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Spectroscop1c AnalysisPart 6 – Spectroscopic Analysis using Fluorescence and
Atomic Absorption Spectrophotometry
Chulalongkorn University, Bangkok, Thailand January 2012
Dr Ron Beckett
Water Studies Centre & School of ChemistryMonash University, Melbourne, Australia
Email: [email protected]
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Fluorescence Analysis Fluorescence
1. Excitation to higher electronic state by absorption
2. Loss of vibrational energy as heat
3. Emission of fluorescence EMR
4. Results in Stokes shift to longer wavelength (lower energy).
5. Lifetime of excited singlet state 10-5 - 10-8 s
Groundstate
Singlet Excited States
Absorption
Fluorescence
Triplet Excited State
Vibrational relaxation
EN
ER
GY
S
S
Stokes Shift
3
Fluorescence Analysisl
CPI P
F
Scan IncidenceEMR
Fluorescence Signal
Absorbance Signal
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Factors Affecting Fluorescence
AnthracenePhenanthrene
Molecular Structure
– Most intense fluorescence from aromatic groups, esp.
multi-ring compounds or highly conjugated molecules,
e.g. PAH's (polycyclic aromatic hydrocarbons).
– Halogen or carboxyl substitution inhibits fluorescence5
CPI P
F
l
Factors Affecting Fluorescence
Relationship between Fluorescence and Concentration of an analyte in solution
F = K c 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
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Origin of an Atomic Emission Peak
Frequency
Intensity
Excitation to a higher electronic state by heat, EMR, etc.
Emission Spectrum
E2
E1
E = hExcitation
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Atomic Emission
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Ba Na
Atomic Emission After Excitation in a Flame
Different atoms give rise to characteristic colours that can be used to identify the elements present
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Mercury (Hg) Neon (Ne)
Atomic Emission Spectra
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Origin of an Atomic Absorption Peak
E2
E1
E = h
Frequency
Intensity
Energy Transition
Absorption Spectrum
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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
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A brief history of Atomic Absorption Spectrometry
• 1802 Wollastone - discovered black lines in Sun's spectrum which were subsequently investigated by Fraunhoffer (1823).
Solar spectrum14
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.
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A brief history of AAS
Hydrogen spectrum
Solar spectrum
Mercury spectrum
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.
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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
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Flame Atomic Absorption Instrumentation
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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
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Analysis by AAS
Concentration
Sample absorbance
c x
Ax
xc xA blA
m
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
Where m is the gradient of the calibration line 21
Analysis by AASStandard Addition Method
– Compensates for chemicachemical interferences– Cannot eliminate spectralspectral interferences
Volume of Standard Added (mL)
Sample
Sample plus Standard Addition
Concentration of Unknown
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Use of AAS in Forensic Science
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Who fired the murder weapon ?
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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
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