INTRODUCTION:

Anothrones are known as carcinogenic compounds. Anthrone derivatives are found to have biological activity¹. These compounds have an azomethine nitrogen atom and this is responsible for their reactivity with the number of transition metal ions which form colored complexes. Its derivatives such as anthralin and anthraquinone, their functions were studied with the inner mitochondrial membrane. Anthralin readily oxidizes to anthralin dimer and anthraquinone; these compounds have also been identified as skin metabolites^2-4. Solubilities of some 9-anthrone derivatives and aminoanthraquinone derivatives in supercritical carbon dioxide were studied using a simple and reliable static method^5-6. Anthranoid laxatives are the most commonly used purgatives in the therapy of acute and chronic constipation and also for the development of colorectal neoplasm region^7-8. Further the metal complexes formed with these reagents are of great medicinal value in the treatment of diseases like colorectal cancer, psoriasis, chronic constipation, tumor related problems^9-15. In this article, the authors present first derivative spectrophotometric method for the simultaneous determination of copper and nickel.

EXPERIMENTAL:

i) Preparation of APH: The reagent anthrone phenylhydrazone was prepared by simple condensation of anthrone with phenyhydrazine by adopting the standard procedure. The structure of compound is given below,

Anthrone phenylhydrazone (APH)

The m.p. of APH is 145-149 ⁰C.

The structure has been established based on IR, mass and NMR spectra.

ii) Solutions preparation:

Buffer solutions are prepared using HCl, CH₃COOH and NaOAC in acidic medium and

NH₄OH, NH₄Cl in basic medium.

iii) Preparation of metal and reagent solutions:

The standard Cu(II) and Ni(II) solutions were prepared using analytical reagent grade CuSO₄.2H₂O, NiCl₂.6H2O. Appropriate quantity of APH is dissolved in DMF for making 0.01 M reagent solution.

Procedure:

a. Preparation of standard derivative spectrum:

5 ml of basic buffer solution of pH 10.5, 2.5 ml of each of the solution of Cu(II) and Ni(II), 6 ml of 1x10^-3M of APH are taken in a 25 ml volumetric flask, the contents are made up to the mark with double distilled water. The amplitudes of these solutions were measured between 350-410 nm against reagent blank.

Shimadzu 160A UV-visible spectrophotometer (Japan) equipped with 1 cm quartz cell was used in these investigations for making amplitude measurements. A pH meter ELICO L₁-120(Hyderabad) is used to make pH measurements.

b. Preparation and analysis of samples:

50 g of the biological material was heated in a 500 ml conical flask with 40 ml of conc.HNO₃ on a steam bath and shaken vigorously until a fine emulsion was formed. The heating was continued with the gradual addition of 6% H₂O₂ (40 ml). The aqueous phase was then transferred to the beaker. The extraction was repeated twice with further addition of 20 ml con.HNO₃ and 20 ml 6% H₂O₂. The combined extracts were evaporated to dryness. The residue was dissolved in minimum amount of dil.HCl and transferred into a 50 ml standard flask quantitatively. The contents were made up to the mark with distilled water. The solution is further diluted as required. A known aliquot of the sample solution, 5 ml of basic buffer solution of pH 10.5 and 6 ml of APH reagent were taken into a 25 ml volumetric flask and mixed well. The derivative spectra of analytes in the sample were recorded using the same procedure. According to the concentrations and amplitudes of standard solutions, the contents of the sample can be calculated from derivative spectrum obtained from sample.

RESULTS AND DISCUSSION:

a. Derivative spectra of Cu and Ni complexes:

The zero order spectrum of a mixture containing copper and nickel in presence of APH results only one peak, no resolution takes place and hence simultaneous determination is not possible and it is presented in the figure-1. Hence we have made an attempt to use a 1^st order derivative spectrum for possible simultaneous determination of the two metal ions. The spectrum is shown in the figure-2. The 1^storder derivative spectra are recorded in one case keeping Cu(II) constant and varying Ni(II) concentration as shown in the figure-3. In the second case the Ni(II) concentration is kept constant and Cu(II) concentration is varied as shown in the figure-4. In order to obtain greater sensitivity, graphs are drawn between Cu(II) concentration and peak amplitude as well as Ni(II) concentration and peak amplitude and presented in the figures-5 and 6. Linear graphs are obtained in both the cases. Hence using the first derivative spectrophotometric method, we can determine Cu in presence of Ni and vice-versa.

b. Effect of pH:

Cu(II) and Ni(II) react with APH forming an intense yellow colored complex. The colored solution shows maximum absorbance at 370 and 364 nm for Cu(II) and Ni(II) respectively in the pH range 7-12 and these are shown in the figures-7 and 8. Further studies reveal that the maximum absorbance is observed at pH 10 for Cu(II) and at pH 11 for Ni(II) and the results are reproducible at this pHs. A solution of pH 10.5 is selected for further detailed investigations and simultaneous determination of both the metal ions. Further Cu(II) and Ni(II) do not form stable complexes in acidic medium. It may be due to the hydrolysis of the reagent or the complex itself. In highly alkaline medium (>pH11) slow turbidity develops which may be due to formation of hydroxides.

The effect of reagent concentration was studied by measuring the absorbances at 370 and 364 nm of solution containing a fixed amount of Cu(II) and Ni(II) respectively by varying concentrations of APH. It was observed that 20-fold excess of APH is sufficient for maximum color development with both the metal ions. However, the excess concentration of the reagent did not show any substantial change in absorbance.

c. Applicability of Beer`s law:

The effect of metal ion concentration on absorbance is studied from 0.402-2.022x10^-5 M for Cu(II) and 0.411-4.811x10^-5 M for Ni(II). The concentration of reagent is kept constant at 1.2x10^-4 M. The absorbance values are measured at 370 nm for Cu(II) and at 364 nm for Ni(II) against a blank solution containing no metal ions and these are shown in the figures-9 and 10. Thus the method can be employed for the determination of Cu(II) in the range 0.397-1.990 µg/ml and Ni(II) in the range 0.4-4.8 µg/ml. The molar absorptivity and Sandell`s sensitivity of Cu(II) and Ni(II)-APH complexes are 5.065x10⁴ L/mol/cm, 2.497x10⁴L/mol/cm and 0.0019µg/cm², 0.0042 µg/cm² respectively.

The color reaction between Cu(II) and Ni(II)-APH is instantaneous at room temperature. The complex is stable for more than 2 hours and hence can be used for analytical applications.

d. Composition and stability of the complex:

The stoichiometry of Cu(II) and Ni(II)-APH complex was studied by Job`s method of continuous variation and also by the mole ratio method. Both the methods indicated the formation of a 1:1 complex between Cu(II) and APH, Ni(II) and APH. The stability constants are calculated as 1.042x10⁵ for Cu(II) and 4.972x10⁴ for Ni(II).

e. Effect of diverse ions:

The effect of diverse ions in the determination of Cu(II) and Ni(II) was examined under the optimum conditions. The extent of interference by various anions and cations was determined by measuring the absorbance of solutions containing a constant amount of Cu(II) and Ni(II) and varying amounts of diverse ions, most of the ions did not interfere in the determination. The tolerance limits for various cations and anions are listed in the table-1.

Table-1 Tolerance limit of foreign ions

Tolerance limit of foreign ions in the determination of 1.141 μg/ml of Cu (II) and 2.845 μg/ml of Ni(II)

pH = 10.5 λ_max = 368 nm

Foreign ion	Tolerance limit (μg/ml)	Foreign ion	Tolerance limit (μg/ml)
Thiourea	104	Co(II)	51
Tartrate	219	Mn(II)	4
Sulfate	855	Zn(II)	2^b
Phosphate	193	V(IV)	170
Fluoride	94	Sr(II)	532
Chloride	215	Ca(II)	209
Iodide	370	Zr(IV)	1
Nitrate	490	Ti(IV)	3
Oxalate	Interferes	Pb(II)	35
EDTA	Interferes	Ba(II)	685
Thiosulfate	1289	Al(III)	2
Bromide	640	W(VI)	198
Citrate	841	Pd(II)	530
Acetate	764	Fe(II)	45^a
		Hg(II)	1605
		Mo(VI)	95

^a Masked in the presence of 200 μg/ml of bromide

^b Masked in the presence of 500 μg/ml of citrate

Fig-1: Zero order spectrum of Cu (II)+Ni(II) in presence of APH

[Cu (II)] = [Ni (II)] = 1x10^-5M;

[APH] = 2.4x10^-4 M; pH = 10.5

Fig-2:

1^st order derivative spectrum of Cu(II)+Ni(II) in presence of APH

[Cu(II)] = [Ni(II)]=1.0x10^-5M; pH=10.5; [APH] = 2.4x10^-4 M;

a) 0.5 ml of Cu(II) and Ni(II) each

b) 1.0 ml of Cu(II) and Ni(II) each

c) 1.5 ml of Cu(II) and Ni(II) each

d) 2.0 ml of Cu(II) and Ni(II) each

Fig-3:

1^st order derivative spectrum of Cu(II)+Ni(II) in presence of APH.

Cu²⁺ concentration is kept constant by varying concentration of Ni^2+.

[Cu(II)] = [Ni(II)]=1.0x10^-5M; pH=10.5; [APH] = 2.4x10^-4 M;

a) 0.5 ml of Cu(II) and 1.0 ml of Ni(II)

b) 1.0 ml of Cu(II) and 1.5 ml of Ni(II)

c) 1.5 ml of Cu(II) and 2.0 ml of Ni(II)

d) 2.0 ml of Cu(II) and 2.5 ml of Ni(II)

Fig-4:

1^st order derivative spectrum of Cu(II)+Ni(II) in presence of APH.

Ni²⁺ concentration is kept constant by varying concentration of Cu^2+.

[Cu(II)] = [Ni(II)]=1.0x10^-5M; pH=10.5; [APH] = 2.4x10^-4 M;

a) 1.0 ml of Cu(II) and 0.5 ml of Ni(II)

b) 1.5 ml of Cu(II) and 1.0 ml of Ni(II)

c) 2.0 ml of Cu(II) and 1.5 ml of Ni(II)

d) 2.5 ml of Cu(II) and 2.0 ml of Ni(II)

Fig-5: First derivative amplitude Vs concentration of Cu(II)

pH=10.5, a=peak; b=valley; c=peak+valley;

Fig-6: First derivative amplitude Vs concentration of Ni(II)

pH=10.5, a=peak; b=valley; c=peak+valley;

Fig-7: Effect of pH on the absorbance of copper (II)-APH system

[Copper (II)]=3x10^-5M, [APH] =2x10^-4M

Fig-8: Effect of pH on the absorbance of nickel (II)-APH system

[Nickel (II)] =3x10^-5M, [APH] =2x10^-4M

A₃₇₀=0.5C-0.01

Fig-9: Effect of metal ion concentration on absorbance (Beer`s law)

λ_max=370 nm, [APH]=2.4x10^-4 M, pH=10

Fig-10: Effect of metal ion concentration on absorbance (Beer`s law)

λ_max=364 nm, [APH]=2.4x10^-4 M, pH=11

APPLICATION:

The present method is applied for the determination of Cu(II) and Ni(II) metal ions simultaneously in sesame seeds, ground nut seeds and grape leaf. The repeatability and precision of the method were satisfied with RSD in the range of 0.0863-0.0888% for five determinations. Therefore, the two metal ions can be directly determined after digestion (as per procedure given in `b`) without any pretreatment by the proposed method. Accuracy of the proposed method was validated using a certified reference material of tea and grape leaves (GBW07605 and GBW08501, Chinese Standard material center) (AAS and APARI, Hyderabad). The values determined by the proposed method and the determined values (n=5) of the certified reference material were within the given guarantee values and shown in the table-2.

CONCLUSION:

In this article, a multi-component analysis with 1^st order derivative spectrophotometry has been developed. The proposed method has been successfully applied for the simultaneous determination of Cu and Ni in certified reference materials and food samples after digestion without further pretreatment. Compared with the traditional spectrophotometry, the proposed method provides good results for two analytes in terms of accuracy and precision and allows 37 determinations per hour for the digested food samples, and the results proved to be satisfactory and meet the criterion of food analysis.

REFERENCES:

1. J.E. Hedge and B.T. Hofreiter, (1962). In Carbohydrate Chemistry, 17 Academic Press, New York.

2. R.L. Eds. Whistler and J.N. Be Miller, (1962). Methods in Carbohydrate Chemistry,

3. B. Thayumanavan and S. Sadasivam, (1984). Qual. Plant Foods Hum. Nutr., 34, p.253.

4. D.M. Updegroff, (1969). Anal. Biochem., 32, p. 420.

5. Ali Reza Karami, Yadollah Yamini, Ali Reza Ghiasvand, Hashem Sharghi and Mojtaba

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7. Mojtaba Shamsipur, Ali Reza Karami, Yadollah Yamini and Hashem Sharghi

8. J. Chem. Eng. Data (2003), 48 (1), pp 71–74 Cancer Gut 1993; 34:1099–101.

9. G.A. Kune, S. Kune, B. Field et al., The role of chronic constipation, diarrhea, and laxative use in the etiology of large-bowel cancer. Data from the Melbourne Colorectal cancer Study Dis Colon Rectum

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12. P.M. Patel, P.J. Selby, J. Deacon, et al., Anthraquinone laxatives and human cancer: An association in one case. Postgrad Med J 1989;65:216–17.

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Received on 29.12.2011 Modified on 14.01.2012

Research J. Pharm. and Tech. 5(3): Mar.2012; Page 357-361

Sample	APARI and AAS (%) method		Amount found (%) By present method		Relative error (%)
Sample	Cu(II)	Ni(II)	Cu(II)	Ni(II)	Cu(II)	Ni(II)
Grape leaf	55.40	33.70	55.30	33.84	-0.18	+0.41
Ground nut seeds	4.275 µg/ml	1.205 µg/ml	4.248 µg/ml	1.189 µg/ml	-0.63	-1.33
Sesame seeds	11.600 µg/ml	1.273 µg/ml	11.468 µg/ml	1.261 µg/ml	-1.14	-0.94