General Chemistry II

Module 6
Fundamentals of
Spectrophotometry

Electromagnetic Radiation

Plane polarized light

-All oscillations of either
the
electric or magnetic field
lie in
a single plane

Electromagnetic radiation is
conveniently
represented as electric and magnetic
fields
that undergo in-phase, sinusoidal
oscillations
at right angles to each other in the
direction of
propagation
Requires no supporting medium for its
transmission and thus passes readily
through a vacuum

Electromagnetic Radiation
C

Characterized by:
Wavelength
Frequency
Velocity
Amplitude

Electromagnetic Radiation
Amplitude (A) of a sinusoidal wave is the length of
the electric field vector at a maximum in the wave
Frequency () is the number of oscillations of the
field that occur per second observed at a fixed point
Determined by the source and independent of the
medium

Wavelength () is the linear distance between any

two equivalent points on successive waves
Like velocity is a function of the medium
Wavenumber (1/) as is directly proportional to the
frequency (and energy of the radiation)

Wave-Particle Duality
Wave model fails to account for phenomena
associated with the absorption and emission
of radiant energy
Particle model views radiation as a stream of
discrete particles, or wave packets, of energy
called photons
The energy of a photon is proportional to the
frequency of the radiation

Dual views of radiation as particles or waves

are complementary for elementary particles

Wave Parameters
In any medium containing matter, propagation of radiation
is slowed down by its interaction with the bound e-s of
the matter.
The refractive index is a measure of the interaction of
radiation with the medium
n = 1 for vacuum
1.3 1.8 for liquids
1.3 2.5 or higher for solids
The speed of light in any medium is c/n where n is the
refractive index of that medium

Electromagnetic Spectrum

Absorption of Light
absorption a process in which e/m energy is transferred
to the atoms, ions, or molecules composing a sample.
In this process, particles are excited to higher energy
excited states.
The lowest energy state of an atom, ion or molecule is
Planks constant
called the ground state.
Absorption occurs at discrete frequencies (E=hv) as a
consequence of the quantum nature of matter.
The frequencies of absorbed radiation are characteristic
of the material and can be used to identify species in
the matter and its environment.

Atomic Absorption
Absorption spectra are simple with absorption at only a
few well-defined frequencies because there are only a
small number of possible energy states for the
absorbing species.
UV and visible radiation can only excite transitions of
valence or bonding e-s.
X-ray radiation must be used to excite core e-s.
Absorption broadens in solvent or solids.

Absorption Processes

absorption spectroscopy the amount of light absorbed as a function of waveleng

ovides both qualitative (identification) and quantitative information about the sa

Emission Processes

Molecular Absorption
Absorption spectra are more complex because more
energy states are available
electronic, vibrational and rotational
Microwave radiation (and longer wavelength IR)
stimulates rotational motion of molecule when it is
absorbed.
Infrared radiation stimulates vibrations of molecules.
Visible and ultraviolet radiation causes electrons to be
promoted to higher energy orbitals, electronic
excitation.
X-rays and short-wave length UV break chemical bonds
and ionize molecules.

What is the wavelength in nanometers of yellow light which had

a frequency of
5.09 x 1014 1/s?

c =

13

The red spectral line of lithium occurs at 671 nm. Calculate the energy of
one photon of this light. E=hv and c = therefore E = hc/

=c/

14

Spectrophotometry
A monochromator is a device that separates the various
wavelengths of light present in a beam of light (prism,
grating, or even a filter) thus allowing the selection of a
single wavelength.
Light of a single wavelength is monochromatic (one
color).
Transmittance, T, is defined as the fraction of the incident
light that passes through the sample
T = P/Po

(0 T 1)

The percent transmittance is simply

100% x T

(0 T 100%)

Absorbance Measurement

Spectrophotometry
Absorbance is defined as

Other way around

A = log10(Po/P) = - log T
T = 10-A
Beers Law (Beer-Lambert Law)
A = bc
b = path length (cm), c = concentration (M)
molar absorptivity () characteristic of the substance, a
measure of how much light is absorbed at a particular
wavelength
17

Example: Find the absorbance, transmittance, and

percent transmittance of a 10.00 M solution of a

molecule with a molar absorptivity of 65,346 M-1cm-1 in
a 1.00 cm cell.

A= bc
= (65,346 M-1cm-1)(1.00 cm)(10.0 M)(1 M/106 M)
= 0.653
T = 10-A = 10-0.653 = 0.222
%T = T x 100% = 0.222(100%) = 22.2 %

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Spectrophotometry
absorption spectrum graph of the variation in A with
The portion of a molecule that is responsible for the
absorption of light is referred to as the chromophore
Substances that absorb light in the visible region appear
colored, the color(s) not absorbed by the substance.

19

Visible Absorption Spectrum of

(ferrozine)3Fe(II) in Analysis of Fe

20

Calibration Curve
In a chemical analysis by spectrophotometry, it is also
important to prepare a series of standard solutions to
establish a calibration curve to be used to determine
the concentration in the unknown samples.
Analysis is valid over the linear region of the calibration
curve.
A least-squares analysis of the calibration curve is used
to determine the unknown concentrations.

21

Calibration Curve

22

Example: Determine the concentration of a solution of

(ferrozine)3Fe(II) that exhibits an absorbance of 0.500
at a wavelength of 562 nm. The molar absorptivity of
the (ferrozine)3Fe(II) complex is 27,932 M-1cm-1 at 562
nm.
c = A/b
= (0.500)/(27,932 M-1cm-1)(1.00 cm)
= 1.79 x 10-5 M

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Limitations of Beers Law (1)

Beers law is an example of a limiting law and applies to
solutions < 0.01M. Solute-solute and solute-matrix
interactions effect the absorption properties of the solute
due to their proximity (average distance)
Apparent deviations arise when an analyte dissociates,
associates, or reacts with a solvent to produce a product
with a different absorption spectrum from the analyte
Most spectrometers produce polychromatic radiation with a
Guassian distribution centered around a particular
wavelength characterized by a band width. If the molar
absorption coefficient varies over this range of wavelengths
then deviations from linearity occur
(Deviations from Beers law are not significant if the
effective bandwidth of the monochromator is less than onetenth of the width of the absorption band at half-height)

Limitations of Beers law (2)

Normally the radiation exiting a
monochromator is contaminated by stray
light with a wavelength outside the nominal
wavelength range for the selected band. At
high concentrations and longer path
lengths stray radiation can be an important
source of non-linearity
Mismatched cells (optical quality) results in
an intercept term in the Beers law
expression. Can usually be easily handled
by linear regression calibration methods

Combined Electronic, Vibrational, and

Rotational Transitions
Changes in the vibrational and rotational states of a
molecule generally accompany an electronic transition.
Electronic absorption bands in molecules are generally
very broad because many different vibrational and
rotational levels are available at slightly different
energies.

What Happens to Absorbed Energy

After an electronic absorption transition occurs
(So S1)
vibrational relaxation (radiationless transition, R1)
vibrational energy is transferred to other
molecules or the solvent by collisions.
part of absorbed photon energy is converted to
heat.
The molecule could enter a highly excited
vibrational state of a lower energy electronic
state having the same energy (internal
conversion, IC), and relaxation may occur
through radiationless transitions, R2 as a result
of collisions.
In this way all absorbed energy is converted to

What Happens to Absorbed Energy

Molecule can cross into a different spin state, T1
(intersystem crossing, ISC), undergo further
radiationless relaxation R3, could then undergo
another intersystem crossing to So and then
radiationless relaxation R4.
In this way all absorbed energy is converted to
heat.

What Happens to Absorbed Energy

Molecule could relax by emission of a photon
S1 So is fluorescence, F
T1 So is phosphorescence, P
These transitions can terminate in any of the
vibrational levels of the So state.
Less common than other relaxation processes in
solution.

What Happens to Absorbed Energy

The lifetime of fluorescence is always very short
10-8 to 10-4 s
The lifetime of phosphorescence is much longer
10-4 to 102 s, and therefore less likely.
The relative rates of these various processes
depend upon
the nature of the molecule
the solvent
the experimental conditions (T, P)

Absorption vs Emission Spectra

In general, fluorescent and phosphorescence have
lower energy than that of the absorbed
radiation as a result of vibrational relaxation
occurring before emission.
molecules emit at longer s than those of the
radiation they absorb.
The emission spectrum is roughly the mirror image
of the absorption spectrum.
Wavelength of maximum absorbance / emission do
not coincide because of geometric relaxation
that occurs after absorption and before
emission (o emission > o absorption).

Excitation and Emission Spectra

Excitation and Emission Spectra

An emission spectrum is obtained by
fixing the excitation
scanning the emission
emission intensity vs emission
An excitation spectrum is obtained by
fixing the emission
scanning the excitation
emission intensity vs excitation
*An excitation spectrum looks very much like an
absorption spectrum.

Luminescence in Analytical Chemistry

If the absorbance of a solution is small at the
excitation wavelength, , then the emission
intensity is proportional to radiant power of
incident radiation and the concentration of
emitting species.
I = kPoc
Sensitivity can be increased by increasing the
incident radiant power.
Luminescence measurements are good for trace
analysis when absorbance is too weak to
measure.

Luminescence in Analytical Chemistry

Applications of Spectrophotometry
Analysis of a Mixture
When a mixture of species are present in a solution being
analyzed by spectrophotometry,
The absorbance detected corresponds to the sum of
the absorbances of all species.
The spectrophotometer is unable to differentiate the
absorbance from different species.

Applications of Spectrophotometry
Analysis of a Mixture
If the species present in the mixture have different
absorptivities at different wavelengths, then the
concentrations of the individual species can be
mathematically determined.
A = Xb[X] +Yb[Y] + Zb[Z} + . . .
where is the molar absorptivity of each species at the
wavelength in question and b is the cell pathlength.

Applications of Spectrophotometry
Case 1. Mixture overlaps significantly at all s
measurements at many are necessary.

Applications of Spectrophotometry
Case 2. Relatively little overlap in some regions,
measurements at 2 where one species dominates
at each.

Applications of Spectrophotometry
Case 2. Relatively little overlap in some regions,
measurements at 2 where one species dominates
at each.

Mixture of Two Compounds

Compound 1 has a dominant absorption at 1 and a
concentration c1
Compound 2 has a dominant absorption at 2 and a
concentration c2
The molar absorption coefficients for both
compounds at both wavelengths are known
AT,1 = 1b c1 + 1b c2 (1)
AT,2 = 2b c1 + 2b c2 (2)
Solve as a simultaneous equation with c 1 and c2 as
unknowns

Optical Instruments
Common components
1. stable source of radiant energy
2. transparent container for holding the
sample
3. device that isolates a restricted region
of the spectrum for measurement
4. detector (converts radiant energy to an
electrical signal)
5. software or electromechanical device
to process and display signal

Absorption Measurments

Double-Beam in Space
Instrument

Photodiode-Array
Spectrometer

General Instrument for Fluorescence

Nearly all fluorescence instruments employ double-beam optics to

compensate for fluctuations in radiant power

Prism Monochromator

Grating Monochromator

Photomultiplier Tubes

Spectrometer