Absorbance and emission
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Transcript of Absorbance and emission
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Absorbance and EmissionA tool to understand and characterize
the system
Tuhin Kumar MajiJRF, SNBNCBSUnder supervision of Prof. Samir Kumar Pal
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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
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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
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ELECTROMELECTROMAGNETIC SPECTUMAGNETIC SPECTUM
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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.
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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
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E -E = h𝜗₁ ₒ Etotal=Eelectronic + Evibrotional + Erotational
The energies decreases in the following order:
Electronic Vibrational Rotational⪢ ⪢
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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
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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
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“ 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
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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
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Know Our Instrument
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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.
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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
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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
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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.
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I. Principles of FluorescenceEnergy level diagram (Jablonski diagram)
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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
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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
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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
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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
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Know your Instrument
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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
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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
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Biological Fluorophores– Endogenous Fluorophores
amino acids
structural proteins
enzymes and co-enzymes
vitamins
lipids
porphyrins– Exogenous Fluorophores
Cyanine dyes
Photosensitizers
Molecular markers – GFP, etc.
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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
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Thank You
Any question ???