BASIC

INSTRUMENTAL
ANALYSIS
CHAPTER
CHM260 1
AN INTRODUCTION TO
SPECTROSCOPIC
METHODS OF
ANALYSIS
NOR AKMALAZURA JANI
 SUBTOPICS
Spectroscopy
Properties of electromagnetic radiations

The electromagnetic spectrum

Spectroscopic measurements

Absorption methods

Beers law

Interaction of electromagnetic radiation

with matter
Absorption spectra

Emission of electromagnetic radiation

 SPECTROS
COPY
Spectroscopy: The study of the interaction
between ELECTROMAGNETIC (EM)
RADIATION and MATTER.
The sample solution absorbs EM from an

appropriate source, and the amount absorbed

is related to the concentration of the analyte
in the solution.
Spectrophotometer/Spectrometer: The
instrument that determines the absorption
spectrum.
 SPECTROSCOPIC
ANALYSIS
covers

ATOMIC MOLECULAR
SPECTROSCOPY SPECTROSCOPY
 ELECTROMAGNETIC
RADIATIONS
Electromagneticradiation is the emission
and transmission of energy in the form of
electromagnetic waves.

EM shows wave like properties and

particle like properties (photon or
quanta) at the same time.
 I. WAVE-LIKE PROPERTIES

Energy radiated in the form of a WAVE caused

by an electric field interacting with a magnetic
field.
Result of the acceleration of a charged particle.

Does not require a material medium and can

travel through a vacuum.
Electric field
(red color)
Magnetic field
(blue color)
 WAVE PROPERTIES
EM radiation is conveniently modeled as waves
consisting of perpendicularly oscillating electric and
magnetic fields, as shown in the diagram.
At 90 to the direction of propagation is an oscillation in
the ELECTRIC FIELD.
At 90 to the direction of propagation and 90 from the
electric field oscillation (orthogonal) is MAGNETIC
FIELD oscillation.
 WAVE PARAMETERS
Period (p) the time required for one cycle to pass a fixed point
in space.

Frequency (v) the number of cycles which pass a fixed point in

space per second.
v = 1/p (s-1 = Hz)
v depends on the source, but is independent of the propagating
(transmitting) material.

Amplitude (A) The maximum length of the electric vector in the

wave (maximum height of a wave).

Wavelength () The distance between two identical adjacent

points in a wave (usually maxima or minima).
Period (p)
 Velocity of propagation (vi)
- The rate at which a wave front moves through a
propagating medium in meters per second (ms-1).
- how fast the wave is moving in the specific
medium.

vi = vi

Velocity of light (c)

c = 3.00 108 ms-1
= 3.00 1010 cms-1

c = v = 3.00 108 ms-1

 Wavenumber (v)
- The number of waves per cm in units of cm-1
v = 1 = v
c

Radiant Power (P)

- The amount of energy reaching a given area per second.
- Unit: watts (W).
- Total power of radiation emitted by a source (lamp, light
emitting diode, etc.), transmitted through a surface or
impinging upon a surface.
 Intensity (I)
- The radiant power emitted per unit solid angle.
- Describes the radiant power of a source emitted in a
certain direction.
- SI unit for intensity: watts per steradian (Wsr-1)
- Solid angle (): the size of area divided by the square
of the radius (r2) of the sphere. Unit solid angle is
steradian (sr).
= A/r2
 II. PARTICLE NATURE OF LIGHT:
PHOTONS
Wave theory failed to explain phenomena
associated with the absorption and emission of
radiation of radiant energy.
Thus, EM is viewed as a stream of discrete

particles, or wave packets of energy called

photons.
Energy of photon is related to the frequency,
wavelength and wavenumber by this equation:

E = hv = hc = hcv

h = Plancks constant = 6.63 10-34 J.s
c = 3.00 108 ms-1
Example:
If the wavelength used in an instrument is changed from
460 nm to 560 nm:
i) has the energy been increased or decreased?
ii) has the frequency been increased or decreased?
iii) has the wavenumber been increased or decreased?

Solution:
(refer to formula)

E = hv = hc = hcv

i) When increase, energy will decreased.

ii) When increase, frequency will decreased.
iii) When increase, wavenumber will decreased.
 SPECTRUM
The electromagnetic spectrum is arbitrarily broken down
into different regions according to the wavelength.
Regions of the UV, Visible and IR
Spectrum

Region Wavelength Range

UV 200 380 nm

Visible 380 780 nm

Near-IR 0.78 2.5 m

Mid-IR 2.5 15 m
 Wavelength Units for Various Spectral
Region

Region Unit Definition (m)

X-ray Angstrom unit, 10-10 m

Ultraviolet/ Nanometer, nm 10-9 m

visible
Infrared Micrometer, m 10-6 m
 Types of spectroscopic methods based on EM
radiation

1 = 10-10 m = 10-8 cm
1 nm = 10-9 m = 10-7 cm
1 m (micron) = 10-6 m = 10-4 cm
QUESTIONS:
Consider an electromagnetic radiation with a
frequency of 5.25 x 1013 Hz.

i) Calculate the wavelength (in microns) of the

radiation.
ii) Name the region in which this
electromagnetic radiation is located.
iii) What is the energy (in Joules) of the
radiation?
ANSWERS:

= 5.71 m
ii) Region: Infrared
iii)E = 3.48 x 10-20 J
 S
HOW DOES MATTER ABSORB RADIATION?

The absorption of radiation can be obtained by

considering the absorption of light in the visible.
The color of an object we see is due to the
wavelengths transmitted or reflected. The other
wavelengths are absorbed.
Polychromatic light (white light) is visible light that
contains all the colors of the rainbow.
If white light is passed through an object, the object
will absorb certain of the wavelengths, leaving the
unabsorbed wavelengths to be transmitted (seen as a
color with our eyes).
 Color of Different Wavelength Regions

Wavelength Absorbed color Transmitted Color

Absorbed (nm) /Observed Color
(Complement)
380-450 Violet Yellow-green
450-495 Blue Yellow
495-570 Green Violet
570-590 Yellow Blue
590-620 Orange Green-blue
620-750 Red Blue-green
 Solution of
KMnO4

Example:
- A solution of KMnO4 absorbs light in the
green region of the spectrum with an
absorption maximum of 525 nm, and the
solution is purple/violet (observed color).
Wavelength Absorbed color Transmitted Color
Absorbed (nm) /Observed Color
(Complement)
380-450 Violet Yellow-green

450-495 Blue Yellow

495-570 Green Violet

570-590 Yellow Blue

590-620 Orange Green-blue

620-750 Red Blue-green

 ABSORPTION
METHODS
ABSORPTION
METHODS

TRANSMITTANCE (T) ABSORBANCE (A)

 TRANSMITTANCE (T)
The fraction of incident radiation (electromagnetic
radiation) that passes through the sample medium.

A = Absorbance
b = path length

Commonly expressed as
a percentage:

% T = P x 100 %
P0
 ABSORBANCE (A)

- A measurement of the amount of radiant power

absorbed by the sample defined as the negative log of
transmittance.
- Absorbance has a linear relationship or directly
proportional with sample concentration defined by
Beers Law.

A = Absorbance
b = path length
QUESTION 1:
Convert the following percent transmittance
data into absorbance:

i) 33.6
ii) 92.1
iii) 1.75

Answer: i) 0.474
ii) 0.0357
iii) 1.76
ANSWER:
i) %T = 33.6
T = 33.6 / 100
= 0.336

A = - log T
= - log 0.336
= 0.474
QUESTION 2:
Convert the following absorbance data into
percent transmittance:
i) 0.375
ii) 1.325
iii) 0.012

Answer:
i) A = - log T
T = Antilog (-A) %T = 0.4212 x 100
= Antilog (-0.375) = 42.12%
= 0.4212
ii) 4.73%
iii) 97.3%
 BEERS LAW
For monochromatic radiation, absorbance is directly
proportional to the path length (b) through the medium
and the concentration (c) of the absorbing species.
These relationships are given by this following
equation.

A = abc
Concentration
Absorbance (g/liter)

Absorptivity Pathlength
(Lg-1cm-1) (cm)
-The term a is a proportionality constant called
absorptivity.
- Absorptivity is a constant for a given chemical species
at a specific wavelength.
* Monochromatic: light of a narrow frequency.
 BEERS LAW
When the concentration is expressed in moles per
liter and cell length is in cm, the absorptivity is
called the molar absorptivity and the symbol is
and the unit for is liter mol-1 cm-1

A = bc
= molar absorptivity, liter mol-1 cm-1
b = sample path length, cm
c = concentration, mol per liter
 EXAMPLES:
1. What is the concentration of an absorbing
species if its molar absorptivity is 1500
L/mol.cm and the measured absorbance in
a 1.00 cm cuvette is 0.742?

Answer:
A = bc
c = A / b
c= 0.742
(1 cm)(1500 L/mol.cm)
= 4.95 10-4 M
2. The measured absorbance of a sample in a 1.00 cm
cuvette is 0.544. If the concentration is 1.40 10-3 M,
what is the molar absorptivity for the species?

Answer:

A = bc
= A / bc
= 0.544
(1 cm)(1.40 10-3 mol/L)
= 389 L/mol.cm
3. A sample in a 1.0 cm cell is determined with a
spectrometer to transmit 80% light at a certain
wavelength. If the absorptivity of this
substance at this wavelength is 2.0, what is the
concentration of the substance?

Answer:
The percent transmittance is 80%. So, T=0.80
A = abc
- log T = 2.0 L/g.cm 1 cm c
- log 0.80 = 2.0 L/g c
c = 0.0969
2.0 L/g
= 0.0485 g/L
PARAMETERS THAT AFFECT ABSORBANCE

A = abc

Concentration, c
Width of cuvette, b

Inherent ability for absorbing species

to absorb light
i) Width of cuvette, b: b
- Wider cuvette consist of more absorbing
species present in the path of the light,
hence absorbance is greater.

ii) Inherent ability for the absorbing species to

absorb light
- Chemical species vary with respect to this
inherent ability since absorption depends on
individual electronic and vibrational transitions
available in a given species.
 APPLICATION OF BEERS LAW
Applying Beers Law to Mixtures

- Beers law also applies to solutions containing

more than one kind of absorbing substance.
- The species do not interact.
- Total absorbance for a multicomponent system at a
single wavelength is the sum of the individual
absorbances.

Atotal = A1 + A2 + + An
= 1bc1 + 2bc2 + + nbcn
 LIMITATIONS TO THE APPLICABILITY OF
BEERS LAW

Deviations are frequently observed from the direct

proportionality between absorbance, A and
concentration, c when pathlength, b is constant.

Deviations may be due to:

1. Real deviations
2. Instrumental deviations
3. Chemical deviations
 1. Real deviations
- fundamental deviations due to the limitations of the law
itself.
- Beers law: the absorption behavior of media containing
low analyte concentration.
- At high concentration (>0.01M) the extent of solute-
solvent interactions, solute-solute interactions, or
hydrogen bonding can affect the analyte environment
and its absorptivity.
- At high concentration, the average distances between
the molecules and ion are diminished where each
particle affects the charge distribution of its neighbors.
- Therefore, this interaction alter the ability of analyte
species to absorb a given wavelength of radiation.
- Causing deviation from the linear relationship between
absorbance and concentration.
2. Instrumental deviations

a) Due to polychromatic radiation

- Polychromatic: describe light that exhibits more
than one color, which also means that it contains
radiation of more than one wavelength.
- Beers Law strictly applies when measurements are
made with monochromatic source radiation.
- In practice, however, it is common to use a
polychromatic source of radiation with continuous
distribution of wavelengths along with a filter or a
grating unit (monochromators) to create a
monochromatic beam from this source.
- Deviations occur if the radiation is polychromatic
since the relationship between A and c is no longer
linear when is differ.
 Why absorption measurements are taken at wavelength
of maximum absorbance max?

- max = Wavelength at maximum absorbance

- If the band of wavelength selected on the spectrometer
is such that the molar absorptivities of the analyte is
essentially constant, deviations from Beers law are
minimal.

- However, if a band is chosen such that the molar

absorptivity of the analyte at these wavelengths
changes a lot, the absorbance of the analyte will not
follow Beers law.

- The deviations in absorbance over wavelengths is

minimal when the wavelength observed is at the max.
Due to this reason, absorption measurements are
taken at wavelengths.
b) Due to presence of stray radiation

- Due to instrument imperfections.

- Stray radiation or scattered radiation is defined as radiation
from the instrument that is outside the nominal wavelength
band selected.
- Usually the wavelength of the stray radiation is very different
from the wavelength band selected. It is known that radiation
exiting from a monochromator is often contaminated with
minute quantities of scattered or stray radiation.
- This stray radiation is due to reflection and scattering by the
surfaces of lenses, mirrors, gratings, filters and windows.
- The wavelength of stray radiation differs greatly from the
principal radiation and may not have passed through the
sample.
- If the analyte absorbs at the wavelength of the stray
radiation, a deviation from Beers law is observed similar to
the deviation due to polychromatic radiation.
- When measurements are made in the presence of
stray radiation, the observed absorbance, A is
given by this equation.

A = log P0 + Ps
P + Ps

Ps - power of non-absorbed stray radiation

% stray radiation = Ps 100

P0
c) Mismatched Cells

If the cells holding the analyte and the blank

solutions are having different path-lengths, or
unequal optical characteristics, it is obvious that
there would be a deviation observed in Beers law.

In such cases when a plot of absorbance versus

concentration is made, the curve will have an
intercept k and the equation will be defined as:

A = bc + k
3. Chemical deviations

- Occur when the analyte undergo dissociation,

association or reaction with the solvent to give
products that absorb differently than the
analyte.

- Example: the ionisation of acidic or basic

indicator
Example:
- Phenol red undergoes a
resonance transformation
when moving from the acidic
form (yellow) to the basic form
(red).

- Due to this resonance, the

electron distribution of the
bonds of molecule changes
with the pH of the solvent in
which it is dissolved.

- The absorption spectrum of

the sample changes with the
change in pH of the solvent.
 INTERACTION OF
ELECTROMAGNETI
C RADIATION WITH
MATTER
 The interaction of radiation with matter can cause
redirection of the radiation and/or transitions between
the energy levels of the atoms or molecules.
A transition from a lower level to a higher level with
transfer of energy from the radiation field to the atom
or molecule is called absorption.
A transition from a higher level to a lower level is called
emission if energy is transferred to the radiation field,
or nonradiative decay if no radiation is emitted.
Redirection of light due to its interaction with matter is
called scattering, and may or may not occur with
transfer of energy, i.e., the scattered radiation has a
slightly different or the same wavelength.
ABSORPTION
SPECTRA
 When radiation pass through a layer of solid,
liquid, or gas, certain frequencies may be
selectively removed by absorption (a process in
which electromagnetic energy is transferred to the
atoms, ions, or molecules composing the sample.

For absorption of radiation to occur, the energy of

the excited photon must exactly match the energy
difference between the ground state one of the
excited states of the absorbing species.
 ATOMIC
ABSORPTION
The passage of polychromatic ultraviolet or visible radiation
through a medium that consist of monoatomic particles, such as
gaseous mercury or sodium, results in the absorption, but a few
well-defined frequencies.
The spectra is simple due to the small number of possible
energy states for the absorbing particles.
Atomic spectra: sharp, well defines lines
Example:
- Sodium vapor exhibits two closely spaced, sharp absorption
peaks in yellow region of the visible spectrum (589.0 and 589.6
nm).
- Reason: Excitation of the 3s electron and two 3p states that
differ only slightly in energy.

Absorbance

Wavelength, nm
 MOLECULAR ABSORPTION
More complex than atomic spectra because
the number of energy states of molecules is
generally large when compared with the
number of energy states for isolated atoms.
Energy associated with the bands of a
molecule is made up of three components:

E = Eelectronic + Evibrational + E rotational

Spectra of benzene vapor:
- a series of closely spaced absorption lines:
rotational energy levels associated with each
vibration state.
Spectra of benzene in hexane:
- broad smooth peaks: using high-resolution
instrument
Spectra of biphenyl in hexane:
- continous spectra: in the condensed state
and in the solvent molecules
 PROCESS OF MOLECULAR TRANSITION
Process of molecule to absorb radiation.
i) Rotational Transition
- Molecule rotates about various axes.
- Erotation being at definite Elevels
- Therefore, the molecule absorb radiation and be raised to a
higher rotational energy level.
- Occurs at very long wavelengths
ii) Vibrational Transition
- Atoms or group of atoms within the molecules vibrate relative
to each other.
- Evibration occur at quantized levels
- Therefore, the molecule absorb radiation and be raised to a
higher vibrational energy level.
iii) Electronic Transition
- Electron of a molecule may be raised to a higher electron
energy .
 ENERGY LEVEL DIAGRAM OF MOLECULAR TRANSITIONS

Energy level:
Electronic > vibrational > rotational
rotational transitions microwave or far-infrared region
vibrational transitions near-infrared region
electronic transitions visible and ultraviolet regions
ELECTROMAGNETI
C RADIATION
 EMISSION OF RADIATION
Electromagnetic radiation is produced when excited
particles (atoms, ions, or molecules) relax to lower
energy levels by giving up their excess energy as photons.

Source of excitation:
i) bombardment with electrons or other elementary
particles. Leads to the X-radiation.
ii) Exposure to an electric current, an ac spark, or an
intense heat source (flame, dc arc, or furnace). Produce
UV, visible or infrared radiation.
iii) Irradiation with a beam of electromagnetic radiation.
Produces fluorescence radiation.
iv) An exothermic chemical reaction that produces
chemiluminescence.
 LINE
SPECTRA
Made up of a series of sharp, well-defined peaks.
Caused by excitation of individual atoms.

Atomic transitions are usually very discrete

changes of electrons from one quantum state to
another energy level (shells, spins, etc).
Only electronic transition is quantized.

No vibrational or rotational transition.

 When an atom changes energy
state, it absorbs or emits energy
equal to the energy difference
E = E1 E0

The wavelength or frequency of

radiation absorbed or emitted
during a transition proportional
to E

E = hv= hc

Produce line spectra

E0 lowest energy electronic level or ground state

E1, E2 higher-energy electronic levels
 BAND
SPECTRA
Encountered in spectral sources when
gaseous radicals or small molecules are
present.
Molecular transition consists of 3 processes:

i) Rotational transition
ii) Vibrational transition
iii) Electronic transition
E = Eelectronic + Evibrational + E rotational

Band spectra is produced due

to vibrational and
rotational
transitions.

Energy
 CONTINUUM SPECTRA
Continuum spectra: A beam of light that contains a broad,
smooth distribution of photon wavelengths.
Continuum radiation is produced when solids are heated to
incandescence.
Thermal radiation is called black body radiation.
- is characteristic of the temperature of the emitting surface
rather than the material of the surface.
- produced by the innumerable atomic and molecular
oscillations excited in the condensed solid by the thermal
energy.
* Energy peaks shift to shorter
wavelengths with increasing
temperature
Emission spectrum of a brine sample obtained with an
oxyhydrogen flame

Consists of the
superimposed
line, band and
continuum
spectra as
constituents of
the sample
 Line spectra

Band spectra

Continuum spectra