Optical Spec 2 - UV Spectroscopy

8/8/2019 Optical Spec 2 - UV Spectroscopy

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Physical Biochemistry UV Spectroscopy

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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

I = Intensity out

l = Path length of cuvette [cm]

I = I010-A A = cl

c = concentration [M]

= 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.

values are given as , where 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) n 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.

Definitions:

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 0.5 1 1.5 2

Path Length

Intensity


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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.

p*


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LUMO Lowest Unoccupied MolecularOrbital

HOMO Highest Occupied MolecularOrbital

E1 (unconjugated) & E2 (conjugated) are different E2 is smaller than E1

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

o Attached rings =o 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 morealternating 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.

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.


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orbitals are hybrid orbitals

UV Spectroscopy of Polypeptides and Nucleic Acids:

Peptide bond ~190nm (vacuum UV region not usually usable)

Tryptophan ~280nm (=5600)

Tyrosine, phenylalanine and cysteine have absorbances over 250nm

RNA/DNA 250-275nm

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:

opH shifts equilibrium to the right

More non-bonding electrons in the phenoxide ion

Higher extinction coefficient()

Greater delocalisation resulting in bathochromic shift

Low pH p (-H+) Higher pH

2 lone pairs 3 lone pairs

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)

nNH2 is an auxochrome its lone

pairs cause some resonance

stabilisation -Aniline

pHq = equilibrium shift to right

No non-bonding e - in anilinium


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Effect of pH on Tyrosine Spectrum

pH has a big influence of Tyr spectrum

Should be similar to that of phenol (similar group)

o pH = o

o 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

* is more polar than in polar molecules

* is better stabilised than in a polar solvent

p * transition undergoes bathochromic shift with increasing solvent polarity

H-bonding stabilises n (non-bonding orbital) more than * in polar solvents

np * undergoes hypsochromic shift

Peak absorbance is reduced due to stabilisation of non-bonding electrons

Effect of Solvent Polarity of Tyrosine Spectrum

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

Redshift: p *

Blueshift np *


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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

Optical Spec 2 - UV Spectroscopy

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Transcript of Optical Spec 2 - UV Spectroscopy