Analytical Chemistry

Lecture 6
Spectroscopic methods of analysis –
Part 1
Principles of spectrometric methods

Instructor: Nguyen Thao Trang

Spectroscopic methods
• Are based on the interactions between light and
matter.

• The science that studies these interactions is called

as spectroscopy

• The interactions can be at molecular or atomic level.

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Light, electromagnetic radiation
• Electromagnetic radiation (EM): a form of energy whose
behavior is described by the properties of both waves and
particles.
• Wave properties:
– EM moves through space as a wave.
– Light waves consist of perpendicular, oscillating electric and magnetic
fields.

– Speed of light: in vacuum, velocity (c) of light is 2.99792 x 108 m s-1 ; in

other medium: velocity (v) is less than c. Difference is small enough to
set c = 3.00 x 108 m s-1 in all purposes. 3
Light, electromagnetic radiation
• Wave properties:
– The interaction of electromagnetic radiation with matter can be
explained using either the electric field or the magnetic field.

– The oscillating electric or magnetic field is described by a sine wave of

the form:

Ae: maximum amplitude

ν: frequency
φ: phase angle

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Light, electromagnetic radiation
• Wave properties:
– Frequency ν: # of oscillations of an electromagnetic wave/s.

– Wavelength λ: distance between any 2 consecutive maxima or

minima of an electromagnetic wave.

– Wave number 𝝂": can be used to describe the wave properties of

electromagnetic radiation.

(cm-1)

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Light, electromagnetic radiation
• Wave properties:
– Effect of medium on light wave:
• Frequency remains the same

• Velocity and wavelength change

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Light, electromagnetic radiation
• Particle properties: consider light as consisting of energetic
particles, called photons
– Energy of a photon is related to its wavelength, frequency, and
wavenumber by:

where, h is Planck’s constant (6.63 x 10-34 J s).

1 erg = 10 –7 J ; 1 eV = 1.6021 × 10 – 19 J

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Light, electromagnetic radiation
• Electromagnetic spectrum: covers an enormous range of
energies (frequencies) and thus wavelengths.
• Boundaries describing the electromagnetic spectrum are not
rigid, and an overlap between spectral regions is possible.

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Light, electromagnetic radiation

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Light, electromagnetic radiation
Designation Wavelength Energy or Transition
range wave number

Cosmic ray
10 – 12 m
γ-ray Nuclear
10 – 11 m >2.5 × 105 eV
X-ray K,L shell electron
10 – 8 m 124 eV
Vacuum UV Middle shell
180 × 10 – 9 m 7 eV
near UV Valence electron
380 × 10 – 9 m 3.3 eV
Visible Molecular electron
780 × 10 – 9 m 1.6 eV
Near IR Molecular vibration
2500 × 10 – 9 m 4000 cm –1
Middle IR Molecular vibration
50 × 10 – 6 m 200 cm –1
Far IR Molecular rotation
10 – 3 m 10 cm –1
Microwave Molecular rotation
0.3 m Electron, & nuclear
Radio wave spin

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Energy state of a chemical species
• Atoms, ions and molecules exist only in discrete states
characterized by definite amounts of energy.
• A species is predominately in its lowest-energy or ground
state.
• Upon being stimulated by absorbing energy, a species can
undergo a transition to a higher-energy or excited state.

Excited state

Ground state

• Species in the excited state can emit energy to return to the

ground state. 11
Energy state of a chemical species
• Absorbs or emits an amount of energy exactly equal to that of
the difference between states.
– Energy absorbed by a species to make that species jumps from the
ground state to the 1 st excited state or energy released when it returns
to the ground state from the 1st excited state is given by:
ℎ𝑐
Δ𝐸 = 𝐸& − 𝐸( = ℎ𝜈& =
𝜆&
– Energy absorbed by a species to make that species jumps from the
ground state to the 2nd excited state or energy released when it
returns to the ground state from the 2nd excited state is given by:
ℎ𝑐
Δ𝐸 = 𝐸- − 𝐸( = ℎ𝜈- =
𝜆-
• Frequency or wavelength are related to the energy difference.
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Spectroscopic measurement
• Use interactions of radiation with mater to obtain information
about a sample.

• A sample is stimulated by applying energy in the form of heat,

electrical energy, light, particles, or a chemical reaction.

• Information about the sample can be obtained by measuring:

– Amount of electromagnetic radiation absorbed as a result of
excitation.

– Measuring the electromagnetic radiation emitted as it returns to the

ground state from the excited state.

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Spectroscopic measurement
• Absorption:
– Sample is stimulated by application of an external electromagnetic
radiation source.
– Some of the incident radiation can be absorbed and promote some of
the analyte species to an excited state.

– Amount of light absorbed as a function of wavelength is measured,

which can give qualitative and quantitative information about the
sample à absorption spectroscopy.

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Nguyen, T. T.; Rembert, K.; Conboy, J. C. J. Am. Chem. Soc. 2009, 131, 1401-1403
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Absorption process
• Absorbance and transmittance
power of the incident power of the
radiation transmitted radiation

Transmittance T is defined as:

𝑃0 𝑃0
𝑇= or %𝑇 = ×100%
𝑃1 𝑃1
Absorbance A is defined as:
𝑃0 𝑃1
𝐴 = − log 𝑇 = −𝑙𝑜𝑔 = 𝑙𝑜𝑔
𝑃1 𝑃0
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Absorption process
• Absorbance and transmittance

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Absorption process
• Absorbance and transmittance:
– Reflection losses at the cell walls and scattering losses at surfaces of
particles in the solution also contribute to the attenuation of the
radiation.

– To correct for these effects, a blank containing no sample must be

used in the same condition. 18
Absorption process
• Beer’s law, relationship between absorbance and
concentration:
𝑃1
𝐴 = 𝑙𝑜𝑔 = 𝑎𝑏𝑐
𝑃0
– radiation.
a is a proportionality constant
called the absorptivity.
b is the pathlength of the absorbing
medium.
c is the concentration of the
absorbing species.
A is unitless.

– If c is the molar concentration and b is in cm, a is called the molar

absorptivity, expressed as ε (L.mol-1.cm-1), thus: 𝐴 = 𝜀𝑏𝑐

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Absorption process
• Beer’s law, relationship between absorbance and
concentration:
𝑃1
𝐴 = 𝑙𝑜𝑔 = 𝑎𝑏𝑐 𝑜𝑟 𝐴 = 𝜀𝑏𝑐 𝐴 = − 𝑙𝑜𝑔 𝑇 → 𝑙𝑜𝑔 𝑇 = −𝑎𝑏𝑐
𝑃0

A
log T

[C] [C]

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Absorption process

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Absorption process
• Beer’s law, relationship between absorbance and
concentration:
Example:

A 5.00 x 10–4 M solution of an analyte is placed in a sample cell that has a

pathlength of 1.00 cm. When measured at a wavelength of 490 nm, the
absorbance of the solution is found to be 0.338. What is the analyte’s
molar absorptivity at this wavelength?

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Absorption process
• Beer’s law, relationship between absorbance and
concentration:
– Calibration curves based on Beer’s law are used routinely
in quantitative analysis.

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Absorption process
• Beer’s law, relationship between absorbance and
concentration:
– 2 simple approaches to use Beer’s law to determine the
concentration of an analyte:
1. The absorptivity is known or has been found using a standard

– Example: A solution of Co(H2O)2+ has an absorbance of 0.20 at 530 nm

in a 1.00 cm cell. ε is known to be 10 L.mol-1.cm-1. What is its
concentration?
– Solution:
Beer’s law: A = εbc
Where: A = 0.20, ε = 10 L.mol-1.cm-1, b = 1cm
C =A/εb = 0.20/ (10x1) = 0.020 M

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Absorption process
• Beer’s law, relationship between absorbance and
concentration:
– 2 simple approaches to use Beer’s law to determine the
concentration of an analyte:
2. Ratio method (ratio of a known and unknown):

– Example: The absorbance of an unknown MnO4– solution is 0.500 at

525 nm. When measured under identical conditions, a 1.0x10-4 M
MnO4– is found to have an absorbance of 0.200. Determine the
concentration of the unknown.
– Solution:
Known MnO4– solution : 𝐴CD(ED = 𝜀𝑏𝐶CD(ED
Unknown MnO4– solution : 𝐴GDCD(ED = 𝜀𝑏𝐶GDCD(ED
HIJKLJ NIJKLJ
= à 𝐶GDCD(ED = 1.0x10-4 . (0.500/0.200) = 2.5x10-4 M
HMJIJKLJ NMJIJKLJ

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Absorption process
• Application of Beer’s law to a mixture:

– Can be applied to solutions containing more than 1 absorbing

substance if no interactions among the various substances:

Atotal = A1 + A2 + … An
= ε1bc1 + ε2bc2 + … + εnbcn
where the subscripts refer to absorbing components 1, 2, …,
n.

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Absorption process
• Limits to Beer’s law:
Concentration deviations:
– Beer’s law describes absorption behavior of dilute solutions only.
– At a more concentrated solution (~ 0.01 M), average distances
between ions or molecules are small enough to make the absorbing
species no longer behave independently of one another à deviations
from the linear relationship.

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Absorption process
• Limits to Beer’s law:
Concentration deviations:

Working range: 15%T(0.824A)~80%T(0.097A)

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Absorption process
• Limits to Beer’s law:
Chemical deviations:
– Absorbing species undergoes association, dissociation, or reaction
with the solvent to give products that absorb differently from the
analyte.

– Typical equilibria that give rise to this effect include monomer dimer
equilibria, metal complexation equilibria where more than one
complex is present, acid/base equilibria, and solvent-analyte
association equilibria.

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Absorption process
• Limits to Beer’s law:
Instrument deviations: Polychromatic radiation
– Beer’s law is only applied for measurements that are made with
monochromatic radiation (single wavelength).
– When a polychromatic radiation (multiple wavelengths) is used:
deviation from linear behavior will be observed.

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Absorption process
• Absorption spectrum:
– A plot of absorbance versus wavelength, wavenumber or frequency.
– A plot of %T versus wavelength produced by older instruments.
– A plot with log A.
– A plot of molar absorptivity ε as a function of wavelength is
independent of concentration.

Typical absorption spectra of

KMnO4 at five different
concentrations.

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