SPECTROSCOPY
Introduction of Spectrometric Analyses
The study of how the chemical compound
interacts with different wavelengths in a given
region of electromagnetic radiation is called
spectroscopy or spectrochemical analysis.
The collection of measurements signals
(absorbance) of the compound as a function of
electromagnetic radiation is called a spectrum.
Energy Absorption
The fundamental principle is the absorption of certain
amount of energy.
The energy required for the transition from a state of lower
energy to a state of higher energy is directly
related to the frequency of electromagnetic radiation
that causes the transition.
THE NATURE OF LIGHT
• Light is composed of electric and magnetic fields,
which are mutually perpendicular and which radiate
out from a source
• It is therefore a form of electromagnetic radiation.
• Electric and magnetic fields are propagated through
space as wave functions which may be characterized
by wavelength, λ (the distance from one part of the
wave to the corresponding position on the next wave)
and frequency, ν (the number of times a wave passes
through a fixed point in space every second).
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• This relationship can be rearranged ;
λ = c / ν , this relationship means that the frequency and wave length
are inversely related ( higher frequency means shorter wave
length).
• thus a red light color of wave length of 650nm and a green color of
540nm wave length , the red light has a higher wave length thus it
has a lower frequency) .
• As for energy: the light with the highest energy will be the one with
the highest frequency - that will be the one with the smallest
wavelength.
• Light of each color has a different wavelength - blue light has a
shorter wavelength than red light. Blue light therefore has a larger
number of peaks per unit of length thus it has higher frequency and
larger energy.
The Electromagnetic Spectrum
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Electronic Excitation
The absorption of light energy by organic compounds
in the visible and ultraviolet region involves the
promotion of electrons in , , and n-orbitals from the
ground state to higher energy states. This is also called
energy transition. These higher energy states are
molecular orbitals called antibonding.


n *
n
n *
Antibonding
*
*
 *
Energy
*
Antibonding
Nonbonding
Bonding
Bonding
Electronic Molecular Energy Levels
The higher energy transitions ( *) occur a
shorter wavelength and the low energy transitions
(*, n *) occur at longer wavelength.
Chromophore is a functional group which absorbs a
characteristic ultraviolet or visible region.
UV
210 nm
233 nm
268 nm
315 nm
Double Bonds
Conjugated Diene
Conjugated Triene
Conjugated Tetraene
Spectrophotometer
An instrument which can measure the absorbance
of a sample at any wavelength.
The essential components of
spectrophotometer
Collimator
Light’s
band
λ1
λ2
λ3
λ4
Light Monochromator
source
Prism
Cuvette
sample
container
Photocell
Slit
Wavelength
selector
Photometer
or detector
Components of a Spectrophotometer
1-Light Source
• Deuterium Lamps－a truly continuous
spectrum in the ultraviolet region
(160nm~375nm)
• Tungsten Filament Lamps－the most
common source of visible and near
infrared radiation.
Components of a Spectrophotometer
2-Monochromator
• Used as a filter: the monochromator will
select a narrow portion of the spectrum
(the bandpass) of a given source
• Used in analysis: the monochromator will
sequentially select for the detector to
record the different components (spectrum)
of any source or sample emitting light
Single and Double Beam
Spectrometer
• Single-Beam: There is only one light
beam or optical path from the source
through to the detector.
• Double-Beam: The light from the
source, after passing through the
monochromator, is split into two
separate beams-one for the sample and
the other for the reference.```````````
Single Beam Spectrophotometer
Lamp
Lens
cuvette
Detector
Dual Beam Spectrophotometer
Sample Cells (Quvettes)
UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass
Plastic
The Beer-Lambert law
•When a beam of radiation (light)
passes through a substance or a
solution, some of the light may be
absorbed and the remainder
transmitted through the sample.
•The ratio of the intensity of the
light entering the sample (Io) to that
exiting the sample (I1) at a
particular wavelength is defined as
the transmittance (T).
z
% T = (Io / I1 ) x 100
A = - log (T)
The Beer-Lambert law
• ε = molar absorptivity or extinction coefficient
of the chromophore at wavelength λ (the
optical density of a 1-cm thick sample of a 1
M solution). ε is a property of the material and
the solvent.
• L = sample pathlength in centimeters
• c = concentration of the compound in the
sample, in molarity (mol L-1)
• Absorbance
• A= -log(I1/I0)=ε·c·l
Transmittance
• The ratio of the intensities of the transmitted
and incident light gives transmittance.
T = I/I0
I0 is the intensity of incident radiation
I is the intensity of transmitted radiation
A 100% value of T represents a transparent substance whereas a zero
value represents a totally opaque.
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Deviation of the Beer’s law
• At high concentrations many molecules form dimers or higher
polymers which have a spectrum that differs than their
monomeric form , which could lead to either positive or
negative deviation.
• Also at high concentrations aggregation can occur which
frequently leads to scattering of light thus decreasing the amount
of light transmitted and thus causing positive deviations.
• Deviations in absorptive coefficients at high concentrations
(>0.01M) due to electrostatic interactions between molecules in
close proximity.
• Stray light ; it is the quantity of light that reaches the detector
that is of a wavelength other than that selected. Therefore, stray
light causes the measured transmittance to be high , thus leading
to negative deviation
Deviations
• Factors affecting absorption properties of a
chromophore;
The spectrum of a chromophore is primarily determined by the chemical
structure of the molecule.
However a large number of environmental factors can cause detectable
changes in λmax and ε these factors are ;
• pH ; The pH of the solvent determines the ionization state of the
chromophore.
• The polarity of the solvent or or neighboring molecules. For polar
chromophores the value of λmax is affected , for example the λmax
For tyrosine is less in polar solvents.
• Orientation effects; Geometric features have strong effects on λmaxand ε
The best example of such an effect is the hypochromism of nucleic acids .
That is the absorption coefficient of a nucleotide decreases when the
nucleotide is contained in a single-stranded polynucleotide in which the
bases are in close proximity .There is a further decrease with a double
stranded polynucleotide because the bases are arranged in an even more
stacked array.
Steps in Developing a Spectrometric Analytical Method
1. Run the sample for spectrum
2. Obtain a monochromatic
wavelength for the maximum
absorption wavelength.
3. Calculate the concentration of
your sample using Beer Lambert
Equation: A = ECL
Wavelength (nm)
Practice Examples
1. Calculate the Molar Extinction Coefficient E at 351 nm for
aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the
following data which were obtained in 1 Cm cell.
Solution
C x 105 M
Io
I
A
2.23
100
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B
1.90
100
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2. The molar extinction coefficient (E) of compound
riboflavin is 3 x 103 Liter/Cm x Mole. If the absorbance
reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is the
concentration of compound riboflavin in sample?
3. The concentration of compound Y was 2 x 10-4 moles/liter and
the absorption of the solution at 300 nm using 1 Cm quartz cell
was 0.4. What is the molar extinction coefficient of compound
Y?