Spectrophotometric reaction rate method for determination of barbituric acid by inhibition of the hydrochloric acid-bromate reaction.

Article abstract of DOI:10.1016/S1386-1425(03)00134-3

A new kinetic-spectrophotometric method was developed for the determination of barbituric acid. The method is based on its inhibition effect on the reaction between hydrochloric acid and bromate. The decolorization of methyl orange by the reaction products was used to monitor the reaction spectrophotometrically at 510 nm. The variable affecting the rate of the reaction was investigated. The method is simple, rapid, relatively sensitive and precise. The limit of detection is 7.9x10(-7) M and calibration rang is 1x10(-6)-6.0x10(-4) M barbituric acid. The linearity range of the calibration graph is depends on bromate concentration. The relative standard deviation of seven replication determination of 5.6x10(-6) M barbituric acid was 1.8%. The influence of potential interfering substance was studied.

Full text of DOI:10.1016/S1386-1425(03)00134-3

Spectrochimica Acta Part A 59 (2003) 3159Á3164
/
www.elsevier.com/locate/saa
Spectrophotometric reaction rate method for determination
of barbituric acid by inhibition of the hydrochloric
acidÁ
/
bromate reaction
Ali A. Ensafi *, H. Movahedinia
College of Chemistry, Isfahan University of Technology, Isfahan 84156, Iran
Received 29 October 2002; received in revised form 29 October 2002; accepted 5 March 2003
Abstract
A new kinetic-spectrophotometric method was developed for the determination of barbituric acid. The method is
based on its inhibition effect on the reaction between hydrochloric acid and bromate. The decolorization of methyl
orange by the reaction products was used to monitor the reaction spectrophotometrically at 510 nm. The variable
affecting the rate of the reaction was investigated. The method is simple, rapid, relatively sensitive and precise. The limit
ꢁ
7
ꢁ6
ꢁ4
of detection is 7.9ꢀ
/
10 M and calibration rang is 1ꢀ
/10
Á
/
6.0ꢀ10 M barbituric acid. The linearity range of the
/
calibration graph is depends on bromate concentration. The relative standard deviation of seven replication
ꢁ
6
10 M barbituric acid was 1.8%. The influence of potential interfering substance was studied.
determination of 5.6ꢀ
/
#
2003 Elsevier Science B.V. All rights reserved.
Keywords: Barbituric acid; Bromate; Methyl orange; Spectrophotometry; Inhibition
1
. Introduction
medical barbiturates which can be lethal in ex-
cessive amounts [5]. Other work has shown that in
mice, barbituric acid will cause liver and kidney
weight increase [6]. Barbituric acid is also a
precursor to derivates that have been shown to
have antibacterial activity [7,8] and for tumor
inhibitory agents [9]. Therefore, determination of
trace amounts of barbituric acid is very important
both in studies of biological and industrial pro-
cesses. Different methods such as chromatography
Barbituric acid {2,4,6(1H,3H,5H)-pyrimidine-
trione} is widely used in preparation of barbitu-
rates, dyes and polymerization catalysts [1],
pharmaceutical preparation and indicators [2],
textile [3] and also been identified as an inter-
mediate in many processes. It is known that
barbituric acid itself has no affect on the central
nervous system [4], however it is a precursor to
[
10Á
phoresis [14], infra-red spectrophotometry [15],
spectrophotometric methods [16Á21] and coulo-
/
12], mass spectrometry [13], capillary electro-
/
*
Corresponding author. Tel.: ꢂ
11-391-2350.
E-mail address: ensafi@cc.iut.ac.ir (A.A. Ensafi).
/
98-311-391-2351; fax: ꢂ98-
/
metric method [22] also have been reported for
determination of barbituric acid. Some of these
3
1
386-1425/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S1386-1425(03)00134-3

3160
A.A. Ensafi, H. Movahedinia / Spectrochimica Acta Part A 59 (2003) 3159Á3164
/
A solution of methyl orange (100 mg lꢁ ) was
prepared by dissolving 0.0250 g methyl orange
(Merck) in water and diluting to 250 ml with
water. Hydrochloric acid was prepared by appro-
priate dilution of concentrated hydrochloric acid
(Merck).
1
methods are time consuming and suffer from lack
of selectivity or good sensitivity and/or have short
linear dynamic range or have higher limit of
detection and/or used reagents not commercially
available. Recently, Medien and Zahran [23] used
1,4-naphthoquinone as a spectrophotometric re-
agent for barbituric acid determination, but the
method is not sensitive and has high limit of
1
2.2. Apparatus
ꢁ
determination (2.7Á61.5 mg ml ) and has many
/
interfering substances for barbiturate determina-
tion. R. Bartzatt [24] used sodium nitrite as a
suitable reagent for colorimetric analysis of barbi-
A Spectronic 20 (Genesys) spectrophotometer
with two matched 1-cm quartz cells was used to get
absorbance graph at various wavelengths. A
Spectronic 20 (Genesys, Model 4001/4) spectro-
photometer with a 1-cm glass cell was used to get
ꢁ
1
turate, with linear range of 18.7Á225 mg ml . D.
/
Nematollahi et al. [22] used controlled potential
coulometric technique for barbituric acid analysis
absorptionÁtime graphs at a fixed wavelength. A
/
in the range of 1Á
/
200 mmol. The method is not
thermostat water bath (Memmert, Model KG
8540, Schwabachw, Germany) was used to keep
very sensitive and has several interferences. There-
fore, the need for a fast, low cost and sensitive
method is obvious, especially for routine quality
control analysis. According to our knowledge,
there is not any kinetic-spectrophotometric proce-
dure for determination of barbituric acid at trace
level.
Kinetic spectrophotometric method [25] is one
of the most attractive approaches for ultra trace
determination of some species. Its advantage is
that only a spectrophotometer is required as the
main instrument. In this present work, a sensitive,
facile, and relatively selective method was devel-
the temperature of solutions at 359
/
0.1 8C.
2.3. Recommended procedure
The inhibited reaction was followed spectro-
photometrically by monitoring the change in
absorbance of the mixed reagents solution at 510
nm. An aliquot of sample solution containing 1ꢀ
/
ꢁ
5
ꢁ3
10
Á
/
6.0ꢀ
/
10
mol barbituric acid was trans-
ferred into a 10-ml volumetric flask, and then 1.4
ml of 2.33 M hydrochloric acid was added,
1
ꢁ
followed by 1.0 ml 100 mg l
solution. The solution was diluted to ca. 8 ml with
methyl orange
oped for the determination of barbituric (1ꢀ
/
ꢁ
6
ꢁ4
ꢁ3
1
0
Á
/
6.0ꢀ/10
M) acid based on its inhibiting
water. Then 1.0 ml 1.44ꢀ
/
10
M bromate was
effect on the reaction of bromate with hydrochlo-
ric acid. The decolorization of methyl orange by
the reaction products was used to monitor the
reaction spectrophotometrically at 510 nm.
added to the solution and the result solution was
diluted to the mark with water. The solution mixed
and a portion of the solution was transferred to
the spectrophotometric cell. The change in absor-
bance with time was measured for a 10Á240 s from
/
initiation of the addition of last drop of bromate
solution. All the solutions were kept at the 35 8C.
2
. Experimental
2
.1. Reagents and chemicals
3. Results and discussion
Doubly distilled water and analytical-reagent
grade chemicals were used. Barbituric acid stock
solution 0.010 M was prepared by dissolving
barbituric acid (Fluka) in water.
Bromate can be reduced by hydrochloric acid as
the following:
ꢁ
ꢁ
3
ꢂ
1
0Cl ꢂ2BrO ꢂ12H X5Cl ꢂBr ꢂ6H O
2
2
2
A 0.010 M sodium bromate solution was pre-
pared by dissolving 1.510 g NaBrO (Merck) in
water and diluting to 100 ml in a volumetric flask.
The produced bromine and chlorine react with
methyl orange and this reaction cause decoloriza-
3

A.A. Ensafi, H. Movahedinia / Spectrochimica Acta Part A 59 (2003) 3159Á
/3164
3161
tion of methyl orange [26] as the following
reaction:
can be calculated as:
T ꢃa ꢁa =b ꢁb
1
ip
1
2
2
Therefore, the calibration graph can be prepared
by plotting of t versus barbituric acid concentra-
tion.
ip
3.1. Influence of variables
This reaction can be monitored spectrophoto-
metrically by measuring the decrease in absor-
bance of the reaction mixture at 510 nm at 35 8C.
Barbituric acid can react with product of the
reaction (bromine and chlorine); therefore, the
induction period increases with increasing barbi-
turic acid concentration (Fig. 1). This inhibitory
effect on the reaction system depends on the
concentration of barbituric acid. The induction
period can be measured mathematically from the
regression equations of the linear part of the
The effect of reagents concentration and tem-
perature on the reaction system was studied to get
the best sensitivity and find optimum conditions.
The influence of hydrochloric acid concentration
on the sensitivity was studied over the range of
0
3
.20Á/0.45 M with and without barbituric acid at
0 8C. Fig. 2 shows the change in absorbance with
time as a function of hydrochloric acid. In addi-
tion, Fig. 3 shows that by increasing HCl concen-
tration up to 0.35 M, the difference in absorbance
change for uninhibited reaction and inhibited
reaction increases, whereas greater amounts of
the acid concentration decrease this difference.
Therefore, 0.35 M HCl was selected for study.
Fig. 4 shows the effect of bromate concentration
on the induction period time. The results show
that by increasing bromate concentration, the
induction period time decreased and the slope of
absorptionÁtime graph. The regression equation
/
for the first linear part of the graph is:
Aꢃa ꢂb t
1
1
And for the second linear part is:
Aꢃa ꢂb t
2
2
By equating these equations the induction period
Fig. 2. Variation of absorbance with time as a function of
hydrochloric acid; (a) 0.20 M; (b) 0.25 M; (c) 0.30 M; (d) 0.35
M; (e) 0.40 M HCl; (f) 0.45 M HCl; and (g) 0.50 M HCl.
Fig. 1. Absorbance change of methyl orangeÁ
/
bromateÁ
/
HCl
M; and (d)
ꢁ
6
ꢁ6
system, (a) 0.00; (b) 2.6ꢀ
/
10
M; (c) 7.2ꢀ
/
10
ꢁ
5
ꢁ1
ꢁ6
ꢁ4
1.2ꢀ/10
M barbituric acid. Conditions: 9.0 mg l
ꢁ
methyl
Conditions: 1.6ꢀ
/
10
bromate, and 10.0 mg l
M barbituric acid, 1.44ꢀ
/
10
M
4
ꢁ1
orange, 1.0ꢀ
/10
M bromate, and 0.35 M HCl.
methyl orange.

3162
A.A. Ensafi, H. Movahedinia / Spectrochimica Acta Part A 59 (2003) 3159Á3164
/
system with and without addition of barbituric
acid (Fig. 5), for the first 10Á240 s from initiation
of the reaction. The results show that the best
/
sensitivity can be achieved in the presence of 1.0ꢀ
/
ꢁ
4
10
M bromate concentration. Therefore, this
concentration was selected for the study.
The influence of methyl orange concentration
on the sensitivity was studied in the range of 2Á
/
20
ꢁ
1
mg l
methyl orange and in the presence of 0.35
4
ꢁ
M HCl and 1.0ꢀ
/10
M bromate concentration
at 30 8C with and without addition of barbituric
acid. The results showed that by increasing methyl
ꢁ
1
orange concentration up to 9.0 mg l , the
sensitivity increases, whereas greater amount of
the dye decreased the sensitivity. Therefore, a 9.0
ꢁ
1
mg ml methyl orange was selected for the study.
The sensitivity of the system dose not depends
on the ionic strength up to 1.0 M ionic strength
Fig. 3. Absorbance change for inhibited reaction (j), unin-
hibited reaction (m), and for their difference (') as a function
ꢁ
6
of HCl concentration. Conditions: 1.6ꢀ
/10
M barbituric
methyl orange.
(using 3.0 M K SO ).
2 4
ꢁ
4
ꢁ1
acid, 1.44ꢀ10 M bromate, and 10.0 mg l
/
The influence of temperature on the sensitivity
was studied over the temperature range of 5Á40 8C
/
in the presence of optimum reagent concentration.
The results showed by increasing temperature up
to 30 8C the sensitivity increased, and after that the
Fig. 4. Effect of bromate concentration on the induction period
ꢁ
5
ꢁ4
ꢁ4
time; (a) 7.2ꢀ
/
10 M, (b) 1.15ꢀ
/
10 M, (c) 1.44ꢀ
M, and (e) 2.16ꢀ
10ꢁ4 M bromate. Condi-
10ꢁ M barbituric acid, 0.35 M HCl, and 10.0 mg
methyl orange.
/
10 M,
ꢁ
4
(d) 1.72ꢀ/10
/
6
tions: 1.6ꢀ
/
ꢁ
1
l
the absorbance change increases for uninhibited
reaction (after initiation of the reaction). In order
to find the optimum concentration of bromate, the
Fig. 5. Absorbance change for inhibited reaction (j), unin-
hibited reaction (m), and for their difference (') as a function
ꢁ6
of bromate concentration. Conditions: 1.6ꢀ10 M barbituric
/
acid, 0.35 M HCl, and 10.0 mg lꢁ methyl orange at 30 8C.
1
change in absorbanceÁ
/
time was plotted for the

A.A. Ensafi, H. Movahedinia / Spectrochimica Acta Part A 59 (2003) 3159Á
/3164
3163
sensitivity decreased. Therefore, 30 8C was selected
for the study.
Table 2
Tolerance limit for diverse ions on the determination of 1.6ꢀ
/
ꢁ
6
1
0
M barbituric acid
Substance
Tolerance limit ratio (mo-
leSubstance/moleBarbituric acid
)
3.2. Calibration graph, precision and limit of
detection
2ꢁ
ꢁ, Kꢂ, Naꢂ
a
C
2
O
4
, ClO
3
2ꢁ
,
1000
ꢁ
2ꢁ
2ꢁ
NO
3
, SO
3
, SO
4
4
, MoO ,
PO , Cd(II), Mn(II), Csꢂ,
3
4
ꢁ
Solutions of known barbituric acid concentra-
Naꢂ, Kꢂ, B
O , acetone,
4 7
tions are utilized for standard curve. Under the
optimum conditions a linear correlation was found
between the induction period and barbituric acid
concentration. The linear dynamic range and
depends on the bromate concentration the results
are presented at Table 1. The linearity of the
calibration curve is demonstrated over a 200-fold
range of the concentration. The limit of detection
urea, ethanol, sucrose, glucose,
fructose, galactose
Pb(II), Cu(II), Mn(II), Al(III),
500
250
100
ꢁ
ꢁ
3 3
ClO , HCO , EDTA, acetate
Citrate, benzoic acid, maleic
acid, tartaric acid, Co(II), Ni(II)
Fe(III), Zn(II), CNꢁ, salicyl
aldehyde, phenobarbital
a
(
divided to slope of the calibration curve) was
3s /m, three of the standard deviation blank
Maximum concentration of substances tested.
b
ꢁ
7
7
.9ꢀ
/
10
M, barbituric acid. The relation stan-
dard deviation for seven-replication determination
Table 3
Determination of barbituric acid in synthetic samples
ꢁ
6
ꢁ6
ꢁ5
of 1ꢀ
/
10 , 5.6ꢀ
/10
and 1.1ꢀ
/
10
turic acid was 2.5, 1.8 and 1.5%, respectively.
M barbi-
Sample number
Barbituric acid (mM)
Added Found (nꢃ
Recovery%
/5)
I
1.50
2.00
2.50
3.00
3.50
4.00
1.46ꢂ
1.96ꢂ
2.48ꢂ
3.04ꢂ
3.40ꢂ
4.12ꢂ
/
0.06
0.05
0.04
0.02
0.02
0.03
97.3
98.0
99.0
101.5
97.0
103.0
4
. Effect of interfering substances
II
/
III
IV
V
/
/
The effect of various substances on the determi-
ꢁ
/
6
nation of 1.6ꢀ
/
10
M barbituric acid was
VI
/
studied and the results are shown in Table 2. The
tolerance was defined as the concentration of
added substance causing a relative error less than
5. Conclusion
3
%. Many substances did not interfere, even when
present in 500-fold excess over barbituric acid. The
results show good selectivity of the method for
barbituric analysis. To evaluate the performance
of the method for analysis of real sample, the
determination of barbituric acid in top water as a
synthetic sample was investigated. The results are
shown in Table 3 with satisfactory results.
With the proposed kinetic method it is possible
to determine barbituric acid at ultra trace levels.
Because the detection limit is dependent on the
concentration of bromate, it is expected that the
limit could be lowered further if lower concentra-
tion of bromate are selected. The method is very
Table 1
Linear regression parameters of calibration data for different concentration of bromate
ꢁ
3
BrO
(M)
Slope (s/M)
Intercept (s)
r (nꢃ
/
13)
Detection limit (M)
Calibration range (M)
ꢁ
ꢁ
4
4
5
ꢁ7
ꢁ6
ꢁ5
ꢁ4
1
.0ꢀ
/
10
217.4ꢀ
/
10
5
65.99
90.33
0.9991
0.9998
7.9ꢀ
/
10
1.6ꢀ
/
10
Á
Á/
/
1.8ꢀ
/
10
ꢁ6
ꢁ4
5
.0ꢀ
/
10
15.2ꢀ
/
10
5.5ꢀ
/
10
8.0ꢀ
/
10
6.0ꢀ
/
10

3164
A.A. Ensafi, H. Movahedinia / Spectrochimica Acta Part A 59 (2003) 3159Á3164
/
[
[
[
6] A. Reid, M.J. Turnbull, Arch. Int. Pharmacodyn. Ther.
11 (1974) 49.
simple, rapid, sensitive and relatively selective for
determination of barbituric acid. By dependence of
the rate of reaction with temperature (log(k) vs. 1/
T), the activation energy of the reaction was equal
2
7] P. Prisyazhnik, G. Palii, Y. Volydnskii, A. Lopushanskii,
E. Opanasenko, Khim. Farm. Zh. 10 (1976) 46.
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1
to 289
/
1.2 kJ mol . The procedure is suitable for
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togr. 620 (1993) 254.
[
[
[
[
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beke, Chromatographia 43 (1996) 599.
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The authors are thankful to the Center of
Excellency in Chemistry Research (IUT) and to
the Research Council of Isfahan University of
Technology for the support of this work.
[
[
14] T. You, X. Yang, E. Wang, Talanta 51 (2000) 1213.
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[
[
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