Absorption Electromagnetic waves and interactions; spectroscopy - - PDF document

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PÉCSI TUDOMÁNYEGYETEM ÁLTALÁNOS ORVOSTUDOMÁNYI KAR www.aok.pte.hu

Absorption spectroscopy

(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

• range

yellow green kék violet far red UV

390nm 422nm 492nm 535nm 586nm 647nm 760nm

increasing energy What is the correlation between frequency, energy and wavelength?

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Spektroscopy

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Absorption in diluted solutions

• transmittance
• absorption

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Transmittance

substance light source detector T = I / I0 Usually given in %

I0 I

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Absorption

• r

light absoprtion

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Absorption

Sample

light intensity distance

I0 I

Exponential function!!!

I = I0 exp (-kx)

• r

I = I0 e-kx

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N = N0 exp (-λt)

• r

N = N0 e-λt

Similar to the radioactive decay low!

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sample

intensity ln(distance)

I0 I

Exponential too!

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+ S+1 Szingulett → S=0; M = 1 Triplett → S=1; M = 3

singlet ground state excited singlet state excited triplet state

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Interpretation of absorption

S0 S1 Ground state S0 – S1 Excited ed state Vibrational relaxation (10-12s) excitation (10-15s) hν T1 T1 → S0 (10-3 – 10-1s ) vibrational levels

1 2 3 4 5

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

intensity 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) absorption

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 detector monochromator

How to measure absorption?

A scheme of a photometer.

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Spektrophotometer

Main componen ents: 1. Ligh ght source ce

• UV lamp (~180-350nm): Deuterium lamp
• Visible source (~350-800nm): Wolfram

2. Monochromat ator: to select wavelength. 3. Sample holder er: e.g. a cuvette 4. photodet etekt ektor: PMT, diode… 5. Other componen ents: 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 the refraction index

λ n λ

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red purple yellow White light Monochromatic light

The wavelength dependence of the refraction 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) absorption fluorescent probe protein

What is the advantage of additivity? (example)

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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!