AN EXPERIMENT ON PRACTICAL SPECTROPHOTOMETRY

Gyle Bennett Pambid¹, Park Sang Keon¹, Kyla Nicole Ponce¹, & Charisse Mae Pondoc¹

¹Department of Biological Sciences, University of Santo Tomas

Abstract: Spectroscopy involves many techniques as it scopes the understanding of electromagnetic

radiation. As it measures mere light quantity, the study then focuses on UV-VIS, a span of region in the
electromagnetic spectrum. The equipment used is a UV-VIS spectrophotometer and samples were
analyzed with the equipment to determine their absorbance. Results show low percent error which
means high accuracy of the equipment. Furthermore, serial dilution was conducted to attain a standard
curve, which showed a direct relationship between the absorbance and the CuCl 2 concentration.
Moreover, results from the water sampling of a given industrial company show that the Cu(II) effluent
violated the standard effluent imposed by the DENR DAO 2016-08. Despite this, possible errors are not
ruled out, which may be caused by systematic or random errors. Uncertainties are inevitable and proper
understanding and enough practice on handling the equipment may minimize said errors.

Keywords: spectrophotometer, electromagnetic radiation, uv-vis, serial dilution

INTRODUCTION This technique, which is called

Electromagnetic radiation is the waves
produced by the motion of electrically charged
particles that travel through space and different
substances. It is characterized by its frequency
(ν) or by its wavelength (λ), both of which are
related by the velocity of light © (Berkeley Lab,
n.d.). Its spectrum ranges from high-energy
cosmic rays to very-low microwaves.
Spectrophotometers use prisms, mirrors and
slits to select light of a desired wavelength. It
directs the light to the sample compartment
and a detector, which measures the intensity of
the light. The sample is placed in the path of the
light and the instrument compares the intensity
of the light going through the sample to the
intensity observed without the sample. The
result is measured either as transmittance,
which is the amount of light that passes through
the sample, or absorbance, which is the amount
of light absorbed by the sample (Bertrand, n.d.).
spectrophotometry, is widely used in different
disciplines.
Furthermore, there are various types of
spectrophotometers, but the one used in this
experiment is the UV-Visible
spectrophotometer, which is more concerned
with the reflectance of a specific spectra of a
given sample. This type of spectrophotometer
uses two kinds of lamps as a light source, a
deuterium lamp for the UV range and a
tungsten lamp for the visible range. It also uses
a monochromator to separate the wavelengths
then place the exit slit where the desired
wavelength is. It is also heavily used in
biological fields to quantify the protein
concentration in a solution or to determine the
number of bacterial cells in a culture (JoVe,
n.d.).
With this, the UV-Visible
spectrophotometer is used to analyze the given

1

sample and will be used to create a standard
curve to determine concentration of the
unknown samples and to investigate a given
problem relating to the concentration and
effluent of a certain body of water.
METHODS
This experiment uses
spectrophotometer to analyze specific samples
and calculate the concentration. The samples
used in the experiment were bromophenol blue
and CuCl2. The first experiment was with
bromophenol blue. Researchers calculated the
absorbance of 5 samples with
spectrophotometer set at 590nm. To the 5
sample cuvettes, 1.0, 1.5, 2.0, 2.5, and 3.0 µL of
bromophenol blue were added along with 1 ml
of distilled water, respectively. The samples
were analyzed from lowest to highest
concentration. Using the Beer-Lambert's Law,
when ε of bromophenol blue is 1.98 x 10 4 L ·
mol-1/cm-1, the expected absorbance
corresponding to the concentration of 5
samples and the experimental absorbance
obtained through the experiment were
graphed.
Consequently, the second experiment
was performed with the CuCl 2 solution. Six
microcentrifuge tubes were prepared for the
experiment. Each tube was labeled No. 1 to 6.
The researchers mixed 10 ml of distilled water
and 1 g of CuCl2 in an Erlenmeyer flask. 1 mL of
the well-mixed solution was transferred to a
microcentrifuge tube labeled No. 1. No. 2 to 6
tubes were filled with 0.9 mL distilled water.
From Tube No. 1, researchers transferred 0.1
mL of CuCl2 solution to tube No. 2 and vortex it.
Same process continued to the rest of the
tubes. The absorbance of these tubes was
measured with a spectrometer at 520nm and
the data was used to construct the graph.
2
Figure 1. Serial dilution
With this, the same mechanism would
be used in the analysis of the waste water
concentration (effluent) of a certain industrial
company in the Philippines upon a certain
complaint was filed regarding the alleged
violations of the said company with the
a
standard effluent mandated by the government.
FINDINGS
The experiment aims to analyze the
sample using the spectrophotometer. Figure 2
a compares the experimental absorbance and
expected absorbance of bromophenol blue.
Graph signifies that absorbance of certain
solutions can be varied with the concentration
of the solution. The absorbance was obtained
using the spectrophotometer and the expected
error was computed with the experimental
absorbance. The low percent error, obtained
from the experimental and expected
absorbance, shows accuracy of the equipment.
Figure 2. Graph for absorbance vs volume,
given the experimental absorbance and
expected absorbance
The graph shown in figure 3 shows that
concentration of solution has correlation with
absorbance. The data was obtained from the
computed values from the serial dilution, as
shown in Appendix A - table 2. The graph shows
a direct relationship between the absorbance
and CuCl2 concentration, in that, the greater the
concentration of CuCl2 in the sample, the
greater the absorbance.

Figure 3. Standard curve for absorbance vs
CuCl2 concentration (ppm)
Moreover, a given company which was
subjected for water sampling (flowchart of the
procedure shown in Appendix B) to check
whether its effluent complies with the standard
mandated by the government was measured to
be 10,500ppm which vividly shows that the
company has violated the standard effluent
amount given by the country’s Department of
Environment and Natural Resources (DENR)
which is 0.04ppm for type C river, as seen in the
DENR Administrative Order (DAO,2016).
DISCUSSION
According to Wenzel (2020), there are
three factors that directly affect the absorbance
of a certain sample which are: 1) the molar
absorptivity of a solution used as a sample for
the spectrophotometer reading; 2) path length
wherein the longer the length is, the more
molecules are in the path of the beam of
radiation which in this experiment is considered
to be at a constant of 1cm; and 3) concentration
of the sample wherein the higher the
concentration is the more radiation will be
absorbed.
As seen in Figure 2, absorbance value
varies with the concentration of the solution
used for the spectrophotometer analysis which
is due to the interaction of molecules within the
sample solution with the light ejected from the
3
instrument (Clark, 2020) which signifies that the
3.0 μL bromophenol blue diluted with 1mL of
dH₂O has a higher concentration due to a higher
measured absorbance of 1.151 (λ=590nm,
ε=1.98x10⁴L/(mol cm)) compared to other
diluted samples with lower volume of
bromophenol blue. This is due to the
logarithmic relationship between absorbance
and transmittance which denotes that only 10
percent of radiation is being absorbed forming a
negative deviation (Wenzel, 2020) and has
yielded a higher percent error of 3.93%.
Moreover, it is also observed that percent error
for the first tube, containing 1mL of both dH₂O
and bromophenol blue is relatively higher
compared to samples with higher
concentrations despite having low absorbance
of 0.394 given that the power from the
spectrophotometer is significantly similar with
the radiation reaching the detector thus the
instrument may measure incoherent noise
rather than the absorb radiation (Wenzel, 2020)
resulting to a higher percent error.
Consequently, the same concept is also
applicable with the analysis using CuCl2 (Figure
3) wherein serial dilution decreases the original
concentration of the CuCl2 solution thus
decreasing the absorbance, as well due to its
direct correlation in accordance with the Beer-
Lambert Law (A = εcl). One variable in the Beer-
Lambert Law correlates with the value of the
molar absorptivity, expressed by an epsilon (ε);
as the larger the molar absorptivity is, the more
probable the electronic transitions are (Clark,
2020). Moreover, the Beer-Lambert Law allows
researchers to measure the absorbance of a
specific sample and to deduce the
concentration of the solution from the obtained
measurement (Hardesty, & Attili, 2010).
With this, the river which is considered
to be Type C due to its vicinity wherein factory
was situated beside it has a standard effluent of
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0.04ppm (DAO, 2016) thus the measured

effluent from the water samples acquired from
the said company shows great violations given CONCLUSION
that it has higher effluent of 10,500ppm The experiment concludes how complex
compared to the standard. This effluent are spectrophotometers are, and how they are
usually diluted with fresh water to be reused as widely used in different aspects, such as on
a source of water (Wilderer, 2011) thus the high measuring how intense a light is in accordance
effluent content from the company contains to its wavelength, and from obtaining the
high biological oxygen demand (BOD) and electromagnetic energy from a sample.
chemical oxygen demand (COD) which may Moreover, absorbances are direct
depletes the oxygen concentration in the river measurements of how much light is absorbed
which is needed by the floras and faunas found by a sample. Thus, the light absorbed by the
within the said body of water (Chakraborty, sample is linear with its concentration.
Dahiya,Srivastav & Mohan, 2019) which may Possible causes of error in
lead to environmental and health risks (Shakir, measurement includes (1) systematic errors,
Zahraw & Al-Obaidy, 2017) and (2) random errors from technical use of
Furthermore, errors accumulated in the equipment. Existing experimental errors and
measurement and analysis of the bromophenol uncertainties are inevitable, and best to assure
blue and CuCl2 using the spectrophotometer much practice and understanding from using
could be due to two possible reasons: (1) the device, and in accumulating future
systematic errors which involves the calibrating measurements and analyses to minimize said
of the instrument which involves the misuse of errors.
the reagent blank, the blank is only used to zero
the spectrophotometer (Cantwel, 2019) and ACKNOWLEDGMENT
serve as the negative control for the experiment The researchers would like to extend
since it only contains distilled water (Sundquist their gratitude to the instructors of the
& Bessetti, 2005); and (2) random errors which BIO4212L course, for providing collated data for
involves using of uncleaned cuvette leading to this formal report.
sample contamination, poorly mixed sample
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 7

APPENDIX A – Data Tables

Table 1. Absorbance readings of different concentrations of bromophenol blue
Expected % Error
Experimental Absorbance (590 nm) Absorbance
Volume (E)

Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Average(A)

1.0 μL 0.392 0.403 0.386 - - 0.394 0.3698 6.54%

1.5 μL 0.499 0.598 0.588 - - 0.562 0.5546 1.33%

2.0 μL 0.699 0.712 0.784 - - 0.732 0.7390 0.94%

2.5 μL 0.954 0.899 0.898 - - 0.917 0.9233 0.68%

3.0 μL 1.023 0.999 1.430 - - 1.151 1.1075 3.93%

Table 2. Absorbance of different concentrations of CuCl2

Concentration Absorbance (520 nm) Average

Tube No.
CuCl2 (ppm) Trial 1 Trial 2 Trial 3 Absorbance

1 1.0 x 105 ppm 2.0826 2.1011 2.0671 2.0836

2 1.0 x 104 ppm 0.1998 0.2083 0.2112 0.2064

3 1.0 x 103 ppm 0.0210 0.0200 0.2080 0.083

4 1.0 x 102 ppm 0.0021 0.0019 0.0018 0.0019

5 1.0 x 10 ppm 0.0001 0.0003 0.0002 0.0002

6 1.0 ppm 0.0000 0.0001 0.0001 0.0001

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APPENDIX B – Flowchart for part C of the experiment