Spectrophotometric Determination of Cu2+ and Monitoring of Hg2+ and Ni2+ in some Iranian Vegetables Using 6-(2-Naphthyl)-2, 3-Dihydro-as-triazine-3-thione

Document Type: Research article

Authors

Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran.

Abstract

Recently, 6-(2-naphthyl)-2, 3-dihydro-as-triazine-3-thione (NDTT) was synthesized in laboratory and used successfully for the spectrophotometric determination of nanogram levels of Cu2+ in aqueous solution. This reagent forms a specific red complex with Cu2+ ions after the extraction by chloroform at alkaline pH. The absorption of the complex in the UV region (313 nm) is about 8 times as strong as in the visible one (510 nm). Mercury and nickel ions form yellow complexes with NDTT under the same conditions which interfere in the UV region and without effect on Cu (II) absorbance in the visible region. The studied vegetables include Mentha pipereta L., Anethum graveolens L., Beta vulgaris L., Coriandrum sativum, Petroselinum hortense H., Ocimum basilicum L., Spinacia oleracea L., Lactuca sativa L., and Brassica oleracea L.

Keywords

Main Subjects


 

 Introduction

   Copper is a mineral that nowadays poses few problems. It is widely distributed, as a component of various enzymes in foodstuffs of all kinds, at levels between 1 and 5 ppm. Milk is notably low in copper, at around 2 ppm, and mammalian liver is exceptionally high, at around 80 ppm. The daily intake in normal adult diets is between 1 and 3 mg, which roughly corresponds to the intake level recommended by most authorities (1). Copper is an essential element in the nutrition of animals and human. It acts as a cofactor in numerous enzymes and plays an important role in protein and cell division. It exerts a crucial influence on the maintenance of cell membrane stability and in the function of immune system. Meanwhile, as a pollutant, copper is of particular concern, due to its high toxicity on aquatic organisms (1-3).

   Determination of trace amounts of copper has received considerable attention and various methods have been developed for this purpose (2-6). These methods are time-consuming, not sufficiently sensitive or narrow range pH-dependent. Therefore, developing specific and sensitive methods without being pH-dependent which rapidly and conveniently detect and determine copper in real samples seems to be desirable (4, 5).

   Previously, 6-phenyl-2, 3- dihydro-as-triazine-3-thione (PDTT) was reported to form a specific red complex with Cu2+, which is easily extractable with chloroform over a wide range of pH. Despite being simple, this method suffersfrom low sensitivity, in a way that the molar absorbance of the complex is 5.0 × 103 (6). In a previous investigation, we have reported the synthesis of 6-(2-naphtyl)-2, 3-dihydro-as-triazine-3-thione (NDTT) as a new sensitive and specific reagent for determining Cu2+ (7). The purpose of the modification was to increase the PDTT sensitivity in determining Cu2+, Hg2+, and Ni2+. The preparation of NDTT was conducted by a one-step synthesis of the as-triazine system (8).

   The analysis of Cu2+ in solution by NDTT could be performed easily and in the presence of many cations and anions (7). Only Hg2+, Ni2+ and Pd2+ form complexes with NDDT. Hg2+ and Ni2+ present natural products in ultra-trace quantities compared with copper. Therefore, the analysis of Cu2+ in vegetable through NDTT could be considered specific by this procedure.

Experimental

Apparatus and reagents

    A Shimadzu 160A UV-VIS spectrophotometer with 1.0 cm quartz cell was used for all absorbance measurements and Atomic Absorption Varian 220 was employed in this study. Sodium hydroxide solution (4 M), tartaric acid solution (2 M) and NDTT in NaOH 4M (1 mg/5 mL) were used. Fresh NDTT/NaOH solution should be used. NDTT reagent itself was prepared according to the reported procedure (6). All reagents used are of analytical reagent grade, unless otherwise stated.

Vegetables under study

    All the vegetables were bought from Tehran daily markets and then approved by Dr. Sh. Rezazadeh. The edible portions of the vegetable under study (1-2 Kg) were separated, weighed, washed thoroughly with distilled water, and left to be dried at room temperature. The weight of the dried samples were recorded and the percentages of the dried to fresh samples were calculated (Table 1). The studied vegetables include: Mentha piperita L., Allium L., Anethum graveolens L., Beta vulgaris L., Petroselinum hortense H., Coriandrum sativum, Ocimum basilicum L., Spinacia oleracea L., Lactuca sativa L. and Brassica oleracea L.

 

Table 1. Weight and percentage of the dried vegetables obtained from the corresponding fresh clean samples.

Vegetable scientific name

Weight of Vegetable (gm)

%D/F*

Fresh

Dried

 

Mentha piperita L.

360

70

19.4

Allium L.

1100

75

6.8

Anethum graveolens L.

415

50

12.0

Beta vulgaris L.

500

30

6.0

Petroselinum hortense H.

450

75

16.7

Coriandrum sativum

550

55

10.0

Ocimum basilicum L.

600

65

10.8

Spinacia oleracea L.

900

80

8.9

Lactuca sativa L.

1160

55

4.74

Brassica oleracea L.

1100

85

7.73

    *D/F = dried/fresh

 

Stock solution of copper nitrate

    Pure elemental copper (exactly 0.5 g) was dissolved in hot con. HNO3. After cooling, 50 mL of HNO3 (1:1) was added and the volume was adjusted to 500 mL by distilled water. Solutions with lower concentrations were prepared by proper dilutions.

Calibration curve for determining Cu2+

    A mixture of 1-20 μg Cu2+, NDTT solution (2 mL of 1 mg / 5 mL) and tartaric acid (1 mL) in a 100 mL separatory funnel was shaken thoroughly. The resulted complex was extracted with 4, 3 and 2 mL of CHCl3. The extracts were collected in a 10 mL volumetric flask and adjusted to volume with CHCl3. The absorbance of the extracts was measured at both UV (313 nm) and visible (510 nm) region vs. a blank. A calibration curve wasplotted for the amount of Cu2+ against the relative absorbance. This curve was used to determine the vegetable Cu2+ content.

 

Table 2. Percentage of ashes obtained from different dried vegetables under study (2.0 gm) after wet digestion at 600°C.

Vegetable scientific name

Ash

Wt. (gm)

%

%in relative to fresh sample

Mentha piperita L.

0.19

9.55

2.0

Allium L.

0.412

20.6

1.4

Anethum graveolens L.

0.32

16.0

1.92

Beta vulgaris L.

0.49

24.5

1.47

Petroselinum hortense H.

0.225

11.25

1.88

Coriandrum sativum

0.303

15.15

1.52

Ocimum basilicum L.

0.308

15.4

1.66

Spinacia oleracea L.

0.275

13.74

1.22

Lactuca sativa L.

0.241

12.06

0.57

Brassica oleracea L.

0.100

5.0

0.39

 

Vegetable sample preparation for Cu2+ analysis

    A portion of the dried vegetable (exactly 2.0 g) was transferred to a crucible and soaked with distilled water (5 mL). The crucible was left for 1 h at 150°C followed by 4 h at 600°C for ignition. The remained ash was dissolved in con. HNO3 (1 mL) followed by distilled water (15 mL), filtered (if necessary), and neutralized by NaOH 4 M. The solution was made to volume in a 25 mL volumetric flask.

Preparation of the blank solution

    Distilled water (15 mL) in a crucible was left for 1 h at 100°C and for 4 h at 600°C. After cooling, con. HNO3 (1 mL) and dis. H2O (15 mL) were added and neutralized in a beaker by NaOH 4M. This solution was transferred to a volumetric flask and made to volume (25 mL) by dis. H2O. A 5 mL portion of this solution was transferred to a separatory funnel, followed by tartaric acid (1 mL), NDTT solution (2 mL) and mixed thoroughly. The complex was extracted by 4, 3 and 2 mL of CHCl3, transferred to a 10 mL volumetric flask and made to volume. The absorption of this solution was measured against CHCl3. This solution was colorless with no absorption at 510 nm, but at 313 nm, it showed insignificant absorption (< 0.02). This solution was used as a blank in determining Cu2+ content of different vegetables.

General procedure for the determination of Cu2+ in vegetables

    A portion (5 mL) of the processed solution of vegetable was transferred to a separatory funnel. Tartaric acid solution 1 M (1 mL), followed by NDTT solution (2 mL) were added and mixed. The extraction of Cu-NDTT complex was performed by 4, 3 and 2 mL of CHCl3, collected in a 10 mL volumetric flask and made to volume by CHCl3. The absorbance at UV (313 nm) and visible (510 nm) was recorded. Using a calibration curve constructed for pure Cu2+, the μg amount of Cu2+ in 5 mL of the processed solution of the vegetable was obtained, and transformed to Cu2+ in the dried and fresh samples (Tables 3 and 4) by proper calculations.

Results and Discussion

     To perform the proposed procedure for determining copper in vegetables, primarily a portion of the edible part was weighed and dried in room temperature. The ratio of the dried to fresh samples was registered (Table 1). As shown in this table, Lactuca sativa L. contains the highest percent of water (95.26%), where Mentha piperita contains the lowest one (70.6%). For preparing the vegetable for analysis, primarily, the dried vegetable (2.0 g) was soaked with distilled water, dried in 150°C and followed by ignition at 600°C for 4 h. Since the vegetable cellulose is not hard, the wet-digestion was undertaken. A white ash which was soluble in concentrated HNO3 was obtained, otherwise the solution was filtered. After neutralizing by NaOH 4M, the volume was made to 25 in a volumetric flask. A portion of this solution (5 mL) was used for the analysis of Cu2+ and monitoring of other cations. In all cases, the chloroform layer was red and similar to a pure Cu2+ solution (except for anethum which was pale orange) (Table 3). Therefore, it is possible to detect Cu2+ in vegetables easily, as NDTT formed a red complex only with Cu2+. The absorption spectra of the complex in chloroform were recorded and the absorbance at UV and visible regions were registered. Using a calibrationcurve, it was possible to find the Cu2+ content in 5 mL of solution. By proper calculations, the content of Cu2+/Kg of fresh vegetable was obtained (Table 4). It was found that Mentha piperita L. contains the highest amount of Cu2+ (3.94 ppm of fresh vegetable) and Lactuca sativa L. contains the lowest amount (0.593 ppm of fresh vegetable) among the studied vegetables. To confirm the applicability of the proposed method for determining Cu2+ in vegetables, the samples were analyzed by atomic absorption method and the results are shown in Table 5. In all cases, there was insignificant difference between the proposed method and the atomic absorption analysis. The difference ranges from2.5 to 11.1%. The cations Hg2+ and Ni2+ formed yellow complexes with NDTT (9, 10), hence, the absence of these two cations could be concluded as the chloroformic layer was red except in the case of Anethum graveolens L. which appeared pale orange. Therefore, Hg2+ and Ni2+ were either absent or present in non-detectable level by NDTT. Finally, it is concluded that the developed spectrophotometric procedure for determining Cu2+ in vegetables using NDTT is very simple, fast and specific.

 

Table 3. Absorbances, λmax, and the color of the chloroformic layers of Cu-NDTT complexes of different vegetables.

Vegetable scientific name

Absorbance/W.L.

Ratio UV/Vis Absorbance

Color of CHCl3 layer

UV

Visible

Mentha piperita L.

0.448/313

0.053/510

8.45

Red

Allium L.

0.528/314

0.062/511

8.52

Red

Anethum graveolens L.

0.306/313

0.058/509

5.28

Pale orange

Beta vulgaris L.

0.599/313

0.075/510

8.00

Red

Petroselinum hortense H.

0.463/313

0.053/509

8.74

Red

Coriandrum sativum

0.467/313

0.057/509

8.2

Red

Ocimum basilicum L.

0.421/313

0.049/507

8.6

Red

Spinacia oleracea L.

0.587/314

0.064/510

9.7

Red

Lactuca sativa L.

0.529/314

0.034/510

15.6

Red

Brassica oleracea L.

0.276/313

0.033/510

8.36

Red

 

 

Table 4. Cu2+ content in different steps of the analytical procedure.

Vegetable scientific name

Absorbance at UV λmax

Cu2+ content

In processed solution μg/5 mL

In 2 g dried sample (μg)*

In 1 Kg fresh sample (mg)** (ppm)

Mentha piperita L.

0.448

8.1

40.5

3.94

Allium L.

0.528

9.5

47.5

1.62

Anethum graveolens L.

0.306

5.51

27.5

1.65

Beta vulgaris L.

0.599

10.8

54.0

1.62

Petroselinum hortense H.

0.463

8.3

41.5

3.47

Coriandrum sativum

0.467

8.4

42.0

2.1

Ocimum basilicum L.

0.421

7.6

38.0

2.05

Spinacia oleracea L.

0.587

10.6

53.0

2.36

Lactuca sativa L.

0.529

5.0

25.0

0.593

Brassica oleracea L.

0.276

5.0

25.0

0.966

*Cu2+/2 g dried sample is obtained from the Cu2+/5 mL test solution multiplied by 5.

**Cu2+(mg)/Kg fresh sample = Cu2+(μg)/2 g dried sample × 0.5 × %D/F ÷ 100.

***Cu2+ content was obtained from the visible absorbance.

 

 Table 5. Copper content (ppm) of fresh vegetables found by the proposed method (NDTT) and atomic absorption method (n = 5).

Sample

NDTT method

Atomic absorption

% difference

Mentha piperita L.

3.94 ± 0.6

4.2 ± 0.72

6.5

Allium L.

1.62 ± 0.32

1.73 ± 0.35

6.8

Anethum graveolens L.

1.65 ± 0.35

1.72 ± 0.4

4.2

Beta vugaris L.

1.62 ± 0.28

1.8 ± 0.3

11.1

Petroselinum hortense H

3.47 ± 0.72

3.67 ± 0.68

5.8

Coriandrum sativum

2.1 ± 0.43

2.2 ± 0.61

4.8

Ocimum basilicum L.

2.05 ±0.41

2.0 ± 0.52

2.5

Spinacia oleracea L.

2.36 ± 0.46

2.30 ± 0.41

2.5

Lactuca sativa L.

0.593 ± 0.08

0.63 ± 0.07

5.0

Brassica oleracea L.

0.966 ± 0.11

1.02 ± 0.12

5.6

 

Acknowledgment

    This investigation was supported by a grant from the Pharmaceutical Sciences Research center, Tehran University of Medical Sciences, and was presented at the 1st International Symposium on Environmental Protection, Fez, Morocco. The authors wish to thank Mr. Khosro Abdi for running the atomic absorption analysis of the samples.

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(4)      Lokhande RS, Nirupa S and Chaudhary AB. Spectrophotometric determination of Cu (II) with 2H-benzopyran-2-acetyl thiosemicarbazone. Ibid (2002) 14: 149-152.

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(8)      Lalezari I. and Golgolab H. A one-step synthesis of the as-triazine system. J. Heterocyclic Chem. (1970) 7: 689-691.

(9)       Maghssoudi RH and Shamsa F. Specrophotometric determination of Hg(II) with 6-phenyl-2,3-dihydro-as-triazine-3-thione. Anal. Chem. (1975) 47: 550-553.

(10)      Shamsa F. and Keeshwardoost F. A new spectrophotometric procedure for the determination of Nickel (II) using 6-phenyl-2,3,-dihydro-as-triazine-3-thione. J. Sci. I. R. Iran (1992) 3: 89-93.