CHEM F313: Instrumental Methods of Analysis: UV-Vis Spectrometry

CHEM F313: Instrumental Methods of Analysis
Lecture 03
UV-Vis Spectrometry
8/13/2024
BITSPilani, Pilani Campus
Outline
Signal to Noise Ratio
Error
Precision and Accuracy
Today’s Class
 Width of Spectral Lines
 Fundamental Law
8/13/2024 2
Electromagnetic waves
1. Wavelength, l
2. Frequency, ν
3. Wave number, ῡ
4. Radiant Power, P
5. Intensity, I
6. Monochromatic/Polychromatic
7. Interference
8. Polarised/non-polarized
8/13/2024 3
Electromagnetic spectrum
Energy Change
Nuclear Inner shell Ionization Molecular Spin Orientation

electrons of Atoms & Vibration
Molecules (stretch, bend)
8/13/2024 Valance Electrons 4

Interaction of EMR and molecule
Electric/magnetic component of EMR may interact with the molecule.
How?
• Electric component may interact with the electric dipole moment of
the molecule: Rotational-, vibrational-, and electronic-
spectroscopy.
• Magnetic component may interact with the magnetic dipole
moment of the molecule: NMR-, EPR- spectroscopy.
Bohr frequency
condition h = 
8/13/2024 5
Representation of spectrum
Y-axis :
Intensity of
Absorption/
Transmission/
Emission
X- axis : λ (wavelength) or related entity

such as wavenumber, frequency etc.
8/13/2024 6
Width of Spectral Lines
Irrespective of the improvement of the instrumentation (thin slit) the
line width of the spectra are often blurred. Natural line width is
governed by,
• Collision Broadening: Due to continual motion in liquid & gas phase,
molecules/atoms collide frequently. These collisions inevitably cause some
deformation of the particle and hence perturb the energies of at least of
outer electrons in each. Spectra of electronic transition affected. Vibrational
& rotational spectra are usually affected. More effective in liquids than
gases.
• Doppler Broadening: Due to collisions. Random motion creates both higher
and lower frequencies. More effective in gases than liquids
• Heisenberg’s Uncertainly Principle: Even in an isolated molecule or atom, the
energy levels are not infinitely sharp due to the operation of the uncertainty
principle of Heisenberg. If a system exists in an energy state for a limited
time Δt seconds, then the energy of that state will be uncertain (fuzzy) to an
extent ΔE.
8/13/2024 7
Intensity of Spectral Lines
ΔE x Δt = ħ/2 ~ 10-34 J.s , which means that lowest energy state is most
certain.
e.g. lifetime of excited electronic state is ~ 10-8 s, so, ΔE ~ 10-26 J
So, a transition between these two states have an energy uncertainty of
ΔE; associated frequency = ΔE/h ~h/2πhΔt~1/2πΔt = 108 Hz
Three important factors controlling the intensity of lines

• Transition Probability: Governed by the selection rules
• Population of States: Higher the population of the lower energy
state, higher is the intensity of the line.
Nupper/Nlower = e-ΔE/kT
• Path Length of sample: The relation can be expressed as
I/I0 = e-kcl This is certainly non-linear expression.
8/13/2024 8
UV-Vis: Instrumentation
8/13/2024 9
Origin of UV-Vis Spectrum
Absorption of EM radiation by a species causes the transitions amongst
the electronic energy levels, and these are accompanied by vibrational
and rotational excitations also.
This results into a spectrum that consists of several closely spaced lines
constituting what is called a band spectrum.
The sample absorbs photons spread over a range of wavelength.

8/13/2024 10
Fundamental Laws
If a monochromatic radiation of radiant power P0 travels an
infinitesimal distance dx, of an absorber then, at a constant
concentration,
• The number of absorbent is proportional to the distance but

independent of P
• k’ depends on the wavelength
or, Lambert’s Law
Also, the relation between radiant power and concentration can be

found out at a constant distance travelled and a fixed wavelength,
8/13/2024 11
Fundamental Laws
 Beer’s Law
• The number of absorbent is proportional to the concentration, C

Combining, and converting to base 10,
or,
“ε” is called absorptivity [Lg-1cm-1] if C is in g.L-1 and l is in cm

Or, molar absorption coefficient [Lmol-1cm-1] if C is in molL-1 and l is in
cm
8/13/2024 12
Numerical problem
 A substance has a molar absorptivity of 14,000 cm-1mol-1dm3 at the
wavelength of its maximum absorption. Calculate the concentration
of the substance whose solution in a cuvette of path length 1 cm has
an absorbance of 0.85.
 The molar absorptivity of a substance is 2.0 × 104 cm‒1mol‒1dm3.

Calculate the transmittance through a cuvette of path length 5.0 cm
containing 2.0 × 10‒6mol dm‒3 solution of the substance.
 A solution containing 36.5 mg of potassium dichromate per 500 cm3

was taken in a cuvette having a path length of 2 cm and its
transmittance was measured at 455 nm. If the percentage
transmittance is found to be 12, calculate the molar absorptivity of
potassium dichromate. (Molecular weight of potassium dichromate =
294 g mol-1)
8/13/2024 13
Quantitative Applications
Quantitative Determination Methodology
The methodology followed for the quantitative determinations have certain
essential steps.
• Forming an absorbing species
• Selection of the measurement wavelength
• Controlling factors influencing absorbance
• Validation of Beer and Lambert’s law
8/13/2024 14
Simultaneous Determination
• The analyte contains two species which have overlapping spectra.
• Need to find two wavelengths where molar absorptivity of two species is
different.
• Measurements are made on the solution of the analytes at two different
wavelengths.
• This gives a set of
simultaneous equations which
could be solved for the
concentrations of the
individual constituents.
• For best results it is desirable
to select two such
wavelengths where the ratio
of molar absorptivity is largest.
8/13/2024 15
Let us select the wavelengths λ1 & λ2 on these two spectra. The molar
absorptivity of spectra 1 and 2 can be found out by taking known amounts of
the substance and recording their absorbance value. Let these values be
(ε1)λ1, (ε2)λ1, (ε2)λ2, (ε2)λ2.
If the absorbance of the solution at the wavelength λ1 and λ2 are Aλ1, and
Aλ2, respectively then we can write, for 1 cm path
The absorbance values of a mixture of K2Cr2O7 and KMnO4 at 440 nm and 545 nm
using a cell of 1 cm path length were found to be 0.405 and 0.712 respectively. The
absorbance values of pure solutions of K2Cr2O7 (0.001 M) and KMnO4 (0.0002 M) in
similar conditions were as follows: A (Cr,440 nm) = 0.374, A (Cr, 545 nm) = 0.009, A
(Mn, 440nm) = 0.019, A (Mn, 545 nm) = 0.475. Using the given data determine the
concentration of Cr2O72- ions and MnO4- ions in the mixture.
8/13/2024 16
Conclusions
 Width of Spectral Lines

 Fundamental Law
 Next Class
 Types of absorption band
 MO involved in electronic structure
 Solvent Effect
8/13/2024 17

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CHEM F313: Instrumental Methods of Analysis

Nuclear Inner shell Ionization Molecular Spin Orientation

8/13/2024 Valance Electrons 4

X- axis : λ (wavelength) or related entity

Three important factors controlling the intensity of lines

The sample absorbs photons spread over a range of wavelength.

• The number of absorbent is proportional to the distance but

or, Lambert’s Law

Also, the relation between radiant power and concentration can be

• The number of absorbent is proportional to the concentration, C

“ε” is called absorptivity [Lg-1cm-1] if C is in g.L-1 and l is in cm

 The molar absorptivity of a substance is 2.0 × 104 cm‒1mol‒1dm3.

 A solution containing 36.5 mg of potassium dichromate per 500 cm3

 Width of Spectral Lines

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