A Laboratory Exercise for Photochemistry and Photobiology: Production of Hydrogen Peroxide

Abstract

Light-excitation of chromophores, changes in redox potential, and transfer of electrons are complex concepts for many students in chemistry and biochemistry. In the proposed experimental work, students can assimilate these concepts by developing a photochemical system to synthesize a product of interest in industry and pharmacy, hydrogen peroxide. The photochemical system has three basic components: (i) the photosensitizer, a molecule that is excited by visible photons allowing the electron flux from (ii) an electron donor to (iii) an electron acceptor. In this laboratory, riboflavin was the photosensitizer and semicarbazide and molecular oxygen were the electron donor and acceptor, respectively. The hydrogen peroxide formed was enzymatically quantified using horseradish peroxidase and o-dianisidine as substrate. Production of hydrogen peroxide can be detected after 20 min of illumination using a simple desk lamp with a bulb of 60 W. The system was developed at pH 7.5 and pH 12.3 to allow discussion about the reactivity of flavin semiquinone forms. As in bacterial and plant photosynthesis, part of the energy absorbed in the photochemical system is used to drive an endergonic reaction.