notes

1. MUNI UNIVERSITY
FACULTY OF EDUCATION
INSTRUMENTAL CHEMISTRY
GROUP MEMBERS
1. MUHINDO AUGUSTINE
2. CHELIMO DAN
3. LULE VINCENT
4. ABUSUNA BAKALI

2. UV-VIS Molecular Spectroscopy
• Introduction
• UV-VIS molecular spectroscopy is a powerful analytical technique that
utilizes ultraviolet (UV) and visible light to study the interaction of
light with molecules.
• It measures the amount of light absorbed by a sample at different
wavelengths, providing valuable insights into the sample's
composition, structure, and concentration.

3. • Ultraviolet–visible spectroscopy or ultraviolet-visible
spectrophotometry (UV-Vis or UV/Vis) refers to absorption
spectroscopy or reflectance spectroscopy in the ultraviolet-visible
spectral region. This means it uses light in the visible and adjacent
(near-UV and near infrared [NIR]) ranges.
• The absorption or reflectance in the visible range directly affects the
perceived color of the chemicals involved. In this region of the
electromagnetic spectrum, molecules undergo electronic transitions.
Fluorescence spectroscopy is based on molecular emission:
• The “UV-Vis” technique is complementary to fluorescence
spectroscopy, in that fluorescence deals with transitions from the
excited state to the ground state, while UV-Vis absorption measures
transitions from the ground state to the excited state.

4. Regions of the electromagnetic spectrum. (For
UV-Vis, λ = 100~500 nm, 500~1000 nm)

5. Note:
• Molecules or parts of molecules that absorb light strongly in the UV-
Vis region are called chromophores.
The visible spectrum ranges from 400 nm to about 800 nm.
The color we see depends on wavelength. The color of a substance is
determined by which color(s) of light it absorbs and which color(s) it
transmits or reflects (the complementary color(s)). Color is an
important property of a substance.
The color of matter is related to its absorptivity or reflectivity.
The human eye sees the complementary color to that which is
absorbed

6. Principle of operation.
 Spectroscopy is based on the interaction between light and matter.
When the matter absorbs the light, it undergoes excitation and de-
excitation, resulting in the production of a spectrum.
 When matter absorbs ultraviolet radiation, the electrons present in it
undergo excitation. This causes them to jump from a ground state (an
energy state with a relatively small amount of energy associated with
it) to an excited state (an energy state with a relatively large amount
of energy associated with it).
 It is important to note that the difference in the energies of the
ground state and the excited state of the electron is always equal to
the amount of ultraviolet radiation or visible radiation absorbed by it

7. ..
• Molecular absorption spectroscopy is based on the measurement of
the transmittance (T), or the absorbance A of solutions contained in
transparent cells having a path length of b centimeters.
• Ordinarily, the concentration (c) of an absorbing analyte is linearly
related to absorbance (A) as given by Beer's law:

8. Important Terms and Symbols for Absorption
Measurements.

9. • Transmittance and absorbance cannot normally be measured in the
laboratory because the analyte solution must be held in a transparent
container, or cell.
• Reflection occurs at the two air-wall interfaces as well as at the two
wall-solution interfaces.
• The resulting beam attenuation (decay) is substantial, where about
8.5% of a beam of yellow light is lost by reflection in passing through
a glass cell containing water.
• In the figure below; Losses by reflection can occur at all the
boundaries that separate the different materials. In this example, the
light passes through the air-glass, glass-solution, solution-glass, and
glass-air interfaces.

10. To compensate for these effects, the
power of the beam transmitted by the
analyte solution is usually compared with
the power of the beam transmitted by an
identical cell containing only solvent.
• An experimental transmittance and
absorbance that closely approximate the
true transmittance and absorbance are
then obtained with the equations

11. Instrumentation
Instruments for measuring the
absorption of U.V. or visible
radiation are made up of the
following components;
1. Sources (UV and visible)
2. filter or monochromator
3. Sample containers or sample
cells
4. Detector

12. Instrumentation conti….
1. Light source: Provides a beam of UV and visible light,
typically a deuterium lamp for UV and a tungsten lamp for
visible light.
A deuterium arc lamp (or simply deuterium lamp) is a low-
pressure gas discharge light source often used in spectroscopy
when a continuous spectrum in the ultraviolet region is
needed.
Hydrogen and deuterium lamps for UV vary in the gases that
are utilized in the discharge. Deuterium lamps also generate a
higher intensity radiation compared to hydrogen lamps

13. Diagram of deuterium lamb and signal
response.

14. • The arc created excites the molecular deuterium contained within the
bulb to a higher energy state.
• The deuterium then emits light as it transitions back to its initial state.
This continuous cycle is the origin of the continuous ultraviolet
radiation.
• This process is not the same as the process of decay of atomic excited
states (atomic emission), where electrons are excited and then emit
radiation. Instead, a molecular emission process, where radiative
decay of excited states in molecular deuterium (D2), causes the effect.

15. …
• Tungsten Halogen Lamps are similar in construction to conventional
gas filled tungsten filament lamps except for a small trace of halogen
(normally bromine) in the fill gas.
• Tungsten Halogen (usually bromine-Br2) Lamps are ideal light sources
for spectrophotometers as they provide broad band spectral radiation
ranging from the ultraviolet, through the visible and into the infrared
out to five microns. Some radiation output can also be obtained at
320 and 340 nanometers.

17. Types of instruments used;
- Single-beam Instruments
In a single-beam instrument, Radiation from the filter or
monochromator passes through either the reference cell or the sample
cell before striking the photodetector
.

18. Double-beam Instruments.
Here, radiation from the filter or monochromator is split into two beams
that simultaneously pass through the reference and sample cells before
striking two matched photodetectors.

20. cont.
2. filters or monochromators:
All monochromators contain the following component parts;
 An entrance slit
A collimating lens
A dispersing device (a prism or a grating)
A focusing lens
An exit slit Polychromatic radiation (radiation of more than one
wavelength) enters the monochromator through the entrance slit. The
beam is collimated, and then strikes the dispersing element at an angle.
The beam is split into its component wavelengths by the grating or prism.
By moving the dispersing element or the exit slit, radiation of only a
particular wavelength leaves the monochromator through the exit slit.

21. Cont.
3. sample containers or sample cells:
A variety of sample cells available for UV region.
The choice of sample cell is based on
a) the path length, shape, size
b) the transmission characteristics at the desired wavelength
c) the relative expense
The cell holding the sample should be transparent to the wavelength region to be
recorded.
Quartz or fused silica cuvettes are required for spectroscopy in the UV region.
Silicate glasses can be used for the manufacture of cuvettes for use between 350
and 2000 nm. The thickness of the cell is generally 1 cm. cells may be rectangular in
shape or cylindrical with flat ends.

22. Cont.
4. Detectors are used to measure the transmitted or reflected
light from a sample and convert it into a signal.
5. Digital display. Displays the signal which can further be
interpreted using the peaks.

23. APPLICATIONS OF UV-VIS SPECTROSCOPY
1. STUDYING THE MAGNITUDE OF MOLAR ABSORPTIVITIES.
• Empirically, molar absorptive (ε values) that range from zero up to a
maximum on the order of 105 L mol-1 cm-1 (100,000 L mol-1 cm-1)
are observed in UV-visible molecular absorption spectrometry.
• For any particular absorption maximum, the magnitude of ε depends
on the capture cross section of the species and the probability for an
energy-absorbing transition to occur.
The relationship between ε and these variables has been shown to be

24. • where P is the transition probability and A is the cross
section target area in square centimeters per molecule.
• The area (A) for typical organic molecules has been
estimated from electron diffraction and X-ray studies to be
about 10-15 cm2/molecule; transition probabilities vary from
zero to one.
• For quantum mechanically allowed transitions, values of P
range from 0.1 to 1 (not 0 to 1), which leads to strong
absorption bands
(εmax = 104 to 105 L mol-1 cm-1).

25. Applications.
• UV-visible spectroscopy is used to identify organic and inorganic
species in a solution.
• It is used to identify the concentration of the unknown solution
• It is used in the study of chemical kinetics. I.e. disappearance of one
functional group and appearance of another functional group
• Used to study isomers i.e. in geometric isomerism, the trans species
absorb a high wave length with large molar absorptivity than the cis
species.
• It helps in the detection or presence of conjugation

26. Applications
• It is used in hospitals and clinics for drug analysis
• It is used in petrochemical industry
• It is used in water quality control labs
• It is used in forensic labs to control crimes
• It is used in the field of chemical and biological plants
• It is used in quantitative analysis, where lambert beers law is used

27. Disadvantages.
• Despite its versatility, UV-VIS spectroscopy has some limitations:
• Limited structural information: While it provides insights into
functional groups, it doesn't offer detailed structural information like
other techniques.
• Interference from other compounds: Overlapping absorption bands
from different compounds can complicate analysis.
• Specificity limitations: It may not be able to differentiate between
structurally similar compounds

28. Disadvantages of uv-visible spectroscopy
• limited sensitivity. It may not be suitable for detecting low
concentrations or trace amounts of substances in a complex matrix.
• Lack of selectivity. If multiple components in a sample absorb light at
similar wavelengths, it can be very difficult to differentiate their
contributions accurately.
• Inability to provide structural information.
• Limited wavelength range. Uv-vis is restricted to the ultraviolet and
visible regions of the electromagnetic spectrum. This limitation
means that it cannot provide information about absorption or
electronic transitions occurring in other regions, such as the infrared
or microwave regions.

29. . UV-VIS Spectra Interpretation
• A UV-VIS spectrum is a plot of absorbance versus wavelength. Peaks
in the spectrum correspond to wavelengths where the sample
absorbs light.
• The position and intensity of these peaks provide information about
the types of functional groups present and their concentrations.

30. REFERENCES:
1. G. W. Ewing, Instrumental Methods of Chemical Analysis, 5th Edition, McGraw-
Hill, New York, 1985.
2. J. Kenkel, Analytical Chemistry - Refresher Manual, Lewis Publishers, Boca
Raton, 1992.
3. D. A. Skoog, F. J. Holler and T. A. Nieman, Principles of Instrumental Analysis,
5th Edition, Thomson Learning, Crawfordsville, 1998.
4. F.W. Fifield and D. Kealey, Principles and Practice of Analytical Chemistry, 5th
Edition, Blackwell Science, Malden, 2000.
5. J. W. Robinson, Eileen M. S. Frame, and G. M. Frame II, Undergraduate
Instrumental Analysis, 6th Edition, Marcel Dekker, New York, 2005.
6. J. D. Ingle and S. R. Crouch, Spectrochemical Analysis, Prentice-Hall Int., New
Jersey, 1988.
7. N. B. Colthup, L. H. Daly and S. E. Wiberley, Introduction to Infrared and Raman
Spectroscopy, 3rd Edition, Academic Press, Boston, 1990.