IJUP08 - Universidade do Porto

Microplate high-throughput methodology for determination of
Folin-Ciocalteu index
F. Santos, L. M. Magalhães, M. A. Segundo, S. Reis and J. L. F. C. Lima
REQUIMTE, Serviço de Química-Física, Faculdade de Farmácia, Universidade do Porto,
Rua Aníbal Cunha, 164, 4099-030, Porto, Portugal
Email: miguel.santos.2@gmail.com
The interest in antioxidant nutrients has increased due to their protective effects against
free radical-induced reactions [1]. In this context, the Folin-Ciocalteu assay [2] has been
proposed recently as a standard method for measurement of antioxidant capacity of food
products and dietary supplements in routine quality control [3]. Therefore, the main goal of
this work is the development of a simple, fast and reliable analytical methodology for the
determination of Folin-Ciocalteu index.
To achieve this objective, the conventional methodology was adapted to a 96-well
microplate format. The reaction volume was then reduced 100 times, from 20 mL to 0.2
mL. Furthermore, the reaction conditions were thoroughly studied, namely the pH of the
reaction media. It was observed that an increase of pH from 10 (usual value applied,
provided by a carbonate buffer) to values around 12-13 changed the reaction kinetics
dramatically. In fact it was possible to decrease the reaction time from 2 hours to 3-5
minutes.
The developed methodology was applied to the determination in several types of food
products, using gallic acid as standard compound. The Folin-Ciocalteu index values
obtained for wines, beers and juices were comparable to those obtained from the previous
batch method, with a significant improvement in the reagent consumption, effluent
produced per assay and determination throughput. These characteristics constitute a step
forward the implementation of greener procedures in the Analytical Chemistry area.
References:
[1] Halliwell, B., Murcia, M.A., Chirico, S., and Aruoma, O.I. (1995), Free-radicals and
antioxidants in food and in vivo –what they do and how they work, Critical Reviews in Food
Science and Nutrition, 35 (1-2), 7-20.
[2] Singleton, V.L., Orthofer, R., and Lamuela-Raventós, R.M. (1999), Analysis of total phenols
and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent, Methods in
Enzymology, 299, 152-178.
[3] Prior, R.L., Wu, X., and Schaich, K. (2005), Standardized methods for the determination of
antioxidant capacity and phenolics in foods and dietary supplements, Journal of Agricultural and
Food Chemistry, 53 (10), 4290-4302.
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