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Intermediate phases during solid to liquid transitions in long-chain n-alkanes

The solid to liquid phase transition of n-alkanes with more than ten carbon atoms is an interesting phenomenon relevant to many fields, from cosmetics to automotive. Here we report Raman spectroscopy of tetradecane, pentadecane and hexadecane as a function of temperature. In order to gain information on the structural changes that the hydrocarbons undergo during melting, and to determine the temperature and the speed at which the phase change occurs, their temperaturedependent Raman spectra are acquired. The spectra are analysed not only with respect to frequency shifts, band widths, and intensity ratio of certain bands, but also using a principal component analysis. The spectroscopic data suggest that the solid to liquid phase transition in hexadecane, differently from tetradecane and pentadecane, is almost instantaneous. Tetradecane shows a slightly faster transition than pentadecane. In addition, a rotator phase as an intermediate state between the liquid and crystalline solid phases is identified in pentadecane. Different characteristic features in the solid spectra of the hydrocarbons relate tetradecane and hexadecane to a tryclinic crystalline structure, and pentadecane to an orthorhombic structure.

Introduction
The crystallisation of n-alkanes is an interesting subject area to explore because of its relevance to important industrial applications, such as the processing of fats, surfactants, and oils. 1 n-Alkanes not only represent the main building blocks of complex organic compounds, such as lipids, surfactants, liquid crystals and polymers, but are also the main constituents of traditional hydrocarbon fuels. The liquid to solid phase transitions in n-alkanes become particularly important for those compounds with more than ten carbon atoms as their melting point is above À30 1C. Consequently, such a transition may occur during winter time when the ambient temperature reaches the melting point leading to significant changes in the macroscopic properties of the fluid in the engine of a car.
In going from the crystalline ordered solid phase to the liquid phase, n-alkanes can show intermediate phases, called rotator phases. A rotator phase is a high-temperature solid phase, which exhibits long-range order in the molecular axis orientation, but lacks long range order in the rotational degree of freedom of molecules about their long axis. 2 Hence, the molecules are free to rotate. Rotator phases have been studied during the last two decades. [3][4][5][6][7][8][9] In general, odd-numbered n-alkanes with 9 o n o 39 and even-numbered alkanes with 20 o n o 38 show at least one rotator phase. 2 A few degrees Celcius below the temperature corresponding to the rotator phase, a solid-solid phase transition occurs where the crystal phase is formed. For n o 30, even n-alkanes undergo a liquid to solid phase transition that results in the formation of a triclinic phase, while odd n-alkanes form an orthorhombic crystal structure. 10 This behaviour was noted to decrease with increasing alkane chain length. A triclinic crystal system has three unequal axes all intersecting at oblique angles. An orthorhombic crystal system has three mutually perpendicular axes, coinciding with the crystallographic axes, all of different lengths. A greater stability is associated with even n-alkanes, which crystallize into the triclinic crystal structure. This is consistent with a heterogeneous freezing mechanism, in which the nucleation starts at the free surface of the sample, leading to surface freezing. 1 X-ray and surface tension measurements have been used by Wu et al. 11 to observe the formation of a crystalline monolayer on the surface of liquid n-alkanes a few degrees above the bulk solidification temperature. Later, Ocko et al. 12 studied the structure of the monolayer in detail determining its existence in alkanes with carbon number (n) of 16 o n o 50. Besides X-ray diffraction, Raman spectroscopy can be used to determine alkyl chain interactions and conformational order. In the Raman spectra of alkanes, for example, the ratio between the intensities of the asymmetric and symmetric CH 2 stretching modes is considered to be an indicator of the rotational and conformational order. 13 The dependance of the ratio of band amplitudes associated with CH 2 asymmetric and symmetric modes in Raman spectra of n-alkanes on the lateral order of extended chains was also demonstrated. 14,15 In this paper we use Raman spectroscopy to investigate and compare the solid to liquid phase transition of three different n-alkanes: tetradecane, pentadecane and hexadecane. In the past, Raman spectroscopy has been mainly used to investigate the solid to liquid transition of n-alkanes due to pressure. [16][17][18][19] A few studies used Raman spectroscopy to observe structural changes in pure n-alkanes due to a systematic change in temperature. Brambilla et al. 20 observed short intermolecular correlations activating the formation of segments of trans-planar chains in different alkanes just above and at the melting temperature. Zerbi et al. 21 observed structural changes taking place at the solid-liquid phase transition and during melting in n-nonadecane and selectively deuterated nonadecanes. Recently, changes in the fingerprint region of the Raman spectra of the n-alkane CH 21 H 44 and of some n-alkanes with 21 o n o 60 during melting were studied. 22,23 Here, for the first time, we present a systematic analysis and comparison of temperature-dependent Raman spectra of tetradecane, pentadecane and hexadecane in order to unveil the difference in the speed of their phase transitions while gaining information about their structural changes. Both the fingerprint (1000-1600 cm À1 ) and the CH stretching (2700-3100 cm À1 ) regions are considered in order to have an independent comparison of the results and determine if information can be obtained equivalently from either region. The two spectral windows are first analysed by combining the detection of frequency shifts, band widths, and intensity ratio of certain bands. Thereafter, the same regions are analysed separately using a Principal Component Analysis (PCA). This chemometric method takes into account all the spectral changes in each spectral window to detect characteristic signatures that may correlated with phase changes.

Hydrocarbons
Tetradecane, pentadecane, and hexadecane were purchased from Sigma-Aldrich and had a purity 499%. They were used as received. The chemico-physical characteristics of these components are summarised in Table 1. Samples were prepared by placing 20 mL of the hydrocarbon under investigation between two microscope glass coverslips (0.16 mm thickness) forming a thin liquid film of B150 mm in height.

Raman spectroscopy
The spectra were acquired using a 180-degree Raman set up, as shown in Fig. 1. A 532 nm laser (Laser Quantum, Ventus Solo) was used as excitation source and it was focused on the sample (with power of B30 mW in the sample plane) through a 50Â air microscope objective (Mitutoyo G Plan, NA = 0.5). In order to slightly overfill the back aperture of the microscope objective, the laser beam was expanded by using a 3.5 : 1 expansion telescope (L1 and L2). The laser irradiated the sample only during the acquisition of the spectra to avoid any possible laser induced heating.
The light scattered from the sample was collected by the same microscope objective used to deliver the excitation beam and then sent to a dichroic mirror (DM). The dichroic mirror acts as reflector at the laser wavelength, but transmits the Raman signal of the sample. The Raman signal then passes through a 514 nm dichroic beamsplitter (DBS). The DBS reflects a very small amount of the elastic light that leaks through the dichroic mirror to a CCD camera and lets the longer Raman shifted wavelengths pass. The signal, after being collimated by 2 achromatic lenses (L3 and L4), is then further filtered by a 532 nm notch filter (NF) and focused by a focusing mirror (FM) onto the entrance slit of a spectrograph (Andor Shamrock, entrance slit 220 mm, focal length 500 mm, grating 1200 lines per mm). The dispersed signal is detected using a CCD camera (Andor Newton DU920P-BEX2-DD). In order to optimise the Raman signal level the exposure time was varied automatically between 1 to 3.6 seconds.
Each sample was initially frozen by placing it in contact with the metallic surface of a cooling stage (PE120 Peltier System, Linkam Scientific) set at a temperature lower than the expected sample melting temperature (see Table 1). The samples were then heated at 0.1 1C min À1 until they became liquid. At each temperature step a pausing time of 2 minutes was set allowing the temperature in the sample to equilibrate at the temperature set in the cooling stage before acquiring the Raman spectra. The spectra, passing from the solid to the liquid state, were recorded each 0.1 1C. At the same time, the temperature of the cooling stage, which was assumed to be the same as the temperature in the thin film sample, was acquired. Using the above system, Raman spectra of tetradecane were acquired in the temperature range between 2 and 5.8 1C. Spectra of pentadecane were acquired between 7 and 9.8 1C and between À4 and À2.2 1C, while spectra of hexadecane were recorded between 15 and 18.4 1C.

Results and discussion
In this section, two different ways of analysing the temperature dependent Raman spectra of tetradecane, pentadecane and hexadecane are used. Firstly, the Raman spectra in the CH stretching and fingerprint region are studied with respect to bands position, frequency shifts, bands widths and intensity ratios of specific bands in order to identify the phase transition temperature and to investigate characteristic changes in the spectra. Secondly, a PCA, which mathematically detects overall spectral changes due to the variation in temperature, is used in the same spectral regions to distinguish between different phases.

Raman spectra
Tetradecane. The Raman spectra of liquid and solid tetradecane (taken at 2 and 5.8 1C, respectively) in the CH stretching and in the fingerprint region are displayed in Fig. 2.
In the liquid spectrum the wavenumbers around 2848 cm À1 and around 2872 cm À1 are attributed to the symmetric and asymmetric stretching vibrations of the CH 2 groups, respectively. The wavenumbers around 2930 cm À1 and around 2957 cm À1 are due to the symmetric and asymmetric CH 3 stretching vibrations, respectively. There appear to be two shoulder bands in the liquid spectrum: one at the high frequency wing of the 2848 cm À1 band and one between the 2884 cm À1 and 2930 cm À1 bands. The Raman intensities of all CH 3 asymmetric stretches are very small compared to the other modes and the spectral bands of these modes overlap with more intensive CH 2 asymmetric modes. The band associated with the asymmetric CH 2 modes at 2872 cm À1 , which merges with the broad Fermi resonance band at 2884 cm À1 , becomes strong and sharp and shifts to 2877 cm À1 in the spectrum of the solid phase. The band associated with the symmetric CH 3 modes, mixed with a second manifold of overtones and combinations of the CH bending modes at 2930 cm À1 , is quite broad and relatively strong in the liquid state and becomes narrower and weaker in the solid state. The band associated with the asymmetric methyl stretch at 2957 cm À1 in the liquid state is split into two bands at 2950 and 2960 cm À1 in the solid state. This split is typical for a triclinic structure. 15 The band intensity of the symmetric CH 2 stretching modes at 2848 cm À1 in the liquid spectrum decreases significantly when the sample becomes solid and red shifts to 2845 cm À1 . On the other hand, the weak shoulder on the high frequency side of the symmetric CH 2 modes in the spectrum of the liquid phase, becomes stronger in the solid state and it can be found at 2856 cm À1 . The intensities of the bands at 2845 cm À1 and at 2856 cm À1 are comparable, which is another feature of a triclinic packing of methylene chains. In the hexagonal alkanes, the band at 2856 cm À1 becomes a weak shoulder. In monoclinic and orthorhombic alkanes, this band contributes to the broadening of the band associated with the symmetric CH 2 modes. 15 Considering the Raman spectra in the fingerprint region, in the liquid state the bands are broader than in the solid state, indicating a reduction in intermolecular interactions, leading to a decreased influence in vibrational motion. In the liquid state, a weak shoulder can be found at the higher frequency of the CH 2 bending band (1434 cm À1 ). In the solid phase, the CH 2 bending band shifts to 1440 cm À1 and the CH 3 bending band becomes a well defined band at 1463 cm À1 .
The Raman spectra of tetradecane in the CH stretching and fingerprint region acquired every 0.1 1C in the temperature range between 2 and 5.8 1C are shown in Fig. 3. By a visual inspection of both regions tetradecane reach the liquid phase at 5.5 1C. This is in perfect agreement with the melting point reported in Table 1.
Different spectral changes can be observed in moving from the solid to the liquid phase in both the CH stretching and the fingerprint region. Focusing on the CH stretching region, in going from the solid to the liquid phase the CH 2 symmetric stretching band shifts to higher wavenumbers as soon as the transition starts (see red dashed line in the CH stretching region in Fig. 3). This blue-shift becomes clearer in Fig. 4(c), which shows the frequency shift of the CH 2 symmetric stretching band (difference of band wavenumber in a spectrum at a given temperature and the band wavenumber of the corresponding band in the spectrum of solid tetradecane at 2 1C) as function of temperature. Such a blue-shift, which indicates a stiffening of the CH covalent bond, could be due to a weakening of the intermolecular interactions during the transition to the liquid phase. Furthermore, a net change in the CH 2 symmetric stretching band intensity in going from the solid to the liquid phase is observed in Fig. 3. Changes in the intensity of the CH 2 symmetric stretching band (at 2848 cm À1 in the liquid spectrum) are due to different chain packing involving different lateral chain interactions. The absolute value of the intensity ratio between the band at 2872 cm À1 (at 2877 cm À1 in the spectrum of the solid tetradecane) and the band at 2848 cm À1 (at 2845 cm À1 in the spectrum of the solid tetradecane) would depend on the order due to lateral crystalline interactions associated with the molecular packing. A higher ratio corresponds to chains in a more crystalline state. In Fig. 4(d) this ratio is plotted as function of temperature. The ratio in the liquid phase is found to be B0.7 (at 5.5 1C), and in the solid phase it is found B1.67. Using such ratios of intensities, a quantitative measure of the lateral order in hydrocarbon systems has been proposed by Gaber and Peticolas,14 as given by the following expression: where r sample and r liquid are the Raman intensity ratios of the bands associated with the asymmetric and symmetric CH 2 stretching modes in the sample in an intermediate phase between the solid and the liquid phase and in the liquid phase, respectively. r crystalline is the value of the intensity ratio in the crystalline state. In terms of this relation, S lat = 1 will correspond to chains in the crystalline state, whereas S lat = 0 corresponds to chains in the liquid phase. Considering the fingerprint region, a blue-shift of the band associated with the twisting vibrational modes (see red dashed line in the fingerprint region in Fig. 3) and a broadening of the twisting band in passing from the solid to the liquid phase is observed. Further changes are visible in the CH bending region.
The data in Fig. 4(a) show the shift of the band associated to the twisting modes (difference of band wavenumber in a spectrum at a given temperature and the band wavenumber of the corresponding band in the spectrum of solid tetradecane at 2 1C) as function of temperature.
The data in Fig. 4(b) show the change in width of the twisting band (difference between the width of the band in a spectrum at a given temperature and the width of the same band in the spectrum of solid tetradecane at 2 1C) as function of temperature. In passing from the solid to the liquid phase a broadening of the band, indicating a reduction in intermolecular interactions, is observed. In conclusion, the data in Fig. 4 confirm that tetradecane is completely liquid at 5.5 1C as visually observed in Fig. 3, and as suggested by the data in Table 1. However, spectral changes in the CH stretching and the fingerprint region can be detected earlier between 4.8 and 5.1 1C. This shows that the transition from the solid to the liquid phase is not instantaneous, but it happens through a series of uncharacterised intermediate phases.
Pentadecane. Fig. 5 shows the Raman spectra of liquid and solid pentadecane (taken at 7 1C and 9.8 1C) in the CH stretching and fingerprint region. The symmetric and asymmetric CH 2 modes in the liquid pentadecane spectrum are observed at 2848 and at 2871 cm À1 , respectively. The CH 3 symmetric and asymmetric modes are observed at 2930 and at 2957 cm À1 , respectively. The band at 2885 cm À1 is due to Fermi resonance, similar to the spectrum of tetradecane. In the solid spectrum the band associated with the CH 2 symmetric modes decreases in intensity and can be found at 2843 cm À1 . The band associated with the asymmetric CH 2 stretching modes becomes narrower and shifts to 2877 cm À1 . Both the symmetric and asymmetric CH 3 modes become weaker and shift to 2931 and 2955 cm À1 , respectively. As for tetradecane, the ratio between the asymmetric and the symmetric CH 2 stretching modes increases when passing  from the liquid to the solid phase. However, contrary to tetradecane, the intensity of the symmetric CH 2 stretching modes, and the weak shoulder observed at 2856 cm À1 in the spectrum of solid pentadecane are not comparable. Furthermore, no splitting of the CH 3 asymmetric modes is observed in the solid phase. This indicates that pentadecane does not solidify in a triclinic phase. It is in fact known that it solidifies in an orthorhombic phase. 25 Typical of this phase is the conservation of the shape of the symmetric CH 2 modes in the liquid and solid spectra. 26 As for tetradecane, the bands in the fingerprint region of the liquid spectrum are broader than in the solid one. In the liquid spectrum, the CH 2 bending band and the Fermi resonance band can be found at 1435 cm À1 and 1445 cm À1 , respectively. Fig. 6 shows the Raman spectra of pentadecane in the CH stretching and in the fingerprint region acquired every 0.1 1C between 7 and 9.8 1C. As in the tetradecane spectra, in the CH stretching region a blue-shift of the CH 2 symmetric stretching band is observed when passing from the solid to the liquid phase. Also a blue-shift and a broadening of the twisting band in the fingerprint region is visible. A visual inspection of both regions indicates that pentadecane reaches the liquid phase at 9.6 1C.
The shifting of the band associated with the twisting vibrational modes, the change in width of the twisting band, the shifting of the symmetric CH 2 band and the intensity ratio of the asymmetric and symmetric CH 2 bands in passing from the solid to the liquid phase are displayed in Fig. 7.
The data in Fig. 7(a) and (b) show that spectral changes can be detected in both the CH stretching and the fingerprint region of pentadecane from 8.9 1C and that pentadecane reaches the liquid phase at 9.6 1C as confirmed by the data in Fig. 7(d). This is in agreement with the melting temperature range given in Table 1. The length of the transition phase in pentadecane is comparable to the one of tetradecane.
An odd-numbered n-alkane with n = 15, pentadecane is expected to show at least one rotator phase in turning from the crystalline ordered solid phase to the liquid phase. 2 The presence of this solid to solid phase transition can be observed in Fig. 8 in which the Raman spectra of pentadecane taken every 0.2 1C between À4.2 1C and À2.2 1C are shown. While changes in the CH region spectra as function of temperature are subtle, in the fingerprint region the CH bending band shows a more prominent change in shape at À3.6 1C, suggesting a transition from the crystalline solid phase to a rotator phase. Pentadecane can be found in this rotator phase until it starts turning to the liquid phase (see Fig. 6). The presence of a solid to solid phase transition is also suggested by a sudden frequency shift of the CH 2 asymmetric stretching band towards lower wavenumbers as the temperature increases (see red dashed line in the CH region).   This red-shift is highlighted in Fig. 9(a), which shows the wavenumber shift of the band associated with the CH 2 asymmetric stretching modes (difference of band wavenumber in a spectrum at a given temperature and the band wavenumber of the corresponding band in the spectrum taken at À4.2 1C) as function of temperature. A net shift can be observed at À3.6 1C. A change in the crystalline phase of the sample at À3.6 1C is also confirmed from the data in Fig. 9(b). It shows the intensity ratio of the asymmetric CH 2 and the symmetric CH 2 stretching bands as function of temperature.
Hexadecane. Fig. 10 shows the Raman spectra of solid and liquid hexadecane in the CH stretching and fingerprint vibrational regions at 15 and 18.4 1C, respectively.
The asymmetric CH 2 band at 2873 cm À1 , which merges with the broad Fermi resonance band at 2886 cm À1 in the liquid spectrum becomes strong and sharp and shifts to 2877 cm À1 in the solid spectrum. The band associated with the symmetric CH 3 modes, mixed with Fermi resonance, at 2930 cm À1 , which is quite broad and relatively strong in the liquid state, becomes narrower and weaker in the solid state. The band corresponding to the asymmetric CH 3 modes at 2957 cm À1 , in the liquid state, increases in intensity and becomes narrower in the solid state. Moreover, it shifts to 2960 cm À1 . The band intensity of the symmetric CH 2 stretching modes at 2848 cm À1 , in the liquid spectrum, decreases significantly when the sample becomes solid and it shifts to 2845 cm À1 . On the other hand, a weak shoulder that can be observed on the high frequency side of the symmetric CH 2 modes in the liquid state, becomes stronger in the solid state and it can be found at 2856 cm À1 . The band at 2856 cm À1 has a slightly lower intensity with respect to the symmetric CH 2 modes. The packing in the solid hexadecane is known to be triclinic and this is confirmed by the comparable intensity of the bands at 2845 cm À1 and at 2856 cm À1 , as in tetradecane. However, the splitting of the asymmetric CH 3 modes observed in tetradecane is not present. In the liquid spectrum of hexadecane in the fingerprint region the Fermi resonance band at 1448 cm À1 is broad and appears only as a shoulder on the high frequency side of the CH 2 bending band at 1435 cm À1 . In the solid spectrum it becomes a well-defined band at 1454 cm À1 and has comparable intensity with the CH 2 band. In the solid state, the CH bending band decreases in intensity. As in the other two n-alkanes the bands in the solid spectrum are narrower than in the liquid one. Fig. 11 shows the spectra of hexadecane in the CH stretching and fingerprint region in the temperature range between 15 and 18.4 1C. As for tetradecane and pentadecane, a blue-shift of the symmetric CH 2 band in the CH stretching region, and of the twisting band in the fingerprint region is observed when passing from the solid to the liquid phase. Visually, the transition to the liquid phase seems to happen quite suddenly at 18.1 1C. Fig. 8 Raman spectra of pentadecane in the temperature range between À4.2 and À2.2 1C. Fig. 9 Changes in the CH stretching region of the Raman spectra of pentadecane in the temperature range between À4.2 and À2.2 1C. (a) Wavenumber shift of the CH 2 asymmetric stretching band (at 2877 cm À1 at À4.2 1C) as function of temperature. (b) Intensity ratio of the CH 2 asymmetric and symmetric stretching bands (at 2877 cm À1 and at 2843 cm À1 at À4.2 1C, respectively). This is confirmed by the data in Fig. 12 that show the shifting of the band associated to the twisting vibrational modes ( Fig. 12(a)), the change in width of the twisting band ( Fig. 12(b)), the shifting of the symmetric CH 2 band (Fig. 12(c)), and the change in the intensity ratio of the asymmetric and symmetric CH 2 bands (Fig. 12(d)) during the transition from the solid to the liquid phase. In hexadecane, in contrast to tetradecane and pentadecane, the transition from the solid to the liquid phase seems to be almost instantaneous without intermediate phases.

Principal component analysis (PCA)
The Raman spectra of tetradecane, pentadecane, and hexadecane acquired at different temperatures were further analysed using PCA. This multivariate method, which has already been described in a previous paper, 27 identifies spectral characteristics that describe the variance of the data set. In other words, the principal components contributing to the individual spectra are extracted. PCA was performed separately on the CH stretching and fingerprint region data sets to have an independent comparison of results obtained from each region. Before running the PCA, each spectrum was processed to remove cosmic rays and normalised to the intensity of the maximum band in the spectrum. Matlab was used to perform all data processing.
Tetradecane. Firstly, the PCA was used to analyse the Raman spectra of tetradecane recorded between 2 and 5.8 1C (see Fig. 3). For the spectra collected in both the CH stretching and the fingerprint region, the most significant source of spectral variability in each data set is represented by the first two PCA components, which are plotted in Fig. 13. In the CH region the first two components explain 97.8% and 1.73% of variability, respectively. In the fingerprint region they explain 68.9% and 23.2% of variability, respectively.
Comparing the data in Fig. 2 with the bands in the PCA components shows that those bands arise from the variability in the Raman intensity of the bands in the original data set; therefore, a molecular origin to the features in the components can be assigned. The PCA components contain positive and negative features. An higher amount of the positive features and a lower amount of the negative ones is present in the spectra to which higher PCA scores are assigned.
The positive features in the first PCA component for the CH stretching region (Fig. 13(a)) correspond to the symmetric CH 2 Fig. 11 Raman spectra of solid and liquid hexadecane in the CH stretching and fingerprint region acquired in the temperature range between 15 and 18.4 1C.  stretching vibrations at 2848 cm À1 and to the asymmetric CH 3 stretching vibrations at 2957 cm À1 in the spectrum of the liquid phase. The negative band corresponds to the asymmetric CH 2 modes in the spectrum of the solid phase.
The positive features in the first PCA component for the fingerprint region (Fig. 13(d)) correspond to the C-C stretching at 1130 cm À1 , the twisting modes at 1291 cm À1 and the bending modes at 1463 cm À1 in the spectrum of the solid phase, while the negative features correspond to the bands in the spectrum of the liquid phase.
The positive and negative features in the second PCA component for the CH stretching region (Fig. 13(b)) are more difficult to assign and they most likely arise from the convolution of the bands in the spectra of the liquid and the solid phase during the transition phase.
The positive features in the second PCA component for the fingerprint region (Fig. 13(e)) correspond, as in the first component, to the bands in the spectrum of the solid phase.
The PCA scores (Fig. 13(c) and (f)) indicate how much of the variability explained by the first components is present in each of the tetradecane spectra in each spectral window. Positive scores associated with the first component in the CH stretching region (Fig. 13(c)) are correlated with increased liquid phase, and negative scores with increased solid phase. Conversely, positive scores associated with the first component in the fingerprint region (Fig. 13(f)) are correlated with increased solid phase, and negative scores with increased liquid phase.
In both Fig. 13(c) and (f), the solid and liquid phase can be clearly distinguished. In both cases, tetradecane seems to reach the liquid phase at 5.5 1C, confirming what was observed in the conventional analysis of the spectra in the previous section. If the CH stretching region is considered, the transition from the solid to the liquid phase clearly starts at 5.2 1C. If the fingerprint region is considered the transition seems starting earlier, at B4.8 1C.
Pentadecane. A PCA was also used also to analyse the Raman spectra of pentadecane recorded between 7 and 9.8 1C, see Fig. 6. Like for tetradecane, the two first principal components explain most of the variability in the pentadecane spectra collected in both the CH stretching and fingerprint region. In the CH region the first two components explain 95.7% and 4.1% of variability, respectively. In the fingerprint region they explain 75.2% and 21% of variability, respectively.
The positive features in the first pentadecane PCA component for the CH stretching region (Fig. 14(a)) retain the same meaning as the tetradecane first PCA component in the CH stretching region. In the first PCA component for the fingerprint region ( Fig. 14(d)) most of the negative features can be assigned to the bands in the spectrum of the solid phase. In particular, the bands at 1056 cm À1 , at 1130 cm À1 and at 1459 cm À1 . The positive features are most likely due to the convolution of the bands in the spectra of the liquid phase and the bands in the spectra taken during the transition from the solid to the liquid phase.
The positive and negative features in the second PCA component for the CH stretching region (Fig. 14(b)) are most likely due to the convolution of the bands in the spectra of the liquid and the solid phase during the transition phase as in the case of tetradecane.
The positive features in the second PCA component for the fingerprint region (Fig. 14(e)) correspond to the bands in the spectrum of the solid phase.
The data in Fig. 14(c) show that positive scores associated to the first component in the CH stretching region are correlated with increased liquid phase, and negative scores with the solid phase. In the same way, positive scores associated to the first component in the fingerprint region ( Fig. 14(f)) are correlated with increased liquid phase, and negative scores with the solid phase. In both regions, the liquid and solid phase are well distinguishable.
In contrast to tetradecane, considering either the CH stretching or the fingerprint region, the transition from the solid to the liquid phase in pentadecane starts at 9 1C, and it results to be liquid at 9.6 1C, confirming the above observations. From the PCA it is even more clear that the transition from the solid to the liquid phase takes longer in pentadecane than in tetradecane.
The data in Fig. 15 show the score plots derived from the PCA of the spectra of pentadecane recorded between À4.2 and À2.2 1C (see Fig. 9). The two different solid phases can be distinguished and the transition between the crystalline solid phase to the rotator phase occurs at À3.6 1C. The two phases can be better distinguished if the PCA is done on the CH stretching region even though a more dramatic change seems to be observed in the fingerprint region.
In order to verify if the rotator phase coincides with the solid phase observed between 7 and 9.8 1C, a PCA considering as input data both the data recorded between À4.2 and À2.2 1C and the data recorded between 7 and 9.8 1C is carried out. In this case, only the CH stretching region, which is the one better predicting the presence of two different solid phases between À4.2 and À2.2 1C is considered. The score plot, shown in Fig. 16, suggests that the rotator phase occurring at À3.6 1C lasts until pentadecane begins to melt at 9 1C.
Hexadecane. Fig. 17 shows the results from the PCA of the Raman spectra of hexadecane acquired between 15 and 18.4 1C (see Fig. 6). As for tetradecane and pentadecane, only two principal components for both the CH stretching and the fingerprint region are retained to explain the variability in the data sets. In the CH region the first two components explain 98.6% and 1.2% of variability, respectively. In the fingerprint region they explain 97.3% and 2.3% of variability, respectively.
Some of the positive features in the first hexadecane PCA component for the CH stretching region (Fig. 17(a)) can be assigned to the vibrational modes identified in the spectrum of the liquid phase (see the band at 2848 and 2957 cm À1 corresponding to the symmetric CH 2 modes and to the asymmetric CH 3 modes, respectively). The negative band at 2877 cm À1 is assigned to the asymmetric CH 2 modes in the spectrum of the solid phase.
In the first PCA component for the fingerprint region ( Fig. 17(d)) most of the positive features can be assigned to the bands in the spectrum of the liquid phase, while the negative ones corresponds to the bands in the spectrum of the solid phase.
The positive features in the second PCA component for the CH stretching region (Fig. 17(b)) corresponds to bands that can be found in both the spectra of the liquid and the solid phase.
The features in the second PCA component for the fingerprint region (Fig. 17(e)) retain the same meaning as the features in the first component for the same region.
The scores plots in Fig. 17(c and f) show as for pentadecane that positive scores associated to the first component in both regions are correlated with increased liquid phase, and negative scores with the solid phase. However, in contrast to the other two hydrocarbons, the transition from the solid to the liquid phase is almost instantaneous (it takes less than 0.1 1C) showing hexadecane to be totally liquid at 18.1 1C if the CH stretching region is considered and at 18.2 1C if the fingerprint region is analysed.

Conclusion
In this paper we investigated the solid to liquid phase transition of tetradecane, pentadecane, and hexadecane analysing their temperature dependent Raman spectra.
Spectra were analysed using conventional spectra analysis, i.e. detecting wavenumber shifts, band widths, and intensity ratio of certain bands together with Principal Component Analysis. Using conventional spectra analysis common characteristics   between the Raman spectra of even and odd n-alkanes have been detected. The spectra of all the hydrocarbons show a blueshift when passing from the solid to the liquid phase. This can be attributed to the fact that the bonds in the solid phase are more rigid making molecules vibrate at lower frequencies.
Another common characteristic is that the ratio between the asymmetric and the symmetric CH 2 stretching modes increases when the n-alkanes become liquid. In addition, specific features of even and odd n-alkanes have also been observed, for example, a net decrease of the band intensity of the CH 2 stretching band when passing from the liquid to the solid phase in even n-alkanes. Furthermore, the weak shoulder on the high frequency side of the symmetric CH 2 modes, in the liquid state, becomes stronger and red-shifted in the solid state. Another important feature in the spectra of even n-alkanes was that the band associated with the asymmetric CH 3 stretching splits into two when passing from the liquid to the solid phase. This split is typical of a triclinic crystal structure. However, the splitting was only observed in the spectra of tetradecane, but not in the ones of hexadecane, even though it is known to have the same triclinic structure. No splitting of the band associated with the CH 3 asymmetric modes was observed in the Raman spectra of pentadecane. Odd n-alkanes, in fact, do not solidify in a triclinic phase, but in an orthorhombic phase. A distinct feature of this phase is the conservation of the shape of the symmetric CH 2 when passing from liquid to solid.
By combining conventional spectral analysis with PCA we correlated spectral changes with molecular changes caused by the variation in temperature identifying the temperatures at which the hydrocarbons start melting. While tetradecane and pentadecane take longer to transition (between B4.8 and 5.5 1C and between 9 and 9.6 1C, respectively), hexadecane shows an almost instantaneous change in phase at 18.1 1C.
The combination of the two different approaches for data evaluation allowed the identification of rotator phases as intermediate states between liquid and crystalline solid. A rotator phase was in fact detected by analysing the spectra of pentadecane in the temperature range between À4 and À2.2 1C.
In conclusion, Raman spectroscopy is a very valuable tool to study phase transitions in hydrocarbons and the combination of conventional spectral analysis and PCA is helpful to gain insights.


extfieldsofstudy: Physics Chemistry

text:
The methods of optical physics as a mean of the objects’ molecular structure identification (on the base of the research of dophamine and adrenaline molecules)

Structural and dynamic models of dopamine and adrenaline are proposed on the basis of ab initio quantum calculations of the geometric and electronic structure. The parameters of the adiabatic potential are determined, a vibrational states interpretation of the test compound is proposed in this work. The analysis of the molecules conformational structure of the substance is made. A method for calculating the shifts of vibrational excitation frequencies in 1,2,4-threesubstituted of benzole is presented. It is based on second order perturbation theory. A choice of method and basis for calculation of a fundamental vibrations frequencies and intensities of the bands in the IR and Raman spectra is justified. The technique for evaluation of anharmonicity with cubic and quartic force constants is described. The paper presents the results of numerical experiments, geometric parameters of molecules, such as the valence bond lengths and angles between them. We obtain the frequency of the vibrational states and values of their integrated intensities. The interpretation of vibration of conformers is given. The results are in good agreement with experimental values. Proposed frequency can be used to identify the compounds of the vibrational spectra of molecules. The calculation was performed quantum density functional method DFT/B3LYP. It is shown that this method can be used to modeling the geometrical parameters molecular and electronic structure of various substituted of benzole. It allows us to construct the structural-dynamic models of this class of compounds by numerical calculations.

Introdution
Detecting the features of compounds spectral identification, their interpretation or their relation to a certain molecular fragment are both fundamental target of mathematical modeling in the theory of molecular system and applied one according to the quantitative and qualitative composition of bioobjects i.e. relations "State-Structure-Spectrum". The achievement of the given problem poccesses its own special features for each composition. The present work demonstrates the possibility of the use of the up-to-date programming software to create structural and dynamical models of the substance and accomplishment on the base of the spectrum identification of the substance. Optical physics methods are the most informative in the studied structures of the discussed types of the molecular objects.
Solving problems of the discussed types is possible due to the high techs of the molecular modeling i.e. the use of quantum method of analyses of parameters of adiabetic potential. These methods in the beginning of the 21 century completely replaced the classical methods of molecular dynamics which are based on the soluteon of inverse problems in theoretical physics.The applied methods are imple-mented as applied programming lay-outs. Dominating one is "Gaussian" according to the frequence use. Its particular feature is the implementation of the method of functional density DFT/B3LYP allowing to model effectively the problems discussed in article.
The interest to the study of the structure and spectrum of molecule of dofamine and adrenaline is understandable ( Figure 1). As an example we refer to the works [1,2,3] where just one of the possible conformers' combinations. Интерес к изучению структуры и спектра молекул дофамина и адреналина (рис.1) понятен. Сошлемся, к примеру, на работы [1,2,3], где рассмотрен лишь один (А) из возможных конформеров соединений. In the work [3] such interpretation for high frequency range is differ from data given in study [4]. In the work [1] there used a classical approach to evaluate the parameters of harmonic force field . In the work [2] the authors researched the range of 570-1880 sm-1. The spectrum modeling calculation are limited by harmonic approximation of the molecular vibrations theory. To measure anaharmonic displacement there used the procedure of scaling [5].
Structurally dopamine and adrenaline are related to 1,2,4benzol substituents(С 6 Н 3 ХYZ).According to the study [6] the number of fundamental vibrations of benzol trisubstitution could be divided into following groups: characterized by frequency,mode and intensity,characterized by frequency and mode , characterized by mode. Theoretical interpretation of fundamental vibrations of spectrum suggested in the work [6] completely agrees with experimental fundamental vibrations spectrum allocation 1,2,4benzol substituted (X=F, CH 3 ), suggested in the work [7]. This gives the possibility to assume that the given interpretation of spectrum skeleton will possess a space for both dopamine molecules and (X,Y=OH, Z=СН 2 -NH 2 )and adrenaline (X,Y=OH, Z=СНОН-NHСН 3 ). The accuracy of such idea could be confirmed by quantum calculations. The given work suggests structural and dynamical models of possible dopamine and adrenaline conformers on the base of model quantum calculations.All calculations are carried out according to the density functional method DFT/B3LYP [8] with the use of the basis 6-311G*, 6-311G**, 6-311+G**.

Methods
The evaluation of the bands' state was carried out by means of the following ratio [9]      (1) There used the equation from the work [10] for anaharmonic constants χ sr .
When testing the method DFT/B3LYP resources , during the primarily calculations of geometrical and electronic structures 1,2,4-trisubstituted benzol , there used relevant difluoro and dihydroxytoluene .The given approach was used in the works [11][12] when constructing structural and dynamical models of a number of trisubstituted benzol.

Results
According to the calculations the geometrical structure of benzol ring when being replaced is not changed greatly. The length of the valence bonds are distinguished by the amount of ~ 0.008 Ǻ, values of the bond angles ~ 1.6º.
The results of model calculations of the vibrational state of the benzol skeleton of the possible conformers of the given test compositions completely agree with the experimental fundamental frequency assignement suggested in the works [1,6,7].
In studied conformers of 1,2-digydro-4-methyl benzol, the great interest is in the band position which are interpreted as valent (q OH ) and non-plane deformational vibrations (χ OH ) of hydroxyl sections. These are the doublets ~ 3710 and 3650 sm -1 in highly frequency range of vibrational spectrum for conformers A and B . The bands are identical for the third conformer (~ 3700 sm -1 ). We can notice that in the works [1,3] the value of frequency being interpreted as valent frequency bonds ОН (q OH ) is transformed into long waved area of the frequency spectrum for ~ 300 sm -1 in comparison with data given above and suggested assignment for the mentioned frequency from the work [8]. Due to the fact that the relevant experiment introduced in the works [1,3] corresponds to the condensing state of dopamine and adrenaline.
The band state interpreted as torsion vibrations χ OH , is characterized by the following doublets : 410 и 210 sm -1 , 420 и 170 sm -1 , 360 и 310 sm -1 . According to the model calculations the bands are the most intensive in a low frequency range of vibrational spectrum .
The most important fact is the vibrational characteristics of the benzol skeleton according to the frequency. There can be observed characteristics according to the intensity for the given type of benzol trisodium while interfusing according to the form for deformational vibrations of hydroxide fragments. При отсутствии смешивания по форме с деформационными колебаниями гидроксильных фрагментов, наблюдается характеристичность по интенсивности для данного типа тризамещенных бензола.The testing results confirm the suggested theory.The intensity band changing values is connected either with the varied electronic substitutes' properties or with the interfusion of ordinary vibrations forms interpreted as deformational vibrations of the relations СН (H=H, F, O) in the model calculations. It can be seen in 1,2 dihydro-4 methyl benzol in the range of е 1550-1100 sm -1 .
The interpretation of vibrational spectrum of dopamine is introduced in the table 1. It was supposed that a molecule belongs to the symmetry group C s (the carboatomic plane is the molecule symmetry plane). Torsional vibrations were produced when the fragments СН 2 С и СН 2 NH 2 of substitutes are in the transconfiguration relatively each other . For the spectrum identification of conformers there can be used IR bands intensity in the range of 1200-1400 см -1 and the bands interpreted as deformational non-flat vibrations of the bonds ОН (χ OH ).The use of the basis 6-311G+** [7] leads to the displacement of calculated values of vibrations (Δ~ 50 см -1 ). But the significant intensity of the given bands in IR spectrum in the range of lower than 400 sm -1 does not create difficulties in the interpretation of low frequency range The determined conformity related to the spectrum bands of the benzol fragment of 1,2,4-trisubstituted benzol allows to interpret the fundamental vibrations of the fragment of СН 2 -СН 2 NH 2 .Their agreement with the experimental data on the characteristic frequency vibrations for the normal paraffins and methylamine [5] confirms the relevance of the harmonic strain constanta from the close molecular fragments system system transfer scheme used in the work of [5] to solve the reverse problems in classical theory of molecular vibrations.
The interpretation of spectrum fundamental vibrations of adrenaline suggested in the table 2 confirms the arguments given above which are related to dopamine spectrum identification. All fundamental vibrations of the compounds could be divided into two groups. The first one is connected with benzol skeleton of С 6 Н 3 Х 3 .The adrenaline symmetry decrease to the group of С 1 does not influence on the bands' position and evaluation of their intensity. The second group is interpreted as the vibrations of the fragment СНOH-СН 2 NHCH 3 . The hydrogen atom substitution into the methyl group in the fragment NH2 leads to the same patterns in the frequency vibrations behavior that happen while transferring from methylamine to dimethylamine [5]. The bands interpreted as valent (q OH ) and nonplane deformational vibration of the bond ОН (χ OH ) of the fragment СНOH-СН 2 NHCH 3 is difficult to use for the conformers compounds spectral identification.

Conclusions
The carried modeling calculations of the vibrational state of rank 1,2,4 replaced benzol particulary dopamine and adrenaline allow us to confirm that the method of the density functional DFT/B3LYP can be used to construct structural and dynamical models of the complex molecule compounds of the given type. The given fundamental vibrations could be relatively divided into two groups. The first group is related to the vibrations of the benzol fragment С 6 Н 3 Х 3 . If to carry out the mass compare (X=F, CH 3 , OH) the vibrations could be considered as characteristic according to the frequency and form. Another group is related to the fundamental vibrations of a molecular fragment which is obtained by 4 varied molecular fragments .substitution of a hydrogen atom of a methyl group in a position.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

text:
Self‐Assembly of G‐Rich Oligonucleotides Incorporating a 3′–3′ Inversion of Polarity Site: A New Route Towards G‐Wire DNA Nanostructures

Abstract Obtaining DNA nanostructures with potential applications in drug discovery, diagnostics, and electronics in a simple and affordable way represents one of the hottest topics in nanotechnological and medical sciences. Herein, we report a novel strategy to obtain structurally homogeneous DNA G‐wire nanostructures of known length, starting from the short unmodified G‐rich oligonucleotide d(5′‐CGGT‐3′–3′‐GGC‐5′) (1) incorporating a 3’–3′ inversion of polarity site. The reported approach allowed us to obtain long G‐wire assemblies through 5′–5′ π–π stacking interactions in between the tetramolecular G‐quadruplex building blocks that form when 1 is annealed in the presence of potassium ions. Our results expand the repertoire of synthetic methodologies to obtain new tailored DNA G‐wire nanostructures.

Introduction
In the development of new nanotechnologies and biomaterials, the possibility of assembling ordered supramolecular structures starting from small building blocks and the exploitation of self-assembly driving forces are very important and much studied topics. [1][2][3] In the bottom-up approach, DNA strands have been demonstrated to be very useful building blocks for the assembly of supramolecular structures of various dimensions and shapes. For example, the DNA nanotechnology known as DNA origami allows the design and the construction of tailored three-dimensional superstructures by exploiting the Watson-Crick base-pairing scheme. [4][5][6][7] Besides the Watson-Crick base-pairing scheme, also the Hoogsteen hydrogen bondingo ne, which allows the formation of G-quadruplex DNA, can be exploited to obtain DNA supramolecular struc-tures. G-Quadruplexes are unusual secondarys tructures of DNA, which form when guanine-rich DNA strandsa re annealed in the presence of suitable monovalent or divalent cations.
The building block of G-quadruplexesi st he G-quartet (Figure 1A), ap lanar arrangement of four guaninesh eld together by ac yclic array of eight Hoogsteen hydrogen bonds. [8][9][10] The onset of p-p interactions among the stackedG -quartets greatly stabilizes the G-quadruplex assembly,w hich is generally more stable than aD NA duplex of the same length. AG -quadruplex can be formed by one, two, or four G-rich DNA strands and can be classified as either parallel, antiparallel,o rh ybrid type depending on the mutual orientation of the strands in-Obtaining DNA nanostructures with potential applicationsi n drug discovery, diagnostics, and electronicsi nasimple and affordable way represents one of the hottest topics in nanotechnological and medical sciences.Herein, we report an ovel strategy to obtain structurally homogeneous DNA G-wire nanostructures of known length, starting from the short unmodified G-rich oligonucleotide d(5'-CGGT-3'-3'-GGC-5')( 1)i ncorporat-ing a3 '-3' inversion of polarity site. The reported approacha llowed us to obtain long G-wire assembliest hrough 5'-5' p-p stacking interactions in between the tetramolecular G-quadruplex building blocks that form when 1 is annealed in the presence of potassium ions. Our results expand the repertoire of synthetic methodologiest oo btain new tailoredD NA G-wire nanostructures. volved in the G-quartets formation. [11,12] Further factorst hat contribute to the wide polymorphism of G-quadruplexes are the length and the base composition of the loops (when present), as well as the nature of the cationsu sed to stabilize the quadrupleh elix structure. [13][14][15] The biological implications of Gquadruplexes in cellular processes [16][17][18] and their use as promising drugs [19][20][21][22] or in drugs delivery [23] and diagnostics [23][24][25] are well known. In addition, G-quadruplexes possess greater conductivityt han DNA duplexes, thus suggesting their use for the obtainment of electronic nano-biomaterials and nanodevices. [26][27][28] These properties indicate the G-quadruplex scaffold as au seful structuralm otif to obtain supramolecular self-assemblies, including the so-called G-wires. [29][30][31][32] G-wire superstructures can reach the length of thousands of nanometers along the axis perpendicular to the G-tetrad planes. Depending on the topological arrangement of the G-rich strandsp articipating in their formation, G-wires can be classified into two categories:i )interlocked G-wires,c haracterizedb yt he cooperative assembly of interlocked slipped strands( Figure 1B)a nd ii) stacked G-wires,c haracterized by the multimerization of Gquadruplexb uilding blocks held together by end-to-end p-p stacking ( Figure 1C). Besides the well-known interlocked Gwires [29][30][31][32][33][34][35] and G-wires containing both topological motifs, [36][37][38] only af ew examples of exclusively stacked G-wires have been reported so far. [39][40][41] In ap revious study,w er eported that in the presenceo fK + cations the 7-mer d(CGGTGGT)c an assemble into the octameric higher-ordered G-quadruplex complex d(CGGTGGT) 8 (Figure S1 in the Supporting Information) through p-p stacking of two unusualG (:C):G(:C):G(:C):G(:C) planar octadsb elonging to two identicalt etramolecularp arallel G-quadruplexes. [42] Later, we demonstrated that the same dimerization pathway is also possible for all the other d(CGGXGGT) DNA strands, with X = A, G, or C. [43] Herein, we report on the achievement of an ew type of stacked G-wire, here indicated as Q n (Figure 2) obtained by exploiting the 5'-5' p-p stacking interactions between the G(:C):G(:C):G(:C):G(:C) octads formed at both ends of the tetramolecular G-quadruplex building block Q 1 (Figure 2). The last forms when the d(5'-CGGT-3'-3'-GGC-5')D NA strand (1, Figure 2), incorporating a3 '-3' inversion of polarity site, is annealed in the presence of K + ions. The resulting G-wires were obtained as ad istribution of quadruplexm ultimers of different length.
The n subscript in Q n indicates the number of tetramolecular G-quadruplex building blocks participatingi nt he G-wirese longation.T he actual formation of the target G-wires was confirmed by polyacrylamide gel electrophoresis( PAGE), HPLC size exclusion chromatography (HPLC-SEC),c ircular dichroism (CD), 1 Hn uclear magnetic resonance (NMR) spectroscopy,a nd atomic force microscopy (AFM) studies. The analytical results allowed us also to gatheri nformation on the G-wires assembly and stability,a nd to isolate and characterize the three shortest Q n species (n = 2-4).

Synthesis of d(5'-CGGT-3'-3'-GGC-5')a nd G-Wires
The d(5'-CGGT-3'-3'-GGC-5')( 1)i ncorporating a3 '-3' inversion of polarity site was synthesized by using as olid-phase automated DNA synthesizer as described in the Experimental Section. The inversion of polarity site wasa chieved by performing the first four coupling cycles with 5'-phosphoramidites and the remaining three with standard 3'-phosphoramidites. The Q n species were obtained by heating 1-dissolved in 1.0 m K + containing buffer at the single strand concentration of 0.1 or 1.6 mm-at 90 8Cf or 10 min and then rapidlyc ooling it to 4 8C (fast annealing procedure). All samples were stored at 4 8Cf or 24 hb efore furtherinvestigation.

PAGE Studies
The annealed 1.6 mm 1 was analyzed by PAGE to obtain information on its propensity to form the tetramolecular G-quadruplex building block Q 1 and/ort he target Q n multimers. For this purpose, we compared the electrophoretic mobility of 1 (lane 3i nF igure 3) with those of the G-quadruplexes d(TGGGGT) 4 (lane 1) and d(CGGTGGT) 8 (lane 2), used as size  markersf or the tetramolecular G-quadruplex building block Q 1 and for its eight-stranded dimer Q 2 ,respectively.Atthe studied conditions, the annealed 1 migrated as al adder of bands, thus confirmingi ts propensity to form G-quadruplex multimers. The electrophoretic mobility of the two fastest bands matched almostp erfectly with that of the size markersi nl anes 1a nd 2, thus confirming the formation of at etra-stranded Q 1 and octastranded Q 2 ;w hereas the remaining slower bands in lane 3 were likely aresult of longer Q n species(n ! 3). The electrophoretic mobility of the isolated Q 1-4 species, obtained by HPLC-SEC fractionation (see the following section), was also assessed (lanes 4-7). Taken together, the PAGE data suggested that the oligodeoxynucleotide( ODN) 1 in the presence of K + ions is capable of self-assembling in ad istribution of G-wire species of differentl engths (Q n )o btained by the sequential stacking of the tetramolecular G-quadruplex buildingb lock Q 1 presenting two "sticky" G(:C):G(:C):G(:C):G(:C) planar octadsa tb oth 5'ends.

SEC Studies
The Q n G-wire population wasa nalyzed by HPLC-SECa tr oom temperature on aR eproSil 200 SEC column 24 ha fter annealing and storage at 4 8C. The HPLC profile of the annealed 0.1 mm 1 showed ad istribution of peaks in which low molecular weight speciesh ad greater retention times ( Figure S2 Ai n the Supporting Information). To obtain information on the M W of the observedp roduct peaks, we used the same HPLC-SEC conditions (see the Experimental Section) to determine the retention times (R t )o fi )the single-stranded dT 7 (panel B), ii)the tetramolecular quadruplexd (TGGGGT) 4 (panel C), and iii)the dimeric quadruplex d(CGGTGGT) 8 (panel D). Based on the HPLC-SEC data, the peak at the highest retention time (R t = 21.45 min) was attributedt ot he single-stranded 1,w hereas the two preceding peaks (at R t = 19.89 and 19.02 min) were assigned to the G-quadruplex buildingb lock Q 1 and to its dimer Q 2 ,r espectively.F rom these data, it was reasonable to hypothesize that each peak in the HPLC-SEC profile belongs to as peciesd iffering from those of the adjacent peaks foraM W corresponding to the tetramolecularG -quadruplexb uilding block Q 1 (i.e. 28 nucleotides). Exploiting the good peak resolution on the HPLC-SEC column, the speciesl ikely corresponding to Q 1-4 were recovered and their purity and electrophoretic mobility assessed by PAGE (Figure3,l anes 4-7). PAGE results confirmed the purity and allowed us to match the speciesr esponsible for the four fast-moving bands in the PAGE (i.e. Q 4 , Q 3 , Q 2 ,a nd Q 1 from the slowest to the fastest band in Figure 3) with the four corresponding peaks in the HPLC-SEC distribution (i.e. Q 4 , Q 3 , Q 2 ,a nd Q 1 from the least to the most retained in Figure S2 Ai nt he Supporting Information). Furthermore, HPLC-SEC re-injection of the isolated Q 1-4 species, performed 24 ha fter their isolation and storage at 4 8C( Figure S3 in the Supporting Information), disclosed that the G-quadruplex buildingb lock Q 1 and the first three Q n species (Q 2-4 )d o not interconverte ach other and can be stored as single G-wire segmentsofknown length for at least 24 h. However,considering the contribution of entropy to the formation of Q n G-wires, different behaviors could be anticipated for samples of 1 annealed at differentO DN concentrations. Thus, we exploited the good chromatographic separation of Q n species to assess the effect of the ODN annealingc oncentration on the formation and size distribution of Q n species. As expected, the HPLC-SEC profile of Q n obtained 24 ha fter the annealing of 1 at 1.6 mm concentration( Figure 4) confirmedt hat the formation of longerG -wiresi ss trongly dependent on the ODN concentration during the annealingp rocedure. Indeed, in Figure 4a ll Q n speciesw ith n 8a re clearly distinguishable and almost equally populated, and an intense envelope peak attributable to longerG -wiresi sp resent at R t = 13.6 min. Conversely,i nt he HPLC-SECp rofile obtained from the sample annealed at 0.1 mm ODN concentration ( Figure S2 Ai nt he Supporting Information), the envelope peak is absenta nd only the Q n species with n 3a re significantly populated.
The effect of the temperature on the distribution of Q n species was investigated by HPLC-SEC2 4h after the annealing of 0.1 mm 1 in 1.0 m K + buffer.H PLC-SEC analyses were performed by injecting the sample 30 min after heating and equilibration at 25, 45, 65, and 85 8C( Figure S4 in the Supporting Information). The profile obtained from the sample injected at 25 8Cs howed almost the same Q n distribution as the sample injecteda t48C, but showed that at room temperature the dimeric Q 2 speciesn ot only is more abundantt han the building block Q 1 (whereas the opposite was observed in the sample stored and injected at 4 8C), but represents the most abundant species. The progressive heatingo ft he sample determined the reduction of the amount of Q n speciesa nd the contextuali ncreaseo ft he single-stranded 1,w hich resulted in the only observable peak at 85 8C. However,t wo significant outcomes emerged from the profiles obtained at 45 and 65 8C: 1) at 45 8C, although the most abundant species was represented by the single-stranded 1,w eobserved that the distribution of Q n speciess hiftedt owards longer G-wires, with the appearance of an ew peak at R t = 13.3 min and with the Q 3 species being even more abundantt han Q 2 ;2 )the analysiso ft he pro- file obtained at 65 8Cs uggested that the Q n speciesm elt throughacooperative process,w hich drives directly to the random coil 1 (the peak of the G-quadruplex building block Q 1 was even less abundant than that of Q 5 species). The higher thermals tability of Q n species relative to Q 1 was also confirmed by CD meltingd ata (see the following section).

CD Studies
CD is aw ell-established diagnostic technique to provide preliminary informationa bout the topologyo fG -quadruplexes in solution.G enerally,t he CD profile of parallel G-quadruplexes, in which all guanosines in the G-tetrads are in the anti glycosidic bond conformation (Type 1stem), is characterized by apositive signal at around 264 nm and an egative signal at around 240 nm;whereas that of antiparallel G-quadruplexes with alternating syn-anti glycosidicb onds along the stem (Type 3s tem) shows ap ositive signal at 245 nm, at rough at around 260 nm, and as econd positive signal at 290 nm. [41,[44][45][46] Considering the formation of stacking interactions between the terminal G-tetrads of Q 1 building blocks (required to obtain the Q n G-wires) and the presence of the 3'-3' inversion of polarity site within the stem of Q 1 and its multimers, the CD profile obtained for the annealed 1 (containing am ixture of Q n of different lengths), showing positives ignals at 246 nm and 300 nm and an egative signal at 270 nm ( Figure 5, dashedl ine), was somewhat unexpected. In fact, although the latter structuralf eatures anticipated for aT ype 2C Dp rofile, [44] the experimental result resembled that of aT ype 3stem. AType 3s tem could be obtainablef rom 1 only if the thymines of two ODN 1 strands would participate in the formationo fapropeller loop, thusa llowing the formation of ab imolecular antiparallel quadruplex stem. However,t his structuralh ypothesis hasb een ruled out by PAGE and HPLC-SECevidence, which indicated the exclusive formationo ft he tetra-stranded quadruplex building block and its multimers.
Indeed, the CD profile of Q n resembles those reported for the G-quadruplexes formed by d(5'TG3'-3'GGT) 4 [47] and d(TGG3'-3'GGT) 4 [48] incorporating a3 '-3' inversion of polarity site and characterizedb yt he presence of all anti G-tetrads. The occurrence of the same system of uninterrupted stacked G-tetrads, including both clockwise and counter-clockwise directionality,i nQ 1 and Q n could explain the CD profiles shown in Figure 5. The resulting structural hypothesis requires that the thymines are projected outside the quadruplex stem to form bulge loops, [49] as depicted in Figure 2. Unfortunately,t he strongp ositive signal at 300 nm induced by the presence of the 3'-3' inversion of polarity site precluded the observation of the negative band at 290 nm, which, in our previous studies, provedd iagnostic for the formation of higher-ordered G-quadruplexa ssemblies obtained by non-covalent p-p stacking of planarG (:C):G(:C):G(:C):G(:C) octads. [42,43] Similar CD profiles were obtained for the isolated Q 1-4 ( Figure 5, colored lines), thus confirming that the CD profile of Q n was the result of the presence of quadruplex assemblies of different lengths incorporatingt he 3'-3' inversion of polarity site. The overall thermal stabilityo ft he species populating the Q n distribution and that of the isolated Q 1-4 complexes was assessedb ym eanso fC D denaturation experiments ( Figure S5 in the Supporting Information). The resulting apparent melting curves clearly provided evidencet hat the thermals tability of the quadruplexm ultimers was higher than that of the parent quadruplex building block Q 1 .

NMR Spectroscopy
The formation of G-quadruplex assemblies from suitable G-rich ODN sequences is usually confirmed by water-suppressed 1 HNMR spectroscopy.W hen the N-1 imino protons of guanine bases are engaged in Hoogsteen-type hydrogen bondingw ith the O-6 carbonyl oxygen atoms of flankingg uaninesi ne ach G-tetrad, they are protectedf rom exchange with the hydrogen atoms of the solvent and are thus visible in the 1 HNMR spectrum as quadruplex-diagnostic slightly broad signals resonating between1 0a nd 12 ppm. [50,51] Dependingo nt he orientation of the strands and on the glycosidic torsiona ngle of guanosines participating in G-tetrads, the four iminop rotons in each G-tetrad resonate as one, two, or four NMR signals. [52] The downfield region of the water-suppressed 1 HNMR spectra of the Q n distribution-obtained by annealing 1 in 1 m K + buffer at 1.6 mm singles trand concentration-recorded at 25, 45, 65, and 85 8Ca re shown in Figure S6 in the Supporting Information. In agreement with the CD evidence,t he observation of the G-quadruplex diagnostic iminop rotons signals in the 11-12 ppm region confirmed the presence in solution of G-quadruplexs pecies at temperatures lower than 65 8C. However,t he intensity and the shape of imino and anomeric protons signals differed significantly from those we observed in the 1 HNMR spectra of the dimeric higher-ordered G-quadruplexes formed by CGGTGGT [42] and CGGAGGT. [43] In the two latter,f our well-resolved imino protons (11.0-12.0 ppm) and intensea nomeric protons signals (5.5-6.5 ppm) werev isible at temperatures up to 65 8C. Conversely,i nt he NMR spectra of 1 recorded at 25 and 45 8C, all iminoa nd anomerics ignals appeared severely broadened, thus suggesting the involvement of imino and anomericp rotons in chemical or conformational exchange phenomena. The line broadening of the imino protonsw as so intense as to require 10 softwarea mplification for signal observation (see insets in Figure S6 in the SupportingI nformation). As far as the low intensity of the imino proton signals is concerned, we hypothesize that the presence of the bulge loops connecting the stacked G3 and G5 tetrads destabilizes the G-quadruplex stem, thusspeeding up the imino protonexchange with water.The anomeric protonsignal broadening, instead, could be the result of side-by-side aggregation of Gquadruplexu nits, as seen by Hu et al. in G-wire assemblies formed by dGMP annealedi nt he presenceo fS r 2 + . [53] As we have showni no ur previousp apers, [42,43] the anomeric protons are located at the outer shell of 2Q-like quadruplexes, thus it is conceivable that lateralc ontacts between flanking G-quadruplex structures participating in the Q n G-wire distribution could be responsible for the observed anomeric signal broadening. As we will see in the followings ection, AFM evidences upports the formation of extended quadruplex layers by lateral aggregation of quadruplex units. AlthoughAFM gives us apictureo f quadruplex aggregates deposited on mica surfaces, we believe that such aggregates could also form in solutionc onsidering the relativelyh igh ODN concentration used for the preparation of the NMR samples (6.0 mm). In the light of this hypothesis, the comparison of the NMR spectra recorded in the 25-85 8C temperature range indicates that the breakdown of quadruplex aggregates and the melting of quadruplex units to the random coil 1 are cooperative processes, which start at temperatures between 45 and6 58Ca nd completeatt emperatures lower than 85 8C.  Figure S7 in the SupportingI nformation). The AFM results are compatible with reported SEC results considering that, differently from heightm easurements, the length and width measurements are affected by the curvature radius of the AFM tip. The preferred orientation can be ascribed to the interaction between G-quadruplexes and the mica surface. The homogeneous values of heights recorded suggested the formation of self-assembled monolayers in the x,y plane owing to the evaporation of the buffer solution, which promoted laterala nd longitudinal aggregation of Gquadruplexes, the first one being favored by side-packing. A1 00-times diluted solution gave different results, as can be noted in Figure S8 in the Supporting Information. Even if apreferred orientation could still be envisaged, the structures seemed sparser and less ordered (see panel A);l engths are in the same intervala sb efore,w hereas the widths are smaller, never exceeding 30 nm, as evident from the measurements in panel B. Also in this case, at ip radius of curvature of about 10 nm must be taken in account. This result confirmed as idepacking mechanism in the assembly of G-structures during evaporation of the solution. The heights are alwaysa bout 2nma sb efore (see measurements in panel C), again endorsing the hypothesis of spontaneousf ormation of am onolayer. In the case of diluted starting solution, the longitudinal aggregation is competitive compared with the lateral one, owing to steric conditions during the evaporation.

Conclusions
In this paper,w er eported the successful results of our study aimed at the obtainmento fanew kind of G-wireD NA by exploitation of p-p stacking interactions between tetramolecular G-quadruplex building blocks incorporating a3 '-3' inversion of polarity site and exposing G(:C):G(:C):G(:C):G(:C) planar "sticky" octads at both 5'-ends. The main findings of the study can be summarized as follows:1 )the obtained G-wiresa re unprecedented and are achievable by using the short 5'CGGT3'-3'GGC5' G-rich ODN as the startingm ateriali nafacile and affordable way;2 )the use of at etramolecularG -quadruplex buildingb lock to obtain DNA G-wire polymers represents am ajor improvement over the use of monomolecular G-quadruplexes, which, possessing differente nd-facesa nd lateral nucleotide loops, could induce the formationo fd ifferent kinds of aggregates thus reducing the amount of target G-wires; 3) the reported G-wiresc an be easily monitored in their formation and length-growth by PAGE and HPLC-size exclusion chromatography.W ea lso demonstrated that the HPLC-SEC technique can be used to determine the number of quadruplex building blocks in each Q n G-wire,a sw ell as to isolatea nd recover the three shortest Q n species (n = 2-4). systems, USA) using au niversal CPG support purchased from Glen Research. The syntheses were performed by adopting the standard b-cyanoethyl phosphoramidite chemistry at 10-15 mm scale and the products were purified as previously described. [54] The synthesis of d(5'-CGGT-3'-3'-GGC-5')( 1)w as performed with the same DNA synthesizer.T he inversion of polarity site within the sequence was achieved by initially assembling the 5'-CGGT-3' tract by using 5'-phorphoramidites and then the 3'-GGC-5' tract with standard 3'phosphoramidites. After completion of the ODN sequence, the support was treated with concentrated aqueous ammonia at 55 8C for 15 h. The combined filtrates and washings were concentrated under reduced pressure and purified through HPLC (JASCO PU2089 pumps equipped with the JASCO 2075 UV detector) with an anion exchange column (Macherey-Nagel, 1000-8/46, 4.4 50 mm, 5 mm) using al inear gradient from 0t o1 00 %Bin 30 min, flow rate = 1mLmin À1 and detection at 260 nm (buffer A: 20 mm NaH 2 PO 4 aq. solution pH 7.0, containing 20 %( v/v) CH 3 CN;b uffer B: 20 mm NaH 2 PO 4 aq. solution pH 7.0, containing 1 m NaCl and 20 %(v/v) CH 3 CN).

Annealing Procedure
The ODN concentrations were determined in water by measuring the absorbance at 260 nm at 90 8Cb yu sing the nearest-neighbor calculated molar extinction coefficient of 5'-CGGTGGC-3' (e = 63 100 m À1 cm À1 ). The 0.1 and 1.6 mm solutions of 1 were obtained by dissolving the lyophilized sample in 900 mm KCl and 100 mm KH 2 PO 4 .T he samples were annealed by heating at 90 8Cf or 10 min and then quickly cooling to 4 8C. After the annealing procedure, the samples were stored at 4 8Cb efore measurements.

PAGE
Native gel electrophoresis experiments were performed on 20 % polyacrylamide gels containing TBE (8.9 mm Tris, 8.9 mm borate, 0.2 mm EDTA, from BIORAD) and 30 mm KCl, at room temperature, 120 Vf or 2h.T he ODN samples, annealed at 1.6 mm single strand concentration in 1.0 m K + buffer,w ere diluted at 0.6 mm loading concentration just before the PAGE runs. Glycerol was added (10 % final) to facilitate sample loading in the wells. The bands were finally visualized by ethidium bromide staining in aB io-Rad Laboratories Gel Doc TM XR + image system. CD CD spectra and CD melting profiles were recorded with aJ asco 715 CD spectrophotometer (Jasco, To kyo, Japan) equipped with aJ asco JPT423S Peltier temperature controller in 1mmo ptical path quartz cuvettes (100 nm min À1 scanning speed, 1s response time). The spectra were recorded in triplicate at 4 8Cf rom 220 to 320 nm. CD samples were prepared in potassium buffer (90 mm KH 2 PO 4 and 10 mm KCl) at 20 mm final single strand concentration. The buffer baseline was subtracted from each spectrum and the spectra were normalized to have zero at 320 nm. CD melting curves were registered at 268 nm, 1 8Cmin À1 heating rate, temperature range 5-90 8C.

NMR Spectroscopy
NMR data were recorded with aV arian UNITY INOVA5 00 MHz spectrometer equipped with ab roadband inverse probe with z-field gradient. The data were processed by using the iNMR software package (http://www.inmr.net). One-dimensional NMR spectra were acquired as 16384 data points with ar ecycle delay of 1.0 sa t 25, 45, 65, and 85 8Ca nd the spectra were apodized with as hifted sine bell squared window function. Water suppression was achieved by including ad ouble pulsed-field gradient spin-echo (DPFGSE) module [55,56] in the pulse sequence prior to acquisition. NMR samples were prepared at the concentration of 1.6 mm single strand in 200 mLo fH 2 O/D 2 O9 :1 containing 900 mm KCl and 100 mm KH 2 PO 4 .
AFM AX E-100 Park Systems instrument was used for the AFM imaging of Q n G-wires. Surface imaging was obtained in non-contact mode by using 125 mml ong silicon/aluminium-coated cantilevers (PPP-NCHR 10 m;Park Systems;tip radius lower than 10 nm), with aresonance frequency of 200 to 400 kHz and nominal force constant of 42 Nm À1 .T he scan frequency was typically 0.5 Hz per line. When necessary,t he AFM images were processed by flattening to remove the background slope, and the contrast and brightness were adjusted. Muscovite mica of about 1cm 2 surface was used as the substrate in the AFM study.M uscovite mica surfaces are typically used as AFM substrates owing to their perfect cleavage along a < 001 > plane, yielding large atomically flat areas. Mica consists of layers of an aluminium phyllosilicate lattice ionically bonded through interstitial K + ions. Upon cleavage, the K + ions are highly mobile and are readily exchanged with divalent cation species at the solid-liquid interface. This exchange results in ap ositive overcharging of the mica surface, which enables the deposition of molecules that hold an et negative charge, such as DNA. [57] Moreover, the positive charge distribution after cleavage enables as uper-hydrophilic surface that guarantees al ower interaction between suspended biomolecules during the evaporation of aqueous solvent. Mica was freshly cleaved by using adhesive tape prior to each deposition to establish its cleanliness. Aliquots (2 mL) of the DNA/imaging buffer were directly deposited by casting onto freshly cleaved muscovite mica. After 2min, every sample was gently washed with deionized water and then dried by evaporation at room temperature under av entilated fume hood.


extfieldsofstudy:
Materials Science
Medicine

text:
The effect of side-chain substitution and hot processing on diketopyrrolopyrrole-based polymers for organic solar cells† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ta01740e

The effects of cold and hot processing on the performance of polymer–fullerene solar cells are investigated for polymers designed to exhibit temperature-dependent aggregation in solution via second-position branched alkyl side chains.

S3
tungsten-halogen lamp. The 1 sun conditions were provided by the use of a 730 nm LED (Thorlabs) at different intensities for appropriate bias illumination. A calibrated silicon cell was used as reference prior to the J−V and EQE measurements. Thermal evaporation of the back electrode and J−V measurements were both performed inside a nitrogen filled glovebox. For the EQE measurements, the photovoltaic devices were encapsulated in a nitrogen filled box with a quartz window. The active layer thickness was determined on a Veeco Dektak150 profilometer.
Hole-only devices with an active area of 0.09 and 0.16 cm² were fabricated in air on patterned indium tin oxide (ITO) glass substrates (Naranjo Substrates). The substrates were cleaned with the same procedure as mentioned before for the solar cell devices. Poly(ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) (Clevios P, VP Al4083) was then spin coated at 3000 rpm to form a 40 nm layer. The active layers were then cast under the same conditions as for the solar cell devices. By varying the spin speed, thicknesses from 120 to 250 nm were obtained. 10 nm of MoO3 and 100 nm of Ag were then deposited by thermal evaporation under high vacuum (~3 × 10 −7 mbar). Current density -voltage (J−V ) characteristics were measured with a Keithley 2400 source meter, after illumination of the cell with UV light to dope the MoO3 for at least 10 minutes, sweeping from 0 to 6 V. For each type of active layer, three layers of different thicknesses were cast and measured. No thickness dependence of the mobility was observed. The mobility was determined by fitting the J−V curves with the Mott-Gurney square law, using an empirical series resistance correction, which varied between 5 and 20 Ω. The published hole mobility is the average of 12 individual devices using three active layer thicknesses.

Synthesis
Scheme S1 shows the synthesis route to the four DPP polymers. In the subsequent sections the synthetic procedures and molecular characterization are provided.

D-PDDP3T-EH
Freshly recrystallized 9 (43.0 mg, 105 μmol), monomer 8a (107 mg, 105 μmol), recrystallized triphenylphosphine (1.65 mg, 6.30 μmol), and Pd2(dba)3 (1.44 mg, 1.57 μmol) were placed inside a dried Schlenk tube and placed under argon. Toluene (1.8 mL) and DMF (0.2 mL) were added and the mixture was degassed with argon at 40 °C. The mixture was then reacted at 115 °C overnight. The viscous polymer solution was dissolved in warm CHCl3 and precipitated in methanol. The polymer was dissolved in chloroform with ethylenediaminetetraacetic acid (EDTA) and refluxed during one hour. Water was added, refluxed for one hour and subsequently the organic layer was washed with water. The organic layer was concentrated under reduced pressure and the polymer precipitated in methanol. The polymer was then subjected to Soxhlet extraction with acetone, hexane, dichloromethane, and chloroform. Finally, the purified polymer was precipitated in methanol. CHCl3 fraction: 98 mg, yield: 99%. GPC (o-DCB, 140 °C): Mn = 39.0 kDa, Mw = 90.0 kDa, PDI = 2.31.

D-PDDP4T-EH
Freshly recrystallized 10 (33.8 mg, 68.7 μmol), 8b (70.0 mg, 68.7 μmol), recrystallized triphenylphosphine (1.08 mg, 4.12 μmol)) and Pd2(dba)3 (0.943 mg, 1.03 μmol) were placed under Ar in a dried Schlenk tube. Toluene (1.8 mL) and DMF (0.2 mL) were added and the mixture was degassed with argon at 40 °C. The mixture was then reacted at 115 °C overnight. The viscous polymer solution was dissolved in warm 1,1,2,2-tetrachloroethane (TCE) and precipitated in methanol. The polymer was dissolved in chloroform with ethylenediaminetetraacetic acid (EDTA) and refluxed during one hour. Water was added, refluxed for one hour and subsequently the organic layer was washed with water. The organic layer was concentrated under reduced pressure and the polymer precipitated in methanol. The precipitated polymer was then subjected to Soxhlet extraction with acetone, hexane, dichloromethane and chloroform. Swollen polymer remaining inside the Soxhlet thimble was dissolved in hot TCE and the solution was filtrated over a heated cellulose filter. Finally, the TCE solution was concentrated in vacuo and the purified polymer was precipitated in acetone.

UV-vis-NIR measurements in o-dichlorobenzene (o-OCB)
UV-vis-NIR spectra were recorded for the four polymers in o-OCB at different temperatures ( Figure S3). o-OCB is a less good solvent for these polymers than chloroform or TCE. With increasing temperature of o-OCB D-PDPP3T-EH, D-PDPP4T-EH, and D-PDPP4T-HD show a more or less gradual loss of the long wavelength peak and its vibronic structure. At ~60 °C D-PDPP3T-EH, D-PDPP4T-HD, reach a state in which the polymer chains are truly molecularly dissolved. For D-PDPP4T-EH a small shoulder around 800 nm remained until 100 °C, which agrees with the lower solubility of this material. For EH-PDPP4T-EH the results ( Figure S3d) were different from the other three polymers. At room temperature the material is sparsely soluble in o-DCB and when conducting the experiment from high to low temperature the polymer precipitated in its aggregated state in the cuvette, resulting in a loss of signal at lower temperatures.   Table 2 of the main text are based on 12 individual devices using three different active layer thicknesses.   The Jsc values were based on integration of the EQE (measured with 1 sun bias illumination) with the AM1.5G spectrum. b The PCE is determined from the EQE integrated Jsc. All devices were made on the same day from the same solutions.


s2fieldsofstudy:
Chemistry
Materials Science

text:
Fabrication of 6FDA-durene membrane incorporated with zeolite T and aminosilane grafted zeolite T for CO2/CH4 separation

In the present work, zeolite T and aminosilane grafted zeolite T are embedded into 6FDA-durene polyimide phase for the fabrication of mixed matrix membranes (MMMs). FESEM images demonstrated that the improvement of interfacial adhesion between zeolite and polymer phases in MMM loaded with aminosilane grafted zeolite T was not significant as compared to zeolite T/6FDA-durene MMM. From the gas permeation test, CO2/CH4 selectivity up to 26.4 was achieved using MMM containing aminosilane grafted zeolite T, while MMM loaded with ungrafted zeolite T showed CO2/CH4 selectivity of 19.1. In addition, MMM incorporated with aminosilane grafted zeolite T particles successfully lies on Robeson upper bound 2008, which makes it an attractive candidate for CO2/CH4 separation.

Introduction
Membrane-based technology has received world-wide attention over the past decades for gas separation applications. Among conventional separation techniques such as absorption, adsorption and cryogenic, membrane process is preferable because of its low operating cost and capital investment, low space requirement, energy efficiency and environmental-friendly [1]. Membrane materials can be categorized into polymeric, inorganic and mixed matrix membrane (MMM). Polymeric membrane has been widely used in industrial application due to its advantages such as cheap and easy to fabricate. However, the upper bound curve reported by Robeson demonstrates that the current polymeric membrane has reached a tradeoff between gas permeability and gas pair selectivity [2]. On the other hand, even though inorganic membrane exhibits higher gas separation performance, it is limited due to poor mechanical strength and high fabrication cost. On the other hand, fabrication of MMMs has been identified as one of the emerging strategies for the enhancement of the performance of membrane materials by embedded inorganic fillers into polymer phases. This type of membrane combines the characteristics of polymer material and properties of inorganic fillers [3].
Zeolites have attracted much attention in the fabrication of MMM due to their narrow pore distribution, well-defined repeating pore structures and great molecular sieving property [4]. Zeolite T exhibits a great potential in CO2/CH4 separation due to its small pores of 0.36nm x 0.51nm [5]. Hence, zeolite T has been used as the selective fillers in the MMMs fabrication for gaseous and organic/aqueous mixtures separations [6,7].
Nevertheless characteristics such as good compatibility and excellent interfacial adhesion between the filler and polymer. Hereafter, some of the techniques have been proposed to enhance the interfacial properties of filler-polymer. Silane coupling agents are commonly proposed for the modification of the filler surface, which covalently bind the filler and polymer phase and subsequently improve the fillerpolymer adhesion properties.
Pechar et al. [8] used 3-aminopropyltrimethoxysilane (APTMS) for the improvement of the compatibility between zeolite ZMS-2 and polyimide phase. They reported that MMM loaded with APTMS-grafted ZSM-2 particles demonstrated the reduction in both gas permeability and gas pair selectivity compared to pristine membrane. The decrement of the separation performance might be due to the partial blockage of zeolite pores by APTMS. Similar results were also obtained by Clarizia et al. [9] when they incorporated silane-grafted NaA zeolite into polyetheretherketone (PEEK). On the other hand, Nik et al. [10] reported that the dispersion of particles enhanced when grafted MOF-199 was incorporated into 6FDA-ODA polyimide phase. They also found that CO2 permeability and CO2/CH4 selectivity increased as compared to ungrafted MOF-199/6FDA-ODA MMM. Li et al. [8] grafted zeolite 3A, 4A and 5A using 3-aminopropylmethyldiethoxy (APMDES). The incorporation of grafted zeolites into polymer phase reduced the rigidification of polymer chain and partial pore blockage, which subsequently enhanced both gas permeability and gas pair selectivity of this membrane as compared to MMM loaded with ungrafted zeolite. Overall, silane grafted zeolite has successfully improved the interfacial contact between the filler and polymer phase and thus, the separation properties of MMM.
To our best knowledge, there is no research work reporting on the fabrication of MMM loaded with aminosilane-grafted zeolite T particles for CO2/CH4 separation. Therefore, in the present work, zeolite T has been grafted with AAPTMS silane coupling agent before incorporated into 6FDA-durene polyimide phase for the formation of MMM. The structure and physicochemical properties of the resultant membranes were investigated by using different analytical tools. In addition, the permeation properties of MMM incorporated with aminosilane grafted zeolite T particles was assessed and compared with ungrafted zeolite T incorporated MMM.

Chemicals and gases
6FDA-durene polymer and zeolite T particles were synthesized from our laboratory. For the preparation of aminosilane grafted zeolite T particles, N-(2aminoethyl)-3-aminopropyltrimethoxysilane (AAPTMS, C8H22N2O3Si, ≥ 97% purity, Merck) and toluene (C6H5CH3, 99.8% purity, Sigma Aldrich) were used as received. For the fabrication of MMM, dichloromethane (DCM, ≥ 99.8% purity) were purchased from Sigma Aldrich and used without further purification. In addition, the purified methane and carbon dioxide gases (purity of 99.9995%) used for gas permeation testing were obtained from Gas Walker Sdn Bhd and used as received.

Synthesis of zeolite T and aminosilane grafted zeolite T particles
Zeolite T particles was synthesized via sonochemical-assisted hydrothermal growth method at molar composition of SiO2 : 0.04Al2O3 : 0.26Na2O : 0.09K2O: 14H2O as reported in our previous work [5]. Meanwhile, aminosilane grafted zeolite T was prepared using the method reported by Li et al. [8]. Zeolite T particles, AAPTMS silane coupling agent and toluene were mixed and stirred for 24 h at room temperature under nitrogen purge. The aminosilane grafted zeolite T particles were then recovered by centrifugation and washed with methanol before dried in an oven at 110 o C for 24 h.

Fabrication of pristine 6FDA-durene and MMMs
Pristine 6FDA-durene membrane was fabricated based on the method reported in our previous work [5]. 2% w/v 6FDA-durene polymer solution was cast on Petri dish prior to dry and thermal anneal at 250°C in the vacuum oven. On the other hand, MMMs contained 1 wt% of zeolite T particles (ungrafted and grafted) in 6FDA-durene polymer phase were fabricated by solution blending method [7]. 6FDA-durene polymer solution was added into zeolite T suspension before casted on Petri dish.
The cast film was then dried and annealed in the vacuum oven for 24 h.

Characterization of particles and membranes
The functional groups presence in ungrafted and aminosilane grafted zeolite T particles were observed using Fourier transform infrared spectrometer (Perkin Elmer Spectrum One). The spectrum of the particles was obtained using KBr method from the acquisition of 50 scans in the wavelength range of 450-4000 cm -1 under the transmission mode.
Subsequently, the resultant membranes were characterized using FESEM, EDX and DSC. The morphology of membrane was attained using field emission scanning electron microscope (FESEM, Zeiss Supra 55 VP) operated at 10 kV under vacuum condition. In addition, the distribution of the particles in the polymer matrix was investigated using Oxford Instrument Inca energy dispersion Xray (EDX) mapping. The glass transition temperature (Tg) of the membrane samples was analyzed using differential scanning calorimetry (DSC), Thermal Analysis (TA) instruments DSC Q2000. The Tg of samples were determined using the midpoint temperature of the transition region in the second heating cycle.

Gas Permeation Measurements
Single gas permeation properties were measured using custom-built gas permeation test rig at pressure of 3.5 bar and temperature of 30°C for pure gases of CO2 and CH4. The detailed explanation on the testing setup and procedure were described elsewhere [1]. The permeability of CO2 and CH4 was determined using equation (1) as follows: where P refers to the membrane permeability (Barrer), Vp is the permeate flow rate (cm 3 (STP)/s), t is the thickness of membrane (cm), Am refers to the membrane area (cm 2 ), ph and pl are the pressure in feed side and permeate side, respectively (cmHg). The gas permeability of the membrane is defined in the unit of Barrer (1 Barrer =1×10 -10 cm 3 (STP).cm/s.cm 2 .cmHg).
The ideal selectivity of the membrane was calculated using equation (2) as follows: where α represents the ideal selectivity of membrane. Figure 1 shows the FTIR spectra of ungrafted and aminosilane grafted zeolite T crystals. Based on Figure 1 (a), both of the zeolite T particles comprise the characteristic peaks at 465 cm -1 , 575-615 cm -1 , 720-775 cm -1 and 1040-1150 cm -1 , which were consistent with those FTIR result reported in the literature for zeolite T structure [11]. The T-O bend in zeolite T topology is characterized by the peaks at 465 cm -1 . The absorbance peaks in the range from 575 cm -1 and 615 cm -1 is attributed to the secondary building units of single 6-ring of zeolite T structure [12]. In addition, the symmetric and asymmetric stretching vibrations of double ring for zeolite T structure is characterized by the absorption bands in the region of 720-775 cm -1 and 1040-1150 cm -1 , respectively. On the other hand, it can be observed that the aminosilane grafted zeolite T demonstrates N-H bending and NH2 scissoring at the characteristic peaks of 1470 cm -1 and in the region of 1620-1650 cm -1 , respectively [13,14]. Furthermore, based on Figure 1(b), the detection of absorption peak at 3250 cm -1 and 3450 cm -1 are contributed from the presence of N-H stretch and O-H stretch of the hydrogen bonded silanol group [15,16]. The grafted zeolite T particles also demonstrates two N-H absorption bands in the region of 3300-3500cm -1 due to the presence of amine primary group in the chemical structure (-NH2) [17]. Overall, the presence of N-H bonds in aminosilane grafted zeolite T particles confirms that the AAPTMS were successfully attached on the zeolite T surface.  Figures 2 and 3 demonstrate the morphology and the dispersion of particles (ungrafted and aminosilane grafted) in the polymer phase, respectively. Based on Figure 2 (b) and (c), it can be observed that both of the membranes exhibit a good dispersion of zeolite T particles in polymer phase with no significant interfacial voids between the particle and polymer phase. The good distribution of zeolite T particles in MMM loaded with ungrafted and aminosilane grafted zeolite T particles are further verified by the homogenous dispersion of Si element in EDX mapping shown in Figure 3(a) and (b), respectively. Overall, the incorporation of aminosilane grafted zeolite T particles into 6FDAdurene polymer phase did not contribute to a significant effect on the improvement of the interfacial adhesion between the particles and polymer matrix.  Table 1. Referring to Table 1, the pristine 6FDA-durene and MMM loaded with ungrafted and aminosilane grafted zeolite T demonstrate glass transition temperature of 410.06ºC, 425.22 ºC and 426.01 ºC, respectively. The enhancement of Tg shown by aminosilane grafted zeolite T/6FDA-durene MMM demonstrates that the incorporation of aminosilane grafted zeolite T in the polymer matrix alters the polymer packing structure of the membrane by restricting the movement of polymer chains. In addition, the increment of Tg might also be attributed to the polymer matrix rigidification.

Gas separation performance
The performance of the resultant membranes was determined by using CO2 and CH4 single gases permeation testing. The permeability of CO2 and CH4 and CO2/CH4 selectivity of the membranes are displayed in Figure 4. Referring to Figure 4, both MMMs demonstrate higher CO2 permeability and CO2/CH4 selectivity compared to pristine 6FDA-durene membrane. On the other hand, CH4 permeability of the MMMs are lower compared to the pristine 6FDA-durene membrane. The increment of gas separation performance of MMMs as compared to the pristine 6FDA-durene membrane might be contributed to the presence of zeolite T particles in the polymer phase which may interrupts the interaction between the polymer chain packing and enhances the free volume in the membrane. Furthermore, the tremendous improvement of CO2/CH4 selectivity upon the addition of zeolite T particles (ungrafted and aminosilane grafted) might be due to the contribution of sieving and adsorption effects of zeolite T particles. Moreover, strong interaction of CO2 with the zeolite T framework may also assist the penetration of gases through the membrane [18,19]. Besides, as compared to ungrafted zeolite T/6FDA-durene MMM (CO2 permeability of 844 Barrer and CO2/CH4 selectivity of 19.1), MMM incorporated with aminosilane grafted zeolite T particles exhibits 2.7% decrement in CO2 permeability with the enhancement of CO2/CH4 selectivity up to 38%. The improvement of separation performance of aminosilane grafted zeolite T/6FDA-durene MMM might be due to the good dispersion of zeolite T particles in the polymer phase as demonstrated in FESEM and EDX-mapping images in Figures 2 and 3, respectively. Besides, as revealed in Table 1, the increment of Tg of this membrane as compared to zeolite T/6FDA-durene MMM also supports its higher separation performance. In addition, the improvement of the separation performance might be also contributed from the ability of aminosilane grafted zeolite T in minimizing the transportation of gases via the unselective voids and consequently increases the selectivity of the membrane [20].

Conclusion
In the present research, zeolite-T and aminosilane grafted zeolite T particles have been incorporated into 6FDA-durene for the formation of MMM. The FTIR spectrum demonstrated that the aminosilane group was successfully grafted onto the zeolite T structure. The FESEM images showed that the grafted zeolite T/6FDA-durene MMM exhibited no significant improvement on the interfacial adhesion between particles and polymer matrix as compared to MMM loaded with ungrafted zeolite T particles. From the permeation results, the incorporation of grafted zeolite T successfully increased CO2/CH4 selectivity up to 38% as compared to ungrafted zeolite T/6FDA-durene MMM and subsequently successfully lies on Robeson upper bound limit 2008.