Synthesis of the New 2-[6-Nitro-2-Benzothiazolylazo]-4-Hydroxy Benzoic Acid Organic Reagent for Spectrophotometric Determination of Copper(II)

Copper plays a different role in the human body. It is an essential nutrient or a toxic element for human beings, depending on the concentration level [1,2]. Some chromogenic reagents have been used in spectrophotometric methods of determination of copper such as Acetophenone-p-chlorophenylthiosemicarbazone [3], Hydrazinecarboxymide2-[(2-hydroxyphenyl) methylene (HC22HPM) [4], Piperazine [5], Chloro(phenyl) glyoxime [6], sodium(I) diethyldithiocarbamate [7], brilliant cresyl blue(BCB)[8], cefixime [9], 4-(6-Bromobenzothiazolylazo)Orcinol [10], 3-(2′-thiazolylazo)-2,6-diaminopyridine [11], 2-[2-(4-methylbenzothiazolyl)azo]-5-dimethylaminobenzoic acid [12], 2[2-(5-bromo thiazolyl) azo]-4-methoxy phenol [13], and 2,6-dichlorophenolindophenol [14]. Thiazolylazo compounds have attracted the attention, as they are sensitive chromogenic reagents in addition to being important complexing agents. These dyes are useful in spectrophotometric determinations due to their good selectivity over a wide range of pH and they are relatively easy to synthesize and purified [15]. In this paper, a new (NO2BTAHB) chromogenic reagent was synthesized for spectrophotometric determination of Cu(II).


Reagents
All reagents were of analytical grade. Freshly distilled and deionized water was used for solutions preparations

Preparation of reagent [16]
To a mixture of {(4.3 gm of para nitro aniline and 3.8 gm of ammonium thiacyanate) in 70 ml glacial acetic acid}, was added drop by drop from burette (1.2 ml Br 2 + 15 ml glacial acetic acid) keeping at temperature >10ºC. After 15 minutes alkaline solution was added to precipitate the thiazole derivative, 1.145 gm of thiazole and in 50 ml glacial acetic acid then add (5 ml conc. HCl + 25 ml water) to the solution. After that drop by drop from burette a solution (0.690 gm NaNO 2 + 50 ml H 2 O) with stirring at 0-5°C to diazonium salt, then (0.1.390 gm of parahydroxy benzoic acid + 50 ml ethanol) is added to diazonium salt and 2-[6-nitro-2-benzothiazolyl azo]-4-hydroxy benzoic acid (NO 2 BTAHB)organic reagent was formed.

Standard solutions
Stock Cu(II) solution; A solution of Cu(II) (100 μg.ml -1 ) was prepared by dissolving (0.0392) gm of CuSO 4 .5H 2 O in (100 ml) distilled water. Other standard solutions of Cu(II) were prepared by dilution of stock solution with distilled water. -1 × 10 -3 M (NO 2 BTAHB) standard solution was prepared by dissolving (0.088) g in 250 ml of absolute ethanol. -Buffer solution [17] (pH=6.0) was prepared by mixing 12.63 ml of (0.2)M Na 2 HPO 4 (which was prepared by dissolving 2.83 gm in 100 ml distilled water) and 7.37 ml of (0.1) M Citric acid (which was prepared by dissolving 1.92 gm in 100 ml distilled water).

Apparatus
Spectrophotometric measurements were made with a Shimadzo (UV-Vis.) scientific equipment with 1.0 cm cell for plot spectra. The pD-303. Spectrophotometer, APEL Japan, was used in the other measurements. The pH-meter, 720 WTW, Germany and FT-IR Spectrophotometer shimadzo, Japan, were used in this work.

Procedure
To an aliquot containing ≤10 μg.ml -1 of Cu(II) in a 10 ml volumetric flask, was added 2 ml of buffer solution, and 3.5 ml of (2 × 10 -4 M) of (NO 2 BTAHB) solution. The solution was diluted to the mark with distilled water, and absorbance was measured at 25°C and wave length of 618 nm against the reagent solution as a blank solution prepared under the same conditions.

FT-IR spectrum of reagent (NO 2 BTAHB)
The following table shows the main vibration frequencies of main absorption bands characteristic of reagent (Table 1 and Figure 1).

Properties of the (NO 2 BTAHB)
(NO 2 BTAHB) reagent is slightly soluble in water, red powder, orange and stable solution for suitable period time, but in basic medium pH ≥ 8.0 the solution being pink. Such behavior may be interpreted by the following equilibria ( Figure 2).

Study of Cu(II)-(NO 2 BTAHB)complex
Absorption spectra: a-Ultra violet-visible absorption spectra of (NO 2 BTAHB) reagent, and Cu(II)-(NO 2 BTAHB)complex solution are shown in Figure 3. The reagent showed an absorption maximum at 439 nm, and the complex at 618 nm.

FT-IR spectrum of Cu-(NO 2 BTAHB) complex
Changing in intensities, shift in peaks positions, and fission in azo peak were seen which indicate to formation of complex as in following Figure 4.

Effect of pH
The effect of pH was studied over the rang (2-9) adjusted by means of dilute HCl and NaOH solution. Figure 5 shows the relationship between absorbance and pH, where the maximum absorbance obtained in the range of pH=(4.0-7.5). At 7.5<pH<4.0 a decrease in absorbance. Therefore, the optimum pH was 6.0, where the absorbance was maximum and constant.

Effect of Time
The stability of complex was studied from (0-120) min with 5 minutes intervals up to 24 hrs. The maximum absorbance was reached at 10 minutes Figure 6 after that the absorbance remains constant.

Effect of Temperature
The effect of temperature on absorbance of complex was studied; the study was performed at temperature between (5-80)ºC. Figure 7 show the maximum absorbance obtained at temperature range (15-40)°C which was regarded as a proper temperature of complex formation. At temperatures higher than 40ºC the absorbance decrease due to dissociation of complex gradually.

Determination of Stoichiometry and Formation Constant
The composing of complex was studied by jobs method of continuous variations and mole ratio method [18]. Figures 8 and 9 both methods indicate that the ratio of metal ion to reagent molecules (M:L) was (1:2) at pH=6.0.

Suggestion of Structural Formula of Cu(II)-(NO 2 BTAHB) Complex
From the obtained results of metal to reagent ratio, and depending on thiazolylazo Compounds properties; the following structure can be suggested;

Precision and Accuracy
The relative standard deviation (R.S.D%), evaluated from seven independent determination of 3.0 μg.ml -1 of Cu(II) was 1.7%, this result show that this method is highly precise. Also the accuracy of this method was determined by calculated the Erel percentage for 3.0 μg.ml -1 standard solution of Cu(II) which was found to be (-1.4) and Re%=98.6.

Interferences
The effect of the ions (Ni 2+ , CrO 2-, Ca 2+ , pb 2+ , Cu +2 , WO 4 -2 , MO 4 -2 , Co 2+ , Mg 2+ , Cd 2+ , Ba 2+ , Bi 3+ ) which form complex with the reagent during its reaction with Cu(II) were studied. On the other hand, suitable masking agents examined for eliminating the effect of the twelve ions, where the mixture of KI, NaF, Na 2 S 2 O 3 , and DMG were found to be a suitable masking agents.