Instrumental analysis
Spectroscopy
Dr. Hisham E Abdellatef
ezzat_hisham@yahoo.com
Definition
• Spectroscopy - The study
of the interaction of
electromagnetic radiation
with matter
Introduction to Spectroscopy
• What to be discussed
– Theoretical background of spectroscopy
– Types of spectroscopy and their working principles in brief
– Major components of common spectroscopic instruments
– Applications in Chemistry related areas and some examples
Electromagnetic Spectrum
Electromagnetic Spectrum
Electromagnetic Spectrum
Visible
200
500
109
106
106
109
1012
Radio
1
1012
Microwave
10-3
1015
Infrared
l (nm)
Ultraviolet
1018
X-ray
1021
Cosmic
Hz
Electromagnetic Radiation
• Electromagnetic radiation (e.m.r.)
– Electromagnetic radiation is a form of energy
– Wave-particle duality of electromagnetic
radiation
• Wave nature - expressed in term of
frequency, wave-length and velocity
• Particle nature - expressed in terms of
individual photon, discrete packet of energy
when expressing energy carried by a
photon, we need to know the its frequency
Definitions
• E = energy (Joules, ergs)
• c = speed of light (constant)
 l = wavelength
• h = Planck’s constant
 n = “nu” = frequency (Hz)
• nm = 10-9 m
• Å = angstrom = 10-10 m
Electromagnetic Radiation
•
Characteristics of wave
–
–
–
Frequency, v - number of oscillations per unit time, unit: hertz (Hz) - cycle per second
velocity, c - the speed of propagation, for e.m.r c=2.9979 x 108 m×s-1 (in vacuum)
wave-length, l - the distance between adjacent crests of the wave
wave number, v’, - the number of waves per unit distance v’ =l-1
c
v   v' c
l
•
The energy carried by an e.m.r. or a photon is directly proportional to the
frequency, i.e.
E  hv 
hc
 hv' c
l
where h is Planck’s constant h=6.626x10-34J×s
Key Formulae
• E = hn
• h = 6.626 x 10-34 J-s


n = frequency in Hz, E = energy
l = c/n
• c = 3.0 x 108 m/s

l = wavelength, n = frequency in Hz
Molecular Absorption
• The energy, E, associated with the
molecular bands:
Etotal = Eelectronic + Evibrational + Erotational
In general, a molecule may absorb energy in three ways:
•By raising an electron (or electrons) to a higher energy level.
•By increasing the vibration of the constituent nuclei.
•By increasing the rotation of the molecule about the axis.
Absorption vs. Emission
hn
En
En
hn
hn
Eo
Eo
Absorption
Emission
Rotational
absorption
Vibrational
absorption
Type of EM Interactions
• Absorption - EM energy transferred to
absorbing molecule (transition from
low energy to high energy state)
• Emission - EM energy transferred from
emitting molecule to space (transition
from high energy to low energy state)
• Scattering - redirection of light with no
energy transfer
Type of electronic transitions:
•Sigma () electrons: represent valence bonds They posses the lowest
energy level (i.e. most stable)
•pi () electrons: pi bonds (double bonds) They are higher energy than
sigma electrons.
•Non bonding () electrons: these are atomic orbital of hetero atom
(N,O, halogen or S) which do not participate in bonding. They usually
occupy the highest energy level of ground state.
*
Antibonding
n
n *
n *
*
 *
Energy
*
Antibonding
non-bonding

Bonding

UV Activity
hn

*
Laws of light absorption
Total light interring
Io
Reflacted part
Ir
absorption
Absorbed part
Ia
Transmitted part
It
transmission
refraction
reflection
Refracted part
If
Scattered part
Is
scattering
Definitions
•
•
•
•
•
Io = intensity of light through blank
IT = intensity of light through sample
Absorption = Io - IT
Transmittance = IT/Io
Absorbance = log(Io/IT)
Io
IT
Absorbance & Beer’s Law
Increasing absorbance
Beer’s Law
Io
IT
pathlength b
Io
IT
pathlength b
Beer-Lambert
Law
Log I0/I = abc
A = ε. B.C
Absorption spectrum
“Molecular” SPECTRUM
•Chromophore: C=C, C=O, N=O….
•Auxchrome: e.g. -OH, NH2,-Cl …
•Bathochromic shift (red shift):
•the shift of absorption to a longer wavelength
•Hypsochromic shift (blue shift):
•the shift of absorption to a shorter wavelength
•Hyperchromic effect: an increase in the absorption intensity.
•Hypochromic effect; an decease in the absorption intensity
Effect of pH on absorption spectra:
Phenol
O
OH
H
acid medium
O
+
alkaline medium
alkaline medium exhibits bathochromic
shift and hyperchromic effect.
aniline
NH2
NH3
NH2
+H
-H
alkaline medium
acid medium shows
hypsochromic shift and
hypochromic effect
acid medium
Complementary Colours
Observed
Absorbed
l
Absorbed colour
Observed colour
400
Violet
Yellow-green
425
Dark-blue
Yellow
450
Blue
Orange
510
Green
Red
550
Yellow-green
Purple
575
Yellow
Violet
590
Orange
Blue
650
red
Blue-green
Visible Light
Red
R
700 nm
Orange
O
650 nm
Yellow
Y
600 nm
Green
G
550 nm
Blue
B
500 nm
Indigo
I
450 nm
Violet
V
400 nm
Single Beam Spectrophotometer
Dual Beam Spectrophotometer
Light source
1. Tungsten halide lamp visible molecular
absorption to deliver constant and uniform
radiant energy from 350 nm up to 2400 nm.
2. High pressure hydrogen or deuterium
discharged lamp are used in the UV
molecular absorption to deliver continuum
source from 160-380 nm.
Monochromator:
wavelength selector
Filter,: absorption, it can be gelatin, liquid and intended glass filters.
Prisms: refraction.
In UV range prism can made
from quartz or fused silica but
in visible range
Grating: diffraction and
interference. it consist of a
large number of parallel line
(15000 -30 000 line per inch)
ruled very close to each other
on a highly polished surface
as aluminum or aluminized
glass.
Cuvettes (sample holder)
• plastic or glass for
determination the
sample in visible
rang,
• or quartz cell for
determination the
sample in UV. Cell
usually take
rectangular (cuvette)
Light detector
transducer
• convert a signal
photons into an
easily measured
electrical signal
such as voltage
or current
Transducer should have the:
•High sensitive
•Linear response
•A fast response time
•High stability
Light detector
transducer
Types of Transducer:
• 1. Barrier layer (photovoltaic cell)
• 2. Phototube
• 3. Photomulriplier
Application of spectrophotometry
1. Quantitative analysis of a single component:
Calibration curve
2. Quantitative analysis of multi-component mixture:
A'  ε 'M bC M  ε 'N bC N
(at λ ' )
A"  ε "M bC M  ε "N bC N
(at λ " )
The measurement of complexation
(ligand/metal ratio in a complex):
1. The mole- ratio method
(Yoe and Jones method)
2. The method of continuous
variations (Job's method)
Deviation from Beer's law
1. Real deviations:
2. Instrumental deviations
– Irregular deviations
– ii. Regular deviations
– Stray light:
3. Chemical deviations:
Practical Applications
• Pharmacy Practice
– Ultraquin (psoriasis med. Needs UV. Act.)
– Pregnancy tests (colorimetric assays)
– Blood glucose tests, Bilichek
• Pharmaceutics
– pH titrations, purity measurement
– concentration measurement
pKa Measurement with UV
n
i
Titration of Phenylephrine
pKa = pH + log
Ai - A
A - An
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