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The Chemical Educator

ISSN: 1430-4171 (electronic version)

Table of Contents

Abstract Volume 19 (2014) pp 006-010

Free Radical Generation and Inhibition With Luminol and Ascorbic Acid (AA)

Charles F. Saladino*, and Adaline Predmore

Chemistry/Biochemistry Department, Misericordia University, Dallas, PA,
Received August 26, 2013. November 26, 2013.

Published: 7 January 2014

Abstract. The overall purpose of these experiments is to provide an undergraduate student taking a biochemistry laboratory the opportunity to demonstrate free radical production and inhibition by a physiologically-relevant antioxidant. Free radicals are initiated by H2O2 reacting with luminol (o-Aminopthalic hydrazide), generating a chemiluminescence detected in a luminometer. The free radical production is inhibited using ascorbic acid (AA) as the antioxidant. The results show a strong dose-response for the inhibition for both single point and kinetic data. pH studies with luminol only show an inverse relationship between chemiluminescence and pH. When K3Fe(CN)6 is used as a catalyst, chemiluminescence increases approximately 16-fold over that without the catalyst; and decay occurs much more rapidly. Thus, this system we describe serves as an interesting and challenging laboratory exercise for students in an undergraduate biochemistry lab (also adaptable for the organic chemistry lab), wherein they can indirectly observe free radical formation and inhibition with a well-known antioxidant. Much of the data generated in developing the protocols are new. The protocols and techniques are accurate, reliable, demonstrate the importance of controlling potentially-confounding variables, and require critical thinking in writing the lab report. They require that the student be precise and accurate in order to achieve high R2-values and a low S.E.M. Also, this series of lab exercises provides the instructor with great flexibility as to how many procedures he/she wishes to have the student perform, while affording the student the opportunity to carry out procedures that are not normally found on-line or in laboratory manuals typically used in undergraduate biochemistry laboratory courses.

Key Words: Laboratories and Demonstrations; biochemistry; student laboratory; free radical inhibition; chemiluminescence

(*) Corresponding author. (E-mail:

Article in PDF format (182 KB) HTML format

Supporting Materials:

One supporting file is available. This contains Appendix I, Single Point Determinations – example of raw data and graph obtained and Appendix II, Raw Data for 10 Second Kinetics – Chemiluminescence of Luminol as a Function of AA Concentration (78 KB).

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