papadopoulos@physik.uni-leipzig.de

Time-resolved Fourier Transform Infrared Spectroscopy (FTIR) in Soft Matter research

2

Outline

Physical processes in the IR spectral range IR spectrometry

Fourier Transform Infrared Spectroscopy (FTIR)

Quantitative information from IR spectra Effects of external fields on the molecular level

Time resolved FTIR Chemical reactions Conformational changes ...

3

Example: CO2 gas

Rotational – vibrational transitions

IR spectral range

-11[cm ]

4

IR spectra of condensed matter

Gases show complex vibrational-rotational spectra In soft matter absorption bands are significantly broader

Martin Chaplin, www.physics.umd.edu

IR spectral range

H2O

CO2

5

Oscillations – selection rules

Covalent bonds can be described by Morse or LJ potential curves

Quantum harmonic oscillator is a good approximation Both stretching and bending modes

Single photon is absorbed by interaction with oscillating dipole – transition dipole moment

Absorption coefficient: No absorption normal to the transition dipole moment

IR spectral range

nmp m n d

Δn=±1Others weakly allowed, due to anharmonicity(overtones)

i iq rd

: dipole operator

2

p E

6

IR spectroscopy as analytical tool

Widely used as analytical tool Easier preparation than NMR, less quantitative

Underestimated! IR and Raman spectroscopy are very powerful techniques

IR spectral range

1-octanol

7

Grating IR spectrometer

Requirements: Well collimated beam Monochromator

Largest part of light intensity is not used Calibration is necessary

IR spectrometry

8

Fourier Transform Infrared Spectroscopy

Michelson interferometer Interferogram: intensity vs optical

path difference Intensity at all wavelengths is

measured simultaneously

-0.01 0.00 0.01-0.2

-0.1

0.0

0.1

0.2

Inte

nsity

(ar

b. u

nits

)

Optical retardation (cm)

0 0det

0ig

, cos 42 2

1 cos 42

I II

II d

IR spectrometry

Optical path difference for each wavelength

γ

9

FTIR spectroscopy

Spectrum is easily obtained from the Fourier transform of the interferogram

IR spectrometry

ig 0

0

0 : 0I I d

igig 0

0

ig0

ig0 ig

0 1cos 4

2 2

0Re

2 2

02Re

2

II I d

IF I

II F I

4000 3500 3000 2500 2000 1500 1000

0.0

0.2

0.4

0.6

0.8

1.0

Inte

nsi

ty (

arb

. un

its)

wavenumber (cm -1)

no sample silk

3500 3000 2500 2000 1500 10000

1

2

3

Abs

orba

nce

wavenumber (cm -1)

-0.01 0.00 0.01-0.2

-0.1

0.0

0.1

0.2

Inte

nsi

ty (

arb

. un

its)

Optical retardation (cm)Fourier transform

Division

solvent solvent

„white light“ position

10

Resolution – Apodization

Problem: impossible to integrate interferogram from - to + Equivalent to multiplying “ideal”

interferogram with a “box” function FT of a product is the convolution of

FT‘s

Resolution depends on maximum mirror path ~ Δ-1

Artefacts! Multiplying with other functions

improves quantitative accuracy, but reduces resolution

Apodization=”removing feet”

Apodizationfunction

Fourier transform of Iig(γ)

Shape ofinfinitely thin lines

IR spectrometry

( ) ( )F f g F f F g

Fourier Transform Infrared Spectrometry,P. R. Griffiths, J.A. de Haseth, Wiley

11

Advantages of FTIR

Jacquinot advantage FTIR not as sensitive to beam misalignment, allowing for

larger aperture – throughput

Fellget advantage (“multiplex”) All frequencies measured together

Connes advantage Built-in calibration, mirror position determined by He-Ne

laser

FTIR is exclusively used nowadays

12

Transmission – reflection modes

Simplified: no interference, etc.

Transmission - absorption Specular reflection

Absorbance

Absorption coefficient αMolar absorption coefficient ε=α/c

Lambert-Beer law:

1

0

logI

AI

1 0 0e el clI I I

ln10 ln10

l clA

Reflectivityref

0

IR

I

Normal incidence in air2

1

1

nR

n

13

Complex refractive index

The imaginary part is proportional to the absorption coefficient

Dielectric function Real and imaginary parts are related through Kramers-

Kronig relations

Example:polycarbonate

n n in

0

0

exp 2

exp 4 exp 4

4

t

t

E x E i n x

I I i n x n x

n

2n

Fourier Transform Infrared Spectrometry,P. R. Griffiths, J.A. de Haseth, Wiley

14

Polarization dependence

Example: salol crystal All transition dipoles (for a certain transition) are perfectly

aligned Intensity of absorption bands depends greatly on crystal

orientation

Dichroism: difference of absorption coefficient between two axes Biaxiality (all three axes different)

IR spectral range

salol

Vibrational Spectroscopy in Life Science, F. Siebert, P. HildebrandtJ. Hanuza et al. / Vib. Spectrosc. 34 (2004) 253–268

15

Order parameter

Non-crystalline solids: molecules (and transition dipole moments) are not (perfectly) aligned Rotational symmetry is common Different absorbance A|| and A Dichroic ratio R= A|| / A

Molecular order parameter

IR spectral range

Reference axis

Molecular segment

Transition dipole

||

2

2

3 cos 1

2molS P

1

0 :2

mol RS

R

1

: 22 2

mol RS

R

“parallel” vibration

“perpendicular” vibration

2

2

1 2cot 2

2 2cot 1mol R

SR

16

1050 1000 950

0.1

0.2

0.3

0.4

Pol

y(al

anin

e)

(Ala

Gly

) n

Pol

y(gl

ycin

e) I

Abs

orba

nce

wavenumber (cm-1)

Pol

y(gl

ycin

e) I

I

Pol

y(al

anin

e)

0°

polarization

90°

0,0

0,2

0,4

0,6

0

30

60

90

120

150

180

210

240270

300

330

0,0

0,2

0,4

0,6

Abs

orba

nce

Smol=0.25

0,0

0,2

0,4

0,6

0

30

60

90

120

150

180

210

240270

300

330

0,0

0,2

0,4

0,6

Smol=0.50

Abs

orba

nce

0,0

0,2

0,4

0,6

0

30

60

90

120

150

180

210

240270

300

330

0,0

0,2

0,4

0,6

Smol=0.80A

bso

rba

nce

0

2

4

0

30

60

90

120

150

180

210

240

270

300

330

0

2

4

Smol=0.93

Abs

orba

nce

High order of alanine-rich crystalsLow order of glycine-rich amorphous chains

Order of crystals and amorphous phase in spider silk

Experimental

2

p E

p: transition dipole moment

Papadopoulos et al., Eur. Phys. J. E, 24, 193 (2007)Glisovic et al. Macromolecules 41, 390 (2008)

17

Examples of structural changes in soft matter

Phase transitions liquid crystals

Conformational changes Protein secondary structure

In many cases these processes take place very fast (< s) Cannot be probed by X-rays or NMR

Lemieux, R. P. Acc. Chem. Res. 2001, 34, 845-853

18

Time-resolved measurements

Two possibilities: Collect interferogram as fast as possible (“rapid scan”) Synchronize spectrometer with external event (“step scan”)

19

Rapid scan - kinetics

Interferograms are collected successively

Time resolution down to a few ms (depending on spectral resolution)

Non-repetitive processes Cannot average scans

noise 0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

Inte

nsi

ty (

arb

. un

its)

time (min)

trigger

-0.01 0.00 0.01-0.2

-0.1

0.0

0.1

0.2

Inte

nsi

ty (

arb

. un

its)

Optical retardation (cm)-0.01 0.00 0.01

-0.2

-0.1

0.0

0.1

0.2

Inte

nsi

ty (

arb

. un

its)

Optical retardation (cm)

Time-resolved FTIR

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Irreversible processes

Rapid scan is useful for studying chemical reactions and phase transitions

Crystallization of a liquid crystal by T-jump

Synthesis of polyurethane

For faster processes:Static measurements at different spots of a flow cell

1t

2tReaction time

Time-resolved FTIR

de Haseth et al., Appl. Spectrosc., 47, 173 (1993)Takahashi et al. J. Biol. Chem. 270, 8405 (1995)

amorphous

crystal

90°C 36°C

21

Step scan

Differences from rapid scan kinetics: Interferograms are not measured successively Triggered event is repeated for every mirror step

Allows study of very fast processes down to ns, ps -> chemical reactions Lower noise than kinetics

Disadvantages: Limited to repetitive processes Sensitive to system instabilities

Time-resolved FTIR

22

-0.01 0.00 0.01-0.2

-0.1

0.0

0.1

0.2

Inte

nsi

ty (

arb

. un

its)

Optical retardation (cm)

-0.01 0.00 0.01-0.2

-0.1

0.0

0.1

0.2

Inte

nsi

ty (

arb

. un

its)

Optical retardation (cm)

Step scan

Stroboscopic technique Mirror moves stepwise All measurements after a certain

dt from trigger are assembled to make a single interferogram

All interferograms are collected in a single scan

One scan takes longer than rapid scan, but much higher time resolution

Time-resolved FTIR

0 200 400 600 800 1000

0.0

0.2

0.4

0.6

0.8

1.0

1.2 rapid scan step scan

op

tica

l pa

th d

iffe

ren

ce (

arb

. u

nits

)

time (arb. units)

23

4000 3500 3000 2500 2000 1500 1000

0.0

0.2

0.4

0.6

0.8

1.0

Inte

nsity

(ar

b. u

nits

)

wavenumber (cm -1)

no sample silk (0 ms) silk (20 ms)

3500 3000 2500 2000 1500 10000

1

2

3

Abs

orba

nce

wavenumber (cm -1)

0 ms 20 ms

1000 9500.3

0.4

0.5

0.6

Abs

orba

nce

wavenumber (cm -1)

0 ms 20 ms

Step scan example: spider silk

Time-resolved FTIR

24

Combined IR and mechanical spectroscopy

polarizer

IR beam

Piezo crystals –DC motors

Force sensor

IR detector

sample

Tracing microscopic effects of strain

Possible to extract order parameter dependence on external fields

Dynamic Infrared Linear Dichroism (DIRLD)

Transmission mode using microscope

Experimental

25

Preparation of Step Scan measurement

Process studied with Step Scan FTIR should be reproducible

Several cycles should be run before actual measurement

Measurement should start at this point to ensure reproducibility

Time-resolved FTIR

26

DIRLD in polymers

Dichroic ratio depends on strain Polymer chains become better oriented Different trend for dipole moments parallel and normal to

the chain

S. Toki et al. / Polymer 41 (2000) 5423–5429I. Noda et al. / Appl. Spectrosc. 42 (1988) 203–216

Natural rubber (polyisoprene)

polystyrene

Time-resolved FTIR

27

External – crystal stress comparison: Phase

The step-scan technique allows IR measurements with high time resolution

Crystal stress can be measured as a function of time under sinusoidal external field

Phase shift < 2°

R. Ene et al. / Soft Matter, 2009, 5, 4568–4574

Time-resolved FTIR

28

What is the origin of frequency shifts?

Vibrational frequency depends on: Atom mass Bond force constant Number of atoms involved in vibration Perturbations

H-bonding Conformation

Anharmonicity Thermal expansion External fields (in this case)

29

C

CH3H

C

O

NH

NH

C

O

-1 -18 cm GPad d

pertV F r -4,68

-4,66

hc

Quantum Perturbation Theory

The shift is ~ 0.3 % QPT is applicable

The bond anharmonicity gives rise to the shift of energy levels

0( ) 20 0 (1 )a r rU U U e

0 1 2 3 4-6

-4

-2

0

2

4

Ene

rgy

(10

-19 J

)

r (Å)

Morse potential

Morse potential+

perturbation

N-C

3 eV

0.12 eV

F r 0.17 eV

disN CE

P. Papadopoulos et al. Eur. Phys. J. E 24, 193 (2007)

Theoretical value

30

Microscopic – macroscopic stress in silk

Crystal stress is equal to the externally applied At time scales from µs to

hours Independent of sample

history

Serial connection of crystals

0 ,0 0 ,2 0 ,4 0 ,6 0 ,8 1 ,0 1 ,2 1 ,4

9 6 1

9 6 2

9 6 3

9 6 4

9 6 5

stress (GPa)

wa

ven

um

be

r (c

m-1

)

S tat ic

-2.6 cm - 1 GPa - 1

Kinetics

Step Scan

PP, J. Sölter, F. Kremer Eur. Phys. J. E 24, 193 (2007)

31

Photoinduced protein folding

Bacteriorhodopsin structure changes after visible photon absorption IR photons do not have enough energy to change structure,

just probe vibrations!

Pulsed laser is synchronized with spectrometer Retinal conformational changes during the complete

cycle (~ms) are observed

reti

nal

R. Rammelsberg et al. Appl. Spectrosc. 51, 558 (1997)

Time-resolved FTIR

32

Folding kinetics of peptides after T-jumps

Alanine-based peptide Secondary structure depends on temperature (coil at higher T) Reaction rate “constants“ can be studied by T-jumps

IR laser pulses synchronized with spectrometer heat the sample by ~ 10°C

The sum ku+kf is determined by kinetics, ratio ku/kf by equilibriumu

f

folded unfoldedk

k

exp u fk k t

T. Wang et al. J. Phys. Chem. B 108, 15301 (2004)

Time-resolved FTIR

33

Summary

Fourier Transform IR spectroscopy is an ideal tool to study fast processes High sensitivity Information for different molecular groups High time resolution

Time resolved measurements Rapid scan Step scan

Effects of external perturbations in various systems: Polymers Proteins Liquid crystals, ...

http://www.uni-leipzig.de/~mop/lectures

Thank you for your attention!

34

N-term. C-term.

GGXGAAAAAAAA

Repetitive pattern

GGXGGX GGX GGX GGX

n

AAAAAAA GPGXX GPGXX GPGXX GPGXX GPGXX

n

MaSp2

MaSp1

Hydrophobic Slightly hydrophilic

Chemical structure of dragline silk and PA6

Block copolymer Two high-MW proteins (MaSp1 and MaSp2) Semi-crystalline High Ala- and Gly- content

PA6 (Nylon):

Spider silk

35

Normal vibrational modes

Simple relations only in diatomic molecules! Vibrations involve more than two atoms

Especially at low frequencies

Example: amide bond C

O

N H

C

k

Amide I Amide II

Amide III

Amide IV

36

Absorption spectrum of silk

Typical protein spectrum Amide vibrations

dominate, but ...

They cannot give aminoacid-specific information

The region 1100 – 900 cm-

1 can be used instead1050 1000 950

0.2

0.3

0.4

Po

ly(a

lan

ine

)

(Ala

Gly

) n

Po

ly(g

lyci

ne

) I

Ab

sorb

ance

wavenumber (cm-1)

Po

ly(g

lyci

ne

) II

Experimental

37

Poly(alanine) segment

Rotondi, K. S.; Gierasch, L. M. Biopolymers 2005, 84, 13-22. Simmons, A.; Ray, E.; Jelinski, L. W. Macromolecules 1994, 27, 5235-5237.

C-terminus

N-terminus

N-terminus

C-terminus

N-terminus

N-terminus

C-terminus

C-terminus

N C

NC

N C

N C

Antiparallel and parallel -sheet structure

38

3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750

0,0

0,3

0,6

0,9

1,2

0,0

0,5

1,0

1,5

-polyalanine

wavenumber (cm -1)

Abs

orb

anc

e

Am

ide

III

Am

ide

II

Am

ide

I

Am

ide

B MA silk ||

Ab

sorp

tion

coe

ffici

ent

(m

-1) A

mid

e A

Polyaminoacid IR spectra

Dragline silk and -polyalanine

A. M. Dwivedi, S. Krimm Macromolecules 15, 186 (1982)

39

Similar findings in PA6

Similar to silk, orientation before crystallization induces the high order

Crystal vibration responds linearly to applied stress Both spider silk and PA6 are

glassy at room temperature

40

Rotational – vibrational transitions

The fine structure of gas vibrational spectra is due to the vibrational transitions

Selection rules: Δn=±1 ΔJ=±1 (and 0 in certain cases)

Relation between integrated molar absorption coefficient and transition dipole moment:

22

2 202

0 0 0

8

2 3 3t t

med

m c he c

H.C. Haken – H. WolfMolecular Physics and Elements of Quantum ChemistryChapter 15