Mass balance was performed on microbeads containing riboflavin
as a validation step to ensure the mass of riboflavin in the
microbeads and, the mass of riboflavin in both the CaCl2 and rinsing
solution (internal method) or the mass of riboflavin remaining
in solution (external method) was equal to the initial mass of riboflavin
used. Microbeads produced by both the internal and external
method were degraded. Riboflavin containing microbeads were
placed in a 10 mM SDS solution containing 0.5% v/v mercaptoethanol
in screw cap containers covered with tin foil for 24 h
at 20 C to degrade, the samples were periodically inverted. SDS
was used as it is a surfactant that can disrupt non-covalent bonds
in proteins causing the protein to unfold (Voet, Voet, & Pratt, 2008).
The subsequent unfolding of the proteins partly disrupts the
microbeads structure and also facilitates the action of mercaptoethanol.
The mercaptoethanol reduces disulphide bonds which
provide structural strength to the microbead (Voet et al., 2008).
The presence of a hydroxyl group on the mercaptoethanol confers
solubility in water allowing it to diffuse into the microbead. The
concentration of riboflavin released from the degraded microbeads
was quantified by HPLC as described in Section 2.8.