Ultraviolet and visible (UV-Vis) absorption spectroscopy measures the attenuation of light passing through or reflected from a sample. When light energy matches an electronic transition in a molecule, some light is absorbed, promoting electrons to higher orbitals. The resulting absorbance spectrum shows absorbance versus wavelength. Fluorescence spectroscopy involves excitation of molecules to higher electronic singlet states followed by emission of light as they relax to ground states. Quantum yield is the ratio of emitted to absorbed photons. Both techniques are useful in characterizing biological systems like proteins, DNA, and fluorophores.

1. Absorbance and Emission
A tool to understand and characterize
the system
Tuhin Kumar Maji
JRF, SNBNCBS
Under supervision of Prof. Samir Kumar Pal

2. Ultraviolet and visible (UV-Vis) absorption
spectroscopy is the measurement of the
attenuation of a beam of light after it passes
through a sample or after reflection from a
sample surface. Absorption measurements can
be at a single wavelength or over an extended
spectral range.

3. When a sample is exposed to light energy that matches the
energy difference between a possible electronic transition
within the molecule, a fraction of the light energy would be
absorbed by the molecule and the electrons would be
promoted to the higher energy state orbital. A
spectrometer records the degree of absorption by a
sample at different wavelengths and the resulting plot of
absorbance (A) versus wavelength (λ) is known as a
spectrum.
The significant features:
 λmax (wavelength at which there is a maximum
absorption)
 єmax (The intensity of maximum absorption)

4. ELECTROMELECTROMAGNETIC
SPECTUMAGNETIC SPECTUM

5. Electronic Spectroscopy
• Ultraviolet (UV) and visible (VIS)
spectroscopy
• This is the earliest method of molecular
spectroscopy.
• A phenomenon of interaction of molecules
with ultraviolet and visible lights.
• Absorption of photon results in electronic
transition of a molecule, and electrons are
promoted from ground state to higher
electronic states.

6.  Ultraviolet absorption spectra arise from
transition of electron with in a molecule from a
lower level to a higher level.
 A molecule absorb ultraviolet radiation of
frequency (𝜗), the electron in that molecule
undergo transition from lower to higher energy
level.
The energy can be calculated by the equation,
E=h𝜗 erg

7. E₁-Eₒ= h𝜗
Etotal=Eelectronic + Evibrotional + Erotational
The energies decreases in the following order:
Electronic ⪢Vibrational ⪢ Rotational

8. In U.V spectroscopy molecule undergo
electronic transition involving σ, π and n electrons.
 Four types of electronic transition are possible.
i. σ ⇾ σ* transition
ii. n ⇾ σ* transition
iii. n ⇾ π* transition
iv. π ⇾ π* transition
8

9. BEER’S LAW
“ The intensity of a beam of monochromatic light decrease
exponentially with the increase in concentration of the
absorbing substance” .
Arithmetically;
- dI/ dc ᾱ I
I= Io. exp(-kc) ---------------------eq (1)
ABSORBANCE LAWS

10. “ When a beam of light is allowed to pass through a
transparent medium, the rate of decrease of
intensity with the thickness of medium is directly
proportional to the intensity of the light”
mathematically;
-dI/ dt ᾱ I
-In . I = kt+b ----------------eq(2)
the combination of eq 1 & 2 we will get
A= Kct
A= ℇct (K=ℇ)
LAMBERT’S LAW

11.  The real limitation of the beer’s law is successfully
in describing the absorption behavior of dilute
solution only.
 In this regarding it may be considered as a
limiting law.

12. Know Our Instrument

13. Know Our Instrument
Light source:
UV - Hydrogen lamp ( hydrogen stored under
pressure) , Deuterium lamp and Xenon lamp-
it is not regularly used because of unstability
and also the radiation of UV causes the
generation of ozone by ionization of the
oxygen molecule.
VIS – Tungsten filament lamp , Tungsten
halogen lamp and carbon arc lamp.

14. Advantage of double beam spectrophotometer
The ratio of the powers of the sample & reference is constantly
obtained.
It has rapid scanning over the wide wavelength region because of the
above factor
DESCRIPTION OF UV- SPECTROPHOTOMETER
samplereference
detector
I
0
I
0
I
0
I
log(I0/I) =
A
20
0 l,
nm
monochromator/
beam splitter
optics
UV-VIS
sources

15. Fluorescence spectroscopy
and basic principle
Luminescence
• Emission of photons from electronically
excited states
• Two types of luminescence:
1.Relaxation from singlet excited state
2.Relaxation from triplet excited state

16. I. Principles of Fluorescence
Singlet and Triplet states
• Ground state – two electrons per orbital; electrons have
opposite spin and are paired
• Singlet excited state
Electron in higher energy orbital has the same spin orientation
with respect to electron in the lower orbital
• Triplet excited state
The excited valence electron may spontaneously reverse its spin
(spin flip). This process is called intersystem crossing.

17. I. Principles of Fluorescence
Energy level diagram (Jablonski diagram)

18. Principles of Fluorescence
Fluorescence process: Non-radiative relaxation
• In the excited state, the electron is promoted
to an anti-bonding orbital→ atoms in the
bond are less tightly held → shift to the right
for S1 potential energy curve →electron is
promoted to higher vibrational level in S1
state than the vibrational level it was in at the
ground state
• Vibrational deactivation takes place through
intermolecular collisions at a time scale of
10-12 s (faster than that of fluorescence
process)
.
So
S1

19. Principles of Fluorescence
Fluorescence process: Emission
• The molecule relaxes from the
lowest vibrational energy level
of the excited state to a vibrational
energy level of the ground state
(10-9 s)
• Relaxation to ground state occurs faster than
time scale of molecular vibration → “vertical”
transition
• The energy of the emitted photon
is lower than that of the incident
photons
So
S1

20. I. Principles of fluorescenceIntersystem crossing
• Intersystem crossing refers to non-radiative transition between states of
different multiplicity
• It occurs via inversion of the spin of the excited electron resulting in two
unpaired electrons with the same spin orientation, resulting in a state with
Spin=1 and multiplicity of 3 (triplet state)
• Transitions between states of different multiplicity are formally forbidden
• Spin-orbit and vibronic coupling mechanisms decrease the “pure” character of
the initial and final states, making intersystem crossing probable
• T1 → S0 transition is also forbidden → T1 lifetime significantly larger than S1
lifetime (10-3-102 s)
S0
S1
T1
absorption
fluorescence
phosphorescence
Intersystem
crossing

21. II. Quantum yield
• Quantum yield of fluorescence, Ff, is defined as:
• In practice, is measured by comparative measurements with reference
compound for which has been determined with high degree of accuracy.
absorbedphotonsofnumber
emittedphotonsofnumber
F f
Quantum yield of fluorescence

22. Know your Instrument

23. Fluorescence Measurements
Typical fluorescence emission spectrum at 340 nm
excitation (the different components)
0
500000
1000000
1500000
2000000
2500000
3000000
300 350 400 450 500 550 600
Wavelength (nm)
FluorescenceIntensity(a.u.)
Raman
Rayleigh (lexc = lemm)
Fluorescence

24. Applications in Biological Systems
Absorbance spectrum of
(a) different DNA bases,
(b) single and double
standard DNA
Absorbance spectrum of amino
acids tryptophan, tyrosine and
phenylalanine and a
representative protein BSA

26. Biological Fluorophores
–Endogenous Fluorophores
amino acids
structural proteins
enzymes and co-enzymes
vitamins
lipids
porphyrins
–Exogenous Fluorophores
Cyanine dyes
Photosensitizers
Molecular markers – GFP, etc.

27. I. Principles of fluorescence
Wavelength
Absorbance
DONOR
Absorbance
Fluorescence Fluorescence
ACCEPTOR
Molecule 1 Molecule 2
• Fluorescence energy transfer (FRET)
Wavelength
Absorbance
DONOR
Absorbance
Fluorescence Fluorescence
ACCEPTOR
Molecule 1 Molecule 2
Non radiative energy transfer – a quantum mechanical process of resonance between
transition dipoles
Effective between 10-100 Å only
Emission and excitation spectrum must significantly overlap
Donor transfers non-radiatively to the acceptor

28. Any question ???