Periklis Papadopoulos
Universität Leipzig, Fakultät für Physik und Geowissenschaften
Institut für Experimentelle Physik I, Abteilung "Molekülphysik“
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
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
6
Grating IR spectrometer
Requirements:
Well collimated beam
Monochromator
Largest part of light intensity is not used
Calibration is necessary
IR spectrometry
7
FTIR 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 (
arb
. u
nits)
Optical retardation (cm)
0 0
det
0
ig
, cos 42 2
1 cos 42
I II
II d
IR spectrometry
Optical path difference for each wavelength
8
FTIR spectroscopy
Spectrum is easily obtained from the Fourier transform of the interferogram
IR spectrometry
ig 0
0
0 : 0I I d
ig
ig 0
0
ig
0
ig
0 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
nsity (
arb
. u
nits)
wavenumber (cm-1)
no sample silk
3500 3000 2500 2000 1500 1000
0
1
2
3
Ab
so
rba
nce
wavenumber (cm-1)
-0.01 0.00 0.01
-0.2
-0.1
0.0
0.1
0.2
Inte
nsity (
arb
. u
nits)
Optical retardation (cm)
Fourier
transform
Division
solvent solvent
„white light“ position
9
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
10
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
11
Transmission – reflection modes
Simplified: no interference, etc.
Transmission - absorption Specular reflection
Absorbance
Absorption coefficient α
Molar 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
12
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
2
n
Fourier Transform Infrared Spectrometry,P. R. Griffiths, J.A. de Haseth, Wiley
13
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
i iq rd : dipole operator
p E
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
2
molS P
10 :
2
mol RS
R
1: 2
2 2
mol RS
R
“parallel” vibration
“perpendicular” vibration
2
2
1 2cot 2
2 2cot 1
mol RS
R
16
1050 1000 950
0.1
0.2
0.3
0.4
Po
ly(a
lanin
e)
(Ala
Gly
) n
Po
ly(g
lycin
e)
I
Ab
so
rba
nce
wavenumber (cm-1)
Po
ly(g
lycin
e)
II
Po
ly(a
lanin
e)
0°
polarization
90°
0,0
0,2
0,4
0,6
0
30
60
90
120
150
180
210
240
270
300
330
0,0
0,2
0,4
0,6
Ab
so
rba
nce
Smol
=0.25
0,0
0,2
0,4
0,6
0
30
60
90
120
150
180
210
240
270
300
330
0,0
0,2
0,4
0,6
Smol
=0.50
Ab
so
rba
nce
0,0
0,2
0,4
0,6
0
30
60
90
120
150
180
210
240
270
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
Ab
so
rba
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
noise0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Inte
nsity (
arb
. u
nits)
time (min)
trigger
-0.01 0.00 0.01
-0.2
-0.1
0.0
0.1
0.2
Inte
nsity (
arb
. u
nits)
Optical retardation (cm)-0.01 0.00 0.01
-0.2
-0.1
0.0
0.1
0.2
Inte
nsity (
arb
. u
nits)
Optical retardation (cm)
Time-resolved FTIR
20
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
nsity (
arb
. u
nits)
Optical retardation (cm)
-0.01 0.00 0.01
-0.2
-0.1
0.0
0.1
0.2
Inte
nsity (
arb
. u
nits)
Optical retardation (cm)
Step scan
Stroboscopic technique
Mirror moves stepwise
All measurements after a certain dtfrom 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
23
Step scan
Mirror position in rapid scan and step scan
0 200 400 600 800 1000
0.0
0.2
0.4
0.6
0.8
1.0
1.2 rapid scan step scan
optical path
diffe
rence (
arb
. units)
time (arb. units)
Time-resolved FTIR
24
4000 3500 3000 2500 2000 1500 1000
0.0
0.2
0.4
0.6
0.8
1.0
Inte
nsity (
arb
. u
nits)
wavenumber (cm-1)
no sample silk (0 ms) silk (20 ms)
3500 3000 2500 2000 1500 1000
0
1
2
3
Ab
so
rba
nce
wavenumber (cm-1)
0 ms 20 ms
1000 950
0.3
0.4
0.5
0.6
Ab
so
rba
nce
wavenumber (cm-1)
0 ms 20 ms
Step scan example: spider silk
Time-resolved FTIR
25
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
26
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
27
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
28
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
29
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
30
C
CH3H
C
O
N
H
N
H
C
O
-1 -18 cm GPad d
pertV F r
1,4 1,6
-4,8
-4,6
Energ
y (
10
-19 J
)
r (Å)
-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( ) 2
0 0(1 )a r r
U U U e
0 1 2 3 4-6
-4
-2
0
2
4
En
erg
y (
10
-19 J
)
r (Å)
Morse potential
Morse potential
+
perturbation
N-C
3 eV
0.12 eV
F r 0.17 eV
dis
N CE
P. Papadopoulos et al. Eur. Phys. J. E 24, 193 (2007)
Theoretical value
31
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
961
962
963
964
965
stress (GPa)
wa
ve
nu
mb
er
(cm
-1)
Static
-2.6 cm- 1 GPa
- 1
Kinetics
Step Scan
PP, J. Sölter, F. Kremer Eur. Phys. J. E 24, 193 (2007)
32
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
retin
al
R. Rammelsberg et al. Appl. Spectrosc. 51, 558 (1997)
Time-resolved FTIR
33
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 unfolded
k
k
exp u fk k t
T. Wang et al. J. Phys. Chem. B 108, 15301 (2004)
Time-resolved FTIR
34
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!
35
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
36
Normal vibrational modes
Simple relations only in diatomic molecules!
Vibrations involve more than two atoms
Especially at low frequencies
Example: amide bondC
O
N H
C
k
Amide I Amide II
Amide III Amide IV
37
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
4000 3500 3000 2500 2000 1500 1000
0.0
0.5
1.0
1.5
Am
ide I
IIAm
ide I
I
Am
ide I
Ab
so
rba
nce
wavenumber (cm-1)
Am
ide A
1050 1000 950
0.2
0.3
0.4
Po
ly(a
lan
ine
)
(Ala
Gly
) n
Po
ly(g
lycin
e)
I
Ab
so
rba
nce
wavenumber (cm-1)
Po
ly(g
lycin
e)
II
Experimental
38
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 b-sheet structure
39
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
b-polyalanine
wavenumber (cm-1)
Abso
rba
nce
Am
ide III
Am
ide II
Am
ide I
Am
ide B MA silk
||
Abso
rptio
n c
oe
ffic
ien
t
(m
-1) A
mid
e A
Polyaminoacid IR spectra
Dragline silk and b-polyalanine
A. M. Dwivedi, S. Krimm Macromolecules 15, 186 (1982)
40
Similar findings in PA6
Similar to silk, orientation beforecrystallization induces the high order
3500 3000 2500 2000 1500 1000
0.000
0.005
0.010
0.015C-N
C=O
CH2
Absorb
an
ce
wavenumber / cm-1
//
N-H
Crystal vibration responds
linearly to applied stress
Both spider silk and PA6 are
glassy at room temperature