Physical Biochemistry UV Spectroscopy
Complementary Colours – The absorbed and perceived
spectral band are diametrically opposite each other in the colour
wheel.
e.g. Chlorophyll absorbs in the deep red / violet regions so
appears yellow / green (colours opposite)
Absorption – Beer Lambert Law:
Can be used for estimating concentrations from spectra
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)
Increasing path length will cause an exponential decrease in absorbance
The extinction coefficient changes with wavelength, as the path length and concentration are
constant. ε values are given as ελ, where λ is the wavelength being used.
If measurements are taken with a constant path length and concentration, then an absorbance
measurement is similar to an extinction coefficient measurement.
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
A = -log10(I/I0) logarithmic relationship
Small I values will result in very high A values due to the logarithmic relationship, making the
measurement of low intensities more error prone.
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Physical Biochemistry UV Spectroscopy
Definitions:
Chromophores – Part of the molecule responsible for absorption
Auxochromes – Groups that modify absorption of neighbouring chromophores
Often have lone pairs (e.g. –OH, –OR, –NR2, –halogen)
Bathochromic shift – Shift towards longer wavelength
Hypsochromic shift – Shift towards shorter wavelength
Hyperchromic shift – Increase in peak absorbance
Hypochromic shift – Decrease in peak absorbance
Unconjugated organic molecules do not contain alternating double bonds.
σ σ* <150nm (vacuum UV) – Deep UV
n σ* / π π* <200nm (vacuum UV) ★
n π* 200-400nm (quartz UV) ★
* Denotes an anti-bonding orbital
★ can be studied in our lab
needs more sophisticated equipment
The UV region can be challenging to study as many materials absorb UV
Glass cannot be used up to 300nm
Quartz can be used, it works down to 200nm, but this is still cant be used for
σ σ*
UV region = 200 – 330nm
Increased conjugation leads to longer absorption wavelengths
In a conjugated
system such as –
C=C– C=C–, the
molecular
orbitals of each –C=C–
will interfere with
one another to form
the molecular
orbital of the conjugated
molecule.
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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)
C is near the visible region
Each ‘little peak’ represents a
transition.
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.
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Physical Biochemistry UV Spectroscopy
3. Bathochromic shift occurs
4. A is increased a little bit as there are more
e- that can conjugate to be excited
Resonance stabilisation shifts the double bond. There
will be a shift to a longer wavelength and there may be
an increase in absorbance due to Boron lone pairs.
Ψ orbitals are hybrid orbitals
UV Spectroscopy of Polypeptides and Nucleic Acids:
Peptide bond ~190nm (vacuum UV region – not usually usable)
Tryptophan ~280nm (ε=5600)
Tyrosine, phenylalanine and cysteine have absorbance’s over 250nm
RNA/DNA 250-275nm
ε can be calculated based on sequence, useful for determining concentration (ProtParam)
If there is a protein / nucleic acid mix, then measure at 280nm for protein conc. then at a
lower wavelength (258nm) to determine nucleic acid content. Useful for determining purity.
Solvent pH:
pH shifts equilibrium to the right
More non-bonding electrons in the phenoxide ion
Higher extinction coefficient (ε)
Greater delocalisation resulting in bathochromic shift
Low pH (-H+) Higher pH
2 lone pairs 3 lone pairs
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Physical Biochemistry UV Spectroscopy
The ring is the main chromophore
Both of the oxygen atoms can participate in resonance stabilisation of the ring
More lone pairs = more resonance stabilisation (longer wavelength)
More lone pairs = more electrons available for excitation higher A / extinction coefficient)
NH2 is an auxochrome its lone
pairs cause some resonance
stabilisation -Aniline
pH = equilibrium shift to right
No non-bonding e- in anilinium
Effect of pH on Tyrosine
Spectrum
pH has a big influence of Tyr spectrum
Should be similar to that of phenol (similar group)
pH = ε
pH = longer wavelength
pH titration can be used to determine whether a Tyr
is in an internal or external environment.
Increasing pH from 6 to 13 causes a redshift.
Polarity Effects of the Solvent
π* is more polar than π in polar molecules
π * is better stabilised than π in a polar solvent
π π* transition undergoes bathochromic shift with increasing solvent polarity
H-bonding stabilises n (non-bonding orbital) more than π* in polar solvents
n π* undergoes hypsochromic shift
Peak absorbance is reduced due to stabilisation of non-bonding electrons
Effect of Solvent Polarity of Tyrosine Spectrum
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Physical Biochemistry UV Spectroscopy
Solid line = H2O
Dashed line =
80% H2O / 20%
ethylene
glycol
Increasing
solvent polarity
results +in a
blueshift (to shorter wavelength)
Increasing solvent polarity will stabilise energy levels by different amounts.
n are stabilised by a greater amount than π
Redshift: π π*
Blueshift n π*
Summary:
Good for rough concentration measurements
Relatively broad spectra – not as useful as many other techniques for the identification of
molecules (e.g. NMR or mass sec)
Conjugation of double bonds leads to a bathochromic shift of the absorption spectra
Environmental factors such as pH or polarity influence the spectrum and can be used as
tools for determining the environment of the absorbing species
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