1. INTRODUCTION:

Thiosemicarbazones have been widely used as analytical reagents. The analytical applications and biological activity of thiosemicarbazones have been reviewed^1-5. These reagents have been used as analytical reagents in spectrophotometry, flurometry, atomic absorption spectrophotometry and as indicators. Thiosemicarbazones with transition metal complexes have great medicinal value. These compounds are used in the treatment of diseases like influenza⁶, protozoa⁷, small pox^8-9and certain kinds of tumour related problems^10-12. These reagents are known for their antitubercular activity¹³. In the treatment of cancer, the active species is the metal chelate of thiosemicarbazone^14-15. Metal chelates of these reagents are used as pesticides¹⁶ and fungicides¹⁷ in agriculture. Thiosemicarbazones with transition metal complexes possess antifungal, antimicrobial and antiviral activity^18-20. In this article, the authors present second order derivative spectrophotometric method for the simultaneous determination of mercury and cobalt.

2. EXPERIMENTAL PROCEDURES:

i) Preparation of FFTSC: The reagent furfuraldehyde thiosemicarbazone was prepared by simple condensation of furfuraldehyde with thiosemicarbazide by adopting the standard procedure. The structure of the compound is given below,

Furfuraldehyde thiosemicarbazone

The m.p. of FFTSC is 131-135⁰C, Yield = 8.10 gm, %Yield = 85.3%.

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

ii) Preparation of buffer solutions:

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 Hg(II) and Co(II) solutions were prepared using analytical reagent grade HgCl₂ and CoCl₂.6H₂O. Appropriate quantity of FFTSC is dissolved in DMF for making 0.01 M reagent solution.

Procedure:

a. Preparation of standard derivative spectrum:

A series of solutions containing varying concentrations of Hg(II) and Co(II) are taken in 25 ml volumetric flasks along with 20 fold concentration of FFTSC reagent, so that it is sufficient to form complexes with both the metal ions. To this 10 ml buffer solution of pH 8.5 is added and made up to the mark with distilled water. The amplitudes of these solutions were measured between 360-420 nm against reagent blank. Shimdzu 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:

Each 15 ml sample of total blood was divided into two parts, one (7.5 ml) was the total blood sample which was stored at 4°C and the other (7.5 ml) was centrifuged to separate the plasma fraction from the heavy red-cell fraction, this was collected and stored at 4 °C. All of the total blood sample and plasma samples were analyzed. A known aliquot of the sample solution, 10 ml of basic buffer solution of pH 8.5 and 5 ml of FFTSC reagent were taken into a 25 ml volumetric flask and made up to the mark with distilled water. The contents of the solution are mixed well to make uniform concentration. 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 are calculated from derivative spectrum obtained from the sample.

One liter of tap, distilled water samples was filtered, the pH adjusted to 1 with concentrated HCl in order to prevent losses by sorption or co-precipitation and preserved in high quality clean plastic containers. To investigate the applicability of the present method to natural water samples, the recoveries of known amounts of Hg(II) and Co(II) are added to these samples and analyzed.

Human urine (5-7 ml from scleroderma patients) sample was taken into a 100-ml volumetric flask. A glass bead and 10 ml of concentrated nitric acid were added, and the flask was placed on a digester under gentle heating. When the initial brisk reaction was completed, the solution was removed and cooled. To this 1 ml of concentrated sulfuric acid was carefully added followed by the addition of 1 ml of 70% perchloric acid and heating was continued until the dense white fumes come. Heating was continued for at least half an hour and then cooling was applied. The contents of the flask were filtered and neutralized with NH₄OH in the presence of 1-2 ml of a 0.01% (w/v) EDTA solution. The resultant solution was then filtered and transferred quantitatively into a 10-ml volumetric flask and made up to the mark with deionized water. A suitable aliquot (1-2 ml) of the final solution was pipetted out into a 25-ml volumetric flask followed by 10 ml buffer solution of pH 8.5 and 5 ml of FFTSC reagent is added. The contents of the solution were made up to the mark with distilled water. The derivative spectra of analytes in the sample were recorded using the same procedure. By comparing the amplitudes of sample and standard solutions the amount of mercury and cobalt were determined.

3. RESULTS AND DISCUSSION:

a. Derivative spectra of Hg(II) and Co(II) complexes:

The zero order spectrum of a mixture containing mercury and cobalt in presence of FFTSC 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, but no fruitful results are obtained. Finally we have made an attempt to use a second order derivative spectrum for possible simultaneous determination of the two metal ions, these are shown in the figures-2 and 3 consist of two peaks and valleys corresponding to the two metal ions. The 2^ndorder derivative spectra are recorded in one case keeping Hg(II) concentration constant and varying Co(II) concentration as shown in the figure-4. In the second case the Co(II) concentration is kept constant and Hg(II) concentration is varied as shown in the figure-5. In order to obtain greater sensitivity, graphs are drawn between Hg(II) concentration and peak amplitude as well as Co(II) concentration and peak amplitude and are presented in the figures-6 and 7. Linear graphs are obtained in both the cases, hence using the second order derivative spectrophotometric method; we can determine Hg in presence of Co and vice-versa.

Fig-1: Zero order spectrum of Hg (II)+Co(II) in presence of FFTSC.

[Hg (II)] = [Co (II)] = 4x10^-5M;

[FFTSC] = 8.0x10^-4 M; pH = 8.5

Fig-2: 2^nd order derivative spectrum of Hg(II)+Co(II) in presence of FFTSC.

[Hg(II)] = [Co(II)]=4x10^-5M; pH=8.5; [FFTSC] = 8x10^-4 M;

Fig-3: 2^nd order derivative spectrum of Hg(II)+Co(II) in presence of FFTSC.

[Hg(II)] = [Co(II)]=1x10^-5M; pH=8.5; [FFTSC] = 2x10^-4 M;

a) 0.5 ml of Hg(II) and Co(II) each; b) 1.0 ml of Hg(II) and Co(II) each

c) 1.5 ml of Hg(II) and Co(II) each; d) 2.0 ml of Hg(II) and Co(II) each

e) 2.5 ml of Hg(II) and Co(II) each

Fig-4:2^nd order derivative spectrum of Hg(II)+Co(II) in presence of FFTSC.

Hg²⁺ concentration is kept constant by varying concentration of Co^2+.

[Hg(II)] = [Co(II)]=1x10^-5M; pH=8.5; [FFTSC] = 2x10^-4 M;

a) 0.5 ml of Hg(II) and 1.0 ml of Co(II); b) 1.0 ml of Hg(II) and 1.5 ml of Co(II); c) 1.5 ml of Hg(II) and 2.0 ml of Co(II); d) 2.0 ml of Hg(II) and 2.5 ml of Co(II); e) 2.5 ml of Hg(II) and 3.0 ml of Co(II)

Fig-5:2^nd order derivative spectrum of Hg(II)+Co(II) in presence of FFTSC.

Co²⁺ concentration is kept constant by varying concentration of Hg^2+.

[Hg(II)] = [Co(II)]=1x10^-5M; pH=8.5; [FFTSC] = 2x10^-4 M;

a) 1.0 ml of Hg(II) and 0.5 ml of Co(II); b) 1.5 ml of Hg(II) and 1.0 ml of Co(II); c) 2.0 ml of Hg(II) and 1.5 ml of Co(II); d) 2.5 ml of Hg(II) and 2.0 ml of Co(II); e) 3.0 ml of Hg(II) and 2.5 ml of Co(II)

Fig-6: Second derivative amplitude Vs concentration of Hg(II)

pH=8.5, a=peak; b=valley; c=peak+valley;

Fig-7: Second derivative amplitude Vs concentration of Co(II)

pH=8.5, a=peak; b=valley; c=peak+valley;

b. Effect of pH:

Hg(II) and Co(II) react with FFTSC reagent form an intense yellow and brown colored complexes. The colored solutions show maximum absorbances at 359 and 364.5 nm for Hg(II) and Co(II) respectively in the pH range 2-11 and these are shown in the figures-8 and 9. Further studies reveal that the maximum absorbance is observed at pH 8 for Hg(II) and at pH 9 for Co(II) and the results are reproducible at this pHs. A solution of pH 8.5 is selected for further detailed investigations and simultaneous determination of both the metal ions. Moreover Hg(II) and Co(II) do not form stable complexes in acidic medium it may be due to hydrolysis of the reagent or the complex itself. In highly alkaline medium (>pH 11) slow turbidity develops which may be due to formation of hydroxides.

Fig-8: Effect of pH on the absorbance of Hg (II)-FFTSC system

[Hg (II)]=4x10^-5M, [FFTSC] =4x10^-4M

ig-9: Effect of pH on the absorbance of cobalt (II)-FFTSC system

[Co (II)] =4x10^-5M, [FFTSC] =4x10^-4M

The effect of reagent concentration was studied by measuring the absorbance values at 359 and 364.5 nm of solution containing a fixed amount of Hg(II) and Co(II) by varying concentration of FFTSC. It was observed that 20-fold excess of FFTSC 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 0.08-0.8x10^-5 M for Hg(II) and 0.018-0.188x10^-5 M for Co(II). The concentration of reagent is kept constant at 4.0x10^-4 M and the absorbance values are measured at 359 nm for Hg(II) and at 364.5 nm for Co(II) against a blank solution containing no metal ions and these are shown in the figures-10 and 11. Thus the method can be employed for the determination of Hg(II) in the range 0.8-8.0 µg/ml and Co(II) in the range 0.18-1.88 µg/ml. The molar absorptivity and Sandell`s sensitivity of Hg(II) and Co(II)-FFTSC complexes are 2.2x10⁴ L/mol/cm, 4.3x10³L/mol/cm and 0.000454µg/cm², 0.00232 µg/cm² respectively.

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

λ_max=359 nm, [FFTSC]=2.5x10^-4 M, pH=8

Fig-11: Effect of metal ion concentration on absorbance (Beer`s law)

λ_max=364.5 nm, [FFTSC]=2.5x10^-4 M, pH=9.0;

The color reaction between Hg(II) and Co(II)-FFTSC is instantaneous at room temperature. The complex is stable for more than an hour and hence can be used for analytical applications.

d. Composition and stability of the complex:

The stoichiometry of Hg(II) and Co(II)-FFTSC 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 1:2 complex between Hg(II) and FFTSC, 1:1 between Co(II) and FFTSC. The stability constants are calculated as 3.495x10⁶ for Hg(II) and 5.176x10⁴ for Co(II).

e. Effect of diverse ions:

The effect of diverse ions in the determination of Hg(II) and Co(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 Hg(II) and Co(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 4.0 μg/ml of Hg (II) and 0.943 μg/ml of Co(II).

pH = 8.5 λ_max = 363 nm

Foreign ion

Tolerance limit (μg/ml)

Foreign ion

Tolerance limit (μg/ml)

Fluoride

Chloride

Iodide

Nitrate

Acetate

Oxalate

EDTA

Thiosulphate

20.54

54.62

253.8

130.53

43.7

8.85

1667

15.5

U(VI)

Ru(III)

W(VI)

Mo(VI)

Se(IV)

Pd(II)

Mg(II)

Cu(II)

Sn(II)

Ni(II)

Zr(IV)

Sr(II)

La(III)

Ti(IV)

Al(III)

Th(IV)

Cd(II)

Cr(VI)

Mn(II)

Fe(II)

82.80

13.26

63.95

19.2

5.45

0.12

32.41

0.45

14.84

0.612

10.73

12.75

52.91

6.96

13.49

64.01

0.804

5.18

19.98

4.68

Table-2: Concentration of selected elements (µg/ml) in the blood Samples, blood plasma and urine from patients with scleroderma.

Sample	CVAAT/AAS value in µg/ml (cold vapor atomic absorption technique and Atomic absorption spectroscopy))		Amount found in µg/ml By present method		Relative error(%)
Sample	Hg(II)	Co(II)	Hg(II)	Co(II)	Hg(II)	Co(II)
Blood	7.4	0.05	7.33	0.047	+0.945	+6
Blood plasma	3.9	0.056	3.94	0.053	-1.02	+5.35
Urine	6.97	6.75	7.1	6.7	-1.86	+0.74

Table-3: Determination of mercury and cobalt in water samples.

Type of water	Hg(II) added	Spectrophotometric method		AAS method
Type of water	Hg(II) added	Found(µg/ml)	Recovery (%)	Found (µg/ml)	Recovery (%)
Tap water (5 ml) Distilled water (5 ml) Well water (5 ml)	- 3.0 4.8 - 3.2 5.6 - 5.4 4.6	ND 2.981 4.779 ND 3.189 5.591 ND 5.1 4.2	- 99.36 99.56 - 99.65 99.83 - 94.44 91.32	ND 2.985 4.782 ND 3.193 5.594 ND 5.2 4.3	- 99.50 99.62 - 99.78 99.89 - 96.29 93.47
Type of water	Co(II) added	Spectrophotometric method		AAS method
Type of water	Co(II) added	Found(µg/ml)	Recovery (%)	Found (µg/ml)	Recovery (%)
Tap water (5 ml) Distilled water (5 ml) Well water (5 ml)	- 100 500 - 100 500 - 113 500	ND 98 494 ND 93 492 ND 100 496	- 98.0 98.8 - 93.0 98.4 - 88.49 99.2	ND 98.3 493 ND 96 494 ND 104 497	- 98.3 99.6 - 96.0 98.8 - 92.0 99.4

4. APPLICATION:

The present method is applied for the determination of Hg(II) and Co(II) metal ions simultaneously in tap water, distilled water and blood samples. The repeatability and precision of the method were satisfied with RSD in the range of 0.346-0.477% for five determinations. Therefore, the two metal ions can be directly determined after digestion (as per procedure given in `iii. b`) without any pretreatment by the proposed method. Accuracy of the proposed method was validated using a certified reference material of natural water and blood samples (ICPAES, ICPMS, AAS, CVAAT and APARI). 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 tables-2 and 3.

5. CONCLUSION:

In this article, a multi-component analysis with 2^nd order derivative spectrophotometry has been developed. The proposed method has been successfully applied for the simultaneous determination of Hg and Co in certified reference materials and biological samples after digestion without any further pretreatment. Compared with the traditional spectrophotometry, the proposed method provides good results for two analytes in terms of accuracy and precision and allows 36 determinations per hour for the digested biological samples, and the results proved to be satisfactory and meet the criterion of biological material analysis.

6. REFERENCES

1. R.B.Singh, B.S.Garg and R.P.Singh, Talanta., 25, 619, 1978.

2. J.M.Cano and Pavon, Microchem.J., 26, 155, 1981.

3. J.M.Cano, Pavon, D.P.Bendito and M.Valcarcel, Quim.Anul., 1, 118, 1982.

4. B.S.Garg and V.K.Jain, Microchem.J., 38, 144, 1988.

5. K.H.Reddy and D.V.Redy, Quart.Chem.Rev., 1, 47, 1985.

6. N.N.Orlova, A.K.Sevova, D.A.Selidovkin and G.N.Bogdonova, Res.Pharm.Toxic., 348, 1986.

7. K.Butler, U.S.Patent, No.3, 382, 266, 1968.

8. D.J.Bauer, L.St.Vincet, C.H.Kempe and A.W.Downe, Lancet., 2, 494, 1968.

9. N.P.Bin-Hol, T.B.Lol and N.D.Xyong, Bull.Soc.Chem. (France)., 694, 1955.

10. H.C.Patering, H.H.Buskrik and G.E. Underwood, Cancer.Res., 367, 1969.

11. E.Hoggerth, N.E.Martin Storey and E.H.P.Yasng, Brit.J.Pharma(O)., 4, 248, 1969.

12. C.I.Zapafonetis and J.P.Kales, Proc.Soc.Expt.Biol.Med., 105, 560, 1965.

13. F.D.Dwyer, F.Maythew and A.Shulman, Brit.J.Cancer., 19, 195, 1965.

14. D.R.Williams, Chem.Rev., 72, 203, 1972.

15. A.Furst and G.Petering, progr.Exp.Tumor.Res., 12, 102, 1969.

16. J.A.Gim and H.G.Patering, Cancer.Res., 27, 1278, 1967.

17. Z.Libermeister, Naturoforsh.B., 5,79, 1950.

18. J.Raj Ranjan and S.Sitaram, Asian.J.Chem., 10,717, 1998.

19. S.Jha and R.P.Varma, Asian.J.Chem., 15, (344) 1719, 2003.

20. Yildiz, B.Deulger, S.Y.Koyuncu and Y.B.S.M.Apici, J.Ind.Chem.Soc., 81(1), 7, 2004.

Received on 07.06.2011 Modified on 15.06.2011

Asian J. Research Chem. 4(9): Sept, 2011; Page 1398-1403