METU Chem. Eng. Dept.

Ch.E. 410 Chem. Eng. Lab. II

EXPERIMENT 2.4
UV-VISIBLE SPECTROPHOTOMETRY (UV)

ATTENTION!!! This is a SHORT-REPORT experiment. You are expected to carry out the
experiment and complete the report by the end of your lab session, or 17:30.

OBJECTIVE

The aim of the experiment is to learn the basic principles of UV-visible spectrophotometry and
how to measure concentration by a UV-visible spectrophotometer.

PRELIMINARY WORK

1. Write down the frequency and wavelength intervals for these electromagnetic wave types: X-
Ray, Infrared, Ultraviolet, Visible.
2. Substances absorb electromagnetic waves only at certain frequencies of the source radiation.
Why do they not absorb at all possible frequencies of the source?
3. What happens to molecules after they absorb UV-Visible light?

4. What is molar absorptivity? Do the absorbed and transmitted fractions of energy always sum
up to give unity?

5. Consider the basic components of a UV-Visible spectrophotometer. What is the quantity that
is measured by the detector?

6. Describe the difference between the wavelength scan and monochromatic modes of UV-
Visible spectrophotometers.

7. What is the function of a monochromator? Define monochromatic light, and explain why it is
necessary in the UV-Visible spectrophotometric analysis.

8. Write down Beer's Law (also known as the Lambert-Beer Law).

9. As a conservative rule of thumb, the absorbance of a sample should be smaller than 2.0 (and
diluted if necessary) for reliable measurements. Inspecting Beer's Law, explain why.

10. Define a blank solution and list the possible errors that can be ameliorated by using a blank
rather than inserting source intensity Io in the absorbance equation.

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INTRODUCTION

Ultraviolet spectrometry is used to measure the concentrations of specific molecules in a

solution. This technique is appropriate for transition metal ions, highly conjugated organic
compounds and biological macromolecules as analytes. It is based on absorption of light in the
visible or ultraviolet region. Absorption of ultraviolet or visible light occurs simultaneously with
an electron jumping into an excited state from its ground state. The energy gaps between ground
and excited states depend on the chemical nature of the molecule of interest and therefore the
wavelength of the light is chosen accordingly for absorption by that molecule.

In UV-visible spectrophotometry, the amount of absorbed light increases with but is not
proportional to concentration (Think about why) and hence another parameter proportional to
concentration is defined as below and named as absorbance, A:

A = log10(I0/I)

where I0 and I are the intensities of light coming from the source and transmitted light,
respectively. Defined as above, absorbance is proportional to concentration according to

A= ϵ . B. C

In this equation; B is the length of the cell parallel to light direction, ε is a constant depending on
temperature, pressure and the molecular nature of the analyte and C is the analyte concentration.
The values of B and ε are not important most of the time, since plotting a calibration line using
standard samples of known concentration is a much more accurate way (Do you agree? why or
why not?).

A UV-visible spectrophotometer consists of a light source, a monochromator, a sample cell and a

detector. Among light sources, the most common are the tungsten filament (300-2500nm),
deuterium arc lamp (190-400nm), xenon arc lamp (160-2000nm) or light-emitting diode (LED)
for visible wavelengths. The prism provides a continuous spectrum using the light coming from
the source so that the light beam at a specific wavelength can be selected by the monochromator
and sent to the solution inside the sample cell. The detector measures the intensities of the light
coming from both the blank sample and analyte solution. Hence the absorbance value is
calculated for each solution.

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 Figure 1. A representative sketch for a UV-Visible Spectrophotometer
http://www.chem.agilent.com/Library/primers/Public/59801397_020660.pdf

EXPERIMENTAL
Equipment and Material:
UV-Visible Spectrophotometer, Shimadzu UV-2550
Methylene-blue
Deionized water
Procedure:
Before performing the experiment you must have studied the MSDS of methylene blue.

Prepare 7 different aqueous methylene-blue solutions in the range between 10 ppm-

100 ppm.

Find the wavelength of the UV-Visible light specific to methylene-blue by

continuous scanning.
Read the absorbance values for each solution.
Plot the absorbance vs. concentration and fit a line on this plot.
Read the absorbance values for the unknown solutions.
Estimate the concentration values for the unknown solutions.

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You will write a short report by hand at the end of the experiment, which will be submitted to
your instructor on the same day during work hours. Bring blank sheets of A4 paper, several
graph papers and a laptop to fit the data points (if you are comfortable using your calculator to fit
lines to data, the laptop is not necessary).

The report must include the following parts: Objective of the Experiment, Procedure, Results and
Discussion. Your report will be maximum three pages.

REFERENCE READING

1. Ewing, W.Galen, “Instrumental Methods of Chemical Analysis”, 4th ed., pp. 34-42,
63-74.
2. Skoog, A.D. West, D.M., “Principles of Instrumental Analysis”, pp. 27-54.

3. Willar, Merritt, Dean, Settle, “Instrumental Methods of Analysis”, 6th ed., pp.66-78.

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APPENDIX: Template for the Laboratory Report

Middle East Technical University Date: ______________

Chemical Engineering Department Group no: __________
ChE410 – Chemical Engineering Laboratory II

Laboratory Report on Experiment 2.4: UV-Visible Spectrophotometry

1. Objective of the experiment (one or two sentences)

2. Procedure (half a page maximum)

3. Results and Discussion (2 pages maximum, including plot)

a. Describe the wavelength scanning result of methylene-blue solution found by

continuous scanning and determine the wavelength of UV-Visible light specific
to methylene-blue.

b. Plot the absorbance vs. concentration graph and obtain the best line for this
plot.

c. Estimate the concentration values for the unknown solutions and state your
reasoning.

d. Discuss the capabilities and limitations of UV-Visible spectrophotometry

analysis and comment on your results.

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