CHM171L Physical Chemistry 2 Laboratory

2nd Quarter, S.Y. 2017-2018

Spectrophotometry
Legaspi, Ranaye V., Lojica, Kate M., Maquiling, Kenth Roger A.1, Calderon, Edna J.2
1Students, 2Professor, Physical Chemistry 2 Laboratory, CHM171L B21, School of Chemical Engineering and Chemistry, Mapúa University,
Muralla St., Intramuros, Manila 1002, Philippines
ABSTRACT
Final Report Information
When light passes through a compound, energy from the light is used to
promote an electron from a bonding or non-bonding orbital into one of
Performed 11 November 2017 the empty anti-bonding orbitals. These energies are associated with
Submitted 25 November 2017
different wavelengths and when certain amount of energy is enough for
an electron to make an energy jump, the corresponding wavelength is
Keywords:
then absorbed. Spectrophotometry is a method of measuring how much
a chemical substance absorbs light by measuring the intensity of light as
Light Waves it passes through a sample solution. Because every chemical compound
Wavelength absorbs and transmits light over a certain range of wavelength,
Absorbance spectrophotometry is widely used in the quantitative analysis of various
Transmittance compounds. In the experiment, a Perkin Elmer Lambda 40
Spectrophotometry Spectrophotometer was used to determine how Transmittance and
Perkin Elmer Lambda 40 Absorbance are related to the wavelength of light. Spectrophotometry
Spectrophotometer was also used to determine the concentration of an unknown by plotting
Beer's Law the absorbance and concentration of various Cobalt (II) Nitrate solutions
Beer-Lambert Plot in a Beer-Lambert Plot. Finding shows that the sample solution has a
wavelength of 510 nm and the unknown with an absorbance of 0.107
has a concentration of 0.0142 M.

INTRODUCTION absorbs certain wavelengths of visible light and

transmits or reflects others.
Each compound exhibits features unique to it and
differentiates it among others. These properties
may include melting, boiling, freezing, ignition
points, solubility and even its color. The
characteristic color of a compound differs from
the other mentioned properties, in that it depends
not on the intermolecular interactions in the said
compound, but in its structure. Furthermore, it is
attributed to the absorbance and reflection of Figure 1. Wavelengths of what we perceive as
particular colors in the visible portion of the spectrum.
light at particular part of the spectrum. Certain
examples would be the yellow color of quinine, The whole molecule does not need to responsible for
green for chlorophyll, and bright yellow to deep the absorption of a particular wavelength of light, in
fact, the bonds and functional groups that give rise to
red color of 2,4-dinitrophenylhydrazine
the absorption of ultraviolet and visible radiation are
derivatives of aldehydes and ketones. Particular called chromophores. Thus, chromophores can be
colors of compounds arise when a molecule treated as well to be responsible for the characteristic
color of a compound (McNaught, 1997).

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Chromophores consist of a conjugated pi-bond
system wherein the energy difference between two
different molecular orbitals falls within the range of the
visible spectrum. As light hits a molecule, this energy
is absorbed by the chromophore resulting in electron
excitation from its ground state, the highest occupied
molecular orbital (HOMO) into an excited state, the
lowest unoccupied molecular orbital (LUMO).
Moreover, the structural information surrounding the
pi electrons such as positions, number, and other
atoms affect the amount of energy the compound
requires for electronic transitions –term for HOMO to
LUMO transitions (Shapley, 2012). This phenomenon
is also applicable to transition metal ions, as they can
be colored due to the fact that d-electrons within the
atoms may be excited from one electronic state to
another.
Figure 2. Electronic transitions involving n, σ, and π
molecular orbitals.
UV/Vis spectrophotometry in particular, a molecular
absorption spectroscopy, is one of the most
frequently encountered quantitative analytical method.
It has been popularly used in the determination of an
analyte’s concentration, which is based on the
analyte’s absorption of ultraviolet or visible radiation
(Harvey, 2000). This technique employs the principle
of Beer-Lambert Law which states that the quantity of
light absorbed by a particular analyte is directly
proportional to its concentration and the path length of
light through the solution, mathematically expressed
as:
𝐴 = 𝜀𝑏𝐶
where ε is the analyte’s molar absorptivity, b is the
path length, and C the analyte’s concentration.
Furthermore Beer’s law applies as well to samples
containing several absorbing components provided
Experiment 4 | Group 6 | November 25, 2017
that there are no interaction between the components.
Additionally, Beer’s law has its limitations as well, in
theory, a calibration curve of absorbance versus the
concentration of analyte in a series of standard
solutions should be a straight line with an intercept of
0 and slope of εb. In various cases however,
calibration curves are found to be nonlinear which is
attributed to fundamental, chemical and instrumental
limitations (Harvey, 2000).
Figure 3. Generalized diagram of a spectrophotometer.
Generalized diagram showing how
spectrophotometer works is depicted on figure 3.
Light from a light source passes through a
wavelength selector, either a filter or monochromator,
with the selected wavelength of light eventually be
projected through the cuvette containing either blank
or the sample for single beam or both for the double
team. The light that would be passing through would
then reach the detector which converts the light signal
into absorbance as output.
The present study revolves on the use of
spectrophotometric method, UV/vis spectroscopy, to
quantify an unknown concentration of cobalt (II)
nitrate. Consequently, the experiment tackled on the
determination of the wavelength at which maximum
absorption occurs, and the subsequent generation of
a standard curve of absorption vs concentration, unto
which the unknown concentration shall be calculated.
EXPERIMENTAL SECTION
a.) Preparation of Reagents
Visible spectrum of a Cobalt (II) Nitrate
solution was examined in the experiment. This
solution is red in color which turns into pink upon
dilution. Six (6) solutions of Cobalt (II) Nitrate with
concentrations ranging from 0.005 to 0.05 M
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were prepared by diluting 1M of Cobalt (II) Nitrate lengths and a six cuvettes rack to absorb light in
in a 25 mL volumetric flask. The following the sample. A lamp emits intense UV radiation
amounts were used: which can damage eyes, hence the students
shall not gaze into the lighted lamp while doing
Concentration Volume of 1M Volume of
the experiment.
of Co(NO3)2 Cobalt (II) Water (mL)
Solution (M) Nitrate (mL) c.) Methodology
0.05 1.25 23.75
The experiment was divided into three
0.04 1.0 24.0
parts: the determination of the color transmitted
0.03 0.75 24.25 of visible light with different wavelengths, the
0.02 0.50 24.50 determination of the absorbance of the visible
0.01 0.25 24.75 spectra of a solution with different wavelengths,
0.005 0.125 24.875 and the determination of the absorbance of the
visible spectra of a solution of different
b.) Apparatus Used concentration. For the convenience of the
A spectrophotometer is an instrument students, only the last part of the experiment was
used for measuring the transmittance or conducted and the values for the first and second
absorbance of solutions, and transparent or parts were obtained from existing literature.
opaque solids. In the experiment, a Perkin Elmer The examination of the visible spectrum
Lambda 40 Spectrophotometer was used in the of a solution can be done using the
determination of the absorbance of the samples, spectrophotometer in a few minutes and brief
connected to it is a monitor that displays the procedures, however the handling of the cuvette
results. is a critical part and any scratches or stains in the
cuvette will yield different results. Proper and
careful handling of the cuvette is also necessary
since the equipment is quite expensive.
To start the experiment, the
spectrophotometer was first calibrated using
distilled water. The liquid samples were
transferred in the cuvette which will be placed in
the cuvette rack inside the spectrophotometer. It
is important not to hold the cuvette in its lower
portion since this is where the light will pass
through. The clear sides of the cuvette were also
positioned sideward with the index mark facing
the front of the instrument. In examining the
Figure 4. Perkin Elmer Lambda 40 Spectrophotometer
solutions, the wavelength of the
The Perkin Elmer Lambda 40 spectrophotometer was set to 200-875 nm range
Spectrophotometer has a wide range of wave and the concentrations of the solutions were

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entered in the computer. The Cobalt (II) Nitrate B. Visible spectrum of a solution
solutions were analyzed one by one after the Table 2. Absorbance readings of 0.05 M cobalt (II)
computer program started, and the cuvette was nitrate solution at 375 nm to 800 nm.
washed with distilled water after every trial and
Wave Wave
then wiped with clean tissue.
Length Absorbance Length Absorbance
d.) Treatment of Results (nm) (nm)
375 1.3997 610 1.1086
Two graphs were plotted in the experiment, an 400 1.19 620 1.1038
410 1.1872 630 1.1002
Absorbance vs. Wavelength graph and a Beer’s
420 1.2275 640 1.0968
Law Calibration Graph. 430 1.31 650 1.0918
440 1.4385 660 1.0854
To determine the concentration of the unknown 450 1.6284 670 1.076
compound in the last part of the experiment, the 460 1.8093 680 1.064
gathered data from the absorbance reading of 470 1.9346 690 1.046
standard cobalt (II) nitrate solutions of different 480 2.0375 700 1.0302
490 2.1246 710 1.0183
concentrations were plotted in a Beer’s Law Plot. 500 2.2488 720 1.0132
Using the least square method, the relationship 510 2.3188 730 1.0147
between the concentration and the absorbance 520 2.2706 740 1.0197
of the solution was expressed using the linear 530 2.1026 750 1.0228
540 1.8574 760 1.0242
equation 550 1.6101 770 1.023
560 1.3936 780 1.0185
A = 2.9578c + 0.0649
570 1.25 790 1.0116
The concentration c of the unknown was 580 1.1668 800 1.0052
590 1.133
determined by substituting its corresponding
600 1.1185
absorbance A into the equation.
C. Beer’s Law Plot
RESULTS
Table 3. Absorbance readings at 510 nm of cobalt (II)
A. Visible light nitrate standard solutions and the unknown.

Table 1. Corresponding color at varying wavelengths. Concentration, M Absorbance

0.05 0.210
Wavelength Color 0.04 0.183
350 Ultraviolet 0.03 0.159
400 Violet 0.02 0.123
450 Blue 0.01 0.098
500 Cyan 0.005 0.075
550 Green Unknown 0.107
600 Yellow Concentration of
0.0142
650 Red Unknown

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Figure 5. Absorbance vs. Wavelength Graph Color is a property of light that depends on the
frequency of light waves and is determined by its
wavelength. Visible spectrum is the observable
region of the electromagnetic wave which is
visible to human eyes. In the electromagnetic
spectrum, the visible spectrum ranges from the
infrared region to the UV region. We can detect
the range of light spectrum from about 400
nanometers (violet) to about 700 nanometers
(red) and after this range, the human eye is
unable to see the rest of the electromagnetic
waves.
Figure 6. Cobalt (II) nitrate solution standard curve.
When visible light strikes an object at a certain
Standard Curve wavelength becomes absorbed, that frequency of
light will never make it to our eyes. However, the
0.25
visible light that will be reflected or transmitted to
0.2
our eyes contributes to the color appearance of
Absorbance

0.15
the object. The light wave transmitted by an
0.1 y = 2.9578x + 0.0649 absorbed light wave appears as its
0.05 R² = 0.9946
complimentary color.
0
0 0.01 0.02 0.03 0.04 0.05 0.06 Table 4. Corresponding light wave appearance for
Concentration, M each absorbed light wave at a certain wavelength

Complimentary
 Region Color
Color
DISCUSSION 400-435 Violet Yellow-green
435-480 Blue Yellow
a. Legaspi, Ranaye V.
480-490 Blue-green Orange
490-500 Green-blue Red
Light is a form of an electromagnetic radiation, a
500-560 Green Purple
form of energy carried by waves. Although the 560-580 Yellow-green Violet
term is often referred to as visible light, the term 580-595 Yellow Blue
actually refers to a vast range of energy waves
which includes gamma rays, X rays, ultraviolet Chemical compounds also undergo the same
rays, visible light, infrared radiation, microwaves, phenomena, and the color appearance of each
radar, and radio waves. Each electromagnetic compound depends on the wavelength of the
radiation has different wavelengths, and the light energies necessary to promote the electrons
retina of our eyes can only detect incident light in their excited stage. Considering that the 0.5 M
that falls in waves 400 to 720 nanometers long. cobalt (II) nitrate solution used in the experiment
Because of this, only the visible light among all appears pinkish red, one can assume that the
the electromagnetic radiation forms can be seen color absorbed by the compound is greenish-blue.
by the human eyes. The absorbance readings of the solution at 375

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nm to 800 nm using the Perkin Elmer Lambda 40
Spectrophotometer verifies that this assumption
is correct by plotting an Absorbance vs.
Wavelength Graph, as shown in figure 5. The
wavelength of the maximum absorbance (peak)
is the characteristic value, which is 510 nm.
Table 1 shows that this wavelength gives a color
in between cyan and green, which is the
complimentary color of a pinkish red.
Spectrophotometry can be also used in
determining the concentration of an unknown
compound. In the experiment, diluted solutions of
1M of cobalt (II) nitrate were prepared and
analyzed in the spectrophotometer. One
observation in this part is that as more water is
added to the solution, the lighter the color of the
solution becomes. This can be explained by the
Beer’s Law, which states that absorbance and
concentration are directly proportional, hence
lower concentration leads to lower absorbance.
This is because a more concentrated solution
has a greater amount of molecules that can block
the light as it pass through, causing a darker
solution.
The Beer-Lambert Law provides a linear
relationship between the absorbance and
concentration. Plotting the obtained absorbance
with their corresponding concentration shows a
linear graph with an r2 of 0.9946 indicating a very
high linearity. Using linear regression, the
relationship of the two quantities was
mathematically expressed as
A = 2.9578c + 0.0649
Substituting the obtained absorbance 0.107 gives
the concentration of the unknown, 0.0142 M.
Experiment 4 | Group 6 | November 25, 2017
b. Lojica, Kate Ann M.
The apparatus that was mainly used in this
experiment was a spectrophotometer and below
is a diagram of how it works. See Figure 7.
Figure 7. Diagram of How Spectrophotometer
Works
A light source is passed through a collimator in
order to produce a beam of parallel rays then
these rays will be transmitted to the
monochromator. The purpose of a
monochromator is to isolate a thin part of the
spectrum. After these, a wavelength slit is
introduced to separate the desired wavelength
from the undesired ones.
The first part of the experiment deals with visible
spectrum of a solution. Before using the standard
solution for UV VIS spectrophotometer, a blank
solution, which is a distilled water, was used and
its absorbance were determined. This blank
solution does not contain any chemical
substance or the analyte. There is a need to first
determine the absorbance of the blank solution
since the absorbance of the species other than
the analyte adds to the total absorbance that will
be measured by the spectrophotometer. The
absorbance of the blank solution shall be
subtracted from the absorbance of the analyte,
which in this case is cobalt (II) nitrate. The device
that was used automatically subtracts the
absorbance of the blank solution to the standard
solution. Subsequently, the absorbance. The
third part of the experiment deals with the
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determination of the unknown concentration of
cobalt (II) nitrate and this was done by using the
Beer’s Law plot. Beer’s Law states that the
absorbance and concentration are directly
proportional with each other. It only means that
darker solutions have higher concentrations and
thus, also higher in absorbance. On the other
hand, light colored solutions have lower analyte
concentrations and lower absorbance value.
Calibration curve method was used in
determining the unknown concentration of cobalt
(II) nitrate. The said method uses a standard with
known amount of samples. An equation can be
generated by linear regression, and the equation
was
𝐴 = 2.9578𝑐 + 0.0649
Main source of error may be due to inaccurate
measurements when preparing solutions of
cobalt (II) nitrate in different concentrations.
c. Maquiling, Kenth Roger A.
The study commenced by scanning for the
wavelength that would give optimum absorbance
readings because this shall be deemed essential for
more accurate results in the determination of the
concentration of the unknown sample solution of
Co(NO3)2. A standard solution of 0.05 M of Co(NO3)2
had its absorbance obtained with varying wavelength,
and a graph was plotted to relate the two quantities
aforementioned. An initial observation would be that
the solution was pinkish red, and an initial hypothesis
is that it would absorb the wavelength of its
complementary color, which is green. Figure 5 shows
the graph obtained relating the absorbance and the
wavelength of the standard solution. As seen, the
optimum absorbance is obtained at a wavelength of
approximately 510 nanometers, which corresponds to
the bluish-green color of light in the electromagnetic
spectrum, thereby proving the initial hypothesis to be
correct. The following figure shows the color wheel,
which shows the complementary colors being
opposite to one another.
Experiment 4 | Group 6 | November 25, 2017
Figure 8. The color wheel, and the complementary
colors.
This phenomenon implies that the solution will absorb
the most Ultraviolet-Visible radiation when the
wavelength of the light that is allowed to strike the
solution is at 510 nanometers (wavelength of green to
blue green light). Therefore, this wavelength is the
optimum wavelength, and was the same wavelength
used in the analysis of the concentration of the
unknown solution. Standard solutions of various
concentrations were prepared and were subjected to
Ultraviolet-Visible light to obtain their absorbance
readings at 510 nanometers. Table 3 shows the data
obtained for the absorbance readings of the standard
solutions.
As one will notice from the trend of the data, there is a
directly proportional relationship with the
concentration and the absorbance of the solution.
Assuming that the solution fully obeys Beer’s Law,
one shall be able to construct a linear relationship
relating the two quantities, and from that relationship,
the concentration of the unknown solution may be
obtained after its absorbance is read. This method is
called the standard calibration method, wherein one
prepares a calibration curve, which is a plot of the
signal (which, in this case, is absorbance) against the
concentrations of prepared standard solutions. From
Beer’s Law,
𝐴 = 𝜀𝑏𝑐
and b, which represents the path length of the cell
used, is kept constant at 1 centimeter. Thus, the
equation is left with
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𝐴 = 𝜀𝑐
showing a linear relationship between the absorbance
and the concentration, with ε, otherwise known as the
molar absorptivity of the solution, is the constant of
proportionality. However, linear plots usually are in
the form of
𝑦 = 𝑚𝑥 + 𝑏
so a correction factor is added to the relationship, but
still maintaining linearity. The equation then becomes
𝐴 = 𝜀𝑐 + 𝑘
and after obtaining the values of the slope and the y-
intercept, the absorbance of the unknown will be read
and then substituted to the linear relationship, readily
yielding a value for the concentration. Linear
regression of the shown data will give a slope of
2.9578 and a y-intercept of 0.0649, therefore giving a
linear relationship of
𝐴 = 2.9578𝑐 + 0.0649
with a correlation constant of 0.9946, indicating a
good linearity. Figure 5 shows the Beer’s Law plot
generated.
Obtaining the absorbance of the unknown
solution yielded a value of 0.107, and substituting it to
the generated linear equation will give a concentration
of 0.0142 M.
CONCLUSIONS AND RECOMMENDATIONS
The objectives of the experiment were achieved.
The laboratory students were able to determine
the maximum absorbance for the ion occurs.
They were also able to determine the
concentration of the unknown sample using the
experimental values for each concentration and
by plotting it to get the linear equation.
Since absorption varies linearly to the
concentration, the property of the absorbing
species, and path length, experiments must be
performed cautiously to prevent any accuracy
and changes in the path length of light during the
experiment or interferences that is caused by
Experiment 4 | Group 6 | November 25, 2017
some other absorbing species in the solution.
Having the same cuvette used for the experiment
makes the path length of light held constant. The
cuvettes provided have standard, reproducible
diameters and volumes.
The possible source of error came from
impurities in the solution. Impurities can affect the
transmittance of light. Another possible error is
the inaccurate dilution of the solutions.
Recommendation for the experiment includes
calibration of the spectrometer. The solvent,
impurities in the solvent, and/or the cuvette glass
can absorb light in the selected region. These
interferences can be corrected or eliminated by
calibrating the spectrometer with the blank in the
sample compartment. In effect, this act instructs
the instrument to ignore any absorbance from
materials in the glass or solvent and to detect
only the absorbance from the particular species
to be measured in the sample.
REFERENCES
McNaught A. and Wilkinson A (1997). IUPAC.
Compendium of Chemical Terminology, 2nd ed.
(the "Gold Book"). Blackwell Scientific
Publications, Oxford.
Shapley, P. (2012) Absorbing light with organic
molecules.http://butane.chem.uiuc.edu/pshapley/
GenChem2/B2/1.html
Harvey, D. (2000). Modern analytical chemistry.
McGraw Hill Companies.
APPENDIX
1. Summary of Results
1.1 Determination of the wavelength of the light
wave absorbed by cobalt (II) nitrate solution
Wave Wave
Length Absorbance Length Absorbance
(nm) (nm)
375 1.3997 610 1.1086
400 1.19 620 1.1038
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410 1.1872 630 1.1002 0.03 0.159

420 1.2275 640 1.0968 0.02 0.123
430 1.31 650 1.0918 0.01 0.098
440 1.4385 660 1.0854 0.005 0.075
450 1.6284 670 1.076 Unknown 0.107
460 1.8093 680 1.064 Concentration of
470 1.9346 690 1.046 0.0142
Unknown
480 2.0375 700 1.0302
490 2.1246 710 1.0183
500 2.2488 720 1.0132 Standard Curve
510 2.3188 730 1.0147
0.25
520 2.2706 740 1.0197
530 2.1026 750 1.0228 0.2

Absorbance
540 1.8574 760 1.0242 0.15
550 1.6101 770 1.023
0.1 y = 2.9578x + 0.0649
560 1.3936 780 1.0185
570 1.25 790 1.0116 0.05 R² = 0.9946
580 1.1668 800 1.0052 0
590 1.133 0 0.01 0.02 0.03 0.04 0.05 0.06
600 1.1185 Concentration, M

Wavelength Color 2. Sample Computation

350 Ultraviolet 2.1 Dilution of 1 M cobalt (II) nitrate
400 Violet
𝑀1 𝑉1= 𝑀2 𝑉2
450 Blue
500 Cyan (1)𝑉1= (0.05)(25)
550 Green
600 Yellow (0.05)(25)
𝑉1= = 𝟏. 𝟐𝟓 𝐦𝐋 𝐂𝐨(𝐍𝐎𝟑 )𝟐
650 Red 1
2.2 Concentration of unknown

A = 2.9578c + 0.0649
0.107 = 2.9578𝑐 + 0.0649
𝒄 = 𝟎. 𝟎𝟏𝟒𝟐 𝑴

1.2 Determination of the concentration of

unknown
Concentration, M Absorbance
0.05 0.210
0.04 0.183

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