EXPERIMENT 1: DETERMINATION OF SACCHARIN, ASPARTAME AND

SODIUM BENZOATE IN SOFT DRINKS USING HIGH-PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)

INTRODUCTION

Artificial sweeteners, preservatives, and colors are commonly used in soft drinks to improve
flavour and appearance, as well as to lengthen shelf life by inhibiting biological decay.
Benzoic acid salts, such as sodium benzoate is commonly used as preservatives in beverages.
Saccharin (1,2-benzisothiazol-3(2H)-one-1,1-dioxide) is a non-glucose, low-calorie substance
with a sweet taste that comes in sodium or calcium salt form. Saccharin has a quick onset, a
brief duration, and a harsh and metallic aftertaste. Aspartame is a widely used artificial
sweetener with a low-calorie content. Aspartame is currently commonly ingested by the
general public in the form of cold drinks, diet soda, low-calorie sweets, sugar-free sweets,
and pharmaceuticals.
HPLC (High-performance liquid chromatography) is a type of column chromatography used
in biochemistry and analysis to separate, identify, and quantify active chemicals. This
approach can be used to analyse materials containing anything from small organic and
inorganic compounds and ions to polymers and proteins with high molecular weights by
using the proper equipment (column and detector). We will use reversed-phase partition
chromatography in this experiment.

https://www.sphinxsai.com/2019/ch_vol12_no4/1/(15-23)V12N4CT.pdf
https://www.researchgate.net/publication/316545292_Aspartame_Effects_and_Awareness
https://www.researchgate.net/publication/
235987484_High_performance_liquid_chromatography_A_short_review
OBJECTIVE

1. To determine amount of saccharin, aspartame, and sodium benzoate in soft drinks by using
HPLC

SCHEMATIC DIAGRAM

b. Stock solution

a. Instrument set up 10 mg of saccharin + 10 mL of distilled

water
10 mg of aspartame + 10 mL of distilled
water
10 mg of sodium benzoate + 10 mL distilled
water

d. Standard mixture c. Working solution

2 mL of saccharin (100 ppm) 1 mL (100 ppm) of saccharin + 10 mL of

2 mL of aspartame (1000 ppm) distilled water
4 mL (400 ppm) of sodium benzoate +
2 mL sodium benzoate (400
10 mL of distilled water
ppm)

e. Sample preparation f. Analyses the standard

mixture and samples
10 mL of soft drinks (Coca
using HPLC.
Cola zero, Sprites, Pepsi
bold)
Experimental Procedure

All the instruments had been set up first before started the experiment. For stock solution,
1000 ppm of stock solution was prepared and 10 mg of saccharin standard was weighed out
approximately into a 10 mL volumetric flask. It was then diluted with distilled water until the
mark. Lastly, step 1 and 2 for aspartame, and sodium benzoate standard were repeated.

For working solution, 100 ppm of standard saccharin solution was prepared and 1 ml of
saccharin stock solution was pipetted into 10 ml volumetric flask. It was then diluted with
distilled water until the mark. It was then diluted with distilled water until the mark. Next,
400 ppm of standard sodium benzoate solution was prepared. 4 ml of sodium benzoate stock
solution was pipetted into 10 ml volumetric flask It was then diluted with distilled water until
the mark.

For standard mixture, 2 mL of saccharin (100 ppm), 2 mL of aspartame (1000 ppm), and 2
mL sodium benzoate (400 ppm) were pipetted and placed in 50 mL beaker. Then, the
retention time and peak area of standard mixture were measured using HPLC.

For sample preparation, 10 mL of soft drinks (Coca Cola zero, Sprites, Pepsi bold) were
poured into 50 mL beaker and were stirred using glass rod to remove carbonated gas. Then, 5
mL aliquot sample was filtered through 0.45 µm pore diameter membrane filter to remove
particular matter before inject into HPLC system. Lastly, all the samples and the standard
mixture prepared were analyzed using HPLC.
DISCUSSION

1. Saccharin

- Advantage: It is good to diabetics and obese persons. Saccharin is extensively used in low-
calorie and sugar-free foods and beverages therefore it aids in the reduction of dental cavities.

https://saccharin.org/facts/benefits-saccharin/

Aspartame

Advantages: - It does not induce tooth decay thus it is a healthy sweetener and taste enhancer
that can be used in place of sugar in the diet,

https://caloriecontrol.org/aspartame/

Sodium benzoate

Advantage: - It has the potential to aid in the treatment of a variety of medical ailments. The
chemical lowers high blood levels of ammonia, a waste product, in persons with liver illness
or urea cycle abnormalities.

https://www.healthline.com/nutrition/sodium-benzoate
EXPERIMENT 2

DATA AND RESULTS

Percentage Yield Formula:

% Yield = Weight of essential oil recovered / Weight of citrus peel x 100%

RESULT IN LEMON SAMPLE

Weight of empty thimble = 2.8823 g

Weight of thimble + lemon zest = 16.7205 g
Weight of lemon sample: 13.8382

Weight of dry empty flask + boiling chips = 121.734 g

Weight of flask + boiling chips + oil = 126.345 g
Weight of essential oil recovered: 4.611
% yield (w/w) of essential oil in sample: 33.32%
Table 2.1 Compounds in Lemon Sample
Table 2.2 Table of limonene (4-isopropenyl-1-methylcyclohexane) in Lemon sample

PEAK RT AREA QUALITY

33 5.2143 37.51 91
34 5.300 6.46 93
35 5.323 2.04 93
36 5.374 5.33 94

RESULT IN ORANGE SAMPLE

Weight of empty thimble = 3.2646 g

Weight of thimble + orange zest = 17.4597 g
Weight of sample: 14.1951

Weight of dry empty flask + boiling chips = 123.354 g

Weight of flask + boiling chips + oil = 126.865 g
Weight of essential oil recovered: 3.511
% yield (w/w) of oil in sample: 24.73%
Table 2.3 Compounds in Orange Sample
Table 2.4 Table of limonene (4-isopropenyl-1-methylcyclohexene) in orange sample

PEAK RT AREA QUALITY

30 5.254 37.47 91
DISCUSSIONS

In this experiment, we use percentage yield (w/w) of oil in sample to determine the result in
lemon and orange samples. Therefore, the percentage yield (w/w) of essential oil in sample is
33.32% compared to the % yield (w/w) of oil in sample is 24.73%. Lemon sample has the
higher percentage of yield than the orange sample because the orange sample only has 1
peak. The lemon sample retention time when peak 33,34,35 and 36 are 5.2143,5.300,5.323
and 5.374 respectively. While for the orange sample, the retention time when peak 30 is
5.254.

The percentage of yield for lemon that we obtained from the experiment is 33.32% compared
the yield of lemon in article is 2.97 w/w %. We also obtained the yield from the experiment
for orange sample is 24.73% compared to the article is 1.63 w/w%. To avoid reading errors,
we should know how to read a gas chromatogram in order to get a satisfactory result. For a
better result, we need then utilize an appropriate substance.

The benefits of limonene intake in daily life are it is possible that it has anti-cancer
properties. Supplementing with limonene reduced the formation of skin cancers by
decreasing inflammation and oxidative stress, according to research. Hence, it is also utilized
as an aromatic oil because of its relaxing and healing effects.

Conclusion

In conclusion, GC-MS is used to determine the amount of limonene in lemon and orange,
yielding 33.32%t and 24.73%, respectively.

REFERENCE

1. Stephen Davidowski, Brian DiMarco. The Extraction and Quantification of Limonene

from Citrus Rinds Using GC/MS. Retrieved from
https://www.perkinelmer.com/PDFs/downloads/APP_Limonene_In_Citrus_Rinds_By_GCM
S.pdf