Unit I Absorption Spectroscopy.pdf

SPECTROSCOPIC METHODS FOR
CHEMICAL ANALYSIS

Unit - I : Absorption spectroscopy :
Colorimetry: theory of colorimetry, derivation of Beer-Lambert’s law, deviations from Beers law.
UV-Visible Spectroscopy: Electronic transition, Solvent effect, Woodward-Fischer rule,
Calculation of λmax using Woodward-Fischer rule, Single and Double beam UV-visible
Spectroscopy-Instrumentation with components, Qualitative and Quantitative applications.
Fluorescence: Basic concept and applications.
Infrared spectroscopy: Molecular vibrations, Degree of freedom, Factors influencing vibrational
frequencies, IR-Instrumentation, Characteristic frequencies of organic molecules, Qualitative and
Quantitative analysis.
Raman spectroscopy: Raman effect and spectra, Differences between Raman spectra and IR
spectra, Raman-Instrumentation.

Spectroscopy & Electromagnetic Radiation
Spectroscopy:
Spectroscopy is the branch of science that deals with the study of the interaction of
electromagnetic radiation with matter. It is the most powerful tool available for the study of
the structure of molecules.
C8H9NO2
XRD
Technique
Paracetamol

Various Spectroscopic Instrumental Techniques
Nuclear Magnetic Resonance (NMR) X-Ray Diffraction Technique (XRD)
UV-Visible Spectroscopic Technique FT-IR Instrument
Raman Spectrophotometer
Electron Spin Resonance (ESR)

Various Spectroscopic Instrumental Techniques
Colorimeter Mass Spectrometry
Fluorescence spectrometer

Electromagnetic Radiation
𝑬 = 𝒉𝝑 =
𝒉𝒄
𝝀

Interaction of Electromagnetic Radiation with Matter

Microwave Radiation Molecular Rotations
Rotational Spectroscopy
Region: 3 x 1010 – 3 x 1012 Hz
Wavelength: 1 cm – 100 μm

Infra-Red Radiation Molecular Vibrations
Vibrational Spectroscopy
Region: 3 x 1012 – 3 x 1014 Hz
Wavelength: 100 μm - 1 μm

Visible & UV Radiation Electronic Transitions
Electronic Spectroscopy
Region: 3 x 1014 – 3 x 1016 Hz
Wavelength: 1 μm - 10 nm

Different types of Transitions in molecules

Absorption / Emission

UV-Visible Spectroscopy
▪ It is also called “electronic spectroscopy”.
▪ It involves promotion of electrons (σ, π, n) from the ground state to higher energy states.

▪ It involves promotion of electrons (σ, π, n) from the ground state to higher
energy states.
▪ UV-Visible spectroscopy is used to measure the
▪ Number of conjugated double bonds;
▪ Aromatic conjugation within the various molecules;
▪ Distinguishes between conjugated and non-conjugated systems;
▪ , -unsaturated carbonyl compounds from , -analogues;
▪ Homo-annular and Hetero-annular conjugated dienes etc.

Examples for conjugated systems:

Examples for conjugate & Non-conjugate systems:
1,3-Cyclohexadiene
1,4-Cyclohexadiene
245 nm
240 nm

Identify the conjugate and non-conjugate diene systems
among the following.

, -unsaturated carbonyl and nitro compounds

Homo-annular and Hetero-annular conjugated dienes

▪ It involves promotion of electrons (σ, π, n) from the ground state to higher energy states.
▪ UV-Visible spectroscopy is used to measure the number of conjugated double bonds and also aromatic
conjugation within the various molecules.
▪ It also distinguishes between conjugated and non-conjugated systems;
▪ , -unsaturated carbonyl compounds from , -analogues;
▪ Homo-annular and Hetero-annular conjugated dienes etc.
▪ The principle involved in the UV-Visible spectroscopy is
Beer-Lambert’s Law.
▪ UV-Visible region extends from 200 nm to 760 nm.
▪ UV region extends from 200 to 380 nm and
▪ Visible region extends from 380 to 760 nm.

E = 71.6 X 4.184 = 299.5744 kJ mole-1

Spectral Shift in UV-Visible Spectroscopy

Qualitative and Quantitative applications
▪ UV-Visible spectroscopy as a quantitative technique be used to determine the concentrations of
substances, to study the rates of reactions, and determine rate equations for reactions, from which a
mechanism can be proposed.
▪ As qualitative technique UV visible spectroscopy is used extensively in teaching, research and analytical
laboratories to identify the structure of organic compounds, detection of different components
present in a mixture and identify compounds in the separation by TLC.
▪ Structural elucidation: presence or absence of absorption band at a particular wavelength for
respective compound may be used to identify the presence or absence of a particular
chromophore in the compound.
▪ Determination of impurities: TLC, Detector for different separation techniques as most of the
organic compounds absorb in UV region.
▪ Determination of isomers

Quantitative applications
▪ Determination of unknown concentration of compound using Beer Lamberts law.
▪ Determination of rate of chemical reaction: (Chemical Kinetics)
Nitroethane Nitroethane anion
𝝀𝒎𝒂𝒙 = 𝟐𝟒𝟎 𝒏𝒎

Quantitative analysis
a. Determination of unknown concentration of compound using Beer Lamberts law.
b. Chemical kinetics- rate of chemical reaction : it involves the measurement of
absorption of either the reactants or products at a fixed wavelength. Once the
wavelength is fixed, measure the absorption as a function of time, from which the rate
of the reaction can be measured.
E.g. reaction of nitroethane with alkali to form nitroethane anion. Here only the anion
absorbs in UV region at 240nm, others don’t show any significant absorption.
To measure the rate of hydroxide ion removing a proton from nitroethane, the instrument
is adjusted to measure absorbance at 240nm as a function of time.

Nitroethane is taken in a cuvette containing a basic solution and the rate of the
reaction is determined by monitoring the increase in absorbance at 240nm.

Similarly, the enzyme lactate dehydrogenase catalyzes the reduction of pyruvate by NADH
(nicotinamide adenine dinucleotide (NAD) + hydrogen (H)) to form lactate.
Here, NADH is the only species in the reaction mixture which absorbs light at 340 nm, hence
by measuring the decrease in absorbance at 340 nm, the rate of reaction can be determined.

Dissociation constants of acids and bases:
pKa of a compound can be determined by UV-Vis spectroscopy if either the acidic
form or the basic form of the compound absorbs UV or Visible light.
For e.g., the phenoxide ion has a maximum absorption at 287nm.
If the absorbance at 287nm is determined as a function of pH, the pKa of the phenol
can be calculated using Henderson Hasselbalch equation.
pH = pKa + log[A-] / [HA]
By plotting a graph between absorbance and wavelength at different pH values, the
ratio of reactant and products can be determined and hence the pKa value can be
determined.

Thermal denaturation of DNA:
UV spectroscopy can also be used to estimate the
nucleotide composition of DNA.
The two strands of DNA are held together by both A–T
base pairs and G–C base pairs.
When DNA is heated, the double stranded DNA breaks
down.
Single-stranded DNA has a greater molar absorptivity
at 260 nm than does double-stranded DNA.
The melting temperature (Tm) of DNA is the midpoint
of an absorbance versus temperature curve.
For double-stranded DNA, Tm increases with
increasing numbers of G–C base pairs because they
are held together by three hydrogen bonds, whereas
A–T base pairs are held together by only two hydrogen
bonds.
And hence can be used to estimate the number of G–C
base pairs.

Thermal denaturation of DNA:
UV spectroscopy can also be used to estimate the
nucleotide composition of DNA.
The two strands of DNA are held together by both A–T
base pairs and G–C base pairs.
When DNA is heated, the double stranded DNA breaks
down.
Single-stranded DNA has a greater molar absorptivity at
260 nm than does double-stranded DNA.
The melting temperature (Tm) of DNA is the midpoint of
an absorbance versus temperature curve.
For double-stranded DNA, Tm increases with increasing
numbers of G–C base pairs because they are held
together by three hydrogen bonds, whereas A–T base
pairs are held together by only two hydrogen bonds.
And hence can be used to estimate the number of G–C
base pairs.

Study of charge transfer complexes:
The formation of charge-transfer complex occurs
between molecules which, when mixed, allow the
transfer of electronic charge through space from an
electron rich molecule to an electron deficient
molecule with molecular orbitals of suitable energy
and symmetry.
The filled π-orbitals in the donor molecule overlap
with the depleted orbital in the acceptor molecule
and generate two new molecular orbitals.
Thus transition between these newly formed orbitals
are responsible for the new absorption bands
observed in the charge transfer complexes.

The brown colour of iodine in benzene or the appearance of deep blue colour when
tetracyanoethylene is added to a chloroform solution of aniline may be explained due to
the formation of charge transfer complex.
The λmax of benzene is 255 nm while for iodine in hexane is 500 nm. The charge transfer
complex (benzene-iodine) displays an intense additional band at 290 nm.
Similarly in the anilinetetracyanoethylene complex, λmax for aniline and tetracyanoethylene
are 280 nm and 300 nm, respectively, while the deep blue complex has λmax at 600 nm.

Other uses:
UV-visible spectroscopy is also used in the quality control in the development and
production of dyeing reagents, inks and paints and the analysis of intermediate dyeing
reagents.
In environmental and agricultural fields the quantification of organic materials and heavy
metals in fresh water can be carried out using UV-visible spectroscopy.
A special type of UV-watermark is kept on many sensitive documents such as credit cards,
driving licenses, passports to prevent forgery. The watermark can only be seen in UV light.
The optical whiteners absorb ultraviolet light and re-emit it in the visible range. This
feature is used in washing powders. Optical whiteners are also added to many toothpastes
and detergent powders.

Infra-Red Spectroscopy
▪ It is also called vibrational spectroscopy
Various vibrational modes in H2O

Infra-Red Spectroscopy
▪ Infra-Red Spectra are mainly used in structure elucidation to determine the
functional groups
▪ The IR region is divided in to Near IR, Mid IR and Far-IR regions.
▪ Near IR region : 0.78 µm – 2.5 µm (12500 cm-1 – 4000 cm-1)
▪ Middle IR region : 2.5 µm – 15 µm (4000 cm-1 – 667 cm-1)
▪ Far IR region : 15 µm – 200 µm (667 cm-1 – 50 cm-1)
Longer wavelength
Shorter Wavelength

IR Spectroscopy – Vibrational frequency
𝑬 = 𝒏 +
𝟏
𝟐
𝒉ഥ
𝒗
𝑬 = 𝒏 +
𝟏
𝟐
𝒉
𝟐𝝅𝒄
𝑲
𝝁
where
Vibrational energy

Vibrational Modes
Scissoring
Rocking
Wagging
Twisting

Vibrational Degrees of Freedom
Translational degrees
of freedom (TN) (3)
Rotational degrees of
freedom (RN) (3 or 2)
Vibrational degrees
of freedom (VN)
Total number of degrees of freedom = 3N, where N is the number of atoms
(TN) + (RN) + (VN) = 3N
(VN) = 3N – {(TN) + (RN) }

Total number of degrees of freedom = 3N, where N is the number of atoms
(TN) + (RN) + (VN) = 3N
(VN) = 3N – {(TN) + (RN) } = 3N – { 3 + (RN) }
For linear molecules, number of rotational degrees of freedom (RN) = 2
For linear molecules, vibrational degrees of freedom
(VN) = 3N – {(TN) + (RN) } = 3N – { 3 + 2 } = 3N - 5
For non-linear molecules, number of rotational degrees of freedom (RN) = 3
For non-linear molecules, vibrational degrees of freedom
(VN) = 3N – {(TN) + (RN) } = 3N – { 3 + 3 } = 3N - 6

The fingerprint region
The functional group region

Factors which decrease/increase the number of IR bands
The following factors decrease the theoretical number of fundamental bands:
I. The frequencies of fundamental Vibrations which fall outside of the region
4000-667 cm-1
II. Fundamental bands which are so weak that they are not observed.
III. Fundamental bands which are so close that they coalesce.
IV. The occurrence of a degenerate band from several absorptions of the same
frequency in highly symmetrical molecules such as carbon dioxide.
V. Certain fundamental vibrational bands which do not appear in the infrared
spectrum due to Iack of the required change in dipole-moment of the
molecule, e.g. in carbon dioxide molecule.

The appearance of the following types of additional (non-fundamental) bands increases the number of bands
as compared to that expected from the theoretical number of fundamental vibrations. All these bands have
one-tenth to one-hundredth intensity of the fundamental bands.
I. Overtone Bands
These may arise if a molecule is excited, e.g. from its first vibrational energy Ievel to the third vibrational energy
Ievel. The energy required is almost twice of that required for the excitation to second vibrational energy Ievel.
In this way, if there are two fundamental bands at x and y cm-1, then the overtone bands can be expected, e.g.
at 2x, 2y, 3x and 3y cm-1. The intensity of the overtone decreases as the order of the overtone increases, e.g. the
second overtone (3x or 3y) is less intense than the first overtone (2x or 2y). Consequently, second and higher
overtones are rarely observed, whereas first overtones are observed only for strong bands.

The appearance of the following types of additional (non-fundamental) bands increases the number of bands
as compared to that expected from the theoretical number of fundamental vibrations. All these bands have
one-tenth to one-hundredth intensity of the fundamental bands.

f
or

f
or
Eq. 3.1

Factors Affecting Vibrational frequency
▪ It should be noted that any factor which affects the force constant of a bond will affect its
stretching frequency.
▪ There are various interrelated factors which shift the vibrational frequencies from their expected
values.
▪ For this reason, the values of vibrational frequencies of the bonds calculated by the application of
Hooke’s law are not exactly equal to their observed values.
▪ The force constant of a bond changes with the electronic and steric effects of the other groups
present in the molecule, and so the vibrational frequencies are shifted from their normal values.
▪ Also, frequency shifts may occur when the IR spectrum of the same compound is recorded in
different states, viz. solid, liquid or vapor.
▪ Usually, a substance absorbs at higher frequency in the vapor state than that in the liquid or solid
state.

Unit I Absorption Spectroscopy.pdf

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Unit I Absorption Spectroscopy.pdf