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Journal of Applied Sciences and Environmental Management
World Bank assisted National Agricultural Research Project (NARP) - University of Port Harcourt
ISSN: 1119-8362
Vol. 10, Num. 3, 2006, pp. 43-46
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Journal of Applied Sciences & Environmental Management, Vol. 10, No. 3, September, 2006, pp. 43-46
Spectrophotometeric Determination of Bromate in Bread
by the Oxidation of Dyes
*1OJEKA E O; 2OBIDIAKU
MC; 1ENUKORAH, C
1Department of Applied Science, C.S.T, Kaduna
Polytechnic P.M.B 20, Kaduna, Nigeria
2K.aduna Refinrery and Petrochemical Company
Limited, Kaduna, Nigeria
Code Number: ja06050
ABSTRACT:A spectrophotometric method for the
determination of bromate based on the oxidation of congo red and crystal violet
dyes in a hydrochloric acid medium is described. The bromate level in bread
samples determined range from 3.70 μg g-1 to 12.10 μg g-1;
with limits of quantification of 0.45 0.78 μg g-1. Results
show the presence of detectable residue levels of potassium bromate in all the
bread samples analysed and the results compare favourably with established AOAC
method. .@JASEM
Bread is food baked from mixed
yeast leavened dough obtained from flour and bromated or phosphate flour or
their combination in the presence of Sacharomyces cerevisae yeast and 1%
bread bean flour. The bean flour activates the whitening of the dough, improves
the quality of the bread, and increases lipoxygenase, which produces
hydroperoxide, an oxidising agent. Ascorbic acid dissolved in water is also
added to the dough mixture to improve the gluten content (de Man, 1990; Alias
and Linden, 1999). Small amount of oxidising agents such as potassium bromate,
potassium iodate, and calcium peroxide are added to flour to enhance the
maturing process and baking. The bromate voluminises the centre of the bread
(produced from low protein wheat), and increases the size of the bread
artificially; as well as producing bread with a pure crumb structure (de Man,
1990). The presence of bromate in bread may cause renal failure, respiratory
depression, hearing loss, breakdown of vitamins and cancer to humans (IPCS,
1994; Field, 2004).
Atkins (1993) analysed bromate in
bread by gas chromatography (GC) based on the formation of a volatile
derivative of bromate and obtained a limit of 12 μg kg1.
Dennis (1994) used inductively coupled plasma mass spectrometry and obtained a
higher C.V of 18% compared to 12% when G.C was used, while Fuller and Ottaway
(1970) reported a kinetic study of bordeaux oxidation and reported that the
rate of reaction was dependent on Bordeaux concentration. Medina-Escriche et
al (1985) determined bromate by the oxidation of pyrogallol red dye in a
sulphuric acid medium and obtained bromate in the range 0.0-3.08 x 10-5
mol dm-3. Prior to their work, a kinetic-spectrophotometric
determination of bromate by the oxidation of dyes has not been described. While
some of these methods are time-consuming, satisfactory results were not
obtained on applying the method of Medina Escriche et al (1985) to
bread samples. The aim of the present study is to modify and highlight the use
of dyestuffs in the detremination of bromate in bread samples as a viable
routine method based on the work of Medina Escriche et al (1985). The
described method involves a simple and readily available instrumentation which
is an advantage to a developing country like Nigeria.
MATERIALS
AND METHODS
Apparatus:
Absorbance measurements were made with a Compec Visible Spectrophotometer Model
201 at λmax of 485 nm for samples containing crystal violet and
452 nm for samples containing Congo red organic reagents respectively. All measurements
were made at room temperature against water as reference.Reagents:
All reagents were of analytical grade unless otherwise specified. 2 M HCl:
43.10 cm3 of concentrated Hcl was diluted with water in a 250 cm3
volumetric flask and made up to mark with distilled water. 5 x 10-4
mol/dm3 Congo red dye solution: 0.348 g of Congo red (m.wt 696.67)
was weighed into a 1000 cm3 volumetric flask, dissolved and diluted
to mark with distilled water. 5 x 10-4 mol/dm3 Crystal
Violet dye solution: 0.216 g of Crystal violet (mwt. 431) was weighed into a
1000 cm3 volumetric flask, dissolved and diluted with distilled
water to mark. Foreign ion solution: Solutions of diverse ions including EDTA,
citrates, halides, ascorbic acid and others were prepared by dissolving the
calculated amount of each compound to give concentrations of 10-100μg-1
and 100 mg/g of the particular ion. 2.5 x 10-3 mol dm-3
potassium bromate: 4.3 g of potassium bromate (m.wt 167) was weighed into a
1000 cm3 volumetric flask, dissolved and diluted with distilled
water to mark. The working standard solutions were prepared in the range, 12,
24, 36, 48, 60 and 72 ppm respectively Sampling: Bread samples
were bought from different retail outlets and bakeries in Kaduna State.
Representative samples were bought from south, north, central, western and
eastern parts of Kaduna metropolis. Basis for sample selection: The samples
analyzed were the most common on popular demand in the respective locations.
The bread samples include; Baabsalam (Tudun Nupawa, Kaduna west), Beta Bread
(Makera, Kaduna south), Central Bread (Kaduna north), Unity Bread (Narayi,
Kaduna East) and Karamah Bread (Ungwan Dosa, Kaduna west).
Sample pretreatment:A circular sample of 2 cm in
diameter from the center of a 15-mm thick slice of each bread sample was taken
and dried in an oven for 72 hours at 55oC. The crust was ground to
a fine powder with mortar and pestle. 5g of each powdered sample was weighed
into a clean 250 cm3 beaker and 50 cm3 of distilled water
was added. The mixture was centrifuged and the liquid fraction was diluted to
100 cm3 in a calibrated flask. The appropriate volume of the aliquot
was taken for treatment under the proposed procedure. The pretreatment of
samples was done in triplicates according to reported method Garcia Sanchez et
al (1989).Proposed procedure: 4 cm3 of aliquot of each of the five bread samples was
measured into 10 separate 25 cm3 calibrated flasks. 5 cm3
of 5x10-4 mol/dm3 solution of Congo red dye or 5cm3
5x10-4 mol/dm-3 solution of crystal violet dye was
added, followed by 10 cm3 of 2M HCl solution . Each flask was
diluted to 25-cm3 marks with distilled water; and shaken gently
prior to colorimetric analysis. Comparison with Established AOAC Method: Bromates can be detected by pouring
over a slab of compressed and wetted flour, a solution containing 0.5 percent
potassium iodide in 2M HCl. Black spots suggest the presence of bromate. An
iodometric titration method for bromate determination described by Armstrong
(1952) and recommended by the AOAC was applied for this work . Statistical Analysis: The
sensitivities of the two methods, the limits of detection and quantification as
well as, precision and accuracy are reported according to modified methods
(IUPAC, 1978; ACS, 1980). The analytical sensitivity(s),
where Sd = standard
deviation of the analytical signal; m = slope of the calibration graph (in
absorbance units per μg bromate per cm3).
Where
Sr = relative standard deviation of the blank signal: m = slope of
the Calibration graph: and Sr = Sd at the detection limit
Where, t = student t-test; S = analytical sensitivity; = average value; and
n = number of determinations
Selectivity and Recovery Studies: Varying amounts of potassium bromate were taken in a 50cm3 calibration flask. Foreign ions were added (cations as sulphate or nitrate
salts) and dissolved with 10cm3 portions of distilled water. 2 cm3
5 x10-4 mol dm-3 crystal violet dye solution was added
followed by 2cm3 of 2 M hydrochloric acid solution with gentle
shaking. The amount of bromate (or foreign ion) was read at 485 nm directly in
concentration units with reference to a solution containing no bromate.
RESULTS AND
DISCUSSION
The use of potassium bromate in
flour milling and baking was banned in Nigeria by National Agency for Food,
Drug Administration and Control (NAFDAC) in 1993; and its use infringes on the
drug and related products (registration) decree 20 of 1999 and NAFDAC Decree 15
of 1993 (Akunyili, 2004).
Joint FAO/WHO (1992) committees
initial recommendation of acceptable level of 0 to 60 mg KBrO3/kg
flour was withdrawn because long term toxicity and carcinogenicity studies in
vitro and in vivo revealed renal cell tumours in hamsters. Also,
toxitcity studies showed that potassium bromate affects the nutritional quality
of bread by degrading vitamins A1, B1, B2, E and niacin - the main vitamins in
bread (FAO/WHO, 1992; PCHRD.com, 2000).
Table 1:
Concentration of potassium bromate in bread samples (μg/g)
Bread
sample
|
Concentration in μgg-1
at 452nm congo red method
|
Concentration
in μgg-1 at 485nm crystal violet dye method
|
A
|
3.70
± 0.01
|
11.60± 0.01
|
B
|
4.20± 0.01
|
12.00± 0.02
|
C
|
4.60± 0.02
|
11.50± 0.03
|
D
|
4.00± 0.01
|
12.10± 0.01
|
E
|
5.20± 0.02
|
12.10± 0.01
|
Table 2:
concentration of potassium bromate in bread samples (μg/g) by the AOAC established method
Bread
sample
|
μg/g- Bromate
found
|
A
|
12.00
± 0.01
|
B
|
12.35± 0.00
|
C
|
11.00
± 0.02
|
D
|
12.60
± 0.00
|
E
|
12.00
± 0.03
|
The
oxidation of the dyes by bromate was carried out in a hydrochloric acid medium
in which bromate is a strong oxidant.
With
irreversible oxidation indicators, the quantity of bromate solution consumed by
the dyestuff indicator is exceedingly small and the indicator is bleached in
the presence of 2M HCl used for this work:
In
the presence of hydrochloric acid, the red colour of congo red (a disazo dye)
changed to blue. The deepening of the colour has been attributed to resonance
among charged canonical structures. Crystal violet is a triarylmethane dye. It
is a leuco compound (colourless) converted to tertiary alcohol (colour base) on
oxidation. In the presence of acid the colourless benzenoid form changed to the
quinonoid dye due to salt formation. The salt is easily reconverted into the
leuco base (Finar, 1973):
It
is purple in weakly acid solution, green in strong acid solution and finally
yellow. The colour changes may be adduced in the light of proton addition
(Finar, 1973). Both dyes were water soluble because of the two sulpuric acid
groups (SO3H) in congo red and dimethylamino groups in crystal
violet dye.
The
molar absorptivity of congo dye is 9.04 x 104 dm3 mol-1
cm-1 at λmax 452 nm;
its wavelength of maximum absorption, while the molar absorptivity of congo
crystal violet dye is 9.70 x 105 dm3 mol-1 cm-1
at λmax, 485 nm.
The proposed crystal violet method is superior to congo red and recovery method
in Table 5 is satisfactory.
Preliminary
investigations show that Beers law was obeyed over the linear dynamic range
(LDR) 0.45 μgg-1 - 5.50 μgg-1,
and the initial reaction rate was found to be a linear function of the bromate
concentration over this range for the two methods. Three determinations were
made on each level of bromate concentration to obtain an average reading ±
standard deviation. Results show that the reaction of bromate with the dyes is
most sensitive with least slope of the calibration graphs
Table 3;
Evaluation of Analytical parameters of the proposed methods
Analytical-mode
|
Slope
(m)
|
Sensitivity
(μgg-1)
|
LOD
(μgg-1)
|
LOQ(μgg-1) |
LDR
(μgg-1)
|
Error
(%)
|
RSD
(%)
|
Crystal
violet method
|
0.28
|
0.052
|
0.68
|
0.45
|
0.45-5.50
|
1.50
|
3.75
|
Congo
red method
|
1.24
|
0.013
|
0.45
|
0.78
|
0.78
4.80
|
6.30
|
8.93
|
Table 4: Selectivity study to estimate the
tolerance limit of diverse ions in the presence of 100mg bromate
Foreign
ion added in μg
|
Concentration
of foreign ion tolerated in μg
|
EDTA;
SO42-
|
100
|
Citrate/
tartrate
|
10
|
Ascorbic
acid
|
5
|
CN‑,
Br-
|
3
|
Fe3+,
Mn7+
|
2
|
Cu2+;
Zn2+, Mn2+
|
100
|
The
crystal violet proposed procedure has a higher sensitive and lower limits of
detection and quantification respectively than the congo red method.
Application of the F-test indicates significant variation in the precision of
the two methods; the congo red method being the more precise (Tables 1 and
3).Ions such as EDTA, and sulphate do not interfere but cations like iron (III)
and manganese (II) which form strong complexes with crystal violet dye even in
the acidic medium interfere seriously andsimilarly,
reductants such as bromide, cyanide and ascorbic acid respectively. A maximum
error of 2% in the absorbance reading was considered tolerable. The effect of
each ion largely depend on the pH of the solution and at high pH, the solutions
were not affected the same way. The reductants as well as Fe(III) and Mn (VI)
accelerate the oxidation of crystal violet by bromate while Cu (II), Zn(II) and
Mn(II) inhibit the oxidation of the dye by bromate.
In
Table 5, the proposed method using crystal violet dye is superior to the use of
congo red, and it (crystal violet) was applied to the determination of
potassium bromate in spiked bread samples. The standard addition method was
done to reduce matrix effects, ascertain the effectiveness of the proposed
method and as a final test of the method. It therefore provides estimates of
the content of bromate in bread, and confirmed by the AOAC established method
Table 5: Determination of bromate in bread
by standard addition method
Bread
|
Bromate
|
Bromate
|
Bromate
|
Endogeneous
|
Recovery
|
Sample
|
Present
(μgg-1)
|
Added (μgg-1) |
Found
(μgg-1)
|
Bromate
(μgg-1)
|
(%)
|
A |
-
|
-
|
11.60±0.01
|
11.60
|
-
|
|
11.60
±0.01
|
3.50
|
15.00
± 0.01
|
0.10
|
99.33
|
B |
-
|
-
|
12.00
± 0.25
|
12.00
|
-
|
|
12.00
± 0.25
|
4.50
|
16.20
± 0.35
|
0.30
|
98.18
|
C |
-
|
-
|
11.50
± 0.03
|
11.50
|
-
|
|
11.50
± 0.03
|
5.00
|
16.00
± 0,01
|
0.50
|
96.96
|
D |
-
|
-
|
12.10
± 0.01
|
12.10
|
-
|
|
12.10
± 0.01
|
5.50
|
17.34
± 0.11
|
0.26
|
98.52
|
E |
-
|
-
|
12.10
± 0.01
|
12.10
|
-
|
|
12.10
± 0.01
|
6.00
|
18.10
± 0.36
|
0.10
|
100.55
|
Conclusion:
Potassium bromate is a mutagen and has shown the potential to cause cancer. The
presence of detectable residue levels of potassium bromate in bread is
therefore undersirable considering the long term effect; although the average
literature value of bromate in bread is 381.25mgg-1. The simplicity of the procedure
adopted herein and the good agreement between our result and the standard AOAC
method further recommends our method for consideration in routine bread
analysis for bromate. Millers and bakers should be encouraged to comply with
set standards of Good Manufacturing Practice (GMP) and Hazard Analysis Critical
Control Points (HACCP) guidelines acceptable world wide. In addition, they
should explore such other natural bread improvers like AMIPAN and unbrominated
bread improver, SS103U made in Malaysia.
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