1. 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
Example: CO2 gas
Rotational – vibrational transitions

4. IR spectra of condensed matter
IR spectral range
IR spectra of condensed matter
Gases show complex vibrational-rotational spectra
In soft matter absorption bands are significantly broader
H2O
CO2
Martin Chaplin,

5. Oscillations – selection rules
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:
No absorption normal to the transition dipole moment
: dipole operator
Δn=±1
Others weakly allowed, due to anharmonicity
(overtones)

6. IR spectroscopy as analytical tool
IR spectral range
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
1-octanol

7. Grating IR spectrometer
IR spectrometry
Grating IR spectrometer
Requirements:
Well collimated beam
Monochromator
Largest part of light intensity is not used
Calibration is necessary

8. Fourier Transform Infrared Spectroscopy
IR spectrometry
Fourier Transform Infrared Spectroscopy
Michelson interferometer
Interferogram: intensity vs optical path difference
Intensity at all wavelengths is measured simultaneously γ
Optical path difference for each wavelength

9. IR spectrometry
FTIR spectroscopy
„white light“ position
Spectrum is easily obtained from the Fourier transform of the interferogram
Fourier transform
solvent
solvent
Division

10. Resolution – Apodization
IR spectrometry
Resolution – Apodization
Fourier transform of Iig(γ)
Shape of
infinitely thin lines
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”
Apodization
function
Fourier Transform Infrared Spectrometry,
P. R. Griffiths, J.A. de Haseth, Wiley

11. Advantages of FTIR Jacquinot advantage Fellget advantage (“multiplex”)
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
Reflectivity
Absorption coefficient α
Molar absorption coefficient ε=α/c
Normal incidence in air
Lambert-Beer law:

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

14. Polarization dependence
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

15. “perpendicular” vibration
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 
Molecular order parameter
Reference axis
Molecular segment
Transition dipole
“parallel” vibration
“perpendicular” vibration || 

16. Order of crystals and amorphous phase in spider silk
Experimental
Order of crystals and amorphous phase in spider silk
p: transition dipole moment
Low order of glycine-rich amorphous chains
High order of alanine-rich crystals
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
Lemieux, R. P. Acc. Chem. Res. 2001, 34,
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

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-resolved FTIR
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

20. Irreversible processes
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
Reaction time
Crystallization of a liquid crystal by T-jump
90°C
36°C
amorphous
de Haseth et al., Appl. Spectrosc., 47, 173 (1993)
Takahashi et al. J. Biol. Chem. 270, 8405 (1995)

21. Step scan Differences from rapid scan kinetics:
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

22. Step scan Stroboscopic technique Mirror moves stepwise
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

23. Step scan example: spider silk
Time-resolved FTIR
Step scan example: spider silk

24. Combined IR and mechanical spectroscopy
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)
polarizer
IR beam
Piezo crystals –
DC motors
Force sensor
IR detector
sample

25. Preparation of Step Scan measurement
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

26. DIRLD in polymers Dichroic ratio depends on strain
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

27. External – crystal stress comparison: Phase
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

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. Quantum Perturbation Theory
The shift is ~ 0.3 %
QPT is applicable
The bond anharmonicity gives rise to the shift of energy levels
Theoretical value
P. Papadopoulos et al. Eur. Phys. J. E 24, 193 (2007)

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

31. Photoinduced protein folding
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)

32. Folding kinetics of peptides after T-jumps
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
T. Wang et al. J. Phys. Chem. B 108, (2004)

33. Thank you for your attention!
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, ...
Thank you for your attention!

34. Chemical structure of dragline silk and PA6
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.
Repetitive pattern
C-term.
MaSp1
AAAAAAA
GGX
GGX
GGX
GGX GA
GGX
GGX n
AAAAAAA
GPGXX
GPGXX
GPGXX
GPGXX
GPGXX
MaSp2 n
Hydrophobic
Slightly hydrophilic
PA6 (Nylon):

35. Normal vibrational modes
Simple relations only in diatomic molecules!
Vibrations involve more than two atoms
Especially at low frequencies
Example: amide bond C H N O C
Amide I
Amide II
Amide III
Amide IV

36. Absorption spectrum of silk
Experimental
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 instead

37. Antiparallel and parallel b-sheet structure
N-terminus
C-terminus N C
C-terminus
Poly(alanine) segment
Rotondi, K. S.; Gierasch, L. M. Biopolymers 2005, 84,
Simmons, A.; Ray, E.; Jelinski, L. W. Macromolecules 1994, 27,

38. Polyaminoacid IR spectra
Dragline silk and b-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:
H.C. Haken – H. Wolf
Molecular Physics and Elements of Quantum Chemistry
Chapter 15