Transcript Document
Spectroscopic ANALYSIS
Part 5 – Spectroscopic Analysis using UV-Visible Absorption
Chulalongkorn University, Bangkok, Thailand January 2012 Dr Ron Beckett Water Studies Centre & School of Chemistry Monash University, Melbourne, Australia Email: [email protected]
Water Studies Centre
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UV-Visible Absorption Spectroscopy Absorption of UV and visible light by a molecule causes electronic excitation
Bond breaking and ionization Electronic excitation Vibration Rotation 10 20
-rays 10 18 X-rays 400 500 10 16 UV 10 14 Infrared 10 12 10 8 Microwave Visible Spectrum 600 700
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UV-Visible spectral peaks result from electronic-vibrational transitions Case (b) in the diagram is most common which gives the typical symmetric peak shape 3
Molecular Orbitals • Bonding in organic molecules is based on overlap between
s
and
p
atomic orbitals .
• This can give rise to bonding orbitals, nonbonding
antibonding
s * and p
n
* s and p molecular molecular orbitals molecular orbitals Two
p
atomic orbitals overlapping to give a s and a s * molecular orbital 4
Molecular Orbitals
Two
p
atomic orbitals overlapping to give a bonding p molecular orbital and a nonbonding p * molecular orbital
A B
p *
A
+
B
p x p x
A B
5 p
Molecular Orbitals and Electronic Jumps s * (antibonding) p * (antibonding)
n
s * n p * n (non-bonding) p p * p (bonding) s s * s (bonding) Electronic energy levels of polyatomic molecules 6
Peak Position and the Type of Electronic Jump Conjugated p bonds 7
Peak Position for Molecules containing Double and Triple Bonds 8
Effect of Conjugation on Peak Position The greater the number of conjugated double bonds the lower the energy jump and higher the wavelength of the UV-visible peak p p * 9
Effect of Conjugation on Peak Position Highly conjugated molecules may be coloured if the absorption peak moves into the visible region 10
Question Time !
Fanta
has
red
and
green
colours !
Will
red
light pass through each of these solutions or will it be absorbed ?
(a) (b) (c) (d)
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Question Time !
Fanta
has
red
and
green
colours !
Will
green
light pass through each of these solutions or will it be absorbed ?
(a) (b) (c) (d)
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Complementary Colours
When white light is absorbed by a chromophore, the eye detects the colours that are
not
absorbed. This is called the complementary colour to the colour absorbed.
V I B G Y O R
Colour Absorbed Colour Observed of maximum absorption 380-440 440-500 500-580 580-680 680-780 violet-blue blue green green yellow orange-red purple green yellow orange-red violet-blue blue green green 13
Colorimetric Analysis
Used for determination of the concentration of analytes in solution when: 1. The analyte is a coloured compound 2. The analyte produces a coloured species when a suitable reagent is added 14
Colorimetric Analysis
Determination of concentration depends on detection of change in colour intensity (absorption) at a particular wavelength.
Photometric measurement (a) visual comparison using colour standards
P o P
Eye 15
Colorimetric Analysis
(b) Colorimeter/Photometer • Filters used to select a wavelength range • Detection with photosensing device
Filter wheel P o P Photodetector
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Spectrophotometric Analysis
(c) Spectrophotometer – Spectral bandwidth ≤ 1 nm, i.e very monochromatic light.
– can operate in both the visible and UV ranges – Colorimetry and spectrophotometry provide
sensitive
methods of analysis, i.e. ppm to ppb ranges.
P o P Monochromator
Prism or Grating
Photodetector
Phototube, photomultiplier 17 or photodiode
•
Single Beam Colorimeter
Single beam spectrometer 18
Quantifying Light Absorption
b
Incident beam
P I
Reflected
P r
beam P a(solute) P a(solvent)
Absorbing solution of concentration,c.
Transmitted beam
P
Incident Light Intensity (
P I
) (sometimes
I i
is used)
P I = P r + P a(solvent) + P a(solute) + P P a(solvent) & P a(solute) are absorbed light intensities P r
≈ 4% for air-glass interface 19
Quantifying Light Absorption Intensity lost due to reflection and solvent absorption are removed by measuring the transmitted intensity of a blank containing only solvent
b
Incident beam
P I
Reflected
P r
beam P a(solvent)
Absorbing solvent
Transmitted beam
P 0
Transmitted Light (
P 0
)
P I = P r + P a(solvent) + P 0 P 0 = P I - P r - P a(solvent)
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Quantifying Light Absorption
Absorbance is defined as P
A = log
P 0
Transmittance
defined as
T = P P 0
Thus A = log (1/T) = log(100/%T) 21
Relationship between Absorbance and Concentration
Beer-Lambert Law
A =
e
l c
Where: •
l
is the
path length in cm
• •
c
is the
concentration
in mol/L e is the
molar absorptivity
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Applications of the Beer-Lambert Law Analysis of a single analyte 1. Measure absorbance of a series of standard solutions 2. Plot a standard curve (should be a straight line ?) 3. Measure absorbance of unknown samples 4. Use standard curve to measure concentrations Assumptions – At fixed and
l
, e constant for a given is solute – the chemical matrix of the standards is the same as the sample.
A 4 A 3 A x A 2 A 1 A 0 C 0 C 1
A =
e
l c
C 2 C C x 3 C 4
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Concentration
Applications of the Beer-Lambert Law Standard Addition Method Used for samples with complex matrix & chemical interferences.
1. Measure A of sample 2. Repeat with known additions of standard to the sample.
Sample plus standard additions Sample
C Add 0
Sample Concentration of Standard added (mL) Concentration 24
Limitations of the Beer-Lambert Law Concentration effects – B-L law applies to dilute solutions (negligible interaction – between solute ions).
Higher concentrations of analyte (i.e. > 10 -2 M) or high electrolyte concentrations, may produce molecular/ionic interactions which result in reduced light absorption at some wavelengths.
Adherence to B-L law Deviation from B-L law (loss of sensitivity) Concentration
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Experimental Considerations Wavelength selection • Choose where A is large to obtain best sensitivity.
• Choose where dA/d = 0 or is small.
1 2 2 4 3
3
Wavelength 26
Experimental Considerations Choice of reagents for colorimetric analysis – Should be stable and pure – Should not absorb at of measurement – Should react rapidly with analyte to give a stable coloured compound (chromophore).
– Absorptivity, e, should
not
be sensitive to minor changes in pH, Temp., electrolyte changes, etc.
– Should be selective for the analyte of interest.
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