Absorption Spectroscopy of Biopolymers

Overview

Visible & near-UV region wavelength (nm)

Microwave & radiowave region frequency (Hz)

Infared region wavenumber (cm-1)

Far-UV, x-ray, -ray energy (=h)

Absorption & Emission

Rapid process(10-15s)

Absorption & Emission

Radiation-Induced Transition

• Absorption

• Stimulated emission

• Spontaneous emission

n

cI

UV-Visible Spectroscopy

• Ultraviolet-visible spectroscopy involves the absorption of ultraviolet/visible light by a molecule causing the promotion of an electron from a ground electronic state to an excited electronic state.

• Ultraviolet/Visible light:

wavelengths () between 190 and 800 nm

UV-visible spectrum

The two main properties of an absorbance peak are:

1. Absorption wavelength

max

2. Absorption intensity

Amax

Housecroft and Sharpe, p. 466

Beer-Lambert Law

Beer-Lambert Law:

log(I0/I) = bc

= A/cb

A = bc

A = c (when b is 1 cm)

I0 = intensity of incident light

I = intensity of transmitted light

= molar absoptivity coefficient in cm2 mol-1

c = concentration in mol L-1

b = pathlength of absorbing solution in cm-1

A = absorbance = log(Io/I)

0.1 cm

ℓ

http://www.hellma-worldwide.de/en/default.asp

Beer-Lambert Law

• A Absorbance or optical density (OD)• absorptivity; M-1 cm-1

• c concentration; M • T transmittance

TblI

IA

t

loglog 0

Transmittance, Absorbance, and Cell Pathlength

http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

Deviations from the Beer-Lambert Law

The Beer-Lambert law assumes that all molecules contribute to the absorption and that no absorbing molecule is in the shadow of another

Low c

High c

http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

Sample Concentrations

Solution too concentrated Diluted five-fold

Molar absorptivities ()

Molar absoptivities are very large for strongly absorbing chromophores ( >10,000) and very small if the absorption is weak (= 10 to 100). The magnitude of reflects both the size of the chromophore and the probability that light of a given wavelength will be absorbed when it strikes the chromophore. A general equation stating this relationship may be written as follows:

= 0.87 x 1020P x a

where P is the transition probability (0 to 1)

a is the chromophore area in cm2

The transition probability depends on a number of factors including where the transition is an “allowed” transition or a “forbidden” transition

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv2

UV-visible spectrum of 4-nitroanaline

Solvent: Ethanol

Concentration: 15.4 mg L-1

Pathlength: 1 cm

NH2

NO2

Molecular mass = 138

Harwood and Claridge, p. 18

UV-visible spectrum of 4-nitroanaline

1. Determine the absorption maxima (max) and absorption intensities (A) from the spectrum:

max = 227 nm, A227 = 1.55 max = 375 nm, A375 = 1.75

2. Calculate the concentration of the compound:

(1.54 x 10-2 g L-1)/(138 g/mol) = 1.12 x 10-4 mol L-1

3. Determine the molar absorptivity coefficients () from the Beer-Lambert Law: = A/cℓ

227 = 1.55/(1.0 cm x 1.12 x 10-4 mol L-1) = 13,900 mol-1 L cm-1

375 = 1.75/(1.0 cm x 1.12 x 10-4 mol L-1) = 15,700 mol-1 L cm-1

UV-visible spectroscopy definitions

chromophore Any group of atoms that absorbs light whether or not a color is thereby produced.

auxochrome A group which extends the conjugation of a chromophore by sharing of nonbonding electrons.

bathochromic shift The shift of absorption to a longer wavelength.

hypsochromic shift The shift of absorption to a shorter wavelength.

hyperchromic effect An increase in absorption intensity.

hypochromic effect A decrease in absorption intensity.

Absorption and Emission of Photons

http://micro.magnet.fsu.edu/optics/lightandcolor/frequency.html

Absorption and Emission

Emission

Absorption: A transition from a lower level to a higher level with transfer of energy from the radiation field to an absorber, atom, molecule, or solid.

Emission: A transition from a higher level to a lower level with transfer of energy from the emitter to the radiation field. If no radiation is emitted, the transition from higher to lower energy levels is called nonradiative decay.

Absorption

http://www.chemistry.vt.edu/chem-ed/spec/spectros.html

Singlet and Triplet Excited States

http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/lumin1.htm

Absorption and emission pathways

McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm

Selection Rules

In electronic spectroscopy there are three selection rules which determine whether or not transitions are formally allowed:

1. Spin selection rule: S = 0

allowed transitions: singlet singlet or triplet triplet forbidden transitions: singlet triplet or triplet singlet

Changes in spin multiplicity are forbidden

http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/lumin1.htm

Selection rules

2. Laporte selection rule: there must be a change in the parity (symmetry) of the complex

Laporte-allowed transitions: g u Laporte-forbidden transitions: g g or u u

g stands for gerade – compound with a center of symmetry u stands for ungerade – compound without a center of symmetry

3. Selection rule of ℓ = ± 1 (ℓ is the azimuthal or orbital quantum number, where ℓ = 0 (s orbital), 1 (p orbital), 2 (d orbital), etc.)

allowed transitions: s p, p d, d f, etc.forbidden transitions: s s, d d, p f, etc.

and* orbitals

http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

and * orbitals

http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

Electronic Transitions: *

McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm

The   * transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”.

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv2

* transitions - Triple bonds

Organic compounds with -C≡C- or -C≡N groups, or transition metals complexed by C≡N- or C≡O ligands, usually have “low-lying” * orbitals

http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

Electronic Transitions: n *

McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm

The n-orbitals do not overlap at all well with the * orbital, so the probability of this excitation is small. The of the n* transition is about 103 times smaller than for the * transition as it is “symmetry forbidden”.

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv2

Lycopene from Tomatoes

http://www.purdue.edu/UNS/html4ever/020617.Handa.lycopene.html

Chlorophyll

B-carotene

hemoglobin

Quantitative Analysis• A plot of absorption versus wavelength is the absorption spectrum

NMNM

MM

NMNM

NN

NM

NM

NMNMNMtotal

AA

l

AA

l

NMl

NMl

NMlNlMlAAA

1221

21

1221

12

222

111

12

21

1N

1M

soA

Ah wavelengt2under smeasurment

systemN and Mcomponent -for two

Solutions containing the amino acids tryptophan and tyrosine can be analyzed under alkaline conditions (0.1 M KOH) from their different uv spectra. The extinction coefficients under these conditions at 240 nm and 280 nm are

A 10-mg smaple of the protein glucagon is hydrolyzed to its constituent amino acids and diluter to 100 mL in 0.1 M KOH. The absorbance of this solution (1 cm path) was 0.717 at 240 nm and 0.239 at 280 nm. Estimate the content of tryptophan and tyrosine in mol (g protein)-1

Mtyr

Mtyr

5

5

1081.219601500538011300

717.01500239.011300

1085.519601500538011300

239.01960717.05380

Isosbestic points Isosbestic wavelength

the wavelength at which two or more components have the same extinction coefficient The occurrence of two or more isosbestics in the spectra of a series of solutions of the same total concentration demonstrates the presence of two and only two components absorbing in that spectra region.

Isosbestic points

NMl

NlMl

iso

isoiso

isoA : isosbestic

isoNM

UV spectrum of BSA UV spectrum of DNA from E. coli

UV Absorption of amino acid

Effect of Secondary structure

Origin of Spectroscopic Changes

1. Change in local charge distribution

2. Change in dielectric constant

3. Change in bonding interaction

4. Change in dynamic coupling between different parts of the molecule

Human Eye

RetinaRetina

RetinaRetina

Outer segmentOuter segmentLight sensitive Light sensitive

proteinprotein

http://www2.mrc-lmb.cam.ac.uk/groups/GS/eye.html

Rhodopsin is a protein in the membrane of the photoreceptor cell in the retina of the eye. It catalyses the only light sensitive step in vision. The 11-cis-retinal chromophore lies in a pocket of the protein and is isomerised to all-trans retinal when light is absorbed. The isomerisation of retinal leads to a change of the shape of rhodopsin which triggers a cascade of reactions which lead to a nerve impulse which is transmitted to the brain by the optical nerve http://www2.mrc-lmb.cam.ac.uk/groups/GS/rmovie.html

1BRD

1BRD

1BM1