Observing Rotaxane Shuttling with Two-Dimensional Femtosecond Vibrational Spectroscopy. M.R. Panman

Transcription

1 Observing Rotaxane Shuttling with Two-Dimensional Femtosecond Vibrational Spectroscopy M.R. Panman November 27, 2007

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

3 Contents 1 Introduction Rotaxanes The principles of Two-Dimensional Ultrafast Infrared Spectroscopy Pump-probe spectroscopy Femtosecond 2D spectroscopy Observing molecular motion Experimental The two-dimensional femtosecond infrared setup The electrochemical setup Results and Discussion Testing the 2-dimensional ultrafast infrared setup Steady-state measurements Vibrational dynamics of the different N-H stretch modes Observing the rotaxane cross peaks Shuttling the rotaxane Conclusions and future measurements Conclusions Future measurements Acknowledgments 34 2

4 Chapter 1 Introduction 1.1 Rotaxanes Rotaxanes are almost always associated with the concept of molecular machines and have received considerable attention from nanoscience disciplines [1]. Rotaxanes are a subclass of mechanically 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 master thesis. is composed of a backbone (the axle ), referred to as the thread; a ring (the wheel ), referred to as the macrocycle, which is mechanically bonded to the thread; and two stoppers which keep the macrocycle from slipping off the thread. Also shown in figure 1.1 are two stations. These are 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 the clipping reaction. Such a reaction involves the binding (referred to as templating) of a precursor of the macrocycle to the thread through one of the stations. This binding site has a higher affinity for the macrocycle than the other. The precursor is then capped to form the macrocycle, this is called the rotaxination. The idea that rotaxanes could form the precursor of molecular machines comes from arguably the most interesting property of rotaxanes: the possibility for the movement of the macrocycle with 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 across membranes [2]. The movement of the macrocycle between two or more stations has to be induced by an external stimulus (figure 1.2). Initially, the macrocycle is bound to the station on which it was 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

5 Figure 1.2: An example of a generic rotaxane shuttling [2]. macrocycle is therefore located predominantly on the former. An external stimulus is applied to the system which disturbs the afore mentioned equilibrium by changing the macrocycle affinities of the stations. There are three main methods of inducing shuttling, each aimed at increasing the affinity of one station over the other [5]. The energy required for shuttling can be supplied via a chemical reaction, an electrochemical redox reaction, or photochemically. An example of a chemically switched rotaxane is the acid-base-switchable rotaxane presented by Garaudée et.al. [3]. Perez et.al. present a photochemically switched rotaxane [6] where the isomerisation of a double bond is the driving force of the shuttling. Nørgaard et. al. present an electrochemically switched rotaxane. The macrocycle dissociates from the first station, moves along the thread by force of Brownian motion [7], and binds to the second station. The system is reset after the effect of the stimulus has decayed (photochemical stimulus), turned off (electro chemistry), or reversed (chemical reaction). This process is shown in figure 1.2. The rotaxane used in the experiments described by this thesis was published by Brouwer et.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 shown in 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. Rotaxane shuttling 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 a radical 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 affinity binds to the ni station. After charge recombination, the hydrogen bond affinities return to the situation before photo excitation, the macrocycle disengages from the ni site and binds to the 4

6 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 %. The process the rotaxane undergoes with electrochemical switching is very similar to that of the photo induced shuttling. Instead of using light to create the radical anion, an electric current is used. 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 coupling between the N-H stretch mode of amide groups in the thread and macrocycle. The principles of 2D femtosecond IR spectroscopy will be discussed in the following section. 1.2 The principles of Two-Dimensional Ultrafast Infrared Spectroscopy The first 2D-IR spectrum was published by P. Hamm et.al. in 1998 [8]. The concept behind 2D femtosecond IR spectroscopy is analogous to 2D NMR spectroscopy [9, 10, 11]. Two-dimensional spectroscopic 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 system dynamics); the 2D-spectrum is 3D-structure sensitive because the coupling between different modes allows for the determination of the relative orientation of the modes with respect to one another; the technique spreads the resonances in two dimensions; and it can discriminate between dynamic and static spectral broadening [12, 13]. The afore mentioned allows the 2D IR spectrum to be decongested; the individual features contributing to the peaks in an IR spectrum can be separated and therefore, more information can be gleaned from the spectrum. The setup of the 2D-IR experiment we will using is discussed in the following chapter. Before we can understand 2D-IR experiments, an explanation of a vibrational 1D (or pump-probe) experiment is in order (following section). We shall begin, however, with a brief overview of the achievements and 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 experiments 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 vibrational states, the couplings between different vibrational modes as well as the intensity and orientation of the transition dipole moments of a quantum system (molecule). The principles of the pulsed frequency- domain will be discussed in greater detail in the following sections. 5

7 Transient 2D-IR (T2D-IR) is an extension to the double-resonance experiment. These techniques involve an extra pulse of UV-VIS light either before the IR pump and probe pulses, in the case of a regular and non-equilibrium spectral diffusion T2D-IR, or in between the two IR pulses, in the case of a 2D-IR (2D-IR-EXSY) experiment [11]. The T2D-IR experiment involves the relative timing of the IR pump and probe to be fixed but the timing between the UV-VIS pulse and the IR pump is variable. This configuration allows one to follow the structural changes of a molecule brought out of equilibrium by the UV-VIS pulse. Experiments of this type have been carried out on photo-switchable cyclic-peptides [14], transition metal complexes [15], and has been used to follow protein folding using the amide I band [16]. The other three experiments will 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 recently been performed by DeCamp et. al. [17, 18]. The spectrometer consists of a broad-band pump pulse and probe pulse that can be delayed with respect to the former to probe the dynamics of the molecule. The frequencies of the pump pulse are dispersed by a grating before interacting with the sample. The probe is dispersed by a grating after it hits the sample. The frequencies of the pump and probe are dispersed perpendicular to one another and are projected onto a 2D CCD array after having been upconverted in a nonlinear crystal with a 800 nm pulse. This setup produces a whole 2D spectrum in just a single shot. The disadvantages of both photon echo and double resonance experiments are the investment in required experimental resources and time. The spectrometer described by DeCamp et.al. requires relatively low cost equipment such as a CCD camera instead of an expensive IR detector and also allows for lower repetition rate lasers, making 2D-IR experiments more widely available. Only the very basics of the 2D-IR photon-echo experiment will be discussed in the following section. The 2D pulsed Fourier-transform technique is derived from its 1D variant [12] which originated from the spin echo NMR experiments in 1950 [19]. An IR-PE experiment requires two laser pulses. The first pulse excites a certain transition in the molecule and places it in what is called a superposition state. This state has a dipole associated with it and initially all the molecular dipoles oscillate in phase. However, due to the inhomogeneous distribution of vibrational frequencies of the dipoles (responsible for the inhomogeneous line shape of a transition band) the dipole moments dephase (free induction decay). After a time τ a second pulse is applied. This pulse initiates the rephasing of the dipole moments by inverting the phase vectors of the dipole moments. The speed at which the individual dipole moments have dephased is of no consequence and all the dipoles will have rephased at time 2τ from the initial pulse 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 excited molecules such that some dipole moments do not rephase. The echo will be less intense and as τ is increased, the echoes decrease in intensity [19]. The intensity of the echo as a function of time gives the homogeneous broadening of the system after a Fourrier transformation. A three pulse PE experiment is also possible. This involves a third pulse which allows for the probing of the dynamics of the system as well as the homogeneous broadening [20, 21]. A variation to the above 3 pulse PE experiment is the heterodyned PE experiment. This involves interfering a fourth pulse with the echo allowing to obtain the phase information as well as the intensity of the photon echo [22, 21]. There are many other types of photon echo experiments such as three pulse 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 and peptide structure [8, 9, 24, 25]. 2D-IR spectroscopy can be used to determine the distance and angle between two oscillators using the through space and through bond coupling between the transition dipoles of the vibrations. There are several examples in which 2D-IR offers insight where other forms of spectroscopy 6

8 cannot. A typically employed absorption band used to probe protein and peptide structure is the C=O stretch of an amide link in the polypeptide backbone (referred to as amide I band or amide I when deuterated) due to the strength of the signal and ease of detection in different solvents. This band is quite sensitive to secondary structure via hydrogen bonding between this moiety and its surroundings [25, 26, 27]. However, this band is a collection of all the C=O stretches 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 modes composing the amide I band [8, 24]. 2D-IR spectroscopy has also been used to complement other spectroscopic techniques, such as vibrational circular dichroism spectroscopy in the determination of the absolute structure of proteins [28]. Vibrational circular dichroism is the differential absorption of right and left circularly polarised light by the sample. This phenomenon is only present in chiral molecules and the absolute configuration of the sample can be determined by comparing the VCD spectrum with calculated spectra [29]. The disadvantage of VCD spectroscopy is that the intensities of the signals are 5 orders of magnitude smaller than IR signals. Additionally, steady state spectroscopy cannot provide information on the dynamics of the system. This disadvantage could be remedied by combining VCD and 2D-IR spectroscopy. A theoretical framework for a 2D-IR VCD experiment has been put forward by Cho et. al. [13] (this technique has not yet been applied in practice). The great advantage of this experimental method is the time resolution 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 by Choi et. al. [30] 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 same spectrum (a third pulse is often used as reference, this is of no importance to the discussion of this subsection but has experimental importance). The pump pulse is of a much larger intensity than the 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 (when chopped, see section 2.1 for a more detailed explanation), the probe pulse measures a normal steady-state spectrum. However, when the pump is present, the probe measures the absorption 7

9 change in the spectrum caused by the pump. Some of the population of the ν = 1 state is excited to the ν = 2 state (excited state absorption) by the probe at a frequency ω 2 = ω 1, where is the frequency difference between the ν = 1 and ν = 2 states due to the anharmonicity of the vibrational potential. Another portion of the population returns to the ground state due to stimulated 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 the result (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 (t 0, t 1, t 2, t 3 ). 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 state compared to the steady-state situation. This is observed as a negative intensity change in the transient absorption spectrum referred to as bleaching. 50 % of the bleaching is due to stimulated emission by the probe. This process induces the release of a photon which is also detected and so it seems there are less molecules in the ground state. Also there is more population in the ν = 1 state when the pump is present than in the steady-state situation. This is observed as a positive signal in the transient absorption spectrum referred to as induced absorption. The ratio between the induced absorption and bleaching of a vibrational mode should be 1:1 because the 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 the induced absorption and bleaching will result in lower observed intensities of both phenomena (if the system is perfectly harmonic, neither bleaching or induced absorption can be observed). The changes between the steady-state and excited-state spectra are, in the order of 10 3 absorption units. The probe can be delayed in time with respect to the probe. This allows the experiment to probe the population of the ν = 1 state at different times after excitation by the pump. The more the probe pulse is delayed with respect to the pump, the less population is present in the ν = 1 state. In this manner a decay curve is constructed from which the lifetime (T 1 ) of the system can be determined (right side figure 1.6) Femtosecond 2D spectroscopy The type of 2D-IR experiment we use is called a double-resonance, or a dynamic hole burning experiment [10]. As with a pump-probe experiment, two pulses are required for a 2D-IR experiment (there are also 2D experiments that require more pulses, such as heterodyned photon 8

10 echo experiments [10]) with one significant change: the pump is spectrally narrow. The central frequency of the pump pulse is changed over the desired range of frequencies whereas the probe pulse is unchanged. For each pump frequency, a pump-probe spectrum is recorded. This can be mapped out on a 2D-spectrum with the spectrum measured by the probe on the x-axis and the pump frequencies on the y-axis. A 2D-spectrum, which is also a difference spectrum, consists of diagonal peaks and cross peaks. The former are the pump-probe signals of the normal modes of the molecule which are also observed in the 1D spectrum. The cross peaks are the result of coupling between two different vibrational modes. The nature of this coupling is a through-space (and some throughbond) transition dipole moment interaction [31, 8, 9]. Shown in figure 1.7 is the vibrational energy-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 mode on the excitation of the other. This situation can be viewed as a combination of the energy of two states: the 11 state. Therefore, the effect of the coupling on the system is a slight change in energy of the 11 level compared to no frequency change when the two oscillators are not coupled (E 11 = E 01 + E 10 ). Imagine a situation where the pump only excites the 10 mode. When the spectrum is measured by the probe, a pump-probe signal is seen at the 10 transition frequency, the same as with a 1D experiment (figure 1.7). The excitation of one of the two modes in a molecule causes a decrease in frequency of the other mode. Therefore, in the presence of the pump the frequency of the 10 to 11 transition is red-shifted compared to the 00 to 01 transition. This is visible in the 2D-spectrum as an off-diagonal signal. This feature is due to the subtraction of two peaks representing the same transition rather than induced absorption and bleaching. The same holds for when the 01 mode is pumped. Structural information can be obtained from the anisotropy of the cross peaks [9]. The anisotropy of a sample is the difference between the difference absorption measured with a pump polarisation parallel and perpendicular to the probe polarisation. This is caused by the preferential excitation of molecules with the transition dipole moment parallel to the pump 9

11 or probe polarisation. Since the coupling is due to the interaction between transition dipole moments of the coupled vibrational states, one can imagine that the orientation of and the distance between the two oscillators with respect to one another can have influence on the observed intensity of the cross peaks. The anisotropy of a system is given by the following equation: R = α α α + 2 α (1.1) where R is the anisotropy, α is the difference absorption intensity with the pump polarisation parallel to that of the probe, and α is the difference absorption intensity with the pump polarisation perpendicular to that of the probe. The anisotropy should ideally be 0.4. Deviations from this number can be caused by several factors: reorientation of the molecule within the timescale of the lifetime of the mode, rapid energy transfer to a different mode, and misalignment for example. In order to measure the anisotropy of the system, the 2D-spectrum has to be measured twice: once with the the pump polarisation parallel to that of the probe and once with the pump polarisation perpendicular to that of the probe. The intensity of a diagonalpeak measured with parallel polarisations ( α ) should be a factor 3 higher than when the polarisations are perpendicular ( α ). This can be mathematically derived [32]. Before using the equation 1.1 to calculate the anisotropy of the cross peaks, the perpendicular spectrum has to be normalised to the parallel spectrum using the diagonal peaks [33]. The anisotropy can then be used in the following relation: R ij = 3cos θ ij (1.2) wherer ij is the anisotropy of the cross peak, θ ij is the angle between the coupled transition dipole 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. used transient UV-Vis absorption to observe the macrocycle motion [2]. Marlin et.al. use steady state UV-Vis spectroscopy to follow indirectly the shuttling motion in a chemically switchable rotaxane [5]. Nørgaard et. al. use X-ray reflectivity measurements to probe the out-of-plane structure and 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 coupling between the stretch modes of different N-H groups in the naphthalimide rotaxane. There are six 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-H groups in the macrocycle are hydrogen bonded to the thread carbonyl groups. The N-H groups in the thread are free. A more detailed discussion of the linear spectrum can be found in section 3.2. The benefit of using double-resonance 2D-IR spectroscopy over the aforementioned methods is that the cross peaks can only originate from the rotaxane. The transient UV-Vis absorption previously 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 is reduced. This would constitute as a direct observation of rotaxane shuttling. We also conducted a proof of principle experiment with a small peptide, Pro-Ac-NH 2 (figure 3.1). Electrochemical switching was chosen above photochemical switching because this manner of switching does not require DABCO in order to function. This avoids possible interference from this molecule when performing the 2D-IR experiments. Secondly, the quantum yield of the photo reduction reaction is too low for our purposes. We wish to observe the disappearance of 10

12 the cross peaks, therefore, if only 20 % of the rotaxanes in solution shuttle, the change in the 2D-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

13 Chapter 2 Experimental This chapter will describe the experimental details of the project. The 2D ultrafast IR setup is the center piece of the experiments and will be presented first. The electrochemical experiments will 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 representation of the setup is shown in figure 2.1. Light Generation and Two-Dimensional Ultrafast IR Setup 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 D4 C FPF BS DL GP -P 2D-Setup FM2 S FM1 F CP Spectograph + MCT Detector LASER OPA C1 BS BS C2 D1 C3 C4 L2 L1 IR generation 2D-setup: DL : Diode Laser GP : Germanium Plate D4 : Delay stage FPF : Fabry-Perot Filter C : Chopper -P : Adjustable lambda plate FM1 : Focusing Mirror S : Sample FM2 : Focusing Mirror F : Flag CP : Calcium fluoride Plate BS D2 D3 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

14 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 by the OPA (4575 cm 1 ) enters the IR generation and is frequency doubled (9150 cm 1 ) by a BBO crystal (C3). A portion of the light produced by the Ti:sapphire amplifier is focused into a KTP crystal (C4) by a lens (L1) where it is difference-frequency mixed with the doubled idler. The resulting broad-band mid-infrared pulses at 3350 cm 1 have an energy of 1 µj, and a bandwidth of 200 cm 1 FWHM (full-width-half maximum). The KTP crystal is not placed exactly in the focus preventing it from being damaged by the high intensity of the beam. The temporal overlap between the doubled idler and 800 nm is regulated by a delay stage (D3). The beam is re-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 a Germanium 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 collimated and can therefore be used for the alignment of the 2D-setup. In addition, the residual 800 nm from the difference-frequency mixing is absorbed by the Ge plate. A small portion of the IR light is split into a reference and probe beam by a CaF 2 beam splitter. The remainder is used as pump beam. The probe can be delayed with respect to the pump by a computer-controlled delay stage (D4). The pump beam, containing the majority of the intensity of the original IR light, is first passed through a computer controlled Fabry-Perot filter (FPF), chopper (C), and a 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 the Fabry-Perot filter. The chopper removes half the pump pulses which allows the setup to probe the sample in the presence and absence of the pump. The half-λ plate allows the polarisation between the pump and probe to be changed. The intensity of the narrow band pump beam is about 40 % of the intensity of the probe beam. All three beams are focused and spatially overlapped in the sample (S) by an off-axis 100 mm parabolic mirror (FM1) and subsequently re-collimated by a similar mirror (FM2). A computer controlled flag (F) is used to block either the pump or both reference and probe beams. All three beams are frequency-dispersed on a 2 32 HgCdTe (MCT) array detector. The pump and probe beams are projected on the same array. During the alignment of the Fabry-Perot filter the reference and probe beams are blocked by the flag such that only the pump beam is detected. We obtain 2D vibrational spectra by recording the absorption change of the sample as a function 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 cooperation with Dr. František Hartl, the schematic of which is shown in figure 2.2. This cell allows us to perform IR spectroscopy on the rotaxane in different redox states. The windows are made CaF 2 and are spaced 150 µm apart. All the electrodes are platinum wires. The working electrode is a wire grid with a circular hole cut into it, this is where the electrochemical reaction takes place and subsequently allows an IR beam to pass through the sample. The electrochemical sample-cell was controlled by a voltage generator and connected to a plotter which plots the current measured at the reference electrode versus the potential on the working electrode. The cell is compatible with standard sample cell holders which allows for taking IR spectra while conducting electrochemical reactions in the cell. 13

15 Figure 2.2: Schematic of the electrochemical IR sample cell. 14

16 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-NH 2 (figure 3.1), a small modified naturally occurring peptide, was chosen for its large signal and, expected clear cross peaks. The molecule has a free amine group (NH 2 ). As shown in figure 3.1, the molecule Figure 3.1: Ac-Pro-NH 2 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 a 7-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 respectively of the unbound peptide. The peaks at 3480 cm 1 and 3315 cm 1 are the the asymmetric and symmetric stretch respectively of the hydrogen bound peptide. The assignment of these peaks was done by Ishimoto et.al. [35]. A simple vibrational model was used to calculate the force constants of the vibrational modes and hence obtain the vibrational transitions. There can however be some discussion about whether one can still talk about symmetric and asymmetric stretch modes of the amine. One of the two hydrogens of the NH 2 group is bonded while the other hydrogen is almost completely free [35]. One could argue that the high frequency peak is not the asymmetric stretch of the amine group, but rather the stretch of the unbound NH. Subsequently, the lower frequency peak could belong to the stretching vibration 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 or bound NH. If this were the case, the free NH stretch would not feel the hydrogen bond and would not be broadened due to this effect. The results published by Tayyari et.al. [36] for a different intra molecularly bound molecule, APO (figure 3.3), could form a useful analogy to Ac-Pro-NH 2. They found that the intensity of certain peaks of APO in CCl 4 in the NH stretch region were concentration dependent. A peak at 3500 cm 1 increased in intensity after dilution and was assigned to the free NH stretching. A peak at 3375 cm 1 was found to decrease 15

17 0,5 0,4 absorption [OD] 0,3 0,2 0,1 0, wavenumbers [cm -1 ] Figure 3.2: The linear spectrum of 40 mm Ac-Pro-NH 2 in CDCl 3 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 bound state. A third peak at 3184 cm 1 was found to remain constant and was assigned to the intra molecularly hydrogen bound NH stretching. Raissi et.al. elaborated on this work and performed DFT calculations and determined the contribution of each vibration to the peaks observed in the spectrum [37]. It was determined that for APO in CHCl 3 the high frequency peak is composed 76 % of the free NH stretching and 21 % of the bound NH stretching. The middle frequency peak was found to be composed of 77 % of the bound NH stretching and 19 % of the free NH stretching. From the example of APO we can cautiously conclude that we can no longer speak of asymmetric and symmetric stretching modes in intra molecularly hydrogen bound Ac-Pro- NH 2. Cautious because the two systems are different in several important aspects, the angle made by the O H-N atoms is different in both cases. Ac-Pro-NH 2, as mentioned before, forms a 7 membered ring in the bound state whereas APO forms a 6 membered ring incorporating an extra double bond. Also, APO exhibits intermolecular hydrogen bonds whereas this is not the case with Ac-Pro-NH 2. Furthermore, APO can undergo tautomerisation. For the sake of simplicity, we will continue to use the assignments of the peaks given by Ishimoto et.al. [35]. 16

18 We expected the cross peaks measured in the 2D spectrum to be strong. This because the coupled transition dipole moments are in very close proximity to one another; the asymmetric and symmetric N-H stretch originate from the same NH 2 group. We also expect cross peaks between the asymmetric and symmetric stretch of the unbound system. We do not expect to see 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 the pump-probe signals of the normal modes, and cross peaks are observed in the perpendicular experiment at the following positions: a negative signal at (3397 cm 1, 3520 cm 1 ) with corresponding positive signal at (3270 cm 1, 3520 cm 1 ); a negative signal at (3515 cm 1, 3406 cm 1 ) with corresponding positive signal at (3626 cm 1, 3408 cm 1 ); a broad negative signal at (3311 cm 1, 3472 cm 1 ), also visible in the parallel spectrum; and a narrow negative signal at (3491 cm 1, 3307 cm 1 ) seen also in the perpendicular spectrum. The first two sets of positive and negative signal indicate coupling between the symmetric and asymmetric NH 2 stretch modes of the unbound conformer. The second two negative signals indicate coupling between the symmetric and asymmetric NH 2 stretch modes of the hydrogen bound conformer. This is what we expected to observe in the 2D spectra. The fact that the cross peaks are better observed in the perpendicular spectrum than in the parallel spectrum suggests that the angles between the coupled oscillators are very close to 90 degrees. This is especially true for the cross peaks associated with the unbound conformer; we do not observe these peaks in the parallel spectrum. The cross peaks associated with the hydrogen bound conformer have intensity in both the parallel and perpendicular spectrum. The shift in energy of the combination band ( 11 ) of the unbound system due to the coupling becomes apparent 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 compared to the corresponding negative signal at (3515 cm 1, 3406 cm 1 ). This is the opposite to what we observe with the other cross-peak and to what one would expect if the configuration of the vibrational energy levels is the same as was described in the introduction. The anharmonicity of the vibrational potential of this particular NH stretch is high. In this is the case, the effect of the coupling translates to an increase in energy of the combination state ( 11 ). The transition from the 10 state to the 11 state is higher in frequency than the transition between 00 to the 01. The sign of the cross-peak signals will therefore be the opposite to what is shown in the 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: The linear spectrum, when fitted, shows three prominent bands: 3438 cm 1, 3375 cm 1, and 3340 cm 1. Comparison of the spectrum of the naphthalimide rotaxane with that of the cyclohexane rotaxane, shown on the left of figure 3.6, allows us to assign the peaks. The cyclohexane rotaxane only has NH groups in the macrocycle and only displays one peak in the NH stretch region which therefore has to belong to the macrocycle NH stretching mode. The mid frequency N-H stretch peak 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 unbound thread N-H; the peak is thin and has the highest energy compared to the other two peaks. The two 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 in the direction of the vibration, weakening the N-H bond and therefore lowering the energy of the vibration. The broadness of the peak is due to a large distribution of possible hydrogen-bond 17

19 Pump frequency cm Probe frequency cm -1 Figure 3.4: Top: 2D-spectrum of Ac-Pro-NH 2 measured at parallel pump polarisation. Bottom: 2D-spectrum of Ac-Pro-NH 2 measured at perpendicular pump polarisation. The measurements were conducted at a probe time delay of 1 ps. Negative signals are in blue and the positive signals are in red. Darker colours indicate more intense signals. 18

20 0,40 0,35 0,30 10 mm 2.5 mm Normalised 2.5 mm 0,10 0,08 absorption [OD] 0,25 0,20 0,15 0,10 0,05 Absorption [OD] 0,06 0,04 0,02 0, , wavenumbers [cm -1 ] wavelength [cm -1 ] Figure 3.5: Left: Linear spectra of the rotaxane at different concentrations. Blue line: 2.5 mm in a 1 mm spaced sample cell. Red line: 10 mm in a 1 mm spaced sample cell. Dashed black line: 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-H stretch modes. 0,5 0,4 absorption [OD] 0,3 0,2 0,1 0, wavelenght [cm -1 ] Figure 3.6: Left: Chemical structure of the cyclohexane rotaxane. Right: Linear spectrum of the cyclohexane rotaxane in the NH stretch region. strengths. The lower frequency peak is indicative of rotaxane clusters. Different concentrations of the rotaxane affects the intensity of the low frequency band (right picture of figure 3.5); the peak increases in intensity with increasing concentration. If indeed the low frequency mode belongs to a rotaxane cluster and this is caused by the interaction between the macrocycle NH groups of one rotaxane and another, it is normal not to observe this cluster peak in the spectrum of 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 absorption bands. This allows us to calculate the coupling strength we can expect for our system using 19

21 the following equation [33]: β 1,2 = 1 4πǫ 0 ( µ1 µ 2 r 3 3 ( r µ ) 1)( r µ 2 ) r 5. (3.1) where the transition dipole moments for the thread NH ( µ 1 ) and macrocycle NH ( µ 2 ) are 0.12 and 0.15 Debye respectively. The distance between the dipoles ( r) is 5 Å and was obtained from the X-ray structure [2]. The coupling was calculated to be -1.3 cm 1. The value of this number should be greater due to through bond interactions, neglected by the calculation. This is nonetheless a very small coupling; it will not be easy to observe a cross-peak in the 2D spectrum of the rotaxane. To obtain an idea of the intensity of the cross-peak we can use two equations put forth by Hamm et.al. [24]: and β2 kl ǫ kl = 4 (ǫ k ǫ l ) 2 (3.2) I kl ǫ kl = ω l, (3.3) Ikk I ll where: β kl is the coupling strength of the coupling between modes k and l; ǫ k and ǫ l are the energies of modes k and l respectively; is the anharmonicity of the NH stretch vibrational potential (separation in cm 1 between the induced absorption and bleaching); ǫ kl is the crosspeak anharmonicity; we take ω l as the average bandwidth (FWHM) of transitions k and l; I kk, I ll, and I kl are the intensities of the k and l peaks (diagonal peaks) and the cross-peak (off diagonal). For our calculation, we take β kl = 1.2 cm 1, ǫ k = 3440 cm 1, ǫ l = 3395 cm 1, and ω l = 38.5 cm 1. This results in the ratio I kl Ikk I ll = The noise of our experimental setup is on the order of 10 5 and the intensity of our signals for the 2D measurements are on the order of This means that if our cross peak is about 10 2 smaller than our diagonal peaks the intensity of the cross peaks will be quite close to the noise value and will therefore be difficult 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 stretching mode of each of the different N-H groups. We used a 10 mm sample of the rotaxane in a 1 mm sample 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 with this setting eliminates the effects of rotational relaxation and resonant energy transfer from the measurement. The transient absorption spectra of the naphthalimide rotaxane in the N-H stretch 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 ps is mostly caused by the residual temperature effects of the macroscopic diffusion of the energy introduced 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 strength between the nitrogen and the hydrogen atoms due to the decreased pull on this bond. When the difference is taken between the spectrum in the presence of the pump and the spectrum in the absence of the pump, this slight shift in frequency translates into a signal. However, the sign of this signal is opposite to what one would expect leading us to believe the signal probably has contributions from the decay of the NH stretch modes. This is not implausible considering the lifetimes obtained from the transient absorption spectrum. To obtain the lifetimes of the separate modes, the individual bands were pumped with a spectrally narrow pump. Using this setup allows us to measure the lifetime of one specific 20

22 Intensity [OD] 0,004 0,002 0,000-0,002-0,004-0,006-0,008-0,010-0,012-0, ps 0.5 ps 1 ps 2 ps 10 ps wavelength [cm -1 ] Figure 3.7: Transient absorption spectrum of the naphthalimide rotaxane at different delay times between pump and probe pulses. mode whilst limiting the contributions to the signal from the other modes (from the linear spectrum we know the bands significantly overlap). The transient spectra of three different pump frequencies are shown in figure 3.8. The shape of the pump-probe signals does not change 0,0005 0,0005 0,0001 0,0000 0,0000 0,0000 absorption [OD] -0,0005-0,0010-0, ps 2 ps 10 ps absorption [OD] -0,0005-0,0010-0, ps 2 ps 10 ps absorption [OD] -0,0001-0,0002-0,0003-0, ps 2 ps 10 ps -0,0020-0,0020-0, wavenumbers [cm -1 ] wavenumbers [cm -1 ] wavenumbers [cm -1 ] Figure 3.8: Pump-probe experiments where the individual NH stretch bands are pumped with a spectrally narrow pump pulse. Left: Pump frequency 3340 cm 1. Middle: Pump frequency 3375 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 the observed time frame. Furthermore, the large anharmonicity of the NH stretch mode becomes apparent from these measurements. The bleaching of the free NH is situated at 3440 cm 1 and the corresponding induced absorption is located at 3300 cm 1. We do not observe the residual temperature signal at 10 ps in the spectra in figure 3.8. This is because the pump is significantly reduced in power after being passed through the Fabry Perot filter. We measured it to be about 40 % of the probe intensity. However, we do see that the transient absorption spectrum of the free NH still contains population in the excited states, the induced absorption of this mode is at the same position where we observe a positive signal in the 10 ps signal (figure 3.7). Finally, this spectrum also shows the importance of using a sufficiently spectrally narrow pump. A portion of the macrocycle NH group is being pumped as well as the intended thread NH group which shows as a lower frequency shoulder in the transient spectrum. The shoulder has disappeared at 10 ps suggesting this contribution has a shorter lifetime and therefore does not belong to the free NH stretch mode. According to Graener et.al. [38], the vibrational excitation of a hydrogen bonded OH group results in the predissociation of this hydrogen bond and subsequent reassociation of it. This 21

23 phenomenon was observed in the transient decay of the OH stretch mode of ethanol. The decay contained two different time constants, the dominant one (T 1 ) is the lifetime of the OH stretch mode itself and and the second lifetime (T eq ) 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 has similarities to the above system and we therefore expect to see the reassociation of the hydrogen bond 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 second time constant in the corresponding decay curve. Figure 3.9 shows transient decay curves obtained from two different measurements. We took 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 0 Intensity [mod] Intensity [mod] 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 Delay [ps] Delay [ps] Figure 3.9: Left: Transient decay curves of the induced absorption of the three different NH stretch modes taken from the transient absorption spectra (figure 3.8). The decay curve of the thread NH stretch mode is shown magnified by a factor of 5. Right: Transient decay curves of the three different NH stretch modes taken from the broad band transient absorption spectra. the induced absorption of the three modes for the determination of T 1 because it only contains information on the relaxation of the NH stretch mode. Any temperature effects or reassociation of the hydrogen-bonds happen at a much longer timescale and are visible in the bleaching part of the non-linear spectrum. This is because the bleaching monitors the population of the ground state whilst the induced absorption monitor the population of the excited state. We took the transient data from 3290 cm 1 when pumping 3427 cm 1 (thread NH stretch), 3233 cm 1 when pumping 3372 cm 1 (macrocycle NH stretch), and 3233 cm 1 when pumping 3320 cm 1 (cluster NH stretch). We fitted the decay curves with a single exponential decay: y = A 1 e x T 1 + y 0 (3.4) where A 1 is the initial amplitude of the time constant T 1, and y 0 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 the thread, macrocycle and cluster NH stretch vibration respectively. We unfortunately were unable to use the data from the bleached frequencies because the noise was too great to determine two time constants. In order to obtain these, we fitted the broad band pumped transient spectrum. The major disadvantage of doing so is that the decay curves of a certain mode will have contributions from the others and the lifetimes we obtain are therefore only indicative. We used a bi exponential decay to fit the data: y = A 1 e x T 1 + A 2 e x Teq + y 0. (3.5) 22

24 We decided to disregard the thread NH absorption band because the corresponding decay curve will have large contributions from the other modes. The T 1 obtained for the broad band data are as follows: for 3396 cm 1 (macrocycle NH stretch) 1.56 ± 0.3 ps and for 3319 cm 1 (cluster NH) 1.48 ± 0.04 ps. The T 1 of the free NH is longer than that of the other two, hydrogen bonded NH groups, the stronger hydrogen bonded low frequency NH stretch mode having the shortest lifetime. The reason for this is that the hydrogen bonded NH groups have a wider range of relaxation pathways due to the distribution of possible hydrogen bond strengths and will therefore relax quicker than the thread NH. The T eq obtained are: for 3396 cm ± 0.7 ps and for 3319 cm ± 5.3 ps. There is a large difference in the Teq of the macrocycle and cluster peak. This discrepancy could be caused by the topological constraints imposed on the macrocycle NH group. Once the hydrogen bond between the C=O of the thread and NH group of the macrocycle is broken, the two cannot travel very far apart because the ring structure keeps them in close proximity. The hydrogen bond 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 displays a much longer T eq. We wished to test whether the T 1 s obtained experimentally are to be expected by comparing them to calculated lifetimes. For this we used a model presented by Staib et.al.[39], originally applied to OH stretch modes in an ethanol dimer system. This model assumes dissociation of the hydrogen bond between an OH group and O group after the excitation of the OH stretch vibration 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 N system. We assume the total nuclear wavefunction of the system can be separated into two separate functions, one dependent on the low frequency OO stretch vibration, and the other dependent on the high frequency OH stretch vibration (referred to as the adiabatic wavefunction): Ψ(q,Q) = ν OH φ νoh ψ(q) νoh, (3.6) where q and Q are the normal mode coordinates of the OH stretch and OO stretch vibrations respectively. Fermi s Golden Rule will be used to calculate the hydrogen bond predisssociation rate of the different modes: T 1 1 = 2π ψ 1(Q) H c (Q) ψ 0 (Q) 2 ρe (3.7) where ρ(e) is the density of continuum states at energy E. This represents all states to which the system can relax to. The total hamiltonian of the system can be expressed by a matrix: ( ) H0 (Q) H H = c (Q) (3.8) H c (Q) H 1 (Q) where H 0 (Q) and H 1 (Q) define the OO potentials when ν OH =0 and ν OH =1 respectively. H c (Q) is the non adiabatic coupling matrix element (or the transition matrix element also observed in 3.7). Essentially, the diagonal matrix elements describe both the vibrationally excited state and ground state of the system with the off-diagonal elements providing the model with a description of how energy transfers from one state to the other. The vibrational potentials used in the diagonal elements are Lippincott-Schroeder potentials first presented in 1955 by Lippincott and Schroeder [40]. This potential was based on the on potential proposed in 1953 which had been successful in predicting bond dissociation energies and anharmonicity constants [41]. 23

25 Staib et.al.[39] found that the curve obtained by calculating lifetime using equation 3.7 can be fitted with a much simpler expression: T 1 (ν OH ) = k (δω OH ) α (3.9) where k is a constant, α = 1.8, and δω OH is the difference between the gas-phase and measured frequency of the OH stretch mode in question (in our case this changes to δω NH ). We take the gas-phase value of an unbound NH stretch mode from a similar rotaxane system to be 3496 cm 1 1. We fill in ω OH with values ranging from 3495 cm 1 to 3340 cm 1 in equation 3.9 to obtain the theoretical lifetimes of the different modes. In figure 3.10 we compare the calculated lifetimes with those obtained with the 1D experiments. We took k = which normalises the 1 ) [ps ] (τ lifetime frequencyofthe NH modes[cm 1 ] Figure 3.10: Comparison between the experimentally observed and calculated lifetimes (T 1 ) curve to the value of T 1 for 3395 cm 1. Unfortunately, the curve does not fit the data very well 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 possible that the theory used to describe the hydrogen bound OH group relaxation cannot be used to fit the 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 probe in a germanium semiconductor material we determined the pulse length to be 0.5 ps. Therefore measuring the 2D-spectra at 0.7 ps would avoid the initial non-linear effects associated with the pump and probe hitting the sample simultaneously and still give us an adequate amount of signal. 1 Personal communication from Dr. A. Rijs 24

26 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, and 10 ps for each pumped frequency. This was done with two pump polarisations: parallel with respect to the probe and perpendicular with respect to the probe. The signal measured with a perpendicular pump polarisation will be a factor of 3 less intense than the signal obtained with a perpendicular polarisation. Ideally, an experiment taken with a perpendicular pump polarisation should be left to accumulate data 9 times longer to obtain the same signal to noise ratio as one taken with a parallel polarisation. Practically, however, the perpendicular experiments were left to accumulate 3 times longer than the parallel experiments. The polarisation of the pump was switched after one complete scan at the parallel polarisation and after three scans at the perpendicular polarisation. This was done to avoid artifacts originating from changes in the sample and laser system over the course of the day. Changing the polarisation frequently ensures that any difference observed between the different polarisations originate from the physical properties of the sample and not from artifacts. The intensity of the pump, as stated in the above section, was typically 40 % of the probe intensity. The result of the above described experiment is shown in figure The 2D spectrum shows the bleaching and induced absorption of the diagonal peaks. The structure visible around the coordinates (3250 cm 1, 3250 cm 1 ) is most likely caused by scattering of the pump in the direction of the probe. We believe this because the 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 measured absorption of the free NH stretch mode in the linear spectrum and the measured absorption change in the nonlinear 2D spectrum. The transient absorption signal is given by the following equation: ( ) Tǫ α = log (3.10) where α is the absorption difference between the situation in the presence of the pump and in the absence of the pump, T ǫ is the transmission of the sample at a certain frequency in the presence of the pump, and T 0 is the transmission of the sample at a certain frequency in the absence of the pump. In the case of the bleaching, α should be divided by a factor of 2 to account for the stimulated emission. The linear absorption of the sample is given by the following equation: ( ) T α = log (3.11) T 0 where α is the absorption of the sample at a certain frequency, T is the transmission of the sample at a certain frequency, and T 0 is the transmission without a sample. If we divide α by α we obtain the percentage of molecules excited by the pump. We chose the free NH stretch mode (3440 cm 1 ) to determine the percentage of molecules excited. α is OD and α is OD. The percentage of the rotaxane excited by the pump pulse in the 2D experiments is 0.26 %. cross peaks are, however, not visible in the 2D spectrum. We enhanced the spectra by increasing the amount of contour lines in the low intensity range, the result shown in figure 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 these coordinates. This suggests there is coupling between the free NH stretch mode and the NH stretch vibration belonging to rotaxane clusters. We had expected a cross-peak between the free NH in the thread and the hydrogen bound NH in the macrocycle; the two modes are closer in energy than the free NH mode and the cluster vibration. However, the nature of the clusters might become apparent if the cross-peak does exist at the location we observe in the 2D spectra. T 0 25

27 Pump frequency cm Probe frequency 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 measurements were conducted at a probe time delay of 0.7 ps. Areas of blue indicate negative signal and areas of red indicate positive signal. Darker colours indicate more intense signals. 26

28 Figure 3.12: Top: An increase in contour lines at the low-intensity signals of the parallel measurement shown in figure Bottom: An increase in contour lines at the low-intensity signals of the perpendicular measurement shown in figure

29 This would suggest that at least one of the thread NH groups is in close proximity and possibly hydrogen-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 section of the 2D spectrum. The NH stretch vibration of the cluster was pumped (3315 cm 1 ) and frequencies from 3150 cm 1 to 3550 cm 1 were probed. The polarisation was switched in the same manner as was described for the 2D spectra above. We accumulated a lot of data in order to improve the signal to noise of the measurement. The difference in intensity between [OD] 0,002 Parallel Perp*3.13 0,000-0,002-0,004 difference 0,0002 0,0001 0,0000-0,0001-0,0002-0,006-0,0003-0, wavenumbers [cm -1 ] -0, wavenumbers [cm -1 ] Figure 3.13: Left: A cross section of the 2D spectrum. The rotaxane was pumped at 3315 cm 1 with the polarisation of the pump parallel (black line) and perpendicular (normalised perpendicular shown by the red line). Right: The difference between the measurement with parallel polarisation and normalised perpendicular polarisation the cluster NH stretch vibration measured with the perpendicular polarisation and the parallel polarisation is found to be a factor of Using equation 1.1 we obtain an anisotropy (R i,j ) of for the naphthalimide rotaxane dissolved in CDCl 3. This does not deviate much from the ideal anisotropy of 0.4 suggesting the rotaxane does not exhibit rotational relaxation, due to its large 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 the right of figure The sharp peaks are due to experimental error; a slight misalignment of the probe frequencies on the detector of one of the measurement at one polarisation with respect to the other. We see a broader negative peak at 3400 cm 1 and a small, but broad positive peak at 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 coupling between the thread NH and macrocycle NH necessary for us to observe a cross-peak in the 2D spectrum. The simulated spectrum with β = 0 cm 1 represents a system without coupling between the thread NH group and the macrocycle NH group and accordingly, shows no cross peaks. 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. The simulated spectrum with β = 5 cm 1 shows a positive cross-peak between the thread NH stretch and macrocycle NH stretch modes. The sign of the cross-peak might seem strange, one would expect to see a negative signal at this position if the vibrational energy levels were of the same configuration as the example shown in section However, like the Ac-Pro-NH 2 peptide, the anharmonicities of the NH stretch modes are very large, as is shown by the non-linear experiments. The sign of the cross-peak signals will therefore be the opposite to what is shown 28

30 a a 3450 b 3450 b pump frequency (cm -1 ) c pump frequency (cm -1 ) c probe frequency (cm -1 ) probe frequency (cm -1 ) a a 3450 b 3450 b pump frequency (cm -1 ) c pump frequency (cm -1 ) c probe frequency (cm -1 ) probe frequency (cm -1 ) Figure 3.14: Top Left: Experimentally measured 2D spectrum. Top Right: Calculated 2D spectrum with no coupling, β = 0 cm 1. Bottom Left: Calculated 2D spectrum with β = 2 cm 1. Bottom Right: Calculated 2D spectrum with β = 5 cm 1. Where in each case a is the linear spectrum of the rotaxane, b the 2D spectrum with pump polarisation parallel to the probe polarisation, and c the 2D spectrum with pump polarisation perpendicular to the probe polarisation. 29