Terahertz and infrared spectroscopy of confined water · spectroscopy measures the intermolecular network vibrations and librational motions while MIR-FTIR spectroscopy probes the

RUHR UNIVERSITY BOCHUM Faculty of Chemistry and Biochemistry

Terahertz and Infrared

Spectroscopy of Confined Water

Dissertation

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Natural Sciences

by

Trung Quan Luong

April 2012

This dissertation presents the results of my PhD study carried out from October 2008 to

April 2012 in the group of Prof. Dr. Martina Havenith, Department of Physical

Chemistry II, Faculty of Chemistry and Biochemistry, Ruhr University Bochum.

First examiner: Prof. Dr. Martina Havenith

Second examiner: Prof. Dr. Hermann Weingärtner

Examination chairman: Prof. Dr. Martin Muhler

Dean: Prof. Dr. Dominik Marx

Publications

Part of the work presented in this dissertation is based on the following publications:

• A. Patra, T. Q. Luong, R. K. Mitra, M. Havenith. Solvent dynamics in reverse

micellar water-pool: A spectroscopic investigation of DDAB/cyclohexane/water

systems. In preparation.

• N. Pérez-Hernández, T. Q. Luong, M. Febles, C. Marco, H. H. Limbach, M.

Havenith, C. Pérez, M. V. Roux, R. Pérez, J. D. Martín. The mobility of water

molecules through hydrated pores. J. Phys. Chem. C 116, 9616-9630 (2012).

• T. Q. Luong, P. K. Verma, R. K. Mitra, M. Havenith. Onset of hydrogen bonded

collective network of water in 1,4-dioxane. J. Phys. Chem. A 115, 14462-14469

(2011).

• T. Q. Luong, P. K. Verma, R. K. Mitra, M. Havenith. Do hydration dynamics

follow the structural perturbation during thermal denaturation of a protein: A

terahertz absorption study. Biophys. J. 101, 925-933 (2011).

(cover paper)

• N. Pérez-Hernández, T. Q. Luong, C. Pérez, J. D. Martín, M. Havenith. Pore

size dependent dynamics of confined water probed by FIR spectroscopy. Phys.

Chem. Chem. Phys. 12, 6928-6932 (2010).

The author also contributed to the following publications:

• T. Q. Luong, S. Hoffmann, E. Bründermann, M. Havenith. Fast detection of

biological agents by terahertz spectroscopy. In preparation.

• A. Arora, T. Q. Luong, M. Krüger, Y. J. Kim, C. H. Nam, A. Manz, M.

Havenith. Terahertz time domain spectroscopy for the detection of PCR

amplified DNA in aqueous solution. Analyst 137, 575-579 (2012).

(cover paper)

i

Contents

1 Introduction .............................................................................................................. 1

1.1 Water in biological systems ................................................................................ 1

1.2 THz and IR spectroscopy .................................................................................... 2

1.3 Models of confined water ................................................................................... 3

1.4 Kinetic THz absorption spectroscopy ................................................................. 5

1.5 Outline ................................................................................................................. 6

2 THz and IR spectrometers ...................................................................................... 7

2.1 THz time domain spectrometer ........................................................................... 7

2.1.1 Overview ..................................................................................................... 7

2.1.2 THz-TDS setup ............................................................................................ 9

2.1.3 Ti:Sa femtosecond laser ............................................................................. 10

2.1.4 Photoconductive generation of THz pulses ............................................... 12

2.1.5 Electro-optic detection of THz pulses ....................................................... 13

2.1.6 Data acquisition and analysis .................................................................... 15

2.2 FTIR spectrometer ............................................................................................ 20

2.2.1 Overview ................................................................................................... 20

2.2.2 FTIR spectrometer setup ........................................................................... 21

2.2.3 Attenuated total reflection FTIR spectroscopy .......................................... 22

2.2.4 Data acquisition and analysis .................................................................... 26

3 Water in nanopores ................................................................................................ 29

3.1 Models for biological channels ......................................................................... 29

3.2 Pore size dependent dynamics of confined water ............................................. 35

3.3 Reversibility of water dynamics in nanopores .................................................. 39

3.4 Discussion and conclusion ................................................................................ 40

ii

4 Water in reverse micelles ....................................................................................... 45

4.1 Water nanopools in DDAB/Cy/water reverse micelles .................................... 45

4.2 Dynamic light scattering measurements ........................................................... 47

4.2.1 Principle of dynamic light scattering ......................................................... 47

4.2.2 Hydrodynamic diameters of reverse micelles ........................................... 49

4.3 THz-TDS measurements ................................................................................... 50

4.4 FTIR measurements .......................................................................................... 51

4.4.1 FIR spectra ................................................................................................. 53

4.4.2 MIR spectra ............................................................................................... 54


5 Water in organic solvents ...................................................................................... 61

5.1 Water confined in 1,4-dioxane .......................................................................... 61

5.2 FTIR spectra ...................................................................................................... 63

5.3 THz-TDS spectra .............................................................................................. 69

5.4 Kinetics of solvolysis reactions of benzoyl chloride ........................................ 74


6 Water in hydration shells ...................................................................................... 81

6.1 Biological water ................................................................................................ 81

6.2 Structure and function of human serum albumin .............................................. 84

6.3 Thermal unfolding and refolding of human serum albumin ............................. 85

6.3.1 CD spectroscopy ........................................................................................ 86

6.3.2 Fluorescence spectroscopy ........................................................................ 91

6.3.3 THz-TDS measurements ........................................................................... 98

6.3.4 p-Ge laser measurements ........................................................................... 99

6.4 Discussion and conclusion .............................................................................. 103

7 Kinetic THz absorption upon T-jump ................................................................ 109

7.1 Focusing of THz beam .................................................................................... 110

iii

7.2 T-jump apparatus ............................................................................................ 113

7.2.1 Overview ................................................................................................. 113

7.2.2 Physical background ................................................................................ 114

7.2.3 Q-switched Nd:YAG laser ....................................................................... 115

7.2.4 Raman shifter ........................................................................................... 118

7.2.5 Experimental parameters ......................................................................... 120

7.3 Kinetic THz measurements upon T-jump ....................................................... 121

7.3.1 Heating pulse profile ............................................................................... 121

7.3.2 Static temperature dependent THz absorption of water .......................... 122

7.3.3 KITA upon T-jump of water ................................................................... 123

7.3.4 KITA upon T-jump of proteins ............................................................... 131

7.3.5 Conclusion ............................................................................................... 137

8 Summary and outlook .......................................................................................... 139

Bibliography ................................................................................................................. 145

List of figures and tables ............................................................................................. 159

List of abbreviations .................................................................................................... 163

Acknowledgements ...................................................................................................... 165

1

1 Introduction

1.1 Water in biological systems

Water (H2O) is the simplest compound of the two reactive elements, consisting of two

hydrogen atoms covalently bonded to a single oxygen atom. Water is a liquid at ambient

conditions, but it also coexists on Earth in solid and gaseous states. Liquid water is the

most extraordinary substance which exhibits a variety of anomalies, such as stable

liquid state, high heat capacity, a density maximum in the liquid state. These anomalies

result mostly from the infinite network of hydrogen bonds (H-bonds) between water

molecules [1, 2]. The oxygen atom of a water molecule is partially negatively charged

and the two hydrogen atoms are partially positively charged. In liquid water, the

hydrogen atoms are not only covalently bonded to the oxygen atom but also attracted

towards other oxygen atoms of the neighboring water molecules. This attraction forms

the H-bonds which are weaker than covalent or ionic bonds, but stronger than a van der

Waals interaction. These H-bonds break and reorganize constantly in picosecond

timescale [3]. Each water molecule can form four H-bonds in a tetrahedral geometry,

involving its two hydrogen atoms and two further hydrogen atoms from neighboring

water molecules. Furthermore, water molecules can establish dipole and induced dipole

interactions with other water molecules [4]. These heterogeneous properties of liquid

water make it flexible in different physical, chemical and biological conditions.

In living systems, water is "more than a bystander", it is called the "matrix of life",

playing essential roles [5, 6]. Many biological processes, such as protein folding and

enzymatic reactions, are inactive in the absence of water. The present of water in a

biological system is not simply to fill up the available space. Water molecules occupy

specific areas, form localized clusters, transport protons, form H-bonds with

biomolecules, mediate and participate in a wide range of biomolecular interactions [6,

7]. These versatile functions of water result particularly from its flexible three-

dimensional H-bond network and its ability to engage in directional, weak bonding with

biomolecules that facilitates structural reconfiguration and reorientation.

A living cell consists of several biomolecules with a concentration up to 400 grams per

litre [8]. Biomolecules occupy about 5 to 40% of the total volume of the cell. Therefore,

1. Introduction

2

the cell is very crowded and biomolecules are typically separated by only 1-2 nm. This

confinement is expected to change the structure and dynamics of water compared to

those of bulk water. The water H-bond network is perturbed as there are interactions

with the surface of biomolecules within a narrow space. The properties of confined

water may vary widely depending on the molecular characteristics of the surface and the

levels of confinement. Different areas in the cell exhibit different levels of confinement,

which is important for various cellular functions. The study of confined water in

different models is thus essential to understand the roles of water in biological systems.

1.2 THz and IR spectroscopy

Figure 1.1: Frequency dependent absorption coefficient of liquid water at

25°C in THz-IR region (data adapted from Ref. [9]). The highlighted

regions show the investigated frequency ranges of the main spectrometers

used in this study. THz-TDS (3-45 cm-1) is sensitive to intermolecular water

network vibrations. FIR-FTIR spectroscopy (50-650 cm-1) measures the

intermolecular network vibrations and libration motions. MIR-FTIR

spectroscopy (3000-3700 cm-1) probes the intramolecular OH stretching

modes.

1. Introduction

3

Terahertz (THz) and infrared (IR) spectroscopy is the study of the interaction between

samples and electromagnetic radiation in the frequency range approximately from 3 to

4000 cm-1. The measurement results are usually the frequency dependent spectra of the

studied samples, showing the absorption coefficient, index of refraction, or other

physical parameters. Figure 1.1 shows the frequency dependent absorption coefficient

of liquid water at 25°C in the THz and IR region. The data are taken from a previous

study [9]. Intermolecular water network vibrations dominate in the low frequency

region below 400 cm-1. The broad band around 600 cm-1 arises from librational motions

of water molecules. Two characteristic intramolecular modes appear in the region 1500-

1700 cm-1 (bending) and 3000-3700 cm-1 (OH stretch).

The main experimental methods used in this study to investigate confined water are

terahertz time domain spectroscopy (THz-TDS) and Fourier transform infrared (FTIR)

spectroscopy. THz-TDS (frequency range 3-45 cm-1) is sensitive to intermolecular

water network vibrations. FTIR measurements were carried out in far-infrared (FIR)

(50-650 cm-1) and mid-infrared (MIR) (3000-3700 cm-1) regions. FIR-FTIR

spectroscopy measures the intermolecular network vibrations and librational motions

while MIR-FTIR spectroscopy probes the intramolecular OH stretching modes of water.

The frequency ranges of the THz-TDS and FTIR spectrometers are shown in Figure 1.1.

These spectrometers cover almost all the important spectral regions of water in the THz

and IR frequency range.

1.3 Models of confined water

In order to study confined water at different levels, this study investigated four different

models: nanopores, reverse micelles, organic solvents, and hydration shells. The first

model (nanopores) is solid crystals while the others are liquid solutions. Figure 1.2

shows a schematic of water confinement in four studied systems. In general, the level of

water confinement is strongest in nanopores, followed by reverse micelles, organic

solvents, and then hydration shells.

In the first model, water molecules are confined in organic nanopores. The nanopores

are tubular crystals formed by water mediated assembly of hydroxyl acids. These

supramolecular structures have hydrated pores with pore diameter of a few Å. Water

1. Introduction

4

molecules can be permanently confined inside the pores at ambient condition. Different

pore sizes give rise to different confinement and dynamics of water.

In the second model, water nanopools inside reverse micelles (RM) are formed in

DDAB/Cy/water (didodecyldimethylammonium bromide/cyclohexane/water) mixture.

In the nonpolar organic solvent (Cy), the cationic surfactant molecules (DDAB)

aggregate into nanoscale RM structures where the hydrophilic heads form the core of

the RM and hydrophobic tails are in contact with the surrounding solvent. Water

molecules in the mixture are trapped inside the RM.

Figure 1.2: Schematic of water confinement in four studied systems:

nanopores, reverse micelles, organic solvents, and hydration shells. In

general, the level of confinement decreases from left to right. These systems

serve as models for water confinement in biological systems.

The third model is mixtures of water and 1,4-dioxane (Dx). In spite of being a nonionic

and relatively nonpolar solvent, Dx solubilizes water from highly diluted to highly

concentrated mixtures. Dx can expose non-interacting hydrophobic sites to water

molecules as well as form H-bonds with water. In water-Dx mixtures with highly

diluted concentration of water, the water-water H-bond network is disrupted, which

resembles the conditions of isolated or confined water molecules with fewer interactions

to other water molecules.

1. Introduction

5

The last model is water in hydration shells around human serum albumin (HSA) in

aqueous solution. Water dynamics in the hydration shells is determined by the structural

organization and perturbation of the protein, and vice versa, the structure and function

of the protein is governed by the water dynamics. Water dynamics in the hydration

shells is generally slowed down by the protein to match the much slower protein

dynamics. In some aspects, water molecules in the hydration shell are more confined

than bulk water and their properties are sensitive to the details of the interactions with

the protein surface.

1.4 Kinetic THz absorption spectroscopy

The kinetics of biomolecules is critical for biological functions. To study a rapid kinetic

process experimentally, a technique to rapidly change the equilibrium of the sample is

required. After being disturbed, the sample relaxes to its original equilibrium.

Temperature jump (T-jump) is the most widely used technique to initiate a kinetic

process because of its sufficient jump size, high speed, and minor disturbance to the

sample environment [10]. A fast increase in temperature can disturb an existing

equilibrium of a protein. T-jump can initiate both the unfolding to heat denatured states,

and the refolding from cold denatured states. Kinetic THz absorption spectroscopy

(KITA) is a high sensitive detection method to observe changes of water dynamics in

the hydration shells around a protein [11]. Upon T-jump, the kinetics structural change

of the protein induces corresponding change in its coupled water dynamics in the

hydration shells, which can be monitored by KITA.

In this study, a new nanosecond time resolution T-jump apparatus was setup. It

generates high power short laser pulses to initiate rapid temperature increase in aqueous

solution. The propagation of the THz beam of the existing THz spectrometer was

improved to optimize the focal size of the beam at the sample position, ensuring that the

probed area of the THz beam is within the heated area of the solution. A data

acquisition process was established to reproducibly record THz signal upon T-jump.

These preparation steps enable a feasible and successful combination between T-jump

and KITA for kinetic studies. Measurement results of the T-jump induced rapid

unfolding to heat denatured states of λ-repressor and human serum albumin, and the

rapid refolding from cold denatured states of ubiquitin reveal an observable difference

between buffer and protein solutions. This is the first study using the combination

1. Introduction

6

between T-jump and KITA to probe the coupled protein-water dynamics in the

microsecond and millisecond timescales.

1.5 Outline

This dissertation presents the study of confined water in different models and KITA

study upon T-jump. Chapter 2 describes in detail the two mainly used experimental

methods, namely THz-TDS and FTIR spectroscopy. Some further complementary

experimental techniques were also used in this study, including dynamic light

scattering, kinetics of solvolysis reactions, circular dichroism spectroscopy, time

resolved fluorescence spectroscopy, and p-germanium laser THz spectroscopy. The

principles of these techniques are given in the sections where their results are discussed.

The investigation of confined water in nanopores, reverse micelles, organic solvents,

and hydration shells are presented in Chapter 3, Chapter 4, Chapter 5, and Chapter 6,

respectively. In Chapter 7, the detailed description of the T-jump apparatus and

experimental results of T-jump induced kinetics probed by KITA are given and

discussed. Finally, Chapter 8 summarizes the studied results and gives an outlook on

possible improvements. The results of this study aim to contribute to a better

comprehension of water dynamics in confined environments which are similar to the

conditions in natural biological systems.

7

2 THz and IR spectrometers

2.1 THz time domain spectrometer

2.1.1 Overview

Terahertz (THz) radiation is electromagnetic radiation in the frequency range

approximately between 0.1 THz and 10 THz. This corresponds to wavelengths between

3 mm (0.1 THz) and 30 µm (10 THz). When converted to other units, 1 THz (1012 Hz)

is equivalent to wavenumber of 33.3 cm-1, photon energy of 0.004 eV, or wavelength of

300 µm. This THz frequency range is located between the microwave part and the

infrared part of the electromagnetic spectrum. THz is still a new research area because

of the difficulty to produce adequate THz sources and efficient detectors [12]. The low

photon energy of THz radiation makes it useful for studying low frequency phenomena

such as vibrational modes of macromolecules like proteins or DNA [13], collective

network motions of water [14], soft lattice vibrations in dielectrics [15], nondestructive

inspection of materials [16]. Furthermore, THz radiation penetrates many materials

which are impervious for visible light, such as paper, wood, textiles and plastics. This

provides opportunities for THz imaging techniques as well as several medical and

security applications [17, 18]. Terahertz spectroscopy explores frequency dependent

optical properties of a material in the THz range. There are different types of THz

spectroscopy, such as Fourier transform spectroscopy, THz narrowband spectroscopy,

p-germanium (p-Ge) laser THz spectroscopy, and THz time domain spectroscopy (THz-

TDS) [19, 20].

THz-TDS is a technique in which the time dependent electric field of a THz pulse is

measured after it interacts with a sample. This is the most recent technique, developed

in the 1980s [21, 22]. After that, several improvements and applications of THz-TDS

were reported, such as sub-ps photoconducting dipole antennas [23], THz imaging

system [24], zinc telluride (ZnTe) crystal for broadband detection [25], de-noising

techniques for THz imaging [26], and planar large-area photoconducting emitter for

impulsive generation of terahertz radiation [27]. THz-TDS technique offers a number of

advantages compared to the others. The transmitted THz electric field is detected

coherently, which provides time resolved information of both power and phase of the

2. THz and IR spectrometers

8

transmitted pulse. Subsequently, both the frequency dependent real and imaginary parts

of optical constants such as the index of refraction and the absorption coefficient can be

measured simultaneously [28]. Furthermore, information about the carrier mobility and

number density in semiconductors can be deduced [29]. THz-TDS is also implemented

in imaging systems to produce accurate spatial information of a sample [19].

The most common techniques to generate THz pulses used in THz-TDS are

photoconductive emission and optical rectification [30]. In the photoconductive

emission, femtosecond laser pulses are used to generate carriers in the conduction band

of a semiconductor under an applied bias voltage. The newly formed carriers are

accelerated by the bias. In an antenna, a time-varying electric current acts as a source

term in the Maxwell’s equations and radiates an electromagnetic pulse. The optical

rectification technique is based on properties of nonlinear crystals. Optical rectification

is a difference frequency mixing that occurs in nonlinear media with large second order

susceptibility. The interaction between femtosecond optical pulses with a nonlinear

medium and wave mixing of two frequencies result in the generation of sum-frequency

and difference-frequency, corresponding to second harmonic and dc pulses. A generated

dc pulse is the envelope of the optical pulse. As incident femtosecond optical pulses

have large bandwidth, the high-frequency components can mix with the low-frequency

components to produce pulses with much longer wavelengths at the different

frequencies from 0 to several THz. The output power of above THz sources is in the

nanowatt to microwatt range [19]. With this low THz power, thermal background

radiation from the environment is a non-negligible noise source. Therefore, coherent

detection is required for THz-TDS systems.

Similar to THz pulse generation, the most common detection methods are

photoconductive sampling and free-space electro-optic sampling. Photoconductive

sampling is similar to photoconductive emission. However, the bias across the antenna

is produced by the electric field of the incident THz pulse instead of being applied

externally as in the photoconductive emission. This bias generates a current in the

antenna which is proportional to the strength of the THz electric field. In electro-optical

sampling, the THz pulse induces birefringence in a nonlinear crystal due to the Pockels

effect. This leads to an induced change in polarization of the optical probe beam which

is the same laser beam as the one used for generation of the THz pulse. The change is

proportional to the electric field of the THz pulse. With changing optical delay, the

waveform of the THz pulse can be determined [25].


9

In practice, there are two widely used experimental setups for THz-TDS: transmission

setup and reflection setup [31]. In the transmission setup, the THz beam generated by

the emitter is focused on the sample position and propagates through the sample. After

that, the beam is focused on the detector system. This transmission setup is used to

measure optically thin samples which allow the THz beam to pass through without

losing almost all its energy. For opaque samples, the reflection setup is required. Instead

of propagating through the sample, the THz pulse is reflected from the sample. Then the

beam is led to the detector system by a beamsplitter.

2.1.2 THz-TDS setup

Figure 2.1: Schematic of the THz-TDS (BS: 25:75 beamsplitter, L: lens,

PM: parabolic mirror, ZnTe: zinc telluride crystal, λ/4: quarter wave plate,

WP: Wollaston prism). The fs laser pulses emitted from the Ti:Sa laser are

split by a beamsplitter. One part is focused on the THz emitter for

generation of THz pulses. The other is focused on the ZnTe crystal together

with the THz pulses for coherent electro-optic detection.

Figure 2.1 shows a schematic of the THz-TDS. The Verdi laser (model Verdi V-10,

Coherent) generates continuous-wave laser radiation at 532 nm and 5 W output power.


10

It is used to pump the Ti:Sa (titanium doped sapphire or Ti:Al2O3) laser (model MTS

Mini Ti:Sa Laser, KMLabs) for generation of femtosecond (fs) laser pulses. The THz-

TDS works in a pump-probe configuration. A beamsplitter is used to split the fs laser

beam into two parts (pump pulse and probe pulse). The pump pulse propagates via a

mechanical delay line for changing the time delay between pump and probe pulse. After

that it is focused on the THz emitter (model Tera-SED, Gigaoptics) for generation of

THz pulses. With the transmission geometry setup, the generated THz beam is focused

by two parabolic mirrors on the sample position and then focused by two other

parabolic mirrors on a nonlinear (110)-ZnTe crystal. The probe pulse is also focused on

the ZnTe crystal for coherent electro-optic detection. In the following sections, the main

components of the THz-TDS will be described in details.

2.1.3 Ti:Sa femtosecond laser

The first prerequisite to generate ultrashort pulses is that a broad spectral bandwidth or a

large number of longitudinal modes are required. When the duration and the spectral

width of the laser pulse are calculated using the standard statistical definitions, they are

related to each other by a universal inequality (Heisenberg’s uncertainty relation) [32]:

∆� · ∆� � 12 (2.1)

where ∆� is the half maximum duration of the pulse and ∆� � 2 · ∆ with ∆ is the

frequency full width at half maximum (FWHM). This relation leads to the quantum

mechanical time-energy uncertainty principle: to generate an ultrashort laser pulse

(small ∆�), a broad spectral bandwidth (large ∆�) is required.

The second prerequisite is that the different modes must be held in phase in order to

achieve constructive interference. When the laser operates in free multimodes, the

competition among the different modes causes large fluctuations in the relative phases

and amplitudes of the modes. In order to generate ultrashort pulses, the competition

between modes must be organized in such a way that their relative phases stay constant,

so that the output intensity of the laser consists of a periodic series of pulses resulting

from the travelling back and forth of a wave packet within the laser cavity. Kerr lens

mode-locking using a nonlinear medium is one of the most widely used methods to


11

meet this requirement. The Kerr effect is a third order self-induced nonlinear index

change in a material. It describes the dependence of the index of refraction � on the

light intensity � [32]:

� � � � �� · � (2.2)

where � is the index of refraction of the material, �� is the nonlinear index coefficient

(or Kerr coefficient) of the material. The nonlinear properties of the amplifying material

naturally enhance the intensity maxima arising within the cavity by inducing a

narrowing of the pulse at each of its round trips through the cavity. This condition is

called self-locking of the modes. The amplifying medium behaves like a converging

lens and focuses the beam like a lens.

The most important advances in generation of ultrashort pulses have been based on the

development of Ti:Sa crystal as an amplifying medium. Firstly demonstrated in 1986,

Ti:Sa crystal has been widely used for generation of ultrashort laser pulses because it

meets both of the above mentioned prerequisites [33]. The crystal is created by doping

sapphire (Al2O3) with Titanium. About a few weight percent of Al3+ ions are substituted

by Ti3+ ions in the crystal structure. Because the ionic radius of Ti3+ ions is about 26%

larger than that of Al3+ ions, a strong distortion of the local environment of the Ti3+ ions

is induced and this distortion creates a strong local electric field. This results in the

unusually wide absorption band of Ti:Sa crystal in the visible spectrum (from 400 to

600 nm). The emission band is also wide and shifted towards lower energies (from 600

to 1000 nm). Using a Ti:Sa crystal, it is easy to get a broad spectral bandwidth when the

Ti:Sa crystal is pumped by a laser beam with a wavelength within its absorption band.

Furthermore, the Ti:Sa crystal has nonlinear properties and can act as the Kerr medium

in a mode-locking process. In the current setup, a continuous-wave laser beam at 532

nm and 5 W output power is used to pump the Ti:Sa crystal. At mode-locked condition,

the Ti:Sa laser emits 20 fs pulses at 94 MHz repetition rate and 500 mW average optical

output power.


12

2.1.4 Photoconductive generation of THz pulses

Figure 2.2: Schematic of the large area THz emitter. It has a metal-

semiconductor-metal structure in gallium arsenide (GaAs) substrate, which

makes charge carriers to be accelerated in the same direction over the whole

excited area to generate THz radiation.

A photoconductive antenna generates THz pulses by transient photocarriers induced by

ultrafast laser pulses. Its main structure consists of two metal electrodes coated on a

semiconductor substrate with a gap between them. With applied bias voltage, the

electric energy is stored in the gap area. When fs laser pulses with photon energy higher

than the band gap of the semiconductor are focused on the gap, electron-hole pairs are

created. These charge carriers are accelerated by the bias field. In this case, the fs laser

pulses act like transient switches to open the stored electric energy and release it in the

form of the emission of a time dependent THz field �� that is proportional to the

derivation of the current density �� [34]:

�� 14� · �� · �� (2.3)

�� (2.4)


13

where � is the vacuum permittivity, � is the area in the gap illuminated by the laser

beam, � is the speed of light in vacuum, � is the distance between the field point and the

THz source, � is density of photocarriers, � is the elementary charge, � is the mobility

of electrons, and � is the bias electric field. In principle, the energy of the THz pulse

depends on the electric energy stored across the gap by the applied bias voltage rather

than the energy of the fs pulses of the excitation laser. However, the stored electric

energy can only be released when the fs laser pulses trigger the generation of the

photocarriers. The conversion of the stored energy into THz radiation is proportional to

the number of generated photocarriers. Therefore, the energy of the fs pulses also

influences the energy of the THz pulse.

In the current setup, a large area emitter (model Tera-SED, Gigaoptics) is used to

generate THz radiation. A schematic of the THz emitter is shown in Figure 2.2. It is a

planar gallium arsenide (GaAs) photoconductor and consists of an interdigitated

electrode metal-semiconductor-metal (MSM) structure. When voltage is applied across

the electrodes, the electric field is reversed between successive fingers. The opaque

metal layer covers every second finger electrode spacing in such a way that optical

excitation is only possible in area exhibiting the same electric field direction. Therefore,

charge carriers are accelerated in the same direction over the whole excited area and the

emitted THz radiation interferes constructively in the far field [27]. The voltage applied

to the emitter is from an external source with the amplitude of 20 V and modulated

frequency of 40 kHz in square wave form. When the stored electric energy in the

emitter is switched by the fs laser pulse, THz pulses of picosecond duration are

generated at a repetition rate of 94 MHz in a beam angle of 7°. The emitted THz beam

has an average optical power of 10 to 20 µW. The modulated frequency applied to the

emitter serves as the reference frequency in a lock-in amplifier which is set, for

example, at an input gain of 20 dB, a sensitivity of 20 mV, and an integration time of

200 ms. As the power of the THz beam is rather low, the lock-in amplifier is required to

separate the THz signals from thermal noise.

2.1.5 Electro-optic detection of THz pulses

Electro-optic detection or electro-optic sampling (EOS) is a highly sensitive coherent

detection method which makes use of the linear electro-optic effect (Pockels effect).

The Pockels effect occurs only in non-centrosymmetric crystals, in which the


14

birefringence (change of the refractive index) of the crystals is proportional to the

strength of an applied electric field. When a sensor crystal is influenced by a low

frequency polarized electric field (THz pulse), it becomes birefringent and induces a

change in the polarization of the fs probe pulse. The change in polarization is

proportional to the electric field of the THz pulse [35]. By varying the relative time

delay between the THz pulse and the probe pulse, the electric field of the whole THz

pulse can be measured.

Figure 2.3: Schematic of the electro-optic sampling. THz beam and fs laser

beam overlap at the birefringent (110)-ZnTe crystal. The linear polarized fs

laser is converted to circular polarized light by the λ/4 wave plate. The

Wollaston prism splits the polarizations into ordinary and extraordinary

parts (parallel and perpendicular to the optical axis). The difference in

intensity of these two parts is proportional to the intensity of the THz

electric field.

Figure 2.3 shows a schematic of the EOS system in the current setup. The (110)-ZnTe

crystal serves as the sensor crystal. The fs probe pulses are focused on the crystal before

being directed to propagate through the quarter wave plate and the Wollaston prism.

The quarter wave plate creates a quarter wavelength phase shift and changes the linearly

polarized probe beam to the circularly polarized beam. The Wollaston prism, which is a

polarizing beamsplitter, splits the polarized beam into two equal polarization

components with perpendicular directions: s-polarized (ordinary beam) and p-polarized

(extraordinary beam). These two beams are focused independently on the auto-balanced

detector (125 kHz Nirvana auto-balanced photoreceiver, Newport). When the THz pulse


15

is also focused on the ZnTe crystal, its electric field makes the crystal birefringent,

which in turn changes the circular polarized beam after the quarter wave plate into

elliptical polarized beam. This means that change in the intensity of s-polarized beam

(�!) and p-polarized beam (�") relative to each other (Δ� � �! $ �") occurs. This change

is proportional to the strength of the THz electric field. The auto-balanced detector

records directly the change of Δ�, thus indirectly measures the THz electric field.

2.1.6 Data acquisition and analysis

2.1.6.1 Data acquisition

Figure 2.4: Schematic of the data acquisition in THz-TDS. The relative

delay between the THz pulse and the fs pulse is varied to record the whole

profile of the THz pulse (left). The recorded data as a function of the

distance delay are converted to the time delay (right).

The temporal intensity of the THz field is recorded by stepwise changing the relative

delay between the THz pulse and the fs laser pulse. The time resolution ∆� of the

measured THz signal is deduced from the minimum step size ∆% by the equation:

∆� � ∆%� (2.5)


16

where � is the speed of light. In the current setup, the usually set step size of the

mechanical delay line is 20 µm, which corresponds to a time resolution of 66.67 fs.

Figure 2.4 shows a schematic of the data acquisition. Upon varying the relative delay,

each point of the THz pulse is probed. When a full scan of the delay line is done, the

THz intensities as a function of the distance delay are recorded. The step size of the

delay line is then converted to time delay. As a result, the THz pulse in time domain can

be constructed. In order to obtain the data in the frequency domain, a fast Fourier

transform (FFT) algorithm is applied to the data in the time domain.

2.1.6.2 Fast Fourier transform

A physical process can be described either in the time domain (the amplitude � of the

process as a function of time �) or in the frequency domain (the amplitude � of the

process as a function of frequency ). These two functions �� and �� are

considered as two different representations of the same function which can be converted

from one to the other by means of the Fourier transform equations [36]:

�� & ��'�()*+%,-

'-

(2.6)

�� & ��()*+%�,-

'-

(2.7)

The data acquired from THz-TDS consists of discrete values. The function �� is

sampled at evenly spaced intervals in time. Therefore, a FFT algorithm must be used to

transform the discrete time domain values into discrete frequency domain values.

Suppose that the Fourier transform of �� has � consecutive sampled values (� is

even) and the sampling interval is ∆�. The sequence of sampled values is:

��.� / ��0 · 1�� (2.8)

with 0 � 0, 1, 2, . . . , � $ 1


17

The reciprocal of the time interval ∆� is called the sampling rate (the number of samples

recorded per second). When FFT is applied, the integral in equation (2.7) can be

approximately replaced by a discrete sum:

��:� ; < ��.��()*=+> · 1� � 1� · < ��.��().:/@ @'A

.B @'A

.B

(2.9)

with : / �� · 1� ; � � $ �2 , … , �2 ; �. � 0 · 1�

It is noticed that there are � � 1 values of � but the two extreme values of � are not

independent (they are equal), this reduces the count to �. Therefore, the FFT maps �

complex numbers ��.� into � complex numbers ��:�.

Figure 2.5: THz pulses of a reference and a sample in the time domain (A)

and their Fourier transform spectra in the frequency domain (B).

Figure 2.5A shows the intensity of the THz pulses of an empty cell (reference) and a

filled cell (sample) as a function of time. The sample pulse has a lower intensity because

of absorption (attenuation of the signal), and it is shifted to the right because of

refraction of the sample (delay of the THz pulse). The corresponding FFT spectra of the

THz pulses as a function of frequency are shown in Figure 2.5B. The spectrum of the

sample has lower intensity than the spectrum of the reference.


18

2.1.6.3 Data analysis

The obtained frequency domain values of ��:� in Equation (2.9) can be expressed in

amplitude �: (absolute value) and phase �E:�:

��:� � F1� · < ��.��().:/@ @'A

.B F · �)G= � �: · �)G=

(2.10)

In THz-TDS, reference measurement (without the sample) and sample measurement

(with the sample) are needed for the analysis. The FFT yields for each frequency

component the intensity �� and phase E�� of the transmitted THz beam. The

intensity is proportional to the absolute square of the electric field:

�� ; |��|� (2.11)

The absorption process of a laser beam passing through a sample is described by the

Lambert‐Beer’s law:

�!�� 'J�*�K (2.12)

where � is the intensity of the reference measurement, �! is the intensity of the sample

measurement, % is the path length (the thickness of the sample) and L is the absorption

coefficient. The conversion of Equation (2.12) gives the value of L:

L�� ln� �� $ ln�!��% (2.13)

The index of refraction �� can similarly be determined from the phase E of the

reference measurement and E! of the sample measurement by [28]:

�� OE!�� $ E ��P2% � 1 (2.14)


19

where � is the speed of light in vacuum. Further physical constants of the sample can be

deduced using the frequency dependent complex index of refraction which is given by:

�Q�� $ R0�� or �Q�� $ R0�� (2.15)

with � � 2 is the angular frequency, � is the real part of the complex index of

refraction, and 0 is the imaginary part of the complex index of refraction. The

imaginary part 0 indicates the amount of absorption loss when the beam propagates

through the sample. It is also called the extinction coefficient which is proportional to

the absorption coefficient:

0�� UL��4 � �L��2� (2.16)

where U is the wavelength. The complex dielectric constant �̂�� is related to the

complex index of refraction by following relations:

�̂�� W�� $ R�WW�� (2.17)

�W�� $ 0�� (2.18)

�WW�� 2��0�� (2.19)

where �W is the real part of the complex dielectric constant, and �WW is the imaginary part

of the complex dielectric constant. Apart from the optical properties of the sample

characterized by absorption coefficient and index of refraction, the complex dielectric

constant gives further insight into the liquid dynamics.


20

2.2 FTIR spectrometer

2.2.1 Overview

Infrared (IR) spectroscopy has a long history for more than a century with broad

application areas. The IR frequency range lays between the microwave and visible

frequency range, covering the wavelength from 780 nm to 1 mm. It is divided into three

ranges: near-infrared (NIR) with the wavelength of 0.78-3 µm, mid-infrared (MIR) with

the wavelength of 3-50 µm, and far-infrared (FIR) with the wavelength of 50-1000 µm

[37]. The IR absorption peaks correspond to the frequencies of intermolecular or

intramolecular rotations and vibrations. As each sample has a unique combination of

atoms, it has a distinctive IR spectrum which represents a fingerprint of the sample.

Therefore, IR spectroscopy has been widely used for qualitative and quantitative

analysis of substances.

In the early days, dispersive IR spectrometers were developed, using prisms or

diffraction gratings as dispersive elements. The dispersive element was used to separate

an infrared light into a continuous range of frequencies. It is a part of a monochromator

which has a narrow slit to select a narrow range of frequencies. By rotating the

dispersive element, each frequency successively passes the slit before reaching a

detector. Therefore, the sample was scanned with the whole infrared frequency range.

Later, the more advanced technique Fourier transform infrared (FTIR) spectroscopy was

introduced. In an FTIR spectrometer, IR light is guided through an interferometer and

all of the IR frequencies are measured simultaneously, instead of recording every single

frequency like dispersive IR spectroscopy. This makes the measurement time of a

spectrum much faster. Multiple scans of a sample can be collected and averaged to

improve the sensitivity. The result of a measurement is an interferogram. A Fourier

transform is applied to the data to obtain a frequency dependent spectrum which is

identical to that from dispersive IR spectroscopy.

The most common interferometer used in FTIR spectroscopy is a Michelson

interferometer. In a simple setup, the interferometer consists of a beamsplitter and two

perpendicularly plane mirrors. One of the mirrors is fixed and the other can move in a

direction perpendicular to the plane. The beamsplitter bisects the planes of the two

mirrors. The moving mirror produces an optical path difference between the two arms

of the interferometer for constructive or destructive interference of the two beams. IR


21

sources and detectors for FTIR spectrometers are different depending on the frequency

range. Globar or Nernst sources are commonly used for the MIR region, tungsten-

halogen lamps for the NIR region, and high-pressure mercury lamps for the FIR region.

Detectors for the NIR and MIR region are usually DTGS (deuterium tryglycine sulfate)

and MCT (mercury cadmium telluride) while bolometers are used in the FIR region.

2.2.2 FTIR spectrometer setup

Figure 2.6: Schematic of the FTIR spectrometer (MM: moving mirror, BS:

beamsplitter, AP: aperture). The light source, the beamsplitter of the

interferometer, and the detector can be changed for measurements at

different frequency ranges.

The current FTIR spectrometer used in the lab is the VERTEX 80v (Bruker Optics).

Figure 2.6 shows a schematic of the spectrometer. The main components are the light

source, the detector and the Michelson interferometer. These components can be

changed to meet the required frequency range. In the FIR region, a mercury-lamp

served as an FIR source, a liquid-helium-cooled silicon bolometer was used as a

detector, and a multilayer beamsplitter is placed in the interferometer. For spectra


22

acquisition in the MIR region, a built-in MIR source, an MCT detector and a KBr

(potassium bromide) beamsplitter are used.

The IR beam from the light source passes through an aperture before arriving the

interferometer. The aperture, whose diameter can vary from 0.2 to 8 mm, is used to

shape the beam. The beamsplitter reflects a half of the beam to a fixed mirror, and

transmits another half to a moving mirror. The beam reflected from these two mirrors is

passed or reflected by the beamsplitter a second time and finally recombined and

focused onto the sample position. In the interferometer, the propagation distance of the

beam reaching the fixed mirror is fixed while that of the beam reaching the moving

mirror changes. This produces an optical path difference between the two beams and the

recombined beam is the result of the interference of these two beams. The detected

signal, which is the intensity as a function of the moving mirror position, is an

interferogram. This interferogram is then analyzed to deduce a frequency dependent

spectrum.

During a measurement, the whole setup, except the sample compartment, is always kept

under vacuum (2 mbar) to avoid external influences. The sample compartment can also

be kept under vacuum if this does not affect the measured samples. The main

advantages of the FTIR spectrometer are wide frequency range, high spectral resolution,

fast spectral acquisition, and simple sample preparation. It can be used for both

qualitative and quantitative analysis of liquid and solid samples. Beside the traditional

transmission spectroscopy, a combination with a reflectance apparatus offers further

sample analyses in reflection spectroscopy.

2.2.3 Attenuated total reflection FTIR spectroscopy

Apart from the traditional transmission spectroscopy, an FTIR spectrometer can be

combined with further components to carry out measurements in reflection geometry.

The attenuated total reflection (ATR) unit is one of the mostly used components for

reflection spectroscopy. The ATR-FTIR spectroscopy has advantages for the study of

optically thick samples, hard samples, slightly curved samples, fibers, and powders.

ATR makes use of the physical phenomenon of total internal reflection. When an IR

beam is directed onto an ATR crystal with a high index of refraction at a certain angle,

it reflects off the internal surface which is in contact with a sample. This internal

reflection creates an evanescent wave which extends beyond the crystal surface into the


23

sample [38, 39]. The sample characteristically absorbs the evanescent wave, which

results in an attenuation of the total reflected light. The absorbance spectra obtained

from the ATR-FTIR spectroscopy are comparable to those from FTIR transmission

spectroscopy.

2.2.3.1 Evanescent wave

Figure 2.7: Schematic of the propagation of an electromagnetic wave

through an interface between two media. The incident medium has higher

index of refraction than the second one (n1 > n2). When the incident angle θ1

smaller than the critical angle θc (left panel), the wave is both transmitted

and reflected at the interface. When θ1 > θc (right panel), the wave is totally

internal reflected and an evanescent wave is generated beyond the interface.

When light strikes an interface between two media of different indices of refraction, it is

partially transmitted and partially reflected (Figure 2.7, left panel). The transmitted

component is refracted at the interface. The relationship between the angles of incidence

XA and the angles of refraction X� follows the Snell’s Law:

�AsinXA � ��sinX� (2.20)


24

where �A is the index of refraction of the incident medium, �� is the index of refraction

of the of the second medium. When light travels from a medium with a higher index of

refraction to a medium with a lower index of refraction (�A Z ��), total internal

reflection occurs if the incident angle XA is greater than the critical angle X[ which is

defined by:

X[ � sin'A \��A] (2.21)

In the case of total internal reflection (Figure 2.7, right panel), a special kind of

electromagnetic field, the evanescent wave, is established beyond the interface between

the two media [38, 39]. The electric field of the transmitted evanescent wave in space

and time ��^, _, �, �� is described as:

��^, _, �, �� 0,0,0, ��)O.`a,.bcP�'.de:fghijgkf':gg (2.22)

where 0 is the wave vector of the transmitted wave, ^– _ plane is the interface between

two media, the origin point (0,0,0) is the cross point between the interface and the

incident light. Equation (2.22) describes the evanescent wave that propagates along the

interface. The magnitude of its electric field decreases exponentially with increasing

distance from the interface.

The penetration depth %" of the evanescent wave into the second medium, which is the

characteristic distance at which the field strength falls off to 1/� of its value at the

interface, can be calculated with the equation:

%" � U2m�A�nR��XA $ ��

(2.23)

where U is the wavelength of the IR light. In an FTIR system utilizing total internal

reflection, the first medium is the crystal of the reflection component and the second

medium is the investigated sample. The index of refraction of the crystal (�A) must be

significantly greater than that of the sample (��). Materials with high index of refraction

which can be used as a crystal include zinc selenide, silicon, germanium, and diamond.


25

The penetration depth %" of the evanescent wave is typically in the range from 0.5 to 5

µm. Therefore, the sample must be in good contact with the crystal. A part of the energy

of the evanescent wave is absorbed by the sample, which results in a corresponding

change in the detected IR light. Consequently, an IR spectrum of the sample can be

probed.

2.2.3.2 ATR unit

Figure 2.8: Schematic of the ATR unit. The diamond crystal with high

index of refraction is used as the sampling crystal to ensure total reflection

of the incident light. The incident angle of the IR beam is 45°. The diamond

crystal is hemispherical, which focuses the beam to the horizontal sampling

surface to produce a 0.5 mm diameter sampling area. The light intensity is

characteristically attenuated by the propagation of the evanescent wave in

the sample.

Figure 2.8 shows a schematic of the ATR unit used in the current setup. It is a single

reflection ATR with a diamond crystal (model MVP-Pro, Harrick). The IR beam with

an incident angle of 45° is focused on the ATR crystal by an aluminum mirror. The

reflected beam is focused on the detector by another aluminum mirror. The ATR crystal

is hemispherical, which additionally focuses the beam to the horizontal sampling

surface to produce a 0.5 mm diameter sampling area. This small sampling area makes

the ATR unit suitable for examining sensitive compounds with little required amount

and minor sample preparation. Solid samples are placed onto the surface of the diamond


26

crystal and are slightly pressed against the crystal using the built-in pressure applicator

to make good contact between the sample and the crystal. For consistent and precise

measurement results, the pressure applicator is equipped with a slip-clutch for

reproducible pressure application. Liquid samples are also placed onto the crystal

surface using a designed liquid cell.

2.2.4 Data acquisition and analysis

Figure 2.9: Measured data of a reference and a sample using a FTIR

spectrometer: (A) interferograms which show the intensity of IR light as a

function of the moving mirror position, (B) Fourier transform spectra of the

interferograms in the frequency domain, (C) calculated absorbance of the

sample, (D) calculated transmittance of the sample.

The data acquired from a measurement is an interferogram, which is the intensity of IR

light versus the moving mirror position. Each position of the moving mirror corresponds

to a temporal delay between the two beams within the interferometer (one reaches the


27

fixed mirror and the other reaches the moving mirror). The measured intensities are

therefore the data in the time domain. To obtain a spectrum in the frequency domain, a

fast Fourier transform algorithm is applied to the time domain data. In order to get a

transmittance or absorbance spectrum of a sample, two measurements must be carried

out: reference measurement (without the sample) and sample measurement (with the

sample). An example of the measured data is presented in Figure 2.9.

From the interferograms of the reference and the sample, the corresponding frequency

dependent spectra are calculated, including the intensity � of the reference and intensity

�! of the sample at each frequency. These analyses are performed by the OPUS

spectroscopic software program of Bruker Optics. The transmittance (o) and absorbance

(�) of a sample are defined by:

o � �!� (2.24)

� � pqrA \� �!] (2.25)

The absorbance can be converted to the absorption coefficient (L) if the thickness of the

sample (%) is known:

L � ln s� �!t% � � · p�10%

(2.26)

IR spectra are useful analytical tools as most of studied samples have characteristic

bands in the IR frequency range.

For measurements using the ATR unit, there are two considerable problems. First, the

penetration depth of the evanescent wave into the sample depends on the frequency as

shown in Equation (2.23), which results in a modified band intensity at different

frequencies. Second, the absorption of the sample relates to its frequency dependent

index of refraction, which causes a shift of the band positions towards smaller

frequencies. Therefore, spectra obtained from ATR-FTIR spectroscopy have to be

corrected, so that the position and the intensity of the absorption bands are similar to


28

those of a spectrum measured with transmission spectroscopy [40, 41]. For a correction

of both mentioned problems, the "Extended ATR correction" command of the OPUS

program is used. This manipulation converts an absorbance spectrum (�) to an ATR

spectrum (�ou) using the equation:

�ou � �� · �q�n�v�� · · m�A�� sin��X� $ 1�� (2.27)

where � is the number of ATR reflections, is the wavenumber (frequency) of the IR

light, �A� is the ratio of the indices of refraction between the ATR crystal and the

sample, X is the incident angle, and � is the middle electric field at the boundary.

The ATR unit used in the current setup is a single reflection ATR with an incident angle

of 45°. The index of refraction of the ATR diamond crystal and the middle electric field

at the boundary are automatically calculated in the OPUS program. In order to perform

an extended ATR correction for ATR-FTIR spectroscopy measurements, the mean

index of refraction of the sample must be input to the program.

29

3 Water in nanopores

The studied nanopores are tubular crystals formed by water mediated assembly of

hydroxyl acids into an H-bond network. These supramolecular structures have hydrated

pores with different pore diameters. Water molecules can be permanently confined

inside the pores at ambient condition. In this chapter, attenuated total reflection (ATR)

FTIR spectroscopy in the FIR frequency range was used to investigate the pore size

dependent dynamics and the thermal stability of confined water inside these nanopores.

The presented results are part of Ref. [42, 43].

3.1 Models for biological channels

Studies of water in confined environment have gained significant interest because of the

implication in a number of biological processes [4, 7]. The permeation of water through

cellular membranes is facilitated by aquaporins, a family of proteins that form

cylindrical transmembrane pores of about 20 Å long and 2.8 Å wide at their narrowest

position [44]. This permeability of water across aquaporin channels is selective with

almost no resistance, while the hydronium ion (H3O+) cannot permeate the channels. A

detailed understanding of the forces that drive the filling of the pores by water

molecules and the functional thermodynamics of these natural pores, which are

predominantly hydrophobic, remains elusive. A full understanding of the transport of

water requires knowledge at the molecular level about structure and dynamics from a

locally well defined area [45, 46].

Water structure in biological channels has been studied using molecular dynamics (MD)

simulations [47]. This theoretical study includes models about the physiological

functioning of natural pores which are characterized by numerous chemical and

structural complexities. For more straightforward evaluations, simpler artificial models

have been investigated, such as single-walled carbon nanotubes (SWCNT) for modeling

the diffusion of water in biological structures [48, 49]. These studies usually modeled

the structural and dynamic properties of water in SWCNT and revealed concerted water

motions and density distribution patterns. However, the application of these nanoporous

structures to study the structure and dynamics of long-lived aggregates of water

3. Water in nanopores

30

molecules is limited [50, 51]. Further MD simulation studies predicted that the structure

and dynamics of water at room temperature are specific to each model and depend on

the inner diameter of the pore and its hydrophilicity [52, 53].

Figure 3.1: Chemical structures of the twelve studied monomers. The

different appendages give rise to hydrated porous compounds (HP1-6), a

hydrated nonporous compound (NP3), and anhydrous nonporous

compounds (NP1-2, NP4-6).

In natural channels, the pore diameter and hydrophilicity (or hydrophobicity) are

different along the pore. This influences the structure and dynamics of water molecules

according to their momentary location. Therefore, water diffusion in the channels will

result from the balances among the transient local structures and dynamics of water

molecules. Theoretical studies showed that water can occupy hydrophobic pores,

although there is a lack of H-bond interactions between water and the inner surface of


31

the pore [54, 55]. However, experimental evidence for the structural nature of water in

hydrophobic pores is lacking and a rigorous understanding of the mechanisms of water

transport through nanopores remains incomplete [56].

To obtain a more thorough understanding of the water transport in natural biological

channels, this study investigated a series of synthetic organic nanopores. The approach

involved the water mediated assembly of organic molecules into porous structures. The

biological formation of a pore is simplified by a general thermodynamically favored

process. For this reason, a set of chemically very similar organic molecules with the

ability of self-assembling into an H-bonded network to form supramolecular structures

was studied. These molecules are different from each other by their appendages. They

were synthesized by changing the number and nature of selected atoms in appendages,

following a previously published synthetic methodology [57]. Figure 3.1 shows the

monomers of the twelve studied compounds. They are hydroxyl acids of general

structure 7,7-appendage1/appendage2-5-hydroxymethyl-6-oxabicyclo[3.2.1]octane-1-

carboxylic acid. These compounds give rise to hydrated porous, hydrated nonporous,

and anhydrous nonporous crystalline structures [58]. The compounds formed by

monomers with the name HP (HP1-6) have hydrated porous structures while the ones

formed by monomers with the name NP (NP1-6) have nonporous structures. Among the

nonporous compounds, five are anhydrous (NP1-2, NP4-6) and one is hydrated (NP3).

The anhydrous nonporous compounds have no water molecules while the hydrated

nonporous compound contains water molecules that form a part of the supramolecular

structure (structural water). On the other hand, the hydrated porous compounds contain

both structural water and water molecules that reside inside the pores. These porous

compounds have served as models of biological water channels where water

confinement has been studied using spectroscopic and calorimetric techniques [59].

Another study has shown that water molecules can successfully occupy permanently the

nonpolar pores at ambient conditions in a thermodynamically controlled manner

without impairing the structure of H-bond networks [60]. The supramolecular structure

is stoichiometrically sustained by the organic monomers and by water molecules

according to the following general equation [61]:

nM � mH�O { Mj · �H�O�| � Mj � �H�O�|

(monomers) (bulk water) (hydrated pore) (anhydrous pore) (water cluster)

(3.1)

where n is the number of organic monomers (

molecules (H2O) which is

general method for the synthesis of long

synthetic approach, the number of water molecules inside the pore is constant and

independent of the inner pore diameter

monomer/water remains unchanged at

Figure 3.2: Crystal structures

[62], (B) narrow porous compound HP6

compound NP2 [58]. Fo

pores are not shown. T

which define the internal pore diameter. The distances a and

maximum distance between the

(HP1·2H2O: a = 9.3 Å, b = 12.2 Å; HP6·2H

From the monomers, crystals from all compounds were slowly grown under identical

conditions using a previously water saturated mixture of carbon tetrachloride/2,2,4

trimethylpentane mixture (ratio of 4:1). Monomers with long, branched, or cyclic

appendages which restrict conformational movements (NP1

anhydrous packing composed mostly of double stranded head

compound formed by NP3 also incorporated water molecules (two monomers with one

water molecule), but the structure is still nonporous. Only monomers with flexible

linear appendages (HP1-


32

is the number of organic monomers (M), and m is the num

is involved in the resulting assembly. This process describes a

general method for the synthesis of long-lived water clusters from bulk water

the number of water molecules inside the pore is constant and

independent of the inner pore diameter [59]. In all hydrated porous models, the

monomer/water remains unchanged at 1/2 (one monomer with two water molecules).

Crystal structures of: (A) wide porous compound HP1·2H

narrow porous compound HP6·2H2O [59], (C) nonporous

. For the porous compounds, water molecules

shown. The white dotted lines show the β-oriented appendages

which define the internal pore diameter. The distances a and b represent the

maximum distance between the β and α oriented appendages, respectively

O: a = 9.3 Å, b = 12.2 Å; HP6·2H2O: a = 7.7 Å, b = 11.3 Å).


previously water saturated mixture of carbon tetrachloride/2,2,4


appendages which restrict conformational movements (NP1-6 in Figure

anhydrous packing composed mostly of double stranded head-to-



6 in Figure 3.1) gave hydrated crystalline structures by

the number of water

This process describes a

lived water clusters from bulk water. With this

the number of water molecules inside the pore is constant and

models, the ratio

2 (one monomer with two water molecules).

: (A) wide porous compound HP1·2H2O

, (C) nonporous

molecules inside the

oriented appendages

b represent the

oriented appendages, respectively

O: a = 7.7 Å, b = 11.3 Å).


previously water saturated mixture of carbon tetrachloride/2,2,4-


Figure 3.1) generated

-tail arrays. The



) gave hydrated crystalline structures by


33

incorporation of two water molecules for each monomer. The compounds HP1·2H2O

(with β-ethyl/α-ethyl appendages), HP2·2H2O (β-ethyl/α-propyl), and HP3·2H2O (β-

ethyl/α-propenyl) have wide pores with pore diameter of about 5.9-9.4 Å [62]. Other

hydrated porous compounds (HP4·2H2O, HP5·2H2O, HP6·2H2O) with longer β-

appendages have narrow pores of about 4.2-6.5 Å [59]. In this study, both the wide and

narrow pores are referred to as nanopores.

Figure 3.2 shows representative front view crystal structures of a wide porous

compound HP1·2H2O [62], a narrow porous compound HP6·2H2O [59], and a

nonporous compound NP2 [58]. The nonporous compound consists mostly of double

stranded head-to-tail arrays. For the porous compounds, the β-ethyl appendage of

HP1·2H2O occupies less space than the β-propenyl appendage of HP6·2H2O does,

which results in wider pore size for HP1·2H2O.

Figure 3.3: Schematic pore profile with typical average diameters of: (A)

wide porous compound HP1·2H2O, (B) narrow porous compound

HP6·2H2O. Water inside the pore of HP1·2H2O is diffuse and less structured

than water inside the pore of HP6·2H2O. (C) Side view of the crystal

structure of HP6·2H2O [59]. Figure courtesy is adapted from Ref. [43].

The conformational deformation by incorporation of water molecules allows an efficient

hexagonal assembly which extends to the third dimension to form tubular H-bond

network. Each puckered channel can be described as interconnected hexagons in chair

conformations as displayed in Figure 3.3C. A schematic of the side view pore profile of


34

HP1·2H2O is shown in Figure 3.3A, and that of HP6·2H2O is shown in Figure 3.3B.

Structural water (represented by blue dots on the wall) helps to fix the tubular structure

in the pore walls. Water inside the wide pore of HP1·2H2O (fat blue ellipse) is diffuse

and less structured than water in the narrow pore of HP6·2H2O (individual blue dots).

The pore diameter is not homogeneous. It changes approximately from 5.9 to 9.4 Å in

the case of HP1·2H2O and from 4.2 to 6.5 Å in the case of HP6·2H2O. To some extents,

these synthetic organic nanopores serve as models for natural biological channels.

In bulk water, the four H-bonding sites are not always fully occupied as the molecules

are mobile in the liquid state. At room temperature, each water molecule coordinates

with 3.5 neighboring water molecules on average [7]. H-bond breaking and reformation

occur continuously in a sub-ps timescale. The average H-bond network rearrangements

(water collective motions) are in the order of 1 ps [3]. When water molecules are

confined inside a nanopore, water collective motions are considerably influenced.

Changes in dynamics of confined water can be directly monitored by FIR spectroscopy.

In this study, pore size dependent dynamics of confined water in nanopores were

investigated by FTIR spectroscopy in FIR frequency range using an attenuated total

reflection (ATR) unit. Furthermore, reversibility of confined water dynamics and

thermal stability of the hydrated nanopores were examined. The study aims to

qualitatively probe the differences in the dynamics of water in nanopores of different

sizes, establish optimal conditions for chemical models of water pores, and contribute to

a better comprehension of water dynamics in nonpolar environments.

ATR-FTIR spectroscopy was used to probe the absorbance spectra of water and the

selected compounds at different temperatures in the FIR region from 20 to 700 cm-1. For

data analysis, the frequency range from 50 to 650 cm-1 was chosen because of its high

signal to noise ratio. The mean index of refraction of the samples was approximately

fixed at 1.5 for the extended ATR correction of the absorbance spectra. A detailed

description of the experimental setup and data analysis is presented in Chapter 2. For

each measurement, the solid sample was placed onto the surface of the ATR diamond

crystal and was slightly pressed against the crystal using the built-in pressure applicator.

Water and ice were also placed onto the surface of the ATR diamond crystal using a

designed liquid cell. The IR source was supplied by a mercury-lamp. A liquid-helium-

cooled silicon bolometer was used for spectra acquisition. All measurements were

carried out under vacuum (2.01 mbar). The temperature in the range from -5 to 40°C


35

was monitored by an external temperature controller. All samples were synthesized by

the collaborators at the Institute for Chemical Research in Seville, Spain.

3.2 Pore size dependent dynamics of confined water

The FIR spectrum of ice and temperature dependent spectra of bulk water in the

temperature range from 0 to 20°C are shown in Figure 3.4. The absorbance spectrum of

ice shows a characteristic maximum at about 200 cm-1, and a broad minimum from 300

to 500 cm-1. For frequencies above 500 cm-1, a rapid absorbance increase is found. The

absorbance of liquid water is distinct from that of ice. Water spectra show a smaller

peak at about 150 cm-1 and a broad absorbance maximum in the frequency range above

400 cm-1. With increasing temperature, the absorbance of water increases. As observed

in the inset of Figure 3.4, the temperature dependence is significant in the high

absorbance frequency region from 400 to 570 cm-1. These FIR spectra of ice and water

serve as a reference to compare with the spectra of confined water in nanopores.

Figure 3.4: Absorbance spectrum of ice and spectra of bulk water in the

range from 0 to 20°C. Inset: spectra of water in the high absorbance

frequency region from 400 to 570 cm-1. The absorbance of water increases

with increasing temperature.


36

The twelve studied compounds are categorized into three groups. The first group

contains hydrated porous compounds with wide pore (from 5.9 to 9.4 Å), including

HP1·2H2O, HP2·2H2O, and HP3·2H2O. The second group contains hydrated porous

compounds with narrow pore (from 4.2 to 6.5 Å), including HP4·2H2O, HP5·2H2O, and

HP6·2H2O. The third group consists of nonporous compounds with one hydrated

(NP3·0.5H2O) and five anhydrous compounds (NP1, NP2, NP4, NP5, NP6). Among the

third group, three compounds have identical α and β-appendages (NP1, NP2, NP3)

while the other three have different appendages (NP4, NP5, NP6).

Figure 3.5: Absorbance spectra at 20°C in the FIR region of: (A) wide

hydrated nanopores, (B) narrow hydrated nanopores, (C) one hydrated

nonporous compound and two anhydrous nonporous compounds with

identical appendages, (D) anhydrous nonporous compounds with different

appendages. In the frequency range from 400 to 570 cm-1 (highlighted

regions), the hydrated porous compounds have significantly higher

absorbance than the nonporous compounds.

The FIR absorbance spectra at 20°C of all compounds in the frequency range from 50 to

650 cm-1 are shown in Figure 3.5. Below 400 cm-1, the spectra of all compounds exhibit


37

relatively small absorbance and no characteristic absorption feature is found. However,

in the region from 400 to 570 cm-1, the absorbance of hydrated porous compounds

(Figure 3.5A, B) absorb significantly more than the nonporous compounds (Figure

3.5C, D). The absorbance of NP3·0.5H2O, which is a hydrated nonporous compound

and only has structural water is similar to that of the anhydrous compounds in region

from 400 to 570 cm-1. A comparison with FIR absorbance of water in Figure 3.4

indicates that it is possibly the water inside the nanopores that accounts for the observed

high absorbance of hydrated porous compounds in this frequency region.

Figure 3.6: Temperature dependent spectra in the range from -5 to 20°C of

three representative compounds: (A) wide hydrated nanopore HP1·2H2O,

(B) narrow hydrated nanopore HP6·2H2O, (C) anhydrous nonporous

compound NP2. The change in the absorbance with temperature of the wide

hydrated nanopore is more significant than that of the narrow hydrated

nanopore while the absorbance of the nonporous compound is almost

unchanged in the entire investigated temperature range.

In order to check the similarities in the behavior of water confined in nanopores and

bulk water, temperature dependent spectra of all compounds in the temperature range

from -5 to 20°C were measured. Figure 3.6 shows temperature dependent spectra in the

range from -5 to 20°C of three representative compounds, one for each categorized

group. These chemically similar compounds show quite different behaviors. The

absorbance of the nonporous compound NP2 (Figure 3.6C) does not change with

temperature in the whole temperature range, while the absorbance of the wide hydrated


38

nanopore HP1·2H2O (Figure 3.6A) and the narrow hydrated nanopore HP6·2H2O

(Figure 3.6B) increase considerably with increasing temperature, especially in the

frequency range from 400 to 570 cm-1. The change in the absorbance with temperature

of the wide hydrated nanopore is more profound than that of the narrow hydrated

nanopore. The observation is a general phenomena as an almost temperature

independent FIR absorbance is found for any anhydrous compound while a clear

temperature dependence is observed for all hydrated porous compounds.

Figure 3.7: Change in absorbance compared with absorbance at -5°C,

averaged over the frequency range from 400 to 570 cm-1 of: (A) six hydrated

porous compounds, (B) one hydrated nonporous compound and five

anhydrous nonporous compounds. The hydrated porous compounds show a

nearly linear increase in absorbance with increasing temperature and the

change for compounds with wide pores (HP1-3) is more significant than the

change for compounds with narrow pores (HP4-6). For the nonporous

compounds (NP1-6), the absorbance is independent with temperature.

For further investigation on the pore size dependent behavior, the integrated absorbance

averaged over the frequency range from 400 to 570 cm-1 of all compounds is calculated.

Figure 3.7 shows the temperature dependent change in the integrated absorbance

compared with the integrated absorbance at -5°C. As previously observed for the

anhydrous compound NP2 in Figure 3.6C, all compounds without hydrated nanopores

show an almost constant absorbance at different temperatures (Figure 3.7B). On the


39

other hand, the less confined water molecules in wide nanopores (HP1·2H2O,

HP2·2H2O, HP3·2H2O) show a significant linear increase in absorbance with increasing

temperature, by approximately 1.0·10-4 per 1°C (Figure 3.7A). A smaller temperature

dependent absorbance is observed for water molecules confined in narrower pores

(HP4·2H2O, HP5·2H2O, HP6·2H2O), in which the slope of the linear increase is

approximately 0.7·10-4 per 1°C (Figure 3.7A).

3.3 Reversibility of water dynamics in nanopores

Figure 3.8: Temperature dependent FIR absorbance of the hydrated

nanopore HP1·2H2O in the temperature interval from -5 to 30°C upon: (A)

heating, (B) cooling. Inset: Integrated absorbance over the frequency range

from 400 to 570 cm-1 compared to the integrated absorbance at -5°C. Upon

heating and cooling within this temperature range, a reversible behavior is

observed.

The thermal stability and reversibility of water dynamics in nanopores were studied by a

series of measurements in which the samples were first heated up and then cooled

down. The thermal behavior of the representative compound HP1·2H2O in the

temperature range from -5 ºC to 30 ºC was investigated. Figure 3.8 shows the

temperature dependent FIR absorbance spectra for the heating (Figure 3.8A) and the

subsequent cooling process (Figure 3.8B). The inset displays the temperature dependent

integrated absorbance averaged over the frequency range from 400 to 570 cm-1


40

compared to the integrated absorbance at -5°C. Upon heating and cooling within the

temperature range, the changes in absorbance are completely reversible. The absorbance

of the compound increases with increasing temperature, then it decreases back upon

cooling.

Figure 3.9: Decrease in FIR absorbance resulted from the release of water

of: (A) HP1·2H2O when being held at 40°C, (B) HP6·2H2O when being held

at 30°C. For both compounds, only mobile water molecules in the pores are

released as the pore structure still maintains.

The decrease in FIR absorbance of compound HP1·2H2O was observed after the

compound was heated at 40ºC for 40 and 90 minutes (Figure 3.9A). After 90 minutes,

the entire content of mobile water in the pore was released and no additional decrease in

absorbance was observed. Similar measurements of the narrow porous compound

HP6·2H2O show a similar trend (Figure 3.9B). For this compound, the release of water

began at lower temperature (30°C), and all mobile water in the pore was released after

120 minutes.

3.4 Discussion and conclusion

The FIR spectrum of ice with a maximum at about 200 cm-1, and a broad minimum

from 300 to 500 cm-1 (Figure 3.4) is in good agreement with a previous study for H2O

ice [63], as well as with the calculated data for D2O ice [64]. The spectra of liquid water


41

at different temperatures show a small peak at about 150 cm-1 and a broad peak in the

frequency range from 400 to 570 cm-1. A FIR study using two-fraction model for low

frequency Debye relaxation [65] and a MD simulation with instantaneous normal mode

theory [66] found similar water absorbance spectra. Experimental data measured with

FTIR transmission spectroscopy in this frequency range also showed the same behavior

[67]. In recent studies, the water absorbance peak at lower frequency is assigned to the

intermolecular H-bond stretching vibrations, while the one at higher frequency is

assigned to the librational motions [9, 68]. The observed increase in absorbance of

water with increasing temperature agrees well with a previous study at similar

temperatures and frequency range [67]. The distinct difference between the spectra of

ice and liquid water indicates that the confined water structure in ice strongly influences

the FIR absorbance.

Compared to the FIR spectra of water, the FIR spectra of the hydrated porous

compounds (Figure 3.5) also show a broad absorption peak in the frequency range from

400 to 570 cm-1, but no peak around 150 cm-1. The absence of the absorbance peak

around 150 cm-1, which is assign to intermolecular H-bond stretching vibrations, can be

explained by the absence of intermolecular water H-bond network when water is

confined in nanopores. This behavior is also observed with confined water in organic

solvents at low water content [69]. In the temperature dependent spectra (Figure 3.7),

the absorption of hydrated porous compounds in the frequency range from 400 to 570

cm-1 increase with increasing temperature, which is similar to the behavior of water,

while the absorption of nonporous compounds is unchanged with temperature. These

observations unambiguously confirm that the observed absorbance in this particular

frequency range is due to the confined water in the nanopores. Furthermore, a smaller

change in the absorbance upon increase of temperature compared to the wide nanopores

shows that water molecules in the narrow nanopores are less dynamic than water

molecules in the wide nanopores as well as water molecules in bulk water. Water

dynamic here refers to their FIR absorbance which is closely connected with the fast

solvation dynamics. A previous theoretical study revealed that the FIR absorbance

spectra of water are related to the fast fluctuations of the water dipole moments [64].

The FIR absorbance describes fluctuations of the H-bond network of water arising

partially from the breaking and reformation of H-bonds between water molecules,

which takes place in the ps timescale [3, 70]. The mentioned dynamic of water in

nanopores is therefore occurs in the ps timescale.


42

The temperature dependent FIR spectra upon heating and subsequent cooling in the

temperature range from -5 ºC to 30 ºC of the hydrated porous compound HP1·2H2O

(Figure 3.8) show a complete reversibility. A previous study using thermogravimetric

analysis and variable temperature powder X-ray diffraction of the same compound

indicated that the loss of some water molecules in nanopores started only when the

temperature reached 40ºC, without the collapse of the pore structure [60]. Therefore, the

observed reversibility is expected as the amount of water molecules remains unchanged

upon tuning within the studied temperature range. The release of mobile water in the

pore observed at 40°C for HP1·2H2O and at 30°C for HP6·2H2O (Figure 3.9) agrees

well with the results observed with thermogravimetric analysis [60]. This study also

found that water is not reincorporated into the pores even when the compounds were

kept under moist conditions for 24 hours. Therefore, this process is irreversible.

Figure 3.10: Temperature dependent change in the integrated absorbance

over the frequency range from 400 to 570 cm-1 compared to the integrated

absorbance at -5ºC of three compounds which are formed by the same

monomer but have different water contents (left panel). Compounds with

lower water content HP1·1.5H2O and HP1·1H2O were obtained by heating

and holding the fully hydrated compound HP1·2H2O for 40 and 90 minutes

at 40°C, respectively. The right panel (Figure courtesy adapted from Ref.

[43]) illustrates the change of the hydration in the pore upon releasing of

water in the pore. Green color represents pore walls and blue circles

represent water molecules.


43

When compound HP1·2H2O was heated and held 40ºC, water in the pore was released

continuously, which also changed the monomer to water ratio. When being held for 40

minutes at 40°C, the ratio changed from 1:2 to approximately 1:1.5, and the compound

became HP1·1.5H2O. After being held for 90 minutes at 40°C, mobile water in the pore

was released completely, and the ratio changed to 1:1 (HP1·1H2O), indicating that only

structural water remained in the compound. To investigate the thermal behavior of the

compounds which are formed by the same monomer but have different water contents,

FIR measurements in the temperature range from -5 to 20°C were carried out for

HP1·2H2O, HP1·1.5H2O and HP1·1H2O. Figure 3.10 shows the temperature dependent

change in the integrated absorbance averaged over the frequency range from 400 to 570

cm-1 compared to the integrated absorbance at -5ºC. The fully hydrated compound

HP1·2H2O shows the highest increase in absorbance with increasing temperature. The

partially dehydrated compound HP1·1.5H2O has less absorbance change with

temperature. The change is approximately a half of the change observed with the fully

hydrated compound. The absorbance of the dehydrated compound HP1·1H2O, in which

mobile water in the pore is fully released, is nearly constant with temperature. This

behavior is similar to that of the hydrated nonporous compound (NP3·0.5H2O) and the

anhydrous nonporous compounds. The change of the hydration in the pore upon

releasing of water in the pore is illustrated in right panel of Figure 3.10. The release of

the mobile fraction of water renders the pores more hydrophobic. A transition of the

inner pore surface from hydrophilic to hydrophobic surface occurs upon increasing the

temperature to 40°C. This may be attributed to the influence of the dynamics and

thermodynamics of confined water molecules on the pore surface affinity. The

differences in the temperature dependent dehydration of the hydrated nanopores indicate

that the behavior of water inside the pores depends on the details of the intermolecular

interactions between water molecules and the inner surface of the pores. The structure

and dynamics of confined water are strongly influenced by the temperature.

The results show that the FTIR spectroscopy in FIR region, especially temperature

dependent studies, can serve as a sensitive method to probe the water dynamics inside

hydrated nanopores. The compounds that contain water inside the pores absorb

significantly more than the anhydrous or hydrated nonporous compounds do in the

frequency range from 400 to 570 cm-1. Furthermore, the temperature dependent FIR

absorbance of water in nanopores is significantly different depending on the inherent

water dynamics in each compound, which is imposed by the size of the pores. Less


44

confined and thus more mobile water molecules inside the wide pores show a large

change in their absorbance upon increase of temperature. In contrast, water confined in

the narrow pores show a smaller increase in absorbance with increasing temperature.

Therefore, FIR-FTIR spectroscopy can reveal and qualitatively characterize distinct

dynamics of confined water molecules inside the hydrated nanopores by their

temperature dependent FIR absorbance. The stability and reversibility at ambient

condition of the hydrated nanopores prove that water molecules can continuously

occupy permanently the nonpolar pores. This confirms that the synthesized nanopores

can serve as model systems for biological channels. Further experimental exploration of

these model systems can be systematically studied, which may open opportunities for a

more comprehensive understanding of water dynamics in biological processes.

45

4 Water in reverse micelles

In a DDAB/Cy/water (didodecyldimethylammonium bromide/cyclohexane/water)

mixture, DDAB molecules aggregate into nanoscale reverse micelles (RM), trapping

water molecules inside the RM. Water confined in RM exhibit significantly different

properties compared to bulk water. This chapter presents the study of DDAB/Cy/water

RM systems at different water concentrations and temperatures using dynamic light

scattering (DLS), THz-TDS, and FTIR spectroscopy. The presented results are part of

Ref. [71].

4.1 Water nanopools in DDAB/Cy/water reverse micelles

RM are water-in-oil droplets stabilized by a surfactant. RM are formed when surfactant

molecules dissolved with water in a nonpolar organic solvent aggregate into nanoscale

RM structures where the hydrophilic heads form the core of the RM and hydrophobic

tails are in contact with the surrounding solvent. Water molecules in the mixture are

trapped inside the RM. Water confined in RM usually exhibit significant differences in

physical and chemical properties compared to its bulk properties. The modification

principally results from the heterogeneous bonding of the water molecules with the RM

interface. RM systems have been widely studied because of their similarity to various

biological systems. There has been a discussion on the effect of confinement on the

water dynamics. The water molecules in the vicinity of the surface are suspected to be

highly structured or retarded in their dynamics [72, 73, 74]. Nuclear magnetic resonance

(NMR) studies of confining proteins within the nanoscale interior of RM found a

significant reduced motion of the hydration water [75, 76]. Most of the previous studies

investigated RM formed by anionic AOT (sodium bis(2-ethylhexyl) sulfosuccinate) or

nonionic surfactants that form spherical RM droplets. In these systems, the curvature of

the interface has an identical contribution towards the water structure and dynamics.

The role of surface topology and the surfactant film curvature on the hydration structure

and dynamics has been neglected. Therefore, it is interesting to investigate how the

morphology of RM interface affects the structure and dynamics of confined water

molecules inside the RM waterpools.

4. Water in reverse micelles

46

Figure 4.1: Structure of DDAB (left) and schematic of a spherical water

nanopool formed by DDAB/Cy/water RM (right). In the nonpolar Cy

solvent, the cationic DDAB surfactant molecules aggregate into nanoscale

RM structures where the hydrophilic heads form the core of the RM and

hydrophobic tails are in contact with the surrounding solvent. Water

molecules in the mixture are confined inside the RM.

DDAB is a double chained cationic surfactant (Figure 4.1). DDAB/alkane/water RM

systems exhibit a rich phase and structural behavior. The maximum water solubilization

capacity of these systems are usually smaller than the conventional surfactant systems

due to the low hydrophilicity of the quaternary ammonium head group of DDAB [77].

At low degree of hydration } (} � ~water�/~DDAB�), the system mainly consists of

cylindrical aggregated structures. With increasing } , the RM changes into discrete

droplets [78, 79]. This gradual cylinder-to-sphere transformation of the RM resulted

from the fact that the elastic property of the DDAB monolayer around water in alkane

solvent undergoes substantial modifications during the process of microscopic phase

transition. A study of DDAB/Cy/water RM systems using small angle neutron

scattering (SANS) indicated that the system shows a cylindrical structure in the }

range from 2 to 8, and at } � 10, discrete droplet type structure prevails [78]. This

transition has successfully been explained on the basis of a disordered open connected

(DOC) model that describes a set of random structures and transitions between them

[80]. Using Fourier transform pulsed-gradient spin-echo (FT-PGSE) NMR, a steady

decrease in the self-diffusion coefficient of water in DDAB RM was detected with

increasing } , which is in contrast to the behavior of RM systems formed by anionic

AOT and nonionic C12E4 (tetraethylene glycol dodecyl ether) [81]. The microscopic

phase transition in DDAB RM has also been confirmed by small angle X-ray scattering


47

(SAXS) and NMR relaxation studies [82, 83]. Different from conventional RM systems,

DDAB RM systems exhibit reverse percolation of conductance which manifests a

transition from connected to discrete structures [84, 85]. The changes in microscopic

phase structure as well as the reverse percolation of conductance result in significant

modification of the properties of the surfactant monolayer at the water-oil interface.

Therefore, DDAB RM systems serve as an interesting platform to understand the

structure and dynamics of water molecules in RM systems with different morphological

states. Figure 4.1 shows the structure of DDAB and the schematic of a spherical

waterpool in DDAB/Cy/water RM. DDAB molecules aggregate into nanoscale RM

structures where the hydrophilic heads form the core of the RM and hydrophobic tails

are in contact with the surrounding nonpolar Cy solvent. Water molecules in the

mixture are trapped inside the RM.

In this study, the structure and dynamics of water molecules in the water nanopools of

DDAB/Cy/water RM systems in dependence of } and temperatures were investigated.

The geometrical dimensions of these systems were estimated using DLS technique. The

collective motions of water molecules in the THz region were investigated using THz-

TDS. FTIR spectroscopy in FIR and MIR region probed the H-bond network structure,

dynamics, and intramolecular OH stretching of water molecules. DDAB and Cy were

purchased from Sigma Aldrich at 98% or higher purity and were used as received. The

concentration of DDAB in Cy was kept constant at 0.1 M. As dry DDAB is insoluble in

Cy [86], a specified amount of water was added in DDAB/Cy mixture to prepare a RM

stock solution with } � 1. Upon further addition of water, RM systems with increased

} were prepared.

4.2 Dynamic light scattering measurements

4.2.1 Principle of dynamic light scattering

DLS, which is also known as photon correlation spectroscopy, was used to determine

the size of small particles in solution by probing the temporal fluctuations of the light

scattered by the particles. When a monochromatic light hits the particles whose size is

much smaller than its wavelength, light scattering occurs in all directions. The intensity

of the scattered light is influenced by the interaction, size and shape of the particles.


48

Furthermore, the scattered intensity fluctuates with time because the particles undergo

Brownian motion, constantly changing their position in the solution [87]. Brownian

motion is the movement of particles due to the random collision with molecules of the

liquid solvent. Assuming that a particle has spherical shape, the relationship between its

size and its speed due to Brownian motion is given by the Stokes-Einstein equation:

� � 0�o3�% (4.1)

where � is the translational diffusion coefficient, 0� is the Boltzmann constant, o is the

temperature in Kelvin, � is the viscosity of the solvent, and % is the diameter of the

particle. As deduced from the equation, large particles move slower than smaller

particles. In a DLS measurement, different movement speeds, or different particle sizes,

results in different effects on the time dependent intensity of the scattered light. An

autocorrelation of the measured intensity yields the dynamic information (diffusion

coefficient) of the particle. When the viscosity of the solvent is known, the

hydrodynamic diameter (%�), which characterizes how the particle diffuses in the

solvent, is estimated from following equation:

%� � 0�o3�� (4.2)

Figure 4.2 shows a schematic of a DLS system. A laser provides the light source to

illuminate the sample which consists of the studied particles. A part of the laser beam

passes through the sample and a part is scattered by the particles. The detector at a

scattering angle θ is used to measure the time dependent intensity of the scattered light.

It is possible to place the detector at any angle because the particles scatter light in all

directions. The autocorrelator compares the scattering intensity at successive time

intervals to derive the variation rate of the intensity. This information is analyzed by a

computer to yield the hydrodynamic diameter of the particles. The fitting of

autocorrelation data of DLS measurements assumes that the particles are spherical. For

particles with other shapes, the fitting usually yields larger sizes.


49

Figure 4.2: Schematic of a DLS system. The laser light is focused on the

sample which scatters light in all directions. The time dependent intensity of

the scattered light is measured by the detector placed at a scattering angle θ.

The measured data are processed and analyzed by the autocorrelator and the

computer to yield the hydrodynamic diameter of the particles in the sample.

In this study, a DLS system (model Nano-S, Malvern) was used to measure the size of

the RM. The system employs a 4 mW He-Ne laser at a wavelength of 632.8 nm as a

light source. All scattered photons were collected at a fixed scattering angle of 173°.

The time dependent scattered intensity were processed using the instrumental software

to obtain the hydrodynamic diameter %�, and the size distribution of the scatterer in

each sample. This experiment was carried out by the collaborators at S.N. Bose

National Centre for Basic Sciences (Kolkata, India).

4.2.2 Hydrodynamic diameters of reverse micelles

Figure 4.3 shows the hydrodynamic diameter %� of DDAB/Cy/water RM systems for

distinct } and temperature values. As observed from the figure, %� of all RM systems

decreases with increasing temperature. For } � 10, %� decreases from about 37-57

nm at 20°C to about 8-15 nm at 50°C. For } � 10, %� is smaller at 20°C (about 14-31

nm) and decreases at higher temperature (about 6-11 nm at 50°C). For DLS

measurements, the conventional determination of the particle size by the fitting of

autocorrelation data assumes that the dispersed particles are spherical. For cylindrical

dispersions, the fitting will yield larger sizes.


50

Figure 4.3: Hydrodynamic diameters of DDAB/Cy/water RM systems at

different temperatures and different w0 measured with DLS. With increasing

temperature, the hydrodynamic diameter of all the RM systems decreases.

This experiment was carried out by the collaborators at S.N. Bose National

Centre for Basic Sciences in India.

4.3 THz-TDS measurements

The frequency dependent absorption coefficients in the frequency range from 0.4 to 1.4

THz (13-45 cm-1) were obtained using a THz-TDS. The detailed descriptions of the

spectrometer and the data analysis are presented in Chapter 2. The samples were placed

in a liquid cell (model A145, Bruker Optics) with z-cut quartz windows and a thickness

of 1 mm. To avoid water vapor absorption, the THz setup is enclosed in a box which is

purged with dry air. All THz-TDS measurements were carried out under thermal

equilibration with humidity at �5 � 0.1� % and temperature at �0.2°C accuracy.

Figure 4.4A shows the absorption coefficient spectra L�� of DDAB/Cy/water RM

systems with different } at 20°C. L�� increases with increasing frequency. With

increasing } , a significant increase of L�� is observed. The integrated absorption

coefficients (� L Z) of the RM systems in the frequency range from 0.4 to 1.4 THz as a

function of temperature are shown in Figure 4.4B. For comparison, � L Z of water in


51

an adjusted scale and � L Z of Cy are also plotted. In this frequency range, � L Z of

water increases with increasing temperature. For Cy, � L Z has negligible small values

(0-0.5 cm-1) and remains almost constant with increasing temperature. For the RM

systems, � L Z increases with increasing temperature for all systems. This indicates the

dominant contribution of the absorption of water for all samples, although the volume

fraction of water is very small. With increasing } , � L Z approaches that of water.

Figure 4.4: THz-TDS spectra of DDAB/Cy/water RM systems at 20°C in

the frequency range from 0.4 to 1.4 THz: (A) absorption coefficient, (B)

integrated absorption coefficient in the frequency range from 0.4 to 1.4 THz

as a function of temperature. The integrated absorption coefficient of pure

water in an adjusted scale and Cy are plotted for comparison.

4.4 FTIR measurements

FTIR spectra were recorded using a VERTEX 80v FTIR spectrometer (Bruker Optics)

at different temperatures under nitrogen gas flow in the sample compartment. The

details of this system are described in Chapter 2. For spectra acquisition in the MIR

region (600-7000 cm-1), the MIR source of the spectrometer and MCT detector were

used. For data analysis, the frequency range from 3000 to 3700 cm-1 was chosen to

investigate the OH stretching modes of water. In the FIR region (20-700 cm-1), a

mercury lamp was used as an FIR source, and a liquid-helium-cooled silicon bolometer

was used for detection. The frequency range from 50 to 650 cm-1 was chosen for data


52

analysis because of its high signal to noise ratio. All samples were measured using a

liquid cell (model A145, Bruker Optics) with a thickness of 52.6 µm (exception: 12.0

µm for pure water). Diamond windows were used for FTIR measurements in FIR region

while z-cut quartz windows were used for MIR measurements. Thermal equilibration at

�0.2°C around the required temperature had to be obtained before experimental data

collection.

Figure 4.5: FTIR spectra of the stock solution (DDAB/Cy/water mixture at

w0=1) in the FIR region (A) and MIR region (B). In the analyzed frequency

regions (marked in orange), the stock solution has negligible absorption.

The strong absorption in the frequency range 2800-3000 cm-1 results from

the CH stretching modes of Cy.

The FTIR spectra of the samples correspond to the difference absorbance spectra

between the measured absorbance of the samples and the measured absorbance of the

stock solution (DDAB/Cy/water system at } � 1). The FTIR spectra in the FIR and

MIR regions of the stock solution are shown in Figure 4.5. In the FIR region, the

absorption of the stock solution is almost negligible. In the MIR region, The CH

stretching modes of Cy are clearly found in the frequency range 2800-3000 cm-1. In the

analyzed MIR region (3000-3700 cm-1), the absorption of the stock solution is

negligible. For the sample at highest water content (} � 12.5), the added volume of

water is only 2% compared to the volume of the stock solution, indicating that the

reduced volume fraction of the stock solution in all investigated RM systems is

negligible. Therefore, the presented FIR and MIR difference absorbance spectra of the


53

RM systems are attributed solely to the partial contribution of water added to the stock

solution.

4.4.1 FIR spectra

Figure 4.6: Difference absorbance spectra of DDAB/Cy/water RM systems

in the FIR region measured with FTIR spectroscopy: (A) spectra of different

w0 values at 20°C, (B) spectra at different temperatures of three

representative RM systems and spectrum of water at 20°C in an adjusted

scale. The inset of (A) shows the relative absorbance intensity between the

libration peak and the intermolecular H-bond stretching peak.

Figure 4.6A shows the FIR difference absorbance spectra of DDAB/Cy/water RM

systems at 20°C. The absorbance significantly increases with increasing } in the entire

investigated frequency range. For all RM systems, there are an absorbance band around

180 cm-1 and a high absorption region above 500 cm-1. This is similar to the absorption

spectrum of water (Figure 4.6B). However, the band around 180 cm-1 is red shifted

compared to that of water which appears at around 200 cm-1. The relative absorbance

intensity between the libration peak (at 600 cm-1) and the intermolecular H-bond

stretching peak (at 180 cm-1) at 20°C is shown in the inset of Figure 4.6A. In water, the

absorbance intensity of the libration peak is 2.4 times higher than that of the

intermolecular H-bond stretching peak. The ratio is relatively higher for the RM

systems. It is 2.9 for } � 10 and increases to 3.4 for } � 2. Figure 4.6B shows the


54

temperature dependent FIR spectra for three representative RM systems. For

comparison, a spectrum of water at 20°C in an adjusted scale was plotted. With

increasing temperature, the absorbance slightly increases in the frequency range below

400 cm-1. In the region of the libration band, the absorbance slightly decreases with

temperature, especially at high } .

4.4.2 MIR spectra

Figure 4.7: Difference absorbance spectra of DDAB/Cy/water RM systems

in the MIR region: (A) spectra of different w0 values at 20°C, (B) spectra at

different temperatures of three representative RM systems. The absorbance

of the RM systems increases with increasing w0 and decreases with

increasing temperature.

Figure 4.7A shows the difference absorbance spectra of DDAB/Cy/water RM systems

at 20°C in the MIR frequency range from 3000 to 3700 cm-1. The difference absorbance

spectra correspond to the absorbance due to the water molecules present in the RM

systems. It is observed that the absorbance strongly increases with increasing } . Figure

4.7B shows the temperature dependent MIR spectra for three representative RM

systems. The absorbance decreases with increasing temperature for all RM systems and

the change is more significant at higher } .


55

Figure 4.8: MIR spectra of DDAB/Cy/water RM systems at w0=2, w0=10,

and bulk water at 20 and 50°C. Each spectrum is deconvoluted into three

sub-bands representing three different types of water in the system: strongly

H-bonded water ν1 (orange dashed curve), distorted structured water ν2

(green dashed curve), and isolated water ν3 (blue dashed curve). The overall

fits are shown in cyan solid curves and the experimental data in black solid

curves. The absorption data of bulk water are adapted from Ref. [88].

In order to understand the change more quantitatively, the MIR spectra of the RM

systems and water are deconvoluted into three distinct Gaussian sub-bands, each of

which defines the population of different water types. Figure 4.8 shows the

representative deconvoluted spectra for } � 2, } � 10, and bulk water at 20 and

50°C. The experimental absorption data of bulk water are adapted from Ref. [88]. There

are different ways to categorize and assign the MIR OH stretching modes of water. For

a quantitative comparison between water in RM and bulk water, the MIR spectrum of

bulk water is deconvoluted to yield three bands centred at 3330 cm-1 (A), 3460 cm-1


56

(�) and 3590 cm-1 (�). These bands correspond to strongly H-bonded, distorted

structured and isolated water molecules, respectively [88, 89]. The spectra of the RM

systems are also deconvoluted into three sub-bands with three fixed peak positions

similar to those of bulk water. With this way, the information on the relative change in

contribution of each sub-bands compared to those of water is obtained. For the RM

system at } � 2, no peak can be found in the region around 3590 cm-1, instead a weak

peak centred at 3400 cm-1 is observed. For the other RM systems, the absorbance

spectra can be fitted well using the peak positions of bulk water.

Figure 4.9: Fraction of area under the curve for the deconvoluted sub-bands

as described in Figure 4.8 as a function of w0 at four temperatures (the

colors blue, green, yellow, orange respectively represent the data at 20, 30,

40, 50°C). ν1 and ν2 represent the sub-bands centred at 3330 and 3460 cm-1.

ν3 represents the sub-band centred at 3400 cm-1 for w0=2 and at 3590 cm-1

for other RM systems. The corresponding values for water at 20 and 50°C

are shown in hollow symbols for comparison.

Figure 4.9 shows the integrated area of each band relative to the total area as a function

of } at four different temperatures. This relative contribution is assumed to be

proportional to the relative abundance of that particular type of water molecules (A, �

or �) present in the systems. For all RM systems and water, the population of isolated


57

water molecules (�) is low (less than 10%) and does not change considerably with

temperature and } . The population of strongly H-bonded water (A) is the most

abundant (about 60%). With increasing temperature, the population of � (distorted

structured water) increases while that of A decreases. A reverse behavior is observed

with increasing } : the population of � decreases while that of A increases. For the

RM systems at low } , A is less abundant and � is more abundant compared to those

of bulk water. The population distribution resembles those of bulk water at } � 7.5.


The observed decrease in the hydrodynamic diameter %� with increasing temperature

(Figure 4.3) of DDAB/Cy/water RM systems is markedly different from the observation

in AOT/hydrocarbon/water RM systems in which %� either remains unchanged or

increases gradually with increasing hydration } and temperature [90, 91]. With

increasing temperature, the droplet size decreases, indicating a transition towards

smaller aggregates. This change in the aggregate size is associated with a corresponding

modification of the elastic property of the surfactant interfacial layer. Using DLS

technique to determine %� of DDAB RM systems is not straightforward as the shapes of

the droplets are not spherical at lower hydration. A previous study of these RM systems

using SANS technique revealed that the system exhibits a cylindrical structure at low

hydration (2 � } � 8) with a length varying from 14 to 20 nm and a radius varying

from 1.5 to 1.6 nm [78]. At increased hydration (} � 10), spherical aggregates with

diameter of about 6 nm are formed. The conventional determination of the size by the

fitting of autocorrelation data assumes that the dispersed particles are spherical. For

cylindrical dispersions the fitting will yield larger sizes. This explains the decrease of

%� of DDAB RM systems with } � 10 compared to that with } � 10 at 20°C.

The investigated frequency range from 0.4 to 1.4 THz of the THz-TDS probes the

intermolecular water network vibrations [30, 92]. In this frequency range, the absorption

coefficient L�� of Cy is negligible (0-0.5 cm-1), while L�� of water is very high (150-

300 cm-1) [93, 94]. This explains the observed increase of L�� with increasing }

(Figure 4.4). These results are in good agreement with the studies of confined water in

water-1,4-dioxane mixtures and water-methanol mixtures [69, 95]. Previous studies of

AOT/heptane/water RM systems using THz-TDS found a large THz absorption

resonance of water confined within the RM which is absent in bulk water [96, 97]. An


58

increase in L�� with increasing } was also observed in these studies. The plots of the

integrated absorption coefficient � L Z in the frequency range from 0.4 to 1.4 THz

show that � L Z increases with increasing temperature for all RM systems. This

behavior is similar to that of water. The absorption contribution of Cy in the RM

systems is negligible as � L Z of Cy is negligible and unchanged with temperature. The

temperature dependent behavior of � L Z of the RM systems is dominated by the THz

absorption of water. With increasing } , � L Z approaches that of water, resulting

from the increasing contribution of water.

In the investigated FIR frequency range, water has two characteristic bands around 200

cm-1 and 600 cm-1 (Figure 4.6B). The band around 200 cm-1 is assigned to the

intermolecular H-bond stretching vibrations, and the one around 600 cm-1 is assigned to

the librational motions [9, 68]. For all studied RM systems, both bands are present

(Figure 4.6A), confirming the presence of an intermolecular water network even at low

} . This agrees well with the anticipated cylindrical structure of the RM at low

hydration. The band arising from the intermolecular H-bond stretching vibrations of

water in RM systems (around 180 cm-1) is significantly red shifted compared to that of

bulk water (around 200 cm-1), especially at low } . This is attributed to a perturbation

of the water H-bonded network by the inhomogeneous H-bonding of water molecules

with the interface. A similar red shift was also observed in AOT RM systems and was

attributed to the weakening of H-bond in the bound water region due to significant

interfacial interaction and high degree of orientation relative to the surface head group

[98, 99]. With increasing hydration, both peak positions show only a very small shift,

indicating the weak dependence of the H-bond network on the surface geometry. In

contrast, a stronger shift of the libration peak was found with increasing hydration in

AOT RM systems, and at } � 40, the peak approaches that of bulk water [99]. In the

present study, the ratio between the absorbance intensity of the libration peak and the

intermolecular H-bond stretching peak of the RM systems at low } is significantly

higher than that of bulk water (inset of Figure 4.6A), showing a modification of the

collective H-bond network. With increasing temperature, the peaks undergo a

progressive red shift (Figure 4.6B). This is due to the weakening of H-bonding resulting

from the large thermal fluctuation upon increasing the temperature [100].

The measurement results of DDAB/Cy/water RM systems in the MIR (Figure 4.9) show

a low fraction of isolated water (�) and a high fraction of strongly H-bonded water

(A). This observation is comparable to the results obtained for anionic AOT RM [101,


59

102], and nonionic RM [103, 104]. The difference in the abundance of different water

types in DDAB RM compared to those of bulk water indicates the inhomogeneity in

bonding of water molecules with the RM interface. At low hydration, water molecules

in contact with the surface are dominating. This can be correlated with the observed

higher abundance of distorted H-bond network water (higher population of �). With

increasing hydration, stronger H-bonds between water molecules start building and the

population of A reaches that of bulk water. Similar structural evaluation of water has

previously been observed in conventional RM systems with increasing } [102, 104].

With increasing temperature, the population of � increases gradually, which is

compensated by the decrease of the population of A. This observation is rather intuitive

as increased thermal energy disrupts a fraction of H-bond water network. The

interconversion of different water types can be correlated with the temperature

dependence of solvent relaxation dynamics.

The experimental results obtained from time resolved fluorescence spectroscopy for the

same RM systems showed that the average solvation time constants of the fluorophore

Coumarin-500 in the RM systems are in the order of a few hundred ps [71]. These

values are about an order of magnitude slower than those of bulk water [105], showing

a retardation of water dynamics in the RM. The slow relaxation dynamics is attributed

to a confinement effect on water dynamics in the RM, similar to what has been

observed in conventional RM systems [90]. In all studied RM systems, the average

solvation time constants decrease with increasing } and temperature, irrespective of

the transition from cylindrical structure (large radius of curvature) to discrete droplet

structure (small radius of curvature). The results also indicated that the bound water

layer at the DDAB interface decreases with increasing water concentration, similar to

the observed behaviors in conventional spherical AOT RM systems [90]. This explains

the accelerated solvation dynamics with increasing } in spite of the decreasing size.

These observations indicate that it is the load of water rather than the surface geometry

determines the water structure and dynamics.

The DDAB/Cy/water RM systems undergo a transition from connected cylindrical

structures at low } to discrete spherical droplets at high } . The structural change

directly results in modifications of the elastic properties and curvature of the surfactant

monolayer [106]. These RM systems offer a wide range of surface geometries at

different } and temperatures. Both THz-TDS and FIR-FTIR studies showed that the

collective H-bond network in water adopts a bulk like configuration with increasing }


60

and temperature. MIR-FTIR measurements found that the population of strongly H-

bonded water molecules (A) increases, while that of the distorted structured water

molecules (�) decreases with increasing } to reach a value comparable to those of

bulk water. In summary, this study reveals a significant change of the water dynamics

with changes in hydration and temperature. However, these changes are independent of

the topological curvature.

61

5 Water in organic solvents

In this chapter, the structure, dynamics and activity of water in the mixtures of water

and 1,4-dioxane (Dx) were investigated using FTIR spectroscopy, THz-TDS, and

kinetics of the solvolysis reactions of benzoyl chloride (BzCl). Dx can expose non-

interacting hydrophobic sites to water molecules as well as form H-bonds with water. In

water-Dx mixtures with low water content, the water-water H-bond network is

disrupted, which resembles the conditions of isolated or confined water molecules with

fewer interactions to other water molecules. The evolution of water H-bond collective

network was studied as the mole fraction of water increased. The presented results are

partially published in Ref. [69].

5.1 Water confined in 1,4-dioxane

The attractive H-bond interactions in water lead to its unique properties, which makes

water an essential partner in a wide range of chemical and biological processes [7, 107].

In hydrophobic environments, water forms clathrate clusters of 2-10 water molecules, in

which entropy and van der Waals forces are thought to play a crucial role [108, 109].

The interaction of polar water molecules with a hydrophobic surface influences the

dynamics of water molecules. H-bond dynamics in bulk water and in water near

hydrophobic molecules have been intensively studied with different techniques, such as

vibrational spectroscopy [110], Raman spectroscopy [111], and MD simulations [112].

However, very little is known about the cooperativity and microheterogeneity in mixed

solvents on a molecular level.

Dx (C4H8O2) is a colorless heterocyclic organic compound. In addition to the most

popular isomer Dx, there are two further rare isomers: 1,2-dioxane and 1,3-dioxane. Dx

is centrosymmetric and has a chair conformation (Figure 5.1), which is similar to the

structure of cyclohexane [113]. The oxygen atoms of Dx are Lewis basic, which makes

it capable to dissolve several inorganic compounds. Dx is a liquid at ambient condition

and can be used as a solvent. Dx is nonionic and relatively nonpolar, but can solubilize

water from highly diluted to highly concentrated mixtures and can expose a substantial

5. Water in organic solvents

62

number of non-interacting sites (hydrophobic segments) to water molecules.

Furthermore, Dx molecules are capable of forming H-bonds with water, which are

weaker than the corresponding water-water H-bonds [114]. This water-Dx bond

formation competitively replaces the water-water H-bonds. In water-Dx mixtures with

highly diluted concentration of water, the three-dimensional water-water H-bond

network is disrupted. This condition resembles that of isolated water molecules with

fewer interactions to other water molecules (Figure 5.1). Therefore, water-Dx mixtures

provide an opportunity to study properties of nearly isolated water molecules which

hardly interact with other water molecules.

Figure 5.1: Structure of Dx (left) and schematic of water molecules

confined in Dx solvent (right). Dx is centrosymmetric, has a chair

conformation, and can form H-bonds with water. In water-Dx mixtures with

highly diluted concentration of water, water-Dx H-bonds partially replace

water-water H-bonds, disrupting the three-dimensional water-water H-bond

network and stimulating the formation of isolated water molecules.

Different techniques have been used to study water-Dx mixtures, such as IR

spectroscopy [115, 116], NMR [117], time resolved fluorescence spectroscopy [118],

and dielectric relaxation [119, 120]. A dielectric relaxation study in the frequency range

of 0.1-10 GHz showed that the formation of cyclic water structures was found at high

water concentrations (mole fraction of water X� Z 0.83) [120]. However, a more recent

study using a wider range of frequencies reported the cooperative network relaxation at

very low water concentrations [119]. A near infrared study identified three different

species of water molecules in Dx and suggested that these form 0, 1 and 2 H-bonds


63

[116]. A recent femtosecond resolved fluorescence spectroscopic study reported very

fast dynamics of water molecules in small water clusters at low X� and slower water

dynamics for higher water concentrations (X� � 0.2) [118]. Using dynamic light

scattering technique, this study also observed a gradual increase of the water cluster size

with increasing X�. The structure and H-bonds of water molecules have been studied

using IR spectroscopy. Symmetric and asymmetric OH stretching bands of water

molecules are found in the frequency range from 3000 to 3700 cm-1 [121]. Coupled

bending and librational motions are characterized around 2100 cm-1, and bending modes

around 1650 cm-1 [9]. Further bands of water in the FIR region are found around 600

cm-1 (libration) and 200 cm-1 (intermolecular hydrogen bond stretching vibrations) [9,

122]. Recently, a combined THz spectroscopy and MD simulation study reported the

collective nature and delocalized character of these low frequency modes of water

which involve correlated particle motions extending several hydration shells [123].

In this study, different concentrations of water in water-Dx mixtures were measured.

The formation of water network upon increasing of water contents in water-Dx mixtures

was studied using FTIR spectroscopy in the FIR and MIR regions. The slow and fast

water dynamics were investigated by using dielectric relaxation data obtained from

THz-TDS measurements. The activity of water is determined by measuring the kinetics

of the solvolysis reactions of BzCl. By investigating the evolution from weakly to

strongly bonded water cluster in Dx, this study aimed to find explanations to three

different aspects: how the water structure changes with X�, how this change in the

structure is related to the water dynamics, and how these changes in dynamics affect the

activity of water. Dx and BzCl were purchased from Sigma Aldrich at 99% or higher

purity and were used as received. Water-Dx mixtures were prepared by dissolving water

in Dx. Water concentration is expressed in terms of mole fraction of water (X�).

5.2 FTIR spectra

FTIR spectra were measured using a VERTEX 80v FTIR spectrometer (Bruker Optics).

The details of this system are described in Chapter 2. All measurements were carried

out at �20 � 0.2�°C under nitrogen gas flow in the sample compartment. The liquid

samples were placed in a liquid cell (model A145 of Bruker Optics). Diamond windows

were used for FTIR measurements in the FIR region while z-cut quartz windows were


64

used for MIR-FTIR. For spectra acquisition in the MIR region (600-7000 cm-1), a built-

in MIR source of the spectrometer and an MCT detector were used. A cell thickness of

52.6 µm was used for X� ranging from 0 to 0.19 and a cell thickness of 28.5 µm for X�

ranging from 0.26 to 0.54. The measured absorbance spectra were then adjusted with

the corresponding cell thickness. For data analysis, the frequency range from 3000 to

3700 cm-1 was chosen to investigate the OH stretching modes of water. In the FIR

region (20-700 cm-1), a mercury lamp served as an FIR source, a liquid-helium-cooled

silicon bolometer was used as a detector. The cell thickness was 52.6 µm for all water-

Dx mixtures and 12.0 µm for pure water. For data analysis, the frequency range from 50

to 650 cm-1 was chosen because of its high signal to noise ratio.

Figure 5.2: FTIR spectra of Dx in the FIR (A) and MIR region (B). The

analyzed regions are marked in orange. In the FIR region, Dx has two strong

absorption peaks at 280 and 610 cm-1. In the MIR region, the CH stretching

modes are clearly shown in the frequency range 2800-3000 cm-1. In the

analyzed MIR region (3000-3700 cm-1), Dx has negligible absorption.

The FTIR spectra of pure Dx in the FIR and MIR regions are shown in Figure 5.2. The

oscillation of the spectra results from the etalon effect of the liquid cell. In analyzed FIR

region (50-650 cm-1), the absorption of Dx is almost negligible, except for two strong

absorption peaks around 280 and 610 cm-1. In the MIR region, The CH stretching

modes of Dx are clearly shown in the frequency range 2800-3000 cm-1. In the analyzed

MIR region (3000-3700 cm-1), Dx has negligible absorption. In order to extract

structural information, the difference between the absorbance spectra of water-Dx


65

mixtures and the absorption spectrum of pure Dx were calculated. This helps to remove

the etalon effect of the spectra. At the regions around 280 and 610 cm-1 where Dx has

strong absorption peaks, the resulting difference absorbance spectra were smoothed to

ensure that they follow a continuous trend of the spectra before and after these regions.

The obtained difference absorbance spectra are attributed solely to the partial

contribution of water in water-Dx mixtures. In the following parts, all presented spectra

of water-Dx mixtures are the difference absorbance spectra.

Figure 5.3: Difference absorbance spectra of water-Dx mixtures in the FIR

region with Xw of 0.005, 0.02, 0.05, 0.11, 0.19, 0.32, 0.42, 0.49 and 0.54

(from bottom to top). The spectrum of water (dashed line) in an adjusted

scale is shown for comparison. The dotted lines are the deconvoluted

spectra of the water spectrum with two peaks centred at 200 and 600 cm-1.

The arrow marks the progressive blue shift of the libration peak with

increasing water content. The inset shows relative absorbance compared to

water for the intermolecular H-bond stretching peak and the libration peak.

Figure 5.3 shows the FIR difference absorbance spectra of water-Dx mixtures and the

spectrum of bulk water in an adjusted scale. The spectrum of bulk water was

deconvoluted into two separate peaks centred at 200 and 600 cm-1, representing two

characteristic peaks around these regions. The peak around 200 cm-1 is assigned to the


66

intermolecular H-bond stretching vibrations, while the one around 600 cm-1 is assigned

to the librational motions [9, 68]. For water-Dx mixtures at low water concentrations

(X� � 0.1), an absorption peak centred at 450 cm-1 (libration peak) is found while

almost no peak can be seen around 200 cm-1. With increasing X�, the libration peak

shows a progressive blue shift, and for X� � 0.54, the spectrum has the libration peak

near 600 cm-1, similar to that of bulk water. The inset of Figure 5.3 shows the relative

absorbances of water-Dx mixtures (measured at the peak) compared to that of bulk

water (measured at the peak) in the region of the intermolecular stretching and libration

peaks. Both of the relative absorbances increase with increasing X� and the change is

more significant when X� exceeds 0.1. The relative absorbance of the libration peak is

always higher than that of the intermolecular H-bond stretching peak.

Figure 5.4: (A) MIR difference absorbance spectra of water-Dx mixtures

and water. The data of water are adapted from Ref. [9] and plotted in an

adjusted scale. The spectra of water-Dx mixtures are blue shifted compared

to the spectrum of water. (B) Deconvoluted spectra of water in MIR region

with two Gaussian sub-bands centred at 3470 cm-1 and 3280 cm-1.

The MIR difference absorption spectra of the water-Dx mixtures in the frequency range

of 3000-3700 cm-1 are shown in Figure 5.4A. The spectrum of bulk water in an adjusted

scale is also plotted for comparison. The absorbance data of bulk water is adapted from

a previous study [9]. This region probes the intramolecular OH stretching modes of

water in the samples. Therefore, the absorbance intensity is roughly proportional to

water content. At low X�, the MIR spectra are clearly blue shifted compared to the


67

spectrum of water. With increasing X�, the MIR spectra get higher and also closer to

the water spectrum. There are different ways to categorize and assign the MIR OH

stretching modes of water. In water-Dx mixtures, water molecules also participate in H-

bonds with Dx, giving rise to several OH stretching modes in the MIR region. As a

result, the partial contribution of bulk-like OH stretches decreases. For a quantitative

comparison between water in water-Dx mixtures and bulk water, the MIR spectrum of

bulk water is deconvoluted into two Gaussian sub-bands centred at 3470 and 3280 cm-1

with comparable intensity as shown in Figure 5.4B. These bands are respectively

assigned to the OH stretch of water arising from weak and strong H-bond

conformations.

Figure 5.5: MIR difference absorbance spectra of four representative water-

DX mixtures and their deconvoluted Gaussian sub-bands. The spectra with

Xw of 0.005 (A) and Xw of 0.05 (B) are deconvoluted into three different

bands centred at 3585, 3505 and 3470 cm-1. For Xw of 0.19 (C) and 0.49

(D), the spectra are deconvoluted into four different bands with one more

band centred at 3280 cm-1.


68

In order to achieve a more quantitative understanding, the spectra of water-Dx mixtures

are deconvoluted into Gaussian sub-bands. At low water concentration (X� � 0.1), the

MIR spectra are properly decomposed into three bands centred at 3585, 3505 and 3470

cm-1 (Figure 5.5A, B). At higher water concentrations (X� � 0.1), the spectra can only

be fitted with an additional band centred at 3280 cm-1 which arises from bulk-like water

having strong H-bonds with the nearby water molecules (Figure 5.5C, D). The band at

3470 cm-1 is assigned to an OH stretch of water molecules which have weak H-bond to

the nearby water molecules. The bands at 3585 and 3505 cm-1 are attributed to

vibrational bands of water molecules that share H-bonds with Dx.

Figure 5.6: Fraction of area under the curve for the deconvoluted bands

centred at 3585, 3505, 3470 and 3280 cm-1 as described in Figure 5.5. A

significant change is observed when Xw exceeds 0.1.

Figure 5.6 shows the partial contributions of the deconvoluted bands centred at 3585,

3505, 3470 and 3280 cm-1 as a function of X�. The contributions are represented by the

respective areas of each curve. This plot correlates the relative population of different

water species present in water-Dx mixtures. The contribution of the band at 3585 cm-1

decreases first slowly and then rapidly with increasing X� while a reverse behavior is

observed for the band at 3470 cm-1. For X� � 0.1, the band at 3280 cm-1 appears and its

contribution grows gradually with X�. The contribution of the band at 3505 cm-1 starts

to decrease at X� � 0.1 and is very low at higher X�.


69

5.3 THz-TDS spectra

The dynamics of water in water-Dx mixtures was investigated with THz-TDS in the

frequency range from 0.4 to 1.4 THz (13-45 cm-1). The details of this system are

described in Chapter 2. All THz measurements were carried out under thermal

equilibration with humidity at �5 � 0.1�% and temperature at �20 � 0.2�°C. The liquid

samples were placed in a liquid cell (model A145 of Bruker Optics). Because of high

water absorption in the THz frequency range, the cell thicknesses were varied

depending on water contents in water-Dx mixtures to guarantee the penetration of the

THz pulse. The cell thickness of 1.54 mm was used for X� ranging from 0 to 0.05, 1.03

mm for X� from 0.023 to 0.26, and 0.21 mm for X� from 0.11 to 0.54. The overlaps of

X� range in different cell thicknesses serve as a calibration tool to ensure correct

spectral comparison for the entire concentration range.

Figure 5.7: Spectra of water and Dx measured with THz-TDS: (A) index of

refraction, (B) absorption coefficient, (C) real dielectric constant, (D)

imaginary dielectric constant. The values of all these parameters of Dx are

significantly lower than those of water in the entire frequency range.


70

From a measurement with THz-TDS, the electric field of the THz beam as a function of

time �� is recorded. A fast Fourier transformation of �� yields the frequency

dependent power and phase of the transmitted pulse. Subsequently, the frequency

dependent index of refraction (�), absorption coefficient (L), real dielectric constant (��) and imaginary dielectric constant (��) can be deduced [28]. Figure 5.7 shows the THz

spectra of these four values of pure water and pure Dx in the representative frequency

range from 0.5 to 1.1 THz. All spectra of Dx are significantly below the corresponding

ones of water. The absorption coefficient L (Figure 5.7B) and the imaginary dielectric

constant �� of Dx (Figure 5.7D) are almost negligible.

Figure 5.8: Frequency and concentration dependent index of refraction n

(A) and absorption coefficient α (B) of water-Dx mixtures. Both n and α

increase with increasing water contents. (C) and (D) show the measured and

calculated (for ideal mixtures) n and α for water-Dx mixtures as a function

of Xw at the frequency of 1 THz. A deviation between the measured and

calculated data is observed.


71

Figure 5.8A shows the frequency dependent index of refraction �� and Figure 5.8B

shows the frequency dependent absorption coefficient L�� of water-Dx mixtures in the

frequency range from 0.4 to 1.4 THz. As observed from the figure, �� decreases

while L�� increases with increasing frequency. Both �� and L�� increases with

increasing water content. In order to check if the change of �� and L�� is only by the

difference in water contents or also by other effects, it is necessary to compare the

measured data with the calculated data when assuming that the mixtures are ideal ones

(no enthalpy or volume change upon mixing). For an ideal mixture of two components,

the index of refraction and the absorption coefficient can be calculated using the

following relation [95]:

�)K�� )K�� ~�A�A�� (5.1)

where � is the index of refraction or the absorption coefficient, � is the volume fraction,

the indices 1 and 2 represent the components 1 and 2 in the mixture, �� is the density

of the real mixture, �)K�� is the density of mixture with assumption that the components

behave ideally when being mixed to form the mixture. The ratio ��/�)K�� is included

to account for small non-idealities in the mixture volume upon mixing. Figure 5.8C

shows the measured and calculated �� and Figure 5.8D shows the measured and

calculated L�� of water-Dx mixtures at different X� values at the frequency of 1 THz.

A considerable deviation between the measured and calculated data is observed.

Further to the obtained �� and L��, dynamic properties of a liquid can be deduced

by analyzing its dielectric relaxation. One of the most commonly used models to

describe dielectric relaxation is the Debye model. This model describes the dynamics in

terms of collective, diffusive, re-orientational motions and has extensively been used for

pure liquids and liquid mixtures [28, 124]. The Debye model describes the dielectric

relaxation as follows:

�̂�� - � < �� $ ��,A1 � R2��:�BA (5.2)

where, is the frequency, �̂�� is the frequency dependent complex dielectric constant, �A � �! is the static dielectric constant, �� is dielectric constant for the �-th relaxation


72

process, �:,A � �- is the extrapolated value for high frequency, �� is the relaxation time

of the �-th process, and � describes the number of relaxation processes which are taken

into account. The Debye model with � � 1 is the simplest case which assumes that a

single relaxation time provides an adequate description. The magnitudes of the induced polarizations are given by the dispersion amplitudes S� � �� $ ��,A. In most of the

cases, the dielectric spectra can be adequately fitted by two independent relaxation

processes (double Debye relaxation fitting with � � 2) with the equation:

�̂�� - � �! $ ��1 � R2�A � �� $ �-1 � R2�� (5.3)

Table 5.1: Double Debye relaxation fitting parameters for water-Dx

mixtures at different water contents.

X� �- �! �� A �ps� �� ps� SA S�

0.005 2.12 2.24 2.20 0.573 0.086 0.04 0.08

0.009 2.12 2.27 2.21 0.793 0.090 0.06 0.09

0.014 2.12 2.30 2.21 1.01 0.090 0.09 0.10

0.019 2.12 2.34 2.22 1.42 0.10 0.12 0.10

0.023 2.12 2.37 2.22 1.72 0.10 0.15 0.10

0.034 2.13 2.46 2.24 1.83 0.11 0.22 0.11

0.05 2.13 2.54 2.24 1.95 0.11 0.29 0.11

0.07 2.13 2.67 2.26 2.19 0.11 0.41 0.13

0.11 2.13 2.98 2.30 2.59 0.12 0.68 0.17

0.19 2.13 3.85 2.38 4.05 0.12 1.47 0.25

0.26 2.14 4.45 2.49 5.74 0.12 1.96 0.35

0.32 2.18 5.57 2.58 6.56 0.13 2.99 0.40

0.42 2.21 7.73 2.71 7.39 0.12 5.02 0.50

0.49 2.25 9.71 2.82 7.90 0.13 6.89 0.57

0.54 2.29 11.7 2.92 8.13 0.13 8.78 0.63


73

This double Debye model was used to fit the dielectric constants of water-Dx mixtures

measured with THz-TDS. The fitted parameters are summarized in Table 5.1. With

increasing X�, the high frequency dielectric constant �- is almost constant while the

dielectric constant of the second relaxation process �� increases slightly. The static

dielectric constant �! increases considerably from 2.24 at X� � 0.005 to 11.7 at

X� � 0.54. With increasing water content, the slow relaxation time constant �A exhibits

significant increase while the fast relaxation time constant �� undergoes marginal

change.

Figure 5.9: Frequency dependent dielectric constant spectra of water-Dx

mixtures with the real part (A) and the imaginary part (B). The solid lines

represent the double Debye relaxation fitting. The fitted spectra match well

with the experimental spectra. On the right are the fitted Debye relaxation

time constant (C) and the related relaxation strength (D) as a function of

water contents. An onset occurs when Xw exceeds 0.1.


74

The measured and fitted data of the dielectric constants at different frequencies are

shown in Figure 5.9A for the real part �� and Figure 5.9B for the imaginary part ��. The

fitted spectra match rather well with the experimental spectra, confirming that the

double Debye model is suitable to fit the measured dielectric constants of water-Dx

mixtures. Similar to the index of refraction and the absorption coefficient, �� and �� increase with increasing water contents, which results from the lower �� and �� of Dx

compared to those of water as shown in Figure 5.7. Figure 5.9C shows the change of �A

as a function of X�. At low water contents in water-Dx mixtures (X� � 0.1), �A values

are very small compared to that of bulk water. The values increase from 0.6 to 2.5 ps

when X� increases from 0.005 to 0.1. At higher water contents (X� � 0.1), �A increases

very rapidly and at high concentration of water (X� � 0.54), �A reaches 8.13 ps, which

is comparable to the value of water [28]. Figure 5.9D shows the strength parameter SA

(SA � �! $ ��) which represents the strength of the cooperative relaxation process of the

H-bond network (the process with the time constant �A). Similar to the plot of �A in

Figure 5.9C, the plot of SA also shows an onset of the intensity when X� exceeds 0.1.

5.4 Kinetics of solvolysis reactions of benzoyl chloride

The activity of water in water-Dx mixtures was investigated by measuring the kinetics

of BzCl solvolysis reaction in each mixture. The kinetics of BzCl solvolysis reaction

was determined by the temporal change in the absorbance of BzCl monitored at 288 nm

using a Shimadzu UV-2450 spectrophotometer. The rate of the reaction was calculated

using a first order exponential fit of the absorbance data. The initial BzCl concentration

was kept constant at 10 µM. Kinetic measurements were carried out in a quartz cuvette

of 1 cm path length. The change of the structure and dynamics of water in water-Dx

mixtures possibly influences its reactivity. A solvolysis reaction is a nucleophilic

substitution where the nucleophile is a solvent molecule. The nucleophilic substitutions

proceed by either S 1 or S 2 mechanism. S 1 reaction involves slow bond breaking to

give an intermediate carbocation or an ion pair which can react rapidly with the

nucleophile. S 2 reaction involves concerted bond making and breaking [125]. In this

study, the solvolysis reaction of BzCl with water was used to investigate the water

activity in the mixtures. This experiment was carried out by the collaborators at S.N.

Bose National Centre for Basic Sciences (Kolkata, India).


75

Figure 5.10: Schematic of the solvolysis reaction of BzCl in water-Dx

mixtures. The mechanism of the nucleophilic substitutions is either SN1

(upper path) or SN2 (lower path).

Figure 5.10 shows a schematic of the solvolysis reaction of BzCl in water-Dx mixtures.

The solvolysis reaction of BzCl is an essentially nucleophilic solvent assisted reaction,

in which there is possibly a complicated competition between S 1 and S 2

mechanisms. Depending upon the environment, C-Cl bond breakage and formation of

acylium cation serves as the rate determining step (S 1 mechanism), or nucleophilic

attack of water acts as the rate determining step (S 2 mechanism) [126]. In confined or

microheterogeneous environment the solvolysis reaction of BzCl exhibits an

intermediate mechanism and either of the two mechanisms or both can play important

roles depending upon the environmental condition. A clear separation between SN1 and

SN2 mechanisms of the solvolysis reaction is difficult because of the gradual nature of

the transition when the ionic character of the transition state changes [125].

Figure 5.11 shows the concentration dependent kinetic results of BzCl solvolysis

reaction in water-Dx mixtures. The inset shows the normalized time dependent

absorbance decay of six representative mixtures. As observed from the figure, the

reaction rate is very slow at the low X� region. For X� � 0.1, the rate constant 0 is

about 10'¡ s'A. For X� � 0.1, the reaction rate increases significantly with increasing

water content, and at X� � 0.54, 0 reaches 4.3 · 10'¢ s'A.


76

Figure 5.11: Concentration dependent rate constant of the solvolysis

reaction of BzCl in water-Dx mixtures. The normalized time dependent

absorbance decay is shown in the inset at six different Xw of 0.02 (1), 0.05

(2), 0.11 (3), 0.19 (4), 0.32 (5) and 0.54 (6). This experiment was carried out

by the collaborators at S.N. Bose National Centre for Basic Sciences in

India.


The FIR spectra of water-Dx mixtures show a progressive blue shift of the water

libration peak with increasing water content X� (Figure 5.3). Similar behavior was

previously observed in water-acetone and water-acetonitrile spectra [95]. In this study,

the most pronounced effect for both mixed systems was observed in the samples with

low content of water. For mixtures with X� � 0.47 (about 10% volume of water), the

maximum peak position was found around 500 cm-1. The H-bond character also

influences the shape and position of the spectrum. In a binary aqueous mixture, the

number of water molecules that act as H-bond donors is nearly independent of the

concentration while the number of water molecules that serve as H-bond acceptors

decreases rapidly with decreasing the water content [127]. In the water diluted region,

more and more water molecules become H-bond donors to solvent molecules [95]. For


77

X� � 0.2, only a small fraction of water molecules have H-bonds to more than two

water molecules because water molecules bind preferentially weaker H-bonds with

solvent molecules. An increase in the number of weak H-bonds results in a shift of the

libration peak to lower frequency. A recent simulation study revealed that the low

frequency modes correspond to collective particle motions with a correlation on the

range longer than 7 Å [123]. The absence of the intermolecular collective vibration

mode at 200 cm-1 in the highly water diluted region can be explained by the absence of

water H-bond network. With increasing water concentration to X� � 0.1, a shoulder

starts to appear around 200 cm-1 and the relative absorbance of the intermolecular H-

bond stretching peak starts to increase (inset of Figure 5.3). This indicates an onset of

the H-bond network dynamics.

In the MIR region, the absorbance spectra of water-Dx mixtures are blue shifted

compared to the spectrum of water (Figure 5.4). This behavior agrees well with the

previous studies for water-Dx and water-acetonitrile mixtures which showed a

progressive blue shift of the OH stretching band with decreasing water content for both

mixtures [115]. When the spectra were deconvoluted to investigate the relative

population of different water species present in water-Dx mixtures, the band centred at

3280 cm-1 arising from bulk-like water having strong H-bonds with the nearby water

molecules is not found for mixtures at X� � 0.1 (Figure 5.5). This indicates that weaker

water-Dx H-bonds dominate in these mixtures, which disrupts the three-dimensional

water-water H-bond network. The band at 3470 cm-1 is assigned to an OH stretch of

water molecules which have weak H-bond to the nearby water molecules. A similar

peak was previously found in water-acetonitrile mixtures [128]. The bands at 3585 and

3505 cm-1 are attributed to OH stretching bands of water molecules that share H-bonds

with Dx. H-bonds between water and Dx are weaker than water-water H-bond, which

results in a strengthening of the OH oscillator or a blue shift of the OH stretch [129].

The relatively high intensity of the band at 3585 cm-1 at low X� indicates a high portion

of such weakly bonded water molecules (Figure 5.6). As X� increases from 0.005 to

0.05, the contribution of the 3470 cm-1 band increases with a consequent decrease in the

contribution of the 3585 cm-1 band. At higher water concentrations (X� � 0.1), the

band centred at 3280 cm-1 appears. This shows an onset of the formation of bulk water

clusters. Upon increasing the water content (X� � 0.49), the absorbance spectrum

resembles that of pure water in which the major contribution results from the bands at

3470 and 3280 cm-1.


78

In the frequency range from 0.4 to 1.4 THz measured with THz-TDS, both the index of

refraction �� and the absorption coefficient L�� of Dx is significantly smaller than

the values of water (Figure 5.7). A previous study in the frequency range from 200 MHz

to 3 THz using a combination of frequency domain reflectometry, travelling wave

interferometry, and THz-TDS also yielded the same results [130]. Because of the big

spectral differences between water and Dx, the spectra of their mixtures are expected to

change significantly with changing water contents. This explains the observation that

both �� and L�� of water-Dx mixtures increase with increasing water content

(Figure 5.8). Previous studies with water-acetonitrile mixtures showed an increase in

�� and a decrease in L�� with increasing water content [131] . For water-acetone

and water-methanol mixtures, both �� and L�� were found to increase with water

content [95], which is similar to the behavior of water-Dx mixtures. The calculated data

when assuming the mixtures are ideal are smaller than the measured data for both ��

and L�� in the entire concentration range. This indicates a significant change in the

network by addition of water. Similar results obtained with FTIR spectroscopy and MD

simulations for water-acetonitrile and water-acetone mixtures also yielded a deviation in

absorption between the real and ideal mixtures [95].

The fit with double Debye model for water resulted in two characteristic relaxation

processes, one slow (�A~ 8 ps) arising from the cooperative relaxation of the H-bond

network, and another faster one (��~ 0.1 $ 0.4 ps) resulting possibly from the non-

separable contribution of the few free water molecules [28, 131]. A previous study

showed that pure Dx has no significant contribution at that timescale [130], so the

probed dynamic processes of water-Dx mixtures are mainly from water. In the

investigated THz frequency range, the slower process (�A) is preferentially probed. At

low X�, �A values are very small compared to that of bulk water (Figure 5.9). As this

mode of relaxation is mostly assigned to the cooperative relaxation of H-bond network,

the low �A value indicates a lack of cooperative network in the mixtures. When X�

exceeds 0.1, �A increases very rapidly. This change shows a rapid onset of the collective

H-bond network motions similar to what was observed in a previous study of solvated

model peptides [132]. The behavior of the parameter SA is similar to that of �A. SA

relatively reflects the number of water molecules that participate in the corresponding

Debye process. With increasing X�, more water molecules participate in the H-bond

network. This leads to an increase in the relaxation strength SA or an increase of the

dipole intensity. The observation agrees well with the results of a previous study using


79

time resolved fluorescence [118]. In this study, a fast solvent relaxation was found at

low water content. Furthermore, the water cluster size measured by dynamic light

scattering was found to increase with increasing X�. The static dielectric constant �!,

which is attributed to water cluster formation and the polarity of clustered water, also

has the same behaviors as �A (Table 5.1). The results of FTIR measurements at low

water contents (X� � 0.1) indicated a negligible bulk water contribution in the mixture.

This correlates with a low polarity or low �! values. With increasing X�, the size of

water clusters increases due to the formation of collective H-bond network of water.

This explains the observed increase in the polarity of clustered water which is

represented by an increase in �! with increasing X�. From these results, it can be

concluded that with the growth in the cluster size, the cooperative relaxation dynamics

of water in the mixtures represented by �A increases gradually and reaches the value of

water at high water content (X� � 0.54).

The measurement results of the kinetics of BzCl solvolysis reaction in water-Dx

mixtures (Figure 5.11) show that the observed rate constants at high X� agree well with

the previously reported rate constants observed in similar systems [133]. The observed

rate constant for water-Dx mixture at X� � 0.54 (0 � 4.3 · 10'¢ s'A) is still considerably slower than that for water (0�g¥ � 1.4 s'A at 25°C) [126]. This behavior

indicates a poor reactivity of water in the mixtures. At low water concentrations

(X� � 0.1), bulk water contribution in water-Dx mixtures is negligible as shown in the

FTIR study. This is coupled with the low polarity of water in the clusters as indicated by

low static dielectric constant in the THz-TDS study. The lower polarity due to cluster

formation destabilizes both the nucleofuge (Cl') and the intermediately formed acylium

cation (Bz,), thereby disabling the SN1 mechanism. As evidenced from earlier dynamic

light scattering measurements, the size of water clusters in Dx increases with increasing

X� due to the formation of collective H-bond network of water [118]. This results in an

increase in the polarity of clustered water, which supports the SN1 reaction pathway as it

stabilizes the acylium cation intermediate as well as the nucleofuge. Therefore, the

reaction rate increases considerably as X� increases, especially with X� � 0.1. A

previous study of similar BzCl solvolysis reaction in AOT reverse micelles also put

forward similar arguments [125, 134]. At low water content, the reaction rate was found

to be low and SN1 mechanism was disfavoured while S 2 mechanism was favoured. At

higher water content, the bonding between the hydrogen atoms of interfacial water

molecules and AOT head groups increased, which resulted in an increased


80

nucleophilicity of water inside AOT RM droplets. As a result, SN1 mechanism was

followed and the reaction rate increased. Therefore, the reaction rate of BzCl solvolysis

reaction is defined by the competition between the two reaction mechanisms. Any

change in the structure and dynamics of the water network in the mixtures can induce a

change in the reaction rate. The observed rapid changes in the water network dynamics

at X� � 0.1 correlate directly with the observed significant increase of the reaction rate

of BzCl solvolysis reaction in water-Dx mixtures. A change in the water network, such

as formation of more rigid water-water H-bonds or change in polarity of water clusters

due to cluster formation, will lead to a corresponding change in solvolysis reaction rate.

This study combines three different experimental techniques with the objective to

understand the evolution of water network structure in water-Dx mixtures. FTIR studies

were aimed to unravel the evolution of the H-bond formation by the shift of

intramolecular OH stretching modes and librational modes of water. The results show a

gradual increase of the collective H-bond network with increasing X�. The hetero-

molecular water-Dx H-bonds dominate in the water diluted region of water-Dx

mixtures. With progressive addition of water, intermolecular three-dimensional H-

bonded water network dynamics appear beyond X� � 0.1 and approach those of water

at X� � 0.54. THz-TDS studies explored the dielectric relaxation of water and

indicated changes in the H-bond relaxation dynamics. A fit of a double Debye model to

the measured data revealed the lack of cooperative water network dynamics for low

water contents and a rapid onset of collective network motions was observed when X�

exceeds 0.1. The kinetic study of BzCl solvolysis reaction showed that the rate constant

is very slow at water diluted region, and it increases rapidly at X� � 0.1, indicating a

change in the reaction pathway as the nucleophilic character of water changes in water-

Dx mixtures. In summary, the study shows a direct correlation between the change in

the structure and dynamics of water with the change in the reaction rate upon increasing

water content in water-Dx mixtures. The investigation of water-Dx system serves as a

model system which shows the influence of hydration on the activity and demonstrates

the importance of the solvent for chemical reactivity.

81

6 Water in hydration shells

Water molecules in the immediate vicinity of a protein form a dynamical hydration shell

around it. Water dynamics in the hydration shells is slowed down by the protein. In

some aspects, these hydration water molecules are more confined than bulk water and

their properties are sensitive to the details of the interactions with the protein surface.

This chapter presents the study of thermal unfolding and refolding of human serum

albumin (HSA) in aqueous solutions using circular dichroism (CD) spectroscopy,

fluorescence spectroscopy, THz-TDS, and p-Ge laser THz spectroscopy to investigate

the correlation between structural changes and changes in the hydration dynamics. The

presented results are published as a cover article in Ref. [94].

6.1 Biological water

Water is considered as a lubricant of life, participating in several functions in living

systems. Water molecules can control the shape, function and dynamics of biomolecules

like DNA and proteins [7]. Water molecules in the immediate vicinity of a biomolecule

form a dynamical hydration shell around it. Water properties in this layer are sensitive

to the details of the interactions with the adjacent surface. Water in hydration shells is

called biological water because its dynamics is determined by the structural

organization and perturbation of the biomolecule, and vice versa, the structural integrity

of the biomolecule is governed by the dynamics and H-bonding network of the

biological water [92, 135]. The flexible H-bond network of water enables it to adapt its

structure and dynamics to biomolecules. Water can interact with biomolecules by

donating and accepting H-bonds, by van der Waals interactions, or via electrostatic

steering. This makes water not only a passive solvent, but also an active partner with

vital functions in most biological processes.

The interaction between proteins and their biological water during protein folding

shows a typical example of the interplay between biomolecules and water. Biological

water makes significant contributions to the structure and energy of proteins and

provides a responsive surrounding for conformational changes. Self-assembly of

proteins is controlled by a delicate interplay between hydrophobic and hydrophilic

6. Water in hydration shells

82

interactions [3]. The hydrophobic effect, which results from the unfavourable

interaction between water and the hydrophobic moiety of amino acids of the protein,

plays an essential role in protein folding. Water at protein interfaces can

thermodynamically stabilize the native structure of proteins, affect protein flexibility,

and contribute to molecular recognition in enzyme catalysis. On the other hand, proteins

also influence the structure and dynamics of surrounding water molecules. The

dynamics of biological water is slowed down to match the much slower protein

dynamics [136]. In the hydration shells around proteins, the H- bonds between water

molecules and proteins have longer lifetimes than bulk water.

Figure 6.1: Schematic of a protein (derived from the PDB file 1UBQ) and

surrounding water molecules. Water molecules near the protein surface

form a dynamical hydration shell (darker area). Water in this shell is called

biological water and has different dynamics compared to bulk water.

Figure 6.1 shows a schematic of a protein and water molecules around it. The structure

of the protein is derived from the PDB file 1UBQ [137]. Water molecules within a

certain distance from the protein form a dynamical hydration shell around the protein.

The hydration shells of proteins contains one to some layers of water. The biological

water within the hydration shell can dynamically exchange with bulk water. The

dynamics of protein surface hydration uniquely bridges fast bulk water motions and

slaved protein fluctuations. This function performs important biological roles in


83

maintaining the intact structure as well as the flexibility of proteins and in mediating

enzymatic reactions [138, 139].

The essential role of biological water has been studied using several techniques, such as

X-ray and neutron diffraction [140], NMR spectroscopy [141], IR spectroscopy [92],

fluorescence spectroscopy [10], Mössbauer spectroscopy [142], and molecular

dynamics simulations [143]. Recently, THz spectroscopy has been introduced as an

innovative and highly sensitive method to measure the rapid change in collective water

network motions [144, 145, 146]. THz spectroscopy detects the collective water

network motions on the sub-ps to ps timescale while other techniques probe longer

processes on the ns to µs timescale like NMR spectroscopy, or measure static properties

like X-ray diffraction. THz spectroscopy probes directly the collective intermolecular

vibrations of the H-bond network of water. Therefore, it is able to detect sensitively any

solute induced changes in solvation dynamics of biological water in the hydration

shells.

Previous studies showed that dynamical hydration shells around proteins extend to

several water layers and reach up to 20 Å from the protein surface [147, 148].

Biological water has a retarded dynamics and they oscillate more in phase compared to

bulk water. As a result, the absorption of biological water in THz frequency range

differs significantly from that of bulk water. A recent combined experimental and MD

simulation study showed that the observed changes in the THz absorption of the

solvated protein can be explained by a blue shift of the THz absorption modes of

biological water [68]. This is directly related to a significant retardation of water

dynamics. The study predicted a decrease of the THz absorption of biological water

compared to that of bulk water at frequencies below 1.7 THz, while an increase in

absorption is predicted for frequencies above 1.7 THz. Both predictions were confirmed

by experimental measurements. These results confirm that THz spectroscopy can probe

the structural change of biomolecules during a kinetic process through the change in the

THz absorption of their biological water. Any structural change of biomolecules will

affect the dynamics of their biological water and any change in water dynamics will be

reflected by a change in the THz absorption. The following sections present the results

from the measurements of HSA in aqueous solutions. The thermal unfolding and

refolding HSA was investigated using combined techniques of CD spectroscopy,

fluorescence spectroscopy, THz-TDS, and p-Ge laser THz spectroscopy.


84

6.2 Structure and function of human serum albumin

HSA is the most abundant protein in blood plasma. It is synthesized and secreted in

liver cells. HSA consists of a single polypeptide chain containing 585 amino acids and

has a molecular mass of 66.5 kDa [149, 150]. Under physiological condition the

polypeptide chain of HSA forms a heart shaped three-dimensional structure with 80 Å

on the side, an average thickness of 30 Å, and a volume of 88,250 Å3 [151]. The

structure, which has approximately 67% α-helix and no β-sheet, is stabilized by 17 pairs

of disulfide bonds performed by 34 cysteine residues. Figure 6.2 shows the X-ray

crystallographic structure of HSA which is derived from the PDB file 1AO6 [150]. The

protein has three homologous domains I-III, each consists of two sub-domains A and B.

The sub-domains A and B are connected by flexible loops and contain six and four α-

helices, respectively.

Figure 6.2: Crystal structure of HSA derived from the PDB file 1AO6. The

protein consists of a single polypeptide chain of 585 amino acids. It has a

heart shaped structure with three domains I-III. Each domain has two sub-

domains A and B.

Previous studies revealed that HSA has a high affinity to a great variety of metabolites,

metals, amino acids, fatty acids, drugs and organic compounds in the circulatory system

[149, 150]. The flexible structure of HSA allows substances to bind from a variety of

binding sites and to be buried within the protein. HSA can bind with various compounds


85

and transport them in the bloodstream to their target organs. HSA also binds, transports

drugs and affects drug distributions in the blood plasma. It can control the active

concentration of drugs [152].

HSA represents the major and predominant antioxidant in plasma. The antioxidant

activities of HSA result from its ligand-binding capacities. It can bind with free

transition metal ions, such as Cu2+ and Fe2+ to prevent them from reacting with oxygen

to form reactive oxygen species [153]. Another antioxidant property of HSA results

from the presence of a free sulphydryl group on the reduced cysteine residue (Cys34),

which acts as an important redox regulator in extracellular compartments and can

scavenge hydroxyl radicals. Furthermore, HSA also regulates the pH, osmotic pressure

of plasma, and distribution of fluid between different compartments.

6.3 Thermal unfolding and refolding of human serum albumin

HSA is one of the most popular model proteins used in biological research. Thermal

denaturation of HSA has been studied using different techniques, such as fluorescence

spectroscopy [154], two-dimensional IR spectroscopy [155], dynamic light scattering

(DLS) and CD spectroscopy [156], and Förster resonance energy transfer (FRET) [157].

A study concluded that thermal denaturation of HSA follows multiple steps: from native

to extended and then to unfolded state (N ¨ E ¨ U) [154]. When being heated to 55°C,

the protein changes from native state to extended state, in which domains II and I move

apart while the native conformation is kept almost intact. At 70°C, the protein turns to

unfolded state, which is accompanied by a disruption of secondary and tertiary

structure.

In this section, the change in the coupled protein hydration dynamics of HSA upon

thermal denaturation is presented. Two processes of HSA thermal denaturation are

studied. Process 1: the temperature of the native protein was increased stepwise from

20°C to 55°C, and the system was subsequently cooled to 20°C (N « E transition).

Process 2: the protein is heated to 70°C and then allowed to cool to 20°C (N « U

transition). Both processes were monitored by far- and near-ultraviolet (UV) CD

spectroscopy, and time resolved fluorescence spectroscopy of the intrinsic fluorescence

of the tryptophan (Trp) moiety of HSA. The change in the hydration dynamics upon

thermal denaturation were determined by the change in THz absorption of the protein


86

solutions compared to that of buffer. THz measurements were carried out using both p-

Ge laser in the frequency range of 2.1-2.8 THz and THz-TDS in the frequency range of

0.1-1.2 THz. This study is the first one to investigate the effect of thermal denaturation

of a protein using THz spectroscopy. For all measurements, HSA purchased from

Sigma Aldrich at 99% or higher purity was used. Protein solutions at different

concentrations were prepared by dissolving the protein in phosphate buffered saline

(PBS) at pH 7.4. The temperatures of the samples were controlled by a thermostat.

Thermal equilibration (temperature at � 2°C around the temperature set value) had to be

established before the data were collected.

6.3.1 CD spectroscopy

6.3.1.1 Principle of CD

A plane polarized light, or linearly polarized light, can be considered as a superposition

of opposite circular polarized light of equal amplitude and phase. Figure 6.3A shows

that the summary of the left circularly polarized light (�¬) and the right circularly

polarized light (�) is linearly polarized light (�¬ � �).

Figure 6.3: (A) Linear polarized light viewed as a superposition of opposite

circular polarized light of equal amplitude and phase. (B) Ellipticity and

optical rotation resulted from different absorption of the left and right

polarized components.


87

In an optically active medium the absorbance of left circularly polarized light (�¬) is

different from the absorbance of right circularly polarized light (�). After propagating

through the sample, each component of light is still circularly polarized and the

polarization direction keeps unchanged. However, the radii of the circles traced out by

the electric vector of each component are different. The combination of these two

circularly polarized light waves provides elliptically polarized light because each wave

has a different amplitude. The occurrence of ellipticity is called CD. At the same time,

optical rotation (OR) also occurs. A difference in the index of refraction of the two

circular components causes a rotation of the polarization plane by a small angle L

(Figure 6.3B).

CD is measured as the difference in absorbance between the left circularly polarized

light and the right circularly polarized light [158]:

CD � ∆� � �¬ $ � (6.1)

Most CD measurements are reported in degrees of ellipticity (θ) which can be

calculated with the approximation:

θ � ∆� \p�104 ] \180 ] (6.2)

Biological molecules, such as proteins and nucleic acids, are chiral and show CD in

their UV absorption bands. CD spectra of proteins are usually used to predict

characteristics of their secondary structure. This is based on the fact that isolated α-

helices, β-sheets, and random coils have distinctly different signatures. The CD spectra

of α-helices are characterized by three peaks: a negative peak at 222 nm, a negative

peak of similar intensity at 208 nm, and a stronger positive peak at 192 nm. CD spectra

of β-sheets are characterized by a negative peak near 217 nm and a positive peak near

195 nm. CD spectra of random coils are characterized by a strong negative peak around

200 nm [158].

The CD spectrum of a complex protein can be determined as a sum of its secondary

structures. For huge globular proteins, it is difficult to get relative contents of the

particular secondary structure element. Therefore, the accurate protein conformation


88

cannot be obtained from the CD spectrum. However, CD spectroscopy provides a

powerful tool to qualitatively follow conformational transitions, such as the structural

transition from α-helix or β-sheet to random coil and vice versa. When a protein

solution is heated above a critical temperature, it undergoes a transition from the native

state to the denatured state. In this transition, H-bonds and hydrophobic interactions are

broken, resulting in a modification of secondary structure, which can be observed in a

CD spectrum. The temperature dependent CD signals at 222 nm (CD222), which is a

characteristic wavelength of α-helices, is usually used to check if the protein structure

changes within the temperature range.

6.3.1.2 CD spectrometer

Figure 6.4: Schematic of the CD spectrometer. The linear polarized light is

modulated to left and right circularly polarized light by the modulator. After

transmitted through the sample, the CD spectrum is detected and analyzed.

In this study, CD measurements were performed with a JASCO-815 spectrometer.

Figure 6.4 shows a schematic of the CD spectrometer. The light source is generated by a

xenon lamp. A monochromator and linear polarizer provide monochromatic linear

polarized light by using two crystal prisms with different axial orientation. The linear

polarized light is modulated to left and right circularly polarized light by a modulator.

The modulated light transmits through a sample and is detected by the detector

(photomultiplier). Finally, the data are processed and analyzed by a computer to yield a

CD spectrum. Far-UV CD spectra were measured in the wavelength range from 200 and

255 nm with a protein concentration of 0.05 mg/ml and scan speed of 50 nm/min. The


89

secondary structural data of the CD spectra are analyzed using CDNN software [159].

Near-UV CD measurements were carried out in the wavelength range from 255 to 300

nm with a protein concentration of 2 mg/ml and scan speed of 20 nm/min. Cell

thickness of 1.0 cm was used for all CD measurements. This experiment was carried out

by the collaborators at S.N. Bose National Centre for Basic Sciences (Kolkata, India).

6.3.1.3 CD spectra of HSA

Figure 6.5: CD spectra of HSA in aqueous PBS buffer at different

temperatures: (A) Far-UV spectra at HSA concentration of 0.05 mg/ml, (B)

near-UV at HSA concentration of 2 mg/ml. The thermal unfolding/refolding

process starts with stepwise forward heating from 20 to 70°C for thermal

unfolding (solid lines), and then continues with backward cooling from

70°C to 20°C for refolding (dashed lines). Upon cooling, both far-UV and

near-UV CD spectra are not reversible. This experiment was carried out by

the collaborators at S.N. Bose National Centre for Basic Sciences in India.

The temperature dependent far-UV CD measurements of HSA were carried out to probe

changes in the secondary structure. The protein solution was heated stepwise from 20°C

to 55°C, then it was cooled to 20°C to observe changes in N « E transition from native

to extended state (Process 1). The CD spectra of HSA in this temperature range stay

nearly unchanged as observed from Figure 6.5A for the CD spectra at 20, 40 and 55°C

of the heating stage. This indicates that there is very little change in the secondary


90

structure in the extended state compared to that of the native state. Upon recooling, the

CD spectra are reversible.

Furthermore, N « U transition from native to unfolded state (Process 2) is investigated,

in which the protein solution is heated from 20°C to 70°C and then cooled back to

20°C. The CD spectra for Process 2 are shown in Figure 6.5A. The CD spectra of HSA

have two characteristic α-helix peaks at 208 and 222 nm. As the temperature is

increased, the CD signals at 208 and 222 nm decrease, indicating a decrease of α-helix

content. A drastic change is only observed when the temperature increase from 55 to

70°C at which the protein changes to unfolded state. When the system is cooled down,

the CD spectra stay closer to the one at 70°C compared to the spectra at the same

temperature in the heating stage. The structure of the refolded protein upon cooling is

therefore irreversible and stays similar to the structure of the unfolded state.

Figure 6.6: Temperature dependent change in the CD signal of HSA at the

wavelength of 222 nm (A), and corresponding change in the α-helix content

of the protein (B). The solid lines are guide to the eyes. Thermal unfolding

was induced by heating from 20 to 70°C and then the system was cooled

back to 20°C to allow refolding. The CD signal and the α-helix content are

both irreversible. This experiment was carried out by the collaborators at

S.N. Bose National Centre for Basic Sciences in India.

Figure 6.6A shows the temperature dependent ellipticity at 222 nm. The change in the

ellipticity at this wavelength serves as a probe for the α-helix content which is shown in

Figure 6.6B. For the native state at 20°C, 63.4% of α-helix, 12.6% of β-sheet and 24%


91

of other structural elements (random coils) are obtained from the CD spectrum. With

increasing temperature, the CD signal at 222 nm decreases, indicating a loss of α-helix

content. At 40 and 55°C, α-helix content decreases slightly to 61.5% and 57.8%

respectively with a corresponding increase in content of random coils while β-sheet

content is stable. Upon increasing the temperature to 70°C, a significant change in the

CD signal is observed. The α-helix content decreases to 41.4% (16% β-sheet and 42.6%

of random coils). When being cooled from 70 to 20°C, the protein structure contains

46% α-helix, 15% β-sheet and 39% of random coils. This indicates the irreversibility of

the unfolding/refolding process.

The structural change of HSA for Process 1 and 2 is further studied with near-UV CD

measurements in the wavelength range from 255 to 300 nm. Figure 6.5B shows the

near-UV CD spectra of HSA at different temperatures. The CD spectrum at 20°C has

two distinct peaks at 262 and 268 nm along with a shoulder at 280 nm. Similar to the

results of far-UV measurements, there is very small change in the tertiary structure for

N « E transition (Process 1). The near-UV CD spectra at 20, 40 and 55°C of the

heating stage in Figure 6.5B are nearly the same. Upon cooling from 55°C, the CD

spectra return to the original ones. For the N « U transition (Process 2), the near-UV

CD spectrum at 70°C is significantly changed in the frequency regions around 262 and

268 nm. Upon cooling 20°C, the CD spectrum is not recovered, which shows an

irreversible structural perturbation during the N « U transition.

6.3.2 Fluorescence spectroscopy

6.3.2.1 Principle of fluorescence spectroscopy

The emission of light is divided into two categories: incandescence and luminescence.

Incandescence is the emission of light from a hot body solely because of its

temperature. Luminescence is the emission of light which does not result from heat. It is

called cold body radiation. Fluorescence is a form of luminescence in which a substance

emits light after having absorbed light or other electromagnetic radiation [160].

Molecules have various states which are referred to as energy levels. In general,

molecules have a ground electronic state of low energy (S0) and an excited electronic

state of higher energy (S1). Each of these electronic states has several vibrational states.

At room temperature most molecules occupy the lowest vibrational state of the ground


92

electronic state. Figure 6.7 shows a schematic of a fluorescence process. A molecule

first absorbs a photon, gains more energy and is excited to one of the vibrational states

in the excited electronic state S1. Then the excited molecule collides with other

molecules, loses its vibrational energy until it reaches the lowest vibrational state of the

excited electronic state. After that, the molecule drops to one of the vibrational state of

the ground electronic state S0 and emits a photon. The emitted photons can have

different energies because the molecule can drop to any level of the vibrational states in

the ground electronic state. At the end, the molecule collides with other molecules and

returns to the lowest vibrational state.

Figure 6.7: Schematic of a fluorescence process. The molecule absorbs a

photon and is excited from the ground electronic state S0 to the excited

electronic state S1. When the molecules release energy through collisions

and comes to lowest vibration state of S1, it emits a photon and return to S0.

The fluorescent photon has lower energy than that of the excitation photon.

Fluorophores, which are molecules with fluorescent characteristics, are used in

fluorescence spectroscopy. Several factors influence the fluorescent characteristics of a

fluorophore, such as structure, concentration, temperature, pH of the solution,

environment around the fluorophore. Molecules with aromatic functional groups and

rigid structures fluoresce better than others [160]. Fluorescence spectroscopy is thus

best utilized for compounds with an aromatic structure. The efficiency of the


93

fluorescence process is called quantum yield. It is defined as the ratio of the number of

emitted photons to the number of absorbed photons. The fluorescence lifetime is the

average time the fluorophore stays in the excited state before emitting a photon. It is

typically in the range from 0.5 to 20 ns.

In a fluorescence spectroscopy experiment, the intensity of the emitted fluorescent light

at different wavelengths is measured while the excitation light has a fixed wavelength.

The obtained emission spectrum gives hints on the local environment around the

fluorophore. Another spectrum which can be measured is the excitation spectrum. In

this case, the excitation light is scanned through many different wavelengths while the

wavelength of the emission is fixed. Time resolved fluorescence spectroscopy is an

advance of fluorescence spectroscopy with promising properties for the study of

structure and dynamics. This technique monitors dynamic events occurring within the

environment of a fluorophore in the ps-ns timescale [161]. The fluorescence of a sample

is monitored as a function of time after the sample is excited. This is used to determine

the fluorescence lifetime of a fluorophore. The lifetime depends on the local

environment around the fluorophore.

6.3.2.2 Fluorescence spectrometer

Figure 6.8: Schematic of a fluorescence spectrometer. Excitation

monochromator selects the wavelengths of the light source for excitation

and the emission monochromator select the wavelengths of the fluorescence

for detection. The measured data are processed and analyzed by a computer

to yield a fluorescence spectrum.


94

Figure 6.8 shows a schematic of a fluorescence spectrometer. The excitation source

emits light in the UV frequency range. The excitation wavelength is selected by the

excitation monochromator. The wavelengths of the fluorescence are scanned by the

emission monochromator to detect signals at different wavelengths and yield a

fluorescence spectrum.

In this study, steady state fluorescence emissions were measured using a Horiba Jobin

Yvon Fluorolog spectrometer. For the time resolved measurements, an fs-coupled

TCSPC (time correlated single photon counting) is combined with the spectrometer.

The excitation source has a wavelength of 300 nm. The details of time resolved

measurements can be found elsewhere [162]. This experiment was carried out by the

collaborators at S.N. Bose National Centre for Basic Sciences (Kolkata, India).

6.3.2.3 Fluorescence spectra of HSA

Figure 6.9: Frequency dependent fluorescence spectra of the Trp in HSA at

different temperatures for two thermal transitions: (A) native to extended

state and reverse to refolded state (Process 1), (B) native to unfolded state

and reverse to refolded state (Process 2). Process 1 is reversible while

Process 2 is irreversible. This experiment was carried out by the

collaborators at S.N. Bose National Centre for Basic Sciences in India.

Fluorescence measurements of HSA were measured intrinsic without modification as

HSA has a single Trp moiety in domain IIA at the 214 position. Trp has an absorption


95

peak around 280 nm. The excitation wavelength at 300 nm was chosen in order to avoid

fluorescence from other amino acids. The emission fluorescence spectra of HSA for

Process 1 (N « E transition) and Process 2 (N « U transition) were shown in Figure

6.9. The protein shows a single emission maximum at about 335 nm at 20°C and the

intensity decreases with increasing temperature. The observed quenching of the

fluorescence intensity is attributed to a decreased quantum yield of Trp in its immediate

environment. For Process 1, the emission maximum position stays almost unchanged

when the protein is heated from native state at 20°C to extended state at 55°C as shown

in Figure 6.9A. Upon cooling to 20°C, the fluorescence spectrum becomes almost the

same as the spectrum of the native state. For Process 2, a different behavior is observed

as shown in Figure 6.9B. When the temperature is increased to 70°C (unfolded state), a

slight blue shift of the emission maximum position is found. Upon subsequent cooling

to 20°C, the obtained fluorescence spectrum of the refolded state is significantly

different from the spectrum of the native state.

Figure 6.10: (A) Time dependent fluorescence spectra of the Trp in HSA of

the native state (20°C), unfolded state (70°C), and refolded state (20°C) of

Process 2. The solid lines represent triexponential fits of the curves. (B)

Average lifetime of Trp in HSA at different temperatures during thermal

unfolding and refolding for Process 1 and 2. The results show a reversible

behavior for Process 1 and an irreversible behavior for Process 2. This

experiment was carried out by the collaborators at S.N. Bose National

Centre for Basic Sciences in India.


96

Further to the frequency dependent spectra, the time resolved fluorescence spectra of

HSA were measured. Figure 6.10A shows three representative time dependent

fluorescence spectra of Trp in HSA for the native state at 20°C, unfolded state at 70°C,

and refolded state at 20°C for Process 2. The significant difference between the

spectrum of the refolded state and that of the native state marks the irreversibility of this

N « U transition. The similar measured results for Process 1 show a reversible N « E

transition. These observations match with those found in the frequency dependent

fluorescence measurements.

For the time resolved fluorescence transients of Trp in HSA, a triexponential fit is a

suitable model which has been frequently applied to the data to yield three lifetimes of

the fluorescence [156, 163]. The fluorescence transients u�� are fitted by using a

nonlinear least square fitting procedure with following triexponential equation:

u�� u¯ � < �)�' +°±�

)BA

(6.3)

where u¯ is the background, �) is the pre-exponential factors, and �) is the characteristic

lifetimes. The relative concentrations v) of the corresponding lifetimes �) in a

triexponential decay are given as:

v) � �)�A � �� (6.4)

The average lifetime of a fluorescence transient � � Z is defined as:

� � Z� < v)�)�

)BA

(6.5)

The fitted parameters of Process 1 and 2 are given in Table 6.1. For the native state at

20°C, the three fitted lifetimes are 0.5, 3.3 and 7.0 ns. The longest component has the

most contribution, which deduces an average lifetime � � Z of 5.2 ns. This average

lifetime represents the decay dynamics of Trp in HSA. Figure 6.10B shows the

temperature dependent average lifetimes for both Process 1 and 2. With increasing


97

temperature, � � Z decreases continuously, indicating a thermal perturbation of the

secondary and tertiary structure of the protein. The N « E transition is reversible as

� � Z of the refolding protein has similar value as that of the native protein. However,

the N « U transition is irreversible. For the unfolded state at 70°C, � � Z decreases

about 3 times compared to the value at 20°C. Upon cooling, � � Z of the refolded

protein is still faster than � � Z of the native protein, which means that only a part of

the native structure is recovered.

Table 6.1: Fitting parameters of the fluorescence transients of Trp in HSA

for Process 1 and 2. The unit for all lifetimes τ is ns. This experiment was

carried out by the collaborators at S.N. Bose National Centre for Basic

Sciences in India.

o �°C� vA �A v� �� v� �� Z

Process 1: 20°C ¨ 55°C ¨ 20°C

20 0.15 0.50 0.21 3.31 0.64 7.00 5.23

40 0.22 0.34 0.26 2.34 0.52 5.85 3.76

55 0.21 0.25 0.44 2.30 0.35 5.50 2.99

40 0.19 0.30 0.30 2.40 0.51 5.99 3.82

20 0.12 0.45 0.28 3.21 0.60 7.22 5.26

Process 2: 20°C ¨ 70°C ¨ 20°C

20 0.15 0.50 0.21 3.31 0.64 7.00 5.23

40 0.22 0.34 0.26 2.34 0.52 5.85 3.76

55 0.21 0.25 0.44 2.30 0.35 5.50 2.99

70 0.24 0.15 0.48 1.18 0.28 3.76 1.66

55 0.35 0.14 0.38 1.57 0.27 4.52 1.88

40 0.37 0.14 0.31 1.59 0.32 4.90 2.12

20 0.38 0.12 0.26 1.58 0.36 5.30 2.35


98

6.3.3 THz-TDS measurements

THz-TDS was used to probe the frequency dependent index of refraction and absorption

coefficient in the frequency range from 0.1 to 1.2 THz (3-40 cm-1). The detailed

descriptions of the spectrometers and the data analysis are presented in Chapter 2. For

all THz measurements, a standard liquid sample cell (model A145 of Bruker Optics)

with z-cut quartz windows and a Teflon spacer was used. The layer thickness of the

samples, which was placed between the two parallel quartz windows, was fixed at

�52.6 � 0.3� �m. The whole THz setup is enclosed in a chamber purged with dry air,

keeping the humidity at �5 � 0.1� % to reduce water vapor absorption.

Figure 6.11: THz-TDS spectra of PBS buffer (pH 7.4) and 1 mM HSA in

PBS buffer at 20°C and 70°C in the frequency range from 0.1 to 1.2 THz:

(A) index of refraction, (B) absorption coefficient. The inset plots the

change in average absorption coefficient over the entire studied frequency

range of 1 mM HSA solution compared to that of the buffer for Process 2.

THz-TDS measurements of 1mM HSA in PBS buffer were carried out for Process 1 and

2 to deduce the frequency dependent index of refraction �� and the frequency

dependent absorption coefficient L��. Similar to the observations of CD and

fluorescence measurements, the unfolding and refolding process is found to be

reversible for the N « E transition in Process 1 and irreversible for the N « U transition

in Process 2. Figure 6.11 shows the representative spectra of Process 2 for the buffer at

20°C and 70°C, and for protein solution at the native state (20°C), unfolded state (70°C)


99

and refolded state (20°C). At 20°C, L�� of the buffer increases from 80 cm-1 to 240

cm-1 in the investigated frequency range while �� decreases with increasing

frequency and reaches about 2.0 at 1 THz. When the temperature is increased to 70°C,

both �� and L�� of the buffer increase. The absorption coefficient L�� changes

from 90 to 350 cm-1 in the frequency range. The spectra of HSA solution are slightly

different from those of buffer. At the same temperature, �� of HSA is higher while

L�� of HSA is lower than the corresponding values of buffer. Both �� and L�� of

the refolded state do not resemble the spectra of the native state.

The inset of Figure 6.11 shows ∆L, which is the change in average absorption

coefficient over the studied frequency range from 0.1 to 1.2 THz of 1 mM HSA solution

compared to that of the buffer for Process 2. ∆L is calculated as:

∆L � ∑ L!�)�.)BA 0 $ ∑ L �)�.)BA 0 (6.6)

where 0 is the number of the measured data points ) of a absorption coefficient

spectrum in the studied frequency range, L! and L are the absorption coefficient of the

protein solution and buffer, respectively. Upon heating, ∆L decreases nearly linearly

with increasing temperature. It is also observed that the refolding process (cooling) is

not reversible as ∆L of the refolded protein is lower than ∆L of the native protein.

6.3.4 p-Ge laser measurements

In addition to the frequency dependent spectra in the frequency range from 0.1 to 1.2

THz probed by THz-TDS, the integrated absorption of protein solutions relative to

buffer in the frequency range from 2.1 to 2.8 THz (70-93 cm-1) was recorded using a p-

Ge laser spectrometer [20]. Different HSA concentrations from 0 to 1.4 mM at four

temperatures (20, 40, 55 and 70°C) were studied. Furthermore, the thermal unfolding

and refolding of 1mM HSA solution in Process 1 and 2 were investigated. Two liquid

cells similar to the one used in THz-TDS were used for all measurements. A schematic

of the p-Ge laser spectrometer is shown in Figure 6.12. The THz radiation emitted from

the p-Ge laser is split by a chopper into two beams. Both are transmitted through two

measurement cells with the same path length (sample cell and reference cell). The

beams are recombined by a second chopper and focused on the detector by a

polyethylene lens. The temperature of the cells is monitored by an external temperature


100

controller. The whole setup is enclosed in a chamber which is continuously purged with

dry air to keep the humidity at �5 � 0.1� % during the measurements.

Figure 6.12: Schematic of the p-Ge laser spectrometer. The THz beam

emitted from the p-Ge laser is split by a chopper. One part probes the

sample absorption and the other probes the reference absorption. Both

beams are recombined by a second chopper and focused on the detector.

The p-Ge measures the integrated THz absorption coefficient averaged over the

frequency range from 2.1 to 2.8 THz where the laser has highest intensity. For each

measurement, 30000 waveforms for each channel are recorded and averaged, which

results in a high accuracy of the measured data. The integrated absorption coefficient L

of a measured sample is obtained by the Lambert‐Beer’s law with following equation:

L � ln� $ ln�!% (6.7)

where % is the thickness of the liquid cell, � is the laser intensity after passing the

empty cell, �! is the laser intensity after passing the filled sample cell.


101

The integrated THz absorption of HSA solutions were measured for different HSA

concentrations (0-1.4 mM) at four temperatures (20, 40, 55 and 70°C). The absorption

coefficient of the protein solution can be calculated according to a two component

model, in which it is assumed that there are only buffer and protein in the solution:

L! � L � �! � L" �"�! (6.8)

where L!, L , L" are the absorption coefficient of the protein solution, the buffer, the

protein, respectively, and �!, � , �", are the total volume of the protein solution, the

volume of the buffer, the volume of the protein, respectively. The water displacement or

change in the absorption coefficient of the protein solution relative to buffer (L��+)¸�)

can be calculated with following equation:

L��+)¸� � L! $ L L � � �! � L"L · �"�! $ 1 (6.9)

Figure 6.13: Concentration dependent THz absorption relative to buffer of

HSA solutions at four different temperatures measured with p-Ge laser in

the frequency range from 2.1 to 2.8 THz. The dashed line indicates the two

component water displacement model. The inset shows temperature

dependent absorption coefficient of water measured with the same system.


102

HSA in aqueous solution can be assumed as a sphere with a radius of gyration of

approximately 3 nm [151, 164]. In the studied frequency range from 2.1 to 2.8 THz, the

absorption of proteins and other biomolecules in solution is much less than the

absorption of buffer, which is similar to that of water [14, 144]. The absorption

coefficient of proteins is less than 40 cm'A while that of water is about 420 cm'A at

20°C and increases nearly linearly with increasing temperature as shown in the inset of

Figure 6.13. For simplicity, L" is assumed to be 20 cm'A. From these assumptions,

L��+)¸� values of each protein concentration can be deduced using Equation (6.9).

With increasing protein concentration, the volume fraction of protein in the solution

increases linearly, resulting in a linear decrease of L��+)¸� as shown in Figure 6.13

(dashed line). The experimental results of HSA solutions are also plotted in Figure

6.13. For all temperatures, the measured L��+)¸� of the solvated protein decreases with

increasing protein concentration. With increasing temperature, a decrease in the

measured L��+)¸� is observed. For all temperatures, the concentration dependent

L��+)¸� of HSA is clearly different from the predicted L��+)¸� (water displacement).

Figure 6.14: Temperature dependent THz absorption relative to buffer of 1

mM HSA solution for Process 1 (A) and Process 2 (B). For Process 1, the

absorption of the refolded protein is similar to that of the native protein. In

contrast, the absorption of the refolded protein is significantly higher than

that of the native protein for Process 2.

Figure 6.14 shows the change in L��+)¸� of 1 mM HSA solution for Process 1 and 2.

The N « E transition in Process 1 (Figure 6.14A) is reversible as L��+)¸� of the


103

refolded protein is almost the same as L��+)¸� of the native protein. However, the

N « U transition in Process 2 (Figure 6.14B) shows a different behavior. It is observed

that L��+)¸� of the refolded protein is significantly higher than that of the native one.


The structural contents of the native HSA at 20°C obtained from far-UV CD

measurements (Figure 6.6B), including 63.4% of α-helix (12.6% of β-sheet and 24% of

random coils), are in good agreement with previously studies [165, 166]. With

increasing temperature, α-helix content decreases slightly up to 55°C and significantly

at 70°C. At 70°C, the α-helix content is 41.4%, which agrees well with the result of

33.6% α-helix content of HSA at 75°C reported earlier [167]. The drastic change in the

α-helix content indicates the N « U transition from native to unfolded state. By

subsequent cooling, a part of the secondary structure is recovered, however, not to the

full extent. When cooled to 20°C, the protein structure contains only 46% α-helix,

indicating the irreversibility of the unfolding/refolding process. The β -sheet content of

the protein does not change much upon unfolding and refolding. The change in the

structure results mainly from the change from α-helix to random coils. Similar results

were previously observed in the case of pH induced denaturation [166].

Near-UV CD spectra of HSA (Figure 6.5B), which is sensitive to the tertiary structure

of the protein, show two distinct peaks at 262 and 268 nm along with a shoulder at 280

nm for the native HSA at 20°C. The results agree well with those observed in previous

studies [165, 166]. The spectra at 20, 40 and 55°C of the heating stage are almost the

same. A slight decrease of the signals at 262 and 268 nm indicates a slight perturbation

of the disulfide bridges [168]. Upon cooling from 55°C, the CD spectra return to the

original ones. The thermal N « E transition is thus reversible. A significant change is

found for the spectra at 70°C, indicating a rupture of several disulfide bonds. Upon

cooling to 20°C, the CD spectrum is not recovered, which shows an irreversible

structural perturbation during the N « U transition. CD experiments clearly suggest a

moderate and considerable modification of the protein secondary and tertiary structure

for N « E transition (Process 1) and N « U transition (Process 2), respectively. Such

changes in HSA structure were also confirmed by a previous dynamic light scattering

study, which probed hydration on the µs-ms timescale [156]. It was found that the

change in the hydrodynamic diameter of HSA (%¹) is negligible for Process 1 while a


104

twofold increase in %¹ at 70°C is observed for Process 2. The considerable increase in

%¹ is attributed to the equilibrium volume expansion of the protein upon thermal

denaturation.

The steady state fluorescence measurements show reversible behavior of the wavelength

dependent fluorescence spectra for Process 1 and an irreversible behavior for Process 2

(Figure 6.9). The fluorescence spectrum of the refolded state of Process 2 upon cooling

from 70 to 20°C is blue shifted and has lower intensity compared to the spectrum of the

native state. This indicates an irreversible rupture of the secondary and tertiary structure

of the protein upon refolding by cooling. The blue shift of HSA fluorescence spectrum

at unfolded temperatures was also observed in a previous report [169]. In the transition

to the unfolded state, domain II of the protein unfolds, which moves Trp214 residue

located at the bottom of a deep crevice to a more hydrophobic environment [170]. This

blue shift indicates an increased exposure of hydrophobic moiety to the protein surface.

The observed average lifetime � � Z of the native HSA at 20°C in the time resolved

fluorescence spectra (Figure 6.10) is in good agreement with previous reports [163,

171]. It is however slower than the observed decay dynamics of aqueous Trp [172]. The

fluorescence transient of aqueous Trp could be fitted to a biexponential curve with fitted

lifetimes of 0.5 and 2.8 ns, of which the latter component dominates. These two

components were also found in most of the Trp containing proteins [173]. In the present

study of HSA, an additional component with a lifetime of 7 ns was found, which is

characteristic for the hydrophobic environment surrounding the buried Trp. At normal

physiological conditions, the heart shaped HSA adopts a globular structure and Trp

residue sits in a 12Å deep water filled crevice in domain IIA [170]. This additional

constrain on the Trp moiety may give rise to the additional 7 ns component. Upon

increasing temperature, the protein starts to melt and Trp is more flexible, results in

faster average lifetime � � Z. For the N « U transition (Process 2), � � Z for the

refolding protein at 20°C is much faster than that of the native protein at the same

temperature, showing that the protein structure is irreversible and Trp is still in a

flexible surrounding.

The spectra at 20°C of �� and L�� of the buffer measured with THz-TDS

measurements (Figure 6.11) are in good agreement with the previously reported results

for water [93, 174]. Both �� and L�� of the buffer increase with increasing

temperature. A previous temperature dependent absorption study of water also showed


105

similar behavior [67]. Compared to the corresponding spectra of buffer, �� of HSA is

higher while L�� of HSA is lower. The absorption coefficient L�� of the unfolded

protein at 70°C is significantly higher than that of the protein at 20°C. However, L��

of the buffer also increases with increasing temperature, which makes the relative

absorption difference between the unfolded protein at 70°C and the buffer at 70°C still

decreased compared to the relative absorption difference between the native protein at

20°C and the buffer at 20°C.

There are three processes which contribute to the observed changes of the unfolded

protein as probed by THz spectroscopy. First, the increase in temperature yields an

overall increased THz absorption in the whole studied frequency range. Second, the

change in protein structure due to the exposure of side chains to the solvent results in a

change in the THz absorption of water in the dynamical hydration shell of the protein.

Third, the increase in volume of the protein causes a decrease in THz absorption as a

volume fraction of highly absorbing water in the solution is replaced by that of less

absorbing protein. The contributions in different directions of these processes make it

difficult to get a direct comparison. For a general comparison, the change in average

absorption coefficient over the entire investigated frequency range from 0.1 to 1.2 THz

of 1 mM HSA solution compared to that of the buffer (∆L) for Process 2 was calculated

(inset of Figure 6.11). With increasing temperature, ∆L decreases nearly linearly. When

being cooled from 70°C, ∆L of the refolded protein is lower than ∆L of the native

protein, indicating a significant change in the hydration dynamics of the refolded

protein.

The absorption of HSA solutions relative to buffer obtained from p-Ge measurements

decreases with increasing protein concentration (Figure 6.13). A decrease in THz

absorption, a "THz defect", is expected when highly absorbing water molecules in the

buffer are replaced by less absorbing protein molecules [144]. However, the measured

L��+)¸� of the solvated protein are clearly different from the predicted L��+)¸� (water

displacement). Similar deviations from the linear two component model measured with

the same system were also found for other proteins such as λ-repressor [147, 175],

ubiquitin [148], and anti-freeze proteins [176]. The observed non-linear behavior is

attributed to the contribution of a third component: water in the dynamical hydration

shell around the protein. Water within this shell is found to have an increased THz

absorption coefficient compared to that of bulk water. This contributes to an increased


106

absorption of the protein solution, which is confirmed by the higher measured L��+)¸�

compared to the predicted L��+)¸�.

With increasing temperature, a decrease in the measured L��+)¸� is observed (Figure

6.14). A change in temperature induces structural changes of the protein. CD

measurements show a considerable rupture of both secondary and tertiary structure of

the protein during transition to unfolded state when the temperature is increased to 70°C

(Figure 6.5). This is coupled with a significant increase in the size of the hydrated

protein as observed with dynamic light scattering measurements in previous studies

[156, 169]. At the thermal unfolded state at 70°C, an increase in the protein volume in

the solution, which is an increased THz defect, directly leads to a decrease in THz

absorption. This explains the observation that L��+)¸� of the unfolded state at 70°C

approaches the predicted water displacement line.

Figure 6.15: Schematic of the reversible N ↔ E transition of Process 1 and

the irreversible E ↔ U transition of Process 2 during thermal unfolding and

refolding of HSA. The protein structure is derived from the PDB file 1AO6.

For the N « E transition in Process 1, previous studies concluded that some buried

residues of HSA get exposed without significantly damaging the protein structure [154,

156], which results in slight perturbation of the water hydration dynamics. The extended

state refolds almost perfectly to the native state as the absorption relative to buffer has

negligible change (Figure 6.14A). This transition thus proves to be kinetically efficient

and thermodynamically inexpensive. For the N « U transition in Process 2 (Figure

6.14B), L��+)¸� of the refolded protein is significantly higher than that of the native

one. Both CD and fluorescence measurements show evidence of a structurally


107

disordered unfolded state in which some of the sub-domains melt to expose their

hydrophobic core to the outer surface. The irreversible structural change induces a

corresponding change in water network dynamics which directly influences the THz

absorption of protein solutions. A schematic of the reversible (Process 1) and

irreversible transitions (Process 2) during thermal unfolding and refolding of HSA is

illustrated in Figure 6.15. The structure of the protein is derived from the PDB file

1AO6 [150].

A recent combined study of MD simulations and THz spectroscopy found that the

vibrational density of states of oxygens in water molecules in the hydration shell of a

protein is blue shifted compared to that of bulk water [68]. The blue shift results in an

increase in THz absorption of water in hydration shells for frequency above 1.7 THz

and a decrease for frequency below 1.7 THz compared to the absorption of bulk water.

This behavior is attributed to a significant retardation of H-bond dynamics on the ps

timescale. The retardation also goes along with a retardation of the rotational motions

and diffusion of water molecules. The blue shift is observed for hydration water around

both hydrophilic and hydrophobic residues. Hydrophilic residues can form H-bonds to

the nearby water molecules, which consequently retards the water dynamics. For water

in the vicinity of hydrophobic residues, the retardation results from sterical hinderance

effects as hydrophobic residues of protein are usually buried in pockets, grooves and

clefts. Any retardation of water network dynamics is found to induce a blue shift of the

vibrational density of states.

THz measurements of HSA show that the THz absorption of the irreversible refolded

protein in Process 2 is lower than the absorption of the native protein in the frequency

range from 0.1 to 1.2 THz measured with THz-TDS (inset of Figure 6.11B) while a

higher absorption is observed in the frequency range from 2.1 to 2.8 THz measured with

the p-Ge laser (Figure 6.14B). The different absorption behaviors in the frequency

ranges show a blue shift of the THz absorption spectrum of the refolded protein

compared to that of the native one with a turning point between 1.2 and 2.1 THz. This

result is in good agreement with the prediction of the MD simulation study on

vibrational density of states which suggests a turning point at 1.7 THz [68]. The

observation indicates a retardation of H-bond dynamics in the hydration shell of the

refolded protein compared to that of the native one. This can be explained by the

exposure of hydrophobic residues to protein surface upon unfolding, which results in

sterical constraints on hydration water.


108

In summary, the thermal unfolding and refolding of HSA was studied with CD,

fluorescence, and THz spectroscopy. Far and near-UV CD spectroscopy indicate a

marginal change in the secondary and tertiary structure of the protein during N « E

transition (20°C « 55°C) and a dramatic change in the structure during N « U

transition (20°C « 70°C). Steady state and ps time resolved fluorescence spectroscopy

show reversible fluorescence spectra and reversible fluorescence average lifetime of

Trp214 moiety in HSA for N « E transition, while the behavior is irreversible for

N « U transition because of the irreversible exposure of Trp to a more flexible

surrounding upon unfolding. The associated change in the solvation of HSA was studied

using THz spectroscopy. Precise p-Ge laser measurements of the THz absorption of

HSA solutions show significant deviation from a two component model. This can be

explained by an increase in THz absorption of water in the dynamical hydration shell,

which results from the retardation of H-bond network dynamics. When the protein

unfolds at 70°C, hydrophobic residues expose to the protein surface. As this process is

irreversible, a part of the structural changes still remain when the protein is cooled down

to 20°C. These structural changes are accompanied by changes in the hydration

dynamics. Further sterical constraints on water molecules in hydrophobic pockets,

grooves, clefts result in retardation of water dynamics and a blue shift in the THz

absorption spectrum. This explains the experimental observations showing that THz

absorption of the irreversible refolded protein from unfolded state is lower than the

absorption of the native protein in the frequency range from 0.1 to 1.2 THz while it is

higher in the frequency range from 2.1 to 2.8 THz. This combined study using CD,

fluorescence, and THz spectroscopy show a clear correlation between structural changes

and changes in the hydration dynamics for HSA protein.

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7 Kinetic THz absorption upon T-jump

The kinetics of biomolecules like proteins is critical for biological functions. One of the

most important subjects which attract scientists from different disciplines is protein

folding. This fundamental biochemical process is still a challenge in science [177]. To

study a rapid kinetic process experimentally, a technique to change the equilibrium of

the sample within a short time is required. After being disturbed, the sample relaxes to

its original equilibrium. The induced kinetic changes are monitored with a spectroscopic

method. In order to do this, the surrounding conditions of the studied molecules have to

be changed. A wide range of techniques have been employed for kinetic studies, such as

stopped-flow and continuous-flow techniques to change the solution condition by

rapidly mixing [178, 179], pressure jump technique to change the pressure applied to

the sample [180], temperature jump (T-jump) technique to change the temperature of

the sample [181], as well as theoretical MD simulations [182].

T-jump is the most widely used technique because of its sufficient jump size, high

speed, and minor disturbance to the sample environment. For the kinetic study of

protein folding, T-jump can be used to initiate both the unfolding to heat denatured

states [183], and the folding from cold denatured states [10]. There are three main

techniques to initiate a T-jump. The first technique was developed in the 1960s, using

electrical current pulses to obtain rapid heating of high conductive solutions. This

technique was used for kinetic studies in the ms times cale [184]. Later, when pulsed

lasers were developed, laser pulses were used for T-jump. This technique offered better

timescales, but a special dye which has high absorption at the laser frequency had to be

added to the solution [185]. Recently, Raman shifted laser pulses at wavelengths

between 1300 and 2100 nm were used to directly heat the water in the aqueous solution

by exiting the OH overtone of water molecules [186]. This approach has a number of

advantages, such as no high conductive solution or dye is required and the timescales

can reach ns and sub-ns.

A number of spectroscopic methods have been combined with T-jump to probe the

generated kinetic processes. Fluorescence technique was used to measure the rapid

refolding dynamics of apomyoglobin by recording the fluorescent signals of tryptophan

[187]. Time resolved IR spectroscopy was used to probe the relaxation kinetics induced

7. Kinetic THz absorption upon T-jump

110

by T-jump of cyclic β-hairpin peptides by measuring the IR absorption of the amide I´

band [188]. Time resolved Raman measurements investigated the thermal unfolding of

ribonuclease A [189]. Nanosecond time resolved optical rotatory dispersion techniques

with high specificity to secondary structure were used for sub-ms unfolding studies of

ribonuclease A and cytochrome c [190]. All these methods probed the kinetics directly

from structural changes of the studied biomolecules. Kinetic THz absorption

spectroscopy (KITA) using the THz-TDS is a high sensitive detection method to

observe changes of water dynamics in the hydration shells around biomolecules [11].

The combination between KITA and T-jump offers a new opportunity to observe the

kinetics of biomolecules like proteins through the kinetics of water molecules in the

coupled hydration shells.

A part of this thesis is to setup a new T-jump apparatus and improve the existing THz-

TDS to enable a feasible combination between T-jump and KITA. The main

improvements of the THz-TDS include the better focus of THz beam at the sample

position and the data acquisition process to reproducibly record THz signal upon T-

jump. In order to probe the kinetics induced by T-jump, the size of the THz beam must

be smaller than that of the heating beam at the sample position. For a significant T-jump

(∆T ≈ 5-10°C), the diameter of the heating beam must be smaller than 2 mm [186].

Therefore, the THz beam size has to be focused as small as possible, at least smaller

than 2 mm. Following sections will give an overview of the setup and experimental

results of the measurements with water and proteins.

7.1 Focusing of THz beam

The propagation of the THz beam is illustrated in Figure 7.1. After being emitted from

the THz emitter, the THz beam is focused on the sample position by two off-axis

parabolic mirrors. Then, two other parabolic mirrors focus the beam on a ZnTe crystal

for detection. The focal lengths of the parabolic mirrors will define the size of the beam

at the sample position. As mentioned previously, an important requirement of T-jump

experiments is that the THz beam must be focused as small as possible at the sample

position. The THz beam pattern from the emitter is approximately a Gaussian beam and

the parabolic mirror with focal length f can be considered as a thin lens with the same

focal length [191]. Therefore, matrix optics for Gaussian beams can be used to calculate


111

the propagation of the THz beam as well as the focal size of the beam at the sample

position [192, 193].

Figure 7.1: Schematic of the THz beam propagation from the THz emitter

to the ZnTe crystal. A symmetric system of four parabolic mirrors (2 x M1,

2 x M2) focuses the THz beam on the sample position and after that on ZnTe

crystal for detection. The relative THz beam size and the distance between

parabolic mirrors are also presented. The distances between the parabolic

mirrors equal the sum of their focus length to form a Gaussian telescope

configuration for optimal focus of the THz beam.

Figure 7.1 shows a schematic of the THz beam propagation in the THz-TDS system.

For optimal focus of the THz beam, a symmetric optical geometry is used and the

distances between the parabolic mirrors equal the sum of their focus length [191]. It

means that the first and last parabolic mirrors have the same focal length (the mirrors

M1 near the THz emitter and ZnTe crystal in Figure 7.1); the second and the third also

have the same focal length (the mirrors M2 near the sample in Figure 7.1). This is a so-

called Gaussian telescope configuration. Beam focusing by a Gaussian telescope is

independent of wavelength. However, the opening angle of the THz beam emitted from

the THz emitter is 7°. This leads to spatial frequency dependence of the THz beam size.


112

Using matrix optics, the beam radius of the THz beam can be calculated at every

position of its propagation way [192, 193]. Figure 7.1 also presents the relative THz

beam size in the THz-TDS system. The system of four parabolic mirrors focuses the

THz beam emitted from the THz emitter on the sample position and after that on ZnTe

crystal for detection.

Figure 7.2: Frequency dependent THz beam radius at the sample position

for three representative mirror systems calculated with matrix optics. The

combination of the new setup (f1 = f2 = 10 cm) is the best solution with

smallest beam radius.

THz beam radius at the sample position is the most important parameter to design the

set up. It depends on the focal length f1, f2, and the frequency. In order to have enough

room for the sample and for the guiding of heating pulses for T-jump, the distance

between two M2 parabolic mirrors near the sample must be at least 20 cm. Based on the

calculation, the optimal solution with four parabolic mirrors of the same focal length of

10 cm (50.8 x 50.8mm, 90 degree off-axis parabolic aluminum mirrors, Edmunds

Optics) was chosen for the new setup. The focus of the THz beam at the sample position

is significantly improved compared to the previous setup where the THz system had f1 =

5 cm and f2 = 15 cm. Figure 7.2 shows the calculated frequency dependent THz beam

radius at the sample position for three representative cases. It is clear that the

combination f1 = f2 = 10 cm (new setup) has the smallest beam radius which is about

twice smaller than the combination f1 = 5 cm, f2 = 10 cm and is about three times


113

smaller than the combination of the old setup (f1 = 5 cm, f2 = 15 cm). The beam radius

is frequency dependent, the higher the frequency, the smaller the beam radius. For the

new setup, the beam radius is below 1.5 mm for the frequency range above 0.5 THz,

and it is smaller than 1.0 mm for the frequency higher than 0.7 THz. This focusing of

the THz beam is sufficient for T-jump experiments.

7.2 T-jump apparatus

7.2.1 Overview

Figure 7.3: Schematic of the T-jump apparatus (TFP: thin-film polarizer,

λ/4: quarter wave plate, L: lens, R: 1.06 µm high reflector, P: prism). The

Nd:YAG laser generates 5 ns laser pulses at 1.06 µm wavelength. The

Raman shifter converts the wavelength of the laser pulses to 1.54 µm which

can directly heat water in an aqueous sample to induce T-jump.

Figure 7.3 shows a schematic of the T-jump apparatus. The main components of the T-

jump setup are a Q-switched Nd:YAG laser (model Surelite III-10, Continuum), and a

one-meter long Raman shifter (model 101 PAL, Light Age). The Nd:YAG laser

generates high power short laser pulses (800 mJ/pulse, 5 ns FWHM) at 1.06 µm

wavelength and serves as a pump source. All optical components used in the T-jump

apparatus are specially designed and have high damage thresholds in order to transmit


114

or reflect the high power laser pulses. Mirrors used to reflect the Nd:YAG laser beam

are special laser line mirrors (model NB1-K13, Thorlabs). An optical isolator consisting

of a thin film polarizer (model 11B00HP.6, Newport) and an achromatic quarter wave

plate (model AQWP05M-950, Thorlabs) are used to prevent the back reflection of the

laser beam. After propagating the optical isolator, the laser beam is collimated to the

Raman shifter by a lens (model LB4374, Thorlabs).

The Raman shifter, containing 25 bar methane gas, converts the laser wavelength from

1.06 µm to 1.54 µm (first order Stokes light) thanks to the effective stimulated Raman

scattering [186]. The conversion efficiency is about 11%, from 400 mJ/pulse at 1.06 µm

wavelength to 45 mJ/pulse at 1.54 µm wavelength. After the Raman shifter, the

remaining radiation at 1.06 µm wavelength is separated from the Raman shifted pulses

by a 1.06 µm high reflector (specially designed by CVI Melles Griot to reflect

wavelength of 1.06 µm and transmit wavelength of 1.54 µm). The Raman shifted pulses

consist not only of first order Stokes light, but also higher order Stokes light as well as

anti-Stokes light [194]. A dispersing prism (model PS853, Thorlabs) is used to separate

the first order Stokes light. Then the heating pulse is focused to the sample position.

With the wavelength of 1.54 µm, the heating pulse directly excites the OH stretching

overtone of water in aqueous samples. The absorption coefficient of water at 1.54 µm

wavelength is about 12 cm-1 at room temperature, which allows smooth and sufficient

T-jumps for kinetic studies [195].

7.2.2 Physical background

When a high power laser pulse is focused on a solution, the temperature change ∆o of

the solution in the absence of thermal diffusion is given by [195]:

∆o�º, �, �� 0��¸ & ��º, �, �W�%��+

(7.1)

where º is the distance from the center axis, � is the axial position, � is the time, 0 is the

absorption cross section, � is the solution density, �¸ is the heat capacity of the solution,

��º� is the intensity profile of the laser pulse, and ��º, �, �� is the instantaneous laser

fluence at axial position � and time �. The laser intensity changes along the spatial


115

coordinate � because of the absorption of the solution. The dependence of the intensity

on � can be calculated by the Lambert-Beer law:

��º, �� º��'J� (7.2)

where L is the absorption coefficient of the solution. For aqueous solutions, the Raman

shifted laser pulse at a wavelength of 1.54 µm is usually used to induce T-jump. The

absorption coefficient L of water at this wavelength is about 12 cm-1 at 293 K. The

calculated temperature increase ∆o at the surface of the cell window is approximately

1°C for the integrated pulse energy of 1 mJ/mm2. As the absorption of water at a

wavelength of 1.54 µm decreases with increasing temperature, ∆o also decreases with

increasing temperature [195].

7.2.3 Q-switched Nd:YAG laser

Nd:YAG lasers are solid state lasers using neodymium doped yttrium aluminium garnet

(Nd:Y3Al 5O12 or Nd:YAG) as a lasing medium. The first Nd:YAG laser was

demonstrated in 1964 [196]. Since then, Nd:YAG crystal has been one of the most

widely used laser media for solid state lasers because of its stability, high gain

efficiency and low threshold pump power. Nd:YAG crystal is a four level gain medium

and has a strong absorption band around 800 nm wavelength. A flashlamp is usually

used to optically pump the crystal. The most common emission wavelength is 1.06 µm.

There are also other emission wavelengths around 0.94, 1.12, 1.32, and 1.44 µm.

Figure 7.4 shows a schematic of the laser process in Nd:YAG crystal. Being pumped by

a flashlamp, Nd3+ ions in the crystal are excited from the ground state (4I9/2) to the

excited state (4F5/2) [197]. Then, the excited ions quickly decay to the metastable laser

state (4F3/2) through nonradiative transitions. The laser state is a relatively long lived

state which allows the ions to build up a large population until population inversion is

achieved. Laser emission at 1.06 µm wavelength occurs through stimulated emission

when the ions decay from the laser state to the lower energy state (4I11/2). If the ions

decay to other energy states (such as 4I9/2, 4I13/2,

4I15/2) laser radiation at other

wavelengths will be emitted. Finally, the ions quickly return to the ground state through

nonradiative transitions.


116

Figure 7.4: Schematic of the laser process of Nd:YAG crystal. The crystal

is a four level gain medium, can be optically pumped by a flashlamp and

commonly emits laser radiation at a wavelength of 1.06 µm.

Nd:YAG lasers operate in both continuous and pulsed mode. Q-switched Nd:YAG

lasers utilizing Q-switching is one of the most common used pulsed lasers with high

power pulse output. In Q-switching, the cavity feedback is blocked or the cavity loss is

increases, which allows the pumping process to build up a much larger than usual

population inversion. The attenuation inside the cavity corresponds to a decrease in the

quality factor (Q factor) of the optical cavity. When the cavity feedback is restored, or

the cavity Q is switched back to its usual high value, the accumulated population

inversion is depleted in a very short time, which results in a short laser pulse of a few ns

with high peak power [198].

Several methods have been developed to achieve Q-switching, such as electro-optical

shutters, rotating prisms, saturable absorbers, acousto-optical switches [199]. In the Q-

switched Nd:YAG laser Surelite III-10 of the T-jump apparatus, a Pockels cell having

Pockels electro-optical effect is used to induce the Q-switching. Figure 7.5 shows a

schematic of the laser cavity. The Pockels cell consists of a nonlinear crystal. With

applied bias voltage, the index of refraction of the nonlinear crystal changes and the

birefringence is proportional to the applied voltage.


117

Figure 7.5: Schematic of the laser cavity of a Q-switched Nd:YAG laser

using a Pockels cell to electro-optically induce Q-switching.

A laser beam propagating from the active medium Nd:YAG crystal is linear polarized

by the polarizer. Then it is incident on the Pockels cell. The electric field of the

incoming light is at 45° to the birefringence axes ^ and _ of the Pockels cell and can be split into �a and �c components. When passing through the Pockels cell, �a and �c

have different phase shifts. With suitable bias voltage applied to the Pockels cell, these

two field components differ in phase by 90° when leaving the Pockels cell, and the light

becomes circularly polarized. It is then reflected by the rear mirror and passes through the Pockels cell once more. The phase difference between �a and �c now becomes

180°, which makes the light linear polarized again but the polarization is rotated 90°

compared to the original polarization. Consequently, the beam is reflected out of the

cavity by the polarizer and cannot reach the threshold for laser oscillation. This

condition indicates that the Q-switch is closed. When the bias voltage applied to the

Pockels cell is removed, the induced birefringence disappears. The light passing through

the Pockels cell experiences no change in polarization and can pass the polarizer on its

way back to initiate laser emission. In this case, the Q-switch is opened. The large

population inversion, achieved when the Q-switch is closed, results in a high peak

power for the emitted laser pulse.

The Q-switched Nd:YAG laser used in the current T-jump apparatus (model Surelite

III-10, Continuum) generates high power short laser pulses at 1.06 µm wavelength. The

power of a single pulse is about 800 mJ and its FWHM is about 5 ns. The maximum

repetition rate is 10 Hz (10 pulses per second). This rate can be varied from 0.1 to 10


118

Hz. Furthermore, the laser can operate in single shot mode, in which a single pulse is

generated when the single shot button is pressed.

7.2.4 Raman shifter

Raman shifters utilize the effective stimulated Raman scattering to convert the

wavelength of a laser [186]. Raman scattering is the inelastic scattering of a photon. The

effect occurs when a photon is inelastically scattered from a molecule leaving the

molecule in an excited state and the wavelength of the photon shifted. Raman effect was

discovered in 1928 by the Indian physicist C. V. Raman who was awarded the Nobel

Prize in Physics in 1930 for this finding [200].

Figure 7.6: The possibilities of light scattering. Elastic scattering (Rayleigh

scattering) occurs when the incident and scattered photons have the same

frequency and kinetic energy. Inelastic scattering (Raman scattering) occurs

where the photon is scattered by an excitation and the scattered photons

have higher (anti-Stokes scattering) or lower (Stokes scattering) frequency

and kinetic energy than those of the incident photons.


119

Figure 7.6 shows a schematic of the different possibilities of light scattering. Rayleigh

scattering is elastic scattering in which the incident and scattered photons have the same

frequency and kinetic energy. A part of the scattering is inelastic (Raman scattering) in

which the photon is scattered by an excitation and the scattered photons have a

frequency higher (anti-Stokes scattering) or lower (Stokes scattering) than that of the

incident photons. Stokes scattering occurs when the molecule absorbs energy of an

incident photon and is excited from the ground vibrational state to the virtual excited

stated, then it relaxes from the virtual excited stated to an excited vibrational state. The

scattered photon has less energy (lower frequency) than the incident photon. The 1st

order Stokes scattering occurs when the molecule relaxes to the 1st excited vibrational

state. Higher order Stokes scattering also occurs when the molecule relaxes to the higher

(2nd, 3rd,…) excited vibrational states.

Apart from being in the ground vibrational state, a small amount of molecules are at the

excited vibrational states before absorbing photons. The excited molecule relaxes from

the virtual excited stated to the ground vibrational state and loses energy compared to its

original state. As a consequence, the scattered photon has more energy (higher

frequency) than the incident photon. This is called anti-Stokes scattering. In general, the

intensity of anti-Stokes scattering is lower than that of Stokes scattering because only a

very small amount of molecules are at the excited vibrational states at ambient

condition. The first-order Stokes scattering has the highest intensity among all Raman

scatterings. The possibility for inelastic scattering is very low, about 10000 times less

likely than elastic scattering. However, with high power incident laser pulses, the

initially scattered photons can further enhance the process, which leads to stimulated

Raman scattering. An effective stimulated Raman scattering can reach conversion

efficiency of up to 40% for first-order Stokes scattering [201].

In the current T-jump apparatus, a one-meter long Raman shifter (model 101 PAL,

Light Age) containing 25 bar methane gas is used. This Raman shifter with two stable

blank windows at two ends can operate at a highest possible pressure of 100 bar. Gas in

the Raman shifter is circulated by a built-in motor. The conversion efficiency from the

wavelength of 1.06 µm (400 mJ/pulse) to the first-order Stokes wavelength of 1.54 µm

(45 mJ/pulse) is about 11%. This energy of the heating pulse is sufficient for a T-jump

of 5-7°C at a heating pulse diameter of about 2 mm.


120

7.2.5 Experimental parameters

Different from normal THz-TDS experiments where a whole THz pulse is probed for

each measurement, in a T-jump experiment, only the signal of one point of the THz

pulse is probed for each measurement. This is the time dependent intensity before and

after T-jump, which shows the kinetic change of THz absorption upon T-jump. In order

to probe the kinetic change of the whole THz pulse, T-jump experiments for each single

point of the THz pulse have to be carried out.

The repetition rate of the heating pulses of the Q-switched Nd:YAG laser is chosen at 2

Hz. This is an optimal rate to ensure both the reproducibility of the aqueous samples

and short measurement time. For better time resolution, the integration time of the lock-

in amplifier is kept as short as possible (100 µs or shorter) because shorter integration

time will results in better (shorter) time resolution for the kinetic change upon T-jump.

However, shorter integration time also requires longer measurement time (average of

more data acquisitions) to reach an acceptable signal to noise ratio. For example, about

1000 data acquisitions (one data acquisition per one heating pulse) need to be collected

and averaged for a lock-in amplifier integration time of 100 µs, and 10000 data

acquisitions are required for an integration time of 10 µs in order to get the same signal

to noise ratio. With the heating pulse repetition rate of 2 Hz, approximately 9 and 90

minutes are required for one measurement with integration times of 100 µs and 10 µs,

respectively. A measurement lasting longer than 90 minutes can lead to instability of the

THz laser and systematic errors. Therefore, integration time of the THz-TDS was

chosen at 10 µs or longer. This spectrometer is designed to probe kinetic events

occurring in the µs to ms timescales.

The parameters of the THz-TDS for T-jump experiments are similar to those described

in Chapter 2. The THz emitter is modulated at a frequency of 200 kHz in square wave

form with an amplitude of 20 V. This modulated frequency serves as the reference

frequency in a lock-in amplifier. For T-jump measurements with integration time of 100

µs or longer, SR844 RF lock-in amplifier (Stanford Research Systems) at an input gain

of 24 dB and a sensitivity of 10 mV is used. For integration time shorter than 100 µs, a

computer controlled 50 MHz lock-in amplifier (model HF2LI, Zurich Instruments) at an

input gain of 48 dB and a sensitivity of 200 mV is used. Data acquisition is at a rate of

200 kHz, which gives a time resolution of 5 µs. For a typical T-jump measurement,


121

kinetic data within 100 ms (10 ms before and 90 ms after the trigger of the heating

pulses) are recorded.

7.3 Kinetic THz measurements upon T-jump

7.3.1 Heating pulse profile

The profile of a heating pulse was measured with laser burn paper (Kodak 1895

Linagraph paper) and a near Gaussian profile was revealed. For further details, the

intensities of the heating pulses were determined by knife-edge measurements. The

power of a heating pulse as a function of position was calculated. Figure 7.7 shows the

normalized profile of the heating pulse. The measured data can be well fitted with a

Gaussian function. The Gaussian beam profile of the heating pulse was also previously

reported for similar T-jump apparatus [181]. The diameter of the heating pulse is about

2 mm. Using a lens, the size of the heating pulse can be adjusted to meet the

requirements of different measurements. However, the diameter of the heating pulse

must be at least 2 mm in order to sufficiently cover the focus of the THz beam at the

sample position.

Figure 7.7: Normalized intensities of a heating pulse determined by knife-

edge measurements and a Gaussian fit of the beam profile.


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7.3.2 Static temperature dependent THz absorption of water

Figure 7.8: Experimental results of static temperature dependent THz

measurements of water in the temperature range from 20 to 34°C for a cell

path length of 90 µm: (A) temporal THz pulses, (B) normalized THz peak

intensity for heating and cooling processes, (C) temperature dependent

absorption coefficient, (D) temperature dependent index of refraction.

In the THz frequency range, the absorption of water increases with increasing

temperature [67, 94]. Therefore, change in water absorption can be used as a

thermometer to measure the temperature increase upon T-jump. In order to set up a

calibration scale, the static temperature dependent THz absorption of water was

measured. Figure 7.8 shows the experimental results of water in the temperature range

from 20 to 34°C for a cell path length of 90 µm. Figure 7.8A displayed the temporal

THz pulses. With increasing temperature, the THz amplitude decreases, which indicates


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an increase in absorption of water as shown in Figure 7.8C. Furthermore, the whole

THz pulse shifts to the right with increasing temperature, which corresponds to an

increase of index of refraction as observed in Figure 7.8D. These results agree well with

a previous report in similar frequency and temperature range [202].

In a T-jump experiment, the time dependent intensity before and after T-jump of a

certain point of the THz pulse is measured. As observed in Figure 7.8A, the peak of the

THz pulse has the most significant change with temperature. Thus, this is the most

suitable position to carry out T-jump experiments as well as to build a calibration scale

for temperature dependent change in THz signal. Figure 7.8B shows the change of the

normalized THz peak intensity with temperature. Upon heating and cooling, the change

in intensity is reversible. With increasing temperature, the intensity decreases nearly

linearly. A linear fit of the data yields a slope of 0.01/°C, which means that the THz

peak intensity decreases by approximately 1% per 1°C. This behavior can be used as a

calibration scale to measure the increase in temperature upon T-jump.

7.3.3 KITA upon T-jump of water

7.3.3.1 Change in intensity at different positions of THz pulses upon T-jump

Figure 7.9A shows THz pulses from static measurements of water at different

temperatures. Different positions of the THz pulse have different changes upon increase

in temperature. At position 3, the THz intensity is nearly unchanged. THz intensity

decreases with increasing temperature at position 1 while a reverse behavior is observed

at position 2. The KITA measurement results of water upon T-jump at position 1, 2, and

3 of the THz pulse are presented in Figure 7.9B in an adjusted scale for comparison

with the temperature dependent static measurements. For all measurements, T-jump was

initiated at time 0. At the THz peak position (position 1), the THz intensity decreases

after the heating pulse meets the sample and it comes back to the original level after

some milliseconds. When compared to the same position of the static measurements in

Figure 7.9A, it is clear that the temperature of water does increase at the heating time,

then the heat is distributed to the surrounding and water is cooled to the original

equilibrium. At position 2, a reverse change upon T-jump is found. This also agrees

with the static measurements where an increase in THz intensity with increasing

temperature at position 2 is observed. Similar to the observations at position 1 and 2, the


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unchanged THz intensity upon T-jump at position 3 is the same as the behavior at the

corresponding position of the static measurements with increasing temperature. These

results confirm that the increase in temperature upon T-jump is observable by KITA.

Figure 7.9: (A) THz pulses obtained from static measurements of water at

different temperatures. Upon increasing temperature, different positions of

the THz pulse have different behaviors, such as decreased intensity at

position 1, increased intensity at position 2, or unchanged intensity at

position 3. (B) KITA upon T-jump of water measured at position 1, 2, and 3

of the THz pulse (in an adjusted scale). After T-jump (initiated at time 0),

the change in the THz intensity at each position is similar to the behavior at

the corresponding position of the static THz measurements with increasing

temperature.

The change in the frequency dependent absorption coefficient of water upon T-jump can

be obtained when the T-jump measurements of all points of the THz pulse are carried

out. Figure 7.10A shows the THz pulses of water before T-jump and at T-jump peak (at

highest water temperature) for a cell path length of 90 µm. Using the calculated

calibration scale, the temperature increase of 5°C is observed upon T-jump. Figure

7.10B shows the corresponding absorption coefficients and a clear increase in THz

absorption of water at T-jump peak is observed. The change of the THz pulse and

absorption with temperature is similar to the change observed by static measurements as


125

presented in Figure 7.8A. Along the THz pulse, the THz peak position shows the most

significant change in intensity upon T-jump. A T-jump of 5°C results in negligible shift

of the THz peak position as observed in Figure 7.8A. Therefore, the THz peak is the

most suitable position for kinetic measurements, enabling the observation of kinetic

events with highest effectiveness. In the following parts, T-jump measurements were

carried out at the fixed THz peak position. The measured THz peak intensity was

normalized, in which it was divided by the average intensity before T-jump. The

presented KITA upon T-jump is the kinetic changes in normalized THz peak intensity.

The time � � 0 was defined as the time when the heating pulses were triggered.

Figure 7.10: Experimental results of KITA measurements of water at the

time before T-jump and at T-jump peak (maximum increased temperature):

(A) THz intensity at different positions of the THz pulses, (B) absorption

coefficients calculated from the THz pulses. The error bars of the data are

smaller than the dot size. The temperature of water increases by

approximately 5°C upon T-jump. Both THz pulses and absorption

coefficient show similar behaviors to those observed with static temperature

dependent measurements.

7.3.3.2 Distinct behaviors of water and other solvents

Figure 7.11 shows the KITA upon T-jump at THz peak position of H2O, D2O and

ethanol (C2H5OH) at 20°C. The heating pulse at a wavelength of 1.5 µm directly excites

the OH stretching overtone of H2O water to generate a sudden temperature increase,


126

which induces an observable decrease in THz intensity. After some ms, the system is

cooled down and the THz intensity returns to its equilibrium value. For D2O and

ethanol, no change in the THz intensity is found. Different from H2O, D2O has

negligible absorption at the wavelength of 1.5 µm and its absorption is nearly constant

with temperature in the range from 10 to 50°C [195]. This results in a negligible

temperature increase in D2O sample when being heated by the heating pulse, which

explains a different behavior of D2O compared to H2O in Figure 7.11. The KITA upon

T-jump of ethanol, which is similar to that of D2O, indicates that ethanol has a

negligible temperature increase upon T-jump or its THz absorption is unchanged with

increasing temperature. The observation shows that water is a suitable solvent for KITA

studies upon T-jump with the current system.

Figure 7.11: KITA upon T-jump at THz peak position of H2O, D2O and

ethanol at 20°C. Only H2O shows a significant change in THz signal when

being heated by high power laser pulses. After T-jump (initiated at time 0),

the THz intensity of H2O decreases due to the increased temperature, then it

increases back to the equilibrium value.

7.3.3.3 Conversion of KITA data into kinetic changes in temperature

As shown earlier in Figure 7.8B, the normalized THz peak intensity of water decreases

linearly with temperature at the rate of about 0.01 per 1°C for a cell path length of 90

µm. This behavior can be used as a calibration scale to convert the KITA data, which


127

are the kinetic changes in normalized THz peak intensity (�� "��.), into kinetic

changes in temperature (∆o) using following approximation equation:

∆o � 100O1 $ �� "��.P (7.3)

Figure 7.12: KITA upon T-jump of water at THz peak position for a cell

path length of 90 µm: (A) measured change in THz peak intensity, (B)

calculated change in temperature. Upon T-jump, water is heated up by about

5°C, then it is cooled down to the equilibrium temperature.

Figure 7.12 shows an example of the conversion of the KITA data of water into kinetic

changes in temperature. At the lowest position, the THz intensity decreases by about 5%

compared to the equilibrium intensity, which means that the maximum increased

temperature is about 5°C. Upon T-jump, water temperature increases to the maximum

temperature very quickly while the subsequent cooling to the equilibrium, resulted from

the heat transfer to the surrounding, occurs much longer. This behavior is in good

agreement with a previous study, in which the change in temperature was measured

with IR spectroscopy [186].

7.3.3.4 Influence of integration time

Previous studies by IR spectroscopy showed that when laser pulses at 1.54 µm

wavelength is used to pump the OH overtone of water, temperature increase in water

occurs within about 100 ps [10]. As mentioned before, the time resolution of a KITA


128

measurement is limited to the order of some µs because the available lock-in amplifier

has a minimum integration time of 1 µs. Furthermore, instability of the THz laser and

systematic errors may occur for long measurements which are required for short time

resolution (e.g. 90 minutes are required for a T-jump measurement with an integration

time of 10 µs). Figure 7.13 shows the KITA upon T-jump of water at 20°C for a cell

path length of 90 µm with three different integration times of the lock-in amplifier (100,

20, and 10 µs). The shorter integration time, which results in better time resolution,

requires longer measurement time (average of more data acquisitions) to reach an

acceptable signal to noise ratio.

Figure 7.13: KITA upon T-jump of water with three integration times of

100, 20, and 10 µs: (A) fast heating and slow cooling processes, (B) zoomed

region of the temperature increase in the heating process. The solid lines are

single exponential fits and τ values are the fitted time constants. The fitted

cooling times in (A) are almost the same while the fitted heating times in

(B) are significantly different with different integration times.

Figure 7.13A shows the KITA upon T-jump of water with three different integration

times. When the heating pulse meets the sample at time 0, water is heated by about 5°C.

After that, water temperature slowly decreases to the equilibrium value. As observed

from the figure, the cooling curves of all measurements are similar and have an

exponential relaxation form. Single exponential fits of the experimental data yield the

average relaxation time for water cooling of about �10.7 � 0.8� ms, which agrees well

with a previous IR spectroscopy study for a similar path length [181]. Figure 7.13B


129

zooms the area of the heating process. At this shorter timescale, the different integration

times result in different fitted time constants. The heating times probed by KITA, which

are obtained from single exponential fits of the experimental data, are nearly

proportional to the integration times. The heating time upon T-jump represents the

temperature rise time from the equilibrium to the highest temperature. The integration

times of 100, 20, 10 µs result in heating times of �270 � 6�, �54 � 3�, �21 � 2� µs,

respectively. The heating time probed by IR spectroscopy is much shorter, about 100 ps,

as reported in a previous study [10]. With the shortest integration time of 10 µs, the

combination between T-jump and KITA is suitable to detect kinetic events in the µs and

ms timescales. Long integration time should be used for measurements of long kinetic

events to reduce measurement time.

7.3.3.5 Influence of temperature

Figure 7.14: KITA upon T-jump of water at 5°C, 20°C and 50°C for a cell

path length of 90 µm. The solid lines are single exponential fits and τ values

are the fitted time constants. With increasing starting temperature, the

maximum decrease in THz peak intensity, corresponding to the maximum

increased temperatures, decreases while the fitted cooling time increases

slightly.

At the wavelength of the heating pulse (1.54 µm), the absorption coefficient of water

decreases by approximately 0.5% per degree, from about 13.5 cm–1 at 5°C to 12.0 cm-1


130

at 20°C and 10.2 cm–1 at 50°C [195]. Therefore, T-jump of water is expected to

decrease when the starting temperature increases. Figure 7.14 shows the KITA upon T-

jump of water at 5, 20 and 50°C for a cell path length of 90 µm. The cooling time

obtained from the single exponential fit of the measured water cooling curve after T-

jump increases slightly with temperature, from �10.5 � 0.3� ms at 5°C to �11.5 � 0.6�

ms at 50°C. The calculated maximum increased temperatures are approximately 5.2, 5.1

and 4.5°C for T-jump at 5, 20 and 50°C, respectively. The decrease in T-jump with

increasing starting temperature is in good correlation with the decrease in absorption

coefficient of water with increasing temperature. At low temperature, the fluctuation of

the measured signals (noise level) is lower than that at high temperature. This

observation can be explained by a significant increase of water absorption with

temperature as shown in Figure 7.8C. Higher absorption of water in the samples allows

less THz signal to reach the detector, and consequently reduces the signal to noise ratio.

Therefore, T-jump measurements at low temperature are preferable.

7.3.3.6 Influence of cell path length

Figure 7.15: KITA upon T-jump at 20°C with three different path lengths.

Water was used for the path lengths of 50 and 90 µm while a mixture of

60% water and 40% ethylene glycol was used for the path length of 210 µm.

The solid lines are single exponential fits and τ values are the fitted time

constants. The fitted cooling time significantly increases with increasing

path length.


131

For measurements of aqueous solutions with KITA at ambient conditions, the path

length of the liquid cell is limited to less than 100 µm due to the high absorption of

water in the THz frequency range. If a thicker path length is required, a certain fraction

of an organic solvent with low absorption in the THz frequency range (e.g. ethylene

glycol, cyclohexane) needs to be added to the measured solutions to ensure that

sufficient THz intensity passes through the sample and reaches the detector. Figure 7.15

shows the KITA upon T-jump at 20°C for three different path lengths. Pure water was

used for the path lengths of 50, 90 µm while a mixture of water and ethylene glycol

(60% volume of water, 40% volume of ethylene glycol) was used for the path length of

210 µm. The change in THz peak intensity upon T-jump is clearly observable,

indicating that the power of the heating pulses is sufficient to generate sufficient T-

jumps for different path lengths. The fitted cooling time obtained from the single

exponential fit of the measured cooling curve after T-jump increases significantly with

increasing path length. The cooling time of the 90 µm path length is �10.9 � 0.7� ms,

about twice longer than that of the 50 µm path length. When the path length increases to

210 µm, the cooling time goes up to �45.6 � 0.2� ms, about four times longer than the

cooling time of the 90 µm path length. These results show that the relation between path

length and cooling time is nonlinear. The increase of the cooling time with increasing

path length can be explained by the fact that more time is required for the heat

distribution of the bigger heated volume (thicker path length) to the surrounding than

that of the smaller heated volume (thinner path length). This observation shows that the

path length can be adjusted to meet requirements of different measurements. With

slower and smoother heat distribution to the surrounding, the thicker path length is

preferable as a sample with thicker path length is more stable than the thinner ones

when being heated several times during a T-jump measurement.

7.3.4 KITA upon T-jump of proteins

KITA measurements of water upon T-jump at different conditions presented in previous

section show that kinetic processes induced by T-jump can be observed with KITA if

they result in significant changes in the collective water network dynamics within µs to

ms timescales. Protein folding dynamics is a promising process to study with this new

system as thermal disturbance upon T-jump can induce structural change, which

consequently modifies the coupled water hydration dynamics. A protein can denature at

high temperature (heat denaturation) as well as at low temperature (cold denaturation)


132

[203, 204]. For the kinetic study of protein folding, an upward temperature increase

upon T-jump can be used to initiate both rapid unfolding to heat denatured states, and

rapid refolding from cold denatured states [10].

Figure 7.16: Structures of the investigated proteins and surrounding water

molecules. The protein structures are derived from the PDB files: 3KZ3 (λ-

repressor), 1AO6 (human serum albumin), and 1UBQ (ubiquitin).

Figure 7.16 shows the structures of three investigated proteins and surrounding water

molecules. The wild type λ-repressor (λ-WT) is a five helix bundle protein λ6-85, which

is the N-terminal residue 6-85 fragment of the λ-repressor [205, 206]. λ-WT consists of

80 residues and has a molecular mass of 9.2 kDa. The λ-repressor is a member of a

large family of prokaryotic DNA-binding proteins [207]. The repressor monitors the

growing of lysogens, controls gene expression, stimulating transcription of its own

gene. Human serum albumin (HSA) is the most abundant protein in blood plasma,

containing 585 amino acids and having a molecular mass of 66.5 kDa [149, 150]. In the

native state, HSA adopts a heart-shaped three-dimensional structure with three domains.

HSA transports metabolites, metals, amino acids, fatty acids, drugs, and other

compounds in the bloodstream to their target organs. It also buffers pH and maintains

osmotic pressure in blood plasma. Ubiquitin (UBQ) consists of a single polypeptide

chain of 76 amino acid residues with a molecular mass of 8.6 kDa [137, 208]. It exists

in all eukaryotic cells and performs its main function as an annihilator of unneeded

proteins. UBQ can be covalently attached to target proteins to signal their subsequent


133

degradation. In this section, KITA measurements upon T-jump of these three proteins at

different conditions are presented.

7.3.4.1 Unfolding to heat denatured state of λ-WT and HSA

The protein λ-WT, which was expressed and purified as described in Ref. [209], was

obtained from the Gruebele group at the University of Illinois at Urbana-Champaign

(Illinois, USA). HSA was purchased from Sigma Aldrich at 99% or higher purity and

was used as received. Both proteins had a concentration of 0.5 mM and were buffered in

50 mM phosphate buffer at pH 7. The cell path length of 90 µm and the integration time

of 100 µs were used for all measurements of λ-WT and HSA at 20 and 60°C. For each

measurement, two thousand KITA signals upon T-jump were collected and averaged.

With the mentioned buffered solution, both proteins have a heat denaturation

temperature at about 60°C [155, 209].

Figure 7.17A shows the KITA upon T-jump of the buffer and λ-WT solution at 20°C.

The maximum increased temperature upon T-jump is about 5°C. The curves of the

buffer and λ-WT almost overlap with each other. The fitted cooling times obtained from

the single exponential fits of the cooling curves after T-jump are similar for both

solutions. Within the range from 20 to 25°C, λ-WT is at its native state, which means

that there is no structural change induced by T-jump. This explains the similar

behaviors of the buffer and λ-WT solution. The observation of HSA at 20°C in Figure

7.17B is also similar to the observation of λ-WT.

A T-jump from 60°C induces a rapid structural change for both proteins from native

state to unfolded state as the thermal unfolding temperature of λ-WT is 61°C and that of

HSA is 60°C [155, 209]. Any change in the protein structure upon T-jump results in

corresponding change in the coupled water dynamics in the hydration shells, which may

be observed with KITA. Figure 7.17C shows the KITA upon T-jump of the buffer and

λ-WT solution at 60°C, and Figure 7.17D shows those of the buffer and HSA solution at

60°C. The noise level of the measurements at 60°C is significantly higher than that at

20°C as the absorption of the aqueous solutions is higher at higher temperature. The

fitted cooling times for all solutions at 60°C are slightly longer than the corresponding

values at 20°C, which is similar to the observation of pure water. For both proteins, the

fitted cooling times of the protein solution and the buffer are slightly different, but they

are still in the same range when the error bars are considered.


134

Figure 7.17: KITA upon T-jump of: (A) buffer and λ-WT solution at 20°C,

(B) buffer and HSA solution at 20°C, (C) buffer and λ-WT solution at 60°C,

(D) buffer and HSA solution at 60°C. The solid lines show single

exponential fits of the cooling curves after T-jump and the fitted cooling

times τ are indicated for each solution.

The results indicate that the unobservable change in KITA upon T-jump of thermal

protein denaturation probably results from the high absorption or the high noise level at

the heat denatured temperature of the proteins. Furthermore, the observed unfolding

rates of λ-WT is in the order of a few µs [209], which is closed to the time resolution of

the KITA system. Therefore, studies of slower kinetic refolding processes of cold

denatured proteins may minimize these problems.

7.3.4.2 Refolding from cold denatured state of UBQ

UBQ was purchased from Sigma Aldrich at 98% or higher purity and was used as

received. UBQ with the concentration of 2 mM was measured at 0°C with the cell path

length of 210 µm. To avoid crystallization of water at 0°C and reduce the absorption of

the protein solution, a mixture of water and ethylene glycol (60% volume of water and


135

40% volume of ethylene glycol, freezing point at about -20°C) with 50 mM magnesium

acetate and 3.5 M guanidinium hydrochloride (GdmCl) at pH 4.0 was used as a buffer.

With this buffered solution, the protein refolds from its cold denaturation state at about

0°C as observed in a previous study [204]. A T-jump from 0°C induces a refolding

process of UBQ from its cold denatured state.

Figure 7.18: KITA upon T-jump of buffer and 2 mM UBQ solution for: (A)

fast heating process in the µs timescale, (B) slow cooling processes in the

ms timescale. The solid lines are single exponential fits and τ values are the

fitted time constants.

Figure 7.18A shows the KITA upon T-jump of the buffer and 2 mM UBQ solution

during the fast heating process in the µs timescale. The integration time of 10 µs was

chosen to probe the fast kinetic change. For each measurement, 10000 KITA signals

upon T-jump were collected and averaged. The fitted heating times obtained from the

single exponential fits of the heating curves are �51.9 � 3.1� µs and �42.8 � 2.8� µs for

the buffer and UBQ solution, respectively. The heating time of UBQ solution is faster

than that of buffer. Figure 7.18B shows the KITA upon T-jump of the buffer and 2 mM

UBQ in a longer timescale. The integration time was fixed at 100 µs as these

measurements focused on the slow cooling process in the ms timescale. For each

measurement, 2000 KITA signals upon T-jump were collected and averaged. Single

exponential fits of the cooling curves yield cooling times of �44.4 � 0.5� ms and

�39.9 � 0.5� ms for the buffer and UBQ solution, respectively. Similar to the behavior


136

in the heating process, the cooling time of the protein solution is typically faster than

that of the buffer.

The KITA upon T-jump shown in Figure 7.18 are the averaged data of several single

measurements. In order to check the reproducibility of the observed results, single

exponential fits of the KITA experimental data upon T-jump in both heating and

cooling processes were carried out for all measurements. The yielded time constants are

shown in Figure 7.19. For both processes, the time constants of the protein solution are

distinctly smaller than those of the buffer.

Figure 7.19: Fitted time constants of different measurements of the buffer

and 2 mM UBQ solution obtained from the single exponential fits of the

KITA experimental data upon T-jump for: (A) fast heating process in the µs

timescale, (B) slow cooling processes in the ms timescale. The averaged

time constants are plotted in hollow symbols at measurement 0. Both the

heating and cooling processes of the protein solution are typically faster

than that of the buffer.

The observed different behaviors between the buffer and the protein solution upon T-

jump probed by KITA indicate that the structural perturbation during

refolding/unfolding process does induce changes in dynamics of hydration water and

these changes are reflected by observable changes in KITA data. However, the

separation of the influence only from protein folding dynamics when comparing the

kinetic change of protein solution to that of the buffer is still difficult because their


137

relative difference falls within the error bar of the measured data. Further developments

are under study to improve the experimental procedure, such as improving data

acquisition and data analysis to deduce kinetic data more precisely, selecting proteins

with suitable folding/refolding rates at low temperature, changing the geometry of the

THz system to enable measurements of a reference and a sample at the same time.

7.3.5 Conclusion

This is the first successful combination between T-jump and KITA, which provides

opportunities to probe the protein folding dynamics through its coupled water dynamics

in the hydration shells. With increasing temperature, the THz pulse of water decreases

in amplitude (increase in absorption) and shifts to the right (increase in index of

refraction) as shown in Figure 7.8. The maximum change in amplitude is observed at

the THz peak position, which indicates that T-jump measurements at THz peak position

are most suitable to optimize the detection of kinetic processes. THz peak intensity

decreases nearly linearly with temperature. This behavior can be utilized to establish a

calibration scale to convert the KITA data upon T-jump at THz peak position into

kinetic change in temperature. Heating pulses with a diameter of 2 mm routinely initiate

T-jumps of about 5°C.

KITA upon T-jump of water shows that the rapid kinetic change can be detected.

Measurements with different parameters offer options to choose optimal conditions for

a certain KITA measurement. First, longer integration times of the lock-in amplifier

require shorter measurement times to obtain a required signal to noise ratio. However, a

long integration time results in a low time resolution, and kinetic processes faster than

the time resolution are not detectable. Second, T-jump measurements of aqueous

solutions at low temperature are preferable because the absorption of water and the

noise level of the measured data decrease significantly with decreasing temperature.

Third, thicker path lengths are better than thinner ones because the heat distribution to

the surrounding of thicker path lengths (with bigger heated volume) during the cooling

process of water temperature is slower and smoother. This makes thicker path lengths

more stable when being heated several times during a T-jump measurement. Depending

on requirements of the studied samples, these parameters can be adjusted to optimize

the observed KITA signals.


138

A protein is only in its native state in a certain temperature range. It unfolds at high

temperature as well as at low temperature. Temperature increase upon T-jump can

initiate both rapid unfolding to heat denatured states when the initial temperature is near

the unfolding temperature, and rapid refolding from cold denatured states when the

initial temperature is near the refolding temperature. The former process was studied

with λ-WT and HSA at 60°C and the later one with UBQ at 0°C. A small difference is

found between the cooling times of the protein solution and the buffer during the heat

unfolding/refolding at 60°C of λ-WT and HSA. High absorption of water in the

solutions at this high temperature limits the signal to noise ratio of the observed signals.

In the case of cold refolding/unfolding at 0°C of UBQ, a considerable difference for

both heating time and cooling time of the protein solution compared to those of the

buffer is observed. The heating and cooling processes upon T-jump of the protein

solution are both faster than the observed values of the buffer. These results show that

changes in dynamics of hydration water induced by kinetic structural change upon T-

jump are observable with KITA. Further improvements are in progress in order to

extract the kinetic information of protein folding dynamics. This first combination

between T-jump and KITA provides opportunities to probe the protein folding

dynamics in the µs and ms timescales through its coupled water dynamics in the

hydration shells.

139

8 Summary and outlook

THz-TDS and FTIR spectroscopy were used as the main experimental techniques to

study confined water. THz-TDS (investigated frequency range 3-45 cm-1) is sensitive to

intermolecular water network vibrations. FIR-FTIR spectroscopy (50-650 cm-1) probes

the intermolecular network vibrations and librational motions of water. The

intramolecular OH stretching modes of water were measured with MIR-FTIR

spectroscopy (3000-3700 cm-1). Four models of confined water with different levels of

confinement were investigated: water in nanopores of tubular crystals formed by water

mediated assembly of hydroxyl acids, water in RM of DDAB/Cy/water system, water in

water-Dx mixtures, and water in hydration shells around HSA. Furthermore, a new T-

jump apparatus was setup and the combination between T-jump and KITA was

optimized to probe the coupled protein-water dynamics in the µs and ms timescales.

In the first model of confined water, temperature dependent measurements of water in

nanopores using ATR-FTIR spectroscopy in the FIR frequency range show that the

frequency range from 400 to 570 cm-1 is sensitive to probe the water dynamics inside

hydrated nanopores. The absorption in this frequency range of the compounds with

water inside the pores is significantly more than that of the nonporous compounds. The

temperature dependent absorbance of water in nanopores considerably depends on the

size of the pores. Less confined and thus more mobile water molecules inside wide

pores show a large increased absorbance upon increase of temperature. In contrast,

water confined in narrow pores show a smaller increase in absorbance with increasing

temperature. The thermal stability and reversibility of water dynamics in nanopores

were studied by a series of measurements in which the samples were first heated up and

then cooled down. The results show that water molecules can permanently occupy

nonpolar nanopores at ambient condition. The release of mobile water in nanopores

occurs at high temperature, changing the inner pore surface from hydrophilic to

hydrophobic. The differences in the temperature dependent dehydration of the hydrated

nanopores indicate that the behavior of water in nanopores depends on the details of the

intermolecular interactions between water molecules and the inner pore surface. The

observations confirm that the synthesized nanopores can serve as model systems for

biological channels. ATR-FTIR study of the OH stretching band of water confined in

8. Summary and outlook

140

the nanopores will be carried out, aiming to extract further information about the

structure of the water arrangement in each pore. Further studies of the hydrated

nanopores with other experimental techniques and MD simulations of the confined

water are planned in order to investigate the water properties at molecular level.

In the second model, the structure and dynamics of water confined in DDAB/Cy/water

RM were investigated using DLS, THz-TDS, and FTIR spectroscopy. The results of

DLS measurements showed that the hydrodynamic diameter of all RM systems

decreases with increasing temperature, indicating a transition towards smaller

aggregates. With increasing degree of hydration (} ), the RM change from connected

cylindrical structures to discrete spherical droplets. The THz spectra measured with

THz-TDS showed that the absorption coefficient of DDAB/Cy/water RM systems

increases with increasing } to approach the values of water. The temperature

dependent THz absorption of the RM systems was found to be similar to the behavior of

water. FIR spectra measured with FTIR spectroscopy showed that the ratio between the

absorbance intensity of the libration peak and the intermolecular H-bond stretching peak

of the RM system is significantly higher than that of water. The peak arising from the

intermolecular H-bond stretching vibrations of water in RM systems is red shifted

compared to that of water, especially at low } . This shows a significant perturbation of

the water H-bond network resulting from the inhomogeneous H-bonding at the

interface. With increasing temperature the peaks undergo progressive red shift due to

the weakening of H-bonding. With increasing hydration, both peak positions show only

a very small shift, indicating the weak dependence of the H-bond network on the

surface geometry. MIR-FTIR measurements found that the relative population of

strongly H-bonded water molecules (bulk-like water) increases, while that of the

distorted structured water molecules (bound water) decreases with increasing } to

reach a value comparable to those of bulk water. This indicates the inhomogeneity in

bonding of water molecules with the RM interface. At low hydration, water molecules

in contact with the surface are dominating. This can be correlated with the observed

higher abundance of distorted H-bond network. With increasing } , the relative

population of bulk-like water increases, irrespective of the decrease in the RM size.

These observations indicate that it is the load of water rather than the surface geometry

determines the water structure and dynamics. This study reveals a significant change of

the water dynamics with changes in hydration and temperature. However, these changes

are independent of the topological curvature. In the next steps, study of hydration water


141

in RM by adding proteins into the water nanopools will be carried out to explore water

properties in a more complex confinement. Previous NMR studies of similar systems

have found a significant reduced motion of the hydration water of proteins in RM [75,

76]. Further to the RM with the cationic DDAB surfactant, other RM systems with

anionic and nonionic surfactants will be investigated with similar experimental

techniques.

In the third model, the evolution of water network structure in water-Dx mixtures was

studied with three experimental techniques. First, FTIR spectroscopy unraveled the

water-water H-bond formation by the shift of intramolecular OH stretching modes and

libration modes of water. FTIR spectra show a gradual increase of the collective water-

water H-bond network with increasing mole fraction of water (X�) in the mixture. The

water-Dx H-bonds dominate in the low X� region. With progressive addition of water,

intermolecular three-dimensional H-bonded water network dynamics appear at X� �0.1 and approach those of water at X� � 0.54. Second, THz-TDS probed the dielectric

relaxation of water and indicated changes in the H-bond relaxation dynamics. The

relaxation time constant arising from the cooperative relaxation of the water H-bond

network is found to be small for X� � 0.1 and increases rapidly for X� � 0.1. The

static dielectric constant �!, which is attributed to water cluster formation and the

polarity of clustered water, is found to be low at X� � 0.1, showing a negligible bulk

water contribution in the mixture. With increasing X�, �! increases because the size of

water clusters increases due to the formation of collective H-bond network of water.

Third, the kinetic study of BzCl solvolysis reaction showed that the rate constant is very

slow at water diluted region and increases rapidly at X� � 0.1, indicating a change in

the reaction pathway as the nucleophilic character of water changes in water-Dx

mixtures. The lower polarity due to cluster formation at low water contents (X� � 0.1)

destabilizes both the nucleofuge (Cl') and the acylium cation (Bz,). As a consequence,

S 1 pathway is disabled while S 2 mechanism is favoured. With increasing water

contents (X� � 0.1), more rigid water-water H-bonds are formed, the size of water

clusters increases, and the intermediate cation and anion are stabilized. As a result, S 1

reaction pathway is followed, which increases the reaction rate rapidly. In summary, the

study shows a direct correlation between the change in the structure and dynamics of

water with the change in the reaction rate upon increasing X� in water-Dx mixtures.

The investigation of confined water in water-Dx mixtures serves as a model system

which shows the influence of hydration on the activity and demonstrates the importance


142

of the solvent for chemical reactivity. For a deeper understanding of the evolution of

water network structure, similar studies with water in organic solvents of different

hydrophilic/hydrophobic properties will be carried out.

In the fourth model, the thermal unfolding and refolding of HSA was studied with CD,

fluorescence, and THz spectroscopy. Far and near-UV CD spectroscopy indicate a

marginal change in the secondary and tertiary structure of the protein during the

transition from native to extended state in the temperature range from 20 to 55°C

(N « E transition), and a dramatic change in the structure during the transition from

native to unfolded state in the temperature range from 20 to 70°C (N « U transition).

Steady state and time resolved fluorescence spectroscopy show reversible fluorescence

spectra and reversible fluorescence average lifetime of Trp214 moiety in HSA for

N « E transition, while the behavior is irreversible for N « U transition. The associated

change of the collective water H-bond network dynamics in the dynamical hydration

shells of HSA was studied using THz-TDS and p-Ge laser THz spectroscopy. The

measurement results of both systems show that the absorption of HSA solution relative

to buffer (L��+)¸�) decreases with increasing temperature. For N « E transition, the

extended state refolds almost perfectly to the native state upon cooling as L��+)¸� has

negligible change. For N « U transition, there is a deviation of L��+)¸� for the final

refolded state compared to the initial native state. In the frequency range from 0.1 to 1.2

THz measured with THz-TDS, the THz absorption of the irreversible refolded protein in

N « U transition is lower than the absorption of the native protein, while a reverse

behavior is observed in the frequency range from 2.1 to 2.8 THz measured with p-Ge

laser. The different absorption behaviors show a blue shift of the THz absorption

spectrum of the refolded protein compared to that of the native one. When the protein

unfolds at 70°C, hydrophobic residues expose to the protein surface. As this process is

irreversible, a part of the structural changes still remain in the refolded protein when the

protein is cooled down to 20°C. These structural changes are accompanied by changes

in the hydration dynamics. Further sterical constraints on water molecules in

hydrophobic pockets, grooves, clefts result in retardation of water dynamics and a blue

shift in the THz absorption spectrum [68]. This combined study using CD, fluorescence,

and THz spectroscopy showed a clear correlation between structural changes and

changes in the hydration dynamics for HSA protein. Investigations of chemical and pH

induced unfolding of HSA using THz-TDS and p-Ge laser THz spectroscopy are in

progress to compare with the thermal unfolding. A suitable fit of the dielectric


143

relaxation measured with THz-TDS using the Debye model is under study to yield the

change in the dynamics of hydration water upon structural changes of HSA. These

further studies will help to obtain a detailed overview of the protein folding process.

The last part of this study describes the setup of a new T-jump apparatus and discusses

KITA studies upon T-jump to measure the kinetic processes of water and proteins. The

T-jump apparatus, consisting of a Q-switched Nd:YAG laser and a Raman shifter, was

successfully setup. It generates high power short laser pulses (45 mJ/pulse, 5 ns

pulsewidth) at a wavelength of 1.54 µm. The heating pulses with a diameter of 2 mm

routinely initiate rapid temperature increase by 5-7°C in aqueous solution. The focal

size of the THz beam at the sample position was improved, ensuring that the probed

area of the THz beam is within the heated area of the solution. A data acquisition

process was optimized to reproducibly record KITA signals upon T-jump. The

temperature dependent THz absorption of water is used as a thermometer to measure the

temperature increase upon T-jump. The intensity of the THz pulse at the peak position

has the most significant change with temperature. It decreases nearly linearly with

increasing temperature. Therefore, the THz peak position is the most suitable position to

carry out T-jump experiments. KITA upon T-jump of water shows that the rapid kinetic

change can be detected. Different measurement conditions, such as integration time of

the lock-in amplifier, temperature and path length of the samples, have influences on the

measured results. Depending on requirements of the studied samples, these parameters

can be adjusted to optimize the observed KITA signals. For the kinetic study of

proteins, a rapid temperature increase upon T-jump initiates both rapid unfolding to heat

denatured states when the initial temperature is near the unfolding temperature, and

rapid refolding from cold denatured states when the initial temperature is near the

refolding temperature [10]. The former process was studied with λ-WT and HSA at

60°C and the later one with UBQ at 0°C. During the heat unfolding/refolding at 60°C of

λ-WT and HSA, a small difference is found between the cooling times of the protein

solution and buffer. High absorption of water in the aqueous solutions at high

temperature limits the observable difference. In the cold refolding/unfolding process of

UBQ at 0°C, a considerable difference for both heating time and cooling time of the

protein solution compared to those of the buffer is observed. The rates of heating and

recooling upon T-jump of the protein solution are both faster than those of buffer. The

observed different behaviors between the buffer and the protein solution upon T-jump

probed by KITA indicate that the structural perturbation during refolding/unfolding


144

process induces significant and measurable changes in dynamics of water in the

hydration shells. This is the first study using the combination between T-jump and

KITA to probe the coupled protein-water dynamics in the µs and ms timescales.

In the next steps, further developments on the experimental procedure are in progress to

improve the data acquisition and the signal analysis. The improvement aims to extract

the precise kinetic information of the protein folding dynamics. The KITA study upon

T-jump of the refolding process from cold denatured state of UBQ will be carried out at

different conditions, such as different denaturants, concentrations, and temperatures.

This will help to obtain a detailed overview of the folding kinetics. A mutant of λ-

repressor protein (λ6-85 Y22W/A37G/A49G), which has a cold denaturation temperature

at 2°C and a folding rate of a few ms as observed in Ref. [210], will be synthesized for

KITA study. A change in temperature from 2 to 6°C yields minimum change in the

density of the aqueous solution as liquid water has the highest density at 4°C. Therefore,

a T-jump of 4°C with the initial temperature at 2°C for a KITA study of this λ-repressor

will minimize the unexpected effects resulted from the density change of the protein

solution.

In summary, the studied results confirm that THz-TDS and FTIR spectroscopy in

combination with the complementary experimental techniques (dynamic light

scattering, kinetics of solvolysis reactions, CD spectroscopy, time resolved fluorescence

spectroscopy, and p-Ge laser THz spectroscopy) successfully explored the structure,

dynamics and activity of confined water in different models. The properties of confined

water strongly depend on the size, structure, surface, hydrophobicity/hydrophilicity of

the confinement models. These models partially mimic the versatile levels of water

confinement in living cells which are crowded with several biomolecules and

transmembrane pores. KITA study upon T-jump of the protein folding/unfolding

process observed the different behaviors between the protein solution and the buffer.

The first combination between KITA and T-jump provides new opportunities to explore

the complicated protein folding process. These studied results contribute to a better

comprehension of a small member of our living systems: water, which is "more than a

bystander", "a matrix of life".

145

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159

List of figures and tables

Figure 1.1: Frequency dependent absorption coefficient of liquid water. ........................ 2

Figure 1.2: Schematic of water confinement in four studied systems .............................. 4

Figure 2.1: Schematic of the THz-TDS. ........................................................................... 9

Figure 2.2: Schematic of the large area THz emitter ...................................................... 12

Figure 2.3: Schematic of the electro-optic sampling ...................................................... 14

Figure 2.4: Schematic of the data acquisition in THz-TDS ........................................... 15

Figure 2.5: THz pulses of a reference and a sample ....................................................... 17

Figure 2.6: Schematic of the FTIR spectrometer. .......................................................... 21

Figure 2.7: Schematic of the propagation of an electromagnetic wave .......................... 23

Figure 2.8: Schematic of the ATR unit ........................................................................... 25

Figure 2.9: Measured data of a reference and a sample ................................................. 26

Figure 3.1: Chemical structures of the twelve studied monomers ................................. 30

Figure 3.2: Crystal structures of: (A) wide porous compound. ...................................... 32

Figure 3.3: Schematic pore profile with typical average diameters ............................... 33

Figure 3.4: Absorbance spectrum of ice and spectra of bulk water ............................... 35

Figure 3.5: Absorbance spectra at 20°C in the FIR region ............................................. 36

Figure 3.6: Temperature dependent spectra in the range from -5 to 20°C ..................... 37

Figure 3.7: Change in absorbance compared with absorbance at -5°C .......................... 38

Figure 3.8: Temperature dependent FIR absorbance ...................................................... 39

Figure 3.9: Decrease in FIR absorbance resulted from the release of water .................. 40


160

Figure 3.10: Temperature dependent change in the integrated absorbance.................... 42

Figure 4.1: Structure of DDAB ...................................................................................... 46

Figure 4.2: Schematic of a DLS system ......................................................................... 49

Figure 4.3: Hydrodynamic diameters of DDAB/Cy/water RM systems ........................ 50

Figure 4.4: THz-TDS spectra of DDAB/Cy/water RM systems at 20°C ....................... 51

Figure 4.5: FTIR spectra of the stock solution ............................................................... 52

Figure 4.6: Difference absorbance spectra of DDAB/Cy/water RM systems ................ 53

Figure 4.7: Difference absorbance spectra of DDAB/Cy/water RM systems ................ 54

Figure 4.8: MIR spectra of DDAB/Cy/water RM systems ............................................ 55

Figure 4.9: Fraction of area under the curve for the deconvoluted sub-bands ............... 56

Figure 5.1: Structure of Dx (left) and schematic of water molecules ............................. 62

Figure 5.2: FTIR spectra of Dx in the FIR (A) and MIR region (B) .............................. 64

Figure 5.3: Difference absorbance spectra of water-Dx mixtures .................................. 65

Figure 5.4: (A) MIR difference absorbance spectra of water-Dx mixtures .................... 66

Figure 5.5: MIR difference absorbance spectra .............................................................. 67

Figure 5.6: Fraction of area under the curve for the deconvoluted bands ...................... 68

Figure 5.7: Spectra of water and Dx measured with THz-TDS ..................................... 69

Figure 5.8: Frequency and concentration dependent index of refraction ....................... 70

Table 5.1: Double Debye relaxation fitting parameters for water-Dx mixtures ............. 72

Figure 5.9: Frequency dependent dielectric constant spectra ......................................... 73

Figure 5.10: Schematic of the solvolysis reaction of BzCl ............................................ 75

Figure 5.11: Concentration dependent rate constant of the solvolysis reaction ............. 76

Figure 6.1: Schematic of a protein .................................................................................. 82


161

Figure 6.2: Crystal structure of HSA .............................................................................. 84

Figure 6.3: (A) Linear polarized light viewed as a superposition .................................. 86

Figure 6.4: Schematic of the CD spectrometer ............................................................... 88

Figure 6.5: CD spectra of HSA in aqueous PBS buffer ................................................. 89

Figure 6.6: Temperature dependent change in the CD signal of HSA ........................... 90

Figure 6.7: Schematic of a fluorescence process ............................................................ 92

Figure 6.8: Schematic of a fluorescence spectrometer ................................................... 93

Figure 6.9: Frequency dependent fluorescence spectra .................................................. 94

Figure 6.10: (A) Time dependent fluorescence spectra .................................................. 95

Table 6.1: Fitting parameters of the fluorescence transients .......................................... 97

Figure 6.11: THz-TDS spectra of PBS buffer (pH 7.4) and 1 mM HSA ....................... 98

Figure 6.12: Schematic of the p-Ge laser spectrometer ................................................ 100

Figure 6.13: Concentration dependent THz absorption ................................................ 101

Figure 6.14: Temperature dependent THz absorption .................................................. 102

Figure 6.15: Schematic of the reversible N ↔ E transition .......................................... 106

Figure 7.1: Schematic of the THz beam propagation ................................................... 111

Figure 7.2: Frequency dependent THz beam radius ..................................................... 112

Figure 7.3: Schematic of the T-jump apparatus ........................................................... 113

Figure 7.4: Schematic of the laser process of Nd:YAG crystal .................................... 116

Figure 7.5: Schematic of the laser cavity of a Q-switched Nd:YAG laser ................... 117

Figure 7.6: The possibilities of light scattering ............................................................ 118

Figure 7.7: Normalized intensities of a heating pulse .................................................. 121

Figure 7.8: Experimental results of static temperature dependent ............................... 122


162

Figure 7.9: (A) THz pulses obtained from static measurements of water .................... 124

Figure 7.10: Experimental results of KITA measurements of water ............................ 125

Figure 7.11: KITA upon T-jump at THz peak position ................................................ 126

Figure 7.12: KITA upon T-jump of water at THz peak position ................................. 127

Figure 7.13: KITA upon T-jump of water with three integration times ....................... 128

Figure 7.14: KITA upon T-jump of water at 5°C, 20°C and 50°C .............................. 129

Figure 7.15: KITA upon T-jump at 20°C with three different path lengths ................. 130

Figure 7.16: Structures of the investigated proteins ..................................................... 132

Figure 7.17: KITA upon T-jump of: (A) buffer and λ-WT solution ............................ 134

Figure 7.18: KITA upon T-jump of buffer and 2 mM UBQ solution .......................... 135

Figure 7.19: Fitted time constants of different measurements ..................................... 136

163

List of abbreviations

λ-WT wild type λ-repressor

AOT sodium bis(2-ethylhexyl) sulfosuccinate

ATR attenuated total reflection

BzCl benzoyl chloride

C-500 Coumarin-500

CD circular dichroism

Cy cyclohexane

DDAB didodecyldimethylammonium bromide

DLS dynamic light scattering

DNA deoxyribonucleic acid

DOC disordered open connected

DTGS deuterium tryglycine sulfate

Dx 1,4-dioxane

e.g. exempli gratia (for example)

EOS electro-optic sampling

FFT fast Fourier transform

FIR far-infrared

FRES Förster resonance energy transfer

FTIR Fourier transform infrared

FT-PGSE Fourier transform pulsed-gradient spin-echo

FWHM full width at half maximum

GaAs gallium arsenide

H-bond hydrogen bond

HSA human serum albumin

IR infrared

KBr potassium bromide

List of abbreviations

164

KITA kinetic terahertz absorption spectroscopy

MCT mercury cadmium telluride

MD molecular dynamics

MIR mid-infrared

MSM metal-semiconductor-metal

Nd:YAG neodymium doped yttrium aluminium garnet

NIR near-infrared

NMR nuclear magnetic resonance

OR optical rotation

PBS phosphate buffered saline

p-Ge p-germanium

Ref. reference (references)

RM reverse micelle (micelles)

SANS small angle neutron scattering

SAXS small angle X-ray scattering

SWCNT single-walled carbon nanotube (nanotubes)

TCSPC time correlated single photon counting

THz terahertz

THz-TDS terahertz time domain spectroscopy (spectrometer)

Ti:Sa titanium doped sapphire

T-jump temperature jump

TRES time resolved emission spectra

Trp tryptophan

UBQ ubiquitin

UV ultraviolet

} degree of hydration (} � [water]/[surfactant])

X� mole fraction of water

ZnTe zinc telluride

165

Acknowledgements

There are many people whose support and advice made a significant contribution to the

completion of this work.

First of all, I would like to thank Prof. Dr. Martina Havenith for giving me the

opportunity to work on interesting research projects at Department of Physical

Chemistry II. Her strong support and encouragement always inspire me with great

motivation.

I am grateful to Prof. Dr. Hermann Weingärtner (Department of Physical Chemistry II)

as the second examiner. I am also grateful for his helpful advice and fruitful

discussions.

I am thankful for the advice and help of Prof. Dr. Christian Herrmann (Department of

Physical Chemistry I) as my second supervisor within the Individual Training and

Supervision Plan of the Ruhr University Research School.

Prof. Dr. Martin Hofmann (Department of Photonics and Terahertz Technology) is

gratefully acknowledged for his useful subsidiary lectures Photonics and Optoelectronic

Devices.

Special thanks goes to Prof. Dr. Martin Gruebele (Center for Biophysics and

Computational Biology, University of Illinois at Urbana-Champaign, USA) for

supporting me to work with the T-jump system in his group.

I would like to thank Dr. Natalia Pérez-Hernández and the collaborators (Institute for

Chemical Research, Seville, Spain) for the cooperation to measure confined water in

organic nanopores.

I am grateful to Dr. Rajib Kumar Mitra and the collaborators (S.N. Bose National

Centre for Basic Sciences, Kolkata, India) for the cooperation to measure confined

water in reverse micelles, in organic solvents, and in dynamical hydration shells around

proteins.

Acknowledgements

166

I would like to thank Dr. Erik Bründermann for his helpful guidance and

encouragement. Without his great support, the KITA experiments upon T-jump would

not have been possible.

I am grateful to Dr. Gerhard Schwaab for his nice support and valuable discussions. I

am indebted to him as the first person to link me with the Physical Chemistry II group.

I am thankful for the cooperation and support of Dr. Arun Arora (Korea Institute of

Science and Technology in Europe, Saarbrücken, Germany), Dr. Stefan Hoffmann

(Department of Photonics and Terahertz Technology), and Dr. Jens Soetebier (Applied

Competence Cluster Terahertz).

I would like to thank Dr. Benjamin Born, Jessica Dielmann, Prof. Dr. Simon

Ebbinghaus, Dr. Stefan Funkner, Dr. Matthias Heyden, Dr. Matthias Krüger, Konrad

Meister, Dr. Gudrun Niehues, Prof. Dr. Diedrich Schmidt, Dr. Konstanze Schröck, and

Vinay Sharma from the terahertz team for the interesting and memorable time working

together.

It’s great to work in the friendly and enjoyable Physical Chemistry II group. I want to

thank all members of the group for their ongoing help and support.

I am grateful to Dr. Erik Bründermann, Konrad Meister, and Dr. Gerhard Schwaab for

the careful proofreading and helpful comments on the manuscript.

Financial support by the Ruhr University Research School and the Heinrich Böll

Foundation is gratefully acknowledged.

Finally, I wish to express my deepest gratitude to my parents and my brothers for their

constant support and continuous encouragement. I dedicate this work to my wife

Phuong who always gives me inspiration and shares my dreams.

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