International Journal of Academic Scientific Research

ISSN: 2272-6446 Volume 4, Issue 4 (November - December 2016), PP 83-96

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SIMULTANEOUS DETERMINATION OF TARTRAZINE

AND CARMOISINE IN FOODSTUFFS BY
SPECTROPHOTOMETRIC METHOD
SAAD ANTAKLI*, LEON NEJEM and WALAA ALSHEIKH AHMAD
Department of Chemistry, Faculty of Science, University of Aleppo, SYRIA.
*
Corresponding author: E-mail: [email protected]

ABSTRACT:
Spectrophotometric method was developed and applied for simultaneous determination of two
colorants: Tartrazine (T) and Carmoisine (C), in Syrian foodstuffs. This study was applied on several
Syrian foodstuffs as: (Custard Powder Strawberry, Crme Caramel Strawberry, Cream Chantilly
Strawberry, Cotton Candy, Yellow Chewing-gums, Strawberry Chewing-gums, Silca Strawberry,
Windmill powder Jelly, Lemon and Strawberry Diva, Samiry Candies, Lemon and Strawberry Ice
Cream). Derivative spectrophotometric (DS) method was applied for the determination of (T) and (C),
respectively. Tartrazine was determined by first derivative spectrophotometry at 370 nm (1D370); and
Carmoisine was determined by first derivative spectrophotometry at 570 nm (1D570). Linearity ranges
were 4-40 g/mL for (T) and 2-40 g/mL for (C), regression analysis showed a good correlation
coefficients R2 = 0.9999 and R2 = 0.9998 for (T) and (C), respectively. The limit of detection (LOD)
and limit of quantification (LOQ) were to be 0.73 and 2.23 g/mL for (T), 0.30 and 0.91 g/mL for (C),
respectively. The proposed derivative method was successfully applied to analysis individual or
mixture of Tartrazine and Carmoisine in foodstuffs. All studied samples showed dye levels conformity
with Syrian legislation.

Keywords: Tartrazine, Carmoisine, Derivative Spectrophotometry.

1. INTRODUCTION
Colorants are the first sensory parameter by which food quality, making food more attractive.
Synthetic colorants are usually added to food to replace the natural colorants that can be lost during
processing or to avoid variations in the color of the final product. The trouble is that some synthetic
Azo dyes such Tartrazine can be toxic to the human health when contact with some drugs which can
cause allergic to some people1. Also researches have shown that it can be linked to asthma,
hyperactivity particularly among children2.

Thus, various methods have been proposed to control the amount of colorants in food such as
capillary zone electrophoresis (CZE)3, differential pulse polarography4-5 and voltammetric methods as
adsorptive voltammetry6. Chromatographic methods have been used for colorants analysis in foods,
high performance liquid chromatography (HPLC)7, high-performance ion chromatography8, reversed-
phase thin-layer plates9, reverse phase high performance liquid chromatographic (RP-HPLC)
method10-11 and micellar electrokinetic capillary chromatography (MEKC)12. Spectrophotometric13-14
and derivative Spectrophotometric methods15-16 are successfully applied to determine other synthetic
dyes.

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ISSN: 2272-6446 Volume 4, Issue 4 (November - December 2016), PP 83-96
The aim of this work is to develop a simple and accurate spectrophotometric method for
simultaneous determination of C (E122) Fig.1, T (E102) Fig. 2 and their mixtures in different
foodstuffs without prior treatment by UV/Vis spectrophotometry and derivative spectrophotometry
(DS), where their mixtures give strawberry shades to products.

Fig. 1. Structural formula of Carmoisine. Fig. 2. Structural formula of Tartrazine.

2. MATERIALS AND METHODS

2.1 Apparatus
All spectral measurements were carried out using a T80+ UV/V spectrophotometer PG
instrument Ltd (UK) connected to computer, quartz cells 1 cm. Ultrasonic bath (Daihan), and
centrifuge (Centurion Scientific Ltd) (UK).

2.2 Chemical regents

Distilled water was used to prepare the solutions. Ethanol from SHAMLAB (Syria). The
standard synthetic colorants were Carmoisine (CI 14720, CAS 3567-69-9) purity 85% and Tartrazine
(CI 19140, CAS 1934-21-0) purity 82%. Both colorants were obtained from Sineset (France).

2.3 Standard preparation

Stock solutions (100 g/mL) of Tartrazine and Carmoisine were prepared by dissolving
appropriate weights of pure colorants in distilled water. The working standard solutions of each
colorant were prepared by appropriate dilutions of stock solutions with distilled water to give
concentrations between 1.2-56 g/mL of (T) and 1.2-56 g/mL of (C), taking the purity of the
colorants on consideration.

2.4 Calibration Curve

To construct the calibration curve, five standard solutions for each concentration were
prepared and the absorbance was measured of each solution five times.

2.5 Sample preparation: Thirteen products were studied:

4 g of Custard Powder Strawberry sample was accurately weighted and dissolved
in mixture of distilled water and ethanol (1:1) v/v, then solution was centrifuged during 15 min. at 5000
rpm for 4 times, then the extracted solution for each time was transferred into a 25 mL volumetric flask
and adjusted to volume with the distilled water and ethanol mixture.
3 g of Crme Caramel Strawberry sample was accurately weighted and dissolved
in mixture of distilled water and ethanol (1:1) v/v, then solution was centrifuged during 15 min. at 5000

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rpm for 4 times then the extracted solution for each time was transferred into a 25 mL volumetric flask
and adjusted to volume with the distilled water and ethanol mixture.

2 g of Cream Chantilly Strawberry sample was accurately weighted and dissolved

in mixture of distilled water and ethanol (1:1) v/v, then solution was centrifuged during 15 min. at 5000
rpm for 4 times then the extracted solution for each time was transferred into a 25 mL volumetric flask
and adjusted to volume with the distilled water and ethanol mixture.

1 g of homogenized Cotton Candy (Strawberry) sample was accurately weighted

and dissolved in distilled water, then solution was transferred into a 25 ml volumetric flask and
adjusted to volume with distilled water.

1 g of homogenized Silca (Strawberry) sample was accurately weighted and

dissolved in distilled water, then solution was transferred into a 25 ml volumetric flask and adjusted to
volume with distilled water.

Twenty Diva Candies pieces (Strawberry) were weighed and crushed. An accurately
weighted crushed sample, equivalent to five pieces was dissolved in distilled water, then solution was
transferred into 25 mL volumetric flask and adjusted to volume with distilled water.

Yellow Diva Candies lemon flavour were prepared by the same precedent way by
weighting crushed sample equivalent to ten pieces, then solution transferred into 25 mL volumetric
flask and adjusted to volume with distilled water .

Five pieces of Strawberry Chewing-gum was taken and extracted the colorant by
dissolving it in distilled water until removing colorant from gum, then solution was transferred into 25
mL volumetric flask and adjusted to volume with distilled water. The result extract was centrifuged
during 15 min at 5000 rpm. Then the supernatant was measured.

Five pieces of yellow Chewing-gum was taken and extracted by the same procedure
as a Strawberry Chewing gum.

1 g of homogenized Windmill powder Jelly lemon flavour sample was accurately

weighted and dissolved in distilled water, then solution was transferred into a 25 ml volumetric flask
and adjusted to volume with distilled water. The sample solution was centrifuged during 15 min at
5000 rpm. Then the supernatant was measured.
Twenty red Samiry Candies pieces were weighed and crushed. An accurately
weighted crushed sample equivalent to one piece was extracted the colorant as a Chewing-gum
procedure.

Ice Cream (Strawberry) was melted. 1 mL equivalent to 1.0650 g was transferred

into a 10 ml volumetric flask and adjusted to volume with distilled water.

Ice Cream (Lemon) was melted. 1 mL equivalent to 1.0650 g was transferred into a
10 ml volumetric flask and adjusted to volume with distilled water.

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3. RESULTS AND DISCUSSION

Absorption spectra of the standard colorants 4 g/mL (T) and 4 g/mL (C) solutions were
recorded within a wavelength range of 350600 nm against distilled water, Fig. 3. As can be seen, (C)
cannot be determined by direct measurement of absorbance at 516 nm, and (T) cannot too, because of
the overlapped spectra. On the other hand, derivative spectrophotometry showed more resolution.
Where it made the determination of (T) and (C) mixture possible without pretreatment.The first
derivative spectrum at zero-crossing point was used to determine (T) in the presence of (C) at 370 nm
(Fig. 4, a).The first derivative spectrum at zero-crossing point was used to determine (C) in the
presence of (T) at 570 nm (Fig. 4, b).

Fig. 3. Zero-order spectra of: Fig. 4. First derivative spectra of:

a- (T), b- (C), c- Mixture of (T) + (C). a- (T), b- (C).

4. METHOD VALIDATION
The validity of the proposed method was assessed by accuracy (reported as recovery
percentage), precision (reported as RSD %), linearity (evaluated by regression equation), limit of
detection (LOD) and limit of quantification (LOQ).

4.1 Linearity
The concentration linearity of (T) was in the range 4-40 g/mL at 370 nm by 1D370 Figs 5, 6
and the concentration linearity of (C) was in the range 2-40g/mL at 570 nm by 1D570 Figs. 7, 8.

Fig. 5: First derivative spectra of (T): Fig. 6: Calibration curve for (T)
C1: 4 g/mL, C2: 8 g/mL, C3: 12 g/mL, n=5 for each concentration.
C4: 16 g/mL, C5: 20 g/mL, C6: 24 g/mL,
C7: 28 g/mL, C8: 32 g/mL, C9: 36 g/mL,
C10: 40 g/mL.

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Fig.7: First derivative spectra of (c): Fig. 8: Calibration curve for (C).
C1: 2 g/mL, C2: 4 g/mL, C3: 8 g/mL, n=5 for each concentration.
C4: 12 g/mL, C5: 16 g/mL, C6: 20 g/mL
C7: 24 g/mL, C8: 28 g/mL, C9: 32 g/mL,
C10: 36 g/mL, C11: 40 g/mL.

4.2 Limit of Detection (LOD) and Limit of Quantification (LOQ):

LOD and LOQ were calculated in Table 1 using the following equations:

Where SD is the standard deviation of y-intercepts of regression lines and m is the slope of the
calibration curve.
Table 1: Statistical data for calibration graphs.
Selected
Linearity rang Correlation LOD LOQ
Method Analyte wavelength
(nm)
g/mL coef. (R2) g/mL g/mL
1
DS T D370 4 - 40 0.9999 0.73 2.23

1
DS C D570 2 - 40 0.9998 0.30 0.91

4.3 Accuracy and precision:

To determine the precision and accuracy of the proposed methods, five replicate
determinations were carried out on three different concentrations of standards (C) and (T). The
validation results are shown in Table 2.

Table 2: Method validation for the simultaneous determination of Carmoisine and Tatrazine by the
proposed methods.
theoretical *observed
Precision Accuracy
Method Colorant concentration concentration
)RSD %) (%)
(g/mL) (g/mL)
12 11.90 0.58 99.17
DS Carmoisine 20 20.10 0.69 100.50
28 27.97 0.28 99.89

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4 4.07 2.21 101.75
DS Tatrazine 12 12.22 1.30 101.83
28 27.60 0.39 98.57

Accuracy (%) = (observed concentration/theoretical concentration) 100. Precision (%) = (standard deviation/mean
Concentration) 100. * Five separate determinations were performed and calculated the mean.

5. ANALYTICAL APPLICATION

The developed method was applied for quantitative determination for food colorants in
different foodstuffs from Syrian local markets. The samples were prepared as described in the section
of samples preparation and analyzed. Quantitative analysis was done by using calibration curves. The
obtained results are summarized in Table 3, for thirteen different products.

In general, the used amounts of (C) were relatively higher than those of (T) in all analysed
products. However, the concentrations of the detected colorants were much lower than the Syrian
limits17 : 500 mg/kg for (T) and 200 mg/kg for (C).

The relative standard deviations RSD % (n=5) of the quantitative results were in the range of
0.71-3.18 % and 0.24-3.46 % for (T) and (C), respectively. Table 3, a-g present the determination
results of colorants (T) and (C), in thirteen Syrian trademarks for five different Batches for each:
(Custard Powder Strawberry , Crme Caramel Strawberry, Cream Chantilly Strawberry, Cotton Candy,
Yellow chewing-gums, Strawberry chewing-gums, Silca Strawberry, Windmill powder jelly, Lemon
and Strawberry Diva ,Samiry candies and Lemon and Strawberry Ice Cream ).

Table 3, a: Results of colorants (T) and (C) in Custard Powder (Strawberry) trademark.
Tartrazine Carmoisine
No. of
Concentration SD Concentration SD
sample RSD % RSD %
g/g g/g g/g g/g
1 45.93 0.85 1.85 196.02 0.47 0.24

2 40.93 1.30 3.18 190.20 3.26 1.71

3 33.30 1.00 3.00 196.21 2.03 1.03

4 32.18 0.85 2.64 193.22 3.34 1.73

5 33.12 0.69 2.08 189.77 4.31 2.27

Range
32.18 - 45.93 189.77 - 196.21
g/g

Table 3, b: Results of colorants (T) and (C) in Crme Caramel (Strawberry) trademark.
Tartrazine Carmoisine
No. of
Concentration SD Concentration SD
sample RSD % RSD %
g/g g/g g/g g/g
1 85.41 2.55 2.99 177.46 0.63 0.36
2 74.58 0.93 1.25 183.59 0.59 0.32
3 62.91 1.74 2.77 168.86 4.25 2.52

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4 68.33 0.93 1.36 191.37 0.63 0.33
5 73.33 0.93 1.27 180.79 1.15 0.64
Range
g/g 62.91 - 85.41 168.86 - 191.37

g/g

Table 3, c: Results of colorants (T) and (C) in Cream Chantilly (Strawberry) trademark.
Tartrazine Carmoisine
No. of
Concentration SD Concentration SD
sample RSD % RSD %
g/g g/g g/g g/g

1 54.37 1.71 3.15 134.10 1.90 1.42

2 50.62 1.39 2.75 157.85 0.89 0.56

3 69.36 1.40 2.02 157.45 1.09 0.69

4 75.62 1.39 1.84 158.65 0.96 0.61

5 96.24 1.39 1.44 176.60 0.89 0.50

Range
g/g 50.62 - 96.24 134.10 - 176.60

Table 3, d: Results of colorants (T) and (C) in Cotton Candy (Strawberry) trademark.
Tartrazine Carmoisine
No. of
Concentration SD Concentration SD
sample g/g g/g RSD % g/g g/g RSD %

1 208.75 3.42 1.64 136.45 1.84 1.35

2 282.50 2.79 0.99 75.80 1.78 2.35

3 383.75 3.42 0.89 54.85 1.90 3.46

4 278.75 3.42 1.23 99.15 1.90 1.92

5 302.50 3.42 1.13 100.80 1.78 1.77

Range
g/g 208.75 - 383.75 54.85 - 136.45

g/g
Table 3, e: Results of colorant (C) in Diva Candy (Strawberry) trademark.
Tartrazine Carmoisine
No. of
Concentration SD Concentration SD
sample RSD % RSD %
g/piece g/piece g/piece g/piece

1 - - - 26.14 0.76 2.91

2 - - - 28.78 0.71 2.47

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3 - - - 31.28 0.43 1.37

4 - - - 22.32 0.40 1.79

5 - - - 19.90 0.22 1.11

Range
- - - 19.90 - 31.28
g/piece

Table 3, f: Results of colorant (T) in Diva Candy (Lemon) trademark.

Tartrazine Carmoisine
No. of
Concentration SD RSD % Concentration SD RSD %
sample
g/g g/g g/g g/g

1 20.24 0.53 2.62 - - -

2 20.96 0.53 2.53 - - -

3 39.22 0.71 1.81 - - -

4 24.74 0.58 2.34 - - -

5 35.24 0.53 1.50 - - -

Range
g/g 20.24 - 39.22 - - -

Table 3, g: Results of colorant (C) in Samiry Candy (Lemon) trademark.

Tartrazine Carmoisine
No. of
Concentration SD Concentration SD
sample RSD % RSD %
g/piece g/piece g/piece g/piece

1 - - - 90.98 0.89 0.98

2 - - - 104.38 2.37 2.27

3 - - - 70.96 0.87 1.23

4 - - - 79.67 0.72 0.90

5 - - - 108.64 0.76 0.70

Range - -
- 70.96 - 108.64
g/piece

Table 3, h: Results of colorant (C) in Silca Candy (Strawberry) trademark.

Tartrazine e: Carmoisine
No. of Samiry
Concentration SD candy Concentration SD
sample RSD %
trademar RSD %
g/g g/g g/g g/g
k.

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1 - - - 20.59 0.47 2.28

2 - - - 19.35 0.54 2.79

3 - - - 17.89 0.47 2.63

4 - - - 16.83 0.47 2.79

5 - - - 18.53 0.47 2.54

Range - -
g/g - 16.83 - 20.59

Table 3, i: Results of colorant (C) in Chewing-gum (Strawberry) trademark.

Tartrazine Carmoisine
e:
No. of Samiry
sample Concentration SD RSD % Concentration SD RSD %
g/ piece g/ piece candy g/ piece g/ piece
trademar
1 - - k.- 177.40 2.19 1.23

2 - - - 165.70 1.78 1.07

3 - - - 110.80 2.32 2.09

4 - - - 122.45 2.32 1.89

5 - - - 129.85 1.90 1.46

Range - - -
g/piece 110.80 - 177.40

Table 3, j: Results of colorant (T) in Chewing-gum (Yellow) trademark.

Tartrazine Carmoisine
No. of
sample Concentration SD RSD % Concentration SD RSD %
g/ piece g/ piece g/ piece g/ piece

1 148.75 2.79 1.88 - - -

2 170.00 2.79 1.64 - - -

3 136.25 2.79 2.05 - - -

4 145.00 2.79 1.92 - - -

5 127.50 3.42 2.68 - - -

Range
127.50 - 170.00 - - -
g/piece

Table 3, k: Results of colorant (T) in Windmill powder Jelly (Lemon) trademark.

No. of Tartrazine Carmoisine

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sample Concentration SD Concentration SD
g/g g/g RSD % g/g g/g RSD %

1 310.00 3.42 1.10 - - -

2 377.50 3.42 0.91 - - -

3 392.46 2.77 0.71 - - -

4 389.98 3.39 0.87 - - -

5 367.50 2.79 0.76 - - -

Range 310.00 - 392.46 - -

g/g -

Table 3, l: Results of colorant (C) in Ice cream (Strawberry) trademark.

Tartrazine Carmoisine
No. of
Concentration SD Concentration SD
sample RSD % RSD %
g/g g/g g/g g/g
- - -
1 67.86 0.87 1.28
- - -
2 63.82 0.72 1.13
- - -
3 59.11 0.71 1.20
- - -
4 48.76 0.71 1.46
- - -
5 55.69 0.87 1.56
Range - - -
48.76 - 67.86
g/g

Table 3, m: Results of colorant (T) in Ice cream (Lemon) trademark.

Tartrazine Carmoisine
No. of
Concentration SD Concentration SD
sample RSD % RSD %
g/g g/g g/g g/g

1 67.60 1.05 1.55 - - -

2 65.05 1.03 1.58 - - -

3 66.19 1.05 1.59 - - -

4 74.17 1.28 1.73 - - -

5 71.35 1.28 1.79 - - -

Range -
65.05 - 74.17 - -
g/g

Recovery:

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The recovery was studied by three addition standards for every product. Table 4 presents the recoveries
results for the thirteen products studied.
Table (4): Recoveries for the thirteen products:

Sample Added Total Recovery RSD Recovery

Product Colorant Found SD
g/mL g/mL % g/mL % Average
g/mL %
7.35 4 11.35 100.00 3.39 3.39
T 7.35 6 13.55 103.33 1.83 1.77 101.73
Custard 7.35 8 15.50 101.88 2.20 2.16
Powder
Strawberry 15.42 2.5 17.91 99.60 0.35 0.35
C 15.42 5 20.56 102.80 1.36 1.32 100.80
15.42 7.5 22.92 100.00 1.09 1.09
8.8 4 12.80 100.00 3.13 3.13
T 8.8 6 14.80 100.00 3.49 3.49 100.41
Crme 8.8 8 16.90 101.25 1.71 1.69
Caramel
Strawberry 21.69 5 26.76 101.40 1.89 1.86
C 21.69 10 31.69 100.00 1.80 1.80 100.93
21.69 12 33.86 101.41 0.65 0.64
4.05 4 8.10 101.25 2.70 2.67
T 4.05 6 10.15 101.66 2.28 2.24 101.59
Cream 4.05 8 12.20 101.87 1.39 1.36
Chantilly
Strawberry 12.62 4 16.62 100.00 1.78 1.78
C 12.62 6 18.59 99.50 1.45 1.46 100.12
12.62 8 20.69 100.88 0.94 0.93
12.10 4 16.15 101.25 3.28 3.23
T 12.10 6 18.05 99.17 1.75 1.76 101.59

Cotton 12.10 8 20.45 104.38 1.41 1.35

Candy 4.03 4 8.12 102.25 1.78 1.74
C 4.03 6 9.96 98.83 1.27 1.29 100.36
4.03 8 12.03 100.00 0.89 0.89
5.10 4 9.05 98.75 2.79 2.83
T 5.10 6 11.20 101.67 1.86 1.83 100.97
Yellow 5.10 8 13.30 102.50 1.62 1.58
Chewing-
gums - - - - - -
C - - - - - - -

- - - - - -
- - - - - -
T - - - - - - -
Strawberry
- - - - - -
chewing-
gums 5.19 4 9.29 102.50 1.90 1.85
C
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5.19 6 11.26 101.17 1.55 1.53 101.63
5.19 8 13.29 101.25 0.95 0.94
- - - - - -
T - - - - - - -

Silca - - - - - -
Strawberry 2.96 4 6.96 100.00 1.90 1.90
2.96 6 8.86 98.33 1.63 1.66 99.02
C
2.96 8 10.86 98.75 0.95 0.96
14.70 4 18.80 102.50 2.79 2.72
T 14.70 6 20.65 99.17 0.11 0.11 100.97
Windmill 14.70 8 22.80 101.25 1.39 1.37
powder
Jelly - - - - - -
C - - - - - - -
- - - - - -
- - - - - -
T - - - - - - -

Strawberry - - - - -
Diva 3.96 4 8.03 101.75 1.78 1.75
C 3.96 6 9.96 100.00 1.26 1.26 100.58
3.96 8 11.96 100.00 0.95 0.95
4.05 4 8.11 101.50 3.42 3.37
T 4.05 6 10.05 100.00 1.86 1.86 100.50

Lemon 4.05 8 12.05 100.00 1.39 1.39

Diva - - - - - -
C - - - - - - -
- - - - - -
- - - - - -
T - - - - - - -

Samiry - - - - - -
Candies 7.09 4 11.26 104.25 2.32 2.23
C 7.09 6 13.22 102.17 2.53 2.48 102.26
7.09 8 15.12 100.38 0.89 0.89
- - - - - -
T - - - - - - -

Strawberry - - - - - -
Ice Cream 5.19 4 9.22 100.75 2.32 2.30
C 5.19 6 11.29 101.67 1.26 1.24 100.34

5.19 8 13.12 99.13 0.83 0.84

7.90 4 12.05 103.75 2.79 2.69
T 7.90 6 14.10 103.33 2.28 2.21

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Cream 7.90 8 16.20 103.75 3.42 3.29 103.61

- - - - - -
C - - - - - - -

- - - - - -

6. CONCLUSIONS
Tartrazine (T) and Carmoisine (C) levels were estimated in individual form and in their
mixtures in different studied local foodstuffs by simple spectrophotometric method. (C) and (T) were
determined by first derivative spectrophotometric method using zero-crossing point.

Colorants levels were lower than their maximum values which established by the Syrian
legislation17. The proposed method of (T) and (C) determination are accurate, simple, sensitive, easy
and directly applicable in quantitative analysis without previous chemical treatment in individual or
binary mixture.

The proportions of mixture of (T) and (C) in the studied products were different from one to
another. It was seen also that Carmoisine level in general was higher than Tartazine in all analyzed
products.The food additives in the different studied products didn't have any interferences on the
analysis of the Tartrazine (T) and Carmoisine (C).

ACKNOWLEDGEMENT
The Ministry of High Education in Syria financially and technically supported this work through
department of Chemistry, Faculty of Science, University of Aleppo, Syria.

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