WHAT WE ARE GOING TO

LEARN?
 PRINCIPLE

 INSTRUMENTATION

 APPLICATIONS
Definition
 Spectroscopy – Spectroscopy is the
measurement and interpretation of
electromagnetic radiation absorbed or
emitted when the molecules or atoms
or ions of a sample move from one
energy state to another.
PRINCIPLE OF
SPECTROSCOPY

Property Excited state

Excitation

Rel
axa SIGNAL
molecule tion
(E m TO BE
issi
on)DETECTED
Electromagnetic
Spectrum
 Electromagnetic
Spectrum

Hz 1021 1018 1015 1012 109 106

λ (nm) 10-3 1 200 500 106 109 1012

Microwave
Ultraviolet
Cosmic

Infrared
Visible

Radio
X-ray
Absorption vs. Emission
hν

En En

hν hν

Eo Eo

Absorption Emission
PRINCIPLE IN COLORIMETRY
 Study of absorption of visible radiation
whose wavelength ranges from 400nm-
800nm.
 Colored substances absorb color of
different wavelength and hence we get
absorption curve by plotting absorbance
vs wavelength.
Beer-Lambert Law
 BEER’S LAW: related to concentration
of absorbing species
 LAMBERT’S LAW: related to
thickness/pathlength of absorbing
species
Beer’s law
 Absorbance & Beer’s Law

Increasing absorbance
 Io = intensity of light through blank
 IT = intensity of light through sample
 Absorption = Io - IT
 Transmittance = IT/Io
 Absorbance = log(Io/IT)

Io IT
Io IT Io IT

pathlength b pathlength b
PRINCIPLE : ULTRA VIOLET
SPECTROSCOPY
 UV radiation ranges 200nm-400nm
 Any molecule has either n,π or σ or
combination of these electrons
 These electrons absorb radiation and
undergo transition from ground state to
excited state
 characteristic absorption peaks are
formed
Types of Electronic
Transitions
1. Transitions involving π , σ , and n
electrons
2. Transitions involving charge-transfer
electrons
3. Transitions involving d and f electrons
Absorbing species
containing π , σ , and n
electrons
 Absorption of ultraviolet and visible
radiation in organic molecules is
restricted to certain functional groups
(chromophores) that contain valence
electrons of low excitation energy. The
spectrum of a molecule containing
these chromophores is complex.
σ − σ *
Transitions

 An electron in a bonding σ orbital is

excited to the corresponding antibonding
orbital. The energy required is large. For
example, methane (which has only C-H
bonds, and can only undergo σ − σ *
transitions) shows an absorbance
maximum at 125 nm.
n − σ *
Transitions

 Saturated compounds containing atoms

with lone pairs (non-bonding electrons) are
capable of n − σ * transitions. These
transitions usually need less energy than
σ − σ * transitions. They can be initiated
by light whose wavelength is in the range
150 - 250 nm. The number of organic
functional groups with n − σ * peaks in the
UV region is small.
n − π * and π − π *

Transitions
 Most absorption spectroscopy of organic
compounds is based on transitions of n or
π electrons to the π * excited state. This is
because the absorption peaks for these
transitions fall in an experimentally
convenient region of the spectrum (200 -
700 nm). These transitions need an
unsaturated group in the molecule to
provide the π electrons.
 Molar absorbtivities from n − π *
transitions are relatively low, and range
from 10 to100 L mol-1 cm-1 . π − π *
transitions normally give molar
absorbtivities between 1000 and 10,000
L mol-1 cm-1 .
Solvent effect
 The solvent in which the absorbing
species is dissolved also has an effect
on the spectrum of the species.
 Peaks resulting from n −π * transitions
are shifted to shorter wavelengths (blue
shift) with increasing solvent polarity.
 The reverse (i.e. red shift) is seen for
π − π * transitions. This is caused by
attractive polarisation forces between
the solvent and the absorber, which
lower the energy levels of both the
excited and unexcited states.
Choice of Solvent
Solvent Minimum Solvent Minimum Solvent Minimum
Wavele-ngth Wavelength Wavelength
(nm) (nm) (nm)

Acetonitrile 190 water 191 cyclohexane 195

hexane 195 methanol 201 ethanol 204

ether 215 methylene 220 Chloroform 237

chloride

carbon 257
tetrachloride
UV spectra and molecular
structure
 The absorbing groups in a molecule are
called chromophores
 A molecule containing a chromophore is
called a chromogen
 An auxochrome does not itself absorb
radiation, but can enhance the absorption
 Bathochromic shift – red shift
 Hypsochromic shift – blue shift
 Hyperchromism – an increase in absorption
 Hypochromism – a decrease in absorption

Chromophore λ max
Transition
○
Alkanes ~ 150 σ to σ ∗
○
Alkenes ~ 175 π to π ∗
○
Alkynes ~ 170
○
Carbonyls ~ 188
○
alcohols, ethers ~ 185 η to σ ∗
○
Amines ~ 195
○
sulfur compounds ~ 195
○
Carbonyls ~ 285 η to π ∗
○
INTERPRETATION OF UV
SPECTRA
 α,β UNSATUREATED KETONES
Structural variation Increment in λ max
α- alkyl substituent +10 mμ

β -alkyl substituent +20 mμ

Exocyclic c=c +5 mμ

Cyclopentenone system -11 mμ

C=C extending +30 mμ

congugatation
II. Conjugated dienes:

Structural variation Increment in λ max

alkyl substituent +5

Exocyclic c=c +5

Presence of homoannular +39

diene

C=C extending +30

conjugatation
Compounds isolated λmax
double bonds
Acetaldehyde 293 mμ

acetone 271 mμ

acetonitrile 160 mμ

ethylene 193 mμ
UV-vis
Spectrophotometer
 Single-Beam UV-Vis Spectrophotometer
 Single-Beam spectrophotometers are often
sufficient for making quantitative absorption
measurements in the UV-Vis spectral
region.
 Single-beam spectrophotometers can
utilize a fixed wavelength light source or a
continuous source.
Single-Beam UV-Vis
Spectrophotometer
The simplest instruments use a single-
wavelength light source, such as a light-
emitting diode (LED), a sample container,
and a photodiode detector.
Instruments with a continuous source have a
dispersing element and aperture or slit to
select a single wavelength before the light
passes through the sample cell.
Dual-Beam uv-vis
Spectrophotometer
 In single-beam
Uv-vis absorption spectroscopy, obtaining
a spectrum requires manually measuring
the transmittance of the sample and
solvent at each wavelength. The double-
beam design greatly simplifies this process
by measuring the transmittance of the
sample and solvent simultaneously.
How Do UV spectrometers
work?
Cuvettes (sample
holder)
 Polystyrene
340-800 nm
 Methacrylate
280-800 nm
 Glass
350-1000 nm
 Suprasil Quartz
160-2500 nm
Array-Detector
Spectrophotometer
 Array-detector spectrophotometers allow
rapid recording of absorption spectra.
 Dispersing the source light after it passes
through a sample allows the use of an array
detector to simultaneously record the
transmitted light power at multiple
wavelengths.
 There are a large number of applications
where absorbance spectra must be recorded
very quickly. Some examples include HPLC
detection, process monitoring, and
measurement of reaction kinetics.
Instrumentation
 These spectrometers use photodiode arrays
(PDAs) or charge-coupled devices (CCDs) as
the detector. The spectral range of these array
detectors is typically 200 to 1000 nm. The light
source is a continuum source such as a
tungsten lamp.
 All wavelengths pass through the sample. The
light is dispersed by a diffraction grating after
the sample and the separated wavelengths fall
on different pixels of the array detector.
 The resolution depends on the grating,
spectrometer design, and pixel size, and
is usually fixed for a given instrument.
 Besides allowing rapid spectral
recording, these instruments are
relatively small . Portable spectrometers
have been developed that use optical
fibers to deliver light to and from a
sample.
Diode Array Detectors
Diode array
alternative puts
grating, array of
photosensitive
Semiconductors
after the light goes
through the sample.
Advantage, speed,
sensitivity,
The Multiplex
advantage
Disadvantage,
resolution is 1 nm,
vs 0.1 nm for
normal UV
 These instruments use only a single
light beam, so a reference spectrum
is recorded and stored in memory to
produce transmittance or absorbance
spectra after recording the sample
spectrum.
Ideal spectrometer has
 Good Scan Speed
 Resolution
 Software Features
 Ease of Operation
 Data Storage
 Customized Calculations
Practical Applications
 Pharmacy Practice
Ultraquin (psoriasis med. Needs UV. Act.
Pregnancy tests (colorimetric assays)
Blood glucose tests,
ELISA’s
 Pharmaceutics
pH titrations, purity measurement
concentration measurement
pKa Measurement with
UV
n

Titration of Phenylephrine

Ai - A
pKa = pH + log
A - An
 Medicinal Chemistry
compound ID (steroids, nucleosides)
monitoring isomerization, chirality
 Pharmaceutical Biotechnology
concentration/purity measurements
monitoring conformation of protein drugs
 Pharmacokinetics/Med. Chem.
HPLC monitoring and purification
Pharmaceutical Apps.
 On Line Analysis of Vitamin A and
Coloring Dyes for the
Pharmaceutical Industry
 Determination of Urinary Total
Protein Output
 Analysis of total barbiturates
 Comparison of two physical light
blocking agents for sunscreen
lotions
 Determination of acetylsalicylic acid in
aspirin using Total Fluorescence
Spectroscopy
 Automated determination of the uniformity
of dosage in Quinine Sulfate tablets using a
Fibre Optics Autosampler
 Determining Cytochrome P450 by UV-Vis
Spectrophotometry
 Light Transmittance of Plastic
Pharmaceutical Containers