Spectroscop1c Analysis
Part 6 – Spectroscopic Analysis using Fluorescence and
Atomic Absorption Spectrophotometry
Chulalongkorn University, Bangkok, Thailand January 2012
Dr Ron Beckett
Water Studies Centre & School of Chemistry
Monash University, Melbourne, Australia
Email: Ron.Beckett@monash.edu
Water
Studies
Centre
1
2
Fluorescence Analysis
Fluorescence
Excitation to higher
electronic state by
absorption
2.
Loss of vibrational
energy as heat
3.
Emission of
fluorescence EMR
4.
5.
Results in Stokes shift
to longer wavelength
(lower energy).
Lifetime of excited
singlet state 10-5 - 10-8 s
Singlet Excited States
Triplet Excited State
T1
S
1
ENERGY
1.
Vibrational
relaxation
Absorption
Fluorescence
Ground
state
S0
l2
l1
l3
l4
Stokes Shift
3
Fluorescence Analysis
l
Scan Incidence
PI
EMR
C
P Absorbance
Signal
F
Fluorescence
Signal
4
Factors Affecting Fluorescence
Molecular Structure
– Most intense fluorescence from aromatic groups, esp.
multi-ring compounds or highly conjugated molecules,
e.g. PAH's (polycyclic aromatic hydrocarbons).
Phenanthrene
Anthracene
– Halogen or carboxyl substitution inhibits fluorescence
5
Factors Affecting Fluorescence
Relationship between Fluorescence and
Concentration of an analyte in solution
l
PI
C
P
F
F = Kc
6
Atomic Absorption Spectrometry
• AAS was invented in 1955 by Sir Alan
Walsh at CSIRO in Melbourne
• It is now used extensively around the
world for elemental analysis of
environmental, industrial and biological
samples
Atomic emission
produces light of the
right frequency
Sample
atoms in
gas phase
absorb
light
Detector
7
Origin of an Atomic Emission Peak
Excitation to a higher
electronic state by
heat, EMR, etc.
E2
DE = hn
Excitation
E1
Emission Spectrum
Intensity
n
Frequency
8
Atomic Emission
9
Atomic Emission After Excitation in a Flame
Different atoms give rise to characteristic colours that
can be used to identify the elements present
Ba
Na
10
Atomic Emission Spectra
Mercury (Hg)
Neon (Ne)
11
Origin of an Atomic Absorption Peak
Energy Transition
E2
DE = hn
E1
Absorption Spectrum
Intensity
n
Frequency
12
Atomic Absorption for a Hydrogen Atom
Electronic energy levels
in a H atom
1s 2s 2p 3s 3p 3d
The lowest energy state
has the single electron in
the 1s orbital
1s1
13
A brief history of
Atomic Absorption Spectrometry
• 1802 Wollastone - discovered black lines in Sun's
spectrum which were subsequently investigated by
Fraunhoffer (1823).
14
Solar spectrum
A brief history of
Atomic Absorption Spectrometry
– 1820 Brewster - suggested black lines in solar
spectrum due to absorption processes in Sun's
atmosphere.
– 1859 Kirchhoff and Bunsen - demonstrated Na D
line absorption in visible spectra.
15
A brief history of AAS
Interpretation of Fraunhoffer lines in terms of atomic
absorption by elements in the suns atmosphere
1859-1955 Astronomers use atomic absorption to
estimate metal concentrations in atmospheres of stars.
Solar spectrum
Hydrogen spectrum
16
A brief history of
Atomic Absorption Spectrometry
• 1955 - Alan Walsh at CSIRO in
Melbourne proposed atomic
absorption spectrometry for
chemical analysis.
• Mid 1960’s - First commercial
AAS manufactured in
Melbourne by Varian-Techtron.
Sir Alan Walsh (d 1998)
17
Atomic Absorption Spectrometer
18
Flame Atomic Absorption Instrumentation
19
Principle of Flame AAS
• Sample solution is sucked into a spray chamber to produce
an aerosol
• The aerosol is introduced into a flame with the fuel gas
• The solution is evaporated and the elements are atomised but
not excited or ionised
• A lamp containing the element being analysed is used to
produce light of the correct wavelength
• Absorbance of this light by the sample atoms is measured
20
Analysis by AAS
1. Measure the absorbance of a blank solution Abl and a
series of standard solutions Ax
2. Plot the calibration line (Ax – Abl) vs Cx
3. Measure the absorbance of the unknown solutions
Calculate the concentrations
of the unknown solutions
A
A
c  m
x
bl
x
Where m is the gradient of
the calibration line
21
Analysis by AAS
Standard Addition Method
– Compensates for chemical interferences
– Cannot eliminate spectral interferences
Concentration
of Unknown
Sample plus
Standard Addition
Sample
22
Volume of Standard Added (mL)
Use of AAS in Forensic Science
23
Who fired the
murder weapon ?
24
Gunshot residue (GSR)
• Bullets contain “primer” and “propellant”
which result in GSR on the firing hand
• Swabbing hands with mild acid will
release barium (Ba) and antimony (Sb)
• Measure by Atomic Absorption
25