Observing Rotaxane Shuttling with Two-Dimensional

Femtosecond Vibrational Spectroscopy

M.R. Panman

November 27, 2007

Abstract

Femtosecond two-dimensional infrared spectroscopy has been used to investigate the couplingbetween the NH stretch vibrational modes of NH groups in the macrocycle and thread of abenzylic amide rotaxane as a potential time-resolved indicator of rotaxane shuttling. The peptidewas used to test the two-dimensional infrared setup. We were able to observe the couplingbetween the asymmetric and the symmetric stretch of the free amine group in two differentconformers proving the system was capable of measuring the interaction between coupled NHstretch modes. Broadband pump-probe experiments have been conducted to probe the dynamicsof the rotaxane in the NH stretch region. Two-dimensional infrared spectra were made of therotaxane. A possible cross-peak was found between the NH stretch modes. A definitive cross-peak has yet to be observed. The performance of a specialised electrochemical IR sample cellwas tested and was found to be suitable for the conducting of 2D IR while performing electrochemistry on the sample.

Contents

1 Introduction 3

1.1 Rotaxanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2 The principles of Two-Dimensional Ultrafast Infrared Spectroscopy . . . . . . . . 5

1.2.1 Pump-probe spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.2.2 Femtosecond 2D spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.3 Observing molecular motion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2 Experimental 12

2.1 The two-dimensional femtosecond infrared setup . . . . . . . . . . . . . . . . . . 122.2 The electrochemical setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3 Results and Discussion 15

3.1 Testing the 2-dimensional ultrafast infrared setup . . . . . . . . . . . . . . . . . . 153.2 Steady-state measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.3 Vibrational dynamics of the different N-H stretch modes . . . . . . . . . . . . . . 203.4 Observing the rotaxane cross peaks . . . . . . . . . . . . . . . . . . . . . . . . . . 243.5 Shuttling the rotaxane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

4 Conclusions and future measurements 32

4.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324.2 Future measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

5 Acknowledgments 34

2

Chapter 1

Introduction

1.1 Rotaxanes

Rotaxanes are almost always associated with the concept of molecular machines and have re-ceived considerable attention from nanoscience disciplines [1]. Rotaxanes are a subclass ofmechanically interlocked architectures. The name rotaxane comes from the Latin rota (wheel)and axis (axle), derived from the general structure of a rotaxane shown in figure 1.1. A rotaxane

Figure 1.1: Cartoon representation of a generic rotaxane similar to the one studied in this masterthesis.

is composed of a backbone (the “axle”), referred to as the thread; a ring (the “wheel”), referredto as the macrocycle, which is mechanically bonded to the thread; and two stoppers which keepthe macrocycle from slipping off the thread. Also shown in figure 1.1 are two “stations”. Theseare sites which bind the macrocycle to the thread through non-covalent bonds.

There are several ways of synthesising rotaxanes, the most commonly used however, is theclipping reaction. Such a reaction involves the binding (referred to as templating) of a precursorof the macrocycle to the thread through one of the stations. This binding site has a higher affinityfor the macrocycle than the other. The precursor is then capped to form the macrocycle, thisis called the rotaxination.

The idea that rotaxanes could form the precursor of molecular machines comes from arguablythe most interesting property of rotaxanes: the possibility for the movement of the macrocyclewith respect to the thread [2, 3]. Perhaps more modest switching applications are in data storage,information processing [3], rearrangement of the structure of surfaces [4] and transport acrossmembranes [2]. The movement of the macrocycle between two or more stations has to be inducedby an external stimulus (figure 1.2). Initially, the macrocycle is bound to the station on which itwas formed. There is an equilibrium of the position of the macrocycle between the two stations,but the templating station has a higher affinity for the macrocycle than the second station. The

3

Figure 1.2: An example of a generic rotaxane shuttling [2].

macrocycle is therefore located predominantly on the former. An external stimulus is applied tothe system which disturbs the afore mentioned equilibrium by changing the macrocycle affinitiesof the stations. There are three main methods of inducing shuttling, each aimed at increasingthe affinity of one station over the other [5]. The energy required for shuttling can be suppliedvia a chemical reaction, an electrochemical redox reaction, or photochemically. An example of achemically switched rotaxane is the acid-base-switchable rotaxane presented by Garaudee et.al.

[3]. Perez et.al. present a photochemically switched rotaxane [6] where the isomerisation of adouble bond is the driving force of the shuttling. Nørgaard et. al. present an electrochemicallyswitched rotaxane. The macrocycle dissociates from the first station, moves along the threadby force of Brownian motion [7], and binds to the second station. The system is reset after theeffect of the stimulus has decayed (photochemical stimulus), turned off (electro chemistry), orreversed (chemical reaction). This process is shown in figure 1.2.

The rotaxane used in the experiments described by this thesis was published by Brouweret.al. [2], and is shown in figure 1.3. This rotaxane can be shuttled electrochemically and

Figure 1.3: Naphthalimide rotaxane [2].

photochemically. The photochemical switching process of the succinamide rotaxane is shownin figure 1.4. In the steady state, the macrocycle predominantly resides on the succinamide(succ) site because the naphthalimide (ni) station is a poor hydrogen bond acceptor. Rotaxaneshuttling is induced by excitation of the ni station with a 355 nm laser pulse. The excited ni

station is reduced by an electron donor, specifically DABCO, resulting in the formation of aradical anion located on the ni station. This station is now a strong hydrogen bond acceptor.The macrocycle travels over the thread and because of this increased hydrogen bond affinitybinds to the ni station. After charge recombination, the hydrogen bond affinities return to thesituation before photo excitation, the macrocycle disengages from the ni site and binds to the

4

Figure 1.4: Photochemical shuttling scheme of the naphthalimide rotaxane [2].

succ site. The reported quantum yield for the photo reduction process is about 20 %. Theprocess the rotaxane undergoes with electrochemical switching is very similar to that of thephoto induced shuttling. Instead of using light to create the radical anion, an electric current isused. The latter will be used to shuttle rotaxane for several reasons discussed in section 1.3

The main focus of the project is to observe the shuttling process through the couplingbetween the N-H stretch mode of amide groups in the thread and macrocycle. The principles of2D femtosecond IR spectroscopy will be discussed in the following section.

1.2 The principles of Two-Dimensional Ultrafast Infrared Spec-

troscopy

The first 2D-IR spectrum was published by P. Hamm et.al. in 1998 [8]. The concept behind 2Dfemtosecond IR spectroscopy is analogous to 2D NMR spectroscopy [9, 10, 11]. Two-dimensionalspectroscopic methods in general have distinct advantages over their 1-dimensional analogs:there are more measurable features (the time resolution allows for the studying of the systemdynamics); the 2D-spectrum is 3D-structure sensitive because the coupling between differentmodes allows for the determination of the relative orientation of the modes with respect toone another; the technique spreads the resonances in two dimensions; and it can discriminatebetween dynamic and static spectral broadening [12, 13]. The afore mentioned allows the 2D IRspectrum to be decongested; the individual features contributing to the peaks in an IR spectrumcan be separated and therefore, more information can be gleaned from the spectrum. The setupof the 2D-IR experiment we will using is discussed in the following chapter. Before we canunderstand 2D-IR experiments, an explanation of a vibrational 1D (or pump-probe) experimentis in order (following section). We shall begin, however, with a brief overview of the achievementsand some of the more recent developments in 2D-IR spectroscopy.

There are several types of two-dimensional experiments available to the modern researcher;these can be separated into pulsed frequency-domain (or double resonance which are the exper-iments presented in this thesis) and time-domain (pulsed Fourier-transform) experiments [11].The former type of experiment has the ability to probe the energies and population of vibrationalstates, the couplings between different vibrational modes as well as the intensity and orientationof the transition dipole moments of a quantum system (molecule). The principles of the pulsedfrequency- domain will be discussed in greater detail in the following sections.

5

Transient 2D-IR (T2D-IR) is an extension to the double-resonance experiment. These tech-niques involve an extra pulse of UV-VIS light either before the IR pump and probe pulses, inthe case of a regular and non-equilibrium spectral diffusion T2D-IR, or in between the two IRpulses, in the case of a 2D-IR (2D-IR-EXSY) experiment [11]. The T2D-IR experiment involvesthe relative timing of the IR pump and probe to be fixed but the timing between the UV-VISpulse and the IR pump is variable. This configuration allows one to follow the structural changesof a molecule brought out of equilibrium by the UV-VIS pulse. Experiments of this type havebeen carried out on photo-switchable cyclic-peptides [14], transition metal complexes [15], andhas been used to follow protein folding using the amide I band [16]. The other three experimentswill not be explained as they are out side the scope of this thesis.

Within the group of the pulsed time-domain experiments the vast majority of these are 2D-IR photon-echo (2D-IR-PE). However, a time-domain double resonance experiment has recentlybeen performed by DeCamp et. al. [17, 18]. The spectrometer consists of a broad-band pumppulse and probe pulse that can be delayed with respect to the former to probe the dynamics ofthe molecule. The frequencies of the pump pulse are dispersed by a grating before interactingwith the sample. The probe is dispersed by a grating after it hits the sample. The frequenciesof the pump and probe are dispersed perpendicular to one another and are projected onto a2D CCD array after having been upconverted in a nonlinear crystal with a 800 nm pulse. Thissetup produces a whole 2D spectrum in just a single shot. The disadvantages of both photonecho and double resonance experiments are the investment in required experimental resourcesand time. The spectrometer described by DeCamp et.al. requires relatively low cost equipmentsuch as a CCD camera instead of an expensive IR detector and also allows for lower repetitionrate lasers, making 2D-IR experiments more widely available.

Only the very basics of the 2D-IR photon-echo experiment will be discussed in the followingsection. The 2D pulsed Fourier-transform technique is derived from its 1D variant [12] whichoriginated from the spin echo NMR experiments in 1950 [19]. An IR-PE experiment requirestwo laser pulses. The first pulse excites a certain transition in the molecule and places it inwhat is called a superposition state. This state has a dipole associated with it and initiallyall the molecular dipoles oscillate in phase. However, due to the inhomogeneous distributionof vibrational frequencies of the dipoles (responsible for the inhomogeneous line shape of atransition band) the dipole moments dephase (free induction decay). After a time τ a secondpulse is applied. This pulse initiates the rephasing of the dipole moments by inverting thephase vectors of the dipole moments. The speed at which the individual dipole moments havedephased is of no consequence and all the dipoles will have rephased at time 2τ from the initialpulse at which point a third pulse of light is emitted from the sample, this is the photon echo[19, 20]. The surroundings of a molecule interacts with it and essentially disturbs the excitedmolecules such that some dipole moments do not rephase. The echo will be less intense and asτ is increased, the echoes decrease in intensity [19]. The intensity of the echo as a function oftime gives the homogeneous broadening of the system after a Fourrier transformation. A threepulse PE experiment is also possible. This involves a third pulse which allows for the probingof the dynamics of the system as well as the homogeneous broadening [20, 21]. A variation tothe above 3 pulse PE experiment is the heterodyned PE experiment. This involves interferinga fourth pulse with the echo allowing to obtain the phase information as well as the intensity ofthe photon echo [22, 21]. There are many other types of photon echo experiments such as threepulse PE peak shift, 6 wave mixing fifth-order, gated PE [23]

The most common application of 2D-IR spectroscopy is in the determination of protein andpeptide structure [8, 9, 24, 25]. 2D-IR spectroscopy can be used to determine the distance andangle between two oscillators using the through space and through bond coupling between thetransition dipoles of the vibrations.

There are several examples in which 2D-IR offers insight where other forms of spectroscopy

6

cannot. A typically employed absorption band used to probe protein and peptide structure isthe C=O stretch of an amide link in the polypeptide backbone (referred to as amide I band oramide I′ when deuterated) due to the strength of the signal and ease of detection in differentsolvents. This band is quite sensitive to secondary structure via hydrogen bonding between thismoiety and its surroundings [25, 26, 27]. However, this band is a collection of all the C=Ostretches of the whole peptide and is therefore very convoluted, especially for large peptides.2D-IR can be used to decongest the amide I band via the coupling between the different modescomposing the amide I band [8, 24].

2D-IR spectroscopy has also been used to complement other spectroscopic techniques, suchas vibrational circular dichroism spectroscopy in the determination of the absolute structureof proteins [28]. Vibrational circular dichroism is the differential absorption of right and leftcircularly polarised light by the sample. This phenomenon is only present in chiral molecules andthe absolute configuration of the sample can be determined by comparing the VCD spectrumwith calculated spectra [29]. The disadvantage of VCD spectroscopy is that the intensitiesof the signals are 5 orders of magnitude smaller than IR signals. Additionally, steady statespectroscopy cannot provide information on the dynamics of the system. This disadvantagecould be remedied by combining VCD and 2D-IR spectroscopy. A theoretical framework fora 2D-IR VCD experiment has been put forward by Cho et. al. [13] (this technique has notyet been applied in practice). The great advantage of this experimental method is the timeresolution which it can provide, necessary to follow the folding of proteins using VCD signals.A similar framework for a 2D-IR VCD photon echo experiment has also been put forward byChoi et. al. [30].

1.2.1 Pump-probe spectroscopy

The pump-probe technique is able to probe the dynamics of a vibrational mode of a molecule.The experiment requires two pulses: a pump pulse and a probe pulse, both having the samespectrum (a third pulse is often used as reference, this is of no importance to the discussion of thissubsection but has experimental importance). The pump pulse is of a much larger intensity thanthe probe pulse and is used to excite a portion of the molecules from the vibrational ground state(ν = 0) to the first vibrationally excited state (ν = 1) as shown in figure 1.5. The probe pulse

Figure 1.5: Schematic representation of the theory of pump-probe spectroscopy.

probes the population of the vibrationally excited states. If the pump pulse is not present (whenchopped, see section 2.1 for a more detailed explanation), the probe pulse measures a normalsteady-state spectrum. However, when the pump is present, the probe measures the absorption

7

change in the spectrum caused by the pump. Some of the population of the ν = 1 state is excitedto the ν = 2 state (excited state absorption) by the probe at a frequency ω2 = ω1 − ∆, where∆ is the frequency difference between the ν = 1 and ν = 2 states due to the anharmonicity ofthe vibrational potential. Another portion of the population returns to the ground state due tostimulated emission by the probe. To obtain the transient absorption spectrum of the system,the excited-state spectrum is divided by the steady state spectrum and the −log is taken of theresult (a generic transient absorption spectrum is shown in figure 1.6). There is less population

Figure 1.6: Left: Theoretical transient absorption spectrum at different delay times (t0, t1, t2,t3). Right: Decay curve corresponding to the bleaching seen in the left picture.

in the vibrational ground state when the pump has excited some molecules to the ν = 1 statecompared to the steady-state situation. This is observed as a negative intensity change in thetransient absorption spectrum referred to as bleaching. 50 % of the bleaching is due to stimulatedemission by the probe. This process induces the release of a photon which is also detected andso it seems there are less molecules in the ground state. Also there is more population in theν = 1 state when the pump is present than in the steady-state situation. This is observed asa positive signal in the transient absorption spectrum referred to as induced absorption. Theratio between the induced absorption and bleaching of a vibrational mode should be 1:1 becausethe cross-section of the ν = 1 to ν = 2 transition is twice that of the ν = 0 to ν = 1 transition.In molecules where the anharmonicity is not big enough the insufficient separation between theinduced absorption and bleaching will result in lower observed intensities of both phenomena (ifthe system is perfectly harmonic, neither bleaching or induced absorption can be observed). Thechanges between the steady-state and excited-state spectra are, in the order of 10−3 absorptionunits.

The probe can be delayed in time with respect to the probe. This allows the experiment toprobe the population of the ν = 1 state at different times after excitation by the pump. Themore the probe pulse is delayed with respect to the pump, the less population is present in theν = 1 state. In this manner a decay curve is constructed from which the lifetime (T1) of thesystem can be determined (right side figure 1.6).

1.2.2 Femtosecond 2D spectroscopy

The type of 2D-IR experiment we use is called a double-resonance, or a dynamic hole burningexperiment [10]. As with a pump-probe experiment, two pulses are required for a 2D-IR ex-periment (there are also 2D experiments that require more pulses, such as heterodyned photon

8

echo experiments [10]) with one significant change: the pump is spectrally narrow. The centralfrequency of the pump pulse is changed over the desired range of frequencies whereas the probepulse is unchanged. For each pump frequency, a pump-probe spectrum is recorded. This can bemapped out on a 2D-spectrum with the spectrum measured by the probe on the x-axis and thepump frequencies on the y-axis.

A 2D-spectrum, which is also a difference spectrum, consists of diagonal peaks and crosspeaks. The former are the pump-probe signals of the normal modes of the molecule whichare also observed in the 1D spectrum. The cross peaks are the result of coupling between twodifferent vibrational modes. The nature of this coupling is a through-space (and some through-bond) transition dipole moment interaction [31, 8, 9]. Shown in figure 1.7 is the vibrationalenergy-level diagram for two coupled oscillators. The coupling of one vibrational state |10〉 and

Figure 1.7: Left: Vibrational energy-level diagram of a system with two coupled oscillators.Right: Corresponding 2-dimensional IR spectrum.

the other vibrational state |01〉 is a manifestation of the dependence of the energy of one modeon the excitation of the other. This situation can be viewed as a combination of the energy oftwo states: the |11〉 state. Therefore, the effect of the coupling on the system is a slight changein energy of the |11〉 level compared to no frequency change when the two oscillators are notcoupled (E11 = E01 + E10).

Imagine a situation where the pump only excites the |10〉 mode. When the spectrum ismeasured by the probe, a pump-probe signal is seen at the |10〉 transition frequency, the sameas with a 1D experiment (figure 1.7). The excitation of one of the two modes in a moleculecauses a decrease in frequency of the other mode. Therefore, in the presence of the pump thefrequency of the |10〉 to |11〉 transition is red-shifted compared to the |00〉 to |01〉 transition. Thisis visible in the 2D-spectrum as an off-diagonal signal. This feature is due to the subtraction oftwo peaks representing the same transition rather than induced absorption and bleaching. Thesame holds for when the |01〉 mode is pumped.

Structural information can be obtained from the anisotropy of the cross peaks [9]. Theanisotropy of a sample is the difference between the difference absorption measured with apump polarisation parallel and perpendicular to the probe polarisation. This is caused by thepreferential excitation of molecules with the transition dipole moment parallel to the pump

9

or probe polarisation. Since the coupling is due to the interaction between transition dipolemoments of the coupled vibrational states, one can imagine that the orientation of and thedistance between the two oscillators with respect to one another can have influence on theobserved intensity of the cross peaks. The anisotropy of a system is given by the followingequation:

R =∆α‖ − ∆α⊥∆α‖ + 2∆α⊥

(1.1)

where R is the anisotropy, ∆α‖ is the difference absorption intensity with the pump polarisationparallel to that of the probe, and ∆α⊥ is the difference absorption intensity with the pumppolarisation perpendicular to that of the probe. The anisotropy should ideally be 0.4. Deviationsfrom this number can be caused by several factors: reorientation of the molecule within thetimescale of the lifetime of the mode, rapid energy transfer to a different mode, and misalignmentfor example. In order to measure the anisotropy of the system, the 2D-spectrum has to bemeasured twice: once with the the pump polarisation parallel to that of the probe and oncewith the pump polarisation perpendicular to that of the probe. The intensity of a diagonal-peak measured with parallel polarisations (∆α‖) should be a factor 3 higher than when thepolarisations are perpendicular (∆α⊥). This can be mathematically derived [32].

Before using the equation 1.1 to calculate the anisotropy of the cross peaks, the perpendicularspectrum has to be normalised to the parallel spectrum using the diagonal peaks [33]. Theanisotropy can then be used in the following relation:

Rij =3cos θij

2 − 1

5(1.2)

whereRij is the anisotropy of the cross peak, θij is the angle between the coupled transitiondipole moments, with i and j being the two coupled normal modes.

1.3 Observing molecular motion

There are several reported observations indicative of rotaxane shuttling. Brouwer et.al. usedtransient UV-Vis absorption to observe the macrocycle motion [2]. Marlin et.al. use steady stateUV-Vis spectroscopy to follow indirectly the shuttling motion in a chemically switchable rotax-ane [5]. Nørgaard et. al. use X-ray reflectivity measurements to probe the out-of-plane structureand determine the position of the macrocycle in different oxidation states of the rotaxane [4].

Here, two-dimensional infrared (2D-IR) spectroscopy was used to investigate the couplingbetween the stretch modes of different N-H groups in the naphthalimide rotaxane. There aresix N-H groups and three different types of N-H groups seen in the linear spectrum (figure 3.5)of the naphthalimide rotaxane. According to Brouwer et.al., either all four or three of the N-Hgroups in the macrocycle are hydrogen bonded to the thread carbonyl groups. The N-H groupsin the thread are free. A more detailed discussion of the linear spectrum can be found in section3.2. The benefit of using double-resonance 2D-IR spectroscopy over the aforementioned methodsis that the cross peaks can only originate from the rotaxane. The transient UV-Vis absorptionpreviously done on the naphthalimide rotaxane could, for example, be due to a solvation effect.The main objective of this thesis is to see the disappearance of the cross peaks as the ni site isreduced. This would constitute as a direct observation of rotaxane shuttling. We also conducteda proof of principle experiment with a small peptide, Pro-Ac-NH2 (figure 3.1).

Electrochemical switching was chosen above photochemical switching because this mannerof switching does not require DABCO in order to function. This avoids possible interferencefrom this molecule when performing the 2D-IR experiments. Secondly, the quantum yield of thephoto reduction reaction is too low for our purposes. We wish to observe the disappearance of

10

the cross peaks, therefore, if only 20 % of the rotaxanes in solution shuttle, the change in the2D-spectrum will not be as pronounced as when almost all have shuttled.

The experimental setup and details will be discussed in the following chapter.

11

Chapter 2

Experimental

This chapter will describe the experimental details of the project. The 2D ultrafast IR setup isthe center piece of the experiments and will be presented first. The electrochemical experimentswill also be treated.

2.1 The two-dimensional femtosecond infrared setup

The optical setup is based on the design reported by Hamm et.al. [34]. A schematic represen-tation of the setup is shown in figure 2.1.

Light Generation and Two-DimensionalUltrafast IR Setup

OPA

IR generation

2D-Setup

LASER

Spectograph+

MCT Detector

BS

BS

BS

BS

C2C1

C3

C4

OPA:

C1 : Saphire plate

C2 : BBO crystal

D1 : Delay stage 1

D2 : Delay stage 2

IR generation:

C3 : BBO ctrystal

L1 : Focusing lens

D3 : Delay stage

C4 : KTP crystal

L2 : Focusing lens

2D-setup:DL : Diode LaserGP : Germanium PlateD4 : Delay stageFPF : Fabry-Perot FilterC : Chopper

-P : Adjustable lambda plateFM1 : Focusing MirrorS : SampleFM2 :F : FlagCP : Calcium fluoride Plate

l

Focusing Mirror

D1

D2 D3

L1

L2

DL

D4

FPFl-P

C

FM1

FM2

S F

CP

GP

Figure 2.1: Schematic representation of the 2D-IR setup. BS stands for beam splitter.

A portion of the output of a commercial amplified Ti:sapphire laser system (1 mJ, 100 fs,

12

repetition rate 1 kHz) is used to pump an optical parametric amplifier (OPA) based on BBO,resulting in signal + idler energies of typically 100 µJ. The frequency of the idler produced bythe OPA (4575 cm−1) enters the IR generation and is frequency doubled (9150 cm−1) by a BBOcrystal (C3). A portion of the light produced by the Ti:sapphire amplifier is focused into a KTPcrystal (C4) by a lens (L1) where it is difference-frequency mixed with the doubled idler. Theresulting broad-band mid-infrared pulses at 3350 cm−1 have an energy of 1 µJ, and a bandwidthof 200 cm−1 FWHM (full-width-half maximum). The KTP crystal is not placed exactly inthe focus preventing it from being damaged by the high intensity of the beam. The temporaloverlap between the doubled idler and 800 nm is regulated by a delay stage (D3). The beam isre-collimated by a second lens (L2) and enters the 2D-setup.

Light from a diode laser (DL) is collimated with the IR light entering the 2D-setup with aGermanium plate (GP) and two irises (not shown). This helps with the alignment of the setup.IR light is not visible, but the diode laser follows the path of the IR exactly if properly collimatedand can therefore be used for the alignment of the 2D-setup. In addition, the residual 800 nmfrom the difference-frequency mixing is absorbed by the Ge plate. A small portion of the IRlight is split into a reference and probe beam by a CaF2 beam splitter. The remainder is usedas pump beam. The probe can be delayed with respect to the pump by a computer-controlleddelay stage (D4). The pump beam, containing the majority of the intensity of the original IRlight, is first passed through a computer controlled Fabry-Perot filter (FPF), chopper (C), anda half-wave plate (λ-P). The former creates narrow-band pump pulses (bandwidth 25 cm−1,pulse duration 750 fs FWHM), the center frequency of which can be tuned by adjusting theFabry-Perot filter. The chopper removes half the pump pulses which allows the setup to probethe sample in the presence and absence of the pump. The half-λ plate allows the polarisationbetween the pump and probe to be changed. The intensity of the narrow band pump beamis about 40 % of the intensity of the probe beam. All three beams are focused and spatiallyoverlapped in the sample (S) by an off-axis 100 mm parabolic mirror (FM1) and subsequentlyre-collimated by a similar mirror (FM2). A computer controlled flag (F) is used to block eitherthe pump or both reference and probe beams. All three beams are frequency-dispersed on a2×32 HgCdTe (MCT) array detector. The pump and probe beams are projected on the samearray. During the alignment of the Fabry-Perot filter the reference and probe beams are blockedby the flag such that only the pump beam is detected.

We obtain 2D vibrational spectra by recording the absorption change of the sample as afunction of the pump and probe frequencies.

2.2 The electrochemical setup

For the electrochemical shuttling experiments a special IR sample cell was built in close coop-eration with Dr. Frantisek Hartl, the schematic of which is shown in figure 2.2. This cell allowsus to perform IR spectroscopy on the rotaxane in different redox states. The windows are madeCaF2 and are spaced 150 µm apart. All the electrodes are platinum wires. The working electrodeis a wire grid with a circular hole cut into it, this is where the electrochemical reaction takesplace and subsequently allows an IR beam to pass through the sample. The electrochemicalsample-cell was controlled by a voltage generator and connected to a plotter which plots thecurrent measured at the reference electrode versus the potential on the working electrode. Thecell is compatible with standard sample cell holders which allows for taking IR spectra whileconducting electrochemical reactions in the cell.

13

Figure 2.2: Schematic of the electrochemical IR sample cell.

14

Chapter 3

Results and Discussion

3.1 Testing the 2-dimensional ultrafast infrared setup

The feasibility of measuring 2D-spectra with our setup was tested. Ac-Pro-NH2 (figure 3.1), asmall modified naturally occurring peptide, was chosen for its large signal and, expected clearcross peaks. The molecule has a free amine group (NH2). As shown in figure 3.1, the molecule

Figure 3.1: Ac-Pro-NH2 in the intra-molecularly hydrogen bound state and unbound state.

resides either in a linear state or can form an intra-molecular hydrogen bonding producing a7-membered ring. This produces four N-H stretch modes in the linear spectrum (figure 3.2).The peaks at 3520 cm−1 and 3408 cm−1 are the asymmetric and symmetric stretch respectivelyof the unbound peptide. The peaks at 3480 cm−1 and 3315 cm−1 are the the asymmetric andsymmetric stretch respectively of the hydrogen bound peptide. The assignment of these peakswas done by Ishimoto et.al. [35]. A simple vibrational model was used to calculate the forceconstants of the vibrational modes and hence obtain the vibrational transitions.

There can however be some discussion about whether one can still talk about symmetricand asymmetric stretch modes of the amine. One of the two hydrogens of the NH2 group isbonded while the other hydrogen is almost completely free [35]. One could argue that thehigh frequency peak is not the asymmetric stretch of the amine group, but rather the stretchof the unbound NH. Subsequently, the lower frequency peak could belong to the stretchingvibration of bound NH (this peak is broader than the corresponding higher frequency peak).These stretching modes are, in any case, not purely the stretches of one of either the free orbound NH. If this were the case, the free NH stretch would not “feel” the hydrogen bond andwould not be broadened due to this effect. The results published by Tayyari et.al. [36] fora different intra molecularly bound molecule, APO (figure 3.3), could form a useful analogyto Ac-Pro-NH2. They found that the intensity of certain peaks of APO in CCl4 in the NHstretch region were concentration dependent. A peak at 3500 cm−1 increased in intensity afterdilution and was assigned to the free NH stretching. A peak at 3375 cm−1 was found to decrease

15

3250 3300 3350 3400 3450 3500 35500,0

0,1

0,2

0,3

0,4

0,5

abso

rptio

n [O

D]

wavenumbers [cm-1]

Figure 3.2: The linear spectrum of 40 mM Ac-Pro-NH2 in CDCl3 in the amine stretch region.

Figure 3.3: 4-Amino-3-penten-2-one (APO).

with dilution and was assigned to the same NH stretching but in the intermolecularly boundstate. A third peak at 3184 cm−1 was found to remain constant and was assigned to the intramolecularly hydrogen bound NH stretching. Raissi et.al. elaborated on this work and performedDFT calculations and determined the contribution of each vibration to the peaks observed in thespectrum [37]. It was determined that for APO in CHCl3 the high frequency peak is composed76 % of the free NH stretching and 21 % of the bound NH stretching. The middle frequencypeak was found to be composed of 77 % of the bound NH stretching and 19 % of the free NHstretching. From the example of APO we can cautiously conclude that we can no longer speakof asymmetric and symmetric stretching modes in intra molecularly hydrogen bound Ac-Pro-NH2. Cautious because the two systems are different in several important aspects, the anglemade by the O· · ·H-N atoms is different in both cases. Ac-Pro-NH2, as mentioned before, formsa 7 membered ring in the bound state whereas APO forms a 6 membered ring incorporatingan extra double bond. Also, APO exhibits intermolecular hydrogen bonds whereas this is notthe case with Ac-Pro-NH2. Furthermore, APO can undergo tautomerisation. For the sake ofsimplicity, we will continue to use the assignments of the peaks given by Ishimoto et.al. [35].

16

We expected the cross peaks measured in the 2D spectrum to be strong. This because thecoupled transition dipole moments are in very close proximity to one another; the asymmetricand symmetric N-H stretch originate from the same NH2 group. We also expect cross peaksbetween the asymmetric and symmetric stretch of the unbound system. We do not expect tosee cross peaks between modes of the bound and modes of the unbound conformations.

The 2D spectrum of the peptide was measured in a similar way to that of the rotaxane(discussed in detail below). The 2D spectra show the diagonal peaks, which are essentially thepump-probe signals of the normal modes, and cross peaks are observed in the perpendicularexperiment at the following positions: a negative signal at (3397 cm−1, 3520 cm−1) with cor-responding positive signal at (3270 cm−1, 3520 cm−1); a negative signal at (3515 cm−1, 3406cm−1) with corresponding positive signal at (3626 cm−1, 3408 cm−1); a broad negative signalat (3311 cm−1, 3472 cm−1), also visible in the parallel spectrum; and a narrow negative signalat (3491 cm−1, 3307 cm−1) seen also in the perpendicular spectrum. The first two sets of posi-tive and negative signal indicate coupling between the symmetric and asymmetric NH2 stretchmodes of the unbound conformer. The second two negative signals indicate coupling betweenthe symmetric and asymmetric NH2 stretch modes of the hydrogen bound conformer. This iswhat we expected to observe in the 2D spectra.

The fact that the cross peaks are better observed in the perpendicular spectrum than in theparallel spectrum suggests that the angles between the coupled oscillators are very close to 90degrees. This is especially true for the cross peaks associated with the unbound conformer; we donot observe these peaks in the parallel spectrum. The cross peaks associated with the hydrogenbound conformer have intensity in both the parallel and perpendicular spectrum. The shiftin energy of the combination band (|11〉) of the unbound system due to the coupling becomesapparent in the perpendicular spectrum. The positive signals of the cross peaks are 127 cm−1

and 111 cm−1 apart. The positive signal at (3397 cm−1, 3520 cm−1) is blue shifted comparedto the corresponding negative signal at (3515 cm−1, 3406 cm−1). This is the opposite to whatwe observe with the other cross-peak and to what one would expect if the configuration of thevibrational energy levels is the same as was described in the introduction. The anharmonicityof the vibrational potential of this particular NH stretch is high. In this is the case, the effect ofthe coupling translates to an increase in energy of the combination state (|11〉). The transitionfrom the |10〉 state to the |11〉 state is higher in frequency than the transition between |00〉 tothe |01〉. The sign of the cross-peak signals will therefore be the opposite to what is shown inthe introduction and fits what we observe in the measurements.

The above 2D measurement shows that our system is able to measure cross peaks.

3.2 Steady-state measurements

The linear spectrum of the naphthalimide rotaxane in the NH stretch region is shown below: Thelinear spectrum, when fitted, shows three prominent bands: 3438 cm−1, 3375 cm−1, and 3340cm−1. Comparison of the spectrum of the naphthalimide rotaxane with that of the cyclohexanerotaxane, shown on the left of figure 3.6, allows us to assign the peaks. The cyclohexane rotaxaneonly has NH groups in the macrocycle and only displays one peak in the NH stretch region whichtherefore has to belong to the macrocycle NH stretching mode. The mid frequency N-H stretchpeak in the naphthalimide rotaxane spectrum is assigned to the macrocycle NH stretching mode.The high frequency peak in the the naphthalimide rotaxane spectrum is assigned to the unboundthread N-H; the peak is thin and has the highest energy compared to the other two peaks. Thetwo are of similar energy and very broad, these features are characteristic of hydrogen bonding.The hydrogen bond decreases the energy of the stretch vibration because it exerts a force inthe direction of the vibration, weakening the N-H bond and therefore lowering the energy of thevibration. The broadness of the peak is due to a large distribution of possible hydrogen-bond

17

3350

3400

3450

3500

3550

3250 3300 3350 3400 3450 3500 3550 3600 3650

3350

3400

3450

3500

3550

Probe frequency cm-1

Pum

p fr

eque

ncy

cm-1

Figure 3.4: Top: 2D-spectrum of Ac-Pro-NH2 measured at parallel pump polarisation. Bottom:2D-spectrum of Ac-Pro-NH2 measured at perpendicular pump polarisation. The measurementswere conducted at a probe time delay of 1 ps. Negative signals are in blue and the positivesignals are in red. Darker colours indicate more intense signals.

18

3200 3300 3400 35000,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40 10 mM 2.5 mM Normalised 2.5 mM

ab

sorp

tion

[OD

]

wavenumbers [cm-1]

3200 3300 3400 35000,00

0,02

0,04

0,06

0,08

0,10

Abs

orpt

ion

[OD

]

wavelength [cm-1]

Figure 3.5: Left: Linear spectra of the rotaxane at different concentrations. Blue line: 2.5 mMin a 1 mm spaced sample cell. Red line: 10 mM in a 1 mm spaced sample cell. Dashed blackline: 2.5 mM normalised to the 10 mM using the free NH stretch absorption as reference. Right:Linear spectrum of the naphthalimide rotaxane fitted with 4 gaussian bands simulating the N-Hstretch modes.

3200 3300 3400 35000,0

0,1

0,2

0,3

0,4

0,5

abso

rptio

n [O

D]

wavelenght [cm-1]

Figure 3.6: Left: Chemical structure of the cyclohexane rotaxane. Right: Linear spectrum ofthe cyclohexane rotaxane in the NH stretch region.

strengths. The lower frequency peak is indicative of rotaxane clusters. Different concentrationsof the rotaxane affects the intensity of the low frequency band (right picture of figure 3.5); thepeak increases in intensity with increasing concentration. If indeed the low frequency modebelongs to a rotaxane cluster and this is caused by the interaction between the macrocycle NHgroups of one rotaxane and another, it is normal not to observe this cluster peak in the spectrumof the cyclohexane rotaxane because of the absence of free NH groups in the thread.

We can also obtain the transition dipole moments of the modes from the integrated absorp-tion bands. This allows us to calculate the coupling strength we can expect for our system using

19

the following equation [33]:

β1,2 =1

4πǫ0

(

~µ1 · ~µ2

r3− 3

(~r · ~µ1)(~r · ~µ2)

r5

)

. (3.1)

where the transition dipole moments for the thread NH (~µ1) and macrocycle NH (~µ2) are 0.12and 0.15 Debye respectively. The distance between the dipoles (~r) is 5 A and was obtainedfrom the X-ray structure [2]. The coupling was calculated to be -1.3 cm−1. The value of thisnumber should be greater due to through bond interactions, neglected by the calculation. This isnonetheless a very small coupling; it will not be easy to observe a cross-peak in the 2D spectrumof the rotaxane. To obtain an idea of the intensity of the cross-peak we can use two equationsput forth by Hamm et.al. [24]:

∆ǫkl = −4∆β2

kl

(ǫk − ǫl)2

(3.2)

and

∆ǫkl = ∆ωlIkl√IkkIll

, (3.3)

where: βkl is the coupling strength of the coupling between modes k and l; ǫk and ǫl are theenergies of modes k and l respectively; ∆ is the anharmonicity of the NH stretch vibrationalpotential (separation in cm−1 between the induced absorption and bleaching); ∆ǫkl is the cross-peak anharmonicity; we take ∆ωl as the average bandwidth (FWHM) of transitions k and l;Ikk, Ill, and Ikl are the intensities of the k and l peaks (diagonal peaks) and the cross-peak (offdiagonal). For our calculation, we take βkl = 1.2 cm−1, ǫk = 3440 cm−1, ǫl = 3395 cm−1, and∆ωl = 38.5 cm−1. This results in the ratio Ikl√

IkkIll= -0.0123. The noise of our experimental

setup is on the order of 10−5 and the intensity of our signals for the 2D measurements are onthe order of 10−3. This means that if our cross peak is about 102 smaller than our diagonalpeaks the intensity of the cross peaks will be quite close to the noise value and will therefore bedifficult to observe.

3.3 Vibrational dynamics of the different N-H stretch modes

Initially, a pump-probe experiment was performed to probe the dynamics of the stretchingmode of each of the different N-H groups. We used a 10 mM sample of the rotaxane in a 1 mMsample cell for the experiment. The angle of the polarisation of the pump is at arctan (

√2),

the magic angle, with respect to the polarisation of the probe. Running an experiment withthis setting eliminates the effects of rotational relaxation and resonant energy transfer fromthe measurement. The transient absorption spectra of the naphthalimide rotaxane in the N-Hstretch region and the decay curves of the different NH stretch modes are shown in figure 3.7.The transient absorption spectra show what one would expect. The signal obtained at 10 psis mostly caused by the residual temperature effects of the macroscopic diffusion of the energyintroduced into the system by the pump pulse. As the sample increases slightly in temperature,the hydrogen bonds decrease in strength. This in turn leads to an increase in the bond strengthbetween the nitrogen and the hydrogen atoms due to the decreased pull on this bond. When thedifference is taken between the spectrum in the presence of the pump and the spectrum in theabsence of the pump, this slight shift in frequency translates into a signal. However, the sign ofthis signal is opposite to what one would expect leading us to believe the signal probably hascontributions from the decay of the NH stretch modes. This is not implausible considering thelifetimes obtained from the transient absorption spectrum.

To obtain the lifetimes of the separate modes, the individual bands were pumped with aspectrally narrow pump. Using this setup allows us to measure the lifetime of one specific

20

3200 3300 3400 3500

-0,014

-0,012

-0,010

-0,008

-0,006

-0,004

-0,002

0,000

0,002

0,004

Inte

nsity

[OD

]

wavelength [cm-1]

0.4 ps 0.5 ps 1 ps 2 ps 10 ps

Figure 3.7: Transient absorption spectrum of the naphthalimide rotaxane at different delaytimes between pump and probe pulses.

mode whilst limiting the contributions to the signal from the other modes (from the linearspectrum we know the bands significantly overlap). The transient spectra of three differentpump frequencies are shown in figure 3.8. The shape of the pump-probe signals does not change

3250 3300 3350 3400 3450

-0,0020

-0,0015

-0,0010

-0,0005

0,0000

0,0005

abso

rptio

n [O

D]

wavenumbers [cm-1]

0.7 ps 2 ps 10 ps

3250 3300 3350 3400 3450

-0,0020

-0,0015

-0,0010

-0,0005

0,0000

0,0005

abso

rptio

n [O

D]

wavenumbers [cm-1]

0.7 ps 2 ps 10 ps

3250 3300 3350 3400 3450

-0,0005

-0,0004

-0,0003

-0,0002

-0,0001

0,0000

0,0001

abso

rptio

n [O

D]

wavenumbers [cm-1]

0.7 ps 2 ps 10 ps

Figure 3.8: Pump-probe experiments where the individual NH stretch bands are pumped witha spectrally narrow pump pulse. Left: Pump frequency 3340 cm−1. Middle: Pump frequency3375 cm−1. Right: Pump frequency 3440 cm−1.

as a function of time. This indicates that there is no energy transfer to other modes within theobserved time frame. Furthermore, the large anharmonicity of the NH stretch mode becomesapparent from these measurements. The bleaching of the free NH is situated at 3440 cm−1 andthe corresponding induced absorption is located at 3300 cm−1. We do not observe the residualtemperature signal at 10 ps in the spectra in figure 3.8. This is because the pump is significantlyreduced in power after being passed through the Fabry Perot filter. We measured it to be about40 % of the probe intensity. However, we do see that the transient absorption spectrum of thefree NH still contains population in the excited states, the induced absorption of this mode is atthe same position where we observe a positive signal in the 10 ps signal (figure 3.7). Finally, thisspectrum also shows the importance of using a sufficiently spectrally narrow pump. A portionof the macrocycle NH group is being pumped as well as the intended thread NH group whichshows as a lower frequency shoulder in the transient spectrum. The shoulder has disappearedat 10 ps suggesting this contribution has a shorter lifetime and therefore does not belong to thefree NH stretch mode.

According to Graener et.al. [38], the vibrational excitation of a hydrogen bonded OH groupresults in the predissociation of this hydrogen bond and subsequent reassociation of it. This

21

phenomenon was observed in the transient decay of the OH stretch mode of ethanol. The decaycontained two different time constants, the dominant one (T1) is the lifetime of the OH stretchmode itself and and the second lifetime (Teq) belongs to the reassociation of the hydrogen bond.The latter was determined to be 20 ps by Graener et.al.. The NH stretch mode of our system hassimilarities to the above system and we therefore expect to see the reassociation of the hydrogenbond in the transient decay curves belonging to the macrocycle and cluster NH stretch modes.The thread NH group is not hydrogen bonded, we therefore do not expect to see the secondtime constant in the corresponding decay curve.

Figure 3.9 shows transient decay curves obtained from two different measurements. We took

0 2 4 6 8 10 -0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Macrocycle NH 3233 cm -1 - T 1 = 1.63 (±0.12) ps

Cluster NH 3233 cm -1 - T 1 = 1.35 (±0.11) ps

Thread NH 3290 cm -1 (x5) = T 1 = 3.15 (±0.48) ps

Inte

nsity

[mO

D]

Delay [ps]

1 2 3 4 5 6 7 8 9 10

-6

-4

-2

0

3319 cm -1 - T 1 = 1.48 (0.04) ps ; T

eq = 8.27 (5.3) ps

3396 cm -1 - T 1 = 1.56 (0.3) ps ; T

eq = 3.57 (0.7) ps

Inte

nsity

[mO

D]

Delay [ps]

Figure 3.9: Left: Transient decay curves of the induced absorption of the three different NHstretch modes taken from the transient absorption spectra (figure 3.8). The decay curve of thethread NH stretch mode is shown magnified by a factor of 5. Right: Transient decay curves ofthe three different NH stretch modes taken from the broad band transient absorption spectra.

the induced absorption of the three modes for the determination of T1 because it only containsinformation on the relaxation of the NH stretch mode. Any temperature effects or reassociationof the hydrogen-bonds happen at a much longer timescale and are visible in the bleaching partof the non-linear spectrum. This is because the bleaching monitors the population of the groundstate whilst the induced absorption monitor the population of the excited state. We took thetransient data from 3290 cm−1 when pumping 3427 cm−1 (thread NH stretch), 3233 cm−1 whenpumping 3372 cm−1 (macrocycle NH stretch), and 3233 cm−1 when pumping 3320 cm−1 (clusterNH stretch). We fitted the decay curves with a single exponential decay:

y = A1e− x

T1 + y0 (3.4)

where A1 is the initial amplitude of the time constant T1, and y0 is the offset of the curve.The lifetimes we obtained are 3.15 ± 0.48 ps, 1.63 ± 0.12 ps, and 1.35 ± 0.11 ps for thethread, macrocycle and cluster NH stretch vibration respectively. We unfortunately were unableto use the data from the bleached frequencies because the noise was too great to determinetwo time constants. In order to obtain these, we fitted the broad band pumped transientspectrum. The major disadvantage of doing so is that the decay curves of a certain mode willhave contributions from the others and the lifetimes we obtain are therefore only indicative. Weused a bi exponential decay to fit the data:

y = A1e− x

T1 +A2e− x

Teq + y0 . (3.5)

22

We decided to disregard the thread NH absorption band because the corresponding decay curvewill have large contributions from the other modes. The T1 obtained for the broad band dataare as follows: for 3396 cm−1 (macrocycle NH stretch) 1.56 ± 0.3 ps and for 3319 cm−1 (clusterNH) 1.48 ± 0.04 ps. The T1 of the free NH is longer than that of the other two, hydrogenbonded NH groups, the stronger hydrogen bonded low frequency NH stretch mode having theshortest lifetime. The reason for this is that the hydrogen bonded NH groups have a widerrange of relaxation pathways due to the distribution of possible hydrogen bond strengths andwill therefore relax quicker than the thread NH.

The Teq obtained are: for 3396 cm−1 3.57 ± 0.7 ps and for 3319 cm−1 8.27 ± 5.3 ps. Thereis a large difference in the Teq of the macrocycle and cluster peak. This discrepancy could becaused by the topological constraints imposed on the macrocycle NH group. Once the hydrogenbond between the C=O of the thread and NH group of the macrocycle is broken, the two cannottravel very far apart because the ring structure keeps them in close proximity. The hydrogenbond could be forced to reassociate much quicker than if it had not been part of the macrocycle.The cluster NH stretch mode does not have such topological constraints and therefore displaysa much longer Teq.

We wished to test whether the T1’s obtained experimentally are to be expected by comparingthem to calculated lifetimes. For this we used a model presented by Staib et.al.[39], originallyapplied to OH stretch modes in an ethanol dimer system. This model assumes dissociation ofthe hydrogen bond between an OH group and O group after the excitation of the OH stretchvibration and subsequent energy transfer to a comparatively low frequency OO stretch vibration(the observations of Graener et.al. [38]). We hope to apply this model successfully to our N-H···Nsystem.

We assume the total nuclear wavefunction of the system can be separated into two separatefunctions, one dependent on the low frequency OO stretch vibration, and the other dependenton the high frequency OH stretch vibration (referred to as the adiabatic wavefunction):

Ψ(q,Q) =∑

νOH

φνOHψ(Q)νOH

, (3.6)

where q and Q are the normal mode coordinates of the OH stretch and OO stretch vibrationsrespectively. Fermi’s Golden Rule will be used to calculate the hydrogen bond predisssociationrate of the different modes:

T−1

1=

2π

~|〈ψ1(Q) |Hc(Q)|ψ0(Q)〉|2 ρE (3.7)

where ρ(E) is the density of continuum states at energy E. This represents all states to whichthe system can relax to.

The total hamiltonian of the system can be expressed by a matrix:

H =

(

H0(Q) Hc(Q)Hc(Q) H1(Q)

)

(3.8)

where H0(Q) and H1(Q) define the OO potentials when νOH=0 and νOH=1 respectively. Hc(Q)is the non adiabatic coupling matrix element (or the transition matrix element also observed in3.7). Essentially, the diagonal matrix elements describe both the vibrationally excited state andground state of the system with the off-diagonal elements providing the model with a descriptionof how energy transfers from one state to the other. The vibrational potentials used in thediagonal elements are Lippincott-Schroeder potentials first presented in 1955 by Lippincott andSchroeder [40]. This potential was based on the on potential proposed in 1953 which had beensuccessful in predicting bond dissociation energies and anharmonicity constants [41].

23

Staib et.al.[39] found that the curve obtained by calculating lifetime using equation 3.7 canbe fitted with a much simpler expression:

T1(νOH) = k (δωOH)−α (3.9)

where k is a constant, α = 1.8, and δωOH is the difference between the gas-phase and measuredfrequency of the OH stretch mode in question (in our case this changes to δωNH). We take thegas-phase value of an unbound NH stretch mode from a similar rotaxane system to be 3496 cm−1

1. We fill in ωOH with values ranging from 3495 cm−1 to 3340 cm−1 in equation 3.9 to obtain thetheoretical lifetimes of the different modes. In figure 3.10 we compare the calculated lifetimeswith those obtained with the 1D experiments. We took k = 6.323 × 103 which normalises the

���� ���� ���� ���� ���� ���� ����]1−mc[sedomHNehtfoycneuqerf

���������������������������

1]sp[

)τ(

emitefil

Figure 3.10: Comparison between the experimentally observed and calculated lifetimes (T1)

curve to the value of T1 for 3395 cm−1. Unfortunately, the curve does not fit the data verywell but does show the same trend: NH groups that have values close to the gas phase value(unbound) have a higher relaxational lifetime than those that are hydrogen bound. It is possiblethat the theory used to describe the hydrogen bound OH group relaxation cannot be used to fitthe NH situation without modification.

3.4 Observing the rotaxane cross peaks

A number of 2D experiments were conducted. From the cross-correlation of the pump and probein a germanium semiconductor material we determined the pulse length to be 0.5 ps. Thereforemeasuring the 2D-spectra at 0.7 ps would avoid the initial non-linear effects associated withthe pump and probe hitting the sample simultaneously and still give us an adequate amount ofsignal.

1Personal communication from Dr. A. Rijs

24

2D-spectrum were measured with a spectrally narrow pump and spectrally broad probe.One scan consists of pumping the sample at 16 different frequencies ranges from 3150 cm−1

to 3550 cm−1 and probing the whole frequency range at time delays of −5 ps, 0.7 ps, and10 ps for each pumped frequency. This was done with two pump polarisations: parallel withrespect to the probe and perpendicular with respect to the probe. The signal measured with aperpendicular pump polarisation will be a factor of 3 less intense than the signal obtained with aperpendicular polarisation. Ideally, an experiment taken with a perpendicular pump polarisationshould be left to accumulate data 9 times longer to obtain the same signal to noise ratio as onetaken with a parallel polarisation. Practically, however, the perpendicular experiments wereleft to accumulate 3 times longer than the parallel experiments. The polarisation of the pumpwas switched after one complete scan at the parallel polarisation and after three scans at theperpendicular polarisation. This was done to avoid artifacts originating from changes in thesample and laser system over the course of the day. Changing the polarisation frequently ensuresthat any difference observed between the different polarisations originate from the physicalproperties of the sample and not from artifacts. The intensity of the pump, as stated in the abovesection, was typically 40 % of the probe intensity. The result of the above described experimentis shown in figure 3.11. The 2D spectrum shows the bleaching and induced absorption of thediagonal peaks. The structure visible around the coordinates (3250 cm−1, 3250 cm−1) is mostlikely caused by scattering of the pump in the direction of the probe. We believe this becausethe feature is positive in the parallel spectrum but negative in the perpendicular spectrum.The percentage of the population excited by the pump can be calculated from the measuredabsorption of the free NH stretch mode in the linear spectrum and the measured absorptionchange in the nonlinear 2D spectrum. The transient absorption signal is given by the followingequation:

∆α = − log

(

Tǫ

T0

)

(3.10)

where ∆α is the absorption difference between the situation in the presence of the pump andin the absence of the pump, Tǫ is the transmission of the sample at a certain frequency in thepresence of the pump, and T0 is the transmission of the sample at a certain frequency in theabsence of the pump. In the case of the bleaching, ∆α should be divided by a factor of 2to account for the stimulated emission. The linear absorption of the sample is given by thefollowing equation:

α = − log

(

T

T0

)

(3.11)

where α is the absorption of the sample at a certain frequency, T is the transmission of thesample at a certain frequency, and T0 is the transmission without a sample. If we divide ∆α byα we obtain the percentage of molecules excited by the pump. We chose the free NH stretchmode (3440 cm−1) to determine the percentage of molecules excited. ∆α is 4.97275 × 10−4 ODand α is 0.19159 OD. The percentage of the rotaxane excited by the pump pulse in the 2Dexperiments is 0.26 % .

cross peaks are, however, not visible in the 2D spectrum. We enhanced the spectra byincreasing the amount of contour lines in the low intensity range, the result shown in figure3.12. The enhanced spectra in figure 3.12 show a possible cross-peak around the coordinates(3450 cm−1, 3275 cm−1). However, we were not expecting a cross-peak to be observed at thesecoordinates. This suggests there is coupling between the free NH stretch mode and the NHstretch vibration belonging to rotaxane clusters. We had expected a cross-peak between the freeNH in the thread and the hydrogen bound NH in the macrocycle; the two modes are closer inenergy than the free NH mode and the cluster vibration. However, the nature of the clustersmight become apparent if the cross-peak does exist at the location we observe in the 2D spectra.

25

3250

3300

3350

3400

3450

3250 3300 3350 3400 3450

3250

3300

3350

3400

3450

Probe frequency cm-1

Pum

p fr

eque

ncy

cm-1

Figure 3.11: Top: 2D-spectrum of the rotaxane measured at parallel pump polarisation. Bottom:2D-spectrum of the rotaxane measured at perpendicular pump polarisation. The measurementswere conducted at a probe time delay of 0.7 ps. Areas of blue indicate negative signal and areasof red indicate positive signal. Darker colours indicate more intense signals.

26

Figure 3.12: Top: An increase in contour lines at the low-intensity signals of the parallel mea-surement shown in figure 3.11. Bottom: An increase in contour lines at the low-intensity signalsof the perpendicular measurement shown in figure 3.11.

27

This would suggest that at least one of the thread NH groups is in close proximity and possiblyhydrogen-bonded to the macrocycle NH groups of a second rotaxane.

We were hoping to increase our chances of observing a cross-peak by making a cross sectionof the 2D spectrum. The NH stretch vibration of the cluster was pumped (3315 cm−1) andfrequencies from 3150 cm−1 to 3550 cm−1 were probed. The polarisation was switched in thesame manner as was described for the 2D spectra above. We accumulated a lot of data inorder to improve the signal to noise of the measurement. The difference in intensity between

3100 3200 3300 3400 3500-0,008

-0,006

-0,004

-0,002

0,000

0,002 Parallel Perp*3.13

[OD

]

wavenumbers [cm-1]

3100 3200 3300 3400 3500-0,0004

-0,0003

-0,0002

-0,0001

0,0000

0,0001

0,0002

diff

eren

ce

wavenumbers [cm-1]

Figure 3.13: Left: A cross section of the 2D spectrum. The rotaxane was pumped at 3315cm−1with the polarisation of the pump parallel (black line) and perpendicular (normalised per-pendicular shown by the red line). Right: The difference between the measurement with parallelpolarisation and normalised perpendicular polarisation

the cluster NH stretch vibration measured with the perpendicular polarisation and the parallelpolarisation is found to be a factor of 3.13. Using equation 1.1 we obtain an anisotropy (Ri,j) of0.415 for the naphthalimide rotaxane dissolved in CDCl3. This does not deviate much from theideal anisotropy of 0.4 suggesting the rotaxane does not exhibit rotational relaxation, due to itslarge size, or rapid energy transfer to another mode when the low frequency NH mode is excited.The difference between the parallel and normalised perpendicular measurement is shown to theright of figure 3.13. The sharp peaks are due to experimental error; a slight misalignment of theprobe frequencies on the detector of one of the measurement at one polarisation with respect tothe other. We see a broader negative peak at 3400 cm−1 and a small, but broad positive peakat 3500 cm−1. This is a possible cross-peak candidate.

2D spectra of the rotaxane have been simulated (figure 3.14) with different coupling strengths(β). The same method was used for the calculations as was presented by Larsen et.al. [33].These calculations were performed in order to obtain an idea of the strength of the couplingbetween the thread NH and macrocycle NH necessary for us to observe a cross-peak in the 2Dspectrum. The simulated spectrum with β = 0 cm−1 represents a system without couplingbetween the thread NH group and the macrocycle NH group and accordingly, shows no crosspeaks. The spectrum with a coupling strength of 2 cm−1 also shows no evidence of cross peaks,this coupling strength is already greater than the expected -1.3 cm−1 coupling strength. Thesimulated spectrum with β = 5 cm−1 shows a positive cross-peak between the thread NH stretchand macrocycle NH stretch modes. The sign of the cross-peak might seem strange, one wouldexpect to see a negative signal at this position if the vibrational energy levels were of the sameconfiguration as the example shown in section 1.2.2. However, like the Ac-Pro-NH2 peptide,the anharmonicities of the NH stretch modes are very large, as is shown by the non-linearexperiments. The sign of the cross-peak signals will therefore be the opposite to what is shown

28

3250 3300 3350 3400 3450probe frequency (cm-1)

3250

3300

3350

3400

3450

3250 3300 3350 3400 3450probe frequency (cm-1)

3250

3300

3350

3400

3450c

pum

p fr

eque

ncy

(cm

-1)

3250

3300

3350

3400

3450

3250

3300

3350

3400

3450b

a

3250 3300 3350 3400 3450probe frequency (cm-1)

3250

3300

3350

3400

3450

3250 3300 3350 3400 3450probe frequency (cm-1)

3250

3300

3350

3400

3450c

pum

p fr

eque

ncy

(cm

-1)

3250

3300

3350

3400

3450

3250

3300

3350

3400

3450b

a

3250 3300 3350 3400 3450probe frequency (cm-1)

3250

3300

3350

3400

3450

3250 3300 3350 3400 3450probe frequency (cm-1)

3250

3300

3350

3400

3450c

pum

p fr

eque

ncy

(cm

-1)

3250

3300

3350

3400

3450

3250

3300

3350

3400

3450b

a

3250 3300 3350 3400 3450probe frequency (cm-1)

3250

3300

3350

3400

3450

3250 3300 3350 3400 3450probe frequency (cm-1)

3250

3300

3350

3400

3450c

pum

p fr

eque

ncy

(cm

-1)

3250

3300

3350

3400

3450

3250

3300

3350

3400

3450b

a

Figure 3.14: Top Left: Experimentally measured 2D spectrum. Top Right: Calculated 2Dspectrum with no coupling, β = 0 cm−1. Bottom Left: Calculated 2D spectrum with β = 2cm−1. Bottom Right: Calculated 2D spectrum with β = 5 cm−1. Where in each case a isthe linear spectrum of the rotaxane, b the 2D spectrum with pump polarisation parallel to theprobe polarisation, and c the 2D spectrum with pump polarisation perpendicular to the probepolarisation.

29

in the introduction and fits what the calculations show. The calculations also make clear that asignificantly higher coupling constant is necessary than the expected -1.3 cm−1 coupling strengthto observe a cross-peak between the thread NH stretch mode and the macrocycle NH stretchmode with our current laser system.

The question which now arises is, what should the distance between the two oscillatingdipoles be if we wish to have a coupling of 5 cm−1. The following calculation uses equation3.1, with β1,2 = 5 cm−1, ~µ1 = 0.12 Debye, and ~µ2 = 0.15 Debye. We have assumed that thegreatest interaction between the dipoles occurs when the angle between ~µ1 and ~µ2 is 0, giving~µ1 · ~µ2 = | ~µ1|| ~µ2|. The dependence of distance r on varying the angle (θ) between vectors ~rand ~µ1 or ~µ2 is shown in figure 3.15: The maximum length r between the dipoles was found

��� ��� ��� ��� ��� ��� ���]dar[θelgna

�������������������������

]01− 0

1×

m[r

Figure 3.15: Angle dependence of the distance r between the NH group in the macrocycle andthe thread for βij = 5 cm−1.

to be 8.98×10−10 m for an angle of 0 degrees which is on the order of magnitude we expect (rfor our rotaxane is 5×10−1 m). The distance r is significantly longer than that of our rotaxane.This is of course because we chose the coupling βij to be significantly higher than that of ourrotaxane and we chose the angle between the dipoles such to obtain the maximum interaction.It is quite interesting to see that r has to be shorter to obtain the same βij with θ = 90 degrees.This means that a greater interaction occurs when the dipoles are oriented anti-parallel. Thisis close to the configuration we see in our rotaxane. The calculation also shows that the angle

(arccos(

~µ1· ~µ2

| ~µ1|| ~µ2|

)

= φ) between the dipoles is the limiting factor of the dipole coupling strength

in our rotaxane.The only method of changing the distance between the dipoles or to change the angle between

them is to synthetically modify the rotaxane. There is of course no guarantee that the newrotaxane will have the same dipole moments as those of the rotaxane investigated in this thesis,especially if it is not a benzyllic amide rotaxane.

30

3.5 Shuttling the rotaxane

The electrochemical experiments were conducted in the afore mentioned electrochemical samplecell (ECSC). We tested the performance of the cell by measuring the electrochemical potentialof 10 mM of the naphthalimide rotaxane in dichloromethane using cyclic voltametry. Theconcentration of the rotaxane was the same as that used in the 2D IR experiments (10 mM). Wetook steady-state IR measurements at different voltages in the N-H stretch range (3200 cm−1

to 3500 cm−1) , and C=O stretch range (1600 cm−1 to 1750 cm−1) (figure 3.16). This allowedus to monitor the state of the rotaxane during the CV experiment. The linear IR spectrum

3200 3300 3400 35001,10

1,11

1,12

1,13

1,14

1,15

1,16

abso

rptio

n [O

D]

wavenumbers [cm-1]

0 V -0.8 V -1.7 V -0.75 V 0 V

1600 1650 1700 17501,0

1,1

1,2

1,3

1,4

1,5

abso

rptio

n [O

D]

wavenumbers [cm-1]

0 V -0.8 V -1.7 V -0.75 V 0 V

Figure 3.16: Left: Infrared absorption spectrum of the rotaxane in the N-H stretch region atdifferent voltages. Right: Infrared absorption spectrum of the rotaxane in the C=O stretchregion at different voltages.

shows changes in both the C=O stretch and the N-H stretch regions at different potentials.The blue and the magenta lines are spectra taken at voltages after the maximum voltage wasreached. Both the spectrum of the N-H stretch region and the C=O region do not return totheir original state after the voltage has been turned off. This suggests that an irreversibleprocess has occurred as well as the reduction of the naphthalimide station. The explanation tothis lies in the high concentration of the sample. At lower concentrations, the reduction of thenaphthalimide occurs at lower voltages than the irreversible process and is well separated fromthe latter process. At higher concentrations the range of voltages over which the two processesoccur start to overlap. We tried to avoid the second process by not fully completing the initialreduction. Unfortunately, if we do not wish to irreversibly reduce any rotaxane, only 10 % of therotaxane at the working electrode can be shuttled. This would not be enough for the experimentin which we would use 2D-IR spectroscopy to monitor the disappearance of the cross peaks aftershuttling.

There are different ways of switching the rotaxane other than electrochemically or photo-chemically. The macrocycle could be detached from the thread chemically. One could use asolvent such as deuterated dimethyl sulfoxide (DMSO-d6) to enter into competition with thethread for hydrogen bonding with the macrocycle. The result would be a macrocycle floatingrandomly along the thread, not being able to bind to the stations. A possible disadvantage ofusing DMSO-d6 to shuttle the rotaxane is its absorption in the N-H stretch region. This problemshould be minimised by using only just enough DMSO-d6 to make a significant portion of therotaxane shuttle. Another, more controlled option could be to use a sacrificial electron donor.The effect of this would be a permanently shuttled rotaxane.

31

Chapter 4

Conclusions and future

measurements

4.1 Conclusions

The steady state studies performed on the naphthalimide rotaxane allowed us to assign the peaksobserved in the NH stretch region of the rotaxane spectrum. From these results it was determinedthat at the concentrations we were measuring at rotaxane clusters were present, possibly dueto the interaction of the NH groups in the thread of one rotaxane and the macrocycle groupsof another rotaxane. Furthermore, from the integrated absorption bands of the linear spectrumthe transition dipole moments of the thread and macrocycle NH stretch vibrations were foundto be 0.12 Debye and 0.15 Debye respectively. This information, along with previously obtainedcrystallographic data [2], allowed for the calculation of a possible coupling strength between thetwo groups. This was found to be -1.3 cm−1.

From the narrow band pump-probe experiments we were able to determine the lifetimes ofthe individual NH stretch modes. These were found to be 3.15 (±0.48) ps, 1.63 (±0.12) ps, and1.35 (±0.12) ps for the free NH, macrocycle NH, and cluster NH stretch modes respectively. Thenarrow band dynamic experiments show that there is no rapid energy transfer from one modeto another. The broad band experiments indicated there was a second time constant and thiscould belong to the reassociation of the N-H· · ·H bond. This led us to use the model by Staibet.al. [39] to fit the T1. This method unfortunately did not fit the data very well which led us tobelieve that the model does not describe the NH- stretch mode as well as the OH-stretch mode.

We were unable to observe the coupling between the NH stretch mode of the macrocycle NHgroups and the NH stretch mode of the thread NH groups. The fact that we did not observethese peaks in either the parallel measurements or the perpendicular measurements suggeststhat the distance between the two groups is too great for us to measure the coupling. A possiblecross-peak candidate was observed at around 3400 cm−1 when the rotaxane was pumped at 3315cm−1. A superior signal to noise to the one we obtained might allow us to better observe thiscross-peak, which is between the low frequency NH stretch mode and the other two NH stretchmodes. This could possibly provide us with information on the nature of the rotaxane clusters.The anisotropy of the system was found to be 0.415 when pumped at 3315 cm−1 and at a delayof 0.7 ps suggesting that there is little rotation of the rotaxane within the lifetime of the clusterNH stretch mode.

Our system was on the other hand able to measure the coupling between the asymmetricand symmetric stretch of the amine group in the peptide Ac-Pro-NH2. We were able to concludethat the angle between the coupled oscillators in the unbound system was close to 90 degrees andthe angle between the coupled oscillators in the intra-molecularly hydrogen bound system are

32

between 45 and 90 degrees. This indicates that, were we able to observe a cross-peak betweenthe NH stretch modes in the rotaxane, we could have used these as a time-resolved indicator ofthe shuttling.

The electrochemical experiments have shown that a second, irreversible, process occurswithin the same range of voltages as the desired reduction of the rotaxane at the high con-centrations of the rotaxane necessary for the 2D IR experiments. Even though we failed toshuttle the rotaxane electrochemically, our experiments show that the electrochemical samplecell performs to our needs and can be used to probe molecular systems with IR light whilstperforming electro chemistry.

4.2 Future measurements

The shuttling, as stated above, could be induced permanently using a sacrificial donor. Anotherpossibility is to use a solvent such as DMSO-d6 to detach the macrocycle from the thread. Ifeverything else fails, a completely different rotaxane could be used. This however means thatwe would have to start from scratch. Possibly, in the far future, the shuttling can be triggeredby a 355 nm laser pulse and the subsequent breaking of the H-bonds between the macrocycleand thread monitored by an IR probe.

Since we found the coupling between the thread NH groups and the macrocycle NH groupsto be too small to observe, the coupling between the NH groups in the macrocycle and theC=O groups in the thread to which they are hydrogen bonded. The coupling between thesetwo groups should be much greater for several reasons: the groups are in much closer proximityto each other than the different NH groups; the groups are hydrogen bonded to one another.The first overtone of the thread C=O stretch mode (fundamental at 1640 cm−1[33]) is close inenergy to the fundamental of the macrocycle NH stretch mode (3395 cm−1). The stretch modeof the C=O groups in the naphthalimide station can also be used (fundamental vibration at1700 cm−1[33]). The macrocycle binds to these groups after being shuttled so we should be ableto observe cross peaks. In both cases, because of the large energy gap between the fundamentaltransitions, a two colour experiment has to be conducted on the rotaxane.

33

Chapter 5

Acknowledgments

The author would like to thank P. Bodis and Dr. S. Woutersen for their daily supervision;Prof. W.J. Buma and Prof. H. Bakker for their supervision; Dr. A. Rijs for donating thenaphthalimide rotaxane used in the experiments; H. Schoenmakers for technical assistance; F.Hartl and T. Mahabiersing for their help with the electrochemical experiments; Y. Rezus andR. Timmer for their help.

34

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