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Improved DPPH determination for antioxidant activity spectrophotometric

assay

Article  in  Chemical Papers- Slovak Academy of Sciences · June 2007

DOI: 10.2478/s11696-007-0022-7

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c 2007 Institute of Chemistry, Slovak Academy of Sciences
DOI: 10.2478/s11696-007-0022-7

SHORT COMMUNICATION

Improved DPPH Determination for Antioxidant Activity

Spectrophotometric Assay

a
M. R. SZABO, a C. IDIŢOIU, a D. CHAMBRE, and b A. X. LUPEA

a Chemical and Technological Research Centre, Food Engineering, Tourism, and Environmental Protection Faculty,
“Aurel Vlaicu” University, Elena Dragoi Street 2, Arad, 310330, Romania
e-mail: ralucaszabo@rdslink.ro

b Chemistry and Organic Technologies Department, “Politehnica” University from Timişoara, FCIIM,
Telbisz Street 6, Timişoara, 300001, Romania

Received 3 August 2006; Revised 1 December 2006; Accepted 4 December 2006

An improved procedure for determination of the residual DPPH (1,1-diphenyl-2-picrylhydrazyl)

free radical concentration was proposed taking into account the absorbance of both DPPH free
radicals and DPPH nonradical (1,1-diphenyl-2-picrylhydrazine) stable form. The calculated residual
DPPH free radical concentrations were compared with those obtained from a calibration curve and
variation coefficients below 10 % were found.

Keywords: DPPH determination, antioxidant activity, spectrophotometric assay, free radicals

Oxidation is one of the most important processes,
which produce free radicals in food, chemicals, and
even in living systems. Free radicals have an impor-
tant role in processes of food spoilage, chemical ma-
terials degradation, and also contribute to more than
one hundred disorders in humans [1—6].
Antioxidants are defined as substances that even at
low concentration significantly delay or prevent oxida-
tion of easy oxidizable substrates [7]. The applications
of antioxidants are industrially widespread in order
to prevent polymers oxidative degradation, autooxi-
dation of fats, synthetic and natural pigments discol-
oration, etc. There is an increased interest of using
antioxidants for medical purposes in the recent years
[8—10].
Several methods are used for the estimation of effi-
ciency of synthetic/natural antioxidants, like the fer-
ric reducing antioxidant power (FRAP) assay [11], β-
carotene/linoleic acid assay [12, 13], Rancimat method
[14, 15], inhibition of low-density lipoprotein (LDL)
oxidation [16], DPPH assay [13, 14, 17—20], etc. This
method diversity is due to the complexity of the an-
alyzed substrates, often a mixtures of dozens of com-
pounds with different functional groups, polarity, and
chemical behavior.
In this paper the attention is focused on the DPPH
assay, which is one of the best-known, frequently em-
ployed, and accurate methods. DPPH (1,1-diphenyl-
2-picrylhydrazyl) is a stable free radical because of its
spare electron delocalization over the whole molecule.
The delocalization causes a deep violet color with λmax
around 520 nm. When a solution of DPPH is mixed
with a substrate acting as a hydrogen atom donor, a
stable nonradical form of DPPH is obtained with si-
multaneous change of the violet color to pale yellow
[21].
All experiments were done at 517 nm, using an Ul-
trospec III (Pharmacia-LKB) single-beam spectropho-
tometer. Using known initial DPPH concentration c0
= 10−4 mol dm−3 in methanol, molar absorptivity of
DPPH free radicals εv = 1.14 × 104 dm3 mol−1 cm−1
was estimated from the Lambert—Beer relation [22],
the value of which is in good agreement with those ob-
tained by other researchers [21]. Similarly, the molar
absorptivity of the stable nonradical form of DPPH,
εy = 430 dm3 mol−1 cm−1 , was determined at the
same wavelength.
Assuming additive character of the molar absorp-
tivity, the overall absorbance could be expressed as

214 Chem. Pap. 61 (3) 214—216 (2007)

DPPH DETERMINATION FOR ANTIOXIDANT ACTIVITY ASSAY

A = Av + Ay + Am = εv cv l + εy cy l + 0.963 (1) 2.05

where A is the experimental absorbance, Av and Ay

the absorbances of the DPPH free radical and its 1.85
stable nonradical form, respectively, cv and cy the
concentrations of the DPPH and its stable nonradi-

Absorbance
cal form, respectively, Am the methanol (solvent) ab- 1.65
sorbance at 517 nm, and l = 1 cm the path length of
the cell. Taking into account
1.45
cy = c0 − cv (2)

and after substitution of all known parameters and 1.25

rearrangement eqn (1) can be written in the form 0 14 28 42 56
time/min


A − 0.963 = 1.14 × 104 cv + 430 10−4 − cv (3) Fig. 1. Variation of the DPPH absorbance with time in the
presence of ascorbic acid (repeated experiments).
that was converted to a calibration relation for the
single-beam spectrophotometer
10
A − 1.006
cv = (4)
10970
8
Eqn (4) has to be valid for any single-beam spec-
cDPPH · 10 /(mol dm )
-3

trophotometer at the same experimental conditions.

Omitting the absorbance contribution of the 6
DPPH nonradical form, the calibration curve (r2 =
5

0.9992) for the DPPH free radical concentration is

given by the relation 4

A − 0.9585
cv = (5)
11462
Validity of eqns (4) and (5) was checked on the ba-
sis of experimentally determined antioxidant activity
of ascorbic acid. During these experiments, 2.9 cm3 of
the DPPH solution was mixed with 0.1 cm3 of the test
solution (ascorbic acid, c = 0.4 × 10−3 mol dm−3 ) in a
4 cm3 quartz cuvette with 1 cm path length. The sam-
ple absorbance was monitored at λ = 517 nm until a
“plateau” was reached. Each experiment was repeated
5 times (Fig. 1).
An average curve of each series of experiments was
used for further calculation of the DPPH free radical
concentrations. In order to compare the accuracy of
calibration relations (4) and (5), the calculated con-
centration vs. time was plotted in Fig. 2.
Variation coefficient (V ), defined as the standard
deviation from the average value ratio [23], was com-
puted for each pair of concentration values. They were
found to vary between 0.2 % (at the reaction start)
and 8.2 % (at the end of the reaction). The change
of the variation coefficient with time was attributed
to the formation of stable nonradical form of DPPH,
which in fact has not been considered in eqn (5). Dif-
ference between the calculated absorbance values be-
low 10 % might be considered as acceptable. Although
the shape of curves shown in Fig. 2 is quite similar, it
2
0 14 28 42 56
tim e/min
Fig. 2. Variation of the residual DPPH concentration with
time as calculated using eqns (4) (dotted line) and (5)
(solid line).
is advisable to use eqn (4) for further calculations be-
cause it is in a better agreement with the experimental
data.
Antioxidant activity of the DPPH free radicals
could be easily evaluated employing one of at least
three methods [21, 22, 24—33]. Thus, more than one
interpretation of the experimental data is possible us-
ing, e.g. the so-called antioxidant inhibition (quench-
ing) or residual concentration [21], as well as the ef-
fective concentration [26, 28, 32, 33]. Recently, the an-
tioxidant activity was expressed in terms of the con-
centration of a known antioxidant such as BHT (2,6-
di-t-butyl-4-methylphenol) [34]. It was found that dur-
ing the evaluation of the antioxidant activity also the
chemical structure of the substrate molecule should be
considered.
The proposed model eqn (4) accounts for both col-
ored forms of DPPH allowing direct estimation of the
antoxidant activity without calibration procedure. On

Chem. Pap. 61 (3) 214—216 (2007) 215


the other hand, one should be aware that the results of
a single assay can give only limited information about
the antioxidant properties of food extracts and, there-
fore, they should be interpreted with some caution.
An approach applying multiple assays in screening the
antioxidant activity is highly advisable and the data
obtained by this improved method still need to be con-
firmed by an independent method.
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