UV-Vis spectra
1. Basic principles
2. QM of interaction between light and molecules
3. Absorption spectroscopy of electronic states
4. Instrumentation - spectrophotometry
5. Spectroscopic analysis of biopolymers
6. Effect of conformation on absorption
Metody biofyzikální chemie
Gauglitz G., Vo-Dinh T.: Handbook of Spectroscopy. Wiley VCH Verlag, Weinheim 2003
Invitrogen Tutorials: www.invitrogen.com/site/us/en/home/support/Tutorials.html
2. Christian Huygens 1692:
Developed a wave theory of light
A very brief history of the study of light
Showed that the component colors of the visible portion of white light can be separated
through a prism, which acts to bend the light (refraction) in differing degrees according
to the wavelength. Developed a “corpuscular” theory of light .
1. Sir Isaac Newton 1672:
3. Hans Christian Oersted 1820
Showed that there is a magnetic field associated with the flow of electric current
4. Michael Faraday 1831
Showed the converse i.e. that there is an electric current associated with a change of
magnetic field
5. James Clerk Maxwell: (1831-1879)
Published his “Dynamical theory of the electromagnetic field” which combined the
discoveries of Newton, Young, Foucault, Oersted and Faraday into a unified theory of
electromagnetic radiation
Light consists of electromagnetic transverse waves of frequency  and wavelength 
related by  = nc where n is the index of refraction of the medium and c is the speed of
the light in vacuum c = 3x1010 cm/s
E
B
E
c
n
B 
we are interested in interactions of the electric field with the matter
6. Max Karl Ernst Ludwig Planck: (1858-1947) NP: 1918
Explained the laws of black body radiation by postulating that
electromagnetic radiation is emitted at discrete energetic quanta E = h ,
where Planck constant h = 6.6256 *10-34 Js.
7. Albert Einstein: (1879-1955) NP:1921
Explained the explained the photoelectric effect by assuming that light is
adsorbed at discrete energetic quanta E = h , photons.
8. Louis Victor Pierre Raymond de Broglie: (1892-1987) NP:1929
Introduced properties of electromagnetic waves to all particles – the wavecorpuscular
dualism of quantum physics. A freely moving particle of
momentum p has wavelength =h/p.
http://www.youtube.com/watch?v=m4t7gTmBK3g
http://www.youtube.com/watch?v=cfXzwh3KadE
The electromagnetic spectrum
Relative brightness sensitivity of
the human visual system as a
function of wavelength
Wavelength and energy scale,
appropriate units
X-ray
UV
Visible light
IR
Microwave
Radio
Wavelength nm
10-4
10-2
100 104102
106 108
1010
Frequency Hz
1021
1019 1017
1015 1013
1011 109 107
Wavenumber
cm-1
1011
109 107
105 103
101 10-1 10-3
Energy Kcal
108
106 104
102 100
10-2 10-4 10-6
Energy eV
107
105 103
101 10-1
10-3 10-5 10-7
What is light?
According to Maxwell, light is an electromagnetic field
characterized by a frequency v, velocity c, and wavelength λ. Light
obeys the relationships:

c
v 
h…….Planck constant 6.63 .10-34 J.s

c
hvhE 
ED 
HB 


1
v~ c.vv ~
112
0 108598 
 Fm..
17
0 104 
 Hm.
c
 00
1
http://www.edumedia-sciences.com/en/a185-transverse-electromagnetic-wave
induction intensity
magneto
electro


permitivity
permeability
Light
Elastic scattering of light
• Rayleigh scattering – small molecules (x<0.3) as a “point
dipole”, Isc ≈ 4 blue sky, red sunset
• Larger scatterers – macromolecules, cells, Mie theory for
spherically symmetrical scatterers
x = 0.07
x = 7

 an
x 
http://omlc.ogi.edu/calc/mie_calc.html
Raman scattering
C.V. Raman
(1888-1970)
• 1923 theoretically predicted by Adolf Smekal using
classical physics
• 1928 observed by C. V. Raman
Stokeselastic anti-Stokes
branch of Raman spectrum
the photon and the molecule
exchange energy
the photon is not absorbed:
scattering is an instantaneous and
coherentv1
v2
0
0-D
hD
0+D
Raman spectrum
intensity of Stokes branch is higher by a factor 




 D






D
D
Tk2
h
exp
2
2
4
0
0 


Stokesanti-Stokes
Interaction of light with matter –
overview of processes
• elastic scattering – no exchange of energy between the
molecule and the photon
• inelastic (Raman) scattering – the photon either gives a
part of its energy to the molecule or vice versa
• absorption or emission of photons by the molecule
1
2
absorption
spontaneous
emission
induced emission
1
2
• induced emission is coherent with incident light
• spontaneous emission by individual molecules is incoherent
• scattering is coherent and instantaneous
Franck-Condon (FC) Principle
• The FC principle states that during an electronic
transition, a change from one vibrational energy level
to another will be more likely to happen if the two
vibrational wave functions overlap more significantly.
• Classically, the FC principle is the approximation that
an electronic transition is most likely to occur without
changes in the positions of the nuclei in the
molecular entity and its environment. The resulting
state is called a FC state, and the transition involved,
a vertical transition.
• The quantum mechanical formulation of this principle
is that the intensity of a vibrational transition is
proportional to the square of the overlap integral
between the vibrational wave functions of the two
states that are involved in the transition.
James Franck (1882-1964) Edward Uhler Condon 1902-1974
lazy nuclei
Since electronic transitions are very fast compared with nuclear
motions, vibrational levels are favored when they correspond to a
minimal change in the nuclear coordinates. The potential wells
are shown favoring transitions between v = 0 and v = 2.
Probability
HIGH
HIGH
MEDIUM
LOW
Energy
Inter-nuclear distance
G
S1
v 0
v 1
v 2
v 3
v1 0
v 11
v 12
v1 3
Electronic transitions from the ground state to the excited state
Probability
Wavelength nm
Inter-nuclear distance
G
S1
v 0
v 1
v 2
v 3
v1 0
v 11
v 12
v1 3
Electronic transitions from the ground state to the excited state
Shaded areas reflects
the probability of
where the electron
would be if it were in
that vibrational band
Most favored
transitions occur From
the
maximum shaded
areas of the ground
state
To the maximum
shaded areas of the
excited state
Interaction of molecules with
photons - quantum description
• Light exists in form of discrete quanta – photons E = h
• Atoms and molecules occupy discrete energetic states,
which can be found as the solution of Schroedinger’s
equation.
electronic states
vibrational states
rotational states
DJ =  1
microwave region
DN =  1
IR – VIS region
UV – VIS region
E
• Exchange of energy with photons is accompanied by
transitions between those states.
Electronic – vibrational spectrum
other transitions (other
vibrational modes, nonfundamental
transitions,…)
effect of room temperature effect of molecular surroundings
The wavelength value of the absorption maximum
and the molar absorptivity
are determined by the degree of Conjugatation of -bonds
Absorption maxima : The importance of conjugation
Increasing the number of double bonds shifts the absorption to lower energy
Wavelength nm
N=5
5 pi-bonds, 10 electrons
N=4
4 pi-bonds, 8 electrons
N=3
3 pi-bonds, 6 electrons
As the degree of conjugation increases
(i.e the number of electrons involved in the delocalized -orbitals)
the absorption energy decreases (> , the energy between the ground and
excited state decreases)
the absorption becomes more intense (>, increased probability of absorption)
Benzene < Naphthalene < Anthracene < naphthacene < pentacene
Abs. Max 262nm 275 nm 375 nm 475 nm 580 nm
Log  3.84 3.75 3.90 4.05 4.20
(Extinction)
275 nm 375 nm 475nm absorption wavelength
Increasing the number of aromatic rings increases the absorption maximum
low resolution CH3(CH=CH)5CH3
Are absorption spectra simple?
Chromofory:
• charakteristická funkční seskupení zodpovědná za barevnost
sloučeniny – obsahují násobné vazby nebo konjugovaný systém
násobných vazeb
• >C=C< -N=N- >C=O- -N=O -C≡N
2 Na
+
O CH3
S
-
O
O
O
CH3
N N
OH
S
-
O
O
O
Absorpční maxima λmax
• Benzen 207 nm
• Naftalen 285 nm
• Antracen 375 nm
• Tetracen 471 nm žluto-oranžový
• Pentacen 580 nm fialový
Biologically useful spectroscopic regions in UV-Vis-IR
Wavelength
(cm-1)
Energy (aprox.)
(J/mol)
Spectroscopic
region
Techniques
/Applications
~ 10-5 1250 vacuum UV electronic spectra
3.10-5 420 near UV electronic spectra
carbon-carbon bond energy
6.10-5 200 visible electronic spectra
~ 10-3 15 IR vibrational spectra
RT at ambient temperature
10-2 1.5 Far IR vibrational spectra
There is no simple way to explain the interaction of light with matter. Why?
Light is rapidly oscillating electromagnetic field. Molecules contain distribution
of charges and spin that have electrical and magnetic properties and these
distributions are altered when a molecule is exposed to light.
Explanation:
1. The rate at which the molecule responds to this perturbation
2. Why only certain wavelength cause changes in the state of the molecule
3. How the molecule alters the radiation
Type of transition
(1) , , and n electrons
(2) d and f electrons
(3) charge transfer electrons
Calculation of properties of molecules by QM
1. The state of a system is described by a wavefunction
2. An observable quantity (E, µ, the location in space) is
governed by a mathematical device known as an operator
3. The result of a measurement on a state can be computed by
taking the average value of operator on that state
4. A transition between two state can be induced by a
perturbation which is measured by an operator (deform the
initial state - resembling final state)
5. The ability of light to induce transitions in molecules can be
calculated according to its ability to induce µ(s) that oscillate
with the light.
6. The preferred directions for inducing dipole moments µ(s) are
fixed with respect to the geometry of the molecule.
INTERACTION BETWEEN LIGHT AND MOLECULES
Interaction of light with molecules (chromophores)
Chromophore ~10 Ǻ Wavelenght of light ~ 3 000 Ǻ
2) … the spacial variation of the electric field of the light within the molecule
1) … the magnetic vector, only the electric vector
For simplicity: I. What can be ignored?
The electric field felt by a molecule:
ti
eE)t(E 
 0
)
c
v(

 22
3) …. the effect of time?
statefinal............
stateoriginal..........a


What we need to compute?.... the rate at which light causes transitions between anda 
For simplicity: II. What can be ignored? …. other states
statefinal............stateoriginal.......... ba 
Is the interaction (light – molecule) dependent on time?.... )t(VˆHˆHˆ ,

 /tiE
bb
/tiE
aa
ba
e)t(Ce)t(C)t( 

Interaction of light with molecules
 /tiE
bb
/tiE
aa
ba
e)t(Ce)t(C)t( 

 )t(Ce)t(Ce)t(Vˆ)dt/dCedt/dCe)t((i b
/tiE
ba
/tiE
ab
/tiE
ba
/tiE
a
baba 
 

Ca and Cb calculation: some approaches
1) … to expand the charge distribution in a multipole series the electric dipole
i
i
i rˆeˆ ie the electronic charge at the position irˆ
Born-Oppenheimer approximation
ti
0eEˆ)t(Vˆ 
The interaction energy: ˆ the dipole operator
abba ˆˆ  
the spatial part from–x to + x, from–y to + y, from –z to + z
Eo is constant
ab EEhv  
spectral bands - light absorption or light-induced transition
only at certain narrow wavelength (or frequency) interval
Interaction of light with molecules
transition dipoles
Bab - the transition probability
2
ab
2
ab ˆ)/)(3/2(B  
the ability of light to distort a molecule
Eˆind 
the transition dipole moment - vector
MATRIX
2
ab ˆ )v(Ifrom intensity electronic distribution within the molecule
0E.ˆ ab )v(I = f (the relative orientation of the molecule with )
ab ˆ  light-induced dipole
not preferred direction, only a preferred orientation
excitation in phase or out of phase
e.g. exempli gratia
In phase Out of phase by 1800
Time = t
Time = t+1/2 v
Induced neighboring chromophores – the interactions can be attractive or repulsive
Electronic structures of simple molecule
S0
S1
T1
Bond length
D
ground state Singlet
excited state
Singlet
excited
state
Triplet
Energy
Dissociated states
Vibrationstates
ABSORPTION SPECTROSCOPY OF ELECTRONIC STATES
(molecular geometry)
Transitions corresponding to:
e – electronic
v – vibrational
r – rotational
Energy S1- S0 ~ 80kcal/mol
335 kJ/mol
Energy v ~ 10kcal/mol
42 kJ/mol
Energy r ~ 1 kcal/mol
4.2 kJ/mol
Interaction between photon and molecule
S0
S1
T1
D
S0
S1
T1
S0 S1 transition
A F
IR
UV-vis
P
Absorption
Fluorescence
Phosphorescence
Interaction between photon and molecule
Jablonski diagram
Kasha's rule is a principle in the photochemistry of electronically excited molecules.
The rule states that photon emission (fluorescence or phosphorescence) occurs in
appreciable yield only from the lowest excited state of a given multiplicity.
Kasha's rule
Electronic absorption spectra of small molecules
line spectrum
atom H
band spectrum
molecule I2
Band broadening: enviromental heterogeneity, Doppler shifts and other effects
01 Sˆrot,vibS 
magnitudes of transition dipoles
absorption spectra of benzene showing solvent-induced broadening
(gas, in solution, benzen in C6H14).
model benzene
modeled as a single
electronic band
Absorbing species
1. excitation
M + h M*
The lifetime of the excited species: 10-8 - 10-9 s
2. relaxation (conversion of the excitation energy to heat)
M* M + heat
The absorption of ultraviolet or visible radiation generally
results from excitation of bonding electrons.
The electrons that contribute to absorption by a molecule are:
(1) those that participate directly in bond formation between atoms
(2) nonbonding or unshared outer electrons that are largely localized
about such atoms as oxygen, the halogens, sulfur, and nitrogen. The
molecular orbitals associated with single bonds are designated as
sigma () orbitals, and the corresponding electrons are  electrons.
Energy
 *, n *, n *, and  *
Methan 125 nm; 150-250nm ; most applications; most applications;
200-700nm; 200-700nm
unshared electrons
Bonding
Bonding
Nonbonding
Antibonding
Antibonding
Energy
σ
π
n
σ*
 * n *
n *
 *
*π*
Chromophores
Compound sample Transition
Chromophores
Effect of Conjugation of Chromophores
1,3-butadiene, CH2=CHCH=CH2: a strong absorption band that is displaced to a longer wavelength by 20 nm
compared with the corresponding peak for an unconjugated diene
Chromophores and conjugated double bonds
 Not only the kind of chromophore is important; the benzene-rings are chromophores with π–π* transitions
but conjugated double bonds have the spectral effect. The environment of the chromophores or the
combination with other chromophores also have a strong influence on the position and values of
absorbance bands
 From naphthalene to anthracene there is an increasing conjugation of the π bonds. The spectral region of
absorbance changes as well as the intensity of absorbance and the form of the absorbance bands
 Anthracene and phenanthrene have the same chemical formula but their spectra are different because
their structure and hence the electronic environment of the π bonds are different.
Energies of Light at the Absorbance Maxima

c
hvhE 
h…….Planck constant 6.63 .10-34 J.s
c…….299 792 458 m.s-1
solvation influences the distribution of energy
levels of the base and the excited state
(effect of surface active substance)
There is a relationship between the
wavelengths of the absorbance maxima and
the polarity of the solvent used (permitivity)
similar effect of pH: protonation-deprotonation
batochrom shift
Solvation effect
Tyrosine
Light
the extinction coefficient
Beer–Lambert–Bouguer law
dlC
I
dI

 the molar extinction coefficient = f (λ or ω)
 
lI
I
dlC
I
dI
00
lC
I
I
ln 0 l)(C
I
I
log)(A  0
3022./
- low concentrations ˂ 10-2 M
- monochromatic light
- stabillity of sample
Absorbance A
Transmittance T
0I
I
)(T 
l)(
eII 
 0
l)(.l)(.elogl)(elog
eI
I
log
I
I
log)(A l)(
l)(
 

1886043430
0
00
the most accurate measurements of A
Absorption: measurement
The Beer Lambert Law
Absorption (Optical Density) = log Io / I =  c l
l is the path length of the sample (1 cm)
Deuterium/
Tungsten
Lamp
PMT sample
PMT reference
I
Io
Mono-
chromator
sample
blank
Detector
• a typical sample: a solution in a cuvette
• the solvent and the reflection from the
cuvette walls contribute to the
extinction of light
• relative measurement of absorption
biopolymers
SS AIlogIlog  0
Reference:
Sample:
RR AIlogIlog  0
RS
S
R
AA
I
I
log 
Double beam spectrophotometer
Differential spectrophotometry
 l)CCC()CC(AA
I
I
log ESESSSEESSEE
 221
1
2
411
10 
]cmmol[max
DNA bases
biopolymers
problems with ε: high and non-correct due to not known molecular weight …………the average ε per residue, for ODN = phosphate residues
ε molar and residue extinction is often implicit
Spectral properties of a simple molecule
Formaldehyde (H2CO)
H………..1s
C………..1s2 2s2 2py 2px hybridization…. 1s2 (2sp2)3 2pz
O………..1s2 2s2 2py
2 2px 2pz
)O(p)C(p zz 22 
)O(p)C(p zz* 22 The absorption intensity
the transition dipole moment
fˆi
initial final
*ˆ  *ˆn 
The π-π* transition is called
an allowed transition
The n-π* transition is called a
symmetry – forbidden transition
 Spectrophotometer
 Single and double beam instruments
Components of optical instruments
1. Sources
2. Wavelength selectors (filters, monochromators)
3. Sample containers
4. Detectors
5. Readout devices
 Applications of Spectrophotometry
Spectrophotometry is more suited for quantitative
analysis rather than qualitative one
45
Instrumentation - spectrophotometry
46
Instrumentation (Spectrophotometers)
A single beam spectrophotometer
Wavelength selector
The above essential features of a spectrophotometer shows that
polychromatic light from a source separated into narrow band of wavelength
(nearly monochromatic light) by a wavelength selector, passed through the
sample compartment and the transmitted intensity, P, after the sample is
measured by a detector
In a single beam instrument, the light beam follows a single path from
the source, to the monochromator, to the sample cell and finally to the
detector
47
Light source
Grating
Rotating the grating
changes the wavelength
going through the sample
slits
slits
Sample
Phototube
The components of a single beam
spectrophotometer
When blank is the sample
Po is determined,
otherwise P is measured
Separates white light
into various colors
detects light &
measures intensity
- white light of constant intensity
Single beam spectrophotometer
Double Beam Spectrophotometer
Range: 200 – 1100 nm
Occuracy: 2 nm
Absorption of air ˂200 nm
Transmittance Transmittance
Absorbance Absorbance
quartz glass
glass
quartz glass
49
Sources used in UV-Vis Spectrophotometers are continuous sources.
• Continuous sources emit radiation of all wavelengths within the
spectral region for which they are to be used.
• Sources of radiation should also be stable and of high intensity.
Continuous
Sources
Visible and
near IR
radiation
Tungsten
Lamp
320-2500 nm
Ultraviolet
radiation
Deuterium
Lamp
200-400 nm
Light sources
Monochromators
Early spectrophotometers used prisms
- quartz for UV
- glass for vis and IR
These are now superseded by:
Diffraction gratings:
- made by drawing lines on a glass with a diamond stylus
ca. 20 grooves mm-1 for far IR
ca. 6000 mm-1 for UV/vis
- can use plastic replicas in less expensive instruments
Think of diffraction on a CD
10m x 10m
What is the purpose of concave mirrors?
Polychromatic radiation enters
2. concave mirror focuses each wavelength at different point of focal plane
Orientation of the reflection grating directs only one narrow band to exit slit
The light is collimated the first concave mirror
Reflection grating diffracts different wavelengths at different angles
51
Selection of wavelength
Absorbance measurements are always carried out at fixed
wavelength (using monochromatic light). When a wavelength is
chosen for quantitative analysis, three factors should be considered
1. Wavelength should be chosen to give the highest possible sensitivity.
This can be achieved by selecting max or in general the wavelengths at
which the absorptivity is relatively high.
λmax
λmax - wavelength where maximum
absorbance occurs
52
Sample compartment (cells)
 For Visible and UV spectroscopy, a liquid sample is usually
contained in a cell called a cuvette.
 Glass is suitable for visible but not for UV spectroscopy because it
absorbs UV radiation. Quartz can be used in UV as well as in visible
spectroscopy
1 cm 1 cm
Opaque
Face
Transparent
Face
Long pathlength
Short pathlength (b)
1 cm pathlength cuvet
53
Detectors
 The detectors are devices that convert radiant energy into electrical
signal.
 A Detector should be sensitive, and has a fast response over a
considerable range of wavelengths.
 In addition, the electrical signal produced by the detector must be
directly proportional to the transmitted intensity (linear response).
h
e-
-V
Photosensitive cathode
amplifier
i- Phototube
anode
Phototube emits electrons
from a photosensitive,
negatively charged cathode
when struck by visible or
UV radiation
The electrons flow through
vacuum to an anode to
produce current which is
proportional to radiation
intensity.
54
Photomultiplier tube
 It is a very sensitive device in which electrons emitted from the photosensitive
cathode strike a second surface called dynode which is positive with respect to
the original cathode.
 Electrons are thus accelerated and can knock out more than one electrons from
the dynode.
 If the above process is repeated several times, so more than 106 electrons are
finally collected for each photon striking the first cathode.
photochathode
anode
high voltage
voltage divider network
dynodeslight
electrons
e-
 The important characteristics of
Spectrophotometric methods
1. Wide applicability to both organic and inorganic systems
2. High sensitivity of 10-6-10-4 M
3. Moderate to high selectivity.
4. Good accuracy the relative error encountered in concentration lie
in the range from 1% to 3%
5. Ease and convenience of data acquisition
55
Spectral properties of biological molecules
 Much more complex than FA
 Major constrains – solvents and solvation
 In water experiments at λ ˃ 170nm, water is strongly polar, electronic
absorption bands are broader than in most other solvents (at various orientations
and distances), problem of temperature (1 – 1000C)
Protein chromophores
peptide bond itself aminoacid side chains any prosthetic groups
model
formamide N-methylacetamide
π electrones are delocalized over C,O,N
n-π* transition – the lowest energy of electronic transition is symmetry – forbidden
210-220 nm, εmax ~ 100
UV spectra of poly-L-lysine in aqueous solutions
UV spectra of aromatic amino acid
AA – in the same spectral region – the strong peptide absorption (π-π*)
AAA – in pH 7 (π-π*) symmetry
Tryptophane
Tyrosine
Phenylalanine
Tyrosine
pH effect
When a DNA helix is denatured to become
single strands, e.g. by heating, the
absorbance is increased about 30 percent.
This increase, called the hyperchromic
effect, reveals the interaction between the
electronic dipoles in the stacked bases of
the native helix.
UV spectra of DNA and its bases
A pure DNA solution appears transparent to the
eye, and absorption doesn't become measurable
until 320 nm. Moving further into the u.v. region,
there is a peak at about 260 nm, followed by a
dip between 220 and 230, and then the solution
becomes essentially opaque in the far u.v.
hyperchromic
hypochromic
(π-π*)
(π-π*)
(n-π*)
Electronic state of nucleobases is more
complex than chromophores of peptides
low symmetry
many nonbonded electrons
several different transition (π-π*) (n-π*)
Physical Chemistry Chemical Physics
The UV absorption of nucleobases: semi-classical ab initio spectra simulations
Absorption spectra of chlorophylls
Effect prosthetic groups
Polypeptide chain – prosthetic group
(apoprotein)
local environment
oxidation-reduction
200 – 300 nm
• Prosthetic group must have a high enough molar
extinction coefficient to be detectable at typical protein
concentrations
• To avoid the formation of intermolecular aggregates
Snímek polární záře na Jupiteru, jak ji v ultrafialovém
oboru spektra zaznamenal Hubbleův vesmírný dalekohled
Podle moderních modelů evoluce je vznik a evoluce prvotních proteinů a enzymů
schopných reprodukce připisován právě existenci ultrafialového záření.