Octa-O-Methoxy Resorcin [ 4 ] Arene Amberlite XAD-4 Polymeric Chelating Resin for Solid Phase Extraction , Preconcentration , Separation and Trace Determination of Ni ( II ) , Cu ( II ) , Zn ( II ) and Cd ( II ) Ions

Synthetic resin, Amberlite XAD-4 was linked covalently with the third generation supramolecule, octa-O-methoxy resorcin [4] arene through -N=Ngroup to form chelating resin, which has been characterized and effectively used for the separation and preconcentration of metal ions such as Ni(II), Cu(II), Zn(II) and Cd(II). Critical parameters such as pH, flow rate, sorption capacity, breakthrough studies, distribution coefficient, preconcentration factor, concentration of eluting agents responsible for quantitative extraction of metal ions were optimized. The synthesized resin showed good binding affinity towards Ni(II), Cu(II), Zn(II) and Cd(II) under selective pH conditions. Good breakthrough capacity and fast exchange kinetics of the resin lead to effective separation of metal ions from their binary and ternary mixture by column method on the basis of pH and eluting agents. The resin could be reused for about 8 10 cycles. The proposed method having the analytical data with the relative standard deviation (RSD) < 2% and with recoveries of analytes higher than 98%, reflects upon the reproducibility and reliability of the method which has been successfully applied in the separation and determination of Ni(II), Cu(II), Zn(II) and Cd(II) ions in synthetic, natural and ground water samples.


Introduction
It is well recognized that several trace elements are essential constituents of enzymes and play a vital role in human metabolism and primarily supplied through diet and drinking water.However, beyond the essential range, deficiency and toxic effects are observed [1].Considering biological research, the role of some trace and ultratrace elements in the body is very important and has diverse functions.Some of the trace elements are essential to life while others are toxic even at low concentrations [2][3][4][5][6].Many metals listed as environmental hazards are essential dietary trace elements required for normal growth and development of animals and human beings.These metals are essential to human life within permissible limits.Toxicity of metal ions such as nickel, copper, zinc and cadmium in human beings is as follows: kidneys and liver [15].Teratogenic properties have been shown [16] whereas carcinogenic properties are suspected [17][18][19].
In this regard, evaluation of trace metals in the environmental samples including natural water has been continuously performed to designate the level of pollution which creates health problems [20,21].Several analytical techniques have been used for the determination of heavy metal ions in the real samples which include Inductively coupled plasma-atomic emission spectrophotometer (ICP-AES) [22], flame atomic absorption spectrometry (FAAS) [23] etc.These techniques are both widely and routinely used for the determination of trace amounts of heavy metal ions.However, the direct determination of heavy metal ions at trace levels is limited not only due to insufficient sensitivity, but also by matrix interference [24,25].
In order to overcome these difficulties, preliminary separation and preconcentration of trace elements from the matrix are frequently used techniques to improve the detection limit and the selectivity [26].Separation technique, in general, deal with the separation of components of mixtures to enhance the purity of substances while in preconcentration technique, one of the analytes or species have been selectively retained on a solid phase and then eluted with an appropriate eluent, and detected and quantified by an appropriate detection technique [27].
Different separation and preconcentration techniques using solid phase extraction (SPE) [28], co-precipitation [29] solvent extraction [30], membrane filtration [31], cloud point extraction [32,33], flotation [34], electrochemical decomposition [35] ion exchange etc. have been developed for the trace metal ion determination.Among these solid-phase extraction technique have been widely used because of its various advantages over other methods, such as higher preconcentration factor, lesser waste generation, lower matrix effect, use of less toxic solvents, saving of time and cost, easy regenerability of a solid phase and thereby more reusability [36][37][38][39].
Calix [4] resorcinarenes possess good capability to form a variety of complexes with organic/inorganic ions, neutral molecules, transition metal complexes and organometallics [72] for the simple reason that they can be functionalized at the upper rim or at C-methyl position or at hydroxyl groups at extra-annular position, enabling them to be used as multifunctional receptors [73].Literature reveals only few reports on the extraction of metal ions using calyx [4] resorcinarene and its derivatives [74][75][76][77][78][79][80].As far as work on polymer supported calyx [4] resorcinarenes is concerned, Merdivan et al. [31,81,82] have reported the use of calyx [4] resorcinarene impregnated on Amberlite XAD for separation and preconcentration of rare earths.
Here, the use octa-O-methoxy resorcin [4] arene Amberlite XAD-4 chelating resin for extraction, preconcentration and sequential separation of metal ions such as Ni(II), Cu(II), Zn(II) and Cd(II) in a column prior to their determination by Spectrophotometry/FAAS/ICP-AES is described.Different factors influencing the separation and preconcentration of these metal ions, such as pH, concentration of eluting agents, flow rate, total sorption capacity, exchange kinetics, preconcentration factor, distribution coefficient, breakthrough capacity, resin stability, effect of electrolytes, and associated metal ions have been investigated.The newly developed method has also been applied for the determination of Ni(II), Cu(II), Zn(II) and Cd(II) metal ions from industrial affluent, natural river and ground water samples of Ahmedabad (Gujarat) City.

Reagents
All the chemicals used in this work were of analytical grade purchased from Sigma-Aldrich and Merck.Amberlite XAD-4 with surface area 750 m 2 •g −1 , pore diameter 50 Å and bead size 20 -50 mesh was procured from Fluka.All aqueous solutions were prepared with quartz distilled deionized water.Glassware were soaked in 5% HNO 3 overnight and cleaned with double distilled deionized water before use.The pH was adjusted with the following buffer solutions: buffer for pH 2.0 and 3.0; CH 3 COO −1 /CH 3 COOH buffer for pH 4.0 to 6.0; condensation reaction of 1,3 dimethoxy benzene and p-hydroxy benzaldehyde as reported earlier [83].Octa-O-methoxy resorcin [4] arene covalently linked with Amberlite XAD-4 through azo (-N=N-) linkage as per Scheme 1.

Instruments
Spectral measurements were carried out using JASCO 570 UV-vis spectrophotometer using 10 mm quartz cells.The metal content determinations were carried out using (FAAS) NOVAA 400 p model with hollow cathode lamps, equipped with an air-acetylene flame without background correction and the operating conditions adjusted were carried out according to the standard guidelines of the manufacturers.When metal ion concentration was too law for its direct determination, inductively coupled plasma-atomic absorption spectrometer (ICP-AES) JY 2000-2 was used.The flow of the liquid through the column was controlled by Miclins Peristaltic pump PP-10 EX.Elico digital pH-meter, model L1 614 equipped with a combined pH electrode was employed for measuring pH.

Synthesis of Amberlite XAD-4-Octa-O-Methoxy Resorcin [4] Arene
The nitration of Amberlite XAD-4 beads, the reduction of the nitrated resin and the subsequent diazotization of the amino resin was carried out by the procedure reported earlier for Amberlite XAD-2 [84].The resulting diazotized resin was allowed to react with octa-O-methoxy resorcin [4] arene (1.0 g dissolved in 50 mL of 10% NaOH) at 0˚C -5˚C for 48 h.The dark brown coloured beads were filtered, successively washed with double distilled water and finally dried for further studies.

General Batch Method
Octa-O-methoxy resorcin [4] arene Amberlite XAD-4 polymeric chelating resin (1.0 gm) was added into a glass stoppered bottle containing 100 mL of each Ni(II)/Cu(II)/ Zn(II)/Cd(II) solutions atan optimum pH conditions and the bottle was shaken for about 30 minutes.The resin was filtered and washed with double distilled water.The amount of metal ions extracted by resin was determined by using suitable eluting agents like HCl or HNO 3 by The water samples from Sabarmati River, ground water from the University area and Vatva industrial zone of Ahmedabad city were collected.

Generalcolumn Method
1.0 gm of octa-O-methoxy resorcin [4] arene Amberlite XAD-4 polymeric chelating resin was added to the mixture of CH 3 OH:H 2 O (1:1) and poured onto the (10 cm long, 1 cm inner diameter) glass column equipped with a stopcock and a porous disk.The resin was then washed with deionized water and conditioned with 15 -20 mL buffer solution of desired pH prior to the passage of suitable aliquot of the sample solution containing metal ions (Ni(II), Cu(II), Zn(II) and Cd(II)) at an optimum flow rate.The amount bound metal ions stripped with suitable eluting agents was determined by spectrophotometry/FAAS/ICP-AES.

Parameters Optimized for Separation and Preconcentration of Ni(II), Cu(II), Zn(II)
and Cd(II)

Effect of pH on Quantitative Absorption
The pH of the solutions plays an important role on ex-traction of metal ions.The influence of pH of the aqueous solution containing 5 µg•mL −1 of each Ni(II), Cu(II), Zn(II), and Cd(II) on 1.0 gm of octa-O-methoxy resorcin [4] arene Amberlite XAD-4 polymeric chelating resin was investigated using the batch method.A 100 mL aqueous solution of each metal ion with buffer solution of desired pH was placed in glass stoppered bottles and stirred slowly for 1 hour.The pH for maximum sorption of Ni(II), Cu(II), Zn(II), and Cd(II) was found to be 3.5, 5.5, 5.0 and 8.0, respectively (Table 1, Figure 2).

Effect of Flow Rate on Metal Sorption
The metal ion sorption on 1.0 gm resin was evaluated at different flow rates (0.5, 1.0, 1.5, 2.0, 2.5 etc., mL•min −1 ), maintained by a peristaltic pump.The rate of flow of the effluent at which 98% sorption took place is consider as the optimum flow rate for the sorption of metal ions.For Ni(II), Cu(II), Zn(II), and Cd(II), the optimum flow rate was found to be 2.5, 2.0, 1.5 and 1.0 ml•min −1 , respectively (Table 1, Figure 3).The obtained flow rates for maximum sorption were employed for the subsequent analysis.

Effect of Type and Concentration of Elutent on Recovery
The choice of a suitable eluent is another important factor to obtain efficient and selective recovery of analytes.In this study, a series of experiments were performed to obtain a reasonable eluent to elute Ni(II), Cu(II), Zn(II), and Cd(II) ions from metal bound resin.The bound metal ions were desorbed with different concentration of eluting agents, (HCl/HNO 3 ) and were determined by FAAS/ICP-AES.Quantitative elution was found possible with 1.5 N HCl for Ni(II), 1.0 N HNO 3 for Cu(II), 1.0 N HCl for Zn(II) and 0.5 N HNO 3 for Cd(II) (Tables 1 and 2).

Sorption Capacity and Distribution Coefficients
The batch method was used for the calculation of the sorption capacity of resin for metal ions.1.0 gm of resin was added to 100 mL of solution containing excess of metal ions (100 mL, 1000 μg•mL −1 ) at their respective pH by shaking for 1 h.The amount of metal ions extracted by resin was calculated from the difference in the metal ion concentration before and after sorption (Table 1).The capacity of resin for Ni(II), Cu(II), Zn(II), and Cd(II) was found to be 26,000, 49,650, 40,499 and 52,310 μg•gm −1 , respectively.
Exchange equilibria arearticulated in terms of the distribution coefficient K d , which is given by the ratio of the Amount of the metal ion taken up by the resin Volume of the solution Amount of the metal ion remaining in the solution Amount of resin taken  the filtrate was subjected to FAAS/ICP-AES for determination of the metal content (Table 1).K d was found to be 4333, 7085, 6733, 8700 for Ni(II), Cu(II), Zn(II) and Cd(II) respectively.

Exchange Kinetics
To determine rate of loading of metal ions Ni(II), Cu(II), Zn(II) and Cd(II) on resin, the kinetics of sorption was studied by batch method in which 1.0 gm resin was stirred with 100 mL of solution containing 260, 496, 404 and 523 μg•mL −1 of Ni(II), Cu(II), Zn(II) and Cd(II) respectively.An aliquot of 1.0 mL was withdrawn each time at apredetermined interval and analysed using FAAS.The time taken (t 1/2 ) for the 50% sorption of Ni(II), Cu(II), Zn(II) and Cd(II) on resin, was found to be 7.0, 4.0, 5.0 and 10.0 minutes (Table 1, Figure 4) respectively, which indicates comparatively good accessibility of Ni(II), Cu(II), Zn(II) and Cd(II) towards chelating sites.

Breakthrough Studies
The capacity of the Moment when analyte starts appearing in the effluent with increased concentration is defined as the breakthrough capacity or actual working capacity.It is more significant than total sorption capacity.Breakthrough studies with 1.0 gm of rein in the column containing 100 µg•mL −1 of metal ions [Ni(II), Cu(II), Zn(II), and Cd(II)] at their optimum pH and flow rates was carried out.For the metal content determination, 20 mL aliquot was collected each time and analyzed by FAAS/

Cd(II)
purpose metal ions from the large volume of sample solution to the relatively small volume.Preconcentration of trace metal ions have been widely used in order to increase the sensitivity of the analytical methods.To study the precocnetration factor, 1000 mL solutions containing 8 µg•L −1 of each Ni(II), Cu(II), Zn(II) and Cd(II) at pH 3.5, 5.5, 5.0, 8.0 respectively, were passed through the column containing 1.0 gm resin.Metal content in the striped solution were determined by ICP-AES.

Concentration of metal in stripping solution PF Initial concentration of metal in feed solution =
The preconcentrating ability of resin was evaluated fr

Stability and Reusability of the Resin
sized resin om the elution profile of metal ions by plotting the concentration of effluents as a function of the volume of stripping solutions: 9.5 mL, 1.5 N HCl for Ni(II); 9.2 mL, 1.0 N HNO 3 for Cu(II); 10.5 mL, 1.0 N HCl for Zn(II) and 9.0 mL, 0.5 N HNO 3 for Cd(II).Under the optimum experimental conditions, the preconcentration factors were found to be 105, 95, 108 and 112 for Ni(II), Cu(II), Zn(II) and Cd(II) with more than 96% recovery (Tables 1 and 3, Figure 6).
The reusability and applicability of the synthe was examined after several loading and elution cycles.It w desorption agreed within 3% -4 % error upto 8 -10 cycles for all the matal ions that shows the efficiency of the sysnthesized resin (Figure 7).
To evaluate the selectivity of possible interference of electrolytes on the recovery of Ni(II), Cu(II), Zn(II) and Cd(II) was investigated and the results are summarized in Table 4.An error of 2% -3% was considered to be within the range of experimental error.It was observed that except Na 3 PO 4 and NaF, all others electrolytes did not interfere between 1.5 to 4.0 M concentrations (ranges), which further expand the potential application of resin for the analysis of real samples.
As observed from experimental practice sy inposses very good sorption capacity, distribution coefficient and exchange kinetics, therefore it was further used for the separation of Cu(II), Zn(II) and Cd(II) from their The mixtures of metal ions can be effectively separated by selective adjustment of the pH and ents.Hence, the following mixtures (each 100 μg in 25 mL buffer solution) were passed through the column at the pH of maximum sorption and optimum flow rate.The column effluents were analyzed for the metal ions by spectrophotometry/FAAS/ICP-AES.

Limit of Quantification and Application
Selectivity and sensitivity are two important factors the extraction and the separation process.To check t capability of the resin to detect trace amounts of metal ions, studies were performed by passing 1000 mL of sample solutions containing metal ions in the range of 5 -10 μg through the optimized column.The quantification limit for Ni(II), Cu(II), Zn(II) and Cd(II) for resin were

Comparison with Other Solid Phase
Co acity and preconcentration

Conclusion
esorcin [4] arene Amberlite XAD-4, a f resin was about 8 to 10 cycles resin was found to be reasonably better than some al-fo indicating the resin's capability to extract the trace metal ions of interest from the real samples.To check the applicability and validity of the proposed method, synthesized resin was subjected to various water sample analyses and the results are compared by the standard procedure.The data is given in Table 5.

Extraction Methods
mparison of sorption cap factor of different adsorbents are summarized in Table 6.
Octa-O-methoxy r newly synthesized polymeric chelating resin was successfully applied for the selective separation, preconcentration and determination of metal ions such as Ni(II), Cu(II), Zn(II) and Cd(II) from real samples.The advantages found in the synthesized resin are its faster exchange rates, better sorption capacity and high preconcentration factors.The resin was found to be highly selective in

Table 1 .
Parameters optimized for sorption and desorption of Ni(II), Cu(II), Zn(II) and Cd(II) on resin.
100 µg each of both Zn(II) and Cd(II) in 25 solution of pH 5.0 at a flow rate of 1.0 m passed through the column.At this pH, Cd(II) was not sorbed on resin and it came out with the effluent while Zn(II) was retained in the column.Zn(II) was eluted with 24.5 mL, 1.0 N HCl.Quantitative separation was achieved in binary mixture as shown in their separation pattern in Figure8(a).4.2.Separatio100 µg each of Cu(II), Zn(II) and Cd(II) i buffer solution of pH 5.0 at a flow rate of 1.0 m were passed through the column.At this pH, Cd(II) was not sorbed on resin and it came out with the effluent, while Cu(II) and Zn(II) were retained in the column.Cu(II) and Zn(II) were separated on the basis of selective eluting agents.Zn(II) was eluted first with 25 mL, 1.0 N HCl followed by Cu(II) with 22.5 mL, 1.0 N HNO 3 .Quantitative separation was achieved in the ternary mixture as shown in their separation patterns in Figure 8(b).