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
„ Student není pohár, který můžeme naplnit, student je pochodeň, kterou můžeme zapálit.“
Biofyzikální chemie II
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
Max Karl Ernst Ludwig Planck (1858-1947)
James Clerk Maxwell (1831-1879)
http://www.edumedia-sciences.com/en/a185-transverse-electromagnetic-wave
Maxwell equations
Maxwell's equations describe how electric and magnetic fields are generated and
altered by each other and by charges and currents. They are the foundation of
classical electrodynamics, classical optics, and electric circuits.
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
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
Transmission and color
The human eye sees the complementary color to that which is absorbed
Oswald circle
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
Ad 1. The state of a system is described by a wavefunction (ψ)
is not a directly measurable quantity and it is often referred to as a probability amplitude
 *P bia  bia* 
22
baP 
  1d*dP  Dirac notation

ba , normalized functions for two states 1aa  1bb 
the maximum value 1ba  the minimum value 0ba 
the functions are ortogonal
bbaa CC  
)s()r()s,r(  
)R()R,r( Ne  
e electron, N nucleus
Born-Opperheimer approximation
)r()r( 2211  the state of two electrons without the interaction
  OˆdOˆ*OOˆ  is a pure number, the eigenvalue
 Hˆdt/di
The Schrödinger equation
time-independent (stationery state)
Hˆ Hamiltonian operator EHˆ  VˆTˆHˆ 
the kinetic energy operator the potential energy operator
 
i i
i
ii
i m
p
vm
22
1 2
222
2
2
  i
i i
i
pˆwhere
m
pˆ
Tˆ systemtheondepends
222
 ip
 EHˆ
time-dependent
/iEt
e)()t( 
 0
22
00 )(ee)()t()t(*P /iEt/iEt
  
22
11
E..........
E..........


2211  CC  
i
iiC
perturbation as a potential Vˆ
statefinal............
stateoriginal..........a

 
i
aiia VˆCVˆ 
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
 
2
02
2
2
1
Eˆdv)t(C
dt
d
dt
dP
abb
b

not only for a – b transition but also b – a transition
!!! the rate at which molecules in stage a are transformed to state b
by light = the rate of change of
2
b )t(C
!!! the rate at which energy is taken up from the incident light
beam )v(IB
dt
dP
ab
b

abB is the transition rate per unit energy density of the radiation
)v(I is the energy density incident on the sample at frequency v
4
E
)v(I
2
0

2
ab
2
ab ˆ)/)(3/2(B  
!!! the rate at which energy is removed from the light depends on the
number of a-b absorption transitions stimulated by light
)v(I)BNBN(hv
dt
)v(dI
bababa  Einstein´s coefficients for
stimulated absorption and
emissioncasessimpleBB baab 
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
Regions of Electromagnetic Spectrum
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
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.
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
d and f electronsconjugation of chromophores >C=C<
-N=N-
>C=O-
-N=O
-C≡N
Lanthanide and Actinide Ions Charge-Transfer Absorption
Donor-acceptor
Donor-acceptor
Donor-acceptor
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
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
the extinction coefficient and cross sections ( molecular size)
A …surface of solution slab (cm2)
dl… thickness of solution slab (cm)
C …molar concentration of solute (M)
r…the radius of the solute molecule
  00010001 0
2
0
2
,/CNrAdl/,/CANrf dlmax 
the number of solute molecules in the slab the number of solute molecules per cm2
maxf the fraction of the cross-sectional area of the slab occupied by solute molecules
P… the probability that light impinging on a molecule is absorbed
Pfmax the fraction of incident light absorbed
2
rP
moleculetheoftionseccrossthe...
 dl,/CN
I
dI
00010
dlC
I
dI

lC
I
I
ln 0
l)(C
I
I
log)(A  0
00010 ,/N
 dl,/CNdlC 00010
3022./
2
rP
30320
2
,/NrP
o
A1ringaromatic... 
the extinction coefficient in calculating molecular properties
)v(I)BNBN(hv
dt
)v(dI
bababa 
the number of excited molecules (Nb) is negligible
)v(I),/BNhv(
dt
)v(dI
ab 00010 the rate of energy uptake for a one-molar solution
Avogadro´s number
dl)v(I
c,
BhvN
dl
dt
)v(dI
c
)v(dI ab



















0001
1 0
dlC
I
dI
 hvN/c,Bab 00001  for both states, integration over a band frequencies
dv
vhN
c,
Bab 


0
00012
ab
2
ab ˆ)/)(3/2(B  
3022./
2
2
009180 )debye(dv
v
.ˆD abab 


Dipole strength
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
36
Instrumentation - spectrophotometry
37
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
38
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
40
Double Beam Spectrophotometer
Slit
(štěrbina)
Beam Chopper
(vrtulník)
Reference
(Blank)
Mirror Mirror
Semi-transparent
Mirror
Tungsten
Lamp
(wolframová lampa)
Grating
(mřížka)
Photo-
multiplier
Quartz
Cuvette
Sample
Mirror
Single Beam vs. Double Beam
(a) single-beam design
(b) dual channel design with beams
separated in space but simultaneous in time
(c) double-beam design in which beams
alternate between two channels."
42
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
Light sources
What is the important properties of a source?
Brightness
Line width
Background
Stability
Lifetime
Black-body radiation for Vis and IR but not UV
- a tungsten lamp is an excellent source of black-body radiation
- operates at 3000 K
- produces  from 320 to 2500 nm
For UV:
- a common lamp is a deuterium arc lamp
- electric discharge causes D2 to dissociate and emit UV radiation (160 – 325 nm)
- other good sources are: Xe (250 – 1000 nm) or Hg (280 – 1400 nm)
44
Wavelength Selectors
Ideally the output of a wavelength selector would be a radiation of a single
wavelength.
No real wavelength selector is ideal, usually a band of radiation is obtained.
The narrower this bandwidth is , the better performance of the instrument.
Wavelength
selectors
Filters Monochromators
45
• Filters permit certain bands of wavelength (bandwidth of ~ 50 nm) to pass
through.
• The simplest kind of filter is absorption filters , the most common of this
type of filters is colored glass filters.
• They are used in the visible region.
• The colored glass absorbs a broad portion of the spectrum
(complementary color) and transmits other portions (its color).
Disadvantage
• They are not very good wavelength selectors and can’t be used in
instruments utilized in research.
• This is because they allow the passage of a broad bandwidth which
gives a chance for deviations from Beer’s law.
• They absorb a significant fraction of the desired radiation.
i- Filters
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
47
• n  = d (sin i + sin r) where n = 1, 2, 3,….
• Since incident angle i = constant; therefore   r
Reflection Grating
Note: For more detail see Skoog text book p. 159-160
For each reflection angle
r , a certain wavelength is
observed
i
r
d
Echellette Grating equation
Monochromators: reflection grating
 Each wavelength is diffracted off the grating at a different angle
 Angle of deviation of diffracted beam is λ dependent  diffraction grating separates the incident beam into
its constituent wavelengths components
 Groove dimensions and spacings are on the order of the wavelength in question
ii- Monochromators
They are used for spectral scanning (varying the wavelength of
radiation over a considerable range ).
They can be used for UV/Vis region.
All monochromators are similar in mechanical construction.
All monochromators employ slits, mirrors, lenses, gratings or prisms.
49
Reflection grating
Grating monochromators
 Polychromatic radiation from
the entrance slit is collimated
(made into beam of parallel
rays) by a concave mirrors
 These rays fall on a reflection
grating, whereupon different
wavelengths are reflected at
different angles.
 The orientation of the
reflection grating directs only
one narrow band wavelengths,
2, to the exit slit of the mono-
chromator
 Rotation of the grating allows
different wavelengths, 1, to
pass through the exit slit
The reflection grating
monochromator
Device consists of entrance and
exit slits, mirrors, and a grating to
disperse the light
50
51
1. The reflection grating
is ruled with a series of closely
spaced, parallel grooves with
repeated distance d.
2. The grating is covered with
Al to make it reflective.
3. When polychromatic light is
reflected from the grating, each
groove behaves as a new point
source of radiation.
4. When adjacent light rays are in
phase, they reinforce one another
(constructive interference).
5. When adjacent light rays are not
in phase, they partially or
completely canceled one another
(destructive interference).
Reflection followed by either constructive
or destructive interferences
Echellette Reflection Grating
1 2
52
Prism monochromators
 Dispersion by prism depends
on refraction of light which
is wavelength dependent
 Violet color with higher
energy (shorter wavelength)
are diffracted or bent most
 While red light with lower
energy (longer wavelength
are diffracted or bent least
As a result, the polychromatic
white light is
dispersed to its individual
colors.
53
Bandwidth Choice The size of the monochromator
exit slit determines the width of
radiation (bandwidth) emitted
from the monochromator.
A wider slit width gives higher
sensitivity because higher
radiation intensity passes to the
sample but on the other hand,
narrow slit width gives better
resolution for the spectrum.
In general, the choice of slit width
to use in an experiment must be
made by compromising these
factors. Still, we can overcome
the problem of low sensitivity of
the small slit by increasing the
sensitivity of the detector.
What are the advantages and disadvantages of decreasing
monochromator slit width?
54
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
55
By performing the analysis at
such wavelengths, it will be
sure that the lowest sample
concentration can be
measured with fair accuracy.
For example, the lowest
sample concentration (10-5 M)
can be measured with good
accuracy at max, while at other
wavelength (1), it may not be
detected at all.
Absorbance
max 1
wavelength
10-2 M
10-3 M
10-4 M
10-5 M
5x10-5 M
56
shoulder
Broad horizontal
bands
 m
Absorbance
A

A
wavelength
Band A
Band B
2. It is preferable to choose the wavelength at which the absorbance will not
significantly change if the wavelength is slightly changed,
i.e., A /  is minimum.
At a wavelength corresponding to broad horizontal band on the spectrum
(band A), the radiation is mainly absorbed to the same extent (A /  zero).
However on a steep portion of the spectrum (band B), the absorbance will
change greatly if the wavelength is changed (A /  is large) . Thus on
repeating the absorbance measurements, you might get different readings
and the precision of the measurements will be poor.
3- If the solution contains more than absorbing species, the
wavelength should be chosen, whenever possible, in region at which
the other species does not absorb radiation or its absorbance is
minimum. By this way, the second species does not interfere in the
determination.
wavelength
Absorbance
sample
m
Interfering
species
57
58
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
59
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.
60
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-
61
Double Beam Spectrophotometer
Signal
Time
B
S
BB
S S
BB
S
100
B
S
T% 
represents P
represents P0
62
Schematic diagram of a double beam scanning spectrophotometer
 In double beam arrangement, the light alternately passes through
the sample and reference (blank), directed by rotating half-sector
mirror (chopper) into and out of the light path.
 When light passes through the sample, the detector measures the
P. When the chopper diverts the beam through the blank solution, the detector
measures P0.
 The beam is chopped several times per second and the electronic
circuit automatically compares P and P0 to calculate absorbance
and Transmittance.
63
Advantages of double beam instruments over single beam instruments
Single beam spectrophotometer is inconvenient because
1. The sample and blank must be placed alternately in the light path.
2. For measurements at multiple wavelengths, the blank must be run at each
wavelength.
In double beam instruments
1. The absorption in the sample is automatically corrected for the absorption
occurring in the blank, since the readout of the instrument is log the
difference between the sample beam and the blank beam.
2. Automatic correction for changes of the source intensity and changes in the
detector response with time or wavelength because the two beams are
compared and measured at the same time.
3. Automatic scanning and continuous recording of spectrum (absorbance
versus wavelength).
64
Applications of Ultraviolet/Visible
Molecular Absorption Spectrophotometry
 Molecular spectroscopy based upon UV-Vis radiation is used
for identification and estimation of inorganic, organic and
biomedical species.
 Molecular UV-Vis absorption spectrophotometry is employed
primarily for quantitative analysis.
 UV/Vis spectrophotometry is probably more widely used in
chemical and clinical laboratories throughout the world than any
other single method.
 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
65
66
Resources and references
Textbook: Principles of instrumental analysis, Skoog et al., 5th edition, chapter
7, 13.
Quantitative chemical analysis, Daniel C. Harris, 6th edition , chapter 20.
Lecture slides partially adopted from Dr. Raafat Aly slides.
Useful links
http://www.youtube.com/watch?v=pxC6F7bK8CU&feature=player_detailpage
http://bio-animations.blogspot.com/2008/04/double-beam-uvvis-
spectrophotometer.html
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.