Absorption Spectroscopy of Biopolymers

Overview

Visible

&

near-UV

region

Microwave

&

radiowave

region

Infared

region

Far-UV

, x-ray, g -ray wavelength (nm) frequency (Hz) wavenumber (cm -1 ) energy ( DE =h n )

Absorption & Emission

Rapid process(10 -15 s)

Absorption & Emission

Radiation-Induced Transition

I



c n

 • Absorption • Stimulated emission • Spontaneous emission

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 ( l ) between 190 and 800 nm

UV-visible spectrum The two main properties of an absorbance peak are: 1. Absorption wavelength l max 2. Absorption intensity

A

max Housecroft and Sharpe, p. 466

Beer-Lambert Law : log(

I

0 /

I

) = e

b

c e = A/

c b

Beer-Lambert Law A = e

bc

A

=

e

c

(when

b

is 1 cm)

I

0 = intensity of incident light

I

= intensity of transmitted light e = molar absoptivity coefficient in cm 2 mol -1

c

= concentration in mol L -1 b = pathlength of absorbing solution in cm -1 A = absorbance = log(

I o

/

I

) 0.1 cm ℓ http://www.hellma-worldwide.de/en/default.asp

Beer-Lambert Law

A

 log

I

0

I t

 e

bl

  log

T

• •

A

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

c

concentration; M • T transmittance cm -1

Transmittance, Absorbance, and Cell Pathlength http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

Low

c

Deviations from the Beer-Lambert Law High

c

The Beer-Lambert law assumes that all molecules contribute to the absorption and that no absorbing molecule is in the shadow of another http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

Sample Concentrations Solution too concentrated Diluted five-fold

Molar absorptivities ( e ) Molar absoptivities chromophores ( e are very large for strongly absorbing >10,000) and very small if the absorption is weak ( e = 10 to 100). The magnitude of e 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: e

= 0.87 x 10 20 P x a

where

P

is the transition probability (0 to 1)

a

is the chromophore area in cm 2 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 NH 2 NO 2 Molecular mass = 138 Solvent: Ethanol Concentration: 15.4 mg L -1 Pathlength: 1 cm Harwood and Claridge, p. 18

UV-visible spectrum of 4-nitroanaline 1. Determine the absorption maxima ( l max ) and absorption intensities (

A

) from the spectrum: l max = 227 nm,

A

227 = 1.55

l max = 375 nm,

A

375 = 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 ( e ) from the Beer Lambert Law: e =

A

/

c

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

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: D S = 0 allowed transitions: singlet  singlet or triplet forbidden transitions: singlet   triplet 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 u

stands for stands for

gerade

– compound with a center of symmetry

ungerade

– compound without a center of symmetry 3.

Selection rule of D 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 forbidden transitions: s   d, d s, d   f, etc.

d, p  f, etc.

s and s * orbitals http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

p and p * orbitals http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

Electronic Transitions: p  p * http://www.cem.msu.edu/~reusch/VirtualText /Spectrpy/UV-Vis/uvspec.htm#uv2 The p  p * transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”.

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

p  p * transitions - Triple bonds Organic compounds with -C ≡C- or -C≡N groups, or transition metals complexed by C≡N lying” p * orbitals or C≡O ligands, usually have “low http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

Electronic Transitions: n  p * http://www.cem.msu.edu/~reusch/VirtualText /Spectrpy/UV-Vis/uvspec.htm#uv2 The n-orbitals do not overlap at all well with the p * orbital, so the probability of this excitation is small. The e of the n times smaller than e p * transition is about 10 for the pp * transition as 3 it is “symmetry forbidden”.

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

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 for two

A total

l  measurment A A so l 1 l   2   

l l

1  

l

    e e

A

component e l

M M

l 1

M

l 2 e l

N

2

M

l 1  s under     e

A

l

N

2

A

l

N

1     e e e e  l

N

l 1

N

2 l

N

1 2

M

l 2  e M

M

l    

A

e

N

2 l

N

1

l

  and       N   e system l

N

wavelengt h

l

  

l

e 

M

l    e l

N

   

l

1     e

M

l 1 e

M

l 1

A

2 e l

N

2   e

M

l 2 e

M

l 2

A

1 e l

N

1    

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    5380 11300  0 .

717  5380     1960 1500  0 .

239  1960    5 .

85  10  5

M

   11300 11300   0 .

239 5380     1500 1500   0 .

717 1960    2 .

81  10  5

M

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

e

M

l  e l

N

 e

iso

l 

isosbestic : A

iso   e

iso l

 e

iso l

e

iso l



   



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.

2.

3.

4.

Change in local charge distribution Change in dielectric constant Change in bonding interaction Change in dynamic coupling between different parts of the molecule

Human Eye

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

Retina Light sensitive protein Outer segment Retina

1BRD http://www2.mrc-lmb.cam.ac.uk/groups/GS/rmovie.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

1BRD 1BM1