Absorbance and EmissionA tool to understand and characterize

the system

Tuhin Kumar MajiJRF, SNBNCBSUnder supervision of Prof. Samir Kumar Pal

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.

UV-VIS SPECTROSCOPY

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)

THE ABSORPTION SPECTRUM

ELECTROMELECTROMAGNETIC SPECTUMAGNETIC SPECTUM

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.

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=h erg𝜗

PRINCIPLE OF UV-VIS SPECTROMETRY

E -E = h𝜗₁ ₒ Etotal=Eelectronic + Evibrotional + Erotational

The energies decreases in the following order:

Electronic Vibrational Rotational⪢ ⪢

TYPES OF TRANSITIONS 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

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

“ 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

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.

LIMITATION OF LAWS

Know Our Instrument

Know Our InstrumentLight 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.

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

sam

ple

refe

renc

e

dete

ctor

I0

I0 I0

Ilog(I0/I) = A

200 l,

nm

monochromator/beam splitter optics

UV-VIS sources

Fluorescence spectroscopyand 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

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.

I. Principles of FluorescenceEnergy level diagram (Jablonski diagram)

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

Principles of FluorescenceFluorescence process: Emission

• The molecule relaxes from the lowest vibrational energy level of the excited state to a vibrationalenergy 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 incidentphotons

So

S1

I. Principles of fluorescence Intersystem 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

absorptionfluorescence

phosphorescence

Intersystemcrossing

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.

absorbed photons ofnumber emitted photons ofnumber

F f

Quantum yield of fluorescence

Know your Instrument

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 600Wavelength (nm)

Fluo

resc

ence

Inte

nsity

(a.u

.)

Raman

Rayleigh (lexc = lemm)

Fluorescence

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

Biological Fluorophores– Endogenous Fluorophores

amino acids

structural proteins

enzymes and co-enzymes

vitamins

lipids

porphyrins– Exogenous Fluorophores

Cyanine dyes

Photosensitizers

Molecular markers – GFP, etc.

I. Principles of fluorescenceIn

tens

ity

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence FluorescenceACCEPTOR

Molecule 1 Molecule 2

• Fluorescence energy transfer (FRET)In

tens

ity

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence FluorescenceACCEPTOR

Molecule 1 Molecule 2

Non radiative energy transfer – a quantum mechanical process of resonance between transition dipolesEffective between 10-100 Å onlyEmission and excitation spectrum must significantly overlapDonor transfers non-radiatively to the acceptor

Thank You

Any question ???