CHM171L Physical Chemistry 2 Laboratory

Vol.2, Issue 1, Submitted: August 2016, Accepted: August 16, 2016

Determination of Concentration of Solution using Spectrophotometry

1

Evidente, Ralph Carlo T.,2 Florendo, Mary Florence F., 3Gapasin, Danica G., Gutierrez, Jomari M. and Marquez, Ariziel Ruth,
School of Chemical Engineering and Chemistry, Mapua Institute of Technology
1

Email: rctevidente@mymail.mapua.edu.ph 2 Email: mflorendo@mymail.mapua.edu.ph 3 Email: dgapasin@mymail.mapua.edu.ph

ABSTRACT

Spectrophotometer is a method to measure how much a chemical substance absorbs light by measuring the
intensity of light as a beam of light passes through sample solution. A spectrophotometer is employed which
measures the amount of light of a specified wavelength which passes through a medium. On the first part, the
spectrophotometer was set to a wavelength of 350-850 nm consisting of ultraviolet ray and visible light. Then, the
cuvette was rinsed thoroughly and filled with distilled water up to full. The standard solution of 0.05 M cobalt (II)
nitrate was then placed on the second cuvette and serve as the sample. The absorbance of the sample was
measured in different wavelengths and was plotted versus the absorbance. For Beers Law plot, absorbance of
different cobalt (II) nitrate concentrations starting from lower concentrations as 0.005 M, 0.01 M, 0.02 M, 0.03 M,
0.04 M, 0.05 M and unknown was measured at specific wavelength that gives the maximum absorbance based
on the wavelength vs. absorbance graph. The amount of light absorbed by the medium is proportional to the
concentration of the absorbing material or solute present. Absorbance changes with respect to concentration.
Therefore, the absorbance is directly proportional to the concentration. The highest concentration has the highest
absorbance because there are many molecules that can absorb light. Lastly, as the concentration decreases, the
molecules in the solution become lesser; hence, the absorbance diminished resulting to a lower absorbance.
Keywords: Spectrophotometer, Absorbance, Concentration, Wavelength, Faradays laws
INTRODUCTION
characteristic absorptions and may indicate their
molecular structures.
One of the important tools for chemists is the
The complexes of octahedral cobalt typically have a
interaction of light (or other electromagnetic
band between 500 to 600 nanometers. The ligand
radiation) with matter. Most of ones learning of
field theory explains the color transition of metal
chemical substances comes from their specific
complexes. Which in the case of the octahedral
absorption or emission of light.
cobalt (II) complex, an octahedral ligand splits the
five d-orbitals into two other types of orbitals. The
From the definition, spectrophotometry is the
first type of orbitals is the t2g orbitals where three of
science of molecules and the characteristics of the
the d-orbitals have lower energy. The other type of
molecules such as absorption, or emission of light,
orbitals is the eg orbitals wherein the two remaining
heat, and other electromagnetic radiation. The
orbitals are raised in energy. Octahedral ligand splits
experiment focuses on the solution of Cobalt (II)
five d-orbitals into two other types of orbitals.
Nitrate. Cobalt Nitrate commonly found as
hexahydrate (hydrate containing 6 molecules of
Spectrophotometry is known to be a quantitative
water) is the inorganic cobalt (II) salt of nitric acid.
measurement of the reflection or transmission
Once these salts were dissolved in water, the
2+
properties of a material as function of wavelength.

Electromagnetic radiation may be absorbed by

[Co ( H 2 O )6 ] ion if formed and appears to have
matter that is responsible for the spectrophotometric
a light ink in color. These colors exhibited by
methods. The scope of level to which the EM
different substances resulted from their
radiation is related to the nature of concentration of

absorbing material present in a sample is likewise

that of wavelength of the radiation employed.
Molecules and atoms can emit specific wavelengths
of light once these have absorbed specific amount of
energies. The particular colors of the visible light
involved are characteristic of the substance. The
amount of substance presence depicts how much
light can be absorbed or emitted. Thus, the
wavelengths emitted can be used to identify the
element emitting the light. The color of the
substance that can be observed by the naked eye
may vary depending on the observer, yet the
absorption of specific wavelengths depends on the
molecular structure of the substance. The intensity
of the light beam as it enters the solution is the
incident intensity, Io. The incident intensity is the
number of photons per second that enters the
sample solution. As the light beam transverses the
solution, some photons may be absorbed by the
components and the wavelength of the light.

Figure 1. Wavelength of Different Spectra of Visible

Light
As an outcome of light absorption, the beam of light
that emerges from the sample has a diminished
intensity symbolized by I. Less photons leave the
sample compared to the number of photons that
I
entered it. The ratio I o are the fraction of light
that actually passes the sample and is called the
transmittance, T.
(Equation 1)

The transmittance of a solution containing a light

absorbing material, the analyte, is related to
experimental conditions by Beers Law.
(Equation 2)
where

is called the molar absorptivity, C is the

concentration of the analyte, b is the length of the

light path through the absorbing solution.
Absorbance depends on the wavelength of light and
on the nature of the substance absorbing the light.
Absorption spectra in the ultraviolet and visible
regions are due to energy transitions of both bonding
and nonbonding outer electrons of the molecule and
usually delocalized electrons are involved.
In this experiment, spectrophotometry was done by
using Perkins Elmer Lambda 40 UV/Vis
Spectrometer. A solution of Cobalt (II) Nitrate was
prepared with different concentrations and these
solutions were used in obtaining their absorbance in
certain wavelengths. The beam of light that passed
through a solution with certain concentration and
some of the light entered were absorbed depending
on the nature of the solution and some of the light
entered were transmitted. Due to the dependence of
the absorbance in the nature of the solution,
spectrophotometry can be used for determining the
concentration of a solution. Lastly, this experiment
aims one to prepare a Beers Law Plot.
EXPERIMENTAL SECTION
The experiment we performed is about
spectrophotometer where we used spectrometer. It
is an equipment used to measure the amount of light
that the sample absorbs or absorbed. The
equipment works by passing a beam light through
the sample and measure the level of intensity of light
reaching a detector.
The figure below was an image of a spectrometer.
This was not the exact equipment that we used to

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perform the experiment because we forgot to take a

picture of it.

Figure 2. Spectrometer
The first thing we did is to perform the beers law
plot. The group prepared 0.05 M Cobalt (II) Nitrate
and dilute it to 0.04M, 0.03M, 0.02M, 0.01M, and
0.005M, and get the absorbance reading of Cobalt
(II) Nitrate solution and an unknown solution that
was given by our zealous and one of the best lab
assistants in this generation.
The second part was to determine the absorbance
reading of a standard solution of Cobalt (II) Nitrate.
Then the lab assistant discussed to the group on
how to use or handle the cuvette properly and to
avoid breaking it. Hence, we wash the cuvette 3
times and make sure to hold it on the blurred part
only because the cuvette may get stain from the
fingers and it may affect the results of the
experiment. Then afterwards we carefully dried it
with a soft tissue to make sure that it is really not wet
or moist. The next thing we do is to transfer the
standard solution to it and carefully put it to the
spectrometer. The cuvette should not have any
bubbles inside that may affect the results. Then
finally, we obtained the absorbance reading through
a monitor and adjusting it to get the absorbance in a
specific wavelength.
The last part which is supposed to be the first part of
this experiment was to determine the color of the
beam in a given amount of wavelength and observe
it from the instrument but our professor told us to
research it because it takes time to perform it.

Experiment 04 Group No. 3 Date Aug. 16, 2016

Figure 3. Washing the cuvette 3 times and carefully

handling it in the blurred part of the cuvette only.

Figure 4. The results in part 2 where we obtain the

absorbance value with a given value of wavelength.
Also, we do adjusting the wavelength to gather the
absorbance value of a standard solution of Cobalt
(II) Nitrate.

Figure

5.

Transferring the diluted solution from the test tube to

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the cuvette and drying the outside part with a soft

tissue.

spectrophotometer is that it allows the light source

shine through a monochromator. An output
wavelength is selected and beamed at the sample.
Finally, a fraction of the monochromatic light is
transmitted through the sample and to the photodetector.
In the experiment, the color of the specific wavelengths
is determined. The absorbance of the analyte is also
determined with varying wavelengths and concentrations.

Figure 6.
Absorbance value for each diluted salt solution and
the unknown.

The color of the specific wavelength in a visible light that

are researched is shown below.
Table 1. Wavelength (nm)
WAVELENGTH
350
400
450
500
550
600
650

Figure
7. Washing
the cuvette 3 times and drying it with a soft tissue.
RESULTS AND DISCUSSION
A spectrophotometer is a photometer that can
measure the light intensity as a function of its
wavelength. Two major classes of the latter are
Single beam and double beam. Linear range of
absorption and spectral bandwidth measurement are
the important features of spectrophotometers.

Figure

8.

The

principle

behind

Experiment 04 Group No. 3 Date Aug. 16, 2016

simple

COLOR
Violet
Blue
Green
Green
Orange
Red

Before using the UV VIS spectrophotometer, the

absorbance of a blank solution must be supplied
first. The blank solution is where a solution does not
contain the analyte yet. Since our solution is Cobalt
Nitrate in water, the distilled water was used for the
blank solution. The total absorbance is contributed
by the absorbance of the cobalt nitrate and the
water. Thus to get the absorbance of the analyte
alone, we must first get the absorbance of the water
then subtract it with the total absorbance. Since the
UV-VIS is a new model, the apparatus subtracted it
already.
After getting the absorbance of the blank solution, we
need to find the wavelength that has is highest
absorbance in a 0.05M of the Cobalt Nitrate. The
absorbance of the different wavelengths is shown at table
2. A graph of the wavelength and the absorbance was
also created for better presentation of the results.

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The values of the absorbance in different wavelengths are summarized in Table 2.

We find that the highest absorbance is at 510 nm, which is 0.34833. The graph shows that before the peak, the absorbance
is steadily increasing and after the peak the absorbance is slowly decreasing.

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Figure 9. A Plot of Wavelength vs. Absorbance- blue dot at maximum absorption.

The last part of the experiment was the determination of the absorbance with varying concentration at the same
wavelength. An unknown sample was also given to know what its concentration is. As said earlier, the Beer-Lamberts Law
states that the absorbance and the concentration is directly proportional to each other. A graph was done to know the
equation of the linear relationship of the absorbance and the concentration of the solution.
In the prepared solutions, the color is darker when the concentration is higher and gets lighter as the concentration
gets lower. We can deduce that the darker the color may give higher absorbance value because it absorbs more light that
gives its color.
In this experiment, the analyte is Cobalt (II) Nitrate. The figure below shows the standard solutions prepared with
concentrations of 0.05 M, 0.04 M, 0.03 M, 0.02 M, 0.01 M and 0.005 M.
Table 3: Beers Law Plot (Wavelength: 510 nm)
CONCENTRATION, M
0.05
0.04
0.03
0.02
0.01
0.005
UNKNOWN

Experiment 04 Group No. 3 Date Aug. 16, 2016

ABSORBANCE
0.375
0.298
0.228
0.139
0.071
0.038
0.077

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Figure10. A plot of Concentration vs. Absorbance -Calibration curve for Cobalt (II) Nitrate
By using the method of linear regression, the concentration of the unknown is calculated by the equation formed from the
linearity of the concentration and absorbance.
The equation formed from the data was given by:

y=7.5666 x0.004

(1)

where x is the concentration in Molarity and y is the absorbance. Using the equation, the computed concentration of cobalt
(II) nitrate was 0.0107 M.
The possible errors of the experiment may be from the inaccurate measurements in preparing the solutions. Also, maybe
from the estimations used in providing the absorbance in the given wavelength. Also, getting smaller increments of the
concentrations or wavelength may give a more accurate answer.

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CONCLUSIONS AND RECOMMENDATIONS

Overall, we have successfully performed the
experiment wherein we are exposed in the
instrument called spectrophotometer. It is a
photometer that can measure the intensity of light as
a function of its wavelength. Single beam and double
beam are the two major classes of
spectrophotometers. Linear range of absorption and
spectral bandwidth measurement are the important
features of spectrophotometers.
In any spectrophotometer, it is used to provide light
of certain energy (wavelength) and to measure the
absorbance of that light. The basic operation of the
spectrophotometer includes a white light radiation
source which passes through a monochromator.
The monochromator is either a prism or a diffraction
grating which separates the light into the colored
components and allows only light of a particular
wavelength to strike the sample. The sample is
poured into a cuvette, which is similar to a small test
tube. It is marked so that it can be positioned in the
light beam the same way each time to avoid
variations due to the differences in the composition
of the glass. The light passes through the sample,
and the unabsorbed portion strikes a photodetector,
which produces an electrical signal proportional to
the intensity of light. The signal is converted to a
readable output that is used in the analysis of the
sample.
Many compounds involving transition elements are
colored. This is because the transition metals
include "d" electrons in its atomic structure. The
spacing of these "d-orbitals" allow for electronic
transitions within the energy range of the visible
region of the electromagnetic spectrum.
Compounds containing the alkali and alkaline earth
metals are white, due to having only "s" electron
transitions. More energy is required to cause this
type of transition and, thus, light of shorter
wavelength is involved.

Experiment 04 Group No. 3 Date Aug. 16, 2016

The amount of light absorbed by a solution is

dependent on the ability of the compound to absorb
light (molar absorptivity), the distance through which
the light must pass through the sample (path length),
and the molar concentration of the compound in the
solution. This relationship is known as Beer's Law
which is the linear relationship between the
absorbance and concentration of the absorbing
sample, i.e. a logarithmic relation exist between the
transmission of light through a substance and the
product of absorption coefficient of a substance and
distance travelled by the light through the
material(path length) The absorption coefficient is
the product of molar absorptivity, the concentration
the material, or an absorption cross section, and the
(number) density.
Thus, the absorbance is measured, if the path length
and the molar absorptivity are known and the
concentration of the substance can be deduced.
According to the experiment, absorbance is
proportional to concentration, so that at dilute
solutions a plot of concentration vs. absorbance
would be straight line, but the Law breaks down for
solutions of higher concentration, and so you might
get a curve under those circumstances.
Some applications of spectrophotometers include
the following: (1) It is directly used to measure light
intensity at different wavelength (2) It is used to
determine the unknown concentration of solution (3)
Spectrometers can be used to determine the
equilibrium constant of a reaction involving ions.
REFERENCES
[1] Castellan, G.W., Physical Chemistry 3rd edition, 1983.
[2] Alberty, R., Physical Chemistry 4th edition, Wiley
Publishing Co.
[3] Klein, David., Organic Chemistry 2nd edition, 2010.
[4] C.W. Garland, J.W. Nibler and D.P. Shoemaker,
Experiments in Physical Chemistry, 4th edition, 2004

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