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Boletín de la Sociedad Chilena de Química

Print version ISSN 0366-1644

Bol. Soc. Chil. Quím. vol.47 no.4 Concepción Dec. 2002 


Carola Henriquez and Eduardo Lissi

Departamento de Química, Facultad de Química y Biología, Universidad de Santiago de Chile,
Casilla 40, Correo 33, Santiago de Chile, Chile.

(Received: January 3, 2002 - Accepted: July 3, 2002)


The exact knowledge of the value of the extinction coefficient of the ABTS derived radical is necessary for its quantitative use in the evaluation of the antioxidant capacity of pure compounds and/or complex mixtures. We have performed experiments in order to stablish its lower and upper limits of its value. The limits obtained,

1.0 x 104 M-1 cm-1 £ e 734 £ 1.6 x 104 M-1 cm-1

are fully compatible with previously reported data. However, the use of these values leads to large stoichiometric coefficients for the reaction of these radicals with simple substrates, such as monophenols or tryptophan.

KEYWORDS: ABTS; extintion coefficient; antioxidants evaluation.


Conocer el valor exacto del coeficiente de extinción del radical catión derivado del ABTS es un pre-requisito para su empleo en la evaluación de la capacidad antioxidante de compuestos puros polifuncionales o mezclas complejas. Nosotros hemos realizado emperimentos que permiten establecer límites inferiores y superiores para su valor. Los límites obtenidos

1.0 x 104 M-1 cm-1 £ e 734 £ 1.6 x 104 M-1 cm-1

son compatibles con los valores reportados en la literatura. Sin embargo, el uso de estos valores conduced a coeficientes estequiométricos anormalmente altos para sus reacciones con comuestos simples como monofenoles y triptofano.

PALABRTAS CLAVES: ABTS; Coeficiente de extinción; evaluación de antioxidantes.

The ABTS derived radical cation (ABTS+* or ABTS-* if the charge of the sulfonate groups is considered ) is widely employed in the evaluation of antioxidant capacities of pure compounds and/or complex mixtures.1-4 In several of these methodologies, the measured property is the loss of absorbance due to the radical cation (measured at 734 nm) elicited by a given amount of the tested sample.1-3 A quantitative interpretation of these data requires the knowledge of the absorption coefficient of the radical cation at the employed wavelength. Since the radical cation has not been prepared as a pure substance, its extinction coefficient has not been directly measured and the values employed have been obtained from consumption and/or production experiments.. The generally employed value is 1.5 x 104 M-1 cm-1.2,3,5,6 However, the use of this value leads to very high stoichiometric coefficients, defined as the number of radicals that can be bleached by a molecule of the added free radical scavenger. For example, it gives an stoichiometric value of two for Trolox,3,4,7,8 , but values four or even higher for p-ethylphenol, and tryptophan.4,7 These high values would imply a very complex reaction mechanism, casting doubts on the validity of the use of this free radical in the evaluation of the amounts of antioxidants present in a given sample. Another possibility is that the true extinction coefficient of the ABTS radical be considerably higher than the accepted value, leading so to meaningful stoichiometric factors.4,7 In order to test this last possibility, we have carried out an estimation of the radical absorption coefficient employing two procedures that allow to establish lower and upper limits to this value.

Lower limit of the extinction coefficient at 734 nm.

The ABTS radical cation can be readily prepared by reaction of ABTS with an excess of activated MnO2. After adding the solid to an aqueous solution of ABTS, the blue-green spectra of the radical cation appears instantaneously (Figure 1). The shape of this band corresponds to that reported for the ABTS derived radical, with a ratio of absorbances at 412 and 734 nm of 2.31, as reported in the literature.5 Exclusive oxidation of the parent ABTS to the radical is compatible with the lack of a band at 518 nm, typical of the species obtained by the two electron oxidation of ABTS.

Fig. 1. Spectra of the ABTS derived radical obtained in the oxidation of ABTS (65 m M) by MnO2

The maximum possible radical concentration is limited by the initial ABTS concentration. The observed absorbance at 734 nm can be expressed then as

A734 = e 734 [ABTS+] £ e 734 [ABTS]

and hence

e 734 ³ A734 / [ABTS]


The data obtained in this type of experiments give then that

e 734 ³ 1.0 x 104 M-1 cm-1

providing a lower limit to the extinction coefficient.

The data obtained in the oxidation by MnO2 can be corrected by considering that the oxidation is not total, and that a fraction of the parent ABTS remains unreacted. This is supported by the presence of a clear peak at 340 nm (see Fig. 1). A precise evaluation of the amount of ABTS is difficult because of the absorbance of the radical cation at this wavelength (See inset of Fig 1 from Ref. 5). However, the amount of ABTS remaining can be estimated if it is assumed that the absorbance of the radical cation is similar at 734 and 340 nm.5 In this case, Eqn. (2) can be reformulated as

e 734 » A734 / ([ABTS]0 - [(A340 - A734)/ e 340]


where e 340 is the extinction coefficient of the parent ABTS at 340 nm. In a typical experiment, applying Eqn. (3) leads to

e 734 » 1.7 x 104 M-1 cm-1

Upper limit of the extinction coefficient at 734 nm.

When a solution of the ABTS radical cation is reacted with a solution of free radical scavengers, such as Trolox or ascorbic acid, takes place a loss of absorbance at 734 nm, indicative of the free radical consumption.2,4,8 This bleaching is linearly related to the amount of added antioxidant. Parallel to the decrease in absorbance at 734 nm, takes place an increase in the absorbance of the parent ABTS, measure at 340 nm (Figure 2).6 When ascorbic acid is employed, these changes can be ascribed to a reaction such as

2 ABTS+ + AH¾® 2 ABTS + A + H+



where A stands for a dehydroascorbate molecule. Since the extinction coefficient at 340 nm of ABTS can be directly determined (3.5 x 104 M-1cm-1 in the present work, 3.6 x 104 M-1 cm-1 from the literature),5,9,10 the increase in absorbance at 340 nm provides a lower limit to the ABTS formation. In fact

D ABTS ³ D A340 / e 340


where the inequality is introduced to take into account the small absorbance of the radical cation at this wavelength. This amount of ABTS formed puts a lower limit to the amount of ABTS+ initially present. Hence

D ABTS = A734 / e 734 ³ D A340 / e 340


where the inequality takes into account Eqn. (5) and the fact that the conversion of the radical cation to the parent compund could be not quantitative. From Eq. (6) it can be obtained that

e 734 £ A734 e 340 / D A340


The data obtained employing both Trolox and ascorbate leads to

e 734 £ 1.6 x 104 M-1 cm-1

putting an upper limit to the ABTS radical extinction coefficient at 734 nm.

Fig. 2. Change in the spectra of a solution of the ABTS radical cation following addition of ascorbic acid. (æ) Prior to ascorbic acid addition; (........) ascorbic acid, 6 mM; (-----) ascorbic acid, 12 mM.

From both sets of experiments it can then be concluded that, for the radical cation

1.0 x 104 M-1 cm-1 £ e 734 £ 1.6 x 104 M-1 cm-1

This inequality is fully compatible with the accepted value of the extinction coefficient of the radical at 734 nm (1.5 x 104 M-1 cm-1), and indicates that the high stoichiometric factors obtained for some compounds are not due to an overestimation of the radical cation concentration. It is difficult then to explain these apparently anomalous results, but they cast doubts on the validity of procedures to determine antioxidant capacities based on the bleaching of ABTS radical cations.


This work has been financed by DICYT (University of Santiago, Chile).


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