In the case of the internal method, riboflavin powder was added
at concentrations of 0.02–0.12 g/L to the denatured whey protein
solution (prepared as per Section 2.2), the riboflavin containing
whey protein solution had a pH of 6.8. The range of concentrations
is converted to a ratio of riboflavin to protein in solution (mg:g).
This solution was dropped into a CaCl2 solution as per Section 2.2
to produce microbeads. The mass of the CaCl2 solution and beaker
was recorded before and after microbead formation to determine
the number of microbeads produced. Microbeads were kept in
100 mM CaCl2 for 24 h to crosslink before rinsing three times in
100 ml of deionised water to remove CaCl2. Samples of the CaCl2
and rinsing water were analysed by HPLC as per Section 2.8 to
determine the loss of riboflavin. A mass balance exercise was performed
on microbeads as per Section 2.4 to confirm the concentration
of riboflavin in the microbeads. All glassware containing
riboflavin was covered with tin foil to prevent photodecomposition.