Title : Spectrophotometric analysis of caffeine and benzoic acid in soft drink

Objective : i. To obtain the absorbance of caffeine and benzoic acid in Sprite

ii. To determine the concentrations of benzoic acid and caffeine in Sprite

Introduction:

Soft drinks usually contain carbonated water, caffeine, sodium benzoate, sweeteners
and flavoring agents. In this experiment we used ultraviolet absorbance to measure two
major species in soft drinks which are caffeine and benzoic acid.

Benzoic acid is a colourless crystalline solid and a simple aromatic carboxylic acid.

Structure of benzoic acid

Salts of benzoic acid are used as food preservatives. Its salts and esters of benzoic
acid are known as benzoates. There are some benefits of benzoic acid such as in
treatment of fungal diseases, topical antiseptics and inhalant decongestant. However,
there are also harmful side of it as its salt, sodium benzoate could cause of DNA
damage and hyperactivity. Taking excessive amounts of benzoic acid may affect liver
and kidney.

Caffeine is a bitter white crystalline purine, a methylxanthine alkaloid. It acts as a

central nervous system (CNS) stimulant and psychoactive stimulant. Caffeine has also
diuretic properties.

Structure of caffeine
 Some of benefits of caffeine are that it temporary wards off drowsiness and restores
alertness. Besides that, the disadvantages are it can cause increases production of
stomach acid and also caffeine induced psychiatric disorders.

Spectrophotometry measures how much a substance absorbs light by measuring the

intensity of light as the light passes through the sample. Each compound absorbs or
reflects light of a certain wavelength, so that is why different compounds have different
colours.

Beer’s law is used in this experiment which relates the attenuation of light to the
properties of the material through which the light is traveling. It also states that quantity
of light absorbed by a substance dissolved in a non-absorbing solvent is directly
proportional to the concentration of the substance and the path length of the light
through the solution. This explains that the higher the concentration of the solution, the
greater would the absorbance be. However, this law only applicable to diluted solutions.

In this experiment, we restricted ourselves to non-diet soft drinks because the sugar
substitute aspartame in diet soda has some ultraviolet absorbance that slightly
interferes in the present experiment. We also avoided darkly colored drinks because
the colourants have ultraviolet absorbance. Sprite was used in this experiment as it is
lightly coloured solution. There is undoubtedly some ultraviolet absorbance from
colorants in this beverage that contributed to systematic error to this experiment.

Chemical reagent:

o Benzoic acid standard

o Caffeine standard
 o 0.1M hydrochloric acid

o 0.01M hydrochloric acid

o Sprite

Apparatus:

o 10ml pipette

o 100ml volumetric flask

o 10ml graduated cylinder

o 5ml graduated cylinder

o UV/Vis spectrophotometer

o Filter paper

o Distilled water

Procedure:

A. Calibration of standards

i. Benzoic acid solutions containing 2,4,6,8 and 10 mg/ml in 0.01M

hydrochloric acid were prepared.

ii. To prepare 2mg/ml solution, 2ml of benzoic acid standard was mixed with
10ml of 0.1M hydrochloric acid in volumetric flask and it was diluted to the
mark with distilled water.

iii. The steps were repeated to produce 4, 6, 8 and 10 ml of benzoic acid

standards.
 iv. Caffeine standards containing 4, 8, 12, 16 and 20mg/ml in 0.01M
hydrochloric acid were prepared in same manner.

B. Soft drink

i. About 20ml of soft drink was warmed up in a beaker on a hot plate.

ii. The concentrated liquid was then filtered through a filter paper.

iii. After it was left to cool down, 4ml of it was pipetted into a 100ml volumetric
flask.

iv. 10ml of 0.1M hydrochloric acid was added and it was diluted to its mark.

C. Spectrophotometric analysis

i. Ultraviolet baseline from 350 to 210nm with water in the sample and in
reference cuvettes was recorded.

ii. The ultraviolet spectrum of each 10 standards with water in the reference
cuvette was recorded.

iii. The wavelength peak for caffeine and benzoic acid were noted.

iv. The ultraviolet absorption spectrums of the unknowns were measured.

v. The concentrations for both caffeine and benzoic acid were determined
based on the absorbance at its respective wavelength peak.

vi. The results from the dilute solutions and the original soft drink were
reported.
 RESULTS AND CALCULATIONS

a) Calibration of standards

Volume of benzoic acid ( ml) Absorbance (A) Wavelength (λ)

2 0.385 230
4 0.900 230
6 1.377 230
8 1.872 230
10 2.831 230
Volume of caffeine ( ml) Absorbance (A) Wavelength (λ)
4 0.354 275
8 0.783 275
12 1.121 275
16 1.556 275
20 1.936 275
 Graph of Absorbance against Concentrati on of Benzoic Acid
3

2.5 f(x) = 0.29 x − 0.29

R² = 0.98

2
Absorbance

1.5

0.5

0
1 2 3 4 5 6 7 8 9 10 11
Concentration of Benzoic Acid (mg/mL)

Graph of Absorbance against Concentrati on of Caff eine

2.5

2
f(x) = 0.1 x − 0.03
R² = 1
1.5
Absorbance

0.5

0
2 4 6 8 10 12 14 16 18 20 22
Concentration of Caffeine (mg/mL)

b) Soft drink

Volume of Sprite ( ml ) Absorbance (A) Wavelength (λ)

2 0.261 230
0.257 275
4 0.574 230
 0.560 275

Volume of Mountain Dew ( Absorbance (A) Wavelength (λ)

ml )
2 0.178 230
0.116 275

4 0.412 230
0.274 275

From the graph plotted,

Molar absorptivity for benzoic acid at 230 nm, ε = 0.2932 cm-1ppm-1
Molar absorptivity for caffeine at 273 nm, ε = 0.0984 cm-1ppm-1
Based on the Beer-Lambert law, A = ε l C

Mountain Dew
a. Benzoic Acid

Dilution Factor = 50 (2:100)

A = ε l CBenzoic Acid
0.178 = 0.2932cm-1ppm-1 x 1cm x CBenzoic acid

CBenzoic acid = 0.6071 ppm

Thus, the concentration of benzoic acid in Mountain Dew = 0.6071 ppm x 50
= 30.36 ppm
Dilution Factor = 25 (4:100)
A = ε l CBenzoic Acid
0.412 = 0.2932cm-1ppm-1 x 1cm x CBenzoic acid

CBenzoix acid = 1.4052 ppm

Thus, the concentration of benzoic acid in Mountain Dew = 1.4052 ppm x 25

= 35.13 ppm
 30.36+35.13 ppm
∴ The average concentration of benzoic acid in Mountain Dew =
2

= 32.75 ppm

b. Caffeine

Dilution Factor = 50 (2:100)

A = ε l CCaffeine
0.116 = 0.0984 cm-1ppm-1 x 1cm x CCaffeine

CCaffeine = 1.1789 ppm

Thus, the concentration of caffeine in Mountain Dew = 1.1789 ppm x 50

= 58.95 ppm

Dilution Factor = 25 (4:100)

A = ε l CCaffeine
0.274 = 0.0984 cm-1ppm-1 x 1cm x CCaffeine

CCaffeine = 2.7846 ppm

Thus, the concentration of caffeine in Mountain Dew = 2.7846 ppm x 25

= 69.62 ppm

58.95+ 69.62 ppm

∴ The average concentration of caffeine in Mountain Dew =
2

= 64.29 ppm
Sprite

a. Benzoic Acid

Dilution Factor = 50 (2:100)

A = ε l CBenzoic Acid
0.261 = 0.2932 cm-1ppm-1 x 1cm x CBenzoic acid

CBenzoic acid = 0.8902 ppm

Thus, the concentration of benzoic acid in Sprite = 0.8902 ppm x 50

= 44.51 ppm
Dilution Factor = 25 (4:100)
A = ε l CBenzoic Acid
0.574 = 0.2932 cm-1ppm-1 x 1cm x CBenzoic acid

CBenzoic acid = 1.9577 ppm

Thus, the concentration of benzoic acid in Sprite = 1.9577 ppm x 25
= 48.94 ppm
44.51+48.94 ppm
∴ The average concentration of benzoic acid in Sprite =
2

= 46.73 ppm
b. Caffeine

Dilution Factor = 50 (2:100)

A = ε l CCaffeine
0.257 = 0.0984 cm-1ppm-1 x 1cm x CCaffeine

CCaffeine = 2.6118 ppm

Thus, the concentration of caffeine in Mountain Dew = 2.6118 ppm x 50

= 130.59 ppm

Dilution Factor = 25 (4:100)

A = ε l CCaffeine
0.560 = 0.0984 cm-1ppm-1 x 1cm x CCaffeine

CCaffeine = 5.6911 ppm

Thus, the concentration of caffeine in Mountain Dew = 5.6911ppm x 25

= 142.28 ppm

130.59+ 142.28
∴ The average concentration of caffeine in Sprite = ppm
2

= 136.44 ppm

Discussion:
 In this experiment, the concentration of benzoic acid and caffeine in soft drink
which in this is Sprite was being determined via spectrophotometer. During the
preparations, diluted solutions of benzoic acid and caffeine were prepared. Next, for
preparation of Sprite, it was first boiled to remove the carbon dioxide, so that carbonic
acid buffer system is also being removed from the sample solution. Besides that, it also
allows later control of pH and ionic strength of analyte solution. After that, the warm
solution was filtered to remove foreign substances. This is to ensure that there is no
solid particles present as it may interrupt the absorbance reading by blocking the
transmitted light pathway. 10ml of 0.1M was added into the sample solution as to
maintain the pH and ionic strength of the solution.

Since, benzoic acid and caffeine exhibit absorbance at peak wavelength of

230nm and 275nm respectively. So, the spectrophotometer was set at wavelength of
230nm to measure the amount of benzoic acid while wavelength of 275nm to measure
amount of caffeine.

A blank solution is a solution containing little to no analyte of interest which in

this case is distilled water and hydrochloric acid. This blank solution was placed into the
spectrophotometer so that it would only give the absorbance of interest. Then, the
absorbance of diluted solutions was measured. Graphs of absorbance against
concentration of benzoic acid and caffeine were plotted according to Beer’s Law.

From the graph of benzoic acid and calculations, the average concentration of
benzoic acid in Mountain Dew is 32.75 ppm. The average concentration of benzoic acid
in Sprite is 46.73 ppm. Since Sprite is made from lemon which is a citric acid, benzoic
acid is widely used to prevent bacteria and fungus growth in the drink under acidic
condition. From the graph of caffeine and calculations, the concentration of caffeine is
64.29 ppm in Mountain Dew. The concentration of caffeine obtained in Sprite is 136.44
ppm.

There were several precaution steps that taken in during the experiment. Before
using the cuvette, it was cleaned with tissue first so that there is no fingerprints on it.
The clear side of the cuvette was not touched because this might affect the absorbance
reading. Besides that, the solution in the cuvette was made sure to be free of bubbles
as again it might the absorbance reading. The cuvette was rinsed with the solution to be
measured first instead of using distilled water as the diluted solutions and sample
solution would be contaminated and thus the absorbance reading would be affected.

Conclusion:

The average concentration of benzoic acid obtained for Mountain Dew is 64.29 ppm
and 46.73 ppm for Sprite. The average concentrations of caffeine obtained for Mountain
Dew is 64.29 ppm and 136.44 ppm for Sprite.

Reference:

1. SPECTROPHOTOMETRICANALYSISOF CAFFEINE AND BENZOIC ACIDIN

SOFT DRINKS. (2011, August 19). Retrieved July 19, 2015, from
http://www.scribd.com/doc/62641560/127-1-Expt-4#scribd
2. Sprite Ingredients. (2013, February 9). Retrieved July 19, 2015, from
http://quake120.hubpages.com/hub/sprite-ingredients
3. (2015). Spectrophotometer analysis of caffeine and benzoic acid in soft drink. In
Chemical Laboratory II (pp. 37-39). Kampar: Universiti Tunku Abdul Rahman.
4. Benzoic Acid. (n.d.). Retrieved July 19, 2015, from Wikipedia:
https://en.wikipedia.org/wiki/Benzoic_acid
5. Blank solution. (n.d.). Retrieved July 19, 2015, from Wikipedia:
https://en.wikipedia.org/wiki/Blank_(solution)
6. Caffeine. (n.d.). Retrieved July 19, 2015, from Wikipedia:
https://en.wikipedia.org/wiki/Caffeine