I.

INTRODUCTION (10%)

Every chemical compound absorbs, transmits, or reflects light (electromagnetic radiation)

over a certain range of wavelength. Spectrophotometry is a measurement of how much a
chemical substance absorbs or transmits. Spectrophotometry is widely used for quantitative
analysis in various areas (e.g., chemistry, physics, biology, biochemistry, material and chemical
engineering, clinical applications, industrial applications, etc). Any application that deals with
chemical substances or materials can use this technique. In biochemistry, for example, it is used
to determine enzyme-catalyzed reactions. In clinical applications, it is used to examine blood or
tissues for clinical diagnosis. There are also several variations of the spectrophotometry such as
atomic absorption spectrophotometry and atomic emission spectrophotometry.

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
while 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.

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.
Spectrophotometry is one of the most useful methods of quantitative analysis in various fields
such as chemistry, physics, biochemistry, material and chemical engineering and clinical
applications.
II. GENERAL OBJECTIVE
The main objective of this experiment is to measure the concentration of solutes in
solution by measuring the amount of the light that is absorbed by the solution in a
cuvette placed in the spectrophotometer
a. Specific objective
1. To measure the absorbance of the sample at different wavelengths.
2. To find out the unknown concentration of the sample.

III. Theoretical Framework/Related Literature

THEORY

It is possible for a ray of light to be absorbed by some material and simply pass
through others without being affected. When a molecule absorbs light, energy is
transferred from the ray of light to the molecule. If the frequency of the electronic and
magnetic fields of a ray of light match the frequency at which molecules will vibrate,
then light will be absorbed, if the frequency does not match, then the light will pass
straight through unaltered. Inert molecules whether solid or liquid appear colored due to
the way they modify light illuminating the object. Thus different objects absorb some
wavelengths and reflect others. For example, if a white light passes through a yellow
solution, it absorbs all colors except yellow. Spectrophotometry is a procedure for
determining how much light is reflected by a chemical material by measuring the strength
of light as a light beam travels through the sample solution. The fundamental theory is
that light is absorbed or emitted over a certain wavelength spectrum by each compound.

A spectrophotometer is made up of two instruments: a spectrometer and a

photometer. The spectrometer is to produce light of any wavelength, while the
photometer is to measure the intensity of light. The spectrophotometer is designed in a
way that the liquid or a sample is placed between spectrometer and photometer. The
photometer measures the amount of light that passes through the sample and delivers a
voltage signal to the display. If the absorbing of light change, the voltage signal also
changes. Spectrophotometers come in a variety of shapes and sizes and have
multipurpose uses to them. The different types of spectrophotometers available are all
different from one another, based on their application and desired functionality. The most
popular spectrophotometers are 45 degrees, sphere and multi-angle spectrophotometers.
Another closely related concept is Spectroscopy that simply measures the absorption of
light from its source and the intensity of light as well.

The basic spectrophotometer instrument consists of a light source, a digital

display, a monochromator, a wavelength sector to transmit a selected wavelength, a
 collimator for straight light beam transmission, photoelectric detector and a cuvette to
place a sample. The intensity of light is symbolized as l0 measure the number of photons
per second. When the light is passed through the blank solution, it does not absorb light
and is symbolized as (l). Other important factors are Absorbance (A) and Transmittance
(T).

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.

IV. Materials and Methodology (10%)

Materials:

 Potassium Permanganate (reagent)

 UV-Vis Spectrophotometry
 Spray Bottle
 Cuvette and cuvette wipes
 Distilled water
 Beaker
 Graduated Cylinder
 Volumetric Flask
 Pipette
 Porcelain spatula
 Aspirator
 Micropipette

Procedure:

1. Prepare a 100 mL stock solution with a concentration of 1000ppm KMnO4.

2. Prepare the stock solution by weighing 0.1 gram of potassium permanganate and
transferring it in a 100 mL volumetric flask using a funnel. Wash the weighing bottle and
pour it again in the volumetric flask to ensure that the reagent is transferred. Do it also in
the funnel.
3. Shake the volumetric flask upside down.
4. Fill the volumetric flask with the distilled water up to the mark and shake it again.
5. Take 10mL of the stock solution and dilute to the following concentrations
1. 250ppm, 100ppm, 75ppm, 50ppm, 0ppm
2. Prepare a calibration curve
a) Set the wavelength at 525 nm (λmax). 
b) Place the blank (sample of just the solvent) and the sample in the cell compartment
and again set the Absorbance to zero. 
 c) Measure and record the absorbance of each of the four standard solutions, starting
with the most dilute standard. 
d) After each measurement, rinse the cuvette with the next standard, not with distilled
water but with the stock solution. 
e) Draw a plot having X-axis as concentration (ppm) and Y-axis as Absorbance at
λmax (525 nm). 
f) Use Beer’s law to calculate ε for KMnO4, given the cell width (path length l ) to be
1 cm. It can be expressed as A = εlc where A is absorbance, ε is the molar
extinction coefficient (which depends on the nature of the chemical and the
wavelength of the light used), l is the length of the path light must travel in the
solution in centimetres, and c is the concentration of a given solution.

1. Take the sample of KMnO4 from your instructor and determine the concentration based
on the prepared calibration curve. 

V. Presentation of Results (10%)

Below are the data gathered during the experiment :

Standards (KMnO4) Absorbance

Trial 1 Trial 2 Trial 3
0ppm 0 0 0

50ppm 0.849 0.845 0.851

75ppm 1.188 1.188 1.182

100ppm 1.545 1.541 1.549

250ppm 3.799 3.809 3.789

Unknown Sample 1.255 1.258 1.248

Wavelength : 525nm

VI. Analysis and Interpretation of Results (30%)

Evaluate the results and relate and compare to the theories and related studies.

VII. Conclusion/Recommendation (10%)

Draw conclusions and give some points for improvement.
VIII. References (5%)
 Vo, Kevin. 2020. Spectrophotometry https://chem.libretexts.org/Bookshelves/
Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Phys
ical_and_Theoretical_Chemistry)/Kinetics/02%3A_Reaction_Rates/2.01%3A_Experi
mental_Determination_of_Kinetics/2.1.05%3A_Spectrophotometry
 https://byjus.com/chemistry/spectrophotometer-principle/#comment-108861
 http://www.rajswasthya.nic.in/RHSDP%20Training%20Modules/CSIO
%20Modules/Lab%20Technicians/SPECTROPHOTOMETER.pdf

IX. Documentation (5%)

You may refer to the video.

Note : Please use Times New Roman, 12pt. 1.15 spacing, margins all sides 1”, paper size - letter