You are on page 1of 4

Introduction light to follow the green path.

However, if
the light hits the black section, it is blocked
Ultraviolet and visible (UV/Vis) shortly to give allowance for any current
absorption spectroscopy measures the generated by the detector in the absence of
attenuation of a beam of light when it light.
passes through or interacts with an
absorbing species. Attenuation can be
brought about by absorption, scattering,
reflection, or interference. To produce
accurate quantitative measurements, the
attenuation measured should come only
from the absorption caused by the
absorbing species (Tissue, 2012).

Figure 2. Rotating disc components. Retrieved

The sample and reference cells contain the
solution with the investigated compound
and pure solvent, respectively. Conversion
of the received light into a current is done by
the detector and finally, the chart recorder
Figure 1. Basic components of a double beam shows the output plot of absorbance against
spectrometer. Retrieved from wavelength (Clark, 2006).
spectrometer.html For light to be absorbed in the
UV/Vis spectral range, the molecule must
Illustrated in Figure 1 is a simple possess chromophores which are groups,
double beam spectrometer. A UV/Vis such as pi bonds or lone pairs, that absorb
spectrometer works in a range from about
light. The wavelength of absorption, λmax ,
190 nm (near UV) to 900 nm (very near
would depend on the presence of these
infrared) (Tissue, 2012). The light source
light-absorbing groups (Clark, 2007).
used is a combination of two – a deuterium
lamp for the UV region and a
The absorbance of light by a sample
tungsten/halogen lamp for the visible region
is directly related to the distance travelled
– which is focused onto a diffraction grating
by the light through the sample and to the
to scatter light of different wavelengths. The
concentration of the absorber according to
slit then permits light of a very small range
Beer-Lambert law, which is expressed as
of wavelengths to enter and hit the rotating
disc, which typically has three components,
namely, the transparent, mirrored, and black
A=ε ×b × c
sections (see Figure 2). If the light hits the
transparent section, it will follow the red where A is the absorbance measured at
path shown in Figure 1. On the other hand, wavelength λ , ε is the wavelength-
hitting the mirrored section will cause the dependent molar absorption coefficient,
b is the path length through the sample, particularly caffeine, and sodium benzoate
and c is the absorber concentration. which will be determined as benzoic acid
Relationships with absorbance are (BA). Caffeine is a white crystalline xanthine
observed to be linear, thus allowing alkaloid which is added to soft drinks as a
accurate concentration measurements of an flavor additive. On the other hand, BA is a
absorbing substance in a provided sample white crystalline solid and its sodium salt,
(Laqua et al., 1988). However, the Beer- benzoate, is used as a food preservative. As
Lambert law is not reliable over certain shown in Figures 3 and 4, the structures of
concentration ranges – i.e. higher caffeine and benzoic acid, respectively, are
concentration. Hence, it is more accurate to aromatic and hence they can absorb light in
measure the concentration via direct the UV/Vis region (Gami & Sarasan, 2016;
calibration method, wherein solutions of McDevitt, Rodriguez, & Williams, 1998;
known concentration of the investigated Patil, 2012).
compound are prepared and measured at
the absorption maximum. Absorbance is
plotted against concentration and from the
graph, the unknown concentration of a
sample can be determined given its
absorbance (Clark, 2007).

UV/Vis analysis usually involves a
straightforward sample preparation. For
absorbing species that can be dissolved, Figure 3. Structure of caffeine. Retrieved from
measurements are done in solution using a
suitable solvent or pH buffer. To make sure
that the absorber is in the same form both in
standard and sample solutions, pH effects
and complexation should be monitored.
Non-absorbing substances can also be
quantified by simply adding a reagent with
which it can form an absorbing complex Figure 4. Structure of benzoic acid. Retrieved
(Dean, 1995). from

In quantitative measurements, it is Hence, the specific objective of this study
necessary that only a few absorbing species was to quantify the concentration of caffeine
in a sample are present because UV/Vis and benzoic acid in 7UP via UV/Vis
spectra typically involve broad bands. absorption spectroscopy.
Samples with many absorbers are to
undergo separation first prior to quantitative
analysis. Likewise, any particles present in
A. Standard Solution Preparation
the sample should be removed via
centrifugation or filtration to prevent light
scattering which interferes with absorbance Stock solutions of BA and caffeine
measurement (Tissue, 2012). having a concentration of 100 ppm were
made. In separate 50-mL beakers, 10.0 mg
In this study, it was aimed to analyze of BA and 10.7 mg caffeine were weighed
commercial soft drink components, and dissolved in a small amount of water.
The solutions were then transferred Plots of absorbance against
quantitatively to separate 100-mL volumetric concentration of the standard solutions for
flasks and diluted to mark with dH2O. each of the two wavelengths were
constructed and using direct calibration
From the stock solutions, working method, the concentration of BA and
standard solutions were prepared in caffeine in the original soft drink was
duplicates. Five clean, 50-mL volumetric calculated.
flasks were secured for each stock solution.
By means of a pipettor, 1, 2, 3, 4, and 10 References
mL of the BA stock solution were transferred
separately to the flasks to make 2, 4, 6, 8, Clark, J. (2006). A double beam UV-visible
and 20 ppm of BA solution respectively. absorption spectrometer. Retrieved from
Similarly, 4, 8, 12, 16, and 20 ppm caffeine
solutions were made by using 2, 4, 6, 8, and ble/spectrometer.html
10 mL of the caffeine stock solution. To all
solutions, 10 mL of 0.10 M HCl was added Clark, J. (2007). Using UV-visible
after which the flasks were filled to mark absorption spectra. Retrieved from
with dH2O.
B. Sample Preparation
Clark, J. (2007). UV-visible absorption
Approximately 20 mL of 7UP was spectra. Retrieved from
placed in a beaker and subsequently
warmed on a hot plate to expel CO2. The ble/theory.html
warm sample was passed through a filter
paper to remove any present particles and Dean, J. A. (1995). Analytical chemistry
then cooled to room temperature. Using a handbook. New York: McGraw-Hill.
pipettor, 2 mL and 4 mL of the sample were
transferred to separate 100-mL volumetric Gami, R., & Sarasan, G. (2016). A
flasks. To both volumes, 10 mL of 0.10 M comparative study of concentration of
HCl was added after which the flasks were caffeine and benzoic acid in various soft
filled to mark with dH2O. drink samples. International Journal of
Science and Research, 5(8), 1655-1659.
C. Absorbance Measurements
Laqua, K., Melhuish, W. H., & Zander, M.
Absorbance measurements were (1988). Nomenclature, symbols, units, and
supposed to be done using a UV-Vis their usage in spectrochemical analysis –
spectrometer via wavelength scan of both VII. Molecular absorption spectroscopy,
standard solutions from 350 to 210 nm to ultraviolet and visible (UV/VIS). Pure
Applied Chemistry, 60, 1449-1460.
obtain the λmax for both BA and caffeine,
followed by the measurement of
McDevitt, V. L., Rodriguez, A., & Williams,
absorbance of each standard and of the two K. R. (1998). Analysis of soft drinks: UV
sample solutions at both maximum Spectrophotometry, Liquid Chromatography,
wavelengths with dH2O as the blank and capillary electrophoresis. Journal of
sample. Instead, experimental data were Chemical Education, 75(5), 625.
given by the instructor for analysis. doi:10.1021/ed075p625
Patil, P. N. (2012). Caffeine in various
samples and their analysis with HPLC - A
review. International Journal of
Pharmaceutical Sciences Review and
Research, 16(2), 76-83.

Tissue, B. M. (2012). Ultraviolet and visible
absorption spectroscopy. In E. N. Kaufmann
(Ed.), Characterization of materials. USA:
John Wiley & Sons, Inc.