Accepted Manuscript

Analytical methods

Dispersive liquid-liquid microextraction for the determination of copper in ce‐

reals and vegetable food samples using flame atomic absorption spectrometry

Kamlesh Shrivas, Nitin Kumar Jaiswal

PII: S0308-8146(13)00513-X
DOI: http://dx.doi.org/10.1016/j.foodchem.2013.04.067
Reference: FOCH 13980

To appear in: Food Chemistry

Received Date: 23 October 2012

Revised Date: 7 April 2013
Accepted Date: 21 April 2013

Please cite this article as: Shrivas, K., Jaiswal, N.K., Dispersive liquid-liquid microextraction for the determination
of copper in cereals and vegetable food samples using flame atomic absorption spectrometry, Food Chemistry
(2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.04.067

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1 Dispersive liquid-liquid microextraction for the determination of copper in cereals and

2 vegetable food samples using flame atomic absorption spectrometry

3 Kamlesh Shrivasa*, Nitin Kumar Jaiswalb

a
4 Department of Chemistry, Guru Ghasidas Vishwavidyalaya, Koni, Bilaspur, CG-495009,

5 India
b
6 Department of Chemistry, School of Engineering and Research, ITM University, New

7 Raipur, CG-493661, India

a
8 *Corresponding author: Email-shrikam@rediffmail.com

9 Phone: +91-7752-260488

10 Abstract

11 Dispersive liquid-liquid microextraction (DLLME) is applied for the determination of copper in

12 cereals and vegetable food samples using flame atomic absorption spectrometry (FAAS). The

13 maximum extraction efficiency of copper was obtained after the optimization of parameters such

14 as extraction and dispersing solvents, pH, concentration of 2,9-dimethyl-1,10-phenanothroline

15 (DPT), N-phenylbenzimidoyl thiourea (PBITU) and salt. The optimized methodology exhibited a

16 good linearity in the range of 0.2–20 ng/mL copper with relative standard deviations percentage

17 (RSD, %) from +1.5 to 3.5%. The method is found to be simple and rapid for the analysis of

18 copper in food samples with the limit of detection (LOD) and quantitation (LOQ) were 0.05 and

19 0.16 ng/mL, respectively. Good recoveries of copper were obtained in the range of 93.5- 98.0%

20 in food samples as well as in Certified Reference Material (99.1%). The application of the

21 proposed method has been successfully tested for the determination of copper in cereals (maize,

22 millet, rice, wheat, gram, lentils, kidney beans and green beans) and vegetable (potato,

23 cauliflower, tomato, spinach, green beans, lettuce, egg plants and bitter gourd) food samples.
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24 Keywords: Dispersive liquid-liquid microextraction, FAAS, Copper, Food samples

25 1. Introduction

26 Copper is a reddish metal that commonly present naturally in rock, soil, water, sediment and air,

27 as well as in plants and animals. The average concentration of copper in the earth crust is 50

28 mg/Kg. Copper is an essential trace element that is very necessary for living beings to perform

29 the enzymatic activity and other functions related to formation of hemoglobin red blood cells and

30 bones (ATSDR, 2004; Kenduzler and Turker, 2003). The average dietary intake of copper in the

31 USA is approximately 1.0–1.1 mg/day for adult women and 1.2–1.6 mg/day for adult men

32 (Tokman, 2007). The intake of copper can occur in human by breathing air, drinking water,

33 eating food, or by skin contact with soil or water. The chronic exposure to excess levels of

34 copper may causes the neurological illness such as schizophrenia, depression, autism and

35 epilepsy (Pfeiffer, 1987). The determination of trace amounts of copper is becoming an essential

36 concern due to the presence of the element as micronutrients in the foods. Thus, the sensitive and

37 selective method is required for separation and preconcentration of copper from the different

38 sample matrixes.

39 Several analytical techniques, including atomic absorption spectrometry (AAS) (Pourreza and

40 Ghanemi, 2006; Taher et al. 2005; Bakireioglu et al. 2004), inductively coupled plasma-atomic

41 emission spectrometry (Guo et al. 2004; Liu et al. 2005), voltammetry (Kumar et al. 2005) and

42 spectrophotometry (Pinto et al. 2002; Isildak et al. 1999; Jankiewicz et al. 1999; Prodromidis et

43 al. 1994) have been reported for the determination of copper in various types of samples. Flame

44 atomic absorption spectrometry (FAAS) is a relatively simple and readily available analytical

45 instrument in many laboratories. It has been widely used for the determination of heavy metals

46 due to its low cost, easy operation, high sample throughput and good selectivity.
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47 Sample preparation plays an important role in analytical process to concentrate and separate

48 the target analytes as well as decrease the interferences from the complex matrix samples. A

49 number of sample pretreatment methods have been established for the determination of trace

50 level of copper from different types of samples. Liquid–liquid extraction (LLE) and solid phase

51 extraction (SPE) are most commonly used conventional separation techniques. However, it

52 suffers from the use of large amounts of high purity organic solvents (in LLE), laborious and

53 time consuming processes. The use of large amount of organic solvents causes a great threat after

54 disposal to the surrounding environment (Marczenko, 1986). Liquid-phase microextraction

55 (LPME) is a novel miniaturized sample preparation technique that has gained an extensive

56 attention in analytical chemistry. It is a simple, low cost and fast extraction technique that

57 incorporates sampling, concentration and sample introduction into a single step. Single-drop

58 microextraction (SDME) (Jeannot & Cantwell, 1996; Liu & Dasgupta, 1996), hollow fiber

59 liquid-phase microextraction (HF-LPME) (Shen & Lee, 2002; Zhao & Lee, 2002), dispersive

60 liquid-liquid solvent microextraction (DLLME) (Mirzei et al, 2011; Gharehbaghi & Shemirani,

61 2011) and drop-to-drop solvent microextraction (DDSME) (Shrivas & Patel, 2011) are examples

62 of LPME techniques. In DLLME, extraction and dispersed solvents were mixes to the sample

63 solution to form the cloudy solutions of fine droplets. The analytes are concentrated in to the fine

64 droplets of extraction solvent which are further separated by centrifugation and used for the

65 instrumental analysis of element.

66 In the present investigation, a novel method is developed for the determination of copper in

67 cereals and vegetable samples. It was based on the separation and preconcentration of copper in

68 extraction solvent containing PBITU and dispersing solvent. Then, the measurement of copper
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69 was performed in FAAS. The parameters that affecting the extraction efficiency of copper, such

70 as dispersive and extraction solvent, pH, concentration of PBITU, DPT and salt were studied.

71 2. Experimental

72 2.1. Reagents and solution preparations

73 All the chemicals used were of analytical grade reagents (AR). Copper sulfate (CuSO4·5H2O),

74 2,9-dimethyl-1,10-phenanothroline (DPT), ascorbic acid, sodium citrate, thiourea and absolute

75 ethanol were obtained from S.D. Fine Chemical Ltd. (Mumbai, India). N-phenylbenzimidoyl

76 chloride was obtained from Aldrich (St. Louis, MO). A fresh stock standard solution 100 µg/mL

77 of Cu(II) was prepared by dissolving a weighed amount of CuSO4·5H2O in deionized water

78 (DW). Working standard solutions (10.0, 1.0 and 0.5 µg/mL copper) were prepared by

79 appropriate dilution of the stock solution (100 µg/mL copper). A 1.0×10−3 M (DPT) in absolute

80 ethanol was used as a chelating agent to form a copper-DPT complex. A 20×10−3 M of N-phenyl

81 benzimidoylthiourea (PBITU) in chloroform solution was prepared for the extraction of the

82 copper-DPT complex. A 0.56 M of ascorbic acid was prepared in DW for the reduction of Cu(II)

83 to Cu(I). A 0.1% solution of sodium citrate was prepared to remove the metal ions inference for

84 the determination of copper in food samples. Acetate and phosphate buffer solutions (pH 2.0-

85 10.0) were prepared for maintaining the pH of the solution. A 1.0% sodium chloride was

86 prepared in DW to evaluate the influence of salt addition in the extraction of copper. The

87 synthesis of PBITU was done in one step by the condensation of 1 mole of N-phenylbenzimidoyl

88 chloride with 1 mole of thiourea (Patel and Mishra 1983). The Certified Reference Material of

89 tomato (NCS ZC85006) was obtained from China National Analysis Center for Iron and Steel

90 used for verifying the validity of the proposed method.

91 2.2. Flame Atomic Absorption Spectrophotometer

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92 Shimadzu model AA6300G atomic absorption spectrometer (Kyoto, Japan) with a copper hallow

93 cathode lamp as radiation source (Hamamatsu Photonics, Japan) at the wavelength of 324.8 nm

94 with slit width of 0.7 nm, current of 30 mA. Deuterium lamp was used for the background

95 correction. The composition of the flame was acetylene and air with flow rate of 2.0 L/min and

96 13.5 L/min. The nebulizer aspiration flow rate was kept between 5.5 and 6.0 mL/min.

97 2.3. Sample preparations for the determination of copper in cereals and vegetable samples

98 The cereals (maize, millet, rice, wheat, gram, lentils, kidney beans and green beans) and

99 vegetable (potato, cauliflower, tomato, spinach, green beans, lettuce, egg plants and bitter gourd)

100 samples were collected from the local market. The cereals and vegetable samples were dried in

101 oven at 100 oC for overnight and stored before the analysis. A 5.0 g of cereals samples were

102 grounded into fine particles with grinder (domestic use) and mortar and pestle. A 2.5 g of

103 crushed sample was placed in a crucible and heated until the sample turned to the ash. Then, the

104 sample was placed in 50-mL beaker containing 7.0 mL nitric acid and 3.0 mL hydrogen

105 peroxide. The sample solution was heated and evaporated till the dryness and residue was

106 dissolved in 10 mL 1.0 M HCl. Similarly, 0.1 g of Certified Reference Material of tomato was

107 treated and obtained sample was used for the measurement of copper.

108 2.4. Procedure for DLLME for the determination of copper

109 A 13.0 mL of standard copper (5.0 ng/mL) aqueous solution was taken into centrifugal

110 polyethylene tube with addition of 0.5 mL each of ascorbic acid, buffer solution, 1.0% NaCl, 0.6

111 × 10−3 M DPT and total volume of the solution was 15.0 mL. Then, 0.5 mL absolute ethanol

112 (dispersing solvent) and 0.2 mL of chloroform containing PBITU (extraction solvent) were

113 injected into the aqueous phase and shaken for few minutes. The cloudy solution was formed that

114 consisted of finely droplets of extraction solvent dispersed in the sample solution. Finally, the
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115 centrifugation (REMI, R4C Centrifuge Machine) was carried out at 754.6 xg for 2 min, and the

116 organic phase at the top of the aqueous phase was carefully withdrawn using a microsyringe and

117 placed into a tube. The obtained volume of the sample volume was found to be small to analyze

118 in FAAS, so it was diluted to 400 µL with ethanol. This solution was directly nebulised into the

119 flame without use of any particular injection system.

120 3. Results and discussion

121 3.1. Optimization of DLLME for extraction and determination of copper

122 The Cu(II) ions present in the sample were reduced to Cu(I) in the presence of ascorbic acid as a

123 reducing agent. Then, Cu (I) ions reacted with DPT organic reagent to form a chelating complex

124 at pH 6.0 followed by the extraction in chloroform (containing PBITU) as a [(Cu-

125 DPT).PBITU]org complex. The reaction is given:

126 Cu(I)+DPT+PBITUorg → [Cu (DPT) . (PBITU)]org

127 The variables like, extraction and dispersion solvents, pH, salts and reagents (DPT and PBITU)

128 concentration were investigated that affected the extraction and preconcentration of copper from

129 the sample solution.

130 3.1.1. Selection of extraction and dispersing solvents

131 The selection organic solvent for the extraction of analytes should be based on the high

132 extraction recoveries of target analytes in to the organic solvent and low solubility into the

133 aqueous phase. On the basis of these considerations, chloroform, dichloromethane, toluene and

134 n-hexane were tested in the present work to extract and determine the copper in sample solution,

135 shown in Fig.1 A. Chloroform was found to be better solvent for the extraction of [(Cu-

136 DPT).PBITU]org complex from sample solution and used for further experiments. The reason

137 may be due to the higher solubility of copper complex in to the chloroform. The choice of the
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138 dispersing solvent was also chosen based on the miscibility into the organic phase (extraction

139 solvent) and aqueous phase (sample solution). Therefore, acetone, methanol and ethanol were

140 tested as a dispersing solvent for the analysis of copper, shown in Fig 1 B. The results indicated

141 that the maximum extraction of copper was achieved by using ethanol as a dispersing solvent

142 and used for further experiments.

143 3.1.2. Effect of pH

144 The pH of the sample solution significantly affects the formation and extraction of the copper

145 complex from the aqueous solution. Therefore, the extraction efficiency was investigated in the

146 pH range of 2.0–10.0 using buffer solution. The extraction efficiency of the complex was

147 increased as the pH of the aqueous solution was increased from 2.0 to 6.0 and after remained

148 constant, shown in Fig 2. Therefore, pH 6.0 was found to be best for the extraction of [(Cu-

149 DPT).PBITU]org complex.

150 3.1.3. Effect of concentration of DPT and PBITU

151 The effect of concentration of DPT was also investigated in the range of 0.2 × 10−3 to 1.0× 10−3

152 M. The result showed that the extraction efficiency of [(Cu-DPT).PBITU]org was increased by

153 increasing the DPT concentration up to 0.6 × 10−3 M and then remained constant, shown in Fig.

154 3. Thus, 0.6 × 10−3 M concentration of DPT was chosen for the subsequent experiments. The

155 effect of PBITU concentration on the extraction of the copper metal was also studied, and 4.0 ×

156 10−3 M of PBITU was found to be the minimum reagent concentration for the extraction of [(Cu-

157 DPT).PBITU]org complex. Fig. 4. An increased concentration up to 10 × 10−3 M of PBITU did

158 not affect the extraction of the complex. Hence, all the experimental work was carried out at 6.0

159 × 10−3 M of PBITU.

160 3.1.4. Effect of salt

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161 The additions of salt to the aqueous sample can significantly improve the extraction of several

162 analytes in LLE. This is possibly due to the salting out effect. Hence, the series of experiments

163 were performed with addition of salt (NaCl) in the range of 0–3.0% to the spiked aqueous

164 solution (5 ng/mL, Copper). The extraction efficiency of metals was increased with increasing

165 the salt concentration from 0% to 1.0% and there after decrease in the trend was observed, shown

166 in Fig. 4. The higher concentration of salt (>1%) might reduced the diffusion rates of the

167 analytes into the organic phase that caused the decrease in extraction efficiency of copper from

168 aqueous phase to organic phase. Therefore, 1.0% of salt was added in order to obtain the better

169 extraction of copper from the sample solution.

170 3.1.5. Effect of interferences

171 The effect of various diverse ions for the determination of 5 ng/mL copper was examined

172 separately as described in the procedure. The tolerable amount of diverse ions in μg/mL causing

173 an error less than +2.0 % is shown in Table 1. According to the data, the major metals ions

174 present in the food samples did not interfere in the determination of copper using DLLME/FAAS

175 method. The tolerance limit obtained for interfering metals were high and these amount of

176 concentration were not supposed to be found in the food samples. Therefore, the chances of

177 inferences of other metal ions were found to be negligible.

178 3.2. Analytical performance for the determination of copper using DLLME/FAAS

179 3.2.1. Linear range, limit of detection, limit of quantitation, precision and robustness

180 Important parameters such as the linear range, precision, detection limit, limit of quantitation and

181 robustness were determined to evaluate the performance of the method. The calculated

182 calibration curves gave a high level of linearity in the range of 0.2–20 ng/mL with the correlation

183 of estimation (r2) of 0.996 (Regression equation, y= 0.077x+ 0.007). The estimated limit of
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184 detection (LOD) for signal to noise ratio (S/N=3) equal to three for copper in sample solution

185 was 0.05 ng/mL. The limit of quantitation (LOQ) was lowest amount of copper determined when

186 the signal to noise ratio (S/N=10) equal to ten, and LOQ found for copper was 0.16 ng/mL.

187 The precision (repeatability and reproducibility) of the method was estimated by making

188 repeat measurements on a sample under specified conditions. The precision can be expressed a

189 relative standard deviation percentage (RSD, %) that was determined by performing six

190 consecutive extractions of copper from sample solution. First, the repeatability of the method

191 was calculated as for the intra-day precision. Intra-day precision was calculated based on the

192 analysis of two concentrations (2 and 5 ng/mL copper) in triplicate analyses (n = 3) in same day

193 and found to be 1.5–3.5%. Next, the reproducibility of the method was calculated by analyzing

194 the concentrations (2 and 5 ng/mL copper) by the different analysts and found to be 1.8-4.2%.

195 The inter-day precision was determined on three consecutive days for analyzing two

196 concentrations (2 and 5 ng/mL copper) and found to be 2.0–3.8%.

197 Robustness of the method was estimated by analyzing the effects of slightly changed

198 parameters used for the determination of copper in food samples. The slightly change in the

199 temperature ((±2°C) and pH (+0.1) of the solution did not show any significant variations for

200 tested ranges proved the robustness of the method.

201 3.2.2. Application of DLLME/FAAS for the determination of copper in cereals and vegetable

202 samples

203 The feasibility of the proposed method was tested for the determination of copper in cereals and

204 vegetable samples using DLLME/FAAS. A 1.0 mL pretreated cereals or vegetable samples were

205 taken in polyethylene centrifuge tube, and 0.5 mL each of ascorbic acid, buffer solution, 0.6 ×

206 10−3 M DPT, 0.1% sodium citrate, 1.0% NaCl and diluted to 15.0 mL DW. After, 0.5 mL ethanol
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207 (dispersing solvent) and 0.2 mL of chloroform (extraction solvent) were injected into the

208 aqueous solution and extraction was performed at the optimized conditions. The enriched

209 extracting solvent containing a copper complex was separated from aqueous phase and diluted to

210 400 µL of ethanol and used for FAAS analysis. The concentration of copper present in the

211 sample was calculated from the calibration curves of standard copper solution. The amount of

212 copper found in cereals and vegetable samples are given in the Table 2. The concentrations of

213 copper in cereals and vegetable samples were found in the range of 1.5–10.2 and 1.6–6.4 mg/Kg,

214 respectively. The higher amount of copper was found in the sample of kidney bean compared to

215 other food samples.

216 3.2.3. Enrichment factors and recovery for the determination of copper

217 The enrichment factor (EF) is defined as the ratio of concentration of copper between the

218 extracted organic phase and the initial concentration of the analyte in the aqueous sample. For,

219 these three replicate extractions were performed at the optimal conditions of sample containing 2

220 ng/mL of copper and EF obtained was 55. In order to evaluate the practical applicability of the

221 present method, the accuracy of method was investigated by performing recovery studies in the

222 vegetable samples. For this study, the known concentration of copper (2, 5 and 10 ng/mL) were

223 spiked in pretreated samples of lentil (copper was not detected in this sample, Table 2) to know

224 the matrix effect in the recovery of element from the sample. The good recoveries were obtained

225 for the determination of copper between 93.5 and 98.0% (in Table 3), which showing the

226 effectiveness for the extraction of copper from the vegetable samples.

227 Further the accuracy of the method was validated by analyzing the Standard Reference

228 Material of tomato. The copper present in Certified Standard Material was 21.1+2.5 (µg/g). The

229 proposed method was used for the determination of copper in given Reference Material and
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230 copper found was 20.92+1.5 (µg/g). Thus, the amount of copper found in the Reference Material

231 using present method was consistent with the certified values.

232 3.2.4. Comparison of DLLME/FAAS with other reported methods for the determination of

233 copper

234 The linear range, LOD, RSD, % and EF obtained by DLLME were compared with other reported

235 methods in order to know the potentiality of the present method for the determination of copper.

236 The results are summarized in Table 4. The LOD and EF values obtained by DLLME were

237 found higher than those with other methods. The RSD,% intended for the determination of

238 copper was found comparable to other methods. Thus, the performance of DLLME was better in

239 the sensitivity and extraction efficiency than other reported methods.

240 4. Conclusions

241 Small amount (200 µL) of an extracting organic solvent is needed to separate and preconcentrate

242 copper from food samples using DLLME prior to FAAS analysis. This method is found to be

243 simple, sensitive and low cost method for the determination of copper in food samples. It is

244 believed that the proposed procedure can be also useful for analysis and monitoring of copper

245 level in other plant products, environmental and biological samples.

246

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264
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267 Figure Captions:
268 Fig.1. (A) Effect of extraction solvent and (B) dispersing solvent for determination of copper (5
269 ng/mL) from spiked sample using DLLME at pH 6.0 with 1% addition of salt.
270
271 Fig.2. Effect of pH on the extraction of copper (5 ng/mL) from spiked sample obtained from the
272 DLLME using 200 µL volume of extracting solvent with 1% addition of salt.
273
274 Fig.3. Effect of concentration of DPT on the extraction of copper (5 ng/mL) from spiked sample
275 obtained from the DLLME using 200 µL volume of extracting solvent at pH 6.0 with 1%
276 addition of salt.
277
278 Fig.4. Effect of concentration of PBITU on the extraction of copper (5 ng/mL) from spiked
279 sample obtained from the DLLME using 200 µL volume of extracting solvent at pH 6.0 with 1%
280 addition of salt.
281
282 Fig.5. Effect of concentration of salt on the extraction of copper (5 ng/mL) from spiked sample
283 obtained from the DLLME using 200 µL volume of extracting solvent at pH 6.0.
284
285

286

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289

290

291

292
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293

294

295

Table 1. Effect of diverse ions for the determination of

copper using DLLME/FAAS

Ions added Tolerance limits, µg/mL

Na+, K+, 50

V3+, Zn2+, Ca2+ 100

Bi3+, Fe3+, Co2+, Sn2+, As5+ 400

Cd2+, Al3+, EDTA, citrate 500

Ni2+, Cr3+, Mn2+, Pb2+ 1000

296

297

298

299

300

301

302

303

304

305

306

307
 18

308

309

310

Table 2. Determination of copper in cereals and vegetable samples, mg/Kg using

DLLME/FAAS.

Cereals samples Copper RSD, +% Vegetable Copper RSD, +%

Maize 1.5 3.5 Potato 3.4 3.5

Millet ND* - Cauliflower 2.8 3.1

Rice 2.5 2.8 Tomato 1.6 2.8

Wheat 2.1 3.5 Spinach 2.0 2.6

Gram 3.2 4.0 Green Beans 4.8 3.3

Lentils 4.0 1.7 Lettuce 3.6 3.0

Kidney beans 10.2 2.5 Egg plants 6.4 2.3

Green beans 4.6 3.1 Bitter gourd 2.0 1.8

311 *ND= not detected

312

313

314

315

316

317

318

319
 19

320

321

322

323

Table 3. Determination of recovery % of copper in food samples using

DLLME/FAAS.

Samples Copper found by Copper added, Total copper Recovery %

present method, ng/mL found, ng/mL

ng/mL

Millet ND* 2.0 1.96 98.0

Millet ND* 5.0 4.75 95.0

Millet ND* 10.0 9.35 93.5

324 *ND= not detected

325

326

327

328

329

330

331

332

333

334
 20

335

336

337

338

Table 4. Comparison of different separating and analytical techniques with present methods for the

determination of copper

Separating/Analytical Linear LOD, RSD, EF Samples References

techniques Range, ng/ ng/mL/g %

mL/g

SDME/Spectrophotometry 5–1000 0.15 3.4 33 Food and Wen et al.

water (2011)

LLE/Spectrophotometry 10–400 2.0–4.0 2.0 5 Water and Shrivas (2010)

soil

DLLME-FAAS 1-600 0.5 1.4 - - Mohammadi et

al. (2009)

DLLME/FAAS 50–2000 3.0 5.1 42–48 water Farajzadeh et

la. (2008)

Coprecipitation/FAAS - 1.32 2.5 20 water Tokahoglu et

al. (2009)

SPE/FAAS 10–340 1.9 2.1 33 Foods Ghaedi et al.

(2010)

DLLME/FAAS 0.2–20 0.05 1.5–3.5 55 Cereals Present method

vegetables
 21

339

340

341

342

343

344

345

346 Fig. 1.

347
 22

348

349

350

351

352

353

354

0.5

0.4
Absorbance

0.3

0.2

0.1

0
0 2 4 6 8 10 12

pH
355

356 Fig. 2.

357

358

359

360

361

362
 23

363

364

365

366

367

368

0.5

0.4
Absorbance

0.3

0.2

0.1

0
0 0.2 0.4 0.6 0.8 1 1.2
Concentration of DPTx10-3 M
369

370 Fig. 3.

371

372

373

374

375

376

377
 24

378

379

380

381

382

383

384

385

0.5

0.4
Absorbance

0.3

0.2

0.1

0
0 2 4 6 8 10 12

Concentration of PBITU, 10-3 M

386

387 Fig. 4.

388

389

390

391

392
 25

393

394

395

396

397

398

0.6

0.5

0.4
Absorbance

0.3

0.2

0.1

0
0 1 2 3 4
NaCl, %
399

400 Fig. 5.
 Small amount (200 µL) of an extracting organic solvent is needed to separate and

preconcentrate copper from food samples using DLLME as separating and

proconcentrating probes prior to FAAS and thus it is an eco- friendly method.

 This method is found to be simple, sensitive and low cost method for the determination of

copper in food samples.

 The performance of the DLLME/FAAS was found better in the sensitivity and extraction

efficiency compared with other reported methods.

 It is believed that the proposed procedure can be also used for analysis and monitoring of

copper level in other plant products or environmental and biological samples.