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PÉCSI TUDOMÁNYEGYETEMÁLTALÁNOS ORVOSTUDOMÁNYI KAR

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Absorptionspectroscopy(Miklós Nyitrai; 2nd of March 2016)

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Content

Light;

Electromagnetic waves and interactions;

Definition of absorption;

Absorption measurements;

Applications.

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Reminder

The light is electromagnetic radiation.

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Reminder

red orange yellow green kék violetfar red UV

390nm422nm492nm535nm586nm647nm760nm

increasing energy

What is the correlation betweenfrequency, energy and wavelength?

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Spektroscopy

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Absorption in diluted solutions

- transmittance

- absorption

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Transmittance

substancelightsource

detector

T = I / I0

Usually given in %

I0 I

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Absorptionor

light absoprtion

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Absorption

Sample

ligh

tin

ten

sity

distance

I0

I

Exponentialfunction!!!

I = I0 exp (-kx)

or

I = I0 e-kx

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N = N0 exp (-λt)

or

N = N0 e-λt

Similar to the radioactive decaylow!

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sample

inte

nsi

ty

ln(distance)

I0

I

Exponentialtoo!

A different presentation

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The Born-Oppenheimer approximation;

Nuclear vs. Electron motions.

What are the bases?

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Example: water molecule

http://www1.lsbu.ac.uk/water/vibrat.html

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Esum = Eelect + Evibr + Erot

The changes can be then written as:

∆Esum = ∆Eelect + ∆Evibr + ∆Erot

The splitting of the energy levels

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Energy scheme: band spectra

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The magnitude of the energy types:

∆Eelect ~ 1,000 * ∆Evibr ~ 1,000,000 * ∆Erot

The energy scale

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Spin multiplicity within the molecules

The number of possible quantum states (n) of a

system based on the spin quantum number S:

M = 2S+1

Szingulett → S=0; M = 1

Triplett → S=1; M = 3

singlet groundstate

excited singletstate

excited tripletstate

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Interpretation of absorption

S0

S1

Ground state

S0 – S1

Excited state Vibrational relaxation (10-12s)

excitation (10-15s)

hν

T1

T1 → S0 (10-3 – 10-1s )

vibrational levels012345

S1 → T1: intersystem crossing(10-10 – 10-8 s)

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Aim:

- easy understanding

- measurable

- additive

Note: transmittance (T = I / I0) is not additive. A solution of 50%transmittance is mixed with a solution of 60% transmittance(keeping the concentrations) will never give a solution of 110%transmittance.

The definition of absorption

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sample

inte

nsi

ty

distance

I0

I

I = I0 10-ε(λ) c x

Define parameters!Why ε(λ) and not only ε ?

Distance dependence

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λ (nm)

abso

rption

ε depends on λ!

Why ε(λ) and not only ε?

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substance

OD = A = - log (I / I0) = ε (λ) c x

I0 I

„optical density” I = I0 10-ε(λ) c x

The definition of absoprtion

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photometry = absorption spectroscopy

Absorption measurements

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light source sample detectormonochromator

How to measure absorption?A scheme of a photometer.

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Spektrophotometer

Main components:

1. Light source

• UV lamp (~180-350nm): Deuterium lamp

• Visible source (~350-800nm): Wolfram

2. Monochromator: to select wavelength.

3. Sample holder: e.g. a cuvette

4. photodetektor: PMT, diode…

5. Other components: lenses, filters, slits.

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How does the prism work?

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How does the prism work?

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The wavelength dependence of therefraction index

λ

n

λ

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red

purple

yellow

White lightMonochromatic light

The wavelength dependence of therefraction index

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Why we use reference?

Photometers

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The absorption of proteins

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The absorption of proteins

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The absorption of proteins

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Measured absorption: A

Extinkciós coefficient: ε

Usual units for ε: M-1cm-1, or (mg/ml)-1cm-1

If A = 0.55 and ε = 1.1 (mg/ml)-1cm-1

c = (A/ ε) in mg/ml; c = 0.5 mg/ml

Applications: protein concentration

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λ (nm)

abso

rpti

on

fluorescent probe

protein

What is the advantage of additivity? (example)

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0 5 10 15 20

0.0

0.2

0.4

0.6

0.8

1.0

S

[mDia3-FH2] (µM)

Application: gel electrophoresis

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- interpretation of absorption;

- definition of absorption;

- absorption measurements;

- applications.

Summary

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Thank you!