Synthesis of amberlite XAD-2-ANSA resin for separation, preconcentration and spectrophotometric determination of trace elements in food samples

A new chelating sorbent has been synthesized using Amberlite XAD-2 resin functionalized with 1-amino2-naphthol-4-sulfonic acid (ANSA) through –N=N– group. This sorbent, characterized by infrared (FTIR) spectra and optimized for sorption-desorption of the studied metal ions then, it was used as packing for the column for separation/ preconcentration of cadmium, cobalt, and nickel prior to their determination spectrophotometrically. Metal ions of Cd, Ni, and Co sorbed in the column were eluted one by one with 0.02 M, 0.10 M, and 0.20 M HCl acid respectively. Under the optimal conditions, the proposed procedure enabled the determination of Cd, Co, and Ni with detection limits of 0.98, 0.30 and 2.10 μg L− 1 respectively. The developed procedure was evaluated by the analysis of mixed metal ions Cd, Co and Ni solution contained 0.30, 30, and 1.25 μg respectively, and the percentage recovery was 95.5 – 104%. The procedure was applied to the analysis of food samples (grains of Sorghum bicolor), and the results obtained were compared with that of FAAS (before and after separation/preconcentration).


INTRODUCTION
Different heavy metals of different samples have been determined, after different preconcentration systems, using different analytical instruments. Cadmium has been determined in river water using visible spectrophotometer (Vis-S) after solid phase extraction [1]; Cadmium and Copper have been determined using FAAS after solid phase extraction [2]; Cobalt using Vis-S after solid phase extraction [3]; Nickel using Vis-S after solid phase extraction [4]; cobalt, copper, and nickel using FAAS after solid phase extraction [5]; lead, Cadmium, Nickel, and Cobalt using ICP-AES and ICP-MS after solid phase extraction [6].
Many studies, after solid phase preconcentration, performed an additional preconcentration step, this carried out by reducing the volume of eluate, by evaporation, before measuring using FAAS [7,8]. However, such this additional step is likely to contribute to loss or contamination and to increase in analysis time and chemicals to be used. Some studies have determined heavy metal by flow injection i.e., online preconcentration and determination [9][10][11]. However, such this system maybe complex, if compared with the off-line system, and on-line is not applicable when there are difficulties to perform the separation/preconcentration and the measurement in the same laboratory. In addition, the off-line system gives options to measure with more than one instrument.
Modern instruments such as ICP-MS, ETAAS or ICP-AES, are more sensitive than FAAS. ETAAS is very expensive, needs a separation and preconcentration procedure [12], and the chemical interference effects are often more severe with electrothermal atomization than with flame atomization [13]. ICP-AES does not have sufficiently high sensitivity than FAAS.
Solid phase extraction has advantages over the other widely technique used for separation/ preconcentration of heavy metals traces. These advantages include simplicity, high enrichment factor, regeneration easiness, and significant reducing the disposal costs and extraction time [21].

Chemicals and reagents
All chemicals and reagents used were analytical reagent grade and used without previous purification. Stopped flasks, 100 mL capacity were used in batch sorption experiments. Glass columns of about 25 cm length and 0.5-0.6 cm internal diameter with stopcock were used in column mode.

Spectrophotometric (Vis-S) determination of heavy metal ions
Each M 2+ ion was determined using Vis-S according to the procedure described in the literature [35]. The absorbance of Cd 2+ was measured (556 nm) at pH 10 using 5-Br-PADAP (0.02% in ethanol) as a reagent. The absorbance of Co 2+ was measured (586 nm) at pH 7 using 5-Br-PADAP (0.03% in DMF) as a reagent. The absorbance of Ni 2+ was measured (558 nm) at pH 5.5 using 5-Br-PADAP (0.02% in ethanol) as a reagent.

Synthesis of the chelating resin
According to literature researches [36-38], 15 g of Amberlite XAD-2 resin were treated with a mixture consisting of 75mL conc. H 2 SO 4 acid and 30 mL of conc. HNO 3 acid. The mixture was placed on a water bath at 60 o C for 1hr with continuous stirring. Then it was poured into a mixture of ice-cold water. Thereafter the nitro resin was filtered and washed with DDW until become free of acid traces.
Nitro resin was mixed, in a rounded bottom flask, with 60 g SnCl 2 , 67 mL conc. HCl and 75 mL ethanol. The mixture was refluxed at 90 o C for 12 h, filtered, washed with DDW and then with 2 M NaOH. The amino resin was washed with 2 M HCl, then with DDW. The amino resin was suspended in 200 mL ice-cold water, then treated with portions each of 2 mL 1 M NaNO 2 and 2 mL 1M HCl acid until a dark blue color with a starch iodine paper obtained, then it filtered and washed with ice-cold water. Diazotized resin was treated with a solution made up of 5.0 g ligand 1-amino-2-naphthol-4sulphonic acid (ANSA) in 8% solution of NaOH at 0-5 o C for 36 h. The produced chelating resin was filtered, washed with DDW until washes become colorless and, finally, it washed with methanol and air-dried.

IR spectra of the prepared new chelating resin (ChR12)
The IR spectra of the prepared new stationary phase and compounds of different preparation stages were recorded using FTIR according to the procedure mentioned in literature [39]. The data obtained and its interpretations will be present in the section of results and discussion.

Batch mode
In a 100-mL stopped flask, 0.1 g ChR12 was shaken, at room temperature, with 25.0 mL aqueous solution of each tested metal ions. Resin material was separated. The amount of metal ion remained in the aqueous phase was determined using Vis-S. The percentage extraction (% E) was calculated by the equation: % E = (C O -C/ C O ) × 100 Where Co and C are the concentrations of metal ion before and after extraction, respectively. Effect of pH was studied in batch mode at 10 mg/L of each tested metal ion, which adjusted at different pH values. Effect of shaking time was studied in batch mode at 10 mg/L of each tested metal ion, which adjusted at optimal pH value. Effect of initial metal ion concentration (Ci) was studied in batch mode at varied Ci of each tested metal ion, which adjusted at optimal pH value.

Colum mode
In this present study glass column of 25 cm length and 0.5-0.6 cm i.d., with stop cock and a small piece of glass wool, was used.

Characterization of the Synthesized Chelating Resin (ChR12) and M 2+ − Retaining ChR12
The recorded infrared spectra were used to confirm the introduction of -NH 2 group into the skeleton of the plain (Ambelite XAD-2 copolymer resin) and then coupled with chelating ligand 1-amino-2-naphthole-4sulfonic acid (ANSA). Infrared spectra were recorded for the plain, nitrated, and reduced nitrated and finally ligand-coupled-resin. The recorded IR spectra of these four types are shown in Fig. 1. As seen from this figure, there are three distinct infrared vibration bands. The first one band at (1555-1485, 1355-1320 cm -1 ), confirming the introduction of the NO 2 group to the skeleton of the plain because of the nitration processes. The second is the disappearance of some these bands assigned to the -NO 2 group (band at 1555-1485, 1355 -1320 cm -1 ), which others were sharply reduced (1480 and 1335 cm -1 ) showing a high reduction percentage was performed during the reduction processes. On the other hand, the clear formation of a new sharp band due to the reduction of -NO 2 group and characteristic to the -NH 2 group was recorded at 3443cm -1 confirming the previous observation related to the -NO 2 group. The third is the appearance (in the ligand-coupled resin) of azo group peak at (≈ 1585cm -1 ), C-S peak at (≈ 684cm -1 ) and -OH group peak at (≈ 1319 cm -1 ), confirming the diazotization and the coupling with ligands (1-amino-2naphthol-4-sulphonic acid). Moreover, the interaction of ligands in the new phase leads to a distinct color change (dark brown) which is no longer obtained with the parent phase. There are some similar IR spectral features have been described in the literature [40][41][42][43][44]. IR spectra, from Table 1, demonstrate metal ions retention on ChR12. It is clear that most of the bands of M 2+ -free ChR12 have undergone changes in their features due to metal retention. There is a similarity between some of these changes and between changes that have been observed and described in many literature types of research [43-46].

Effect of different parameters on extraction efficiency of metal ions
The effect of pH on the extraction of Cd 2+ , Co 2+ , and NI 2+ ions, by the newly prepared stationary phase ChR12, has been critically investigated.
The obtained results are summarized in Fig. 2a-c. It is obvious from these curves that the percentage extraction increases with increasing pH value from 3 to 8-9 for all studied metal ions. It has been observed that there is a maximum percentage of extraction at pH 8-9 for all studied metal ions.

Effect of extraction time and sorption kinetics.
The loading rate of Cd 2+ , Co 2+ , and Ni 2+ ions on ChR12 resin was studied in batch mode at optimal pH value and shaking for different periods (10-120 min) at about 25 ºC. The sorption kinetics of the metal ions is shown in Table 2. It was observed that the shaking time required to reach equilibrium state were 60-120 min for 73-80% E of Cd 2+ , 20-30 min for 99 -100% E of Co 2+ and 60-120 min for 86-98% E of Ni 2+ . However, all of the studied metal ions were of very good sorption rates. The good rates of extraction of the studied metal ions with ChR12 resin may indicate good accessibility of metal ions to one or more chelating sites (OH, -N=N-or NH 2 ). The loading half time t 1/2 which is defined as the time required for reaching 50% of the total uptake, was estimated. The Lagregren firstorder rate equation is the simplest rate equation for the sorption in liquid/solid system based on solid capacity [47], dqt/t = k 1 (qe-qt) where k 1 is the equilibrium rate constant of first-order (1/min), qe (µg/g) is the amount (µg) of metal ions sorbed, with one gram of sorbent (ChR12), at equilibrium, and qt (µg/g) is the amount (µg) of metal ions sorbed, with one gram of sorbent (ChR12), at any time t. Integrating this Equation, for the boundary conditions t = 0 to t and qt = 0 to qt, yields log (qe-qt) = log (qe) -(k 1 /2.303) t. Plotting log (qe-qt) versus t gives a straight line and, therefore, slope = -k 1 /2.303, and t 1/2 = 0.693/k 1. Lagregren plot for Cd 2+ , Co 2+ and Ni 2+ are shown in figure 3.8.a-c. The values for k 1 and t 1/2 are found in Table 3.

Effect of initial concentration (Ci) on Static Sorption capacity (Qs) of ChR12
The sorption capacity of metal ions was studied by batch mode at different initial concentration C i (mmol/L) solution at optimal pH and shaking time. The total sorption capacity Qs (mmol/g ChR12) was calculated using the equation: Where C 0 is the initial concentration (mg/L), V is the sample volume (L) of the metal concentration C 0, mol wt. is the weight of mole atoms of the metal, and m is the weight of ChR12 (g). Fig. 3a-c, show the effect of the initial concentration (C i ) on static sorption of ChR12 for Cd 2+ , Co 2+ and Ni 2+ respectively. The calculated total static capacity (Qs) at a higher initial concentration (C i ) are summarized in Table 4. These results show that ChR12 has sufficient high Qs for the studied metal ions. Co 2+ and Ni 2+ ions appeared to have higher affinity than Cd 2+ to be sorbed with ChR12 and their approximate capacity is 0.20 whereas of Cd 2+ ion is 0.15 mmol/g ChR12.

Effect of sample flow rate on sorption of metal ions with ChR12
The influence of sample flow rate on the sorption of each metal ion with ChR12 was studied by percolating, at 0.5-4.5 mL min -1 flow rate, 25 mL 10 mg/L metal ion solution at optimal pH value and, finally, measuring the The obtained results indicated that the optimum flow rate for quantitative metal ions retention is about 2 mL min -1 or less for Co 2+ and Ni 2+ , and about 0.6 mL min -1 for Cd 2+ . The later one metal ion exhibited its need for a long contact time to reach adequate equilibration

Breakthrough curve and dynamic capacity (Qd)
A break-through curve is obtained by plotting column effluent concentration versus volume treated or time of treatment. Breakthrough point is defined as the time when the cation of the feed first appears in the effluent [48]. Furthermore, the volume of solution percolated from the breakthrough point to the point of leveling of the loading curve for a given solution flow rate also depends upon the kinetics of exchange [49]. The breakthrough curve of each M 2+ ion was carried out by percolating, up ChR12 saturation, a suitable volume of metal ions at optimal condition and, finally, measuring the remains concentration in the effluents. where V50% is the effluent volume (mL) at 50% breakthrough, C o is the concentration of the influent solution (μmol mL -1 ), and m is the mass (g) of ChR12. Breakthrough and dynamic capacities (mmol g -1 ) for the studied metal ions are listed in Table 5. Breakthrough capacities for all of the studied metal ions are lower than their dynamic capacities. This is normal as the breakthrough capacity is calculated at throughput volume i.e., at first detection of metal ions in the effluent, whereas dynamic capacity is calculated at 50% breakthrough. Dynamic Capacities for all of the studied metal ions are lower than their Static (batch) Capacities. This is also usual and it relies on several parameters such as the column bed height, sample flow rate, temperature of the solution, particle size, and concentration of the feeding solution [49]. High linear velocity may result in premature breakthrough [51]. In addition, the difference between static and dynamic capacities of a particular metal ion depends on the required time for binding with the chelating sites. From Table 4, and Table 5, it is seen that there is no large difference between the static and dynamic capacity for Co 2+ and Ni 2+ whereas this difference is too large for Cd 2+ , this maybe because that Cd 2+ ion needs longer contact time for binding in the chelating sites.

Effect of the eluting agent on elution of metal ions
Elution step is one of important solid phase extraction steps and, here, in this present study, elution gains additional importance and interest since this study, in it's the main part, aimed for determining toxic heavy metals using analytical instruments of good availability as Vis-S and FAAS. So, wide varieties of eluting agents types, concentration and flow rate were examined for each metal ion. The important results we got for a particular eluting agent, are depicted in one figure for comparison purpose. From Fig. 6a, it is obvious that the eluting agent 0.5 M HCl acid can elute all of the studied metal ions. From Fig. 6b, it is clear that 0.5 M HCl acid in acetone can elute Cd 2+ and Co 2+ , but cannot elute Ni 2+ . From Fig. 6c, 1 M HNO 3 acid can elute Cd 2+ , Ni 2+ quantitatively, and Co 2+ partially. From Fig. 6d and e

Evaluation and application of the Developed Separation/preconcentration Procedure
Validation must always be carried out for newly developed or modified procedures. This can take place either by comparison of the results of the analyses with those of a validated or an independent analytical procedure or by analysis of reference materials or of control samples prepared in the laboratory itself [52]. The procedure TTEST (one tail, two tails) can be used for two results comparison [151].
In this present study, the developed separation-preconcentration method has been evaluated and validated by analysis of metals mixture solution and analysis of food samples (grains of sorghum bicolor) and results in comparison with results of another instrument as follows:

Separation of mixture metal ions solution
In order to separate Cd 2+ , Co 2+ and Ni 2+ ions from a mixture using the ChR12 resin packed in column, the solution containing all metal ions was adjusted to pH 8.5 using buffer solution and suitable masking agents, then the solution was percolated through the column packed with ChR12 resin at flow rate 0.6-0.2 mL/min, analyses of three replicates were carried out. the tested ions were eluted from the column by the suitable eluting agent and, then, determined by Vis-S. The obtained results are summarized in Table 6. From Table 6, it is very clear that the Proposed separation, a method is valid (accurate and precise) for all tested metal ions. Since the %R average means were excellent, 104, 95.5 and 97, and the RSD were also very good, 3.9, 0.7 and 7.2 for cadmium, cobalt, and nickel respectively.

Comparison between results of sorghum grains analysis for metal ions content using Vis-S and FAAS
It is clear from Table 7, that the Cadmium analysis result using FAAS (before separation) was less precise as its RSD was somehow high (15.7), n= 1 whereas those of both Vis-S and FAAS (after separation) were more precise as their RSD were somehow lower (4.1), n= 2 for FAAS, and (8.2), n= 3 for Vis-S. In addition, these of Vis-S and FAAS (after separation) are not significantly (p, 2-tails= 0.21) different. Nickel analysis results of Vis-S and FAAS (before and after separation) are not significantly (p, 2-tails= 0.10) different.

Advantages of the newly Developed solid phase Procedure
From Table 8, it can be concluded that there are similarities in the accuracy (%R), precision (RSD) and the metal amounts added to be recovered, between the present developed solid phase Procedure and most of the other preconcentration systems. The advantage of our developed separation/preconcentration procedure is that it enables analysis of three important toxic heavy metals (Cd 2+ , Co 2+ , and Ni 2+ ) using one simple column in a single run, reasonable volumes of gradient low concentration one eluting agent type, and measuring using more than one available and inexpensive Analytical instruments.

CONCLUSION
Based on this work, the following conclusions can be illustrated: The synthesized chelating resin12 (ChR12) is a solid phase capable efficiently to preconcentrate traces of Cd 2+ , Co 2+ and Ni 2+ ions from a reasonable volume of sample solution using applicable one simple column.
The developed procedure is suitable for determination of Cd 2+ , Co 2+ and Ni 2+ ions using low sensitive and inexpensive analytical instruments.
Analyses results of our developed separation/ preconcentration method using Vis-S were comparable to that of high sensitivity analytical instruments.
The developed procedure has advantages since it involves working on three important toxic metals, using one column simultaneous extraction and separative elution, reasonable volumes of gradient low concentration one eluent type, and measuring using available analytical instruments as Vis-S, FAAS; these advantages are not together involved in another separation/preconcentration method according to our good knowldgement.
Separation and/or preconcentration is important in trace determination of cadmium and lead regardless of the sensitivity of the instrument to be used.