Absorption spectrometry – summary
Rehearsal:
Properties of light (electromagnetic radiation), dual nature
light‐matter interactions (reflection, transmission, absorption, scattering)
Absorption phenomena, change in light intensity: exponential and logarithmic function → Lambert‐
Beer law,
absorbance, optical density, extinction coefficient, concentration, optical path (sample width) and
their relationship
Absorption (and emission):
matter = sample = absorbent (synonyms)
phenomena: excitation‐deexcitation, absorption‐emission, interaction of photon and electron
atomic energy levels, energy difference of levels, photon energy, resonance condition
atomic line type, molecular band type and highly heated matter’s continuous (emission) spectra
Interpretation of molecular BAND type spectra: origin, characteristics
Born‐Oppenheimer approximation
electronic, vibrational, rotational energy levels and their independence → Jablonsky diagram (term
scheme)
interpretation of absorption phenomena on the Jablonsky diagram
Absorption spectrometry – fotometer:
setup and arrangement: light source, monochromator, sample, detector (PMT), data aquisition and
data handling unit (PC)
principle of operation, one‐/two way fotometer, sample and reference
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Absorption photometryElectron-spectroscopy
Biophysics 2nd semester
Febr. 2012
József Orbán
University of Pécs, Department of Biophysics
Rehearsal
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Light – electromagnetic radiation
photon (quantum of radiation energy) : E = h f
Dual nature of light:Dual nature of light:
Electromagnetic wave(propagation)Maxwell
• Diffraction
Particle (photon)(reaction)Einstein
• Photoeffect• Interference• Polarisation
• Compton-effect
A b s o r p t i o nR e f l e c t i o n
Propagation of electromagnetic wave
x
direction ofpropagationIf = 600 nm, then
f = 5·1014 Hz
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Propagation of electromagnetic wave
wavelength
electric fieldstrengthvector
E
x
magneticfield strengthvector
x
B
c = f
transversalwave
The electric- and the magnetic field strength vectors are perpendicular to each other and to the direction of propagation, as well!
x
x
Total spectra of electromagnetic radiations
Energy, frequency (E=hf Wavelength (=1/f)
E = hf
c = f
Gamma
X-ray (Röntgen)
Visible range: light
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Radiation – matter interaction
Interaction of Light and Matter
electromagnetic (or radioactive) radiation
matter = substance = absorber
Radiation – matter interaction
substanceI0 (initial intensity) I
Reflection
I ~ nI: intensityn: photon number
ReflectionTransmissionAbsorption
Scattering – Rayleigh-type
Which properties of thesubstance define theabsorption?
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matter
Absorption
light sourceI
I0
I0 Iwidth
0
1 2 3
Which function fitsto the plot?Exponential function!!! to the plot?Exponential function!!!
xx eII 0)(
xx eNN 0)(photon number:
intensity:
Light absorption in a substance
Always true:I ≥ I’ > I
homogenous
sampleI0’
• reflected: R = I0’-I0
• absorbed : A = I0-I• transmitted : T = I
I0 ≥ I’0 > I
I
Inte
nsit
y
I0
In general reflection is negligible! Or it is eliminated by appropriate measurement setup.
x width / optical path
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matter
I0 I
Definition: ABSORBANCE
DETECTOR
light source
OD = A = - log10 (I / I0) = () · c · x
I = I0 10-() c x Why () and not only ?
Lambert-Beer equation (insolutions)
I = I0 e-xgeneral equation:
optical densityabsorbance No dimension, additive quantity!
(): extinction coefficient (depends on constitution), c: concentration of solution, x: optical path (width)
E x e r c i s eE x e r c i s e
• A substance transmits 10% of incoming light. Calculate the absorbance.
A = OD = - log (I/I0) = - log (0.1) = 1
• If transmittance is 1%, the absorbance is?
A = 2
• Calculate the total absorbance if we use thesetogether (10% and 1% transmittance)!together (10% and 1% transmittance)!
A = 2+1 = 3, T = 0.01·0.1 = 0.001 = 0.1 %
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S p e c t r a
(line type emission spectra)
state:
Absorption and Emission of atoms1. absorption
ground state excited state
phenomena:
+
e-
Eabs = h +
e-
+
e-
(photon)Absorption
Absorption of thephoton and the
(electron)Excitation
Absorption photon and theexcitation of theelectron occurssimultaneously!
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state:
Absorption and Emission of atoms2. emission
ground state excited state
phenomena: (electron)De excitation
+
e-
Eem = h+
e-
+
e-
The de-excitation of l d h
(photon)Emission
De-excitation electron and theemission of the photonoccurs at the same time!
The energy of the absorbed and emitted photon is equal and matches withthe energy difference of the electron’s ground and excited states’ energy!
Electronic energy levels of atomsBohr- and quantummechanic atommodel
energy (level) energy difference
Electrons have quantised (defined) energy→ energy levels!
Figures are only for demonstration!
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Spectrum
Spectrum:• (light) intensity or analogous quantity - plotted against( g ) y g q y p g• wavelength or frequency.
Types:• line ( atoms )( )• band ( molecules )• continuous ( any material at high temperature:
black body radiation )
Spectral typesI
emission
absorption
Continuous, emission
Line emission
I
Source of images: http://csep10.phys.utk.edu/astr162/lect/light/absorption.html
See: continuous emission radiation, black body, Planck, Stefan-Boltzman
Line, emission
Line, absorption
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2,5
Band type (absorption) spectrumof ACTIN molecule
How can you explain the shape?
1,0
1,5
2,0
Ab
sorp
tion
The line spectra of atomsand band spectra of molecules are characteristic!(depends on their chemicalconstitution)
260 270 280 290 300 310 320
0,0
0,5
Wavelength (nm)
actin
Absorption of proteins - aminoacids
There are 3 aminoacids thatabsorbs in UV.
(Measuring the absorptionspectra of protein solution,the concentration can bedetermined.)
tinc
tion
coef
fici
ent
wavelength
Ext
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Spectra of molecules
Born-Oppenheimer approximation:
Atomic nuclei moves much slower than electrons, becausethe nucleus is much more heavy,
therefore the simplified model of molecular motionconsiders the nucleus as not moving object. The electron-nucleus can be considered as two objects at two ends of anucleus can be considered as two objects at two ends of a spring, with electric charge.
Dynamic molecular structureWhy band, and not line spectra?
http://en.wikipedia.org/wiki/Infrared_spectroscopy
Etotal = Eelectronic + Evibrational + Erotational
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Molecular energy-levels (terms)
Etotal = Eelectronic + Evibrational + Erotational
E = E + E + E
• All the energy levels are below the zero level!• More vibration level can appear at one electronic energy level. These vibrational energy levels are superimposed on each electronic level equidistantly separated from each
Etotal = Eelectronic + Evibration + Erotation
Eelectronic ~ 1000 * Evibrational ~ 1000000 * Erotational
each electronic level equidistantly separated from each other.• One vibrational level can consist several rotational levels.• As electron transitions can take place between severalenergy levels with not much different energy → line spectra widens to band spectra!
Jabłonsky-type term-scheme
• All the energy levels are below the zero level!
0
S2
• Each line represents a well defined(electronic, vibrational, rotational) energy state of the molecule.
• Ground state: the electrons stay atone vibrational level of the lowestelectronic level (S0).
Ene
rgy
S1
S2
rotational levels• S stands for singlet state
S0vibrational levels
electronic levels: S0, S1, S2
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0
S2
1. Excitation & Absorption2. De-excitation & Emission
l & h
Jabłonsky-type term-schemeE
nerg
y
S1
2 electron & photon
E = hf(resonance condition)
AbsorptionhfEmission hf
Depending on the energy of the photon:• electronic
ibrational
S0
Emission hf • vibrational• rotational• mixedtransition happens.
Detector 1monochromator
(prism or grating)light source Sample
How do we determine absorbance?Setup of a (absorption) photometer
Referencepath
(prism or grating) p
Reference (blank)
Detector 2
Data aquisition(PC)
LightElectric signal
To measure an absorbance spectra the absorbance values aredetermined from to , step by step) in a wide (spectral) range.
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Basic concepts
Light source
PMT:
Detection of light (photons):Converts electromagnetic radiation to electric signal.
Photoelectron multiplier tube (PMT)
Photoeffect (photon force the cathode to eject an electron!
on
photocathodedynodesph
oto
elec
tron If: 1e- → 2 e-
amplification:2n
(n: number of
accelerating high voltage
current/voltmeterelectron push other (1-6) electrons
Total amplification: 1 photon (photoelectron) → 1-10 million electrons!
dynodes)
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Two forensic investigator speaks regarding at a blood sample:- I am sure that he died of hampered respiration.- Cyanosis? How do you know it?- From the color of the blood!
(How) is it possible?
Oxy- and carboxy-hemoglobinThe difference in absorption in the range 600-800 nm explains the different appearent color.
Two peaks of HbO2 measured on practical: „Spectroscopy and spectrometry”
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Interaction of Electromagnetic Radiation and Matter(absorbtion)
Phenomenon to study Spectral Range Wavelength
Inner electron shells X-rays 0 01-1 0 nmInner electron shells,ionization
X rays 0.01 1.0 nm
Valency (outer) electrons Ultraviolet
Visible
0-400 nm
400-800 nm
Molecular vibrations (bond stretching), rotation
Infrared 800 nm –
0.8 mm
Rotation & electron spin orientation in magnetic field
Microwaves 0.8 mm - 30 cm
Nuclear spin orientation in magnetic field
Radio waves >100 cm
Supplement• http://en.wikipedia.org/wiki/
/Spectroscopy/Spectrum
White light can be split to several colours (with prism or optical grating)
RedOrangeYellowGreen
ROYGGreen
BlueViolet
GBV
We may understand the colours of our colourful environmentif we keep in mind that only photons reaching our eye candefine the colour of the percepted objects.
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SupplementSurface colours defined by reflection
RedOrangeYellowGreenBlueVi l
What is the colour of this object?
Violet
Search for the term:Additive / constructive colour mixinge.g.: colour of the furniture, wall, ink,paper, hair, iris
Supplement„body” colours defined by absorption (transmission)
RedOrangeYellowGreenBlueVi l t
What is the colour of this object?
Violet
e.g.: colour of the red blood cells, leaves, lead glass windows