Experiment 5: Introduction to Ultraviolet-Visible Spectrophotometry

Experiment No. 5: INTRODUCTION TO ULTRAVIOLET-VISIBLE SPECTROPHOTOMETRY
I. INTRODUCTION
Spectroscopy is the study of matter’s light absorption and emission. Light is electromagnetic radiation
ranging from Radio waves, Microwaves, Infrared (IR), Visible light, Ultraviolet (UV) light, X-rays, and Gamma rays
in a spectrum known as the Electromagnetic (EM) spectrum [1] shown in Figure 1.
Figure 1. Electromagnetic Spectrum (EM)
A spectrophotometer is a photometer that directly measures transmitted light intensity at a specific
wavelength. It has two main classifications, single beam and double beam spectrophotometers. Its essential
features are linear absorption range and spectral bandwidth. The incident light passes through the blank sample
or analyte in a single beam spectrophotometer. In contrast, incident light separates into two, towards the blank
sample and analyte, in the double beam spectrophotometer.
Other Spectrophotometer types are Visible Light (400-700 nm), UV, and IR. A Visible Light
Spectrophotometer’s light sources are incandescent, halogen, LED, or combination. This type varies in accuracy.
Its sample holders are either plastic or glass cuvettes. Contrary to Visible Light Spectrophotometer, A UV
Spectrophotometer’s sample holders are quartz cuvettes. IR Spectrometers analyze molecules’ chemical
structures and vibrations as the two are dependent on each other due to different energies at different
wavelengths. Mid-range and near IR elicit rotational and harmonic vibrations, respectively.
University of Santo Tomas
Faculty of Engineering
Department of Chemical Engineering
CHE 216L:
PHYSICAL CHEMISTRY FOR
ENGINEERS 1 LABORATORY
Individual Written Report
Name: AGAWIN, AARON D.
Date Performed: May 5, 2021
Date Submitted: May 26, 2021
Section: 2ChE-C Group No: 1 Instructor: Ma’am Rose Mardie Pacia

AGAWIN, AARON D. 2
Experiment 5: Introduction to Ultraviolet-Visible Spectrophotometry
Beer-Lambert’s Law states the linear relationship between Absorbance (A) and Concentration (C). This
linear relationship occurs at dilute solutions as physiochemical interactions between the solute particles increases
when increasing Concentration. Spectrophotometers require Absorbance values below 1 for the Absorbance-
Concentration linear relationship to occur. Most spectrophotometers precisely measure Absorbance values
between 0.1 and 1. Hence, Absorbance values higher than one or too high concentrations result in curved
Absorbance-Concentration graphs [2].
The Beer-Lambert Law assumes two external scenarios, (1) Absorbance is directly proportional to
Concentration, shown in EQ 1, and (2) Absorbance is directly proportional to the light path’s length (ℓ) [3], shown
in EQ 2. If the cuvette is a rectangular prism, the light path’s length is the rectangular prism’s width.
A ∝ C (EQ 1)
A ∝ ℓ (EQ 2)
Combining these two proportionality relations leads to EQ 3.
A ∝ Cℓ (EQ 3)
Expressing the relation EQ 3 into an equation EQ 4 results to a proportionality constant Molar Absorptivity
(ε). It is a substance’s ability to attenuate specific wavelengths. Attenuation is the Absorbance of light through a
solution. Similar to Absorbance, it is wavelength (λ) dependent. Additionally, it is unique to each substance.
Hence, it identifies an unknown sample’s identity.
A = εCℓ (EQ 4)
Where:
A = Diluted Solution’s Absorbance
C = Diluted Solution’s Molar Concentration
ℓ = Light Path’s Length or Cuvette width
ε = Solute’s Molar Absorptivity, Molar Extinction Coefficient, or Molar Attenuation Coefficient
Since the spectrophotometer converts the transmitted light intensity (It) into Voltage (ΔV) then into an
Absorbance reading, Absorbance is related to the incandescent light intensity (Ii) and transmitted light intensity.
This relation’s equation is shown in EQ 5.
A = log (
Ii
It
)
(EQ 5)
Where:
Ii = Incident Light Intensity
It = Transmitted Light Intensity
Another property is Transmission or Transmissivity (T), the ratio between transmitted light intensity and
incandescent light intensity, as shown in EQ 6. It physically defines the ratio of incident light that becomes
transmitted light from passing through the sample and cuvette [4].

AGAWIN, AARON D. 3
T =
Ii
It
(EQ 6)
Where:
T = Incident Light’s Transmission or Transmissivity
Single-Beam Spectrophotometer consists of a Light source, Collimator (Lens), Monochromator (Prism or
Grating), Wavelength Selector (Slit or Adjustable Aperture), Sample Holder (Cuvette), Detector
(Photocell/Photoresistor), Digital Display or Meter [5].
Figure 2. Single-Beam Spectrophotometer Parts
The collimator bends the divergent incident light into collimated light with parallel light rays and no
parallax. Parallax is the angle between divergent light rays.
Figure 3. Collimator Mechanism; Collimated Light (Left), Divergent Light (Right)
The Monochromator refracts the collimated light into divergent light with separate wavelengths. The
wavelength selector passes through wavelength bands or single wavelengths through a slit [6]. The number of
wavelengths passing through is adjustable with the Aperture. In contrast, the wavelengths passing through are
adjustable with the Monochromator’s rotation as it changes the wavelengths’ direction.

AGAWIN, AARON D. 4
The Detector converts the transmitted light intensity into the circuit’s voltage change. Hence, it essentially
transforms the light signal into an electrical signal, which the software interprets. Finally, the Digital Display or
Meter shows the Absorbance reading.
Absorbance, Light Path’s Length, and Molar Extinction Coefficient are knowable with the Beer-Lambert’s
Law in a known diluted solution. However, determining an unknown diluted solution’s Concentration requires a
calibration curve. It is a method of determining unknown concentrations by plotting a linear graph of known
concentrations and the knowable parameter in the known and unknown concentrations, then express a
relationship between the unknown Concentration and its known parameter with the linear graph [7]. The unknown
diluted solution’s expected Concentration should be within the linear graph’s concentration values to calculate
the most accurate approximation possible.
Since the calibration curves require different known concentrations, serial dilution is applicable. It
sequentially dilutes an aliquot of the stock solution into decreasing concentrations. However, the sequentially
diluted aliquot is constant in volume. Hence, the Concentration of the succeeding diluted solutions is knowable
with a factor known as the dilution factor [8].
fd =
Vi,aliquot
Vi+1,solution
(EQ 7)
Where:
fd = dilution factor
Vi, aliquot = Previous Diluted Solution’s Aliquot
Vi+1, solution = Blank Solution Added with Previous Diluted Solution’s Aliquot
This dilution factor relates to the stock solution’s Concentration that is useful for calculating the
Concentration of the successive serial dilutions as shown in EQ 8.
CDS,n = fd
n
CSS (EQ 8)
Where:
n = Order of Serial Dilution
CDS,n = nth Serial Diluted Solution’s Concentration
CSS = Stock Solution’s Concentration
Different substances have different absorption maxima (λmax) as the distance between their orbitals tells
the maximum energy they absorb during excitation. This energy corresponds to the λmax as a higher energy
difference yields lower λmax [9]. The Molecular Orbital Theory best describes organic compounds’ λmax. These
conjugated π bonds decrease the energy gap between the Highest Occupied Molecular Orbital (HOMO) and the
Lowest Unoccupied Molecular Orbital (LUMO) by increasing the number of π electrons available for occupying
Molecular Orbitals [10]. The number of π electrons and non-bonding electrons available for electronic transitions
depend on the extent of conjugation and the number of free electrons. The transitions that a π electron and non-
bonding electron undergo during excitation is π-π* transition and n-π* transition, respectively [11].

AGAWIN, AARON D. 5
Figure 4. Transitions of non-bonding, π, and σ electrons (Left); π-π* transition (Right)
Crystal Field Theory best describes complex ions’ λmax. Most complex ions are Transition metals bonded to
ligands. This ligand bonding changes a transition metal’s electron configuration. Since transition metals are d-
block elements, changes in electron configurations occur at the d orbitals. When bonding with ligands, the
degenerate d-orbitals separate to two higher energy d-orbitals (eg), dz
2 and dx
2
-y
2, and three lower energy d-orbitals
(t2g) - dxy, dxz, and dyz [12}.
Figure 5. d-orbitals of Metal cation (Left), Spherical uniformly distributed negative charge around Metal cation
(Middle), Octahedral vertex distribution of negative charge around Metal cation (Right)
The repulsive energy between the electrons varies in the different d-orbitals. The dz
2 orbital’s electrons repel
localized electrons along the octahedral ligand field’s z-axis. Meanwhile, the dx
2
-y
2 repels the localized electrons
on coordinating ligands’ x and y axes. The t2g orbitals’ do not repel the octahedral ligand field’s localized electrons
as those orbitals are between the x, y, and z axes and do not intersect them. Hence, they have lower energy than
the eg orbitals [13].

AGAWIN, AARON D. 6
Figure 6. d-orbitals around coordination ligand fields
The energy associated with the distance between eg and t2g is the Crystal Splitting Energy. It is also the
energy that an electron from the t2g absorbs to excite towards e2. Note that the orbitals’ Crystal Splitting Energy
depends on the central atom’s shape. Its shape dictates where its electron orbitals are along the x, y, and z axes.
These positions tell where electron repulsion occurs [14].
The ligands also contribute to varying Crystal Splitting Energies as complex ions with the same metal ion.
However, different ligands have different Crystal Splitting Energies. The Spectrochemical Series ranks their
contribution to Crystal Splitting Energies as their contribution is independent of the metal ion [15].
Figure 7. Spectrochemical series
Different experiments were performed with different aims:
• To gather Absorbance versus Wavelength graphs of six different solutions
• To determine Cobalt (II) Chloride’s Molar Absorption Coefficient
• To calculate six other Cobalt (II) Chloride unknown concentrations with Beer-Lambert’s Law
• To tabulate Cobalt (II) Chloride’s Absorbance versus Concentration
• To determine unknown concentrations of Blue Dye #1 POWERADE Mountain Berry Blast and Red Dye #40
Gatorade Tropical Fruit with a DIY Spectrophotometer
II. METHODOLOGY
A. AMRITA WEB-BASED SPECTROPHOTOMETRY
Solution Scanning and Molar Absorption Coefficient Determination
https://vlab.amrita.edu/?sub=2&brch=190&sim=338&cnt=4 was visited and logged in with Google account.
Cobalt (II) Chloride solution was chosen and the Concentration was set to 0.1 M. A blank cuvette was selected,
placed in a spectrophotometer, and the lid was closed. 0 ABS 100%T button was clicked, after which A reads to
0.00000 A. The lid was opened and the blank cuvette was removed. The known Concentration was selected,
placed in a spectrophotometer, and the lid was closed. The Absorbance versus Wavelength was measured
between 350-700 nm by clicking the up and down arrows. The Absorbance versus Wavelength was recorded and

AGAWIN, AARON D. 7
plotted in MS Excel. The procedure was repeated with the Absorbance versus Wavelength of the other five
solutions. The wavelengths with maximum Absorbance were recorded for each solution. The Molar Absorption
Coefficient of the Cobalt (II) Chloride solution was calculated at the wavelength with maximum Absorbance – 510
nm.
Figure 8. Amrita Web-based Spectrophotometer Set-up
Unknown Concentration Determination with Beer-Lambert’s Equation
The wavelength was set to 510 nm. The cuvette with the unknown concentration Cobalt (II) Chloride
solution was placed. The Absorbance for this wavelength was read. The Concentration was calculated. The
calculated concentration value was entered in the given box in four decimal places. The same procedure was
repeated for a second solution.
Absorbance Versus Concentration at Different Concentrations
The procedure was repeated but at 0.05, 0.025, 0.013, 0.007, and 0.003 M concentrations of Cobalt (II)
Chloride solution. Each Concentration’s Absorbance was measured at 510 nm.

AGAWIN, AARON D. 8
B. DIY SPECTROPHOTOMETER
Preparation of Samples and DIY Spectrophotometer
The light source is a screen where Red-Orange (628 nm) and Light Green (510 nm) Virtual Background
from https://academo.org/demos/wavelength-to-colour-relationship/ was set up. A circle was cut on the top of a
cardboard box for placing the sample holder. A small rectangle was cut on the front side for the Detector
(Smartphone Camera). The back was cut for the light source. The DIY Spectrophotometer was taped for a stable
set-up. The Real-time RGB value detector Application Colorimeter RGB (for Apple Devices) was downloaded.
Stock solutions of Blue Dye #1 POWERADE Mountain Berry Blast and Red Dye #40 Gatorade Tropical Fruit were
prepared.
Figure 9. DIY Spectrophotometer Set-up; Blue Dye #1 (BD#1) POWERADE Mountain Berry Blast (Left), Red
Dye #40 (RD#40) Gatorade Tropical Fruit (Right)
Absorbance Versus Concentration
Five (4 diluted solutions) of each stock solution were prepared by serial dilution with Absolute Distilled
Drinking Water as the solvent. The concentrations were halved by adding 85 mL of the previous diluted solution
to the next 85 mL initially solute-free solution. Another diluted solution was prepared from the stock and was
labeled as unknown. However, its Concentration was noted as it was compared with the Concentration calculated
from the data analysis (linearization). Three trials of each five diluted solutions with Blue Dye #1 POWERADE
Mountain Berry Blast and Red Dye #40 Gatorade Tropical Fruit separately were prepared for the subsequent
runs. Another blank plastic cup with only 170 mL of Absolute Distilled Drinking Water was also prepared. The
corresponding Absorbance (R, G, or B) of the blank, stock, the four diluted solutions, and the unknown was read
for each trial and dye.

AGAWIN, AARON D. 9
Figure 10. DIY Spectrophotometer Completed Trials; Blue Dye #1 (BD#1) POWERADE Mountain Berry Blast
(Left), Red Dye #40 (RD#40) Gatorade Tropical Fruit (Right)
III. RESULTS AND DISCUSSION
Figure 11. Summary of 0.1M Solutions’ Wavelength vs Absorbance
The maximum Absorbance for Cobalt (II) Chloride, Hexaaqua Cobalt (II) ion, Ferrocene, Crystal Violet,
Rose Bengal, and Coumarin 0.1M solutions are 16.6100, 10.1200, 30.2318, 25.6898, 23.8214, and 30.0836 A,
respectively. The wavelengths corresponding to these maximum Absorbances were 510 (Green), 510 (Green),
445 (Bright Blue), 590 (Yellow), 560 (Yellow-Green), and 445 (Bright Blue) nm, respectively. Meanwhile, the
-45.7204
-36.2264
-26.7324
-17.2383
-7.7443
1.7497
11.2438
20.7378
30.2318
350 400 450 500 550 600 650 700
Absorbance
[A
(A)]
Wavelength [λ (nm)]
Cobalt (II) Chloride Hexaaqua Cobalt (II) ion Ferrocene
Crystal Violet Rose Bengal Coumarin

AGAWIN, AARON D. 10
complementary colors of these maximum Absorbance wavelengths are Pink, Pink, Yellow-Orange, Bright Blue,
Purple, and Yellow-Orange, respectively.
In aqueous solutions, Cobalt (II) Chloride forms complexes with water. It dissolves to its constituent ions
in aqueous solutions and form Hexaaquacobalt (II) complex (Co(H2O)6
2+) and Pentaaquachlorochromium(II)
chloride ([Co(H2O)5Cl]+) [16]. The complementary color of the 0.1 M Cobalt (II) Chloride’s λmax is closest to pink,
confirming that the solution consists of the two complex ions. As Cobalt (II) Chloride forms complex ions in
aqueous solutions, Crystal Field Theory best describes the potential energy difference between the orbitals of
Cobalt chloride hexahydrate and Pentaaquachlorochromium(II) chloride.
Hexaaquacobalt (II) complex is a Cobalt monoatomic di-cation (Co2+) that forms octahedral ligand fields
with water molecules. Relative to complex ions, Hexaaquacobalt (II) complex has a low crystal field splitting
energy. It is a high-spin complex ion from complexing with water molecules that are weak field ligands [17]. Its
crystal field splitting energy is 232.076 kJ/mol in most available literature and 234.562 kJ/mol in this experiment
[18].
The Pentaaquachlorochromium(II) chloride’s crystal field splitting energy was lower than the Cobalt
chloride hexahydrate. This is because the single Chloride ligand reduces the crystal field splitting energy as
Chloride ions are weaker field ligands than water [19]. However, there are more water ligands in
Pentaaquachlorochromium(II) chloride, and the solution consists of both Hexaaquacobalt (II) complex and
Pentaaquachlorochromium(II) chloride. Hence, the 0.1 M Cobalt (II) Chloride’s absorption spectrum is not affected
significantly by the other complex ion Pentaaquachlorochromium(II) chloride.
Figure 12. Structures of Pentaaquachlorochromium(II) (Left) chloride and Hexaaqua Cobalt (II) ion (Middle);
Electronic transition of Cobalt (II) ion
Ferrocene’s λmax is relatively low compared to the other solutions as it has few conjugated π bonds with
six π bonds, totaling to twelve π electrons [20]. These contribute to π-π* transitions wherein the twelve π electrons
occupy the molecular orbitals. Since there are no free electrons in Ferrocene, there are no n-π* transitions that
further decrease the potential energy change and increase the λmax.

AGAWIN, AARON D. 11
Figure 13. Ferrocene’s Structure
Similar to Ferrocene, Coumarin has a relatively low λmax that is also due to the few conjugated π bonds
that total to five π bonds and ten π electrons [21]. These ten π electrons contribute π-π* transitions wherein they
occupy the molecular orbitals. Even with fewer π electrons for π-π* transitions, Coumarin has the same λmax as
Ferrocene. Its λmax substantially increases to Ferrocene’s as its two oxygen atoms with four lone pairs contribute
n-π* transitions [22].
Figure 14. Coumarin’s Structure
Rose Bengal is tied with Crystal Violet with the relatively most extensive conjugation, contributing to π-
π* transitions. It has ten conjugated π bonds, totaling to twenty π electrons that occupy the molecular orbitals.
Additionally, it has numerous non-bonding electrons across the Chlorine, Oxygen, and Iodine atoms, having more
n- π* transitions than Crystal Violet [23]. However, even with the most extensive conjugation and n- π* transitions,
Rose Bengal does not have the longest λmax. This phenomenon is due to steric hindrance from the bulky groups
on either side of the double bonds. These prevent charge transfer between atoms, lowering conjugation and λmax
[24].
Figure 15. Rose Bengal’s Structure

AGAWIN, AARON D. 12
Crystal Violet has the highest λmax as it has a relatively high extent of conjugation with ten π bonds,
totaling to twenty π electrons available for occupying Molecular orbitals [25]. However, besides conjugation, two
non-bonding lone pairs of electrons from the Nitrogen atoms contribute another electronic transition, namely n-
π* transition [26]. This transition has higher energy than the highest energy bonding p-orbital. This state leads to
n- π* transition’s narrower energy gap than π-π* transition, hence n- π* transition lengthens the λmax more than
π-π* transition [27]. Thus, these two transitions, π-π* and n- π* contribute to Crystal Violet’s highest λmax relative
to the other solutions.
Figure 16. Crystal Violet’s Structure
Table 1. Gathered Data from Web-based Simulation
Cuvette Length (cm) Maximum Absorbance
Wavelength (nm)
Calculated Molar Absorption
Coefficient (A⋅M-1
⋅cm-1
)
1 510 166.1
Table 2. Gathered Data from Determination of Unknown Concentrations of Cobalt (II) Chloride
Known Concentration
(M)
Known
Concentration’s
Maximum
Absorbance (A)
Unknown
Concentration’s
Maximum
Absorbance (A)
Calculated
Concentration (M)
0.1 16.6100 2.5081 0.0151
0.05 8.3050 7.9562 0.0479
0.025 4.1525 6.2786 0.0378
0.013 2.1593 16.0951 0.0969
0.007 1.1627 1.0132 0.0061
0.003 0.4983 8.6870 0.0523
Table 2 shows that decreasing the known Concentration decreases the known Concentration’s maximum
Absorbance as the number of analyte molecules, ions, or atoms decreases, passing more light through the
solution. This phenomenon is foreseeable as Absorbance depends on Concentration, Cuvette Length, Molar

AGAWIN, AARON D. 13
Absorptivity, Solvent, Temperature. As mentioned above, Molar Absorptivity depends on the analyte. This
experiment is set to Cobalt (II) Chloride Solution, 510 nm Wavelength, and Cuvette length of 1 cm. Hence, Cuvette
length and Molar Absorptivity are constant, and only Concentration affects Absorbance.
Figure 17 shows the Blue Dye #1 POWERADE Mountain Berry Blast’s Concentration vs. Absorbance
Graph. As shown, the graph is not linear as the DIY spectrophotometer’s set-up is not ideal. Positive and negative
deviations from the regression line occur at different regions of the Absorption versus Concentration Curves.
Figure 17 shows negative deviations at 0.0000, 0.2688, and 2.1500 µM and shows positive deviations at 0.5375
and 1.0750 µM. Meanwhile, Figure 18 shows negative deviations at 3.7770 and 7.5541 µM and shows positive
deviations at 0.0000, 1.8885, and 15.1082 µM. Ideally, the graph is entirely linear as the Beer-Lambert Law
assumes a linear relationship between Absorbance and Concentration at λmax [28].
The sample holder’s shape is essential as any variations in path length change the measured Absorbance
[29]. The increasing diameter from the bottom to the top cylindrical shape of the cuvette replacement contributes
to pathlength variation. In addition, changes in the Detector or phone camera position and cup position change
the path length corresponding with the phone camera. The centermost of the plastic cup from the lateral view has
the most extended path length as it has the longest diameter. In contrast, the shortest path length is the leftmost
or rightmost from the lateral view.
Since the DIY spectrophotometer is not entirely enclosed and does not have a monochromator, multiple
wavelengths can interfere with the wavelength shown on the screen. Although a monochromator is not absolutely
ideal as it still passes a band instead of a single wavelength of light [30], the DIY spectrophotometer set-up has
multiple light sources for them to interfere with the chosen wavelengths [31].
Multiple reflections from the plastic cup might have contributed to errors because instead of light
transmitting through the cup, the light reflects off the plastic cup and is not absorbed by the analyte. Additionally,
reflections from the screen can contribute to unwanted light bands being detected by the phone camera [32].

AGAWIN, AARON D. 14
Figure 17. Blue Dye #1 POWERADE Mountain Berry Blast’s Concentration vs Absorbance
Figure 18. Red Dye #40 Gatorade Tropical Fruit’s Concentration vs Absorbance
y = 0.0354x + 0.0048
R² = 0.9556
0
0.0188824
0.0377649
0.0566473
0.0755298
0 0.5375 1.075 1.6125 2.15
Absorbance
[A
(A)]
Concentration [C (µM)]
y = 0.0068x - 0.0089
R² = 0.9551
0
0.0250896
0.0501793
0.0752689
0.1003586
0 3.7770436 7.5540873 11.331131 15.108175
Absorbance
[A
(A)]
Concentration [C (µM)]

AGAWIN, AARON D. 15
Table 3. Results of the DIY Spectrophotometer
Sample
Theoretical
Concentration (µM)
Calculated
Concentration (µM)
Percent Error (%)
Blue Dye #1
POWERADE Mountain
Berry Blast’s
0.1344 0.0273 -79.6588
Red Dye #40 Gatorade
Tropical Fruit
0.9443 1.5729 66.5767
This experiment’s decreasing order of the parameters’ error contribution is Path-length, Multiple
reflections, and Stray radiation. Several literatures shows that the three can contribute to -10 to +10 A (parts per
thousand), +0.5 to +3 A (parts per thousand), 0 to -1 A (parts per thousand) deviations from the actual Absorbance
[33].
Blue dye #1 has higher molar absorptivity than red dye #40, with the former having a molar absorptivity
of 130,000 L·cm-1·mol-1 while the latter is 25,900 L·cm-1·mol-1 [34]. This difference in Molar Absorptivity Constant
significantly increases the Blue Dye #1’s susceptibility to change its Absorbance with different path lengths.
Decreasing path length decreases the Absorbance as they are directly proportional to each other. Similarly,
decreasing Absorbance decreases Concentration. Since the Absorbance values in this experiment correspond
to apparent Absorbance and not True Absorbance, the concentration corresponding to the former is the calculated
Concentration, whereas the latter is theoretical Concentration.
IV. ANSWERS TO QUESTIONS
1. What is the significance of the λmax of a spectrum?
The λmax of a spectrum is the wavelength at which the substance has the highest Absorbance [35]. This
parameter is unique to different substances. Different functional groups and groups of atoms or molecules
contribute different Absorbance across the EM spectrum, which affects the substance’s λmax [36]. It also tells the
absorbed energy to induce electronic transition that a substance undergoes under electromagnetic radiation.
Other wavelengths have a chance of exciting the electrons, but the λmax is the wavelength that best excites the
electron [37]. Moreover, λmax tells a substance’s different parameters, such as the extent of conjugation, the
number of free electrons [11], oxidation states [38], geometry [39], and magnetic [40] properties.
2. How did you decide on the wavelength at which you will read the Absorbance of the diluted solutions? What
is the importance of reading the Absorbance of diluted solutions at λmax? What will vary if you chose a
wavelength 20 nm lower or higher than your chosen wavelength?

AGAWIN, AARON D. 16
The wavelength at maximum Absorbance is the wavelength to read diluted solutions’ absorbances.
Knowing beforehand the λmax and taking stepwise measurements of Absorbance at increasing wavelengths is
best to determine the λmax of the analyte solution [41].
Reading the diluted solution’s Absorbance at λmax prevents unwanted complications in the
spectrophotometric analysis, increases the experiment’s reproducibility, and decreases percent error.
Complications in the spectrophotometric analysis decrease as most monochromators transit a narrow wavelength
band. There is minimal difference in Absorbance around λmax and a pronounced difference around the sloping
region.
It increases reproducibility as minor errors that slightly change the wavelength do not significantly affect
Absorbance. It occurs as the λmax region has a minimal difference in Absorbance. The percent error decreases
as larger Absorbances generally have more minor percent errors than smaller Absorbances as precision errors
become more minimal with increasing values [42].
Choosing a wavelength 20 nm higher or lower than the λmax decreases the Absorbance reading.
Depending on how narrow the Wavelength versus Absorbance curve is around λmax, the Absorbance versus
Concentration trend might deviate significantly from λmax’s Absorbance versus Concentration trend as the
spectrophotometer loses sensitivity at lower Absorbances.
3. Why do you have to use the same cuvette after doing the baseline correction?
The baseline correction subtracts the Absorbance that instrument noise and solvent’s light-scattering
particles induce around the absorption spectrum. This method ensures that the spectrophotometer only reads the
Absorbance of the analyte solute and not the solvent, other light-scattering particles, nor the instrument noise.
Changing the blank cuvette after the baseline correction makes the baseline correction inconsistent as the
spectrophotometer calibrates different concentrations and identities of light-scattering particles from the solvent
[43]. Similarly, different cuvettes have different compositions that are transparent to specific bands of the EM
spectrum [44]. Using the same blank cuvette ensures that the spectrophotometer reads the Absorbance from the
specified spectral bands.
4. What are the other uses of the spectrophotometer?
Spectrophotometers have uses beyond measuring incident light intensity and estimating dye
concentrations. They have broad applications across numerous industries because of their different types. UV-
Visible spectrophotometers can measure nucleic acid and protein concentrations and bacterial cell densities,
which are applications for Biochemistry and Microbiology [45]. IR spectrophotometers analyze a substance’s
identity and structure [46]. Fluorescence spectrophotometers analyze the amount and identity of fluorescent dyes
and luminescent materials and analyze carbon nanotube’s structure [47]. Atomic Absorption spectrophotometers

AGAWIN, AARON D. 17
determine metal and electrolyte amounts in tissue samples, measure heavy metal content in water, and for
geological analysis such as measuring cadmium and lead content in the lake and river sediments [48]. More
examples of spectrophotometer’s uses include the following [49]:
i. Identify impurities
ii. Identify protein’s characteristics
iii. Quantify dissolved oxygen content
iv. Analyze respiratory gas in hospitals
v. Functional group detection
vi. Determine molecular weight
vii. Identify compound classes
V. CONCLUSION
The Absorbance versus Wavelength graphs of the six different solutions showed no discrepancies in the
relationship between their molecular structure and λmax. The extent of conjugation and the number of free
electrons are directly proportional to λmax for organic compounds. Meanwhile, there are no significant observable
trends in the spectrochemical series of ligands’ field strengths and λmax as there are only two complex ion samples
in the Amrita Web-based Spectrophotometry with near field strengths.
Cobalt (II) Chloride’s calculated unknown concentrations agree with the Beer-Lambert Law as the
calculated Concentration from the calibration curve and the actual Concentration are equal. Similarly, Cobalt (II)
Chloride’s Absorbance versus Concentration follows the trend predicted by the Beer-Lambert Law as decreased
Concentration and decreased Absorbance is observable.
The calculated and theoretical concentrations of Blue Dye #1 POWERADE Mountain Berry Blast and Red
Dye #40 Gatorade Tropical Fruit are not equal, suggesting discrepancies in the experiment. Since the set-up is a
DIY Spectrophotometer, there are likely multiple systematic errors. Varying path lengths, multiple reflections, and
stray radiation might have contributed to the discrepancies. Additionally, the Colorimeter RGB Application for
Apple Devices is not stable in its RBG Value detection. The Red and Green values can be spread out to as high
as 20 units.
It is recommendable to use rectangular prism plastic cups with smooth surfaces to ensure even path
lengths. The environment should also be dim to remove unwanted reflections from the plastic cups and the laptop
screen. Lastly, external light sources should be minimum to ensure that only the light emitted by the laptop screen
transmits into the solution.

AGAWIN, AARON D. 18
VI. REFERENCES
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Visible%20Molecular%20Absorption%20spectrophotometry.pdf.
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37. Ashenhurst, J. (2020, January 25). What is UV-Vis Spectroscopy? And How Does It Apply To
Conjugation? Master Organic Chemistry.
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38. Awan, A., Truong, H., & Lancashire, R. J. (2021, May 7). Crystal Field Theory. Chemistry
LibreTexts.
Chemistry)/Crystal_Field_Theory/Crystal_Field_Theory.
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Complexes. JoVE. https://www.jove.com/science-education/11462/crystal-field-theory-tetrahedral-
and-square-planar-complexes.
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pplemental_Modules_(Physical_and_Theoretical_Chemistry)/Physical_Properties_of_Matter/Atomi
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for Analytical Chemistry Instruction. https://terpconnect.umd.edu/~toh/models/BeersLaw.html.
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43. DeNovix. (2018, October 29). Baseline Correction: Technical Note 119. DeNovix.
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44. Raja, P. M. V., & Barron, A. R. (2021, March 21). 4.4: UV-Visible Spectroscopy. Chemistry
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Visible_Spectroscopy.
45. Schmid, F. X. (2001). Biological macromolecules: UV‐visible spectrophotometry. e LS.
46. Osibanjo, R., Curtis, R., & Lai, Z. (2020, August 15). Infrared Spectroscopy. Chemistry
LibreTexts.
pplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Vibrational_Spectrosc
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47. Wilkinson, J. (2017, May 18). Applications of Advanced Fluorescence Spectroscopy. AZoM.com.
https://www.azom.com/article.aspx?ArticleID=13958.
48. Raja, P. M. V., & Barron, A. R. (2021, March 21). 1.4: Introduction to Atomic Absorption
Spectroscopy. Chemistry LibreTexts.
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Book%3A_Physical_Methods_in_Ch
emistry_and_Nano_Science_(Barron)/01%3A_Elemental_Analysis/1.04%3A_Introduction_to_Ato
mic_Absorption_Spectroscopy.

AGAWIN, AARON D. 22
49. Ramzy. (2021, March 28). 5 Main Types of Spectrophotometers + Application. linquip.
https://www.linquip.com/blog/types-of-spectrophotometers/.
50. Saddleback College. (n.d.). EXPERIMENT 8. Determination of Red Dye #40 in Fruit Punch Gatorade
INTRODUCTION. PDF Free Download. http://docplayer.net/82916540-Experiment-8-
determination-of-red-dye-40-in-fruit-punch-gatorade-introduction.html.

AGAWIN, AARON D. 23
APPENDIX
A. DIY Spectrophotometer Dilution Calculations (Alternative Method for Dilution Calculations)
CDye,DSf
= (
CiVi
VDSf
)
Dye
(EQ 9)
Where:
CDye,DSf
= Final diluted solution’s Concentration
VDye,DSf
= Final diluted solution’s volume
Ci = Initial solution’s concentration
Vi = Initial solution’s volume
BD#1 = Blue Dye #1
RD#40 = Red Dye #40
SS = Stock solution
DS1 = First diluted solution
DS2 = Second diluted solution
DS3 = Third diluted solution
DS4 = Fourth diluted solution
UDS = Unknown diluted solution
a. Blue Dye #1 POWERADE Mountain Berry Blast
CBD#1,DS1 = (
CSSVSS
VDS1
)
BD#1
CBD#1,DS1 =
(4.3μM)(0.085L)
2(0.085L)
CBD#1,DS1 = 2.15μM
CBD#1,DS2 = (
CDS1VDS1
VDS2
)
BD#1
CBD#1,DS2 =
(2.15μM)(0.085L)
2(0.085L)
CBD#1,DS2 = 1.075μM
CBD#1,DS3 = (
CDS2VDS2
VDS3
)
BD#1
CBD#1,DS3 =
(1.075μM)(0.085L)
2(0.085L)
CBD#1,DS3 = 0.5375μM
CBD#1,DS4 = (
CDS3VDS3
VDS4
)
BD#1
CBD#1,DS4 =
(0.5375μM)(0.085L)
2(0.085L)
CBD#1,DS4 = 0.26875μM
CBD#1,UDS = (
CDS4VDS4
VUDS
)
BD#1
CBD#1,UDS =
(0.26875μM)(0.085L)
2(0.085L)
CBD#1,UDS = 0.134375μM
b. Red Dye #40 Gatorade Tropical Fruit
CRD#40,Gatorade Tropical Fruit =
1.5 ∙ 10−2
g
L
[50]
CRD#40,SS = (
1.5 ∙ 10−2
g
L
) (
1mol
496.42g
) (
106
μmol
1mol
) CRD#40,DS1 = (
CSSVSS
VDS1
)
RD#40

AGAWIN, AARON D. 24
CRD#40,SS = 30.22μM
CRD#40,DS1 =
(30.22μM)(0.085L)
2(0.085L)
CRD#40,DS1 = 15.11μM
CRD#40,DS2 = (
CDS1VDS1
VDS2
)
RD#40
CRD#40,DS2 =
(15.11μM)(0.085L)
2(0.085L)
CRD#40,DS2 = 7.55μM
CRD#40,DS3 = (
CDS2VDS2
VDS3
)
RD#40
CRD#40,DS3 =
(7.55μM)(0.085L)
2(0.085L)
CRD#40,DS3 = 3.78μM
CRD#40,DS4 = (
CDS3VDS3
VDS4
)
RD#40
CRD#40,DS4 =
(3.78μM)(0.085L)
2(0.085)L
CRD#40,DS4 = 1.89μM
CRD#40,UDS = (
CDS4VDS4
VUDS
)
RD#40
CRD#40,UDS =
(1.89μM)(0.085L)
2(0.085L)
CRD#40,UDS = 0.94μM
B. DIY Spectrophotometer Collected Data
Table 4. Collected Data on Blue Dye #1 POWERADE Mountain Berry Blast’s DIY Spectrophotometer
Solution Concentration (µM) R value Absorbance (A)
Trial 1 Trial 2 Trial 3 Mean
Blank 0 255 252 251 252.67 0
DS4 0.27 247 245 247 246.33 0.011
DS3 0.54 242 237 232 237 0.028
DS2 1.08 224 224 224 224 0.052
DS1 2.15 210 220 207 212.33 0.076
SS 4.3 125 100 128 117.67 0.33
Theoretical UDS 0.13 252 246 250 249.33 0.0058
Calculated UDS 0.027
% Error -79.66%

AGAWIN, AARON D. 25
Table 5. Collected Data on Red Dye #40 Gatorade Tropical Fruit’s DIY Spectrophotometer
Solution Concentration (µM) G value Absorbance (A)
Trial 1 Trial 2 Trial 3 Mean
Blank 0 246 241 240 242.33 0
DS4 1.89 246 238 234 239.33 0.0054
DS3 3.78 239 235 235 236.33 0.011
DS2 7.55 228 225 225 226 0.030
DS1 15.11 200 194 183 192.33 0.10
SS 30.22 136 122 121 126.33 0.28
Theoretical UDS 0.94 248 238 238 241.33 0.0018
Calculated UDS 1.57
% Error 66.58%
C. Unknown Concentrations in Web-based Simulation
M =
AM
εℓ
(EQ 10)
Where:
A = Unknown Concentration’s Absorbance
M = Unknown Concentration
M1 =
2.5081A
(
166.100A
M ∙ cm
) (1cm)
M1 = 0.0151M
M2 =
7.9562A
(
166.100A
M ∙ cm
) (1cm)
M2 = 0.0479M
M3 =
6.2786A
(
166.100A
M ∙ cm
) (1cm)
M3 = 0.0378M
M4 =
16.0951A
(
166.100A
M ∙ cm
) (1cm)
M4 = 0.0969M
M5 =
1.0132A
(
166.100A
M ∙ cm
) (1cm)
M5 = 0.0061M
M6 =
8.6870A
(
166.100A
M ∙ cm
) (1cm)
M6 = 0.0523M
D. Blue Dye #1 POWERADE Mountain Berry Blast’s Absorbance
An alternative form of EQ 5 is shown
𝐴 = −log (
I𝑡
I𝑖
)
(EQ 11)
ADS4 = − log (
246.33
252.67
)
ADS4 = 0.01102A
ADS3 = − log (
237
252.67
)
ADS3 = 0.02780A
ADS2 = − log (
224
252.67
)
ADS2 = 0.05230A
ADS1 = − log (
212.33
252.67
)
ADS1 = 0.07553A
ASS = − log (
117.67
252.67
)
ASS = 0.3319A
AUDS = − log (
249.33
252.67
)
AUDS = 0.005768A

AGAWIN, AARON D. 26
E. Red Dye #40 Gatorade Tropical Fruit’S Absorbance
ADS4 = − log (
239.33
242.33
)
ADS4 = 0.005410A
ADS3 = − log (
236.33
242.33
)
ADS3 = 0.01089A
ADS2 = − log (
226
242.33
)
ADS2 = 0.03030A
ADS1 = − log (
192.33
242.33
)
ADS1 = 0.1004A
ASS = − log (
126.33
242.33
)
ASS = 0.2829A
AUDS = − log (
241.33
242.33
)
AUDS = 0.001796A
F. 0.1M Solutions’ Wavelength vs Absorbance
Figure 19. 0.1M Cobalt (II) Chloride Solution’s Wavelength vs Absorbance
Figure 20. 0.1M Hexaaqua Cobalt (II) Ion Solution’s Wavelength vs Absorbance
2.4600
4.2288
5.9975
7.7663
9.5350
11.3038
13.0725
14.8413
16.6100
350 400 450 500 550 600 650 700
Absorbance
[A
(A)]
0.0000
1.2650
2.5300
3.7950
5.0600
6.3250
7.5900
8.8550
10.1200
350 400 450 500 550 600 650 700
Absorbance
[A
(A)]

AGAWIN, AARON D. 27
Figure 21. 0.1M Ferrocene Solution’s Wavelength vs Absorbance
Figure 22. 0.1M Crystal Violet Solution’s Wavelength vs Absorbance
-41.6614
-32.67475
-23.6881
-14.70145
-5.7148
3.27185
12.2585
21.24515
30.2318
350 400 450 500 550 600 650 700
Absorbance
[A
(A)]
-45.7204
-36.79413
-27.86785
-18.94158
-10.0153
-1.089025
7.83725
16.763525
25.6898
350 400 450 500 550 600 650 700
Absorbance
[A
(A)]

AGAWIN, AARON D. 28
Figure 23. 0.1M Rose Bengal Solution’s Wavelength vs Absorbance
Figure 24. 0.1M Coumarin Solution’s Wavelength vs Absorbance
-45.3252
-36.68188
-28.03855
-19.39523
-10.7519
-2.108575
6.53475
15.178075
23.8214
350 400 450 500 550 600 650 700
Absorbance
[A
(A)]
-44.046
-34.7798
-25.5136
-16.2474
-6.9812
2.285
11.5512
20.8174
30.0836
350 400 450 500 550 600 650 700
Absorbance
[A
(A)]

AGAWIN, AARON D. 29
G. Calculated Concentrations with Reference Cobalt (II) Chloride Solutions at varying Concentrations
Figure 25. Calculated Concentration with Reference 0.1 M Cobalt (II) Chloride

AGAWIN, AARON D. 30

AGAWIN, AARON D. 31

AGAWIN, AARON D. 32
H. Blue Dye #1 POWERADE Mountain Berry Blast’s DIY Spectrophotometer Trials
Figure 31. Blue Dye #1 (Trial 1)
Figure 32. Blue Dye #1 (Trial 2)

AGAWIN, AARON D. 33
Figure 33. Blue Dye #1 DIY (Trial 3)
Figure 34. Complete set of trials conducted for Blue Dye #1; Trial 1 (Left column), Trial 2 (Middle column), Trial
3 (Right column)

AGAWIN, AARON D. 34
I. Red Dye #40 Gatorade Tropical Fruit’s DIY Spectrophotometer Trials
Figure 35. Red Dye #40 (Trial 1)

AGAWIN, AARON D. 35
Figure 38. Complete set of trials conducted for Redd Dye #40; Trial 1 (1st row), Trial 2 (Middle row), Trial 3
(last row).

Experiment 5: Introduction to Ultraviolet-Visible Spectrophotometry

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