Spectrophotoll'letric Determination Of

Saccharin In Food And Pharmaceutical Products

 Spectrophotometric Determination Of
Saccharin In Food And Pharmaceutical Products

Summary

5'l simple and higlify sensitive metfwa is descrweif for tlie

spectropfwtometric determination of saccftarin..'lne method is 6ased on' the
6romination of saccharin to form 9I£-6romoaerivative, wliich reacts with
potassium ioaide to [werate ioaine. 7fie liberated iodine is tnen reacted with
feucocrysta{ viofet and tlie crystal viokt aye formed sfwws ma;dmum.
a6sor6ance at 593 nm. 'l1ie colour system 06eys tJ3eer's WW in the
concentration range of 0.05 - 0.5J19 mflof saccliarin and lias apparent
mowr a6sorptivity of 2.93 .t 10 5 [mof1cm-1. 7fie metfipd is free from
interferences of common diverse ions and many of tlie ingretfients commonfy
found in fOOd prodll;cts ana lias 6een satisfactorily app{iea to the
aetermination of saccharin in fooa andpliarmaceuticafs. .

III Press,}. Indian cliern. Soc., MsS '}.[p. 226/97.

85
Introduction

Saccharin (o-benzoic sulfamide, C.H.COSO.NH) and its salls are white crystaline
powders, odourless and In diluted solution are obout 400 - 500 times sweeter than sucrose
(1). Due to its characteristics properties, it is one of the most consumed artificial sweetner in
some countries. It has been widely used in medicine and in variety of food products as a non-
nutritive sweetner (2). It is used primarily in disease such as diabetes, where it is harmful for
the patient to use sugar and persons dieting to lose weight often use saccharin in place of
sugar (3 - 4). In order to avoid excessive use, several countries have set tolerance limit (5).
Saccharin is very sweet in dilute solution but is bitter in concentrated solution (3).

The artificial sweetner saccharin is a weak bladder carcinogen and causes risk to human
and animal (4 - 8). When saccharin is given in diet it causes cancer of the urinary tract, when
it is inserted in bladder as an implant saccharin, it causes bladder cancer (9). Recent studies
suggested that high doses of saccharin and sodium saccharin produces cytotoxicity of the
urothelium and consequently regeneration hyperplasia (10).lt enhanced celi proliferation and
promote tumor (11). Due to its possible carcinogenic effects its use in food products is prohibited.
So, due to medical and legal aspects the determination of saccharin and other non-fattening
artificial sweetners in dietary products has an economical and social relevance (4).

Due to its wide use in food products such as canned fruit juices, vegetables, cookies,
other backery products, flavouring extracts, gelatin, pudding, frozen desserts, imitation jams,
jellies, marmalades and salad dressing etc. (12) and beverages, literature contains numerous
methods for the determination ot saccharin in various food products, beverages and
pharmaceuticals. Various techinques such as gas chromatography (13-15), thin layer
chromatography (16-20), high performance liquid chromatography (21-24), gas liquid
chromatography (25 -26), polarography (27-28), fluorimetry (29), ultraviolet spectrophotometry
(30 -32), infrared spectrophotometry (33), gravimetry (34-35), molecular emission cavity analysis
(36), ion selective electrode (37). flow injection analysis (38), volumetric (39). potentiometric
(40-41) etc. are being used for its determination.

A number of methods have also been reported for the spectrophotometric determination
of saccharin (5. 28. 42-49). The common reagents reported include azure B (42), tris-{1:10
phenanthroline)-iron II chelate (43), methylene blue (44), chlorophenothiazine (45),
phenothiazine (5), azure C (46). ferroin (47), phenol-sulphuric acid (28, 48) and starch-Cdl.
(49), Nevertheless most of these methqds are time consuming and laborious. While others
either need costly equipment or have limitations in sensitivity, selectivity and instability of
colour.

Here a simple and non expensive manual method is proposed for the determination of
saccharin. The method is based on the bromination of saccharin to form N-bromoderivatives

86
which on reaction with potassium Iodide liberates iodine. The liberated Iodine selectively oxidises
leucocrystal violet and the crystal violet dye so formed shows maximum absorbance at 593
nm. Various analytical parameters were investigated for optimum sensitivity. The method is
free from the interference of a number of substances commonly found In food products and
has been applied to the determination of saccharin in food and pharmaceuticals.

Experimental
Appratus - A Systronics UV-VIS Spectrophotometer 108 with matched silica cells was
used for all spectral measurements. A Systronics pH meter model 331 was used for pH
measurements.
Reagents - All chemicals used were of AnalaR grade and double distilled water was
used throughout the experiment.
Saccharin - A stock solution containing 1mgml-1 of saccharin was prepared by dissolving
100 mg of saccharin in 100 ml of dDuble distilled water and standardized volumetrically (39).
Bromine water'- A saturated solution of bromine in water was prepared. The solution
was prepared daily.
Formic acid - A 50% aqueous solution.
Potassium iodide (E. Mercl(, Germany) - 0.1 % aqueous solution.
Leucocrystal violet (LCV) (Eastman Kodak Co., New York) - To a 1 I volumetric flask
200 ml water, 3 ml 85% phosphoric acid and 200 mg of leucocrystal violet were added and
shaken gently until the dye gets dissolved. The content of the flask was then diluted to 11 with
water. This solution was stable for several months when kept in dark.
Sodium hydroxide - 1 M aqueous solution.

Procedure
Preparation of Calibration Curve - An aliquot of working standard solution containing
1.25-12.5 119 of saccharin was taken in 25 ml calibrated tubes. To it 0.5 ml of bromine water
was added and the mixture was shaken gently for 2 min The excess of bromine was removed
by dropwise addition of formic acid (30) after which 1 ml of potassium iodide was added. The
yellow solution obtained was shaken gently for few seconds. Then 1 ml of LCV solution and
4-5 drops of 1 M sodium hydroxide were added. The solution was kept in a thermostat maintained
at 55°C for 5 min. The solution was cooled diluted to 25 ml with deionised water and kept for
10 min for full colour development and measured at 593 nm against a reagent blank.

Determination of Saccharin in Soft Drinks - Samples of different soft drinks were

decarbonated by repeated shaking and pouring from one beaker to another. 10 ml of the
solution was transferred into a 60 ml separating funnel. 1 ml of 10% sulphuric acid was added

87
and the mixture was equilibrated with 2 x 6 ml of diethy1 ether. The lower aqueous layer was
discarded. The upper ether layer was equilibrated with 2 x 2 ml of 2"10 sodium hydrogen
carbonate solution. The ether layer was discarded and aqueous layer was acidified with 2 ml
of 5% hydrochloric acid and then extracted with 2 x 5 ml of diethyl ether into a conical flask. All
of the ether was evaporated from the-extract on a hot water bath. The saccharin residue was
dissolved in 10 ml of water and transferred completely into a calibrated flask and made upto
25 ml with water (42). Aliquots were then analysed as described above. Results are shown in
Table 1.

Determination of Saccharin in Jam, Condensed Milk and Ice-Creams - 1 gm of

each sample such as jam, condensed milk and ice creams were dissolved in 10 ml of water
and deproteinised by adding 25% lead acetate (51) and acidified with 2 ml of 5% hydrochloric
acid then extracted with 3 x 6 ml of diethyl ether. The etheral solution was then washed with 5
ml of water. The ether layer was separated and evaporated on a hot water bath. Residue was
,
dissolved in 10 ml of water and transferred completely into a calibrated flask and the volume
was made upto 25 ml (51) and aliquots were then analysed as described above. Results are
given in Table 1.

Determination of Saccharin in Pharmaceuticals - All drug samples tested were

purchased from the local pharmacy. A saccharin tablet was ground. into a fine powder. weighed
and stirred for 2-3 min with 50 ml of deionised water. 1 ml of 5% EDTA was added and the
solution was filtered through Whatman no. 40 filter paper. The insoluble mass was washed
with three successive 5 ml portions of water and the filtrate plus washing were diluted to
volume in a 250 ml calibrated flask (42). A known volume was further diluted depending upon
the saccharin content and the colour of the sample. Aliquots were analysed by recommended
procedure. Results are shown in Table 2.

Determination of Saccharin in Cosmetic Samples - Various samples of tooth paste

(1 gm of each) were dissolved in 25 ml of hot water. The solution was cooled and centrifuged
at 1850 g for 5 min. The supernatant solution was acidified with 10% hydrochloric acid and
saccharin was extracted into 3 x 6 ml of ethylacetate. The combined extract was evaporated to
dryness and the residue was dissolved in 25 ml of water (52). Aliquots were then analysed as
described above. The results are shown in Table 1.

Results and Discussion

Spectral characteristics - The absorption spectra of crystal violet dye formed in the
proposed reaction shows maximum absorbance at 593 nm. The reagent blank gave negligible
absorbance at this wavelength (fig 1).

88
 Adherence to Beer's law, Molar absorptivity and Sandell's sensitivity· The colour
system obeys Beer's law over the concentration range of 1.25-12.5119 of saccharin per 25 ml
of final solution (0.05-0.5 ppm) at 593 nm (fig 2). The molar absorptivity and Sandell's sensitivity
were found to be 2.93 x 10' I mol-'em-' and 0.0006 119 em-' respectively.

Effect of reagent concentration ·It was found that under optimum condition 0.5 ml of
bromine water. 1 ml of 0.1 % potassium iodine, 1 ml of LCV and 4-5 drops of 1 M sodium
hydroxide were required for full colour development. A few drops of formic acid was sufficient
for the removal of excess of bromine. It was also found that if the proposed reagent concentration
is reduced or increased the absorbance value decreases (fig 3).

Effect of time· It was found that about 2 min were sufficient for bromination and iodine
was liberated on addition of potassium iodide immediately. Full colour development required
about 15 min. It was found that once developed the colour was stable for several days (fig 4).

Effect of temperature· 550C-650C temperature was found to be most suitable for colour
reaction. The temperature was obtained by keeping the solution in a thermostat maintained at
about 55°C. At higher and lower temperature the colour gradually faded (fig 4).

Effect of pH • pH of 4.5-5.5 was found to be best for obtaining constant and maximum
absorbance I'alues. Increase of pH above 5.5 severely affected the stability and sensitivity of
Ihe dye. Colour development did not take place belOW pH 4.5.

. Reproducibility - Reproducibility of the method was found by analysing 10 I1g per 25 ml

of the dye over the period of seven days. Standard deviation and relative standard deviation
were found to be ±0.011 and 1.83% respectively.

Interference studies· The validity of the method was assessed 'by investigating the
effect of common foreign species added to a solution containing 10 I1g per 25 ml of saccharin,
Table 3, shows that saccharin can be selectively determined in the presence of many substances
that are likely to be present in the soft drinks and the saccharin tablets, Most of the common
Ions did not interfere in the procedure.

Colour Reaction
The colour reaction involves the following steps (Scheme A) -
(1) Formation of N-bromoderivatives with bromine water (49),
(2) Liberation of iodine by the reaction of N-bromoderivative with potassium iodide in acedic
medium,
(3) Liberated iodine oxidises the leucocrystal violet to form crystal violet dye,

89
Application

The method has been satisfactorily applied to the determination of saccharin in food
products, pharmaceuticals and cosmetic products. Table 1-2. The results of pharmaceuticals
analyses obtained by the proposed method also agreed with the claimed value on the labels in
all instances. Table 2.
To check the validity of the method known amount of saccharin was added to various
saccharin free samples such as icing sugar, fresh apple, oranges and grapes juices and then
determined by the proposed as well as reported method (28, 48) Table 4. The recoveries of
saccharin added to different samples were found to be 95.8-98.4% which is in agreement with
the results obtained by the established methods indicating the accuracy of the proposed method.

Conclusion
It can be concluded that the proposed method is simple, rapid, accurate and reproducible
\

and can be further automated for the routine measurements. The method has been compared
favourably with most of the other methods and found to be highly sensitive and selective.
Table 5.

90
 (jJrco
(1)
~NH
+ Br. > ~~~r
SO. SO.
Saccharin
N-bromoderivative

co
(2) PllT
.Jy-NBr
+ KI ------7» 12
Iodine
S02

o
N+(CH3}2

tJ( 'tIl
G
(3)
)
(CH 3)2N N(GH')2
Crystal Violet dye
I..max 593 nm
Leucocrystal Violet

Scheme A - Colour Reaction of Saccharin_

91
 --.-~

AMOUNT OF POTASSIUM IODIDE, ml, B

035
0' o. 08
". I.
" I.

03

E
C 025
M
0>

'"ill 0'
U
Z
...: 015
ro
II:
0 01
(f)
ro
<
005

0
0 0" 04 06 08 12 14 '6

AMOUNT OF BROMINE WATER. ml, A

CONCENTRATION OF SACCHARIN: 5 I1g/25 ml
fig 3. - EFFECT OF AMOUNT OF BROMINE WATER AND
POTASSIUM IODIDE ON COLOUR REACTION

TIME IN MINS. B

5 f) 15 '0 ,s 30 35 40 45
07

OS

E
C
05
M
0>
It)

ill 0'
U
Z
...:
ro 03
a:
0 "A
(f) 02
ro
...:
01

0
0 t) '0 30 '0 50 60 70 80 90

TEMPERATURE °C. A
CONCENTRATION OF SACCHARIN: 10 IIg/25 ml

fig 4. - EFFECT OF TEMPERATURE AND TIME ON COLOUR REACTION

. ------- --- ___ ...J

93
 II .

08

E 06
c:
'"'" ,
It) ,
uj
()
Z OA
«
!D
a:
0
(J)
(IJ 02
«

0
2 3 4 5 6 7 8

,
pH
CONCENTRATION OF SACCHARIN: 10119/25 ml
lig 5 .• EFFECT OF pH ON COLOUR REACTION

I,
,,
,
I

!
,

94
 Table· 1. Determination of saccharin In food and cosmetic samples.

Sample Saccharin found (lIg)'

mass/volume P R"
Soft drinks8
A 4.94(±O.O20) 4.73
B 5.41 (±O.O25) 5.10
C 3.07(±0.021) 2.76
Ice-creamsb
A 6.56(±0.017) 6.25
B 6.61 (±O.O20) 6.45
C 6.09(±O.O23) 5.88
Condensed milkc 10.05(±0.035) 9.89,
Jamd 6.77(±0.020) 13.71
Tooth pastee
A 8.64(±0.011 ) 8.48
B 8.12(±0.020) 8.02
C 8.75(±0.020) 8.54

P"= Proposed method R = Reported method

'Mean of three replicate analysis.
a = 10 ml of sample was treated as described in the procedure section and then
1 ml of aliquot was analysed. '
b, c, d, e = 1 gm of sample was treated as described in the procedure section and
then 1 ml of aliquot was analysed.

,
Table·2. Determination of saccharin in pharmaceuticals.

Samples Saccharin mg/tablet a Claimed value

P R mg/tablet

Tablet
A 11.80 (1.66%) 11.60 12.00
B 51.20 (0.58%) 51.00 51.50
C 12.40 (0.83%) 12.30 12.50

a = Mean of three replicate analYSis.

p = Proposed method, R = Reported method I3.)

95
 Table - 3. Effect of foreIgn species.
=
Concentration of saccharin 10 ~ 125 ml (0.4 ppm)

Foreign species Tolerance limit' (f.lg/ml, ppm)

Ascorbic acid 80
Citric acid 500
Tartaric acid 600
Gelatin 750
Sodium bicarbonate 850
Benzoic acid 1000
Malic acid 1500
Sucrose 3000
Glucose 4000
C02+, Cu2... 400
Cr"+, AI'+, Zn2+, Be2., Fe'" 850 .
PO4 3- • Acetate SO42-
I
2500
Ca2+ 4000

'The amount causing ±20/0 error in absorbance value.

96
 Table· 4. Recovery of saccharin In saccharin
free food product and fresh fruit JUices.

Sample Saccharin Total saccharin % Recovery

added found' (lIg)
p R'· P R'·

Icing sugar" 10.00 9.73 (±0.020) 9.53 97.30 95.30

Apple juiceb 10.00 9.58 (±O.030) 9.52 95.80 95.20
Orange juice' . 10.00 9.68 (±O.011) 9.60 96.80 96.00
Grapes juice" 10.00 9.73 (±O.017) 9.65 97.30 96.50

= =
P Proposed method, R Reported method
=
Size of sample: a 1 gm, =
b, c, d 10 ml.
* Mean of three replicate analysis.

Table· 5. Comparison with spectrophotometric methods.

Method/Reagent/ReI. Amax Beer's law range! Remarks

(nm) detection limit (ppm)

Phenol-Sulphuric acid2• 575 0.4 Long waiting time (2h),

corrosive reagent used

42
.
less sensitive .
Azure 8 685 2-68 Less sensitive.
Phenothiazine5 510 20-400 Less sensitive and
selective.
Starch-Cdl 249 600 0.4-1.6 Less sensitive.
LCV (Proposed method) 593 0.05-0.5 Sensitive, free from
most of the interferants.

97
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99