INTRODUCTION TO

SPECTROSCOPIC METHODS OF

ANALYSIS

1
 Introduction

 Spectroscopic methods of analysis are powerful tools for the study of

atomic and molecular structures.

 Analysis is rapid, nondestructive and highly sensitive.

 Used for the analysis of wide range of samples:

2
 Introduction

 Spectroscopic techniques are based on an atom or compound’s interaction with

electromagnetic radiation (EMR) of specific wavelength ().

 Spectroscopy can be used to determine the:

(a) presence or absence of an element such as a metal

(Flame Tests and Emission spectroscopy)
(b) amount of an element present
(Colorimetry, Atomic Absorption Spectroscopy (AAS), UV-Vis Spectroscopy)
(c) structure of a compound
(Mass Spectroscopy (MS), 1H NMR Spectroscopy, 13C NMR Spectroscopy,
Infrared Spectroscopy (IR))

 Spectroscopic technique may rely on either:

(a) emission of energy from the substance being investigated

(Flame Tests and Emission spectroscopy)
(b) absorption of energy by the substance being investigated
(Colorimetry, Atomic Absorption Spectroscopy (AAS), UV-VisSpectroscopy)
3
 Flame Tests
 Performed by introducing a sample into the blue flame of a Bunsen burner and
noting any change in the color of the flame.

 The heat the flame causes electrons in the element to move from their lower
energy ground state to a higher energy excited state. When these excited
electrons fall back to their lower energy levels,
 they emit energy in the form of light of a characteristic wavelength which
is the color of the flame we observe.

4
 Flame Tests

Features Uses

1. Inexpensive. Identification of metals in

2. Fast, but some colors may be masked by some salts
others making it unreliable. • Li+ crimson
3. Qualitative technique (Not quantitative) • Na+ yellow
4. Limited application as many common metals • K+ lilac
(e.g., Al & Mg) do not color a flame. • Ca2+ brick red
5. A type of emission spectroscopy which • Sr2+ scarlet
can be observed by the naked eye. • Ba2+ green
• Cu2+ green-blue

5
 Colorimetry
 Used to measure the absorbance of light by a colored solution.
The amount of light absorbed is measured and recorded as the
"absorbance (A)".

 The wavelength is selected for maximum absorption for the colored solution.
We can use a "color wheel" to determine the color of light to use.

 The intensity of the color of a solution is related to the concentration of the

solution.
The more concentrated the solution is, the more intense the color will
be.
6
 Colorimetry

Features Uses

1. Relatively inexpensive • copper(II) sulfate in garden

2. Fast, requiring a set of standard sprays.
solutions • iron(III) concentration using
3. Qualitative & quantitative KSCN to form the colored
4. A type of absorption spectroscopy Fe(CNS)2+ complex
limited to colored solutions or • colored solutions such as food
compounds colorings

 wavelength of light is selected for maximum absorbance &

 calibration curve is constructed of A = f [concentration].

7
Colorimetry

8
 Determining the Concentration of a Solution of CuSO4(aq)
1. Determine  (color) of light to use for the colorimetric analysis.

2. Prepare a stock solution of aqueous copper sulfate.

3. Prepare dilute copper sulfate solutions, calculating the concentration of each solution.

4. Use the colorimeter to measure the absorbance of each of these solutions of known concentration.

[CuSO4(aq)] (mol /L) Absorbance (λ = 635 nm)

0.00 0.000
0.10 0.280
0.20 0.560
0.30 0.830
0.40 1.110
0.50 1.390

5. Next we plot these results on a graph and draw a "line of best fit" through the data points.

6. Now we measure the absorbance of our copper sulfate of unknown concentration.

Example: Absorbance of unknown solution = 0.950

9
Reading off the graph we see that an absorbance of 0.950 corresponds to a concentration
of 0.34 mol / L.
 UV-visible Spectroscopy

 UV-visible spectroscopy is considered an important tool in analytical

chemistry.

 This tool is used for identification of chemicals (qualitative analysis) and

for determination of different organic and inorganic compounds in solution
(quantitative analysis).

Spectroscopy is the measurement of the interaction of light (or

electromagnetic radiation) with matter.

 Electromagnetic Radiation (EMR) is a radiant energy that is transmitted

through space in the form of waves. It has both wave and particle
10
properties.
 Wave Properties

EMR is conveniently modeled as waves consisting of perpendicularly

oscillating electric and magnetic fields
y

x
z Electric Field
Magnetic Field

Direction of
propagation

a. Wavelength (λ) is a linear distance between successive maxima or

minima of a wave.
b. Frequency (ν) is the number of waves or cycles per second.

+ Wavelength ()
Electric Field

Amplitude (A)
0
11

- Time or Distance
 Wave Properties
The relationship between λ and ν:
Speed of light = Wavelength x Frequency
C = λν
λ = C/ν
ν = C/λ
For electromagnetic waves, the speed of light (C) is a constant (3.00x108 m/s).

Electromagnetic spectrum

12
 Definitions

Period (p): the time required for one cycle to pass a fixed point in space.

Frequency (): the number of cycles which pass a fixed point in space per
second.

Amplitude (A): The Maximum height of a wave.

Wavelength (): The distance between two identical adjacent points in a wave
(usually maxima or minima).

Wavenumber (): The number of waves per cm in units of cm-1.

Radiant Power (P): The amount of energy reaching a given area per second.
(Unit in watts)

Intensity (I): The radiant power per unit solid angle. 13

 Particle Properties of Light Photons

• EM is viewed as a stream of discrete particles, or

wave packets, of energy called photons.
• We can relate the energy of photon to its wavelength,
frequency and wavenumber by
E = h  - frequency
=hc  - wavelength
 υ - wavenumber
= hcυ

 h: Planck’s constant = 6.63x10-34 J·s

14
 Particle Properties of Light Photons

Example: What is the energy of a 500 nm photon?

 = c/ = (3 x 108 m/s)/(5.0 x 10-7 m) = 6 x 1014 s -1
E = h  = (6.626 x 10-34 J•s)(6 x 10 14 s-1) = 4 x 10-19 J

REGIONS OF THE UV, VISIBLE AND IR SPECTRUM

Region Wavelength Range

UV 180 – 380 nm

Visible 380 – 780 nm

Near-IR 0.78 – 2.5 μm

Mid-IR 2.5 – 50 μm 15
 Spectroscopic Measurements

Absorption Methods
 Absorption is the measure of the decrease in radiant power.
 Quantitative absorption methods require 2 power measurements:
- Po= incident radiant power
- P = transmitted radiant power
Po P
Transmittance Absorbing solution

 The fraction of incident radiation transmitted through the sample

medium.
T = P/Po

Transmittance is commonly expressed in %.

% Transmittance: % T = (P/Po) x 100

 A measurement of the amount of radiant power absorbed by the

sample defined as the - log of T.
16

A = - log T = - log P/Po

Question: Convert the following absorbance data into percent
transmittance:
i) 0.375
ii) 1.325 % T = (P/Po) x 100
iii) 0.012

Answer: A = - log T = log P/Po

i) 42.2%
ii) 4.73%
iii) 97.3%

17
 Absorption of UV-Vis Radiation

Different molecules will absorb different wavelengths of light. The

wavelengths of light for UV-visible absorption are (200 nm to 800 nm).

1. The energy of a certain wavelength in UV-visible region being absorbed

to a molecule.

2. The light/energy excites the ground state (E0) valence electrons to an

excited state (E1).

3. The energy from the absorbed UV light is equal to the energy difference
between the higher energy state and the ground state.

18
 Basic Principle of UV-Vis Spectroscopy

 UV-Vis spectrophotometer principle follows the Beer-Lambert Law.

Beer-Lambert Law
 Shows linear relationship between absorbance (A), concentration (C) of
the species measured, sample path length (b) and the absorptivity (a)
 Beer-Lambert Law is expressed through this equation:
A=εbC
Where:
A: Absorbance
ε : Absorptivity (L/mol cm)
b: Path length (cm)
C: Concentration (mol/L) 19
 Applications of Beer’s Law

 Determination of unknown concentration by measuring absorbance.

Example: A tea solution in a 0.5 cm sample cell gave an absorbance

reading of 0.64 in blue light (420-440 nm). If the molar absorptivity for the tea
is 120 L / mol cm.
Using the same light, calculate the concentration and the transmittance
of the solution.

Answer:
i) C = A / ε b = 0.64 / 120x0.5 = 0.011 M
ii) T = 10 –A = 10 -0.64 = 0.23
23% of the power of the incident beam is transmitted. 20
 Instrumentation
Components of Spectrophotometer
Detector
Light source
Mono-chromator
Sample cell
Recorder

Types of source, sample holder and detector for various EM region

REGION SOURCE SAMPLE HOLDER DETECTOR

Ultraviolet Deuterium lamp Quartz/fused silica Phototube, diode array

21
Visible Tungsten lamp Glass/quartz Phototube, diode array
 Example
Toluene is an important industrial chemical

The concentration of toluene in a solution can be determined using

UV-visible spectroscopy.

1. Choose an appropriate wavelength: a wavelength at which there is

significant absorbance.
The UV-visible spectrum of a solution of 7.5 ppm toluene is
shown below.
It can be seen that the maximum absorbance of toluene occurs
at 265 nm.
265 nm is the best wavelength to choose in order to determine the
concentration of toluene in solution.

22
 Example
Toluene is an important industrial chemical

2. Prepare a set of standard solutions of toluene.

A standard solution is a solution with a known concentration.
3. Measure the absorbance of each standard solution at the selected
wavelength (265 nm).

4. Plot the calibration graph.

concentration average
(ppm) absorbance
5. Measure the absorbance, at the same
wavelength, of the sample solution of 0.0 0.0
unknown concentration. 5.0 0.5
At 265 nm the solution of 7.5 0.8
unknown toluene concentration had 10.0 1.1
an absorbance of 1.28. 12.5 1.3
15.0 1.6
6. Plot the absorbance of the sample
solution on the calibration graph and read
off the concentration.
23
The concentration of toluene in
the sample solution is 12.0 ppm.
 Applications

1. Quantitative analysis (Using Beer’s law)(A = ε b C).

2. Detection of impurities (Additional peaks observed).
3. Qualitative analysis (comparing absorption spectrum with the spectra of known compounds).
4. Chemical kinetics (UV passed through reaction cell and A changes can be observed)

24
 Atomic Absorption Spectroscopy (AAS)

 Light with specific frequencies is absorbed by different metals when they

vaporize in a flame.
The energy absorbed excites electrons, moving them from their ground
state to a higher energy state.

 It uses hollow cathode lamps to emit light with these frequencies which is then
absorbed by the sample containing the metal ion.

 The amount of light absorbed is proportional to the concentration of the metal

ion in solution.
Concentrations are often expressed as mg/L or ppm.

 The amount of light absorbed by the sample is compared to the amount of light
absorbed by a set of standards of known concentration. 25
 Atomic Absorption Spectroscopy (AAS)

Features Uses

1. Expensive •Metals in the soil, blood, air,

2. Fast, requiring a set of standard solutions water, and food.
3. Quantitative
- mercury in shellfish
4. Reliable, sensitive (ppm, ppb)
- lead in soil samples
5. A type of absorption spectroscopy

correct hollow cathode tube selected & a calibration curve constructed of

absorbance versus concentration.

26
AAS Instrumentation

27
AAS Instrumentation

28
 CALIBRATION CURVE

Calibration Curve:
 Used to determine the unknown concentration of an element in a
solution. The instrument is calibrated using several solutions of known
concentrations. The absorbance of each known solution is measured and
then a calibration curve of concentration vs absorbance is plotted.

 The sample solution is fed into the instrument, and the absorbance of
the element in this solution is measured. The unknown concentration of the
element is then calculated from the calibration curve

Applications:
 Determination of even small amounts of metals (lead, mercury, calcium,
magnesium, etc) as follows:

 Environmental studies: drinking water, ocean water, soil.

 Food industry.
 Pharmaceutical industry.
29
 Example of a Calibration curve

 AAS was used to determine the concentration of lead ions (in ppm) in
fish.

 AAS was set up with a lamp that emitted light with a wavelength that is
absorbed by lead atoms.

 AAS was calibrated using different solutions containing known

concentrations of lead ions.

 The graph on the next slide shows the variation of absorbance with
the concentration of lead.

 A 2 g sample of the fish was ground up and heated on a hot plate

with 10 ml of nitric acid. This mixture was filtered and then sprayed into
the flame of the AAS. The absorbance reading was 6. Determine the
concentration of lead ions in the fish.

30
 Example of a Calibration curve

For A = 6 → 12 ppm or 12 mg/L or 12 g/mL

12 g → mL
? → 10 mL

120 g/2g

60 g/g or 60 ppm (1 ppm = 1 g/g) 31

End of Chapter

32