Time-resolved Fourier Transform Infrared Spectroscopy (FTIR) in Soft Matter research
papadopoulos@physik.uni-leipzig.de
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

...
2
IR spectral range
Example: CO2 gas

Rotational – vibrational transitions
 
1

[cm -1 ]
3
IR spectral range
IR spectra of condensed matter

Gases show complex vibrational-rotational spectra

In soft matter absorption bands are significantly broader
H2 O
CO2
Martin Chaplin, www.physics.umd.edu
4
IR spectral range
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:

pnm  m d n
2
No absorption normal to the transition
dipole
moment

 pE
d   qi ri
: dipole operator
Δn=±1
Others weakly
allowed, due to
anharmonicity
(overtones)
5
IR spectral range
IR spectroscopy as analytical tool

Widely used as analytical tool

Easier preparation than NMR, less quantitative
1-octanol

Underestimated!

IR and Raman spectroscopy are very powerful techniques
6
IR spectrometry
Grating IR spectrometer

Requirements:

Well collimated beam

Monochromator

Largest part of light intensity is not used

Calibration is necessary
7
IR spectrometry
Fourier Transform Infrared Spectroscopy

Michelson interferometer

Interferogram: intensity vs optical path difference

Intensity at all wavelengths is measured simultaneously
γ
Intensity (arb. units)
0.2
0.1
I det   ,  
0.0
I ig     
-0.1
-0.2
-0.01
0.00
Optical retardation (cm)
0.01
I 0   I 0  

cos  4 
2
2
I 0  
1  cos  4   d
2
Optical path difference for
each wavelength
8
IR spectrometry
FTIR spectroscopy
„white light“ position
Spectrum is easily obtained from the Fourier transform of the
interferogram

  0 : Iig  0   I 0   d
Intensity (arb. units)
0.2
I ig    
0.1

0.0
-0.1
-0.2
-0.01
0.00
0.01
Optical retardation (cm)
1.0
I 0   
Fourier
transform
I ig  0 
2
I ig  0 
2
0



1
I 0   cos  4  d
2 0


Re F  I 0 
2
 
 
I  0  
Re  F  I ig     ig
 


2

 
2
solvent
solvent
3
no sample
silk
0.8
Absorbance
Intensity (arb. units)

0.6
0.4
Division
0.2
2
1
0.0
4000
3500
3000
2500
2000
-1
wavenumber (cm )
1500
1000
0
3500
3000
2500
2000
1500
1000
-1
wavenumber (cm )
9
IR spectrometry
Resolution – Apodization
Fourier transform of Iig(γ)

Problem: impossible to integrate interferogram from - to
+




Apodization
function
Shape of
infinitely thin lines
Equivalent to multiplying “ideal” interferogram with a “box”
function
FT of a product is the convolution of FT‘s
F  f  g   F ( f )  F (g)
Resolution depends on maximum mirror path ~ Δ-1
Artefacts!

Multiplying with other functions improves quantitative
accuracy, but reduces resolution

Apodization=”removing feet”
Fourier Transform Infrared Spectrometry,
P. R. Griffiths, J.A. de Haseth, Wiley
10
Advantages of FTIR

Jacquinot advantage


Fellget advantage (“multiplex”)


All frequencies measured together
Connes advantage


FTIR not as sensitive to beam misalignment, allowing for larger aperture – throughput
Built-in calibration, mirror position determined by He-Ne laser
FTIR is exclusively used nowadays
11
Transmission – reflection modes

Simplified: no interference, etc.
Transmission - absorption
Absorbance
A   log
Absorption coefficient α
Molar absorption coefficient ε=α/c
Lambert-Beer law:
Specular reflection
I1
I0
Reflectivity
R
I ref
I0
Normal incidence in air
I1  I0 el  I0 e cl
l
 cl
A

ln10 ln10
 n  1 
R  
 n 1 
2
12
Complex refractive index
n  n  in
 The imaginary part is proportional to the absorption coefficient
Et  x   E0 exp  i 2 n x  
I t  I 0 exp  i 4 n x  exp  4 n x  


  4 n
Dielectric function
    n 
Real and imaginary parts are related through Kramers-Kronig relations
2
Example:
polycarbonate
Fourier Transform Infrared Spectrometry,
P. R. Griffiths, J.A. de Haseth, Wiley
13
IR spectral range
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)
salol
Vibrational Spectroscopy in Life Science, F. Siebert, P. Hildebrandt
J. Hanuza et al. / Vib. Spectrosc. 34 (2004) 253–268
14
IR spectral range
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 
Reference
axis
Molecular order parameter
Molecular
segment
S mol  P2   
“parallel” vibration
  0 : Smol 
“perpendicular” vibration


2
3 cos2   1
R 1
R2
R 1
R2
R  1 2 cot 2   2

R  2 2 cot 2   1
: Smol  2
S mol
Transition
dipole
2
||

15
Experimental
Poly(alanine)
(AlaGly)n
0.4
p: transition dipole moment
Poly(glycine) II

2
Poly(alanine)
pE
Absorbance


Poly(glycine) I
Order of crystals and amorphous phase in spider silk
0.3
polarization
0.2
0°
0.1
90°
1050
1000
950
-1
wavenumber (cm )
90
120
60
mol
Absorbance
150
S =0.25
30
0,2
0,0 180
0
0,2
330
210
Absorbance
0,4
0,4
150
S =0.50
0,0 180
30
0,4
0
0,2
330
210
0,4
0,6
240
300
270
240
4
60
120
150
S =0.80
0,0 180
30
0
0,2
330
210
60
mol
mol
0,2
0,4
0,6
120
0,6
0,2
0,4
90
90
60
mol
Absorbance
0,6
120
0,6
Absorbance
90
2
150
S =0.93
30
0 180
2
0
330
210
300
0,6
270
240
300
270
Low order of glycine-rich amorphous chains
4
240
300
270
High order of alanine-rich crystals
Papadopoulos et al., Eur. Phys. J. E, 24, 193 (2007)
Glisovic et al. Macromolecules 41, 390 (2008)
16
Examples of structural changes in soft matter

Phase transitions


liquid crystals
Conformational changes


Lemieux, R. P. Acc. Chem. Res. 2001, 34, 845-853
Protein secondary structure
In many cases these processes take place very fast (< s)

Cannot be probed by X-rays or NMR
17
Time-resolved measurements

Two possibilities:

Collect interferogram as fast as possible (“rapid scan”)

Synchronize spectrometer with external event (“step scan”)
18
Time-resolved FTIR
Rapid scan - kinetics
trigger

Interferograms are collected successively

Time resolution down to a few ms (depending on spectral
resolution)
Non-repetitive processes

Cannot average scans
0.8
0.6
0.4
0.2
noise
0.0
0
1
2
3
4
5
time (min)
0.2
Intensity (arb. units)
0.2
Intensity (arb. units)

Intensity (arb. units)

1.0
0.1
0.0
-0.1
0.1
0.0
-0.1
-0.2
-0.01
0.00
Optical retardation (cm)
0.01
-0.2
-0.01
0.00
0.01
Optical retardation (cm)
19
Time-resolved FTIR
Irreversible processes

Rapid scan is useful for studying chemical reactions and phase transitions
For faster processes:
Static measurements at different spots of a
flow cell
Synthesis of polyurethane
crystal
t1
Reaction time
Crystallization of a liquid
crystal by T-jump
90°C
36°C
t2
amorphous
de Haseth et al., Appl. Spectrosc., 47, 173 (1993)
Takahashi et al. J. Biol. Chem. 270, 8405 (1995)
20
Time-resolved FTIR
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
21
Time-resolved FTIR
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
rapid scan
0.2
0.2
0.1
0.1
step scan
1.0
0.8
0.6
0.4
Intensity (arb. units)
1.2
Intensity (arb. units)
optical path difference (arb. units)

0.0
-0.1
-0.1
0.2
0.0
0.0
0
200
400
600
time (arb. units)
800
1000
-0.2
-0.2
-0.01
0.00
Optical retardation (cm)
0.01
-0.01
0.00
0.01
Optical retardation (cm)
22
Time-resolved FTIR
Step scan example: spider silk
Intensity (arb. units)
1.0
0.8
no sample
silk (0 ms)
silk (20 ms)
0.6
0.4
0.2
0.0
4000
3500
3000
2500
2000
1500
1000
-1
wavenumber (cm )
0 ms
20 ms
0.6
Absorbance
Absorbance
3
2
1
0 ms
20 ms
0.5
0.4
0
3500
3000
2500
2000
-1
wavenumber (cm )
1500
1000
0.3
1000
950
-1
wavenumber (cm )
23
Experimental
Combined IR and mechanical spectroscopy
Transmission mode using microscope



Tracing microscopic effects of strain
Possible to extract order parameter
dependence on external fields
Dynamic Infrared Linear Dichroism
(DIRLD)
IR detector
Force sensor
sample
polarizer
Piezo crystals –
DC motors
IR beam
24
Time-resolved FTIR
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
25
Time-resolved FTIR
DIRLD in polymers

Dichroic ratio depends on strain

Polymer chains become better oriented

Different trend for dipole moments parallel and normal to the chain
Natural rubber (polyisoprene)
polystyrene
S. Toki et al. / Polymer 41 (2000) 5423–5429
I. Noda et al. / Appl. Spectrosc. 42 (1988) 203–216
26
Time-resolved FTIR
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
27
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)
28
Quantum Perturbation Theory
The shift is ~ 0.3 %

QPT is applicable

CH3
O
H
C
C N
C N
H
O
H
The bond anharmonicity gives rise to the shift of energy
levels

ENdisC  3 eV
  0.12 eV
Theoretical value
F  rN-C  0.17 eV
-4,6
J)
-4,66
Morse potential
0
Morse potential
+
perturbation
-2
-4
-6
0
1
2
r (Å)
3
4
-19
2
Energy ( 10
-19
J)
4
Energy ( 10
d d  8 cm-1 GPa -1
hc
U  U0  U0 (1  ea ( r r0 ) )2
-4,68
V pert   F  r
-4,8
1,4
1,6
r (Å)
P. Papadopoulos et al. Eur. Phys. J. E 24, 193 (2007)
29
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
Static
Kinetics
-1

965
wavenumber (cm )

Step Scan
964
963
-1
-1
-2.6 cm GPa
962
961
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
stress (GPa)
PP, J. Sölter, F. Kremer Eur. Phys. J. E 24, 193 (2007)
30
Time-resolved FTIR
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
retinal

R. Rammelsberg et al. Appl. Spectrosc. 51, 558 (1997)
31
Time-resolved FTIR
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 equilibrium
ku
folded
unfolded
kf

exp   ku  k f  t
T. Wang et al. J. Phys. Chem. B 108, 15301 (2004)

32
Summary

Fourier Transform IR spectroscopy is an ideal tool to study fast processes




Time resolved measurements



High sensitivity
Information for different molecular groups
High time resolution
Rapid scan
Step scan
Effects of external perturbations in various systems:



Polymers
Proteins
Liquid crystals, ...
Thank you for your attention!
http://www.uni-leipzig.de/~mop/lectures
33
Spider silk
Chemical structure of dragline silk and PA6

Block copolymer

Two high-MW proteins (MaSp1 and MaSp2)

Semi-crystalline

High Ala- and Gly- content
N-term.
C-term.
Repetitive pattern
MaSp1
AAAAAAA GGX GGX GGX GGX GA GGX GGX
n
MaSp2
AAAAAAA GPGXX GPGXX GPGXX GPGXX GPGXX
n
Hydrophobic
Slightly hydrophilic
PA6 (Nylon):
34
Normal vibrational modes

Simple relations only in diatomic molecules!

Vibrations involve more than two atoms


 k 
Especially at low frequencies
Example: amide bond
C
O
N H
C
Amide I
Amide II
Amide III
Amide IV
35
Experimental
1.0
Amide III
Amide vibrations dominate, but ...
Amide I

1.5
Amide A
Typical protein spectrum
Absorbance

Amide II
Absorption spectrum of silk
0.5
0.0
4000
3500
3000
2500
2000
1500
1000
-1
Poly(alanine)
(AlaGly)n
0.4
Poly(glycine) I
The region 1100 – 900 cm-1 can be used
instead
Poly(glycine) II

They cannot give aminoacid-specific
information
Absorbance

wavenumber (cm )
0.3
0.2
1050
1000
950
-1
wavenumber (cm )
36
Antiparallel and parallel b-sheet structure
N
C
N-terminus
C-terminus
C-terminus
N-terminus
C
N
N
C
C-terminus
N-terminus
Poly(alanine) segment
N-terminus
C-terminus
N
C
Rotondi, K. S.; Gierasch, L. M. Biopolymers 2005, 84, 13-22.
Simmons, A.; Ray, E.; Jelinski, L. W. Macromolecules 1994, 27, 5235-5237.
37
Polyaminoacid IR spectra
Amide II

0,5
0,0
1,2
Absorbance
MA silk
||
Amide III
1,0
Amide I
1,5
Amide B
Amide A
Dragline silk and b-polyalanine
Absorption coefficient
-1
(m )

b-polyalanine
0,9
0,6
0,3
0,0
3500
3250
3000
2750
2500
2250
2000
1750
1500
1250
1000
750
-1
wavenumber (cm )
A. M. Dwivedi, S. Krimm Macromolecules 15, 186 (1982)
38
Similar findings in PA6
0.015
Similar to silk, orientation before crystallization
induces the high order

Absorbance
C-N
C=O
0.010
0.005
N-H
CH2
//
0.000
3500

3000
2500
2000
wavenumber / cm

1500
1000
-1
Crystal vibration responds
linearly to applied stress

Both spider silk and PA6 are
glassy at room temperature
39
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:
 e2 8 2 m0 2
 2


d




t


t
2

2m0 0c 3he
3 0 c
H.C. Haken – H. Wolf
Molecular Physics and Elements of Quantum Chemistry
Chapter 15
40