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
Excited state

Property
Excitation

molecule

Rel
axa

tion

SIGNAL
TO BE
(E m
issi DETECTED
on)

Electromagnetic
Spectrum

200

500

106

106

109

1012

Microwave

109

Infrared

10-3

1012

Visible

(nm)

1015

Ultraviolet

1018

X-ray

1021

Cosmic

Hz

Radio

Electromagnetic
Spectrum

Absorption vs. Emission

h

En

En
h

Eo

h
Eo

Absorption

Emission

PRINCIPLE IN COLORIMETRY

Study of absorption of visible radiation

whose wavelength ranges from 400nm800nm.
Colored substances absorb color of
different wavelength and hence we get
absorption curve by plotting absorbance
vs wavelength.

Beer-Lambert Law

BEERS LAW: related to concentration

of absorbing species
LAMBERTS LAW: related to
thickness/pathlength of absorbing
species

Beers law

Absorbance & Beers 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

pathlength b

Io

IT

pathlength b

PRINCIPLE : ULTRA VIOLET

SPECTROSCOPY

UV radiation ranges 200nm-400nm

Any molecule has either n, or 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.
2.
3.

Transitions involving , , and n

electrons
Transitions involving charge-transfer
electrons
Transitions involving d and f electrons

Absorbing species
containing , , and n
Absorption of ultraviolet and visible
electrons

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.

 * Transitions

An electron in a bonding 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 *
transitions) shows an absorbance
maximum at 125 nm.

n * Transitions

Saturated compounds containing atoms

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

n * and *
Transitions

Most absorption spectroscopy of organic

compounds is based on transitions of n
or electrons to the * 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 electrons.

Molar absorbtivities from n*

transitions are relatively low, and range
from 10 to100 L mol-1 cm-1 . *
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* transitions
are shifted to shorter wavelengths (blue
shift) with increasing solvent polarity.

The reverse (i.e. red shift) is seen for

* 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
Wavelength (nm)

Solvent

Minimum
Wavelength
(nm)

Solvent

Minimum
Wavelength
(nm)

Acetonitrile

190

water

191

cyclohexane

195

hexane

195

methanol

201

ethanol

204

ether

215

methylene 220
chloride

Chloroform

237

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

max Transition

Alkanes

~ 150
~ 175
~ 170
~ 188
~ 185
~ 195

to
to

~ 285

to

Alkenes
Alkynes
Carbonyls
alcohols, ethers
Amines
sulfur compounds
Carbonyls

to

~ 195

INTERPRETATION OF UV
SPECTRA

, UNSATUREATED KETONES
Structural variation

Increment in max

- alkyl substituent

+10 m

-alkyl substituent

+20 m

Exocyclic c=c

+5 m

Cyclopentenone system

-11 m

C=C extending
congugatation

+30 m

II. Conjugated dienes:

Structural variation

Increment in max

alkyl substituent

+5

Exocyclic c=c

+5

Presence of homoannular
diene

+39

C=C extending
conjugatation

+30

Compounds isolated
double bonds

max

Acetaldehyde

293 m

acetone

271 m

acetonitrile

160 m

ethylene

193 m

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 singlewavelength light source, such as a lightemitting 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,
ELISAs

Pharmaceutics
pH titrations, purity measurement
concentration measurement

pKa Measurement with

UV
n

Titration of Phenylephrine
pKa = pH + log

Ai - A
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