Extractionof Lead ( II ) with Functionalization of Nano Graphene oxide with Prior to determination by Flame Atomic Absorption Spectrometry MOSTAFA

A simple, highly sensitive, accurate and selective method for determination of trace amounts of Pb2+in water samples .In this paper, a nano Graphene oxide with covalently linked porphyrin (GO–H2P) based new sorbent was prepared. The modified nano Graphene oxide (GO–H2P) was used for preconcentration of Pb(II) from aqueous solutions. The analytical variables were optimized by central composite design (CCD). Flame atomic absorption spectrometer was utilized for determination of Pb(II). The analytical features for sorption were found to be 5.2; 4.3 mL min-1 and 55.0 mL for pH, flow rate and sample volume, respectively. HNO3 was used as eluent and maximum preconcentration factor was found to be 200.Elution parameters were also determined as 4 mL min-1; 0.6 mol L-1 and 5.1 mL forflow rate, eluent concentration and eluent volume, respectively.Limit of detection and limit of quantification were found to be 49.6 and 163.7 ng L-1, respectively. Validation of the developed method was performed using certified reference material (TMDA-53.3). The methodology was applied for determination of Pb(II) in natural water samples and satisfactory results were obtained.


INTRODUCTION
nanoGraphenes are attracting renewed interests owing to recent advances in micromechanical exfoliation and epitaxial growth methods that make macroscopic 2D sheets of sp 2carbon atoms available 1 .A variety of simple yet elegant physics relating to its zero-gap semiconductor character has thus been demonstrated [2][3][4][5] .It would be very desirable to make these materials solution (or more accurately, dispersion) processable by coating or printing, which will open applications for large and/or flexible substrates.Graphite oxide (GO) is a possible candidate for this because it is a precursor to nano Graphene through deoxidation either thermally or by chemical reduction.[6-8] Although GO itself has been studied for over a century, [9] its structure and properties remain elusive, and progress has been made only recently to give materials with limited dispersability and electronic quality [10][11][12][13][14] .
Lead at trace concentrations acts as both a micronutrient and a toxicant in marine and fresh water systems 8 .This element is needed by plants at only very low levels and is toxic at higher levels.At these levels, Lead can bind to the cell membrane and hinder the transport process through the cell wall.Lead at nearly 40ng mL -1 is required for normal metabolism of many living organisms 9,10 .On the other hand, Lead is an important element in many industries.Thus, the development of new methods for selective separation, concentration and determination of it in sub-micro levels in different industrial, medicinal and environmental samples is of continuing interest.The determination of Lead is usually carried out by flame and graphite furnace atomic absorption spectrometry (AAS) 11,12 as well as spectrometric methods 13,14 .However, due to the presence of Lead in medicinal and environmental samples at low levels, its separation from other elements presents and also the use of a preconcentration step prior to its determination is usually necessary.
Different methods, especially Liquid-Liquid extraction of Lead in the presence of various classical 15 and macrocylic 16 co-extractant ligands has attracted considerable attention.However, the use of classical extraction methods for this purpose is usually time-consuming, labor-intensive and requires large amounts of high purity solvents for extraction.Nevertheless, several other techniques for the preconcentration and separation of Lead have been proposed including liquid chromatography 17 supercritical fluid extraction 18,19 , flotation 24 , aggregate film formation 25 , liquid membrane 26 , column adsorption of pyrocatechol violet-Lead complexes on activated carbon 27 , ion pairing 28 , ion pairing 29 , preconcentration with yeast 30 , and solid phase extraction using C 18 cartridges and disks [31][32][33] .
Solid phase extraction (SPE) or liquid-solid extraction is poplar and growing techniques that are used to sample preparation for analysis.It is an attractive alternative for classical liquid-liquid extraction methods that reduce solvent usage and exposure, disposal costs and extraction time for sample separation and concentration purposed 34- 36 .In recent years, the octadecyl-bonded silica SPE disks have been utilized for the extraction and separation of different organic compounds from environmental matrices [37][38][39][40] .Moreover, the SPE disks modified by suitable ligands are successfully used for selective extraction and concentration of metal ions [41][42] .
In a recent series of papers, [43][44][45] we have described the application of metal-DNA conjugates to nucleic acid sequence determination with catalytic signal amplification; the assay relies on the esterase activity of a DNA-linked Pb complex.For optimization of the system and exploration of structure-activity relationships, a sensitive probe would be useful, which allows straightforward detection of esterase activity of ligated Pb 2+ in low concentration.The structure of nano Graphene oxide with covalently linked porphyrin(GO-H 2 P)is shown in Scheme 1.The chelated ions were desorbed and determined by FAAS.The modified solid phase could be used at least 50 times with acceptable reproducibility without any change in the composition of the sorbent, GO-H 2 P. On the other hand, in terms of economy it is much cheaper than those in the market, like C 18 SPE mini-column.
In the present work, nano Graphene oxide with covalently linked porphyrin (GO-H 2 P) was employed for production of solid phase.The synthesized and characterized new sorbent (GO-H 2 P)was utilized for preconcentration of Pb(II) from water samples.Determination of Pb(II) concentration was achieved by FAAS after preconcentration procedure.The experimental conditions were optimized by CCD.

Reagents and Chemicals
The analytical grade deionized water was obtained by reverse osmosis system.All containers and glassware were kept overnight in 10% nitric acid and rinsed three times with water before use.1,000mg L-1stock Lead standard solution was prepared from Pb(NO 3 ) 2 .4H 2 O (Merck) and diluted as required to the µg L -1 levels.In interference study, cations were added as nitrates and the anions were added as sodium salts.Feasibility of the suggested method was tested with Lake Ontario water certified reference material (TMDA-53.3,lot 0310).

Synthetic procedures Preparation of GO-H 2 P
GO (15 mg) was stirred in 20 mL of oxalyl chloride at 80 °C for 24 h to activate the carboxylic units by forming the corresponding acyl chlorides.Then, the reaction mixture was evaporated to remove the excess oxalyl chloride and the brownish remaining solid (GO-COCl) was washed with anhydrous tetrahydrofuran (THF).After centrifugation, the resulting solid material was dried at room temperature under vacuum.For the covalent coupling between the free amino function of H 2 P and the acyl chloride of GO, 15 mg of GO-COCl was treated under anaerobic, dry conditions with 7 mg of H2P dissolved in 6 ml of dry THF at room temperature for 72 h.The hybrid material, namely GO-H 2 P, was obtained as a brown-graysolidby filtration of the reaction mixture through 0.2 mm PTFE filter and the filtrate was sufficiently washed with methylene chloride (4× 20 ml) to remove nonreacted free H 2 P and then with diethyl ether (2 × 20 mL) before being dried under vacuum.

Apparatus
A Philips X Pert-Pro diffracrometer (Pb Kal=1.54060A°, 30 mA, 40 kV), and Perkin Elmer Spectrum 65 FTIR-ATR spectrometer were used to confirm the synthesized GO-H2P.Determination of Pb(II) in solutions were carried out by Perkin Elmer AAnalyst200FAASequipped with deuterium background correction.All measurements were performed inan air/acetylene flame.GFL 3005 orbital shaker having speed and time control was used forpreparation of the sorbent.During the solid phase extraction experiments, Velp Scientific a SP311 peristaltic pump with Tygon tubes was used.A Thermo Orion 5 Star model pH meter,Heidolph MR 3001 K model magnetic stirrer, Sartorius TE214S electronic balance,Eppendorf Research micro pipettes wereused for the present work.Funnel tipped glass tube(10x100 mm) equipped with stopcock was used as a column for the preconcentration experiments.

Preparation of Solid Phase
The commercially available nano Graphene oxide with covalently linked porphyrin (GO-H2P) was activated by refluxing with 0.5 mol L -1 HNO 3 for 1 h to remove any adsorbed metal ions.Then it was filtered, washed with deionized water until the filtrate was neutral.
In order to synthesize physically bonded GO-H2P, 10.0 g of nano Graphene oxide with covalently linked porphyrin (GO-H2P) was added to 50.0 mL acetone containing 50 mg MSE and refluxed for 24 h.The product was filtered off and washed with deionized water and dried at room temperature.

General Enrichment Procedure
1.0 g of GO-H2P was filled in the column and the effective factors on the preconcentration including pH, flow rate andselection of eluent type were investigated using the model solutions containing 5µg Pb(II).The determination of Pb(II) in eluates were achieved by FAAS.According to the precipitation of the metal hydroxides at alkaline environment, pH effect was evaluated within the range of 3-7.Experiments of flow rate for sorption and elution were performed at 3-20 mL min -1 .In order to choose a proper eluent for desorbing the Pb(II) from the sorbent surface, different mineral and organic acids were tested .The proposed analytical procedure for Pb(II) preconcentration was optimized using three level full factorial CCD.The optimization procedure was performed separately for sorption and elution.Three variables; pH, sample volume, flow rate and eluent volume, eluent concentration, flow rate were regarded as factors for sorption and for elution, respectively.

RESULTS AND DISCUSSION
The treatment of nano Graphene oxide with covalently linked porphyrin (GO-H 2 P) can lead to the derivatization of both the edge carboxyl and surface hydroxyl functional groups via formation of amides 20 or carbamate esters 21 , respectively.
The formation of GO-H 2 P was followed by ATR-IR spectroscopy.Initially, in the spectrum of GO, the carbonyl vibration appears at 1716 cm -1 ,

Table 1: Levels and the real values of factors utilized in CCD Table 2: Experiments and recovery values for the sorption of Pb(II)
while there are fingerprints at 3616 cm -1 and 3490 cm -1 due to the presence of hydroxyl species at the basal plane of nano Graphene.The covalent linkage of H 2 P with the acyl chloride activated GO is evident from the presence of a band at 1630 cm -1 , which is characteristic for the carbonyl groups of the amide units 23 .
The amount of porphyrin attached onto the nano Graphene sheet was evaluated by thermogravimetric analysis.As compared with the TGA results of pure graphite, which is thermally stable up to 900 °C under nitrogen, and GO which decomposes above 600 °C,after having lost the oxygenated species at 240 °C (i.e.14.7% weight loss), the 6% weight loss occurred in the temperature range 250-550 °C for the GO-H 2 P material, is attributed to the decomposition of H2P (Fig. 2).
The GO-H 2 P material forms a stable dispersion in DMF at a concentration not exceeding 1 mg mL -1 .The electronic absorption spectrum of GO-H 2 P in DMF (Fig. 3), shows (i)a broad signal monotonically decreasing from the UV to the visible region, which is attributed to GO and (ii) a characteristic band at 420 nm (Soret-band) corresponding to the covalently grafted H 2 P units (the Q-bands at 516, 557, 589 and 648 nm wereflattened to the base line in the GO-H 2 P material).Interestingly, the absorption of porphyrin in the GO-H 2 P material is broadened, shortened and bathochromically shifted (ca. 2 nm) as compared to that of the free H 2 P, a result that corroborates not only the linkage of porphyrin with the GO sheets but also electronic interactions between the two species (i.e.GO and H 2 P) in the ground state.These results are in agreement with studiesbased on other hybrid systems consisting of porphyrins covalently grafted to carbon nanotubes and nanohorns 20 .

Effect of pH
The pH of the sample solution plays important role in retention of metals on sorbent.The pH of the model solutions containing 5µg Pb(II) were adjusted to certain value using diluted HNO 3 and NaOH.As shown in Fig. 4,the recovery results were not dramatically affected by the change in pH between 4 and 7.According to this, pH=2.9 was chosen as center value for the optimization procedure.[52]  1 mol L "1 HBr 50 FAAS Hexathia18 crown-6 tetraone/ Empore disk [53]  1 mol L "1 HBr 100 FAAS 1,5-Diphenylcarbazone/SDS coated alumina [54]  HNO 3 -ICP-AES 1,5-Bis(2-pyridyl)-3sulfophenylmethylene) thiocarbonohydrazide/Dowex anion exchange resin Desorption Reagent 0.5 mol L -1 of HNO 3 , HCl, CH 3 COOH, H 2 SO 4 and H2O 2 were tested for desorption of Pb(II) from GO-H2P.The recovery percentages were varied between 5.7-87.9% except HNO 3 elution experiments.It was observed that HNO3is the best as an eluent and the recovery percentage is 94.2±1.5 %.

Effect of Flow Rate on Sorption and Elution
The retention of a metal ion on the sorbent also depends on the flow rate of the sample solution.Quantitative results (> 95%) were obtained up to 10 and 8 mL min-1 for sorption and elution, respectively.In order to avoid an abrupt change in adsorption and increase the contact time of the sample solution with the sorbent,flow rate was selected as 4 mL min -1 for sorption and elution.

Optimization of Variables
Optimization of the preliminary studies was achieved by a three level full factorial CCD with20 runs.Table 1lists the maximum, minimum andcentervaluesof the variables for sorptionand elution.The metal concentration in solutions was determined with external standardcalibration method by FAAS.The experimental design matrix and the results for sorptionand elution are given in Fig. 3: The UV-vis spectra of GO-H 2 P (black) and free H 2 P (red),obtained in DMF Table 2and 3.Quadratic Eq. ( 1) and Eq. ( 2), which were obtained byusing data of response values, are given below for sorption and elution, respectively.
x 1 , x 2 and x 3 represents the 1st, 2nd and 3rd factors.Derivatives of the equations in terms of x 1 , x 2 and x 3 were equalized to zero and solved using Microsoft ® Excel.The real values presented in Table 4 obtained from CCD and used as optimal conditions for further experiments.

Interference Effects
The preconcentration procedures of trace metal ions can be strongly affected by other ions.For this reason, the effects of matrix ions were investigated under optimal conditions.The results revealed that the GO-H 2 P behaves as a neutral ionophore in the pH range 3.0 [49,50] so that the Lead ions are retained as ion pair complexes by the membrane disks.As seen, acetate ion is the most efficient counter anion for the SPE of Pb(II) ions.The influence of the concentration of sodium acetate ion on Lead recovery was investigated, and the results are shown in Table 4.As seen, the percent recovery of Pb 2+ increasedwith the acetate concentration until a reagent concentration of about 0.1 M is reached, beyond which the recovery remained quantitative.Moreover, acetate ion acts as a suitable buffering agent, while it effectively contributes to the ions-pair formation; thus, in the SPE experiments, there was no need for the addition of any buffer solution.The tolerance limit of coexisting ions was given in Table 5.The experiments indicated that, no further sample treatment or masking reagents are needed.

Effect of Sample Volume
The preconcentration studies were applied to solutions within the range of 25-1000 mLcontaining 5 µg amount of Pb(II) to explore the possibility of enriching at low concentrationwith high enrichment factor.The recovery value was obtained as 98.7 % at 1000 mL samplevolume by analyzing 5 mL eluate and the highest preconcentration factor was found to be200.

Analytical Figures of Merit
The accuracyand precision (RSD, %)of the proposed solid phase extraction procedure underoptimal conditions were investigated (n=10) as 102.0±0.2 % and 2, respectively.Limits ofdetection (LOD) and the limits of quantification (LOQ) were obtained by using a criterionsignal-tonoise ratio of 3 and 10, respectively.The results were calculated 49.6ng L -1 for LOD and 163.7ngL - 1 for LOQ.

Validation and Application of the Improved Method
The developed procedure was validated by Pb (II) determination in certified reference material.The results are given in Table 6.A statistical evaluation was performed by Student'st test and t value was calculated as 2.95.Criticaltvalue (4.30) is higher than the calculatedone at 95 % confidence level.This test showed no significant difference between Pb(II)concentration obtained from the presented method and the certified value.

Real Sample Analysis
To assess the applicability of the method to real samples, it was applied to the extraction and determination of Lead from different water samples.Tap water(Tehran, taken after 10 min operation of the tap),rain water(Tehran, 20 January, 2014), Snow water (Varamin , 6 February ,2014)and Sea water(taken from Caspian sea, near the Mahmoud-Abad shore) samples were analyzed(Table 7).As can be seen from Table 4 the added Lead ions can be quantitatively recovered from the water samples used.As is seen, the recovered Lead ion reveals that the results are quite reliable and are in satisfactory agreement with those obtained by ICPAES (Tables 7).

Fig. 4 :
Fig. 4: Influence of sample pH and dissolving solvent of GO-H 2 P on the percentage recovery of Pb(II)

Table 4 : Optimum preconcentration conditions for Pb(II) by GO-H 2 P Diverse ion Amounts taken(mg) % Found %Recovery of Pb 2+ ion
a Initial samples contained 10µg Pb 2+ and different amounts of various ions in 100 mL water(0.1 M acetate ion).b Values in parentheses are RSDs based on five individual replicate analysis.

Table 5 : Separation of Lead from binary mixtures aTable 6 : Analysis of standard reference materialTable 7 : Recovery of Lead added to 1000mL of different water samples (contaning 0.1Macetate at pH= 5.0) Sample Pb 2+ added (µg) Pb 2+ determined(ng.mL -1 ) ICP-AES
a Values in parentheses are %RSDs based on five individual replicate analysis b Not detected.