Spectrophotometric Determination of Some Phenolic Compounds Using N,N- diethyl –p-phenylenediamine (DE – PPD) and Benzoyl peroxide

A rapid and sensitive spectrophotometric method has been developed for the determination of some phenolic compounds (phenol, oaminophenol and α-naphthol) and was applied for determination of amoxicillin drug. The method is based on oxidative coupling reaction of these compounds with N,N-diethyl-p-phenylenediamine in the presence of benzoyl peroxide as oxidizing agent. The formed blue indophenol dyes have maximum absorptions at 627, 612.5, 611.5 and 628.5 nm. for phenol, o-aminophenol, α-naphthol and amoxicillin respectively. The Spectrophotometric Determination of Some Phenolic Compounds Using ... 9 molar absorptivities are 1.72× 10, 1.92× 10, 1.65 × 10. l. mol ١ . cm for concentrations obeyed Beers law in the ranges 0.2 -4.0, 0.5 -11.0, 0.6 – 16.0 and 0.8 – 25.0 μg ml for the above compounds respectively. The average recovery % was ranged between 98.99% 100.45 with relative standard deviation ≤ 1.04 for all the studied compounds. The method is applied successfully to the assay of amoxicillin. INTRODUCTION Phenolic compounds have great importance in the nutritional organoleptic and commercial properties of plant-derived food and beverages. Furthermore their consumption has been associated with positive health benefits such as antioxidant, antiviral, antiallergenic, cardioprotective, and anticarcinogenic effect. [1] Several spectrophotometric methods have been proposed for the determination of present phenolic compounds using several reagents such as hydroxylamine and cerium (IV) [2], hydroxylamine and sodium nitroprusside [3], p-phenylazoaniline [4],4-aminoantipyrine and potassium ferricyanide [5], nitrous acid and resorcinol [6], 3-methyl –2benzothiazoline hydrazone [7] 4-amino N, N-dimethyl aniline and dichromate [8] p-phenylene diamine [9] Anisidine [10] N,N-diethyl–p– phenylene diamine and N-bromosuccinimide [11], copper tetramine and Triiodite [12] Folin ciocalteu reagent [13]. The present paper describe a simple, sensitive and accurate spectrophotometric method for the determination of some phenolic compounds using N,N–diethyl–p-phenylene diamine (DE-PPD) in the presence of benzoyl proxide in alkaline medium. EXPERIMENTAL Apparatus All spectral absorbance measurements were carried out on single beam spectrophotometer CECIL (UV – VIS) using 1-cm silica cell. Reagents All chemicals used were of analytical reagent grade. N,N-diethyl–p-phenylenediamine hydrochloride (DE-PPD) solution (1x10 M). Prepared by dissolving 0.3995 g of DE PPD in 200 ml ethanol. Working solution was prepared by further dilution of stock solution Standard solutions of phenolic compounds (100 μg/ ml). Prepared by dissolving 0.01 g of each phenol, o-aminophenol, αnaphthol and amoxicillin in 100 ml ethanol. Benzoyl peroxide solution (1x10 M) Prepared by dissolving 0.4845 g of benzoyl peroxide in 200 ml ethanol. Sodium hydroxide solution 0.05 M Saladdin M. AL–Talib & Elham S. Salih & M. M. Alsharook 10 Prepared by dissolving 0.5gram in 250 ml. distilled water. Recommended procedure for calibration: Into a series of 25-ml calibrated flasks 3ml of 1x10 M DE-PPD solution was transferred. Add increasing volumes of phenolic working solutions (100 μg /ml), followed by 1.5 ml of 1x10 M benzoyl peroxide and 1.0 ml of 0.05 M sodium hydroxide. Dilute the solutions to the mark with ethanol and allow the reaction mixture to stand for 5 minutes. Measure the absorbance at 627, 612.5, 611.5, and 628.5nm for phenol, oaminophenol α-naphthol and amoxicillin respectively against the reagent blank. Procedure for the assay of amoxicillin in capsules: Ten capsules were weighed and the amount of the powder containing 500 mg of amoxicillin was dissolved in distilled water and made up to 1000 ml with distilled water. The solution was filtered and then 100 μg ml of amoxicillin solution was prepared, An aliquot of this solution was treated as described above under recommended procedure. Result and discussion A blue colour oxidative coupling products with an absorption maximum at 627, 612.5, 611.5 and 628.5 nm are formed when phenol, oaminophenol, α-naphthol and amoxicillin were allowed to react with DE – PPD in the presence of benzoyl peroxide. The absorption spectra of the resulting products are shown in Fig [2]. Fig2; Absorption spectra of the products from the reaction of DE-PPD and benzoyl peroxide with 3μg.ml of Aphenol, Bo-aminophenol, Cα-naphthol, Damoxicillin, EAbsorption spectraum of blank vs distilled water Spectrophotometric Determination of Some Phenolic Compounds Using ... 11 Optimum reaction condition The influence of various reaction variables were tested to establish the optimum conditions for the proposed procedure and the most suitable values of the variables tested based on absorbance obtained in each case. The phenol was used in all reaction. Effect of diluents’ agents on the absorbance and stability of the product: The effect of distilled water and ethanol absolute as diluants on the colour intensity of the reaction product were studied. The results obtained in (Table 1) indicated that ethanol absolute which gave an stable product at room temperature is a suitable diluents agent. It was found that the product was developed immediately and remained stable for at least 90 min; there after a slight decrease was observed. Table 1: Effect of diluents on the absorbance and stability of the Product. Diluent Ethanol absolute Distilled Water λmax Absorbance λmax Absorbance Time (min) 627 627 627 627 627 627 627 627 627 0.817 0.811 0.813 0.814 0.814 0.813 0.814 0.812 0.809 670 669 668 665 663 659 654 636 621 1.119 0.838 0.633 0.483 0.417 0.342 0.294 0.231 0.196 0 5 15 30 45 60 75 90 120 Effect of DE – PPD concentration The effect of changing the DE–PPD concentration on the absorbance was studied. Table 2 show that the absorbance increased with increasing DE–PPD concentration and reached maximum when using 3 ml of 1x 10 M DE – PPD. Therefore, this concentration was using in all subsequent experiments. Saladdin M. AL–Talib & Elham S. Salih & M. M. Alsharook 12 Table 2: Effect of DE – PPD concentration 3.5 3.0 2.5 2.0 1.0 ml.of DE-PPD 1X10M 0.813 0.811 0.707 0.579 0.323 Absorbance Effect of benzoyl peroxide concentration. The effect of changing the benzoyl proxide concentration on the absorbance was studied. Table 3 show that the absorbance reached maximum when using 1.5 ml of 1x10 M benzoyl peroxide.Therefore, this volume was using in all subsequent experiments. Table 3: Effect of benzoyl peroxide concentration. 2.5 2.0 1.5 1.0 0.5 0.1 ml of 1x10 -2 M benzoyl peroxide 0.748 0.746 0.813 0.730 0.609 0.286 Absorbance Effect of alkaline medium The effect of different alkaline was studied. The color product is formed in The use of 1 ml of 0.05 M sodium hydroxide solution gave a maximum intensity (Table 4). Table 4: Effect of alkaline

The present paper describe a simple, sensitive and accurate spectrophotometric method for the determination of some phenolic compounds using N,N-diethyl-p-phenylene diamine (DE-PPD) in the presence of benzoyl proxide in alkaline medium.

EXPERIMENTAL Apparatus
All spectral absorbance measurements were carried out on single beam spectrophotometer CECIL (UV -VIS) using 1-cm silica cell.

Reagents
All chemicals used were of analytical reagent grade.

N,N-diethyl-p-phenylenediamine hydrochloride (DE-PPD) solution (1x10 -2 M).
Prepared by dissolving 0.3995 g of DE -PPD in 200 ml ethanol.Working solution was prepared by further dilution of stock solution

Benzoyl peroxide solution (1x10 -2 M)
Prepared by dissolving 0.4845 g of benzoyl peroxide in 200 ml ethanol.Sodium hydroxide solution 0.05 M Prepared by dissolving 0.5gram in 250 ml.distilled water.

Recommended procedure for calibration:
Into a series of 25-ml calibrated flasks 3ml of 1x10 -2 M DE-PPD solution was transferred.Add increasing volumes of phenolic working solutions (100 µg /ml), followed by 1.5 ml of 1x10 -2 M benzoyl peroxide and 1.0 ml of 0.05 M sodium hydroxide.Dilute the solutions to the mark with ethanol and allow the reaction mixture to stand for 5 minutes.Measure the absorbance at 627, 612.5, 611.5, and 628.5nm for phenol, oaminophenol α-naphthol and amoxicillin respectively against the reagent blank.

Procedure for the assay of amoxicillin in capsules:
Ten capsules were weighed and the amount of the powder containing 500 mg of amoxicillin was dissolved in distilled water and made up to 1000 ml with distilled water.The solution was filtered and then 100 µg ml -1 of amoxicillin solution was prepared, An aliquot of this solution was treated as described above under recommended procedure.

Result and discussion
A blue colour oxidative coupling products with an absorption maximum at 627, 612.5, 611.5 and 628.5 nm are formed when phenol, oaminophenol, α-naphthol and amoxicillin were allowed to react with DE -PPD in the presence of benzoyl peroxide.
The absorption spectra of the resulting products are shown in Fig [2].

Optimum reaction condition
The influence of various reaction variables were tested to establish the optimum conditions for the proposed procedure and the most suitable values of the variables tested based on absorbance obtained in each case.The phenol was used in all reaction.

Effect of diluents' agents on the absorbance and stability of the product:
The effect of distilled water and ethanol absolute as diluants on the colour intensity of the reaction product were studied.The results obtained in (Table 1) indicated that ethanol absolute which gave an stable product at room temperature is a suitable diluents agent.
It was found that the product was developed immediately and remained stable for at least 90 min; there after a slight decrease was observed.

Effect of DE -PPD concentration
The effect of changing the DE-PPD concentration on the absorbance was studied.Table 2 show that the absorbance increased with increasing DE-PPD concentration and reached maximum when using 3 ml of 1x 10 -2 M DE -PPD.Therefore, this concentration was using in all subsequent experiments.

Effect of benzoyl peroxide concentration.
The effect of changing the benzoyl proxide concentration on the absorbance was studied.Table 3 show that the absorbance reached maximum when using 1.5 ml of 1x10 -2 M benzoyl peroxide.Therefore, this volume was using in all subsequent experiments.

Effect of alkaline medium
The effect of different alkaline was studied.The color product is formed in The use of 1 ml of 0.05 M sodium hydroxide solution gave a maximum intensity (Table 4).

Effect of temperature and reaction time.
The reaction time was studied by following the colour development at room temperature and different temperatures in thermostatically controlled water-bath and ice bath.The absorbance were measured and the reagent blank treated similarly.It was observed that formation of colored complex was achieved maximum immediately at room temperature and it remains stable for at least 90 min (Table 5).

Effect of order addition
To obtain optimum results the order of addition of reagents should be followed as given under the recommended procedure.Otherwise a loss in color intensity was observed (Table 6).

Analytical Application:
Typical calibration data for the four phenolic compounds investigated from linear regression analysis of absorbance reading vs concentration of each compound gave the slopes, intercepts and correlation coefficients present in Table 8.

Accuracy and Precision:
To estimate the accuracy and precision of the method, six replicated determinations were made at three different concentration of Amoxicillin each phenolic compound.The results indicated that the present method is accurate and precise (Table 9).The nature of reaction product DE-PPD oxidized in different oxidizing agent to p-benzoquinone diimine and then to p-benzoquinone monoimine which react with some phenolic compounds to produced indophenol dye in alkaline medium [9,14].
A similar mechanism has been sngested by Al -Esawati.[11] for the reaction of DE-PPD with some phenolic compounds in the presence of N-bromosucinimide.
The stoichiometry of the reaction between DE-PPD and phenol in the presence of benzoylperoxide was investigated by mole -ratio method [15].The obtained results showed the existent of 1:1 DE-PPD: phenol.Therefore the suggested mechanism for the formation of the dye can be written as follows: .

O
The apparent stability constant was estimated by comparing the absorbance of a solution containing stoichiometric amounts of phenolic compound and DE-PPD to one containing an excessive of DE-PPD reagent.The average conditional stability constants of the dye are shown in Table 10.

Interferences
Amoxicillin is usually formulated in a capsule form therefore; the effect of some exceipients which often accompanied pharmaceutical preparation were studied.It was found that the exceipients do not interfer on the assay amoxicillin (Table 11).
The proposed method was applied by analyzing commerial formulation of amoxicillin (capsule) and comparing the results obtained with AL-Esawati method [11].Satisfactory agreement between the results was obtained with an acceptable range of error (Table 12)

Conclusion
A simple, rapid and sensitive spectrophotometric method for the determination of some phenolic compounds have been devised, based on the oxidative coupling reaction with DE -PPD and benzoylperoxide.The proposed method needs neither temperature control nor solvent extraction and it can be applied successfully for determination of amoxicillin in capsules.

Table 1 :
Effect of diluents on the absorbance and stability of the Product.

Table 5 :
Effect of temperature on the absorbance and stability of the product.

Table 6 : Order of addition
However; the optimum reaction conditions for developing the color intensity of the products are summarized in Table7.

Table 7 : Optimum reaction conditions for the determination of phenolic compounds.
* Room temperature = 29 ± 3

Table 9 : Accuracy and precision of the method
*Average of six determinations