migration velocity depended on their degree of ionization and molecular size. On the other hand, vitamins B3 (nicotinamide) and B12 were non-charged analytes at this pH, and a comigration with EOF peak was evidenced. It should be noted that thiamine (B1) was the only vitamin
that has a positive charge at this pH, and therefore migrated faster than EOF peak. As shown in Fig. 2, the resolution between C and B5
vitamin peaks increased when increasing borate content. Thus, as a compromise between resolution and analysis time, a 40 mM borate
buffer was chosen for the next studies.