ADAMSON UNIVERSITY

College of Engineering
Chemical Engineering Department
M a ni l a

EXPERIMENT NO. 8
SPECTROPHOTOMETRY

51080/ F/ 7:00 10:00/OZ404

Submitted by:

Agoto, Ariadna L.
(201312884)
Roderno, Karl Philip G.
(201513016)

Santiago, Dana Michiko M.

(201314338)

Submitted to:
Engr. Robert Delfin

Date of the Experiment: September 22, 2017

Date of Submission: September 29, 2017
ABSTRACT

In this experiment, it shows the analysis of a substance based on their absorbance of light.
Every atom and compound absorbs a distinct set of light wavelengths. Cobalt (II) nitrate and
Potassium Permanganate are the samples used in this experiment. Using analytical instrument,
which is spectrophotometer, the amount of light of specific wavelengths absorbed by the samples
was measured. The absorption of radiant energy by matter can be described using Beers Law.
Readings from the spectrophotometer can be expressed in terms of either % absorbance (amount
of light absorbed by the sample) or % transmittance (the amount of light passing through the
sample).Then, finally the gathered data were plotted, absorbance vs. wavelength.

The objective of this experiment is to determine the absorption of spectrum of a solution

at different wavelengths.

INTRODUCTION

Colors are the way of our brain, by the use of our eyes, to interpret electromagnetic
radiation of a wavelength within the visible spectrum. Every chemical compound absorbs,
transmits, or reflects light (electromagnetic radiation) over a certain range of wavelength.
Spectrophotometry 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.

If a beam of light passes through a glass container filled with liquid, the emergent
radiation is always less powerful than that entering. If the energy absorbed is greater for some
visible wavelengths than for others, the emergent beam will appear colored. The absorption of
radiant energy by matter can be described using Beers Law.

THEORETICAL BACKGROUND

Light is defined by the Merriam-Webster dictionary as simply something that makes

vision possible. In physics terms, it is a transverse, electromagnetic wave that can be seen by
humans. The wave nature of light was first illustrated through experiments on diffraction and
interference. Like all electromagnetic waves, light can travel through a vacuum.

Technically speaking, colors are the way our brain, by use of our eyes, interprets
electromagnetic radiation of a wavelength within the visible spectrum. Visible light lies between
400 and 700 nanometers. The different wavelengths are seen as different colors, as in the
spectrum below. You see a spectrum like this everything you see a rainbow. Frequency
determines color, but when it comes to light, wavelength is the easier thing to measure. A good
approximate range of wavelengths for the visible spectrum is 400 nm to 700 nm (1 nm = 109
m) although most humans can detect light just outside that range. Since wavelength is inversely
proportional to frequency the color sequence gets reversed. 400 nm is a dull violet (but violet
always appears dull). 700 nm is a dull red.

Figure 1: Wavelength changes for monochromatic light (nm)

Spectrophotometry

Spectrophotometry 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. The basic
principle is that each compound absorbs or transmits light over a certain range of wavelength.
This measurement can also be used to measure the amount of a known chemical substance.

A spectrophotometer is an instrument that measures the amount of photons (the intensity

of light) absorbed after it passes through sample solution. With the spectrophotometer, the
amount of a known chemical substance (concentrations) can also be determined by measuring
the intensity of light detected. Depending on the range of wavelength of light source, it can be
classified into two different types:

UV-visible spectrophotometer: uses light over the ultraviolet range (185 - 400 nm) and
visible range (400 - 700 nm) of electromagnetic radiation spectrum.
 IR spectrophotometer: uses light over the infrared range (700 - 15000 nm) of
electromagnetic radiation spectrum.

In visible spectrophotometry, the absorption or the transmission of a certain substance

can be determined by the observed color. For instance, a solution sample that absorbs light over
all visible ranges (i.e., transmits none of visible wavelengths) appears black in theory. On the
other hand, if all visible wavelengths are transmitted (i.e., absorbs nothing), the solution sample
appears white. If a solution sample absorbs red light (~700 nm), it appears green because green is
the complementary color of red. Visible spectrophotometers, in practice, use a prism to narrow
down a certain range of wavelength (to filter out other wavelengths) so that the particular beam
of light is passed through a solution sample.

A spectrophotometer is a device which measures the absorbance of a solution as light of a

specified wavelength is passed through it.

Figure 2: Basic structure of a spectrophotometer (

A spectrophotometer, in general, consists of two devices; a spectrometer and a

photometer.

Spectrometer: It produces a desired range of wavelength of light. First a collimator (lens)

transmits a straight beam of light (photons) that passes through a monochromator (prism) to split
it into several component wavelengths (spectrum). Then a wavelength selector (slit) transmits
only the desired wavelengths, as shown in Figure 2.
 Photometer: After the desired range of wavelength of light passes through the solution of
a sample in cuvette, the photometer detects the amount of photons that is absorbed and then
sends a signal to a galvanometer or a digital display, as illustrated in Figure 2.

The amount of photons that goes through the cuvette and into the detector is dependent
on the length of the cuvette and the concentration of the sample. Once you know the intensity of
light after it passes through the cuvette, you can relate it to transmittance (T). Transmittance is
the fraction of light that passes through the sample. This can be calculated using the equation:

=
0

Where: T = Transmittance
It = the light intensity after the beam of light passes through the cuvette
I0 = the light intensity before the beam of light passes through the cuvette
Transmittance is related to absorbance by the expression:

= log() = log ( )
0

With the amount of absorbance known, the unknown concentration of the sample can be
determined by using Beer-Lambert Law.

Beer-Lambert Law

Beer-Lambert Law, or also known as Beers Law, states that the amount of light of a
particular wavelength absorbed by a substance across a constant distance is proportional to the
concentration of that substance. Mathematically, it can be expressed as:

Where: A = Absorbance (no units)

= molar extinction coefficient or or absorption coefficient
l = path length (as presented in Figure 2)
c = concentration
 Figure 3: Transmittance

METHODOLOGY

This experiment aimed to determine the color imparted at different wavelengths and the
absorption spectrum of the given solutions at different concentrations. A spectrophotometer was
used to determine the absorption spectrum of the solutions. It was provided by the Adamson
University Engineering Laboratory located in Ozanam building at fourth floor. A
spectrophotometer is a device which measures the absorbance of a solution as light of a specified
wavelength is passed through it.

The results of this experiment were determined by using different concentrations of

Cobalt (II) nitrate and Potassium permanganate solutions. For this configuration, the whole class
was divided and given certain parts of the whole experiment in order to finish gathering data.

Materials, Reagents, and Instrumentation

This experiment required distilled water, cobalt (II) nitrate, and potassium permanganate
as the solution samples. The samples provided were diluted in different amounts of distilled
water in order to obtain different concentrations. For the materials and instrumentation, the
experimenters used cuvettes, beaker, droppers, graduated cylinder, wash bottle, white papers, an
acid burette for disposal of chemicals, and a spectrophotometer.
A. Initial Set-Up

The set-up of the spectrophotometer was already made by the laboratory faculties.

B. Color of Visible Radiation

Place the given samples in a cuvette. Place it near a light source and a blank piece of
white paper on the other side. Observe the color of the sample and compare it to the colors
presented in the electromagnetic spectrum scale for colors from 460 nm to 700 nm.
C. Visible Spectrum of a Solution

The spectrophotometer was calibrated by placing 2 clean and dry cuvettes were filled
with distilled water with at least 3/4 of the total volume of the cuvette on the spectrophotometer
and the wavelength was set to 375 nm and 100% transmittance. Then, one of the cuvettes were
replaced with the 0.1 M Co(NO3)2 placed in a cuvette filled with around 2/3 of the total volume.
The measurement of absorbance was then recorded by a printer directly connected to the
spectrophotometer. The measurement of absorbance was gathered from 400 nm to 780 nm with
10 nm intervals. The experiment was repeated with 0.001 M KMnO4 as the sample.

D. Determination of Concentration using Absorbance Values

The absorbance values of the 0.050 M, 0.030 M, 0.010 M, and 0.005 M of Co(NO3)2 and
0.01 M, 0.001 M, 0.0001M, and 0.00001 M KMnO4 were obtained through the same process in
part C. The data gathered were then tabulated and plotted with respect to the absorbance and
concentration.

Results

The results observed during the experiment spectrophotometry were obtained using a
spectrophotometer. The gathered data of absorbance were the tabulated and plotted with respect
to its concentration in order to present the data in a clear and concise form.

I. COLOR OF VISIBLE RADIATION

COUMPOUND COLOR WAVELENGHT RANGE

0.01 M Co(NO3)2 Pinkish-red 620 800 nm
0.001 M KMnO4 Violet 400 - 450 nm
II. VISIBLE SPECTRUM OF A SOLUTION

Table 1. Using a 0.01 M Co(NO3)2

WAVELENGTH ABSORBANCE WAVELENGTH ABSORBANCE WAVELENGTH ABSORBANCE

375 0.3531 520 0.4116 670 0.1479
400 0.3331 530 0.3778 680 0.1431
410 0.3246 540 0.3309 690 0.1392
420 0.3237 550 0.2865 700 0.1341
430 0.3262 560 0.2466 710 0.1320
440 0.3401 570 0.2189 720 0.1274
450 0.3612 580 0.2025 730 0.1245
460 0.3815 590 0.1884 740 0.1221
470 0.3912 600 0.1744 750 0.1195
480 0.3978 620 0.1665 760 0.1171
490 0.4054 640 0.1600 770 0.1140
500 0.4172 650 0.1563 780 0.1115

Table 2. Using 0.001 M KMnO4

WAVELENGTH ABSORBANCE WAVELENGTH ABSORBANCE WAVELENGTH ABSORBANCE

375 1.1813 520 2.8721 670 0.2872
400 0.6053 530 3.0103 680 0.2565
410 0.5112 540 2.5331 690 0.2278
420 0.4829 550 2.4817 700 0.2021
430 0.4942 560 1.8486 710 0.1799
440 0.5364 570 1.7321 720 0.1615
450 0.6173 580 1.0425 730 0.1471
460 0.7385 590 0.5997 740 0.1329
470 0.9940 600 0.5065 750 0.1207
480 1.2697 620 0.4291 760 0.1106
490 1.6704 640 0.3812 770 0.1011
500 2.1210 650 0.3512 780 0.0927
II. DETERMINATION OF CONCENTRATION USING ABSORBANCE VALUES

POTASSIUM PERMANGANATE
CONCENTRATION (M) ABSORBANCE
0.01 0.1976
0.001 0.1673
0.0001 0. 0342
0.00001 0.0120

COBALT (II) NITRATE

CONCENTRATION (M) ABSORBANCE
0.05 0.8823
0.030 0.8125
0.010 0.5544
0.005 0.3525

DISCUSSION OF RESULTS

Based on the data, the absorbance of each concentration of the sample has its maximum
y-value, means has its highest peak. In other concentrations, the absorbance reach above 1.0 (too
much high). This means that the sample is need to be diluted because the linear absorbance range
of most spectrometers is between 0.1 and 1.0. Greater than 2.0 absorbance means 99% of
available light is being blocked (absorbed) by the sample. And greater than 3.0 means 99.9% of
the available light is being blocked by the sample.

Nevertheless, the absorbance vs. concentration of the solutions has direct proportionality
and inverse logarithmic relationship between the concentration and light transmittance as proven
in Beers Law.

CONSLUSIONS AND RECOMMENDATIONS

The wavelength absorbed by a substance in the visible part of the spectrum is
complementary to the color that we perceive. The "color" is a function of human perception, but
absorption of specific wavelengths of light is a function of molecular interaction with light. If a
substance absorbs blue and red light, but not green light, we will see it as green since that is the
only light that reaches us from that substance. Therefore, when you look at the absorption
spectrum of a green solution, it should show low absorbance of wavelengths in the green part of
the spectrum but high absorption of light in the red and blue parts of the visible spectrum. While
the "color" of a substance depends on the person observing it, absorption of specific wavelengths
depends on the molecular structure of the substance.

And base on the data gathered, the application of spectrophotometry is that the proportion
of light that is absorbed by a solution of a particular compound is a function of the concentration
of that compound. And according to the Beer-Lambert Law, 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.

To obtain more accurate results and data for every prepared concentration, the proper
way of conducting and using the spectrophotometry must be followed. In putting the solution in
the cuvette, always make sure it is bubble-free because it may affect the wavelength of the
solution. And also during the preparation of the given concentrations of solutions, the volume
must be carefully measured. Generally, controlling the extraneous conditions that may affect the
results like temperature and measuring out quantities of the samples may help to obtain better
results, if followed.
APPENDICES

0.01 M Co(NO3)2 Absorbance

0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
375 410 430 450 470 490 520 540 560 580 600 640 670 690 710 730 750 770

Figure 4: graph of absorbance with respect to wavelength for 0.01 M Co(NO3)2

0.001 M KMnO4 Absorbance

3.5

2.5

1.5

0.5

0
375 410 430 450 470 490 520 540 560 580 600 640 670 690 710 730 750 770

Figure 5: graph of absorbance with respect to wavelength for 0.001 M KmnO4

Figure 6: Placing the cuvette in the spectrophotometer

Figure 7: Different concentrations of Co(NO3)2

NOMENCLATURE

nm measure of length in nanometers (1 x 10-9 m)

Abs absorbance

M concentration in molality

LITERATURE CITED

Elert, G. (n.d.). The Physics Hypertextbook. Color. Retrieved September 26, 21017 from:
https://physics.info/color/.

Elert, G. (n.d.). The Physics Hypertextbook, The Nature of Light. Retrieved September 26, 2017
from: https://physics.info/light/.

Light. (n.d.). Merriam-Webster Dictionary. Retrieved September 26, 2017 from

https://www.merriam-webster.com/dictionary/light.

Spectrophotometry. (2017). The University of Queensland. Retrieved September 26, 2017, from:
https://di.uq.edu.au/community-and-alumni/sparq-ed/sparq-ed-
services/spectrophotometry.

Vo, Kevin. (2015). Spectrophotometry. University of California (UCDAVIS). Retrieved

September 26, 2017, from Libretexts:
https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kinetics/Reaction_
Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry.