Application of the Langmuir Technique to Study the Response of C-dec-9-en-1-ylcalix[4]resorcinarene and C-undecylcalix[4]resorcinarene Ultra-thin Films' Interactions with Cd2+, Hg2+, Pb2+, and Cu2+ Cations Present in the Subphase

The calix[4]resorcinarene macrocycles are excellent oligomers for the design of amphiphilic derivatives; they can form self-assemblies and stable sensing networks. Owing to their favorable properties, they are the focus of many exploitations and studies ranging from biological controls to heavy metal ion sensing. In this perspective, two calix[4]resorcinarene derivatives, namely: C-dec-9-en-1-ylcalix[4]resorcinarene (ionophore I) and C-undecylcalix[4]resorcinarene (ionophore II) were used to form stable ultra-thin Langmuir monolayer films at the air/water interface; their interactions with different harmful metal cations (Cd2+, Pb2+, Hg2+, and Cu2+) were studied and highlighted via the pressure-area (Π-A) isotherms. The obtained results in the current investigation showed a dependence of both macrocycle interactions on the metal cation concentration in the subphase, confirming their complexation. In addition, the ionophore (I) exhibited high selectivity towards Pb2+ and Cu2+ cations, whereas the ionophore (II) showed tendency to bind with Cu2+ cations over others, approving the potential applicability of these macrocycles as ion selective chemical sensors.


Introduction
Heavy metal (HM) ion poisoning is a vital threat to the environment, affecting hazards ranging from environmental pollution to human toxicity. HM ion toxic exposure occurs by means of environmental contaminations, medical treatments, industrial production, and accidents (Eddaif et al. 2019a). The low atomic mass metals (Cu and Zn) frequently play important roles in cell physiology, thus are required for the human body. HM such as Pb, Hg, and/or Cd forms stable coordination complexes with thiol groups, leading to important biological perturbations including DNA alterations, biotransformations, renal, cerebral, or even liver poisoning, accordingly; as a result, the necessity of special early detection and monitoring techniques is of enormous importance in environmental analysis (Eddaif et al. 2019a). Currently, a variety of metal ion detection methods are employed including conventional (ICP-MS, AAS, or ICP-OES) and non-conventional (ion selective electrodes, chemical sensors…etc.), the later techniques tend to be more practical in terms of cost, time, sensitivity, and selectivity, as well as usage simplicity (Eddaif et al. 2019a).
The first validation step of the sensing platform's (e.g., calix[4]resorcinarene macrocycles) applicability manifests in performing some preliminary examinations (e.g., Langmuir monolayers and surface pressure-area isotherms) in order to improve its surface activities from one side and to be sure of the potential interactions, which will occur between it and the target detection analytes from another side. Therefore, it is of huge importance to initially investigate the calix[4]resorcinarenes monolayer behavior on subphases containing various HM (in case of metal ion monitoring), before direct applications for detection purposes, since studying such ultrathin films can offer perceptions on the intra or even intermolecular interactions happening between the sensing platform and the metallic toxicants (Davis et al. 1998;Davis and Stirling 1996;Moreira et al. 1994;Shahgaldian et al. 2005;Supian et al. 2010;Torrent-Burgués et al. 2012;Turshatov et al. 2004).
I n o u r p r e s e n t i n v e s t i g a t i o n , t w o calix[4]resorcinarene ionophores which have alkyl chain different substituents, namely: C-dec-9-en-1y l c a l i x [ 4 ] r e s o r c i n a r e n e ( I ) a n d Cundecylcalix[4]resorcinarene (II), were utilized to study the interactions and complexation behavior against various heavy metals (Cd, Hg, Pb, and Cu) in producing stable Langmuir monolayers and surface pressure-area isotherms.

Ionophores synthesis
The cyclocondensation synthesis, the chemical, and the structural characterization of the ionophores (I) and (II) were described in our previous work (Eddaif et al. 2019d); additionally, the synthetic approaches are presented in Fig. 2.

Langmuir Compression Isotherms
A rectangular (length: 30 cm, width: 20 cm, depth: 0.5 cm) Langmuir-Blodgett trough (Model 611, NIMA Technology Ltd., Coventry, England) equipped with a Wilhelmy-type surface pressure gauge by means of filter paper (20 mm length) was used to produce all the Langmuir isotherms, the trough was thermostated to 20°C with a 0.5°C precision, and was enclosed in a box.
The calix[4]resorcinarene solutions were prepared using chloroform as solvent with a 1mg/ml concentration, and the Langmuir films were produced by d r o p p i n g u n i f o r m l y a 2 0 μ l v o l u m e o f calix[4]resorcinarene solution onto the subphase's surface using a Hamilton microsyringe, and a time of 10 min was allowed for the solvent evaporation. Then, the barrier was closed to record the surface pressure-area isotherms (the average compression speed was of 100 cm 2 /min).

Results and Discussion
The aim behind the Langmuir isotherm studies was to evaluate the influence of different metal ions on the Langmuir films and on the interactions occurring between those ions and the ionophores. The ionophore surface pressure-area isotherms were acquired at the air/water interface via the surface pressure (Π) recording (expressed in mN/m) against the molecular area variations (Å 2 ). To ensure both the stability and the reproducibility of all Langmuir isotherms, each experiment was repeated at least 3 times; the result comparison showed a good stability, as well as noble reproducibility (Azahari et al. 2014)

Langmuir Studies: Surface Pressure (Π-A) Isotherms
Tables 1 and 2 summarize all the (Π-A) isotherm data of both ionophore monolayers (I) and (II), whereas Figs. 3a, b, c, d and 4a, b, c, d show the Langmuir isotherm evolution of (I) and (II) using pure water and different (Cd 2+ , Pb 2+ , Cu 2+ , and Hg 2+ ) concentrations as aqueous subphases. The results revealed interesting changes in terms of the limiting area/molecule, as well as selective binding to some metals over others supporting the possibility of using these oligomers as selective chemical sensors. Moreover, further descriptions are stated in the coming paragraphs.

Cadmium Ion Subphase
In a general manner, and due to the amphiphilic character of the calix[4]resorcinarene molecules, they can form both well-ordered (stable) and insoluble monolayers at the air/water interface, and consequently, leading to precise Langmuir isotherms. In addition, the calix[4]resorcinarenes ring is assumed to be parallel with the water/air interface's plane, and mostly, they are stable in a cone conformation; precisely, the alkane or alkene chains (substituents) are in front of the air, whereas the resorcinols hydroxyl groups are tending to form hydrogen bounds with the water molecules (in the aqueous subphase), as mentioned in the literature (Supian et al. 2010). Figures 3a and 4a show the Langmuir isotherm evolution of both C-dec-9-en-1-ylcalix[4]resorcinarene and C-undecylcalix[4]resorcinarene monolayers using pure water and different Cd 2+ concentrations as aqueous subphases. At the compression's commencement, both ionopohres (I) and (II) undergone a gas-liquid transition for all subphases at~107,~173,~167, and~194 Å 2 / molecule for (I) and at~135,~160,~230, and~340 Å 2 /molecule for (II), respectively, for 0, 5, 25, and 250 ppm concentration. A second phase transition is observed for the 25 and 250 ppm concentrations at1 05 and~115 Å 2 /molecule correspondingly for (I) and at about~135 and~170 Å 2 /molecule for (II). This is attributed to a liquid-quasi solid or a liquid-liquid transition. However, for pure water and 5 ppm Cd 2+ , the Π value increased without an obvious phase transition reaching the collapse pressure.
The isotherms were not similar considering the phase transitions, which can be explained either by some inter/ intra molecular changes, or else by the strong interaction occurring between the monolayers and the ions while increasing their amounts in the subphase. This circumstance was confirmed by the increase of the A lim (limiting area per molecule): obtained by extrapolating the linear part of the isotherm on the 'x' axis; the extrapolated A lim values were about~110,~130,~160, and1 85 Å 2 /molecule for (I), and about~105,~150,~220, and~320 Å 2 /molecule for compound (II), sequentially for pure water, 5, 25, and 250 ppm of Cd 2+ ions, therefore demonstrating the incorporation of the cadmium ions into the Langmuir monolayers.

Copper Ion Subphase
By examining Figs. 3b and 4b presenting the monolayers (Π-A) isotherms by means of pure water and different Cu 2+ concentrations, it is obvious that upon compression, a gas-liquid transition is observed at about 107,~169,~230, and~330 Å 2 /molecule (I) and at1 35,~190,~385, and~330 Å 2 /molecule (II), respectively, for 5, 25, and 250 ppm). Furthermore, in the case of (I), a second-phase transition is observable for all copper concentrations (~87,~91, and~160 Å 2 /molecule correspondingly for 5, 25, and 250 ppm), mostly explained by a liquid-liquid transition. Considering the pure water-calix (I) monolayer, only one phase transition was seen while increasing the pressure up to collapse (A c = 65 Å 2 /molecule, Π c = 32 mN/m), equally the (II) monolayer went through one principal phase transition for all added copper concentrations (i.e., the pressure increased up to collapse without extra phase transitions).
Evaluating the A lim values, which had an increase with the rise of copper amounts, they were about~110, 150,~200, and~320 Å 2 /molecule for (I) and~105,1 40,~370, and~450 Å 2 /molecule in case of (II) for 0, 5, 25, and 250 ppm of Cu 2+ ions; consecutively, a significant difference in terms of the average area upon addition of various copper amounts is observed, attesting the strong interaction occurring between the Cu 2+ ions and the calix[4]resorcinarene monolayers.

Mercury Ion Subphase
The corresponding Langmuir compression isotherms of pure water and various Hg 2+ solutions for monolayers of (I) and (II) are displayed in Figs. 3c and 4c. Inspecting both graphs shows that Langmuir films presented two major phase transitions for the mercury containing solutions for (I) while increasing the compression. However, the calix monolayer (II) went through one phase transition only, although there was a little slope variation in the 250 ppm isotherm at about~171 Å 2 /molecule which is probably attributed to some intermolecular changes rather than a liquid-liquid transition. In both cases, the primary phase transition is a gas-liquid one and could be obtained at~107,~225,~240, and3 10 Å 2 /molecule for (I), also at~135,~230,~300, and~330 Å 2 /molecule in the case of (II) for 0, 5, 25, and 250 ppm. Afterward, the pressure increased reaching collapse without any other phase transitions in case of (II), as well as the pure water isotherm of (I). The second phase transition shown  at~130,~135, and~180 Å 2 /molecule for 5, 25, and 250 ppm of mercury ions (in case of (I)) is mainly a liquid-liquid transition. The A lim values were about~110,~220,~240, and3 00 Å 2 /molecule for (I), as well as~105,~220,~290, and~310 Å 2 /molecule in case of (II) for 0, 5, 25, and 250 ppm of Hg 2+ ions, a noteworthy increase is observed, demonstrating the strong interaction occurring between the Hg 2+ and the calix[4]resorcinarene monolayers.

Lead Ion Subphase
The analogous Langmuir isotherms formed by the calix[4]resorcinarene-Pb 2+ monolayers at air/ water interface are presented in Figs. 3d and 4d.
After the beginning of compression, the (I) Langmuir monolayers showed two phase transitions for lead solutions (25 and 250 ppm), the first one is seen at~290, and~360 Å 2 /molecule for 25 and 250 ppm, and the second is observed at~147 and1 83 Å 2 /molecule for 25 and 250 ppm of Pb 2+ ions, the first phase transition is mainly attributed to a gas-liquid transition, whereas the second is explained by a liquid-liquid transition. Though the pure water-calix and the 5 ppm isotherms revealed only a gas-liquid, or properly a gas-condensed liquid transition at~107 and 150 Å 2 /molecule. However, with increasing the Π up to collapse of pure water (A c = 65 Å 2 /molecule, Π c = 32 mN/m) and 5 ppm subphase (A c = 70 Å 2 /molecule, Π c = 38 mN/m), no Fig. 3 Surface pressure-area isotherms of the ionophore (I) on subphases of a cadmium, b copper, c mercury, and d lead cations additional phase transition was seen. In case of the c-undecylcalix[4]arene monolayer, only one phase transition was shown for all lead solutions at1 35,~165,~190, and~315 Å 2 /molecule for 0, 5, 25, and 250 ppm, it corresponds to a gas-liquid or a gas-condensed liquid transition. Though with increasing the Π up to collapse, no additional phase transition was seen.

Ion Discrimination and Selectivity Within (I) and (II) Ionophores
The increase in terms of the average area (limiting area) is a strong indicator of the potential interactions occurring at the water/air interface while studying Langmuir isotherms, in case of using ionophores for complexing, extracting, or else sensing metal ions, the changes happening to the A lim while adjusting the ions amounts in the examined subphases, is a proof of the interaction (complexation) concerning both the ionophores and the metals. However, each molecule has a tendency to one or more metal ions over others; thus, the Fig. 5 is showing the dependence of A lim on the metal ions concentrations , the A lim increased systematically with increasing the ions amounts in the studied subphases for both ionophores, specifically and most rapidly for (I)-Cu 2+ , (I)-Pb 2 , (I)-Hg 2+ , and (II)-Cu 2 + indicating that the C-dec-9-en-1ylcalix[4]resorcinarene is more selective to lead and copper ions over others, whereas the Cundecylcalix[4]resorcinarene is more selective to copper cations over others (Fig. 6).

Adsorption of Heavy Metal Ions Within Ionophores (I) and (II) Monolayers
In this work, we explain the adsorption of various heavy metals ions within the Langmuir monolayers of both ionophores (I) and (II) by means of the Gibbs Eq. (1).
Bearing in mind the fact of working in diluted solution conditions, consequently, the heavy metal ion activities are considered equal to their concentrations (a HM~CHM ), besides their adsorption factor Γ HM is roughly close to the maximum adsorption of ions Γ max HM . Furthermore, the integral of Eq. (1) is presented by the Gibbs-Shishkovsky empirical Eq. (2) (Turshatova et al. 2004).
The collapse pressure differences between the blank and the heavy metals ion subphases (П c H 2 O − П c HM ) can be plotted against the ln(C HM ). Generally, the slope of  Hence, an idea about the interaction occurring between each ion and ionophore can be established. In both cases, the collapse pressure was affected by the metals ions present in the subphase. Besides, it is figured out that ionophores (I) and (II) have interactions with all ions, nevertheless with a preference to copper and lead over others at higher concentrations, this fact is translated by positive slope signs, and maximum adsorption values (Table 3).

Conclusion
The C-dec-9-en-1-ylcalix[4]resorcinarene (I) and Cundecylcalix[4]resorcinarene (II) formed stable monolayers at the air/water interface; consequently, stable surface pressure-area isotherms were gotten while studying the interactions between those ionophores and various heavy metals, the interactions were strong enough, which supports the prospect of the potential use of these ionophores as sensing platforms. A concentration dependence was seen in both cases; moreover, the ionophore (I) was selective to copper and lead ions, whereas the (II) showed tendency to bind with copper ions. The interactions taking place between the Langmuir monolayers and the heavy metals ions were explained by means of the Gibbs-Shishkovsky adsorption empirical equation.
A further investigation is currently under way, it consists on developing selective piezogravimetric and electrochemical sensing platforms based on the calix[4]resorcinarene derivatives for the selective detection of heavy metal ions.