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 

1

Absorption photometryElectron-spectroscopy

Biophysics 2nd semester

Febr. 2012

József Orbán

University of Pécs, Department of Biophysics

Rehearsal

2

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

3

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

4

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?

5

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

6

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 %

7

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