¹H-NMR spectra were recorded in CDCl₃ on a Bruker-ARX 400 (Switzerland) instrument, using TMS as reference. The UV-Vis measurements were conducted on UV-1800 Shimadzu (Kyoto, Japan) Spectrophotometer. Sulphonated dyes i.e. RR-2, AB, B-19 and D-2 were purchased from Acros Organics, China. The HR-FABMS (positive or negative-ion mode) were recorded from JMS HX-110 with a data system and JMS DA-500 mass spectrometers, resp.; m/z (rel. %). Dichloromethane (CH₂Cl₂) and Dimethyl sulphoxide (DMSO) of analytical grade were used.

Calixarene derivatives i.e. calix-1, calix-3 and compound (1) were synthesized according to previously published method [27] [28] , while the calix-2 was synthesized as follows:

5,11,17,23-tetrakis (N’-methyl-N-piperazino)methyl)-25,26,27,28-tetrahydroxycalix[4]arene (calix-2)

In a 25 ml round bottom flask 0.8 g (0.00188 mol) of compound (1) was dissolved in THF (18 ml). After stirring for 5 minutes 1.8 ml CH₃COOH, 1.4 ml (0.00752 mol) 1-methyl piperazine and 0.4 ml (0.00752 mol) HCHO was added to it. Then the reaction mixture was stirred at ice cooled condition for 24 h. The reaction was monitored by TLC (10% CH₃OH in CH₂Cl₂) which showed that almost the reaction was completed. Then the

reaction was stopped and THF was removed through rotary evaporator till a viscous material was appeared. The viscous material was dissolved in water and extracted 2 - 3 times with (CH₃CH₂)₂O. Then 10% K₂CO₃ solution was added to aqueous content in order to neutralize it and caused precipitation. The precipitates were filtered and recrystallized with CHCl₃ and the yield obtained was 76%. The recrystallized material was kept in desiccators for drying. FAB positive: 874, ¹H-NMR (DMSO-d₆): δ = 6.74 (s, 8H, ArH) 4.22 (d, 4H, J = 12Hz, Ar-CH₂- Ar), 3.14 (d, 4H, J = 12Hz, Ar-CH₂-Ar) 3.10 (s, 8H, ArCH₂N), 2.26 (br s, 32H, NCH₂CH₂N), 2.12(s, 24H, NCH₃)

By following the Pederson procedure 10 mL of 2.5 × 10⁻⁵ aqueous dye solution was agitated with 10 mL of 1 × 10⁻³ M calixarene derivatives at room temperature [29] . The dye concentration was determined using UV-Vis spectrophotometer, the maximum wavelength at which RR-2, AB, B-19 and D-2 absorbs are 539 nm, 618 nm,

590 nm, and 536 nm respectively. The percentage of extraction was calculated using the following Equation,

where C_o and C are the initial and final concentrations of dye in aqueous solution before and after extraction, respectively. Each reading is taken in triplicate while estimated errors come within range i.e. ±0.2.

The complexation ability of calix[4]arene derivative for dyes is depended on functionalization at its upper and lower rim, since cavity of calix[4]arene is small in comparison to dye molecules. Thus calix[4]arene derivatives with amine functionality at their upper rims were selected because chemical properties of amines are quite renowned. Seemingly, calix-1 and calix-3 exhibits two types of tertiary amine functional groups, one cyclic and other non-cyclic i.e. N-piperidine and dimethylamine, respectively (Figure 1). To further examine the effect of calix[4]arene functionality on dyes, it was planned to synthesize 1-methyl piperazine derivative of calix[4]arene. The 1-methyl piperazine functionality is related in structure with piperadine by the substitution of one NR group by CH₂. Synthetic route for 1-methyl piperazine derivative of calix[4]arene is elaborated in Scheme 1. The compound (1) was treated with 1-methyl piperazine by following Mannich reaction, in the presence of CH₃COOH and HCHO. Subsequently, calix-2 was obtained in 76% yield and characterized through FAB -MS and ¹H NMR .

The selectivity of calixarene derivatives i.e. calix-1, calix-2 and calix-3 for H-shaped, anthraquinone based and heterocyclic based dyes were explored by initially performing conventional liquid-liquid extraction experiments. The structure of dyes (RR-2, AB, B-19 and D-2) is shown in Figure 2. The results from extraction experiment are shown in Figure 3 and Table 1. The extraction efficiency of calix-1, calix-2 and calix-3 was observed for D-2, B-19 and AB respectively, which clearly depict the variation in extraction selectivity of dyes with calix[4]arene derivatives of different functionality. Calix-1 shows highest % extraction for synthetic dye i.e. D-2 that belongs to heterocyclic class of dyes. However, it extracts dyes of H-shaped class secondly and has less affinity with B-19 dye from anthraquinone based class. The chemistry of complexation involves ionic, hydrogen bonding, dipole-dipole interactions and van der waals interaction between dye and calix[4]arene molecules [16] -[18] . Thus presence of piperidine functionality at the upper rim of calix-1, make it more hydrophobic and suitable for inclusion with D-2.

Furthermore calix-2 reveals appealing facts as well. The % extraction of D-2 and RR-2 are badly diminished, however AB shows nominal extraction while it possess superior selectivity for B-19. The hydrophobic interaction in case of piperazine functionality becomes two fold, because of having tertiary amine group (?NR₂) twice. Consequently, selectivity of calix-2 with B-19 is highest relative to other dyes. Likewise to calix-2, the % extraction with calix-3 for D-2 and RR-2 are also reduced. Conversely, both AB and B-19 have fairly close % extraction, however values are not higher as in the case of calix-1 and calix-2. Probably non-cyclic tertiary amine functionality in calix-3 could not develop enough interaction with the dyes. This observation substantially supports the significance of appropriate functionality at the skeleton of calix[4]arene that can make strong complexation linkages with dyes.

To further study the impact of time on the extraction process, liquid-liquid extraction experiment was repeated with prolong times (Figure 3). The extraction was complete after 3 hrs and no noticeable change was seen further. Clearly, the time has more influence on the % extraction of less extracted dyes relative to the dye

Scheme 1. Synthetic pathway for calix-2.

which was already extracted more within 1 hr. Apparently, the interaction of dyes with calixarene derivatives increases with the passage of time that results in subsequent enhancement in the % extraction.

The pH influence on the complexation process is imperative. The surface charge on the functional groups of calixarene moiety and dissociation of dye molecules are fairly dependent on the pH of solution. Thus to understand the complexation phenomena between calixarene derivatives and dyes in detail, extraction experiment was accomplished by varying pH of aqueous layer from 2 to 12. Comparative graphical results of all dyes are combined in the Figure 4 and Table 1 and are quite interesting with respect to each dye. The % extractions of dyes at pH 4 and pH 8 are approximately equal to those at neutral pH while lower in extreme conditions i.e. pH 2 and pH 12 [17] . This complexation phenomenon of dyes with calixarene derivatives can be justified in terms of electrostatic interaction. At mild acidic conditions, the amine functionality on each calixarene derivative i.e. calix-1, calix-2, and calix-3 become protonated and interact effectively with the negatively charged sulphonated dyes. However, in extreme acidic condition negative charge on all dyes is neutralized by H⁺ ions with subsequent reduction in complexation. Similarly in moderate basic medium, sulfonate groups on all dyes are dissociated and negatively charged dyes involve in complexation with amines of calixarene derivatives. Conversely, under high basic pH both calixarene derivatives and dye molecules are repelled owing to negative character. Hence, nominal pH is suitable for maximum complexation between calixarene derivatives and dye molecules which also enlighten green aspect of chosen complexation system.

The nature of solvent has immense influence on the process of extraction. The solvent properties i.e. viscosity, dipole moment, volatility and water immiscibility actually provide stability to the complexation. To estimate the impact of solvent on the extraction ability of calixarene derivatives extractions were carried in CH₂Cl₂, CHCl₃, and CCl₄. The results are collected in Figure 5. It can be seen that the maximum % extraction for all dyes is in CH₂Cl₂ whereas the trend is as follows CH₂Cl₂ > CHCl₃ > CCl_4. The comparison of solvent properties is shown in the Table 2, to better understand the linkage between solvent type and extraction phenomenon. CH₂Cl₂ is proved appropriate solvent for the calixarene derivatives to extract dyes effectively since it possess high dipolar moment, dielectric constant and low viscosity relative to CHCl₃, and CCl₄ [17] . More specifically polarity in CH₂Cl₂ participates in maximum extraction of dyes from aqueous to organic phase owing to stabilization of electrostatic interaction between dye molecule and calixarene derivative.

dielectric constant (20˚C)

refractive index (20˚C)

dipole moment (D)

viscosity (cP)

molar volume (M⁻¹)

Basically, the prominent characteristics for dyes complexation in calixarenes are cyclic structure, cavity size and functional groups having ability to make hydrogen bonding and/or hydrophobic interactions. Conversely the hydrophobicity and ionic property of dyes are influential factor for their complexation with calixarenes. Owing to

wider central cavity, calix[6]arenes and calix[8]arenes were proved previously excellent adsorbents for azo dyes relative to calix[4]arenes [22] -[25] . Apart from the size of calix[4]arene cavity, introduction of appropriate functionality in calix[4]arene structures could enhance its complexation ability towards dyes magnificently. Concomitantly, Yang et al. presented successful synthesis of calix[4]arene derivative with large s-triazine conjugate group, exhibiting strong complexation affinity towards dyes [18] -[26] . Conclusively, calix[4]arene after adding desired different functional groups could preferentially be utilized for the inclusion complexation with dyes.

Thus the complexation phenomena between calixarene derivatives (calix-1, calix-2, calix-3) and sulphonated dyes (RR-2, AB, B-19 and D-2) were analyzed theoretically. In UV-visible spectroscopy, complex formation is generally assured by shifting in bands of complexes to higher or lower intensity or longer wavelengths in comparison to free ligand (here calixarene derivatives). Moreover, new bands may also appear in complex spectrum depending on the nature of interaction with complexating species, as in the present case. The complexation of calixarene derivatives with dyes was examined in DMSO and is displayed in Figure 6(a), Figure 6(c), Figure 6(e). The concentration of both calixarene derivatives and dyes were kept same i.e. 1 × 10⁻⁵ M. The strong bands associated with calix-1, calix-2 and calix-3 can be observed at 290 nm, 289 nm and 288 nm, respectively in Figure 6(a), Figure 6(c), Figure 6(e). The band in this region correspond to π→π* transition in calixarene derivatives. The sharp prominent peaks at 536 nm, 590 nm, and 618 nm are obvious in Figure 6(a), Figure 6(c), Figure 6(e) for D-2, B-19 and AB, respectively. In the spectrum of calix-1: D-2 complex, the intensity of calixarene band is augmented at 290 nm with subsequent reduction in intensity of D-2 band at 536 nm Figure 6(a). Similarly, band shifts for calix-2: B-19 and calix-3: AB complexes are 3 nm and 5 nm respectively. Moreover noticeable reduction in the bands of B-19 (590 nm) and AB (618 nm) are seen as well from the spectra of both complexes Figure 6(c), Figure 6(e). These apparent changes in the spectra of complexes are clear indication of successful complexation among calixrene derivatives and sulphonted dyes. Thus, the stoichiometric ratio between calixarene derivatives and dyes was planned to investigate using conventional protocols such as Jobs plot and Benesi-Hildebrand equation.

Dyes can make specific interaction with calixarene derivatives through endo- or exo-complexation, owing to the nature of interaction. The active groups in dyes are sulfo, hydroxyl and azo while cavity in calixarene also play an important role for making particular interaction with dyes. Figure 8 illustrates the graphical representation of calix-1: D-2, calix-2: B-19, and calix-3: AB complexes. The active piperadine, piperazine and tertiary amine functionality in calix-1, calix-2 and calix-3 along with central aromatic cavity encapsulate dyes among themselves.

(a) Molar absorptivity for Calixarene-dye complexes, (b) Plot 1/[G]ⁿ against 1/∆A with high correlation coefficient value, and K_s is association constant from B-H equation, whereas K_a, and K_d represents association and dissociation constants from scatchard plots respectively.