N Mofaddel

H Krajian

D Villemin

P L Desbène

Enantioseparation of binaphthol and its mono derivatives by cyclodextrin-modified capillary zone electrophoresis 1

Keywords: binaphthol derivatives, capillary electrophoresis, enantioseparation, atropisomers, cyclodextrins, chiral ionic liquid

Chiral binaphthols belong to the group of most effective ligands for asymmetrical catalysis. In this context, various binaphthols presenting original substituents have been synthesized. Their study through capillary electrophoresis is the object of this work. The literature dedicated to the separation of atropisomers by capillary electrophoresis, corresponding only to binaphthol, reveals that its enantioseparation is always delicate because of the influence of many factors and the resolutions obtained are weak. Therefore, for a structured optimization, we first successfully evaluated the acidity constants of different binaphthols by means of capillary electrophoresis. With these known physicochemical characteristics, we could successfully carry out enantiomeric separations of the different binaphthols at pH=11.5, practically in completely ionized form, in phosphate medium, and in the presence of cyclodextrin (CD), with analysis times lower than 8 minutes. The nature of CDs (α-CD, β-CD, γ-CD, hydroxypropyl-α-cyclodextrin (HP-α-CD), HP-β-CD, HP-γ-CD and trimethyl-β-CD (TM-β-CD)) and other factors in relation to enantiomeric resolution (applied voltage, nature and concentration of the electrolyte, and concentration of cyclodextrin) were optimized. These studies allowed us to determine the optimal conditions of separation (concentration and nature of CD) for each of the studied binaphthols. It is necessary to mention that, for the 1,1'binaphthyl-2,2'-diol (Binol) at pH=11.5, the S atropisomer always migrated first, regardless of the nature and concentration of the cyclodextrin used. Moreover, an inversion in elution order of the two atropisomers as a function of pH was observed with γ-CD (pH range: 10-11.5). The R atropisomer migrated first at pH=10. At pH=10.8 the migration order of the two

Introduction

Over the last two decades, research in the area of asymmetric synthesis has expanded. The final aim of enantioselective synthesis or catalysis is to obtain enantiomeric pure chiral compounds from achiral substrates in the presence of chiral reactants. The role of the latter is to induce the selective formation of the desired enantiomer. In reality, the situation is more complex. Indeed, both the substrate and the reactant can exist in the form of a mixture of conformational isomers, in non-negligible proportions and in different states of solvation, imposing distinct reactivities for each species present. The result is a weighted average, which generally leads to weak stereoselectivity according to the distribution and the reactivity of the implied species. A reasoned approach of stereoselectivity is based on the use of molecules that only have pure rotation elements like, for example, chiral atropisomers and, particularly, binaphthol systems [1,2]. Structurally, the binaphthols or Binols, according to the authors, are composed of two naphthyl groups carrying a hydroxyl function in position 2 and linked by a simple C-C liaison in positions 1 and 1'. As a result of the restricted gene at the level of the rotation around this C-C liaison, two conformations (S-and R-Binol) exist, hence the appellation atropisomer. Used since 1990, the atropisomers of the 1,1'-binaphthyl-2,2'-diol (or Binol) are among the most widely used ligands for asymmetric catalytic reactions. They can also be used as chiral hosts in the enantiomeric separation of racemic mixtures of "guest" compounds or even as chiral reagents during the optical purity study of enantiomers by 1 H NMR [3]. A recent paper [4] identifies all the preparation methods for Binol synthesis, as well as its uses as a reactant or ligand in various reactions.

Since 1993, the enantiomeric separation of Binol has been the subject of several studies in capillary electrophoresis (CE), the method of choice for enantioseparation. Indeed, in addition to the advantages of high efficiency and short analysis time, only very small quantities of samples and separation electrolytes are required. Under these conditions, the study of chiral compounds, which are generally extremely expensive in their pure enantiomeric form, and their use as electrolytes becomes less problematic. Finally, the potential presence of impurities within the chiral compound does not handicap their separation since they have different electrophoretic mobilities and therefore do not interfere upon separation. The majority of the possible modes in CE are applicable to the separation of enantiomers, but micellar electrokinetic chromatography (MEKC) is by far the most used. More particularly, concerning the enantioseparation of Binols and their derivatives in CE, many studies have been undertaken in capillary electrokinetic chromatography (EKC), generally by using native or derivative cyclodextrins (CD-CZE) [5][6][7][8][9][10][11] or even with surfactants such as sodium N-(4-ndodecyloxybenzoyl)-L-Valinate (MEKC) [12]. These studies showed the complexity of Binol enantioseparation. Indeed these separations require the control of many parameters, on the one hand the background electrolyte (electrolyte nature, pH, ionic strength, presence or not of cosolvent) and on the other hand the chiral selector (cyclodextrin (CD) type, substitution, concentration, degree of substitution and use of charged CD or dual CD system). All studies and optimization of various factors were not always carried out systematically. The aim of this work is first to determine the acidity constants of Binol and its mono derivatives. We will also study the effect of pH on the enantioseparation of Binol in order to determine the most favorable pH of this enantioseparation. To our knowledge, such a study has never been reported. Finally, after the determination of this principal parameter, we seek to establish optimal conditions for the enantioseparation of each of the binaphthol derivatives studied using CD.

Experimental

Instrumentation

Capillary electrophoretic experiments were performed using a P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Fullerton, CA, USA) fitted with a diode array detector from 190-600 nm. An uncoated fused silica capillary (Thermo Electron SA, Courtaboeuf, France), 31.2 cm long (21 cm to the detector), 50 µm i.d. (internal diameter), was used for the pKa determinations and one 40.2 cm long (30 cm to the detector), 50 µm i.d., was used for the enantioseparations. The capillaries were thermostated at 20.0 ± 0.1°C. The samples were pressure-injected by 0.5 psi (34 mbar) for 3 s at the inlet side of the capillary (anode). Electrophoretic runs were performed with a 10 kV potential. The UV detection was cathodic (λ=214 nm). The System 32 Karat software (Version 4.0, Beckman Coulter) piloted the electrophoretic system and was used for controlling the data. The pH of the running solutions was measured before each experiment by using a Model IQ240 pH meter (IQ Scientific Instruments Inc., San Diego, CA, USA). A Branson 2510 sonication apparatus (Branson, Danbury, USA) was used for degassing all solutions.

The data examination of the experimental design was performed with JMP software, version 5.1 (SAS institute, Cary, NC, USA).

Chemicals and reagents

The racemate and R enantiomer of Binol (1,1'-binaphthalen-2,2'-diol or 1,1'-bi-2-naphthol) were provided by Sigma-Aldrich (Sigma-Aldrich France, Saint Quentin Fallavier, France).

Native α-CD was purchased from Sigma-Aldrich, β-CD (98%) from Interchim (Interchim, Montluçon, France) and γ-CD (98%) from Acros Organics (Acros Organics France, Noisy-le-Grand, France). The compounds 2-hydroxypropyl-α, β and γ-CDs (HP-x-CDs) (d.s.≈0.6) were obtained from Fluka Chemie (Sigma-Aldrich-Fluka France, L'Isle d'Abeau, Chesne, France). Heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD) was purchased from Sigma-Aldrich. Sodium dihydrogenphosphate (NaH2PO4), disodium hydrogenphosphate (Na2HPO4), trisodium phosphate (Na3PO4), sodium hydroxide (+98% purity), and absolute ethanol (98.8%) were obtained from Sigma-Aldrich.

The Binol derivatives studied in this work were synthesized in house; the syntheses of 6bromo-1-(2-hydroxynaphthalen-1-yl)naphthalen-2-ol (BN2) [13], 1-(2-(allyloxy)naphthalen-1-yl)naphthalen-2-ol (BN3) [14], 1-(2-(benzyloxy)naphthalen-1-yl)naphthalen-2-ol (BN4) [14] and 1-(2-metoxynaphthalen-1-yl)naphthalen-2-ol (BN5) [14] have already been reported in the literature. Their structures are shown in Table 1.

Table 1

For chiral ionic liquids, the syntheses of (R)(-)-2-hydroxy-N,N,N-trimethyl-1-phenylethanaminium bis(trifluoromethylsulfonyl)imide (R-Phenylcholine NTf2) and (R)(-)-1-hydroxy-N,N,N-trimethylbutan-2-aminium bis(trifluoromethylsulfonyl)imide (R-Etcholine NTf2) were reported previously [32]. (S)(+)-tetrabutylammonium camphorsulfonate (S-tbcamphor) was prepared by neutralization of (S)(+)-camphorsulfonic acid with tetrabutylammonium hydroxide.

Running electrolyte and sample preparation

All running electrolytes were prepared, without any prerequisite purification, using ultrapure water produced by means of a Milli-Q-System water purification apparatus (Millipore France, Montigny-le-Brotonneux, France). The solutions were sonicated just before use for 10 min.

For the pKa determinations, the running electrolytes contained phosphate salts (according to the target pH value (6<pH<12)) with an ionic strength equal to 40 mM. For the enantioseparations, a phosphate electrolyte (Na2HPO4-Na3PO4) with an ionic strength equal to 80 mM, and cyclodextrin concentrations ranging from 0 mM to 20 or 25 mM were used. All samples were prepared in ethanol at a concentration equal to 10 -4 M. New uncoated capillaries were activated by performing the following washing process: water for 2 min, NaOH (1 M) for 30 min and water for 10 min. The capillary was conditioned for 15 min with the electrolyte before running and for 2 min between each run.

Results and discussion

Determination of the acidity constant

Although Binol is a well-known compound and has been used for many years, it is necessary to mention that, to date, the determination of its acidity constants has never, to our knowledge, been reported in the literature. Nevertheless, the knowledge of these dissociation constants is a key parameter, especially in transport phenomena and more particularly for electrophoretic mobility. For these reasons, it is essential to first determine these constants for Binol and its derivatives before undertaking the enantioseparation.

During the past several years, capillary electrophoresis has been demonstrated to be a convenient and precise method for the determination of acidity constants (Ka) in aqueous [15,16] and non-aqueous mediums [17,18]. Several experimental approaches, linear or nonlinear, are applicable. In order to determine the acidity constants we only need to follow the evolution of the effective electrophoretic mobility (µeff) of the target sample as a function of the pH of the running electrolyte, the ionic strength of which is kept constant. The experimental Ka value is then determined from the slope of the graph obtained; the inflection point of the graph µeff = f (pH) is equal to the pKa. The pKa is then deduced thermodynamically by using the equation (1):

I r 0.328 1 I 0.5085 pK pK i exp a thermo a × × + - = ( 1
)
where I is the ionic strength of the running electrolyte and ri is the hydrated radius of the species studied. A value of 0.5 nm is generally recommended [19].

Despite the fact that Binol and its derivatives have high pKa values because they are very weak acids, the use of CE allowed us to determine their pKa values without difficulties, as the pH value of the running electrolytes can reach about 12. The results obtained for the different Binols studied are grouped in Table 2.

Table 2 As was expected, Binol and its derivatives have high acidity constants: Binols with two hydroxyl functions have values of values 9/9.4 for the first acidity and 10.9 for the second,

and Binols with only one hydroxyl function have a value of approximately 10.2, Table 2.

Before starting the optimization of the enantioseparation of binaphthol and its four derivatives in the presence of cyclodextrins as chiral selectors, in the case of Binol, we studied the evaluation of the enantioseparation as a function of the pH by using a native cyclodextrin as the chiral selector.

Study of the enantioseparation of Binol

The enantioseparation depends directly on the affinity of the two enantiomers to the cyclodextrin (CD) used. Therefore, the use of different cyclodextrins (native or derivative, neutral or charged) strongly affects the results. For the enantioseparation of Binol racemate and its derivatives, generally denoted as BN in the remainder of this text, the study was limited to the case of neutral cyclodextrin. To our knowledge, the enantioseparation of Binol and its derivatives has never been carried out in a genuinely satisfactory way (electrophoretic enantioselectivity αexp>1), with the exception of the use of trimethyl-β-CD (TM-β-CD, αexp=1.22 [5]), hydroxypropyl-α-cyclodextrin (HP-α-CD) and TM-β-CD, (HP-α-CD, Rs=1.2 and TM-β-CD, Rs=1.2 [6]). With other neutral CDs, either no enantioseparation was systematically observed (in the case of α-CD and HP-γ-CD) or, according to the authors, no enantioseparation was obtained at the pH used [5,6]. On the contrary, specific enantioselectivity was noted (in the case of β-CD, γ-CD and HP-β-CD at pH 10.5) [10].

Although enantioselectivities are weaker than those reported above, K. Kano et al. [5] and H.

Nishi [6] did not observe a chiral resolution of the Binol racemate at pH 10.5, while at this same pH, O. Zerbinati et al. obtained certain enantioselectivity [10].

All of the CDs studied are naturally exempt from all charge, so it proved imperative to charge the analytes in order to confer a charge to the BN/CD complexes, and thus ensure different velocities of the experimental electrophoretic migration for the S and R enantiomers. Taking into account the obtained pKa values of Binol and its derivatives (the range of the pKa corresponding to the first acidity is between 9 and 10.2, see Table 2), a significant negative charge for the Binol racemate can only be obtained at pH ≥ 10.5. A weak acid presents a charge equal to -1 when the pH of the electrolyte used is higher or equal to pKa + 2.

Consequently, we decided to examine a range of pH between 9.5 and 12. However, it should be noted that the experiments conducted at pH=12 showed to be delicate due to problems encountered concerning the repeatability as well as the stability of the electrophoretic current.

Therefore, we were forced to limit the range of the studied pH between 9.5 and 11.5. Using the Binol racemate as a sample test, we studied the influence of the ionic strength and the pH of the electrolyte on the enantioseparation.

Ionic strength optimization

For the pH range studied, the charge of the two enantiomers of Binol was maximal at pH 11.5, with a charge slightly higher than -1.5. The influence of ionic strength on the enantioseparation was therefore studied at this pH. Indeed, the electroosmotic flow is cathodic and the greatest resolution with neutral CDs should be achieved under these conditions. In fact, at weak ionic strength, the electroosmotic flow is very significant and consequently the electrophoretic peaks corresponding to the two enantiomers of Binol were hardly differentiated from that of the electroosmotic flow due to their electrophoretic migration direction, which is diametrically opposed to that of the electroosmotic flow. Despite the possibility of operating under very intense electric fields, consequently obtaining very high efficiencies, i.e. the joule effect was perfectly dissipated, we did not obtain a satisfactory resolution regardless of the size of the cyclodextrin used (α-CD, β-CD or γ-CD).

Progressively reducing the electroosmotic flow by increasing the ionic strength of the electrolyte, at a constant pH equal to 11.5, we noticed a simultaneous increase in the enantioresolution. Thus, we obtained a resolution superior to 1.5 for an ionic strength equal to 80 mM, while, in such conditions, the applied voltage could not exceed 10 kV. Consequently, for the remainder of the study, the ionic strength was maintained at 80 mM and the applied voltage was fixed at 10 kV.

Choice of the pH of the medium

The electrophoretic enantioselectivity (αexp: the ratio of the electrophoretic mobilities of the two enantiomers) proved to be slightly greater with γ-CD (αexp=1.05) than with the other two native CDs (αexp=1.04 with α-CD as well as with β-CD). Therefore, we studied the influence of the pH on the enantioseparation of the Binol racemate with γ-CD. The ionic strength and applied voltage were fixed at 80 mM and 10 kV, respectively. In fact, the study of the evolution of the resolution was simultaneously conducted as a function of the pH and γ-CD concentration, in the pH range between 9.5 and 11.5 and γ-CD concentrations between 0 and 20 mM, in order to determine the best experimental conditions.

Figure 1 reports the electropherograms obtained at the end of the analysis of Binol racemate spiked with pure R enantiomer at pH 10 (the upper line) and pH 11.5.

A comparison of the two electropherograms showed that the migration order of the two enantiomers of Binol was inversed at pH 11.5 compared with that obtained at pH 10. The R enantiomer of Binol migrated initially at pH 10 and eluted a second time at pH 11.5.

Figure 1 Such an observation could originate in the progressive ionization of γ-CD as the pH is increased. In fact, in the pH range between 10.2 and 10.5, we observed the coelution of the two enantiomers. If we refer to the literature, we find that the pKa of γ-CD is equal to 12.08 [20].

At pH 10, γ-CD is neutral and therefore is carried by the electroosmotic flow, which is cathodic. Consequently, the enantiomer with more affinity towards the hydrophobic torus of CD migrates first, since the two non-complexed enantiomers migrate towards the anode at this pH (pKa1 Binol=9.04 and pKa2 Binol=10.90, see Table 2). Thus, in light of the electropherograms reported in Figure 1, we conclude that the R enantiomer of Binol has the greatest affinity for γ-CD. On other hand, at pH 11.5, γ-CD is partially ionized (charge << -1/2) and thus, under such conditions, it presents an electrophoretic mobility that is diametrically opposed to that of the electroosmotic flow, and, consequently, it slows down the migration of the enantiomer that has the greatest affinity with it, that is to say the R enantiomer.

Figure 2 summarizes the evolution of the resolution of the Binol racemate as a function of the pH in the range explored. As this graph highlights, the resolution value at pH 11.5 (Rs=2.4) is greater than at pH 10 (Rs=0.9), that is to say, before the inversion of the enantiomer migration order. Such an observation was made not only in the case of γ-CD as the chiral selector, but was systematically noticed in this study regardless of the cyclodextrin used.

Figure 2 Furthermore, it should be noted that when the pH of the electrolyte is lower than 10, the resolution drops rapidly (Rs is about 0.6 when the pH of the electrolyte reaches 9.5). Indeed, the two enantiomers of Binol present small charges, while γ-CD is neutral. Thus, their migration becomes differentiated from that of the electroosmotic flow and, consequently, the difference in their migration velocity is enough only to be displayed as the beginning of the separation. Secondly, it appears that the optimal concentration of γ-CD is approximately the same at the two pH values considered. In fact, at pH 10, the optimal concentration of γ-CD is equal to 10 mM, where the enantiomeric resolution reaches its maximum value, while, at pH 11.5, this concentration is equal to 6 mM.

We tried to find if such an inversion could be observed as a function of pH with the other two native cyclodextrins (α-CD and β-CD), as well as with different neutral cyclodextrins, either hydroxypropylated (HP-α-CD, HP-β-CD and HP-γ-CD) or trimethylated (TM-β-CD). In fact, such an inversion in the migration order of the Binol enantiomers was observed with β-CD as well as with two out of three hydroxypropylated cyclodextrins (HP-β-CD and HP-γ-CD). On the other hand, no inversion was observed in the case of α-CD, HP-α-CD or TM-β-CD. However, we noticed that in the case of α-CD, a coelution could be observed as a function of the pH, unlike in the case of HP-α-CD and TM-β-CD.

The migration order observed at a pH greater or equal to 10.6 corresponds to the migration order observed by O. Zerbinati and F. Trotta [10] at pH 10.5, during a partial resolution of the Binol racemate in the presence of γ-CD. In that study, the authors determined the association constants of the two Binol enantiomers with γ-CD and acceded in that way to the thermodynamic enantioselectivity for the Binol/γ-CD couple. The treatment used is based on non-charged-CDs. In fact, such a hypothesis seems reasonable, as, at this pH, the charge of γ-CD is very weak when taking into account its pKa (12.08). Such an observation is also applied in the present study, and so the origin of the migration order reversal of the Binol enantiomers as a function of pH must found elsewhere, approximating the charge of γ-CD to zero. In reality, in a very interesting study, G. Vigh et al. [21][22][23] modeled the enantiomeric separation of chiral weak acids and bases in CE. They showed that according to the magnitude orders of (1) the electrophoretic mobilities of (dissociated analytes / CD) complexes (µRCD -and µSCD -), and (2) the complex formation constants of the analytes in their dissociated and nondissociated forms with CD (on the one hand KRCD -, KSCD -and on the other hand KHRCD, KHSCD), three kinds of separation could be envisaged as a function of the pH and CD concentration, while the latter is systematically neutral. The three kinds of separation are desionoselective separation or Type I (µRCD -= µSCD -, KRCD -= KSCD -and KHRCD ≠ KHSCD), ionoselective separation or Type II (µRCD -≠ µSCD -, KRCD -≠ KSCD -and KHRCD = KHSCD), and duoselective separation or Type III (µRCD -≠ µSCD -, KRCD -≠ KSCD -and KHRCD ≠ KHSCD).

The two latter types allow for, if so desired, the reversal of the migration order of the enantiomers as a function of the electrolyte composition (pH, CD concentration).

Thereby the origin of the reversal migration order of Binol enantiomers as a function of pH in the presence of γ-CD is more likely be found in the behavior of Type II or III, concerning the enantioseparation, than in the progressive ionization of γ-CD. In fact, the latter method remains modest even at pH 11.5.

A fortiori in the present case, the reversal of the migration order of the enantiomers as a function of the pH probably results from the behavior reflected either in an ionoselective separation (Type II) or a duoselective separation (Type III). In fact, if we observe the pKa values of the previously studied cyclodextrins, they prove to be even greater than that of γ-CD. They increase from γ-CD (pKa=12.08) to α-CD (pKa=12.33). The pKa of β-CD is intermediary and is equal to 12.20 [20]. On the other hand, the most acidic sites must be the most reactive and we can therefore suppose that the pKa values of the derivative cyclodextrins are appreciably higher than the corresponding native CDs. Consequently, there is a likelihood that, in the considered pH range, the charge of the cyclodextrin is very weak, even null concerning HP-β-CD and HP-γ-CD, and that the second hypothesis for the explication of the reversal of the migration order of the enantiomers as a function of pH is more plausible than the first one. Therefore, we consider that the possible charge of CD is negligible in the pH range considered.

Finally it should be noted that when no inversion of the migration order was observed as a function of the pH (HP-α-CD and TM-β-CD), we systematically remarked that the enantioselective resolution of Binol racemate increased regularly with the pH of the electrolyte and that it was much higher at pH 11.5 than at other any pH. Consequently, this pH value (11.5) was used for the remainder of the study.

Optimization of cyclodextrin concentration as a function of its nature

The optimal conditions were thus defined in the case of the (R,S Binol/γ-CD) couple. We then determined the optimal concentrations of the other six neutral CDs studied, namely the two natives (α-CD and β-CD), the hydroxypropylated (HP-α-CD, HP-β-CD and HP-γ-CD) and the trimethylated (TM-β-CD). The pH and the ionic strength were fixed at 11.5 and 80 mM, respectively. Table 3 reports (1) the optimal concentration of cyclodextrin (where the electrophoretic enantioselectivity, also consigned in this table, is maximal), (2) the literature data concerning the enantioseparation of the Binol racemate when the same CD is used as the chiral selector, and (3) the pH for each of the CD used in this study.

Table 3 In fact the optimal concentrations of CDs were experimentally deduced from Δµ eff = f([CD]) curves (Δµ eff = µ2-µ1 the difference in electrophoretic mobilities of the two atropisomers, µ1 and µ2).

It appears that, at pH 11.5 and contrary to what has been observed in the literature at pH 9

[24], 10.5 [5,6] and 11 [25], we can obtain a resolution of the Binol racemate with all of the cyclodextrins studied. In fact, the enantioseparation of the Binol racemate has only been reported with TM-β-CD, either at pH 10.5 [5,6] or at pH 11 [5,25], with HP-α-CD at pH 10.5 [6], with HP-β-CD at pH 10.5 [10], with γ-CD either at pH 9 [24] or at pH 10.5 [10], and with β-CD at pH 9 [24] and 10.5 [10].

Such an observation clearly demonstrates the definite advantages of working in a more basic medium and thus at pH 11.5.

Secondly, it appears that, with TM-β-CD, the enantioselectivity is maximal (αexp=1.27 at optimal concentration of TM-β-CD equal to 10 -2 M). Otherwise, the three hydroxypropylated cyclodextrins led approximately to the same enantioselectivity (αexp about 1.13) with a slight advantage for HP-α-CD (αexp=1.14). However, the optimal concentrations of the three hydroxypropylated cyclodextrins are not identical, and the highest concentration is for HP-α-CD (Copt=1.5.10 -2 M) against only 8.10 -3 M for HP-γ-CD and only 4.4.10 -3 M for HP-β-CD, which proved to be the most interesting among the three hydroxypropylated CDs when taking into account the concept of consummated quantity.

Finally, the three native cyclodextrins led to a weaker enantioselectivity than the other CDs studied. Once again, with a first approximation, we can say that they led more or less to the same enantioselectivity, but for concentrations of CD varying from single to triple. For γ-CD, which presents the lowest optimal concentration of CD among the three native CDs (Copt=6.10 -3 M), a slightly greater enantioselectivity is obtained (αexp=1.05 against αexp=1.04 with α-CD as well as with β-CD).

In the case of γ-CD, an inversion in the migration order of the two enantiomers of Binol was observed at pH 10.8 as a function of cyclodextrin concentration; this case is reported below.

However, before discussing this important case and taking into account all of these positive results, we evaluated the capacity of the seven CDs, implemented previously, towards the resolution of the racemic mixtures of various Binol derivatives (structures given in Table 1) at pH 11.5 with an ionic strength of 80 mM.

Enantioseparation of Binol derivatives

For the four Binol derivatives, we alternatively used each of the seven CDs studied as chiral selectors. For each (racemate of Binol derivative/CD) couple we studied the evolution of the electrophoretic enantioselectivity (αexp) as a function of the CD concentration. The range of the explored concentration was between 0 and 2.5.10 -2 M taking into account the previously obtained results for the Binol racemate. Such a study allowed us to determine the various optimal concentrations of cyclodextrins for each of twenty-eight (Binol derivative/CD) couples. Copt depended on the CD structures, where the enantioselectivity was maximal and thus the resolution was maximal. The results obtained are given in Table 4.

Table 4

Examination of Table 4 confirms the first conclusions of the experiments conducted with Binol. α-CD proved to be the least favorable CD among the seven cyclodextrins studied. In fact, it did not allow the enantioseparation of two out of four Binol derivative racemates, that is to say BN2 and BN3.

On the other hand, the cyclodextrins that are constituted by seven or eight glucose units (β-CD and γ-CD natives or derivatives) provided better results than their α homologue, which is constituted by six glucose units. In fact, taking into consideration all the CDs studied with seven glucose units (native β-CD and its derivatives HP-β-CD and TM-β-CD), only one out of four Binol derivative racemates, namely BN2, could not be separated with this CD family.

On the contrary, taking into consideration the studied cyclodextrins with eight glucose units (native γ-CD and its derivative HP-γ-CD), all of the Binol derivative racemates could be separated using this CD family as the chiral selector.

Finally, in general the cyclodextrin derivatives led to higher enantioseparations than that observed with their homologous native CD. However, we note one exception to this rule:

HP-β-CD proved to be less satisfactory than β-CD towards the resolution of the BN2 racemate, since it did not lead to any enantioseparation, while we observed an enantioselectivity (αexp=1.05) with β-CD. Moreover, except for the case of BN5, in general TM-β-CD led to the highest enantioselectivity observed during this study.

Reversal of enantiomer migration order as a function of γ-CD concentration

At pH 10.8, when the γ-CD concentration progressively increased from 2.10 -3 M to 6.10 -3 M, the electrophoretic peak corresponding to the S enantiomer of Binol (enantiomer migrates firstly) gradually approached the peak of the R enantiomer. We then observed the coelution of the two enantiomers at γ-CD concentrations ranging from 7.10 -3 M to 15.10 -3 M. The concentration range of the observed coelution appears to be relatively important. Finally when the γ-CD concentration became higher than 1.5.10 -2 M, we once again observed a resolution of the Binol racemate, but in this case the R enantiomer migrated first. Figure 3 shows three electropherograms concerning the analysis of the Binol racemate effectuated at pH 10.8. In the presence of a variable concentration of γ-CD, these three electropherograms show enantioseparations in each of the three above-mentioned zones of γ-CD concentration.

Figure 3 This phenomenon of inversion in enantiomer migration order was widely interpreted by B.

Chankvetadze [START_REF] Chankvetadze | Capillary Electrophoresis in Chiral Analysis[END_REF]. The author presented and discussed the different parameters responsible for the origin of such an inversion, such as the pH, the inversion of the direction of electroosmotic flow or even the change of the chiral selector nature. However, there are very few examples of inversion reported in the literature where the experimental parameter of the inversion origin is the cyclodextrin concentration directly [START_REF] Schmitt | [END_REF][28][29]. For example, T. Schmitt and H. Engelhardt [START_REF] Schmitt | [END_REF] reported the case of dansylated phenylalanine racemate with HP-β-CD, where the migration order of the two enantiomers is reversed at low and high concentrations of HP-β-CD. The authors explain this reversal migration order by the modifications of the electrophoretic mobilities as a function of CD concentration: at low concentrations, the electrophoretic mobilities reflect the differential interactions with HP-β-CD, while at high concentrations the migration order is directly controlled by the electrophoretic mobility of the diastereomeric formed complexes.

The case described in this paper follows principally the same rationalization. Thus, we can postulate that at low γ-CD concentrations, the two Binol enantiomers are separated according to their affinity with γ-CD, while at high γ-CD concentrations the principle of the resolution of Binol enantiomers is due to the electrophoretic mobilities of diastereomeric complexes formed between each of the Binol enantiomers and γ-CD. However, such a hypothesis could be validated by the evaluation of the complexation constants and the complex mobilities according to B. Chankvetadze [30].

Finally in very recent works, the utilization of chiral ionic liquids is reported for the first time either as a single chiral selector [31] or by combination with cyclodextrins [32] for the enantioseparation of alcohols, amine, acid, and amino acids, and 2-arylpropionic acids. We decided to evaluate the effect of adding a chiral ionic liquid in the case of atropisomer enantioseparation, a problem that has not yet been reported in the literature.

Simultaneous adding of chiral ionic liquids

P. Gareil et al. [32] showed weak synergistic effects concerning the enantioseparation of racemic mixtures of 2-arylpropionic acids by simultaneously using cyclodextrins and (R)(-)-2-hydroxy-N,N,N-trimethyl-1-phenylethanaminium bis(trifluoromethylsulfonyl)imide (R-Phenylcholine NTf2) or (R)(-)-1-hydroxy-N,N,N-trimethylbutan-2-aminium bis (trifluoro-methylsulfonyl) imide (R-Etcholine NTf2). Firstly, we envisaged the successive addition of these chiral ionic liquids for the enantioseparation of binaphthol. Unfortunately, R-Phenylcholine NTf2 has a chromophore, which generates a strong absorbance at the detection wavelength, and consequently it was impossible to carry out such experimentation.

On the other hand, R-Etcholine NTf2 is transparent and well adapted for the test. In order to evaluate the potential of R-Etcholine NTf2 in combination with γ-CD at pH 11.5 for the enantioseparation of binaphthol, we resorted to the experimental design methodology. The aim of this study was to highlight the potential interactions between the operation parameters, i.e. γ-CD concentration and R-Etcholine NTf2 concentration. In order to model the response surface (electrophoretic enantioselectivity αexp) in the explored experimental domain (γ-CD concentration between 0 and 3.10 -2 M and R-Etcholine NTf2 concentration between 0 and 2.10 -2 M) we resorted to a central composite design with uniform precision (CCD-UP). This design consists of 13 runs and 2 factors with 2 levels. The factors and their levels are reported in Table 5, where α is equal to 1.41 in adequacy with the theory ( ) 2 ( n 4 / 1 = α with n=2) [START_REF] Sado | Les plans d'expériences[END_REF].

Table 5 The evolution of the electrophoretic enantioselectivity (αexp) of the two atropisomers of binaphthol as a function of γ-CD and R-Etcholine NTf2 concentration is presented in Figure 4a.

Figure 4 As has been highlighted previously, if there is an effect during the addition of R-Etcholine NTf2 in the presence of γ-CD, then it is weak. In order to objectively analyze this potential effect, we substituted lithium NTf2 in the second series of runs for R-Etcholine NTf2 and studied the response surface (αexp) in an identical experimental domain using CCD-UP. The response surface (electrophoretic enantioselectivity αexp) values after the end of the new series of 13 runs is shown in Figure 4b. A comparison between Figures 4a and4b clearly shows that there is neither a synergistic nor an antagonistic effect from adding R-Etcholine NTf2 to the implemented electrophoretic system during the enantioseparation of atropisomers.

At this stage in the study, we tried to find an ionic liquid without chromophore among the chiral ionic liquids already reported in the literature. If this material exists, its synthesis will not be based on the choline derivative already imployed. With this reasoning, we next evaluated the effect of the simultaneous addition of S(+)-tetrabutylammonium camphorsulfonate (S-tb-camphor) to the experimental mixture.

Resorting again to the previously exployed experimental design (CCD-UP), we were able to plot the response surface (αexp) after 13 runs, Figure 4c.

As Figure 4c clearly demonstrates, the addition of S-tb-camphor is not neutral to the resolution of the Binol racemate under the operation conditions established previously, contrary to what we observed with the addition of R-Etcholine NTf2. In fact, Figure 4c highlights a weak antagonistic effect for S-tb-camphor. Thus the degree of the enantioselectivity, obtained by using γ-CD at pH 11.5, is progressively decreased as the S-tbcamphor concentration is increased, regardless of the γ-CD concentration.

Finally, it must be noted that no enantioseparation of the Binol racemate was observed with R-Etcholine NTf2 as the only chiral selector. Unfortunately, a similar situation was also observed with the other chiral ionic liquids tested. In fact, no separation was found when only S-tb-camphor was used as the electrophoretic medium in the range of explored concentrations (0 -3.10 -2 M).

Conclusion

The aim of this work was to study the evolution of the enantioselectivity of various racemates of Binol and four of its mono substituted derivatives in the presence of different cyclodextrins (seven native or derivative cyclodextrins).

The result of this study is that the enantioselectivity of Binol atropisomers and its mono substituted derivatives increases systematically with pH, regardless of the Binol/cyclodextrin couple being considered. Consequently, it was interesting to carry out such separations at pH 11.5, where no difficulty was met in realizing the enantioseparations.

On the other hand, this study highlighted an inversion in the enantiomer migration order as a function of the pH, in the case of the Binol and the γ-cyclodextrin. Thus, in this specific case, there is a pH value where no resolution could be observed. Based on experimental results, this pH value is situated between 10.3 and 10.5.

Having defined the pH, we studied the evolution of the enantioselectivity as a function of the cyclodextrin concentration. Once again, we were able to highlight a particular behavior in the case of the Binol/γ-cyclodextrin couple by observing an inversion in the enantiomer migration order as a function of the γ-cyclodextrin concentration at pH 10. Globally, α-CD proved to be the least favorable CD among the seven cyclodextrins studied.

On the other hand, the cyclodextrins that are constituted by seven or eight glucose units (β-CD and γ-CD natives or derivatives) provided better results than their α homologue, which is constituted by six glucose units. In fact, taking into consideration all the CDs studied with seven glucose units (native β-CD and its derivatives HP-β-CD and TM-β-CD), only one out of four Binol derivative racemates, namely BN2, could not be separated with this CD family.

On the contrary, taking into consideration the cyclodextrins studied with eight glucose units (native γ-CD and its derivative HP-γ-CD), all of the Binol derivative racemates could be separated using this CD family as chiral selector.

Finally, the addition of the chiral ionic liquid (R-Etcholine NTf2 or S-tb-camphor) did not perceptibly reinforce the enantioselectivity of the system considered.

We envisage, in the near future, studying the enantioseparation of other Binol derivatives in order to discover if the observed inversion of the enantiomer migration order can be observed in these systems as well.

  8. The S enantiomer migrated before the R enantiomer at low cyclodextrin concentration. Consequently, at concentrations between 7.10 -3 M and 15.10 -3 M, no enantioselective separation could be observed in the case of Binol and γ-cyclodextrin. This double inversion in the enantiomer migration order, on the one hand as a function of the pH and on the other hand as a function of the concentration of chiral selector, is remarkable. It is an illustration of the Type II enantioseparation (ionoselective) or the Type III enantioseparation (duoselective), according to the terminology established by G. Vigh following his theoretical treatment of modeling the enantioseparation of weak chiral acids.

Figure 1 :

 1 Figure 1: Enantioseparation of Binol racemate in presence of γ-CD. Experimental conditions: uncoated silica capillary (40.2 (30) cm × 50 µm ID); hydrodynamic injection: 3s at 0.5 psi; T=20°C; λ=214 nm; voltage: 10kV; electrolyte: Na2HPO4 / Na3PO4, ionic strength: 8.10 -2 M; [γ-CD]=4.10 -3 M; sample: Binol racemate spiked with the R enantiomer; (A) at pH=10 and (B) at pH=11.5.

Figure 2 :

 2 Figure 2: Resolution of Binol racemate as a function of the pH of the running electrolyte. Experimental conditions: uncoated silica capillary (40.2 (30) cm × 50 µm ID); hydrodynamic injection: 3s at 0.5 psi; T=20°C; λ=214 nm; voltage: 10kV; electrolyte: phosphate with ionic strength=8.10 -2 M; [γ-CD]=4.10 -3 M; sample: Binol racemate spiked with the R enantiomer.

Figure 3 :

 3 Figure 3: Electropherograms recorded during the enantioseparation of Binol with variable concentrations of γ-CD. Experimental conditions: uncoated silica capillary (40.2 (30) cm × 50 µm ID); hydrodynamic injection: 3s at 0.5 psi; T=20°C; λ=214 nm; voltage: 10kV; electrolyte: Na2HPO4 / Na3PO4; pH=10.8; ionic strength: 8.10 -2 M; [γ-CD]: variable; sample: Binol racemate spiked with the R enantiomer.

Figure 4 :

 4 Figure 4: Response surface plots for the electrophoretic enantioselectivity of Binol racemate (αexp) as a function of γ-CD and ionic liquid concentrations a) R-Etcholine NTf2, b) LiNTf2 and c) S-tb-camphor.


Quanbo Jiang

Benoît Rogez

Jean-Benoît Claude

Antonin Moreau

Julien Lumeau

Guillaume Baffou

Jérôme Wenger
email: jerome.wenger@fresnel.fr

Adhesion Layer Influence on Controlling the Local Temperature in Plasmonic Gold Nanoholes

Keywords: thermoplasmonics, nanoaperture, zero-mode waveguide, temperature measurements, nanooptical trapping

Gold films do not adhere well on glass substrates, so plasmonics experiments typically use a thin adhesion layer of titanium or chromium to ensure a proper adhesion between the gold film and the glass substrate.

While the absorption of light into gold structures is largely used to generate heat and control the temperature at the nanoscale, the influence of the adhesion layer on this process is largely overlooked.

Here, we quantify the role of the adhesion layer in determining the local temperature increase around a single nanohole illuminated by a focused infrared laser. Despite their nanometer thickness, adhesion layers can absorb a greater fraction of the incoming infrared light than the 100 nm thick gold layer leading to a significant increase of the local temperature. Different experimental designs are explored, offering new ways to promote or avoid the temperature increase inside nanoapertures. This knowledge further expands the plasmonic toolbox for temperature-controlled experiments including single molecule sensing, nanopore translocation, polymerization, or nano-optical trapping.

Introduction

While plasmonics offers many opportunities to control the light fields into nanoscale dimensions, it is also intrinsically bound to the occurrence of absorption and Ohmic losses into the metal. [START_REF] Novotny | Principles of Nano-Optics[END_REF] These losses eventually lead to Joule heating of the surrounding environment, 2 hereby increasing the local temperature. This new ability to generate local temperature distributions has driven the emergence of thermoplasmonics to control heat generation at the nanoscale, [3][4][START_REF] Baffou | Thermoplasmonics[END_REF] which is fruitful for various applications in different fields of physics, biology and chemistry. [START_REF] Qiu | [END_REF][7][8] Evaporated gold films, which are commonly used in plasmonics, generally require the presence of an additional adhesion layer (typically a few nanometers of titanium or chromium) to promote the adhesion of the gold structure onto the glass substrate. 9,10 Despite its small thickness in the nanometer range, this adhesion layer may have a significant influence on the plasmonic response of the gold structure. For instance, the adhesion layer was shown to shift and broaden the plasmon resonance, 11-14 affect the transmission through nanohole arrays, 15 damp the enhancement factor in surface-enhanced fluorescence 16,17 and Raman scattering, [18][19][20] and affect the nano-optical trapping potential. 21 However, in the context of heat generation in thermoplasmonics, the role of this supplementary adhesion layer remains largely unexplored and very little is known about the quantitative influence of various gold adhesion layers in the temperature increase around illuminated gold nanostructures. 22 Here, we experimentally and numerically quantify the role of the gold adhesion layer in determining the local temperature increase around a single nanohole (SNH) illuminated by a focused infrared laser beam.

We also investigate supplementary approaches to control the temperature by properly selecting the illumination geometry and the substrate material. Nanoholes are chosen to benchmark the influence of the adhesion layer, as these structures have received a large interest owing to their ability to confine light into attoliter volumes, 23,24 enhance the luminescence of single quantum emitters, 16,25 and trap single nano-objects. 26,27 Our experimental configuration reproduces the typical arrangement used for nanooptical trapping using nanoholes, [27][28][29][30][31][32][33][34][35] and therefore our results can be directly applied to these experiments. The choice of a single nanohole of fixed diameter is not a limitation: we have recently shown that the temperature increases inside a single or a double nanohole are quite comparable and largely independent of the plasmonic resonance for aperture diameters below 350 nm. 36 Thus, the results reported here for a 300 nm diameter SNH directly apply also to other nano-optical trapping experiments. [27][28][29][30][31][32][33][34][35] More generally, adjusting the adhesion layer and the experimental configuration bring new options to control the local temperature with plasmonics. This further expands the thermoplasmonics toolbox for several heat-controlled applications including polymerization, 37 enzymatic activity monitoring, 23,24 nanopore translocation, [38][39][40] or thermophoretic trapping. 41,42

Results and Discussion

Our experiments focus on a single nanohole (SNH) of 300 nm diameter milled by focused ion beam (FIB) in a 100 nm thick gold film (Fig. 1a,b). A 1064 nm infrared (IR) laser beam focused by a high NA microscope objective is used to heat the gold SNH. We have recently developed a technique to measure the temperature inside SNH structures based on monitoring the fluorescence lifetime of Alexa Fluor 647 molecules. 36 This technique has also been validated against two other independent experimental measurements based on fluorescence intensity and diffusion time, and against numerical simulations. 36 The key concept in the fluorescence lifetime approach is that the nonradiative decay rate constant of the Alexa Fluor 647 dyes is temperature dependent and can be calibrated to measure locally the temperature around a SNH. The Methods section briefly summarizes the approach and main equations needed to compute back the temperature increase from the fluorescence lifetime measurements. In addition to the infrared heating laser, we overlap a 635nm pulsed red laser with 20 µW/µm² average intensity to excite the fluorescence and record the lifetime. We neglect the heating effect induced by the red laser as its intensity remains always small compared to the IR laser intensity in the mW/µm² range.

We have recently shown that the temperature increase inside SNHs does not significantly depend on the nanohole diameter as long as the diameter is below 350 nm. 36 Similarly, the temperature increase is quite comparable between single and double nanohole structures, independently of the plasmonic resonance, and is mainly dictated by the absorption and Joule losses into the metal layer. Therefore, the results discussed here for the 300 nm diameter SNH can be readily extended to other SNH diameters, and even double nanohole structures (DNH). Titanium, chromium or chromium oxide layers are typically used in plasmonics to ensure a proper adhesion between the gold film and the glass substrate. [11][12][13][15][16][17][18][19][20][21] Here we use a representative selection of these materials and test different thicknesses for the adhesion layer. For example, the 9 nm thick chromium oxide (Cr2O3) adhesion layer can be seen by scanning electron microscopy (SEM) after half the SNH structure has been cut by FIB to show a cross-section image of the SNH (Fig. 1c).

For each adhesion layer, we record the temperature increase as a function of the IR heating intensity.

Figure 1d-f briefly presents typical experimental results for a 9 nm Cr2O3 adhesion layer. The starting point is the fluorescence decay histograms (Fig. 1d) measured on a 300 nm diameter SNH filled with the Alexa Fluor 647 water solution. The three histograms on Fig. 1d for increasing IR intensities readily show a clear fluorescence lifetime reduction as the IR intensity raises. From the fluorescence decays, we extract the fluorescence lifetime and the nonradiative decay rate which are temperature dependent (Fig. 1e). Using the calibrated temperature dependence and the technique described in the Methods section, we can compute back the temperature increase for each IR illumination intensity (Fig. 1f). For all cases, we observe a linear relationship between the temperature increase and the IR intensity, as expected from the linearity of the heat-diffusion equation. 5 The main goal of our paper is to explore how the range of temperature increase can be tuned by properly choosing the nature and thickness of the gold adhesion layer, the illumination geometry and the substrate material. Figure 2 summarizes our results for different adhesion layers. We also show the results obtained when only gold is illuminated (all other experimental parameters are identical). Although the same SNH diameter, same gold thickness, and same IR power are used, the fluorescence decay histograms recorded for different adhesion layers directly show a pronounced difference, which indicates a different temperature elevation (Fig. 2a). When no infrared beam is present, the fluorescence lifetime is reduced by the gold SNH from the 1.0 ns confocal value to 0.8 ± 0.05 ns (Fig. 2b, blue bars). This moderate lifetime reduction is ascribed to the SNH size and quite independent of the choice of the adhesion layer, confirming that the Alexa Fluor 647 molecules experience similar nanophotonic environments. However, when the IR heating laser is turned on, the fluorescence lifetime is then clearly different depending on the adhesion layer material (Fig. 2b, red bars). This observation directly shows that the adhesion layer plays a key role in the local temperature increase inside a SNH.

Based on the lifetime reduction measured for each adhesion layer compared with the case without IR illumination, we compute back the temperature increase following the same procedure as for Fig. 1f.

Figure 2c summarizes our main results. The 13 nm Cr adhesion layer yields the highest temperature increase for a given IR power, and can reach a gain up to +40°C at 5 mW/µm² IR illumination. Here we have used a relatively thick adhesion layer in order to see how large the temperature increase can be despite the presence of the gold layer acting as a heat sink. 43 Conversely, the gold film without any adhesion layer features a ten-fold reduced temperature gain with only +4.3°C at the same 5 mW/µm² IR intensity. This impressive difference reveals the major role played by the adhesion layer in controlling the local temperature increase upon SNH laser illumination. Additionally, our measurements for the gold film with no adhesion layer match very well two completely independent temperature assessments based on ionic current measurements 40 or numerical simulations 35 (pink cross markers in Fig. 2c). This further validates our experimental results and methodology.

Comparing between the different adhesion layers, we find that the temperature increases with 6 nm Ti and 6 nm Cr layer are about twice less than with 13 nm Cr case. This effect is mainly a consequence of the reduced thickness used, as the infrared absorption of Cr and Ti layers at 1064 nm are very similar (Supporting Information Fig. S1 andS2). Furthermore, for thicknesses below 20 nm, the IR absorption and the temperature increase are linearly proportional to the adhesion layer thickness (Supporting Information Fig. S1). Chromium oxide Cr2O3 is significantly less absorbing the IR than pure metals like Cr or Ti. Therefore, despite its 9 nm thickness, the temperature increase with the Cr2O3 layer remains slightly lower than with a 6 nm Ti or Cr layer (Fig. 2c). Altogether, these experimental results show that the temperature increase inside a SNH can be largely controlled by the material chosen for the gold adhesion layer, its thickness and the IR intensity used. Our experimental findings suggest that the temperature increase is mainly ascribed to the IR Ohmic losses into the adhesion layer instead of the gold layer. This is quite surprising given the small thickness used for the adhesion layers (below 13 nm) as compared to the 100 nm thick gold film. To confirm this effect, we have conducted a series of numerical simulations using COMSOL Multiphysics (Fig. 3). Spatial maps of the heat source density and temperature increase on a 300 nm gold SNH without (left) and with the 13 nm Cr adhesion layer (right) are presented in Fig. 3a,b respectively. In the absence of the adhesion layer, the heat source essentially accumulates at the aperture edges, where the power dissipated by the laserinduced electron current is maximum. 44 In the presence of the 13 nm Cr layer, the absorption into the thin Cr layer generates a dominating additional contribution to the heat source (Fig. 3a). The maximum heat source density is increased by 8 fold, and now spreads over a much larger area. As a consequence of the Joule heating, the temperature rises in the nanohole. Due to the large difference of heat source densities between the cases with and without adhesion layers, the temperature increase in the nanohole with the 13 nm Cr layer is almost ten times higher than the case without any adhesion layer (Fig. 3b), in excellent agreement with our experimental results (Fig. 2c). In both cases (with and without the adhesion layer), the 100 nm thick gold film serves as a heat sink to dissipate the temperature. Therefore the thermal gradients in both cases follow very similar spatial distributions, despite the ten-fold reduced amplitude in the absence of Cr layer.

The same numerical simulations are performed to reproduce the other experimental configurations, and the temperature increase is averaged over the SNH volume. This allows a direct comparison with the experimental results (Fig. 3c), for which we find an excellent agreement for all the different cases. This set of simulations clearly confirms that the nature and thickness of the gold adhesion layer largely control the temperature inside nanohole structures. Other approaches can be used to further tune the temperature gain inside the SNH. Instead of the borosilicate glass, the metal layers can be deposited on a sapphire substrate, which features a much better thermal conductivity than standard glass. This introduces a supplementary heat dissipation channel, still choosing a transparent thermally-conductive substrate. Figure 4a shows our experimental results for a 300 nm diameter SNH milled in a 100 nm gold film with 9 nm Cr2O3 adhesion layer. The only difference is the borosilicate glass or sapphire used for the 150 µm thick substrate material. In the case of sapphire, the supplementary heat dissipation brought by the substrate yields a four times lower temperature increase than with a glass coverslip.

To further damp the temperature elevation, the sample can be reversed to illuminate the gold film from the side opposite to the adhesion layer (Fig. 4b). In this case the temperature gain is similar to the case of the gold film without any adhesion layer (as the adhesion layer is no longer directly illuminated by the infrared beam). Again, we find that the use of the sapphire substrate reduces the temperature increase by an additional 4-fold factor as compared to the glass substrate. Altogether, the results from Fig. 4 show that under 5 mW/µm² IR excitation, the temperature increase can be damped from +17°C (glass, illumination from the adhesion layer side) to merely +1°C (sapphire, illumination from opposite side) by properly choosing the substrate and illumination conditions while keeping the same adhesion layer properties. This information can be particularly useful if one has to mitigate the temperature increase in conditions of nano-optical trapping, 27,29 especially as the adhesion layers for gold cannot be fully avoided for most of the applications. Lastly, a closer look at the fluorescence and IR transmission time traces show that under conditions of large temperature gains above +30°C, additional time-dependent fluctuations appear on top of the shot noise and are anti-correlated between the fluorescence and IR transmission signals (Supporting Information Fig. S3-S4). These fluctuations are related to temperature changes by ±1.5°C (Supporting Information Fig. S5) and cannot be ascribed to mechanical drifts of the microscope focus. As irreversible damage to the SNH structure occurs for IR intensities above 5 mW/µm², the additional noise could be due to some thermally-induced expansion and collapse of the SNH aperture edges, which in turn affects the IR transmission, the local temperature and the fluorescence emission. This also sets an upper limit to about +40°C for the maximum temperature increase reachable with a 13 nm thick Cr adhesion layer.

Conclusions

While many plasmonics applications cannot avoid using an adhesion layer between the gold film and the substrate, 9,10,16,18 the influence of this supplementary adhesion layer remained largely overlooked in the context of the temperature increase of plasmonic nanostructures illuminated by a laser beam. In excellent agreement with numerical simulations, our measurements demonstrate that the adhesion layer plays an essential role in determining the local temperature. If a temperature increase has to be avoided, a thin adhesion layer of Cr2O3 together with a sapphire substrate provides an effective way to reduce the heat generation and evacuate the heat while maintaining good adhesion properties. On the opposite, if a maximum temperature gain is searched, then a thicker chromium or titanium adhesion layer can significantly increase the temperature change in nanoapertures above +40°C. Altogether, these different dedicated designs pave the way for heat-controlled experiments in nanoapertures taking advantage of their well-defined attoliter volumes, high background rejection and accurate local temperature tunability.

A broad range of applications can be envisioned, including crystallization and polymerization, 37 biosensing and enzymatic activity monitoring, 23,24 DNA and protein nanopore translocation, [38][39][40] or thermophoretic trapping. 41,42

Methods

Nanohole fabrication

Cleaned borosilicate or sapphire coverslips of 0.15 mm thickness are first coated an adhesion layer (either 6 nm Ti, 6 nm Cr, 13 nm Cr or 9 nm Cr2O3) prior to 100 nm thick gold deposition assisted by electron-beam evaporation (Bühler Syrus Pro 710). Single nanoholes with 300nm diameter are then milled by galliumbased focused ion beam (FEI dual beam DB235 Strata) using 30 keV energy and 10 pA beam current.

Numerical simulations

Numerical simulations are performed using COMSOL Multiphysics combining "Electromagnetic Waves, Beam Envelopes" and "Heat Transfer in Solids" via the "Electromagnetic Heat Source" module in 3D geometry. The thermal parameters for glass, sapphire, titanium, chromium, chromium oxide, gold and water are taken from the COMSOL material library and assumed to be temperature independent. The complex refractive index for Au (100 nm thick), Ti (6 nm thick), Cr (13 nm) and Cr2O3 (9 nm) are taken from Johnson, Christy 45,46 and Oldham 17 for bulk materials (i.e. nAu=0.25846 + i6.9654, nTi=3.4654 + i4.0085, nCr=3.5408 + i3.5777, nCr2O3=2.0583 + i0.1123 at 1064 nm). In the case of chromium oxide, our simulations include a residual 2 nm thick layer of pure chromium underneath the oxide layer. This supplementary residual amount of chromium was found necessary to match with our experimental observations, as a pure chromium oxide layer would feature even lower absorption (Supporting Information Fig. S1). The refractive index of water, glass and sapphire are taken as 1.33, 1.52, 1.77 respectively. The waist of the incident IR laser beam is 1 µm, the polarization is linear and the amplitude follows a Gaussian profile.

Optical microscope

Our inverted confocal microscope contains two overlapping laser beams: one 635 nm pulsed red laser (Picoquant LDH-P-635) at 80 MHz repetition rate and one continuous wave 1064 nm infrared laser (Ventus 1064-2W). The red and infrared laser beams are designed for exciting the fluorescence of Alexa Fluor 647 molecules and heating the sample respectively. Both lasers are focused by a high NA microscope objective (Zeiss Plan-Neofluar 40x, NA 1.3, oil immersion). We first estimate the laser spot sizes at the objective focus by recording images of a single nanohole with 300nm diameter, which are 0.6 µm and 1 µm in diameter for the red and infrared lasers respectively. Thus, the power intensity of two lasers could be calculated. The illumination power intensity of the red laser is fixed at 20 µW/µm², and we confirmed that no photobleaching and no saturation of the fluorescence of Alexa Fluor 647 molecules occur under this power. The intensity of the infrared laser ranges from 0 to 5 mW/µm², and corresponds to the conditions used for the plasmonic nano-optical trapping under the damage threshold for the gold film (typically around 50 mW/µm²). Please note that throughout our study, we take into account a 50% transmission of the microscope objective at 1064 nm, and express the IR illumination intensity at the objective focus Throughout our work, we carefully checked that our observations upon infrared illumination were fully reversible, i.e. we could retrieve the same results after switching off the 1064 nm laser than what we found before turning it on. For IR intensities below 5 mW/µm² at the microscope focus spot, we could not detect any sign of photodamage. Since we are using relatively low laser power of 20 µW for red excitation of the fluorescent dyes and since the dyes are constantly diffusing across the nanoaperture (providing a constant source of "fresh" fluorescent molecules), we did not monitor any sign of fluorescence saturation or photobleaching.

Temperature measurement based on fluorescence lifetime

The temperature measurements inside the SNH follow the fluorescence lifetime approach introduced in ref 36 and briefly summarized here. The non-radiative decay rate of many fluorescent molecules, such as Alexa Fluor 647 used in this article, is very sensitive to the temperature change. We calibrated the change in the non-radiative decay rate of Alexa Fluor 647 in a homogeneous water medium with temperature increasing above the room temperature (21°𝐶):

Δ𝑇 = 91.31 -91.22 × ( 𝛤 𝑛𝑟𝑎𝑑 (21°𝐶) 𝛤 𝑛𝑟𝑎𝑑 (𝑇) ) 0.42 (1)
where 𝛤 𝑛𝑟𝑎𝑑 (21°𝐶) and 𝛤 𝑛𝑟𝑎𝑑 (𝑇) are the non-radiative decay rates at room temperature and any temperature above 21°𝐶. By considering the radiative rate and plasmonic loss rate constant when the plasmonic nanohole is introduced, 36 the non-radiative decay rate inside the nanohole can be determined by:

𝛤 𝑛𝑟𝑎𝑑 (𝑇) 𝛤 𝑛𝑟𝑎𝑑 (21°𝐶) = 1 + 1 𝛤 𝑛𝑟𝑎𝑑 (21°𝐶) ( 1 𝜏 𝑇 * - 1 𝜏 21°𝐶 * ) (2)
where 𝜏 𝑇 * and 𝜏 21°𝐶 * are the fluorescence lifetime measured in the nanohole at any temperature above 21°𝐶 and room temperature which are expressed in nanoseconds. 𝛤 𝑛𝑟𝑎𝑑 (21°𝐶) is used as 0.67 ns -1 by knowing the 1.0 ns lifetime and 33% quantum yield of Alexa Fluor 647 at room temperature. By combining equations ( 1) and ( 2), we finally deduce the relationship between the temperature increase in a single nanohole and the fluorescence lifetime reduction: An advantage of this approach is that it only requires two fluorescence lifetime measurements, which do not depend on the local concentration of fluorescence molecules and can even be performed if the sample is slightly defocused on the microscope. Let us also point out that we have carefully checked in ref. 36

Δ𝑇 =

Supporting Information

Linear relationship between thickness and infrared absorption, comparison between Cr and Ti adhesion layers of 6 nm thickness, fluorescence and infrared transmission time traces analysis. Cr and Ti films share a similar IR absorption (Fig. S1). Here our experimental data confirms that both Cr and Ti adhesion layers (of same thickness) lead to a comparable temperature increase within the experimental uncertainties.

S3. Fluorescence and infrared transmission time traces analysis

Having established how to control the local temperature inside a SNH, we take a closer look at the fluorescence and IR transmission signals under conditions of large temperature gains up to +40°C. First, we confirm that the optical microscope setup is stable, leading to a steady fluorescence time trace where the noise fluctuations are dominated by shot noise (Fig. S3a). We also check that the transmitted IR signal is stable in the absence of gold structure (Fig. S3b). The situation becomes clearly different when a SNH is illuminated, leading to a temperature raise of +32°C (Fig. S3c,d nm Cr layer. When we performed experiments at values higher than this level, we could not retrieve the initial values anymore when the IR intensity was reduced. This indicates the occurrence of irreversible damage to the SNH structure, and sets an upper limit for the maximum temperature increase reachable.

Therefore, we believe that the IR transmission fluctuations could be due to some thermally-induced expansion or collapse of the SNH aperture edges, which in turn induces the modifications of the local temperature and fluorescence properties. We have also checked that convection fluxes in the water medium do not play a significant role in our observations. Similar results were obtained with two different liquid heights above the SNH: one height of a few micrometers (where convection is prohibited) and one of several hundreds of micrometers (where convection may occur).

Figure 1 .

 1 Figure 1. Temperature measurement in a single nanohole. (a) Sketch of the experimental setup with a 1064 nm focused laser beam to heat the nanostructure and a 635 nm laser beam to probe the fluorescence of Alexa Fluor 647 solution filling the nanohole. In this configuration, the adhesion layer between the gold film and the glass substrate is illuminated and heated. (b) Top view SEM image and (c) cross-section with 52° tilted view SEM image of a 300 nm diameter aperture milled in gold. The 9 nm Cr2O3 adhesion layer is visible between the Au film and the glass substrate. (d) Normalized fluorescence decay curves of Alexa Fluor 647 in the 300 nm diameter SNH with 9 nm Cr2O3 adhesion layer recorded with 3 different IR intensities. From top to bottom, blue, orange and red curves correspond to the IR intensities of 0, 2 and 4 mW/µm² respectively. (e) Normalized lifetime (solid curve) and nonradiative decay rate (dashed curve) deduced from the fluorescence decay curves in (d) as a function of the IR intensity. (f) Temperature increase estimated from the lifetime data in (e) as a function of the IR intensity.

Figure 2 .

 2 Figure 2. Influence of the adhesion layer for the gold film on the temperature increase. (a) Normalized fluorescence decay curves in 300 nm diameter gold SNHs with different adhesion layers at 5 mW/µm² IR illumination. (b) Fluorescence lifetime values for different adhesion layers. The case "no film" is the reference for a glass coverslip without any metal. The blue and red bars indicate the cases without and with a 5 mW/µm² IR illumination. (c) Temperature increase measured on a 300 nm SNH for different gold adhesion layers. All the other experimental conditions are identical. The pink cross markers refer to independent measurements and simulations in Ref 40 and 35 respectively.

Figure 3 .

 3 Figure 3. Numerical simulations confirming the role of the adhesion layer on the temperature increase. (a,b) Spatial maps of the heat source density (a) and the temperature increase (b) around a 300 nm diameter gold SNH. The left part of the images shows the case without any adhesion layer (gold directly on glass substrate), while in the right part includes a 13 nm Cr layer between the Au film and the glass substrate. In both cases, the IR intensity is 5 mW/µm² with the polarization perpendicular to the plane. In (b), the temperature increase with no adhesion layer is multiplied by 10× to display on the same color scale as with the Cr adhesion layer. (c) Comparison of the temperature increase for different adhesion layers at 5 mW/µm² IR intensity: the solid top bar corresponds to the experimental measurements while the bottom dashed bar shows the simulations results.

Figure 4 .

 4 Figure 4. Comparison of the temperature increase with different illumination geometries and substrates. (a,b) Temperature increase inside a 300 nm gold SNH deposited on a glass (red markers) or sapphire (blue) substrate. In (a), a 9 nm Cr2O3 adhesion layer is illuminated by the IR beam, while in (b) the IR light is incoming from the top gold surface and does not reach the adhesion layer underneath the 100 nm gold film.

(

  photodiode. All fluorescence and infrared transmission time traces are analyzed with the Symphotime 64 software (Picoquant) enabling to compute the intensity time trace and TCSPC decay histogram for the fluorescence lifetime measurement.

  provides the way to measure the temperature increase Δ𝑇 for any SNH structure by simply measuring the Alexa Fluor 647 fluorescence lifetime inside the SNH with (𝜏 𝑇 * ) and without (𝜏 21°𝐶 * ) the heating infrared laser. The experimental errors for the temperature measurement based on the fluorescence lifetime mainly stem from the uncertainties induced by the noise during the curve-fitting process, and are about 7% of the temperature increase.

  that the 1064 nm infrared beam alone does not noticeably influence the Alexa Fluor 647 photophysics for IR intensities below 5 mW/µm². The fluorescence lifetime is obtained by fitting the fluorescence decay histograms using a Levenberg-Marquard optimization performed on a commercial software (PicoQuant SymPhoTime 64). The fitting model is implemented by an iterative reconvolution including the instrument response function (IRF) with more than 96% of all detected photons being included. The fluorescence decay curves of Alexa Fluor 647 in the nanoapertures are well fitted with a biexponential function. The first short lifetime component is fixed at 150 ps to account for the non-radiative losses into the metal and has a moderate influence. The dominating second lifetime component provides the relevant fluorescence lifetime.

S2.

  Figure S2. Temperature increase as a function of the infrared intensity at 1064 nm measured on a 300 nm diameter gold nanoaperture for chromium and titanium adhesion layers of similar 6 nm thickness. The experimental conditions are identical to Fig. 2 of the main document. Numerical simulations indicate that

  ). Additional fluctuations appear on top of the shot noise with a time scale of a few seconds, and are anti-correlated between the fluorescence and IR transmission signals. More time traces for increasing IR intensities are shown in Fig. S4.

Figure S3 .

 S3 Figure S3. Time traces of fluorescence intensity and infrared transmission in a 300 nm diameter SNH with 13 nm Cr adhesion layer. (a) Alexa Fluor 647 fluorescence intensity on a SNH with no heating infrared beam: the signal is stable and within ±3 standard deviation (SD) as indicated by the horizontal dashed lines. The thin blue trace corresponds to a binning time of 10 ms while the thick black trace is smoothed over a 500 ms period. (b) Infrared transmission when no metal film is present, again the trace is stable and within ±3 SD. (c,d) Fluorescence and IR transmission when the SNH is illuminated at 4 𝑚𝑊/µ𝑚 2 IR intensity, leading to an average temperature increase of +32°C. Additional fluctuations are clearly visible on top of the shot noise, and are anticorrelated between fluorescence and IR transmission. (e,f) Analysis of the fluctuation amplitude normalized by the standard deviation defined as (max-min)/SD for the fluorescence (e) and IR transmission (f) time traces as a function of the IR intensity.

Figure S5 .

 S5 Figure S5. Fluorescence lifetime measured on a selected time interval corresponding to high/low fluctuations of the fluorescence intensity. (a) Fluorescence time trace (same as Fig. S2c). The lower (red) and higher fluorescence (blue) parts are selected for the partial lifetime measurement instead of the average of the overall time trace. (b) Normalized fluorescence decay curves of Alexa Fluor 647 solution corresponding to the red and blue parts in (a). The temperature gains extracted from the measured lifetimes are indicated on the graph.

P. B. Johnson and R. W. Christy, Phys. Rev. B, 1974, 9, 5056-5070.

Acknowledgements

The authors thank Olivier Hector for help with the thin film deposition.

Funding Sources

This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 723241 TryptoBoost) and from the Agence Nationale de la Recherche (ANR) under grant agreement ANR-17-CE09-0026-01 and ANR-18-CE42-0013.

Conflict of Interest

The authors declare no competing financial interest.

Supporting Information for Adhesion Layer Influence on