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I0 = Intensity in 1
0.9
I = Intensity out 0.8
l = Path length of cuvette [cm] 0.7
0.6
0.5
0.4
-A
I = I010 A = εcl 0.3
0.2
0.1
c = concentration [M] 0
0 0.5 1 1.5 2
ε = molar absorption coefficient [M-1cm-1]
A = absorbance (optical density)
Limitations
Only valid when particles are acting independently (i.e. at low concentrations)
Scattering and fluorescence
Finite bandwidth of detector & measurable intensity is exponentially related to conc.
High values (>1.5) are generally unreliable
[Page 1]
Physical Biochemistry UV Spectroscopy
Definitions:
[Page 2]
Physical Biochemistry UV Spectroscopy
π 1 π3 = constructive interference
π2 π4 = destructive interference
LUMO – Lowest Unoccupied Molecular Orbital
HOMO – Highest Occupied Molecular Orbital
ΔE1 (unconjugated) & ΔE2 (conjugated) are different – ΔE2 is smaller than ΔE1
This reduced energy results in the absorbance spectrum taking place at a longer wavelength
(bathochromic / red-shift)
Increasing conjugation within a molecule results in a bathochromic shift.
This applies for linear molecules as well as ring molecules
Attached rings = extended conjugated system (more alternating double bonds)
Resulting in a shift towards longer wavelengths (bathochromic shift)
More rings = more extended conjugated system (there are more alternating double bonds)
Many molecules are tailored to absorb in the visible wavelength have three or four attached rings
Auxochromes:
Located next to the chromophore and influence its absorption
Auxochromes with lone pairs often lead to increased delocalisation (and conjugation)
Therefore leading to a bathochromic shift
1. The lone pair of the auxochrome (B) can delocalise, leading to resonance stabilisation
2. Conjugation is extended. A resonance state is added.
[Page 3]
Physical Biochemistry UV Spectroscopy
Solvent pH:
[Page 4]
Physical Biochemistry UV Spectroscopy
[Page 5]
Physical Biochemistry UV Spectroscopy
Increasing
solvent polarity
results +in a
blueshift (to shorter wavelength)
Redshift: π π*
Blueshift n π*
Summary:
[Page 6]