Spectroscopic Methods of Analysis

Dr. M. Coley
 Introduction to Spectroscopy
Spectroscopy is the study of the interaction of
electromagnetic radiation with matter.
Spectroscopy involves the use of spectra, that is, the various
wavelengths of electromagnetic radiation.
Different types of spectroscopy may be identified based on the
wavelength range of Electromagnetic radiation used.

Electromagnetic radiation refers to a continuum light

source which ranges from gamma rays (λ = 10-12 nm),
which are of extremely short wavelength to radio waves
(λ = 103 nm) which have much longer wavelengths.
The Electromagnetic Spectrum
Interaction of EM Radiation with Matter
The Electromagnetic Spectrum
The wavelengths that comprise the electromagnetic
spectrum (EM) may be defined into specific regions.
EM of different wavelength range will interact with matter in
different ways.
These interactions may give information about the:
structure of molecules
nature of their bonds
functional groups present
concentration of species in solution

Different types of interaction with electromagnetic radiation

give rise to a different types of spectroscopy.
 Types of Spectroscopy

Infra-red spectroscopy ᴥ UV/VIS spectroscopy

X-Ray Spectroscopy ᴥ Mass Spectroscopy

 Properties of EM Radiation
EM radiation can undergo reflection, refraction & diffraction.
EM radiation can be characterized by wave properties, eg:
Wavelength
Amplitude
Frequency
Velocity
Wave number.

EM radiation can be characterized by particle properties, eg:

can be absorbed, emitted & transmitted
Particles of light are referred to as ‘photons’.
Photons can be considered as particles of energy that move through
space in a wave-like manner.
 Particle Properties of EM Radiation
The energy of a photon may be represented as:
E = hv
Since: c = vλ and v = c/λ
Hence: E = hc/λ but 1/λ = v

Therefore: E = hc/λ = hcv

Also: E = E1 – Eo = hc/λ

E is energy for transition of an electron from a ground state (Eo) to an excited state (E1).
Particle Properties of EM Radiation
Problem:
An organic molecule absorbs light of λ = 225 nm. Calculate:
(a) the energy of absorption per molecule;
(b) The energy of absorption per mole in kJ.
E = hc = 6.626 x 10-34 x 2.998 x 108 = 7.790 x 10-19 J/molecule
λ 255 x 10-9

E = 6.626 x 10-34 x 2.998 x 108 x 6.02 x 1023 x 1 kJ = 469 kJ/mol

255 x 10-9 1000 J
 Types of Spectroscopy
UV/VIS:
• Absorption of radiation causes
electronic transitions.
• Electronic transitions are from a ground
state to an excited electronic state.

Infrared:
• Absorption of radiation causes
vibrational transitions.
• Radiation absorbed causes bonds to:
vibrate, rotate, stretch, wag, bend, twist,
rock, etc.
• Different functional groups show
characteristic behaviour depending on
structure of molecule.
Infra-red Spectroscopy
 Interacts with molecules to cause
stretching, bending, rotation or
vibration of bonds.
 Characteristic vibrations are used to
identify various functional groups.

 useful for probing the structure of

organic molecules.
 Vibrational Transitions

C – H symmetrical C – H Asymmetric C – H In-plane scissoring

stretching 2925 cm-1 stretching: 2850 cm-1 1480 - 1440 cm-1

Out-of-plane twisting In-plane rocking Out-of-plane wagging

IR Spectrum of Formaldehyde
 How to Analyze IR Spectra
 Observe peaks left of Finger print region (>1250 cm-1)

 Pay attention to strongest absorptions:

C=O ; -OH ; -NH2 ; -C ≡N

 Stretching frequencies for bonds to carbon:

C-H > C-C > C-N > C-O
 C=O absorption suggests presence of aldehyde,
ketone, carboxylic acid, ester, amide;
• Stronger the bond - higher the stretching frequency.
• C≡C > C=C > C-C
• C≡N > C=N > C-N
Summary of IR Absorptions

Increasing wavenumber (energy, frequency)

IR Data Correlation Table
Functional Group Wavenumber, cm-1
Alcohol O-H 3650-3200
Acid O-H 3300-2500*
Phenol O-H 3600-3000*
Acid C=O 1725-1700
Ketone C=O 1725-1705
Aldehyde C=O 1740-1720
Ester C=O 1750-1730
Amide C=O 1690-1680
Primary amine N-H (stretch) Two bands 3500 and 3300
N-H (deform) 1650-1580
Secondary amine N-H (stretch) 3450-3300
N-H (deform) 1650-1550
Aromatic C=C (stretch) 1600
Alkene C=C (stretch) 1680-1620**
Alkyne CC 2260-2100
Nitrile CN 2400-2200
Aromatic C-H 3150-3000
Aliphatic C-H 3000-2850
* O-H stretches are broad due to hydrogen bonding
** weak bands
 IR Spectra - Alkene

C=C

sp2 C-H
sp3 C-H

Increasing wavelength
Increasing wavenumber (energy, frequency)

SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/
 IR Spectra – Alkyne

C≡C - 2120 cm-1

sp3 C-H str

sp C-H str

SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/
IR Spectra – Carboxylic acid
acid O-H str

sp3 C-H str

Acid C=O str

SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/
 IR Spectroscopy – Advantages &
Limitations
• Inexpensive & easy to use
• Used to identify functional groups of
organic compounds
• IR alone cannot determine a structure.
Other techniques required.
• Can identify unknown through
correspondence with known sample.
IR Spectroscopy – Sample Identification
IR Determination of Air Contaminants
 UV/VIS Spectroscopy
 is the application of visible or ultraviolet light in the
identification or quantification of analyte species

 applicable to a wide range of species:

– Metals – Fe, Mn, Cr
– Non-metals – PO43-, NO3-, SiO4 ,organics
– Any species that can absorb a measurable amount of UV or visible
radiation resulting in an atomic or molecular electronic transition
 Electronic Transitions
Eo E1

Photon of
energy:
E = hv
E = hc/λ
 Particle Properties of EM Radiation
The energy of a photon may be represented as:
E = hv
Since: c = vλ and v = c/λ
Hence: E = hc/λ but 1/λ = v

Therefore: E = hc/λ = hcv

Also: E = E1 – Eo = hc/λ

E is energy for transition of an electron from a ground state (Eo) to an excited state (E1).
The intensity of the energy, E, absorbed by species in a homogeneous solution is proportional to the number
 Beer-Lambert’s Law
“The amount of light absorbed by a homogeneous solution of absorbing
species that is exposed to monochromatic light is directly proportional to
the number of absorbing species present in the light path.”

ie: A = Ecl

A = Ecl
compare:
y = mx

Higher sample concentration, greater absorbance produced.

Calculating Absorbance & Transmittance
A = ─Log T = Log 1/T
If transmittance, T = 0.25, If absorbance is 0.015,
(i.e. 25%) calculate the calculate transmittance.
absorbance. A = 0.015
T = 0.25 0.015 = -Log T
A = log 1/T Log T = -0.015

A = log 1/0.25 T = 0.966

T = 96.6 %
A = 0.602
Determining Concentrations by UV-Vis spectroscopy
Prepare a series of standards of known  For coloured species, simply dilute
concentrations. standards and samples.

Measure absorbances of standards at λmax. CrO42- Cr2O72-

 For colourless species, add suitable
Measure absorbance of unknown at same colouring agent, eg. KSCN.
wavelength.
 Intensity of coloured complex is then directly
proportional to analyte concn.

λmax  Standards & samples should contain

same amount of colouring agent &
other additives (acids, etc.)

 Beer’s law requires pathlength in units

of cm & concentration: mol L-1.
 Using Beer’s law to determine Sample Concentration

Spectrophotmetric Determination of Iron

Fe(II) conc.
Solution %T Absorbance
(mg/L)

blank 0.000 100 0.000

1 0.102 94 0.056

2 0.511 77 0.119

3 1.02 60 0.194 Sample =

1.63 mg/ml
4 2.55 23 0.509

5 5.11 9 1.046

unknown ? 42 0.377
 Beer’s Law - Applications
Example:
A series of standard solutions containing Fe(III) concentrations as shown
in the table below, were made up in the presence of excess potassium
thiocyanate (KSCN) solution. A sample of unknown Fe(III) concentration
was developed similarly and when measured in a 1 cm cuvette, gave an
absorbance of 0.652.
i. From a graph, determine the concentration of the sample solution.
ii. Estimate the value of the molar absorptivity, E.

0.652 unknown
 Beer’s Law - Applications
Absorbance of
1.5 Sample = 0.652
y = 0.7792x
Absorbance

1
R2 = 0.9995 [Sample] =
0.836 mol L-1
0.5

0
0 0.5 1 1.5
Concentration Mol/L

From graph: [Fe(II)] = 0.836 mol L-1

Recall: A = Ecl
 0.652 = E x 0.836 mol L-1 x 1cm
 E = 0.652/(0.836 mol L-1 x 1cm)
 E = 0.779 mol-1 L cm-1
 Problem
Following a 1:10 dilution, a solution was measured at 820 nm to
determine its PO43- concentration. If the absorbance, A is 0.500, the
path-length, l is 20mm and the molar absorptivity, E is 100 M-1cm-1,
what is the concentration of the original solution?
A = Ecl
c = A/(E x l)
= 0.500/100 M-1cm-1 x 2 cm
= 0.0025M
Considering dilution factor:
Original PO43- Concn: = 10 x 0.0025M
= 0.025M
 Chromophores
A chromophore is the part of a molecule responsible for its colour.
The colour arises when the molecule absorbs certain wavelengths of
visible light and transmits or reflects others.
The chromophore is a region in the molecule where the energy difference between
two different molecular orbitals falls within the visible range.

Visible light that hits the chromophore can thus be absorbed by exciting an electron
from its ground state into an excited state.
Instrumentation:
Single-beam spectrophotometer
Spectrophotometer–
Double Beam