Análisis de la variación de compuestos en extractos crudos de corteza, hoja y rama.

5.2.1 Determination of the CMC by ST and DLS

The CMCs of the AzoTABs in water at 20 °C were determined for both isomers using surface tensiometry and dynamic light scattering. As a representative example, Figure 5.1a shows the variation in surface tension as a function of concentration for C4AzoOC6TAB. The CMC is reached when the surface tension becomes independent of

surfactant concentration,12 _{at 0.40 mM and 1.05 mM for the trans- and cis-forms,}

respectively. The CMC was confirmed by DLS, whereby the intensity of scattering light increases significantly upon the onset of micelle formation (Figure 5.1b).

89 Figure 5.1. Determination of the CMC of C4AzoOC6TAB in water at 20 °C. Variation of (a)

surface tension and (b) light scattering intensity as a function of concentration. The data points show the trend before (trans-form, black circles) and after irradiation with UV light at λex = 365 nm

for 5 minutes (predominantly cis-form, blue squares). The CMC values are obtained from the intersection between the two trends (solid lines). The error bars show the standard deviation of the mean values obtained over 5 (ST) or 3 (DLS) measurements. The dashed lines serve only to guide the eye.

The CMCs determined for all AzoTABs by both ST and DLS are summarized in Table 5.1. It is worth noting that the Krafft temperature of alkyltrimethylammonium bromide surfactants (e.g. C16TAB) is known to be around 25 °C in water.13 Interestingly, among the

AzoTABs studied here, only C6AzoOC4TAB displays low solubility in water at 20 °C, and

flocculates, suggesting that the Krafft temperature and formation of micelles is not achieved for this AzoTAB.14 _{The solubility experiment showed that the complete}

solubilisation of trans-C6AzoOC4TAB in water (Concentration = 1 mM) was achieved at

27-28 °C. The values for the CMC obtained are in good agreement with those previously reported for AzoTAB surfactants, as indicated in Table 5.1 The absolute values of the surface tension isotherm above the CMC are in good agreement with reported values for azobenzene photosurfactants,3 with the longest hydrophobic segment showing the lowest surface tension (i.e. R1 + R2 = 14). Table 5.1 shows that three key trends can be identified. For a given surfactant, the CMC of the cis-isomer is always higher than that of the trans-isomer, which can be rationalized based on the increased dipole moment, and thus hydrophilicity, upon photoisomerisation. Secondly, the hydrophobic driving force for micellisation is driven by the total length of the hydrophobic segment of the structure, with a larger length (i.e. R1 + R2) leading to a lower CMC. This has been previously demonstrated for short hydrophobic segments (R1 + R2 ≤ 8, Table A.5.1)1, 15 and is confirmed here for much longer hydrophobic segments (up to R1 + R2= 14), enabling the CMCs to be tuned from mM to µM. Finally, for a fixed hydrophobic segment length, a

0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 500 1000 1500 2000 CMC trans-form cis-form kc ou nt s Concentration (mM) CMC (b) 1E-3 0.01 0.1 1 10 30 40 50 60 70 80 CMC trans-form cis-form S ur fa ce T en si on ( m N m -1 ) Concentration (mM) CMC (a)

90 longer alkyl tail (and thus a shorter spacer) leads to a smaller CMC, for example C4AzoOC6TAB> C8AzoOC6TAB (Table 5.1 and Figures A.5.1-4). Similarly, for a fixed

alkyl chain length, the CMC decreases as the spacer length increases.

Table 5.1. Summary of the CMCs obtained for AzoTABs in water by surface tensiometry (ST) and dynamic light scattering (DLS) at 20 °C.

AzoTAB trans-CMC (mM) cis-CMC (mM) trans-/cis-CMC (mM)

Literature ST DLS ST DLS C 4AzoOC4TAB 1.10 ± 0.02 1.2 ± 0.1 2.50 ± 0.02 2.7 ± 0.2 1.2/2.7 b _{; 1.0/2.0}c C 4AzoOC6TAB 0.40 ± 0.02 0.4 ± 0.1 1.05 ± 0.02 1.1 ± 0.1 ~0.5/NR d C 6AzoOC4TAB a 0.19 ± 0.03 0.2 ± 0.1 0.62 ± 0.03 0.7 ± 0.1 C 8AzoOC2TAB 0.34 ± 0.02 0.3± 0.1 0.82 ± 0.03 0.8 ± 0.1 0.3/0.8 b C 8AzoOC6TAB 0.09 ± 0.02 0.1 ± 0.1 0.15 ± 0.03 0.2 ± 0.1

a_{Determined at 30 °C.} b_{From Hayashita}

et al.1 – Determined by conductivity (T = 25 °C). c From McCoy et

al.3 – Determined by pendant drop tensiometry (T = not specified). d From Zakrevskyy et al.16 – Determined

by isothermal titration calorimetry (T = 25 °C), cis-isomer not reported (NR).

It has been shown that the logarithm of the CMC of classical alkyl trimethylammonium surfactants such as CTAB decreases linearly as the number of carbon increases (Figure 5.2).17 Yang et al. have determined the equivalent hydrophobicity of trans-C2AzoOC2TAB to correspond to an alkyl chain of 12.6 atoms of carbon.2 Figure 5.2

shows that a predictable trend can be drawn based on the number of carbons in the hydrophobic segment for alkyl trimethylammonium surfactants.

Figure 5.2. Evolution of the logarithm of the CMC as a function of the number of carbons in the alkyl chain of alkyl trimethylammonium surfactants (black squares), redrawn from Ref. 17. The trend shows that the CMC decreases as the number of carbon atoms increases. The equivalent hydrophobicity of the trans-azobenzene core can be estimated from this trend. The CMC of the trans-AzoTABs reported in the literature (open red squares) are obtained from Refs. 1 and 2, and of

10 12 14 16 18 20 22 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

CMC of AzoTABs in this report

CMC of reported AzoTABs Lo g (C M C ) Number of carbons CMC of Alkyl-TAB surfactants

91 the trans-AzoTABs obtained by this study (blue squares) shows that the equivalent hydrophobicity of the trans-azobenzene core corresponds to an alkyl chain of 9.0 ± 1.0 carbon atoms, on average.

Based on these studies and on the obtained CMC of the AzoTABs, the equivalent hydrophobicity of trans-C4AzoOC6TAB has been estimated to be equivalent of that of an

alkyl trimethylammonium surfactants of 18 carbons. This means that the trans-azobenzene core possesses an equivalent hydrophobicity of 8 atoms of carbon as the hydrophobic segment of C4AzoOC6TAB contains 10 carbon atoms.

The difference in the CMC (Δ_{CMC) is an important parameter as it represents the}

range of concentrations in which micelles exist in the trans-form only but are expected to return to unimers upon photoisomerisation to the cis-isomer (Figure 5.3). This property has previously been exploited for the reversible encapsulation and release of small molecules, for example for catalysis18 or drug release.19

Figure 5.3. Schematic representation of the formation of micelles upon increasing unimer concentration, for trans- and cis-isomers. The concentration range where trans-isomers form micelles, while cis-isomers remain as unimers, is called the ∆CMC. The ∆CMC can be tuned by the design of the photosurfactants.

The Δ_{CMC for the cis- and trans-isomers as a function of the total number of}

carbons in the hydrophobic segment (R1 + R2) is shown in Figure 5.4. There is an apparent sweet spot for obtaining the maximum Δ_{CMC, which occurs for a total hydrophobic}

segment length of six carbons, irrespective of the tail and spacer lengths from which the segment is composed. As the total hydrophobic segment length is increased or decreased, the magnitude of the Δ_{CMC decreases significantly. If we consider the extreme cases, i.e.}

a very short or very long hydrophobic segment, the low Δ_{CMC can be rationalised. For}

trans-isomer cis-isomer Concentration scale (µM) ΔCMC micelles unimers

92 short hydrophobic segments, the azobenzene core dominates both the length and the volume, and the difference between the hydrophobic driving forces for micellisation (dominated by the alkyl tail) for the two isomers is small. Similarly, for long hydrophobic segments, the longer alkyl tail and/or spacer increases the flexibility of the hydrophobic segment in both isomers, leading to an increase in the free volume irrespective of photoisomerisation. For a hydrophobic segment length of R1 + R2= 6, there is an apparent compromise between these two factors, whereby the free volume of the hydrophobic segment, and therefore packing of the molecules in the micelle, is controlled predominantly by the photoisomerisation of the azobenzene core.

Figure 5.4. Comparison of the difference in CMC (∆CMC) for cis- and trans-isomers of AzoTABs as a function of the total number of carbons in the hydrophobic segment (R1 _{+ R}2_{).Data are}

presented from this study (coloured circles) and previous reports (black circles). The errors bars result from different reports of the CMC in the literature.

Analysis of the absolute values reveals that an apparently larger Δ_{CMC is obtained}

for a longer spacer length, i.e. C2AzoOC4TAB > C4AzoOC2TAB. If we consider the

homologous series of (R1 _{+ R}2 _{= 10) AzoTABs, the same trend is observed, with the}

absolute value of Δ_{CMC increasing with the spacer length, i.e. C4AzoOC6TAB >}