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: Ron.Beckett@monash.eduWaterStudiesCentre

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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

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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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