Name: Nomasonto Khumalo Student Number 213523727

Date of Practical: 19 February 2018

Spectrophotometric Analysis of a Mixture-Practical 2

Aim
Provide a brief aim for this experiment [2]
 The aim of the experiment was to analyse the soft drink, Mountain Dew, by using the double beam
spectrophotometer (UV-vis NIR spectrophotometer) to measure the absorbance of caffeine and benzoic
acid at two wavelengths.
 To use the Beer-Lambert law for determining the concentration of caffeine and benzoic acid in the soft
drink.
Abstract
Provide an abstract for this experiment [3]
In the instrumental analysis lab, a group of students analysed the soft drink, Mountain Dew, in order to
determine the concentration of benzoic acid and caffeine. The use of UV-vis spectrophotometer’s software
allowed students to easily obtain the absorbance values at two different wavelengths from the spectra of graphs
labelled absorbance against wavelength. The first λmax was found to be 230 nm and the second λmax was 275
nm. The simultaneous equation was manually used to determine the concentration of benzoic acid = 2.019 mg
L-1 and caffeine = 6.506 mg L-1 after their calibration curves were plotted with intention of finding the molar
absorptivity values.

Introductory Questions
Discuss why using UV/Vis is suitable for this analysis. [5]
The UV-vis spectrophotometry is suitable for this analysis because the instrument was developed to maintain
the optimum levels of caffeine and benzoic acid (S.A. Bhawani et.al, 2015). The monitoring of caffeine and
benzoic acid is important to avoid harmful effects such as caffeine-induced anxiety disorder and sodium
benzoate, possibly causingAbsorbance ceand hyperactivity (N. Meya, 2012).

The UV-vis spectrophotometry is also suitable for this analysis because the spectrophotometric determination
in UV-vis region is often less expensive, less time consuming, follows a simple procedure and provides high
accuracy and reproducibility from a small number of samples (S.A. Bhawani et.al, 2015).

The Beer-Lambert law relates the absorbance to concentration. Define molar absorptivity and
give the units you have used. [5]
The concentrations of caffeine and benzoic acid were determined using the Beer-Lambert law:, where A was
the absorbance measured, was the molar absorptivity and b was the path length which is usually 1 cm in most
cases. In this experiment, the molar absorptivity was determined using the linear plot of absorbance against
concentration (absorbance α concentration), where the slope of the graph was equivalent to molar absorptivity,.
Molar absorptivity is defined as the measurement of how strongly a chemical species absorb light at a given
wavelength and it is known to depend on chemical composition and chemical structure of material, but is
independent on concentration (P. Atkin and J. de Paula, 2002). Molar absorptivity is an important property
which allows us to estimate the molar concentration of solution from its measured absorbance. The SI units for
molar absorptivity are M-1 cm-1.

Data/Results [20]
Provide the plotted UV spectra for the analyte and samples.

0.18 0.6
0.16
0.5
0.14
0.12 0.4
Absorbance

0.1
Absorbance

0.3
0.08
0.06 0.2
0.04
0.1
0.02
0 0
-0.02 0 100 200 300 400 0 100 200 300 400
-0.1
-0.04 Wavelength (nm)
Wavelength (nm)

Figure 1: The UV spectrum of 2.00 ppm benzoic Figure 2: The UV spectrum of 4.00 ppm benzoic
acid prepared. acid prepared.

0.9
1.2
0.8
1
0.7
0.6 0.8
Absorbance

Absorbance

0.5
0.6
0.4
0.3 0.4

0.2 0.2
0.1
0
0 0 100 200 300 400
0 100 200 300 400 -0.2
-0.1
Wavelength (nm)
Wavelength (nm)

Figure 3: The UV spectrum of 6.00 ppm benzoic Figure 4: The UV spectrum of 8.00 ppm benzoic
acid prepared. acid prepared.
 0.7
0.7
0.6
0.6
0.5 0.5
Absorbance

Absorbance
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
0 100 200 300 400 0 100 200 300 400
Wavelength (nm) Wavelength (nm)

Figure 5: The UV spectrum of 10.0 ppm benzoic Figure 6: The UV spectrum of 4.0 ppm caffeine prepared.
acid prepared.

1.2 2

1
1.5
Absorbance
Asorbance

0.8
0.6 1

0.4
0.5
0.2
0 0
0 100 200 300 400 0 100 200 300 400
Wavelength (nm) Wavelength (nm)

Figure 7: The UV spectrum of 8.00 ppm caffeine prepared. Figure 8: The UV spectrum of 12.0 ppm caffeine prepared

2.5 3

2.5
2
Absorbance

2
Absorbance

1.5
1.5
1
1
0.5 0.5

0 0
0 100 200 300 400 0 100 200 300 400
Wavelength (nm) Wavelength (nm)

Figure 9: The UV spectrum of 16.0 ppm caffeine prepared. Figure 10: The UV spectrum of 20.0 ppm caffeine prepared.
 1.8
1.6
1.4
1.2
Absorbance

1
0.8
0.6
0.4
0.2
0
0 50 100 150 200 250 300 350 400
Wavelength (nm)

Figure 11: The UV spectrum of 2.00 mL soft drink diluted to 50.0 Ml.

What are the λmax for each of the components of your sample?
 The maximum wavelength for benzoic acid is approximately ≈ 230 nm.
 The maximum wavelength for caffeine is approximately ≈ 275 nm.
 The maximum wavelength for soft drink is approximately ≈ 229 nm.

Plot the 4 calibration curves for caffeine and benzoic acid from the standards you have
prepared.

Table 1: Concentrations of benzoic acid standards and their corresponding absorbance at a given wavelength.
Concentration (mg L-1) Absorbance
λmax = 230 nm λmax = 275nm
2.00 0.1396 0.0047
4.00 0.3994 0.0313
6.00 0.5462 0.0433
8.00 0.7106 0.0583
10.0 0.4411 0.0532
 0.8
0.07
0.7
0.06
0.6 y = 0.0457x + 0.1731
ABSORBANCE

R² = 0.4745 0.05

ABSORBANCE
0.5
0.04
0.4
0.3 0.03
y = 0.0062x + 0.001
0.2 0.02 R² = 0.8426
0.1 0.01
0 0
0 5 10 15 0 5 10 15
CONCENTRATION (MG L-1 ) CONCENTRATION (MG L-1)

Figure 12: The curve showing concentration versus Figure 13: The curve showing concentration versus
absorbance for benzoic acid at λmax = 230 nm. absorbance for benzoic acid at λmax = 275 nm.

Table 2: Concentrations of caffeine standards and their corresponding absorbance at a given wavelength.
Concentration (mg L-1) Absorbance
λmax = 230 nm λmax = 275nm
4.00 0.1309 0.2231
8.00 0.2269 0.4047
12.0 0.3329 0.6171
16.0 0.4040 0.8290
20.0 0.5359 1.0262

0.6
1.2
0.5
y = 0.0247x + 0.03 1
0.4 R² = 0.9935
y = 0.0508x + 0.0109
ASORBANCE

0.8
ABSORBANCE

R² = 0.9993
0.3
0.6
0.2 0.4

0.1 0.2

0 0
0 5 10 15 20 25 0 5 10 15 20 25
CONCENTRATION (MG L-1) CONCENTRATION (MG L-1)

Figure 14: The curve showing concentration versus Figure 15: The curve showing concentration versus
absorbance for caffeine at λmax = 230 nm. absorbance for caffeine at λmax = 275 nm.
Showing ALL your calculations, calculate the concentration of caffeine and benzoic acid in
your sample of Mountain Dew. Express your values in mg L-1.
Table 3: The absorbance readings of soft drink at λmax = 230 nm and λmax = 275 nm
Wavelength (nm) Absorbance Molar absorptivity Molar absorptivity
of benzoic acid of caffeine
(M-1 cm-1) (M-1 cm-1)
230 0.6843 0.0457 0.0247
275 0.3180 0.0062 0.0508

Absorbance is additive
Atotal = A1 + A2
Atotal = 𝜀 1bc1 + 𝜀 2bc2
NB: 1 = Benzoic acid
2 = Caffeine
Assuming that path length, b = 1
A230nm = A1 + A2
0.6843 = 0.0457C1 + 0.0247C2....(1)
A275nm = A1 + A2
0.3180 = 0.0062C1 + 0.0508C2...(2)
From equation (1): C1 = 1.497 - 0.5405C2...(3)
Substitute (3) into (2)
0.3180 = 0.0062(1.497 - 0.5405C2) + 0.0508C2
0.3180 = 0.0092814 - 0.0033511C2 + 0.0508C2
0.30872 = 0.04745C2
C2 for caffeine /mg L-1 = 6.506 mg L-1
Substitute C2 on equation (3)
C1 Benzoic acid /mg L-1 = 1.497 - 0.5405(6.506 mg L-1) = 2.019 mg L-1

Data Analysis [15]

Explain why HCl was added to your samples and standards.
 The 10 mL of 0.010 M was added to samples and standards in order to maintain the protonated form of
the compounds.
State the wavelengths chosen for the analysis and provide reasons for your answer.
 The wavelengths chosen for this analysis were 200 nm and 350 nm. These wavelengths were chosen
because the spectrum of this range presents more than one (2 or 3) absorption peaks with λmax,1 = 230
nm and λmax,2 = 275 nm.
  Caffeine and benzoic acid absorb UV light at many of the same wavelengths and the UV-vis absorption
spectrum of caffeine and benzoic acid are usually found in the region of 230 nm – 275 nm because they
have the common chromophore, C = O. The chromophore (C = O) for both benzoic acid and caffeine is
associated with the n → π⃰ electronic transition.

Why are caffeine and benzoic acid UV active?

 According to the molecular orbital theory, benzoic acid and caffeine are aromatic compounds with
conjugated systems (i.e. alternated single and double bonds) as part of their structure. They are both UV
active because they poses π → π⃰, which is the lowest energy electronic transition in molecules with an
aromatic ring and is assigned to the great transition in the UV, thus, creating a broad band because
vibrational and rotational energies cause the energies of the molecular orbitals to change slightly.

Explain why, in this case, the total absorbance of the solution is additive.
 Absorbance is additive because the soft drink solution is the sample, containing more than one kind of
absorbing substance. It contains caffeine and benzoic acid. Furthermore, Beer –Lambert law can be
applied to determine the concentrations of the absorbing substances using the simultaneous equation and
inserting the values of measured absorbance and molar absorptivity at two different wavelengths.

Compare your results, using the relevant stats, to typical values for Mountain Dew. Comment
on the similarities and differences to your data.
Basic stats of absorbance for benzoic acid at 230 nm.
Mean = 0.447
Standard deviation = 0.210
Related standard deviation (%RSD) = (Standard deviation) / (Mean) x 100
= 0.210/0.447 X 100
= 46.98%
Basic stats of absorbance for benzoic acid at 275nm.
Mean = 0.0382
Standard deviation = 0.0214
Related standard deviation (%RSD) = (Standard deviation) / (Mean) x 100
= 0.0214/0.0382 x 100
= 56.02%

Basic stats of absorbance for caffeine at 230 nm

Mean = 0.326
Standard deviation = 0.157
Related standard deviation (%RSD) = (Standard deviation) / (Mean) x 100
Related standard deviation (%RSD) = 0.157/0.326 x 100 = 48.16%

Basic stats of absorbance for caffeine at 275 nm

Mean = 0.620
Standard deviation = 0.321
Related standard deviation (%RSD) = (Standard deviation) / (Mean) x 100
Related standard deviation (%RSD) = 0.321/0.620 x 100 = 51.77%

The related standard deviation (%RSD) was used to compare precision of the experimental results. The values
of %RSD range from 45% - 57% and they indicated that the quality of results were not satisfying. According to
the nutritional information on the 335 ml can of Mountain Dew, the content of caffeine is 152 mg L-1, but in
this experiment a 6.506 mg L-1 of caffeine was found. This suggest that the precision was poor indicated by
high values of (%RSD) which were due to errors during the analysis.

NB: The mean and standard deviation values were calculated using the basic stats procedure on casio
calculator model fx-82ZA PLUS.

Identify and discuss potential sources of error in the analysis.

 The sources of errors for this experiment were as follows:
1. Error could have occurred during sample preparation. For example, adding too much/little of the
reagents required to make the solutions.
2. Instrumental error: this error occurs due to the misuse of the instruments and loading effect (D.
Amposah, 2014). For example, if a good instrument is used in an unintelligent manner then it results in
the misuse of the instruments, producing poor results.

References

 D. Amposah (2014). Determination of the amount of caffeine and benzoic acid in selected soft drinks.
International Journal of Scientific and Engineering Research. Volume 5 (6), pp 1375 – 1378.
 N. Meya (2012). Spectrophotometric Analysis of Caffeine and Benzoic Acid in Soft Drink. University
of Tunku Abdul Rahman, Faculty of Science.
 P. Atkins and J. de Paula (2002) Physical Chemistry. 7th Edition. Chapter 13: Molecular Spectroscopy,
pp 330 – 450.
 S.A. Bhawani, S.S. Fong and M.N.M. Ibrahim (2015). Spectroscopic Analysis of Caffeine.
International Journal of Analytical Chemistry. Volume 2015 (2015), pp 1 – 7.