Cyclodextrins (CDs) are water soluble cyclic, non reducing oligosaccharides

consisting of D-(+)-gluocopyranose units linked through -1,4-glycosidic linkages.

The major and most common three cyclodextrins, -cyclodextrin, -cyclodextrin and
-cyclodextrin consists of six, seven and eight D-(+)-glucopyranose units,
respectively. In CDs every gluocopyranose units has three free OH groups, two of
which (C-2 and C-3) are secondary, and one (C-6) is primary. Cyclodextrins are
modified for a wide variety of reasons. In CD derivative family, CD polymers reserve
a special position. In recent years attention has been expanding from supramolecular
chemistry of cyclodextrins to supramolecular chemistry of cyclodextrins based
polymers because more sophisticated structures and advanced functions have been
achieved by the formation of supramolecular cyclodextrin polymers. Compounds
consisting of covalently linked cyclodextrin rings are called cyclodextrin polymers.
The cyclodextrins fixed into polymeric structures behave differently from their
monomeric derivatives. CD polymers have the inclusion ability and the controlled
release ability, like their parent CDs. They have fine mechanical intensity and
excellent stability. Therefore, CD polymers have been studied for a long time and
received much attention in pharmaceutical industry, biomedical areas and
environmental chemistry.
Cyclodextrins (CDs) are water soluble: therefore they cannot be used as an adsorbent
in the uptake of dyes and various organic pollutants. The only possible way is to
cross link the cyclodextrin either by using a suitable cross-linking agent or by
covalently binding the cyclodextrin to the backbone of existing polymers.
Chapter 3 deals with the synthesis of -Cyclodextrin polymer using epichlorohydrin
as the cross-linker and also characterization by SEM and elemental analysis and
removal of some food dyes using -Cyclodextrinepichlorohydrin polymer as a solid
phase extractant.

69

3.1 PROCEDURE
3.1.1 Synthesis of -cyclodextrinepichlorohydrin polymer
-CDP was synthesized by method given in Literature (1a). A brief procedure is given
here : 40g of -CD, 10 g of soluble starch and 100 mL of 20% sodium hydroxide were
added into a beaker. The mixture was vigorously stirred at 50-60C until the reactants
dissolved. Total 60 mL of epichlorohydrin was added drop wise into the solution, and
-CDP was formed in 30 min. Filtered with pressure through Buchner funnel and then
washed with distilled water 5-6 times, the polymer was dried at 100C and then stored
at room temperature in the desiccators for further use.

70

Fig. 3.1.1 Represents generalized form showing cavity, toroidal form of Cyclodextrin (-CD) & -Cyclodextrin polymer (-CDP).

Fig.

3.1.2.

Schematic

representation

cyclodextrinepichlorohydrin polymer

71

of

the

Synthesis

of

3.1.3 Characterization of -CDP

The synthesized -CD polymer was characterized by following techniques:
(i) Scanning Electron Microscope (SEM) Analysis
(ii) FT-IR Analysis

3.1.3.1 Scanning Electron Microscope (SEM) Analysis

The synthesized polymer was subjected to SEM analysis to study their morphological
aspects. SEM Scans of the polymers were carried out at Sophisticated Instrumentation
centre (SIC), Punjabi University, Patiala on JEOL (Tokyo, JAPAN) JSM-6510/LVSEM Instrument. For the above experiment 1mg. of the sample was mounted on a
glass slide and subjected to scan at an acceleration voltage of 10Kv. The SEM images
were taken at five different magnifications from X200, X1000, X2500, X3000 and
X5000. From the SEM micrographs it is obvious that the structure of -CD is rock
like and -CDP is relatively fibrous which shows that cross-linking has taken place.

72

Fig. 3.1.3 SEM scan of -CD at X200

Fig. 3.1.4 SEM scan of -CD at X1000

Fig. 3.1.5 SEM Scan of -CD at X2500

X3000

Fig. 3.1.7 SEM Scan of -CD at X5000

73

Fig. 3.1.6 SEM Scan of -CD at

Fig. 3.1.8 SEM scan of -CDP at X100

Fig. 3.1.9 SEM scan of -CDP at X1000

Fig. 3.1.10 SEM scan of -CDP at X2500 Fig. 3.1.11 SEM scan of -CDP at X3000

Fig. 3.1.12 SEM scan of -CDP at X5000

74

3.1.3.2 Fourier Transform Infra-red (FT-IR) Analysis

The reactant -Cyclodextrin (-CD), the synthesized -Cyclodextrin polymer (CDP) were subjected to IR analysis on a Pro resolution FTIR system as KBr pellets in
the range 4000-400 cm-1region. From FT-IR (KBr) of -CD, -CDP it can be seen
that: for -CD, 3600- 3000 cm-1 (OH), 2900 cm-1 (CH); while for -CDP a sharp band
at 3400 cm-1 (OH), 3000 cm-1 (C-H stretch) and 1040 cm-1 (C-O-C stretch) is
obtained.

75

Fig. 3.1.10 FT-IR Spectrum of native -Cyclodextrin

76

Fig. 3.1.11 FT-IR Spectrum of cross-linked -Cyclodextrin polymer (-CDP)

77

3.2 DETERMINATION OF BRILLIANT GREEN DYE

Color [1] is a vital constituent of food and probably the first characteristic perceived
by the human senses. Food colorant is any substance that is added to food or drink to
change its color probably, for economic reasons. Brilliant green dye is a cationic and
triarylmethane dye of the malachite green series. This has been used in dyeing paper,
pulp, textile, plastics, leather, cosmetics and food (2). The present work describes cyclodextrinepichlorohydrin polymer (-CDP) used as a solid support for the
preconcentration and determination of the Brilliant green dye.
3.2.1 Materials
3.2.1.1 Equipment
A Shimadzu UV-1800 spectrophotometer (Shimadzu Ltd., Japan) equipped with the
matched 10-mm quartz cells was used to measure absorbance. Digital century pHmeter CP-901 with a combined glass electrode was used to carry out pH
measurements. A thermostatic shaking water bath (Perfit India Ltd.) was used to carry
out all the inclusive procedures.
3.2.1.2 Reagents
All reagents used were of analR grade unless otherwise stated. Double distilled water
was used throughout the experiment. A 0.01M stock solution of Brilliant green dye
(Loba chem.) was prepared in double distilled water and further diluted as and when
required.
Buffer solution in the pH range of 1.0-3.5 were made by mixing equimolar solutions
of hydrochloric acid/Sodium Acetate and buffer solutions in the pH range of 4.0-6.5
were made by mixing 0.2 M sodium acetate and 0.2 M acetic acid solutions in the
different proportions. While those in the pH range of 7.0-11.0 were made by mixing
0.5 M ammonia and 0.5 M ammonium chloride.
The glass wares were washed with chromic acid and soaked in 5% nitric acid and then
cleaned with double distilled water before use and dried in an electric oven.

78

3.2.2. Procedure
200 mg of -CDP and 2.5 mL of buffer solution (pH 4.0) were added to a 100 ml
stoppered conical flask at room temperature. The mixture was allowed to stand for 15
min. so that -CDP should swell sufficiently and an appropriate amount of dye was
added and made up to 90 ml with double distilled water. The mixture was shaken in
the thermostatic shaking water bath for 120 min. at the rate of 120 r.p.m. agitation
speed, 5.0 ml of supernatant solution was taken and the absorbance was measured
spectrophotometrically.
3.2.3. Optimization of various parameters
3.2.3.1 Effect of pH
The formation of inclusion complex of the dye in the polymer depends on the pH of
the sample solution which was studied in the range of (1.0-7.0.) using different buffer
solutions. The % uptake ( 95) was obtained at pH 4.0 (Fig. 3.2.3.1). Therefore, the
working pH was chosen as 4.0 for the subsequent studies.
3.2.3.2. Effect of shaking time
Shaking time is an important factor in determining the possibility of application of the
-CD polymer for the selective uptake of brilliant green dye. Different shaking time
(ranging from 15 to 135 min.) were studied for the % uptake of brilliant green dye by
-CD polymer. The results of Brilliant green dye show that the % uptake of (95%)
was attained within 120 min. (Fig. 3.2.3.2). Therefore, the shaking time of 120 min.
was selected for further studies.
3.2.3.3. Effect of sample volume
Enriching low concentration of dye from large volume of sample the effect of sample
volume is an important factor in determining the possibility of application of polymer
for the % of uptake of brilliant green dye. For this purpose 15, 30, 45, 60,75, 90 and
105 ml of sample volumes containing a fixed amount of dye were taken and uptake
of brilliant green dye were studied (Fig. 3.2.3.3). The maximum % uptake (95%) of
brilliant green dye was obtained at sample volume of 90 ml. Therefore, 90 ml of
sample volume was used for further studies.
79

3.2.3.4. Effect of agitation speed

Shaking speed is an important factor in determining the possibility of application of
polymer for the quantitative % uptake of brilliant green dye. The driving force i.e
Shaking speed could help in mass transfer and facilitate the concentration gradient
between the sample solution and the polymer. Different speeds (ranging from 40 to
140 r.p.m.) were studied for the % uptake of brilliant green dye by polymer. The
results of brilliant green dye uptake (Fig. 3.2.3.4) show that the % uptake reach
maximum ( 95%) at 120 r.p.m. Therefore, the shaking speed of 120 r.p.m. was
selected for further studies.
3.2.3.5. Effect of amount of polymer
The amount of the -CD polymer is another important parameter that affects %
uptake of dye. A quantitative removal ( 95%) cannot be achieved when the -CD
polymer is less than the optimum amount. In order to optimize the smallest amount of
polymer, 100 mg, 200 mg, 300 mg, 400 mg and 500 mg of the polymer were added to
the solution containing known amount of dye. The quantitative recoveries were
obtained at 200 mg of -CDP (Fig. 3.2.3.5). Therefore, 200 mg of the -CDP has been
used for further studies.

80

Fig. 3.2.3.1. Effect of pH on the % uptake of the Brilliant green dye by the -CD
polymer

Fig. 3.2.3.2. Effect of shaking time on the % uptake of the Brilliant green dye by the
-CD polymer

Fig. 3.2.3.3. Effect of sample volume on the % uptake of the Brilliant green dye by
the -CD polymer
81

Fig. 3.2.3.4. Effect of agitation speed on the % uptake of the Brilliant green dye by
the -CD polymer.

Fig. 3.2.3.5. Effect of amount of adsorbent on the % uptake of the Brilliant green by
-CD the polymer.

82

3.2.4. Application
3.2.4.1 Determination of Brilliant green dye in Mouth freshener
Green color tablets was grounded with a mortar and pestle, 3.0 g sample (mouth
freshener), added in double distilled water and was heated for some time to dissolve
the tablets properly and then cooled. After cooling double distilled water was added
to the sample and mixed thoroughly. The residue was filtered and filtrate was diluted
with double distilled water and made volume up to the mark in a standard flask. The
sample solution was then analyzed by the developed procedure. The results are given
in (Table 3.2.4.1.)
3.2.4.2 Determination of Brilliant green dye in Jelly
5.0 g green color jelly was crushed and was heated for some time to dissolved
properly and then cooled. After cooling double distilled water was added to the
sample and mix thoroughly. The residue was filtered and filtrate was diluted with
double distilled water and made the volume up to the mark in the standard flask. The
sample solution was then analyzed by the developed procedure. The results are given
in (Table 3.2.4.2)
3.2.4.3 Determination of Brilliant green dye in khus syrup
5 mL of the sample of khus syrup was transferred to a standard flask and made the
volume up to mark with double distilled water and solution was analyzed by
developed procedure. The results of analysis are given in (Table 3.2.4.2)

83

Table 3.2.4.1 Results for the analysis of determination of Brilliant green dye in
Mouth Freshener.
Food Samples
a

Pizzle

mouth

Added, g/ml
0

Found, g/ml

Recovery, %

0.012

0.268

0.266

95.00

0.536

0..536

97.76

1.072

1.072

99.25

Freshener

Pizzle mouth Freshener - locally available in market

Table 3.2.4.2 Results for the analysis of determination of Brilliant green dye in Jelly
and Khus syrup.
Food samples
a

Jelly

Khus syrup

Added, g/ml
0

Found, g/ml

Recovery, %

0.025

0.268

0.267

95.35

0.536

0.536

97.38

1.072

1.072

99.19

0.014

0.268

0.270

96.42

0.536

0.536

95.69

1.072

1.088

95.53

Jelly - locally available in market, bKhus syrup locally avaiable in market

84

3.3 DETERMINATION OF SUDAN I DYE

Sudan I is a synthetic azo dye that is typically used in many industrial applications
including solvents, oils, fats, waxes, plastics, printing inks, and floor polishes (3,4).
The main reason for the widespread usage is its colorfastness and low price. Azocolorants are biologically active and have been associated with increased occurrence
of bladder cancer in textile and leather dyers, painters, and hairdressers (5-8). Due to
their potential carcinogenicity, many countries have banned the use of most azo dyes
at any level in products for human consumption. The present work describes cyclodextrinepichlorohydrin polymer (-CDP) as a solid support

for the

preconcentration and determination of the Sudan I dye.

3.3.1 Materials
3.3.1.1 Equipment
Equipments are same as described in section 3.2.1.1
3.3.1.2 Reagents
All reagents used were of analR grade unless otherwise stated. Double distilled water
was used throughout the experiment. A 0.01M stock solution of Sudan I dye (Loba
chem.) was prepared in double distilled water and further diluted as and when
required.
Buffer solutions are same as described in section 3.2.1.2
3.3.2 Procedure
200 mg of -CDP and 2.5 mL of buffer solution (pH 3.0) were added to a 100 ml
stoppered conical flask at room temperature. The mixture was allowed to stand for 15
min. so that -CDP should swell sufficiently and appropriate amount of dye was
added and made up to 75 ml with double distilled water. The mixture was shaken in
the thermostatic shaking water bath for 90 min. at the rate of 100 r.p.m., 5.0 ml of
supernatant solution was transferred into a test tube and the absorbance was measured
spectrophotometrically.

85

3.3.3 Optimization of various parameters

3.3.3.1 Effect of pH
The formation of inclusion complex of the dye in the polymer depends on the pH of
the sample solution which was studied in the range of (1.0-7.0) using different buffer
solutions, % uptake ( 95) was obtained at pH 3.0. (Fig.3.3.3.1). Therefore, the
working pH was chosen as 3.0 for the subsequent studies.
3.3.3.2. Effect of the shaking time
Shaking time

is an important

factor in determining the possibility of

application of the -CD polymer for the selective uptake of Sudan I dye. Different
shaking time (ranging from 15 to 120 min) were studied for the % uptake of Sudan I
dye by -CD polymer. The results of % uptake of Sudan I dye vs. the shaking time
show that the % uptake of (95%) was attained in 90 min.(Fig. 3.3.3.2). Therefore,
the shaking time of 90 min. was selected for further studies.
3.3.3.3 Effect of sample volume
Enriching low concentration of dye from large volume of sample the effect of sample
volume is an important factor in determining the possibility of application of
polymer for the % of uptake of Sudan I dye.For this purpose 15, 30, 45, 60, 75 and 90
mL of sample volumes containing a fixed amount of dye were taken and uptake
of Sudan I dye was studied (Fig. 3.3.3.3.). The maximum % uptake (95%) of
Sudan I dye was obtained at sample volume of 75 mL. Therefore, 75 mL of sample
volume was used for further studies
3.3.3.4 Effect of agitation speed
Speed of shaking is an important factor in determining the possibility of
application of polymer for the quantitative % uptake of Sudan I dye. The driving
force i.e Shaking speed could help in mass transfer and facilitate the concentration
gradient between the sample solution and the polymer. Different speeds (ranging from
40 to 140 r.p.m) were studied for the % uptake of Sudan I dye by the polymer. The
results of % uptake of Sudan I vs. agitation speed (Fig. 3.3.3.4) show that the %

86

uptake reach maximum (95%) at 100 r.p.m. Therefore, the shaking speed of 100
r.p.m. was selected for further studies.
3.3.3.5. Effect of amount of polymer
The amount of the -CD polymer is another important parameter that affects %
uptake of dye. A quantitative removal ( 95%) cannot be achieved when the -CD
polymer is less than the optimum amount. In order to optimize the smallest amount of
polymer, 100 mg, 200 mg, 300 mg, 400 mg and 500 mg of the polymer were added to
the solution containing known amount of dye. The quantitative recoveries were
obtained at 200 mg of -CDP (Fig. 3.3.3.5). Therefore, 200 mg of the -CDP has been
used for further studies.

87

Fig. 3.3.3.1. Effect of the pH on the % uptake of the Sudan I dye by -CD polymer

Fig. 3.3.3.2.Effect of shaking time on the % uptake of the Sudan I dye by -CD
polymer

Fig. 3.3.3.3. Effect of sample volume on the % uptake of the Sudan I dye by -CD

polymer
88

Fig. 3.3.3.4. Effect of agitation speed on the % uptake of the Sudan I dye by -CD
polymer.

Fig. 3.3.3.5.Effect of amount of adsorbent on the % uptake of the Sudan I dye by CD polymer

89

3.3.4. Applications
3.3.4.1 Determination of Sudan I in Chilli Powder/ Chilli Sauce
5.0 g of the Chilli powder/ Chilli sauce sample was dissolved in hot double distilled
water. After mixing, the residue was filtered, and filtrate was diluted with double
distilled water and made the volume up to the mark in a standard flask. The sample
solution was then analyzed by the developed procedure. The results of analysis are
given in (Table 3.3.4.1)
3.3.4.2 Determination of Sudan I dye in Tandoori masala mix
3.0 g of the Tandoori masala mix sample was dissolved in hot double distilled water.
After mixing, the residue was filtered, and filtrate was diluted with double distilled
water and made the volume up to the mark in a standard flask. The sample solution
was then analyzed by the developed procedure. The results of analysis are given in
(Table 3.3.4.2).

90

Table 3.3.4.1. Results for the analysis of determination of Sudan I dye by developed
method in Chili Powder / Chili Sauce samples.
Food Samples
a

Chilli powder

Chilli Sauce

Added, g/mL

Found, g/mL

Recovery, %

0.017

0.331

0.334

95.97

0.662

0.662

97.49

0.892

0.890

97.90

0.015

0.331

0.332

95.95

0.662

0.650

96.01

0.892

0.895

98.45

Chilli powder - locally available in market, bChilli Sauce - locally available in market

Table 3.3.4.2. Results for the analysis of determination of Sudan I dye by developed
method in Tandoori masala mix sample.
Food Samples

Tandoori

masala

Added, g/mL

Found, g/mL

Recovery, %

0.006

0.350

0.348

97.75

0.700

0.698

98.86

0.932

0.930

99.14

mix

Tandoori masala mix locally available in market

91

3.4. DETERMINATION OF SUDAN III DYE

The most widely used synthetic organic azo dyes have been studied for their toxicity
risk (9). The chromophoric azo group on reduction forms suspected carcinogenic
aromatic amines under certain conditions (10). The Sudan III dye was used in food
industry with the regulation of maximum permissible levels in a particular foodstuff
(11). Based on toxicity data, various azo dyes are unauthorized and are sometimes
illegally used in food preparations either to enhance or to maintain the appearance of
food products (12-14). The adulteration of hot chili products with Sudan I, II, III and
IV led the EU to adopt emergency measure (15). The present work describes the use
of -cyclodextrinepichlorohydrin polymer (-CDP) as a solid support for the
preconcentration and determination of the Sudan III dye.
3.4.1 Materials
3.4.1.1 Equipment
Equipments are same as described in section 3.2.1.1
3.4.1.2 Reagents
All reagents used were of analR grade unless otherwise stated. Double distilled water
was used throughout the experiment. A 0.01M stock solution of Sudan III dye (Loba
chem.) was prepared in double distilled water and further diluted as and when
required.
Buffer solutions are same as described in section 3.2.1.2
3.4.2 Procedure
300 mg of -CDP and 2.5 mL of buffer solution (pH 4.0) were added to a 100 ml
stoppered conical flask at room temperature. The mixture was allowed to stand for 15
min. so that -CDP should swell sufficiently and an appropriate amount of dye was
added and made up to 75 ml with double distilled water. The mixture was shaken in
the thermostatic shaking water bath for 75 min. at a rate of 120 r.p.m. agitation speed.
5.0 ml of supernatant solution was transferred into a test tube and the absorbance was
measured spectrophotometrically.
92

3.4.3. Optimization of various parameters

3.4.3.1 Effect of pH
The formation of inclusion complex of the dye in the polymer depends on the pH of
the sample solution which was studied in the range of (1.0-7.0) using different buffer
solutions. % uptake ( 95) was obtained at pH 4.0. (Fig.3.4.3.1). Therefore, the
working pH was chosen as 4.0 for the subsequent studies.
3.4.3.2 Effect of the shaking time
Shaking time

is an important

factor in determining the possibility of

application of the -CD polymer for the selective uptake of Sudan III dye. Different
shaking time (ranging from 15 to 120 min) were studied for the % uptake of Sudan
III dye by -CD polymer. The results of % uptake of Sudan III dye vs. the shaking
time show that the % uptake of (95%) was attained within 75 min. (Fig. 3.4.3.2).
Therefore, the shaking time of 75 min. was selected for further studies.
3.4.3.3 Effect of sample volume
Enriching low concentration of dye from large volume of sample the effect of sample
volume is an important factor in determining the possibility of application of
polymer for the % uptake of Sudan III dye. For this purpose 15, 30, 45, 60, 75 and 90
mL of sample volumes containing a fixed amount of dye were taken and uptake
of Sudan III dye was studied (Fig. 3.4.3.3). The maximum % uptake (95%) of
Sudan III dye was found with sample volume of 75 mL. Therefore, 75 mL of sample
volume was used for further studies
3.4.3.4 Effect of agitation speed
Shaking speed is an important factor in determining the possibility of application
of polymer for the quantitative % uptake of Sudan III dye. The driving force i.e
Shaking speed could help in mass transfer and facilitate the concentration gradient
between the sample solution and the polymer. Different speeds (ranging from 40 to
140 r.p.m) were studied for the % uptake of Sudan III dye by polymer. The results of
% uptake of Sudan III vs. agitation speed (Fig. 3.4.3.4) shows that the % uptake was

93

maximum (95%) at 120 r.p.m. Therefore, the shaking speed of 120 r.p.m. was
selected for further studies.
3.4.3.5 Effect of amount of polymer
The amount of the -CD polymer is another important parameter that affects
%uptake of dye. A quantitative removal (95%) cannot be achieved when the -CD
polymer is less than the optimum amount. In order to optimize the smallest amount of
polymer, 100 mg, 200 mg, 300 mg, 400 mg and 500 mg of the polymer were added to
the solution containing known amount of dye. The quantitative recoveries were
obtained at 300 mg of -CDP (Fig. 3.4.3.5). Therefore, 300 mg of the -CDP has
been used for further studies.

94

Fig. 3.4.3.1. Effect of the pH on the % uptake of the Sudan III dye by -CD polymer

Fig. 3.4.3.2.Effect of shaking time on the % uptake of the Sudan III dye by -CD
polymer

Fig. 3.4.3.3 Effect of sample volume on the % uptake of the Sudan III dye by -CD
polymer
95

Fig. 3.4.3.4. Effect of agitation speed on the % uptake of the Sudan III dye by -CD
polymer

Fig. 3.4.3.5. Effect of amount of adsorbent on the % uptake of the Sudan III dye by
the -CD polymer

96

3.4.4. Applications
3.4.4.1. Determination of Sudan III in Curry Powder/ Curry Paste
3.0 g of the Curry powder/ curry paste sample was dissolved in hot double distilled
water. After mixing, the residue was filtered, and filtrate was diluted with double
distilled water and made the volume up to the mark in a standard flask. The sample
solution was then analyzed by the developed procedure. The results of analysis are
given in (Table 3.4.4.1)
3.4.4.2. Determination of Sudan III in Navratan oil
5.0 mL of Navratan oil, dissolved in Dimethylformamide (DMF). After mixing, the
residue was filtered, and filtrate was diluted with double distilled water and made the
volume up to mark in a standard flask. The sample solution was then analyzed by the
developed procedure. The results of analysis are given in (Table 3.4.4.2).

97

Table 3.4.4.1 Results for the analysis of determination of Sudan III dye by developed
method in Curry powder and Curry paste sample.
Food Samples

Curry powder

Curry paste

Added, g/mL

Found, g/mL

Recovery, %

0.015

0.254

0.256

95.16

0.440

0.440

96.70

0.920

0.918

98.18

.010

0.260

0.262

97.03

0.450

0.452

98.26

0.950

0.948

98.75

Curry powder Locally available in market

Curry paste locally available in

marke
Table 3.4.4.2 Results for the analysis of determination of Sudan III dye by developed
method in some Navratan cosmetic samples.
Food Samples

Navratan oil

Added, g/mL

Found, g/mL

Recovery, %

0.019

0.274

0.286

97.61

0.469

0.470

96.31

0.939

0.950

99.16

Navratan oil locally available in market

98

3.5 DETERMINATION OF SUDAN IV DYE

Sudan dyes are a class of lipophilic synthetic organic colorants that are widely used
in industry because of their colorfastness and low price. These azo-dyes are known to
be biologically active through their metabolites (16), and they represent a potential
risk to public health if they enter the food chain. Sudan IV dye have been associated
with the occurrence of bladder cancer in textile and leather dyers, painters, and hairdressers (17,18). Sudan IV is considered a possible human carcinogen and mutagen
(19), classified as a category 3 carcinogen by the International Agency for Research
on Cancer (IARC) (20). Sudan IV dye has been found to cause a high frequency of
bladder

carcinomas

in

mice

(21).

The

present

work

describes

cyclodextrinepichlorohydrin polymer (-CDP) as a solid support

for the

preconcentration and determination of the Sudan IV dye.

3.5.1 Materials
3.5.1 Equipment
Equipments are same as described in section 3.2.1.1
3.5.1.2 Reagents
All reagents used were of analR grade unless otherwise stated. Double distilled water
was used throughout the experiment. A 0.01M stock solution of Sudan IV dye (Loba
chem.) was prepared in double distilled water and further diluted as and when
required.
Buffer solutions are same as described in section 3.2.1.2
3.5.2 Procedure
200 mg of -CDP and 2.5 mL of buffer solution (pH 3.0) were added to a 100 ml
stoppered conical flask at room temperature. The mixture was allowed to stand for 15
min. so that -CDP should swell sufficiently and an appropriate amount of dye was
added and made up to 90 ml with double distilled water. The mixture was shaken in
the thermostatic shaking water bath for 120 min. at the rate of 120 r.p.m. agitation

99

speed, 5.0 ml of supernatant solution was transferred into a test tube and the
absorbance was measured spectrophotometrically.
3.5.3 Optimization of various parameters
3.5.3.1 Effect of pH
The formation of inclusion complex of the dye in the polymer depends on the pH of
the sample solution which was studied in the range of (1.0-7.0) using different buffer
solutions

(Fig. 3.5.3.1), % uptake (95) was obtained at pH 3.0. Therefore, the

working pH was chosen as 3.0 for the subsequent studies.

3.5.3.2 Effect of the shaking time
Shaking time

is an important

factor in determining the possibility of

application of the -CD polymer for the selective uptake of Sudan IV dye. Different
shaking time (ranging from 15 to 135 min) were studied for the % uptake of Sudan
IV dye by -CD polymer. The results of % uptake of Sudan IV dye vs. the shaking
time show that the % uptake of (95%) was attained within 120 min.(Fig. 3.5.3.2).
Therefore, the shaking time of 120 min. was selected for further studies.
3.5.3.3 Effect of sample volume
Enriching low concentration of dye from large volume of sample the effect of sample
volume is an important factor in determining the possibility of application of
polymer for the % of uptake of Sudan IV dye.For this purpose 15, 30, 45, 60, 90 and
105 mL of sample volumes containing a fixed amount of dye were taken and
uptake of Sudan IV dye was studied (Fig. 3.5.3.3). The maximum % uptake
(95%) of Sudan IV dye was obtained at sample volume of 90 mL. Therefore, 90 mL
of sample volume was used for the further studies.
3.5.3.4.Effect of agitation speed
Shaking speed is an important factor in determining the possibility of application
of polymer for the quantitative %uptake of Sudan IV dye. The driving force i.e
Shaking speed could help in mass transfer and facilitate the concentration gradient
between the sample solution and the polymer. Different speeds (ranging from 40 to
100

140 r.p.m) were studied for the % uptake of Sudan IV dye by polymer. The results of
% uptake of Sudan IV vs. agitation speed (Fig. 3.5.3.4) show that the % uptake reach
maximum (95%) at 120 r.p.m. Therefore, the shaking speed of 120 r.p.m. was
selected for further studies.
3.5.3.5 Effect of amount of polymer
The amount of the -CD polymer is another important parameter that affects %
uptake of dye. A quantitative removal (95%) cannot be achieved when the -CD
polymer is less than the optimum amount. In order to optimize the smallest amount of
polymer, 100 mg, 200 mg, 300 mg, 400 mg and 500 mg of the polymer were added to
the solution containing known amount of dye. The quantitative recoveries were
obtained at 200 mg of -CDP (Fig. 3.5.3.5). Therefore, 200 mg of the -CDP was
used for further studies.

101

Fig. 3.5.3.1. Effect of the pH on the % uptake of the Sudan IV dye by -CD polymer

Fig. 3.5.3.2. Effect of shaking time on the % uptake of the Sudan IV dye by the -CD
polymer

Fig. 3.5.3.3. Effect of sample volume on the % uptake of the Sudan IV by the -CD
polymer
102

Fig. 3.5.3.4. Effect of agitation speed on the % uptake of the Sudan IV dye by the CD polymer

Fig. 3.5.3.5. Effect of amount of adsorbent on the % uptake of the Sudan IV dye by
the -CD polymer

103

3.5.4. Applications
3.5.4.1. Determination of Sudan IV in Hot Chili Tomato Sauce/ Chili pepper
3.0 g of the Hot chili tomato sauce/ Chili pepper sample was dissolved in hot double
distilled water. After mixing, the residue was filtered, and filtrate was diluted with
double distilled water and made the volume up to the mark in a standard flask. The
sample solution was then analyzed by the developed procedure. The results of
analysis are given in (Table 3.5.4.1)
3.5.4.2. Determination of Sudan IV in Red Sausage
3.0 g of the Red Sausage sample was dissolved in hot double distilled water. After
mixing, the residue was filtered, and filtrate was diluted with double distilled water
and made the volume up to the mark in a standard flask. The sample solution was then
analyzed by the developed procedure. The results of analysis are given in (Table
3.5.4.2)

104

Table 3.5.4.1. Results for the analysis of determination of Sudan IV dye by developed
method in Hot Chilli Tamoto Sauce and Hot Chilli Pepper food samples.
Food Samples

Hot Chilli Tamoto

Added, g/mL

Found, g/mL

Recovery, %

0.012

0.258

0.260

96.29

0.550

0.548

97.50

0.936

0.930

98.10

0.014

0.258

0.264

97.05

0.550

0.552

98.87

0.936

0.938

97.73

Sauce

Hot Chilli Pepper

Hot Chilli Tamoto Sauce - locally available in market, bHot Chilli Pepper - locally

available in market
Table 3.5.4.2 Results for the analysis of determination of Sudan IV dye by developed
method in Red Sausages sample.
Food Samples

Red Sausage

Added, g/mL

Found, g/mL

Recovery, %

0.011

0.260

0.262

97.03

0.568

0.570

98.44

0.945

0.950

99.37

Red Sausage locally available in market

105

3.6 DETERMINATION OF SUNSET YELLOW DYE

Synthetic dyes are widely used for improving the color and enhancing the visual
aesthetic appeal of some foods, and this effect is maintained throughout the
production process and during storage. They present high stability to light, oxygen,
and pH changes, and have lower prices compared to natural dyes (22-23). These dyes
are added as small amounts in food, drugs or cosmetic products (24-26). Sunset
yellow is widely used as additives in soft drinks and other non-alcoholic drinks, the
highest dose of each in soft drinks being 70.0 mg 1-1 (27). The present work describes
that -cyclodextrinepichlorohydrin polymer (-CDP) as a solid support for the
preconcentration and determination of the Sunset yellow dye.
3.6.1 Materials
3.6.1.1 Equipment
Equipments are same as described in section 3.2.1.1
3.6.1.2 Reagents
All reagents used were of analR grade unless otherwise stated. Double distilled water
was used throughout the experiment. A 0.01M stock solution of Sunset Yellow dye
(Loba chem.) was prepared in double distilled water and further diluted as and when
required.
Buffer solutions are same as described in section 3.2.1.2
3.6.2. Procedure
200 mg of -CDP and 2.5 mL of buffer solution (pH 3.0) were added to a 100 ml
stoppered conical flask at room temperature. The mixture was allowed to stand for 15
min. so that -CDP should swell sufficiently and an appropriate amount of dye was
added and made up to 75 ml with double distilled water. The mixture was shaken in
the thermostatic shaking water bath for 90 min., at the rate of 140 r.p.m., 5.0 ml of
supernatant solution was transferred into a test tube and the absorbance was measured
spectrophotometrically

106

3.6.3. Optimization of various parameters

3.6.3.1 Effect of pH
The formation of inclusion complex of the dye in the polymer depends on the pH of
the sample solution which was studied in the range of (1.0-7.0.) using different buffer
solutions (Fig. 3.6.3.1), % uptake ( 95) was obtained at pH 3.0. Therefore, the
working pH was chosen as 3.0 for the subsequent studies.
3.6.3.2 Effect of shaking time
Shaking time is an important factor in determining the possibility of application of the
-CD polymer for the selective uptake of sunset yellow dye. Different shaking time
(ranging from 15 to 105 min.) were studied for the % uptake of sunset yellow dye by
-CD polymer. The results of % uptake of Sunset yellow dye vs. the shaking time
show that the % uptake of (95%) was attained within 90 min.(Fig. 3.6.3.2).
Therefore, the shaking time of 90 min. was selected for further studies.
3.6.3.3 Effect of sample volume
Enriching low concentration of dye from large volume of sample the effect of sample
volume is an important factor in determining the possibility of application of polymer
for the % of uptake of sunset yellow dye. For this purpose 15, 30, 45, 60, 75 and 90
ml of sample volumes containing a fixed amount of dye were taken and uptake of
sunset yellow dye was studied (Fig. 3.6.3.3). The maximum % uptake (95%) of
sunset yellow dye was at sample volume of 75ml. Therefore, 75ml of sample volume
was used for the further studies.
3.6.3.4. Effect of agitation speed
Shaking speed is an important factor in determining the possibility of application of
polymer for the quantitative % uptake of sunset yellow dye. The driving force i.e
Shaking speed could help in mass transfer and facilitate the concentration gradient
between the sample solution and the polymer. Different speeds (ranging from 40 to
140 r.p.m.) were studied for the % uptake of Sunset yellow dye by polymer. The
results of % uptake of sunset yellow vs. agitation speed (Fig. 3.6.3.4) shows that the

107

% uptake reach maximum ( 95%) at 140 r.p.m. Therefore, the shaking speed of 140
r.p.m. was selected for further studies.
3.6.3.5 Effect of amount of polymer
The amount of the -CD polymer is another important parameter that affects %uptake
of dye. A quantitative removal ( 95%) cannot be achieved when the -CD polymer is
less than the optimum amount. In order to optimize the smallest amount of polymer,
100mg, 200mg, 300mg, and 400mg of the polymer were added to the solution
containing known amount of dye. The quantitative recoveries were obtained at 200
mg of -CDP shown in (Fig. 3.6.3.5). Therefore, 200 mg of the -CDP has been used
for further studies.

108

Fig. 3.6.3.1. Effect of pH on the % uptake of the Sunset yellow dye by the -CD
polymer

Fig. 3.6.3.2. Effect of shaking time on the % uptake of the Sunset yellow dye by the
-CD polymer

Fig. 3.6.3.3. Effect of sample volume on the % uptake of the Sunset yellow dye by
the -CD polymer
109

Fig. 3.6.3.4. Effect of agitation speed on the % uptake of the Sunset yellow dye by
the polymer

Fig. 3.6.3.5 Effect of amount of adsorbent on the % uptake of the Sunset yellow dye
by the -CD polymer

110

3.6.4. Applications
3.6.4.1. Determination of Sunset yellow in Santra Goli (orange flavor)
8.0 g sample of Santra goli was grounded in mortar with pestle and then dissolved in
hot double distilled water. The solution was cooled and filtered. Filtrate was diluted
with double distilled water and made the volume up to the mark in a standard flask.
The sample solution was then analyzed by the developed procedure. The results of
analysis are given in (Table 3.6.4.1)
3.6.4.2. Determination of Sunset yellow in Mirinda (orange flavor)
5.0 mL of Mirinda sample was dissolved in double distilled water. After mixing, the
residue was filtered, and filtrate was diluted with double distilled water and made the
volume up to the mark in a standard flask. The sample solution was then analyzed by
the developed procedure. The results of analysis are given in (Table 3.6.4.1).
3.6.4.3. Determination of sunset yellow in Dry Syrup (ZIFI 100)
5.0 mL of Dry syrup sample was dissolved in double distilled water. After mixing, the
residue was filtered, and filtrate was diluted with double distilled water and made the
volume up to the mark in a standard flask. The sample solution was then analyzed by
the developed procedure. The results of analysis are given in (Table 3.6.4.2).

111

Table 3.6.4.1 Results for the analysis of determination of Sunset Yellow dye by
developed method in Santra Goli and Mirinda food samples.
Food Samples
a

Added, g/ml

Santra Goli

Mirinda

Found, g/ml

Recovery, %

0.010

0.304

0.302

96.17

0.603

0.590

96.24

1.206

1.208

99.34

0.022

0.304

0.301

95.85

0.603

0.595

95.20

1.206

1.201

97.80

Santra goli - locally available in market, bMirinda - locally available in market

Table 3.6.4.2 Results for the analysis of determination of Sunset yellow dye by
developed method in Dry Syrup (ZIFI 100) pharmaceutical sample.

Food Samples
a

Dry Syrup (ZIFI

Added, g/ml

Found, g/ml

0.011

Recovery, %
-

100)

0.228

0.232

97.07

0.454

0.458

98.49

0.808

0.810

98.90

Dry Syrup locally available in market

112

Table

3.7

SHOWING

DETERMINATION

OPTIMIZED

CONDITIONS

FOR

THE

OF FOOD DYES USING -CDEPICHLOROHYDRIN

AS SOLID PHASE EXTRACTANT

Food dyes

Shaking

Sample

Agitation

Amount of

time (min.)

volume (mL)

speed (r.p.m.)

adsorbent (mg)

4.0

120

90

120

200

Sudan I

3.0

90

75

100

200

Sudan III

4.0

75

75

120

300

Sudan IV

3.0

120

90

120

200

Sunset

3.0

90

75

140

200

Brilliant

pH

green

yellow

113

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