Spectrophotometric Determinations of Sulfacetamide Following Simple Diazotization and Coupling with Chromotropic Acid Samar A. Darweesh Ibdul Mohsin A. Al-Haidari Alaa K. Mohammed Sarmad B. Dikran Dept. of chemistry/College of Education for Pure Science(Ibn Al-Haitham / University of Baghdad Received at: 27 May 2013, Accepted at 24 September 2013 Abstract A simple, sensitive, accurate and economic spectrophotometric method has been developed for the determination of sulfacetamide (SFA) in pure form, synthetic sample and urine. The method is based on diazotization of primary amine group of sulfacetamide with sodium nitrite and hydrochloric acid followed by coupling with chromotropic acid in alkaline medium to obtain a stable orange colored chromogen which exhibit a maximum absorption (λmax) at 511.5 nm. Different variables affecting the completion of reaction have been carefully optimized following the classical univariate sequence and modified simplex method (MSM). Under optimized conditions, Beer’s law obeyed in the concentration range of (0.5- 20.0 µg.ml-1) with molar absorptivity of 3.2186*104 L.mol-1.cm-1. The limit of detection was 0.054 µg.mL-1 and Sandell’s sensitivity value 7.8989 µg.cm-2. The proposed method was successfully applied to the determination of (SFA) in synthetic sample and urine. Key words: Spectrophotometric determination, Sulfacetamide, Diazotization reaction, Coupling reaction, Chromotropic acid. 281 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Introduction Sulfacetamide (SFA) Figure (l) is a member of the sulfonamide family of antibiotics and chemically it is N-(4-Aminobenzenesulfonyl) acetamide (M.wt.=214.24). Sulfonamides are widely used in the treatment of urinary tract infection, acne vulgaris, bacterial infection of eye, superficial infection including conjnnctivitis, bacterial infection of skin, seborrhea including seborrhoeic dermatitis and seborrhoeic sicca, and trachoma; adjnnct. They are also the drugs of choice for the treatment of nocardiosis toxoplasmosis, severe diarrhea and meningococcal infections[1]. Sulfacetamide has been determined by the development of several analytical techniques such as flow injection analysis [2,3], sequential injection analysis [4], HPLC[5-7], micellar electro kinetic capillary chromatography (MEKC)[8,9], spectrophotometric method [10-14]. The aim of the present work is to provide an optimized spectrophotometric method using the univariate and multivariate simplex method. In the simplex method three-interest factors concentration of sulfamic acid, chromotropic acid, sodium hydroxide were designated as independent variables and absorbance as response. Experimental Instruments The absorption spectra were recorded on a double-beam (shimadzu 1800), and all spectrophotometric measurements were carried out on (CECIL 1011)UV-Visible single beam spectrophotometer with 1cm matched quartz cells. Materials and reagents Pharmaceutical grade sulfacetamide received as gift sample powder in pure form (99.99%) the State Company for Drug Industries and Medical Appliances Samara-Iraq (SDI). All chemicals and reagents used were of analytical grade. Preparation of solutions Sodium nitrite [0.5 % (wt/v)]: was prepared by dissolving 0.5 g of NaNO2 in distilled water and diluting to l00 mL in a volumetric flask. Sulfamic acid [2 % (wt/v)]: was prepared by dissolving 2 g of sulfamic acid in 100 mL of distilled water. Chromotropic acid (CTA) [2 % (wt/v)]: was prepared by dissolving 2 g of CTA in l00 mL of distilled water. Sodium hydroxide [2 M]: was prepared by dissolving 8 g of NaOH in 100 mL of distilled water. Hydrochloric acid [5 M]: 85mL of concentrated HCl was taken and diluted to 200 mL with distilled water. Hydrochloric acid [2 M]: was prepared by taken 16.72 mL of concentrated HCl and diluted to 100 mL with distilled water. Standard drug preparation (100 μg.mL-1) The standard solution of SFA was prepared by dissolving accurate weighted 0.01 g of pure drug in 100 mL with distilled water. Preparation of synthetic drug sample 1- To 20 mg of the bulk drug, 5 mg of interfering substances mixture (consisting of equal weights of each substance, namely, glucose, lactose, soluble starch, and vanillin) was added. 2- 12.5 mg of the resulted mixture was dissolved in 100 mL of distilled water in the same manner as used for the preparation standard drug to obtain 100 μg.mL-1. Preparation of drug solution in urine 282 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Solution of drug in urine was prepared by dissolving 0.01 g of (SFA) in a little amount of distilled water, then the volume was complete to 100 mL urine in volumetric flask to obtain 100 μg.mL-1 of spiked synthetic sample solution. General standard procedures Two procedures were recommended for the determination of SFA via the proposed methods. The first was carried out following the conditions obtained by univariate optimization, while the second base of those conditions was obtained by chemometric multivariate simplex optimization. Univariate To aliquots the standard solution (l00 μg.mL-1) containing (5-150 μg) of SFA were transferred into a series of 10 mL volumetric flasks and cooled in an ice bath, 1.0 mL of 0.05 % (w/v) sodium nitrite solution and 1.0 mL of 0.5 M HCl were added. Each solution was shaken thoroughly, and then 1.5 mL of 0.5 % (w/v) sulfamic acid was added.The solutions were swirled and the resulting diazotized product was coupled with CTA by the addition of 1.0 mL of 1 % (w/v) of this reagent followed by 2.0 mL of 0.05 M sodium hydroxide solution. The mixtures allowed to stand for 10 min., after then were made up to the mark with distilled water. The absorbance of orange colored chomogen was measured at 511.5 nm against the reagent blank. The constructed calibration curve was used to compute the amount of SFA in given samples. Multivariate (simplex method) Aliquots of the standard solution (100 μg.mL-1) containing (5-200 μg) of SFA were transferred into a series of 10 mL volumetric flasks. After cooling in an ice bath, to each flask, l.0 mL of 0.05% (w/v) sodium nitrite solution and l.0 mL of 0.5 M HCl were added. Each solution was shaken thoroughly and l.5 mL of 0.25 % (w/v) sulfamic acid was added. The solutions were swirled and the resulting diazotized product was coupled with CTA by the addition of l.0 mL of 0.5 % (w/v) the reagent solution followed by 2.0 mL of 0.075 M sodium hydroxide solution and allowed to stand for 10 min. The solutions were making up to the mark with distilled water. After mixing the solution well, the absorbance of orange colored chomogen was measured at 511.5 nm against the reagent blank. The amount of SFA was computed from calibration curve. Result and discussion Absorption spectra and reaction scheme Primary aromatic amine upon treatment with nitrous acid in an ice-cooled solution forms diazonium salt. Ar-NH2 + NaNO2 + HCl → Ar-N2+Cl-+ H2O Under proper conditions, diazonium salts react with certain aromaticcompounds to yield products of general formula Ar-N=N-Ar', called as azo compounds and the reaction is called as diazo coupling reaction [15]. Ar-N2+ + Ar'H → Ar-N=N-Ar' + H+ The proposed method involves coupling reaction of the diazotized sulfacetamide with chromotropic acid in an alkaline medium to yield an azo dye according to Scheme 1. The former orange colored product shows a maximum absorption at 511.5 nm (Figure 2). Optimization of reaction variables Various parameters (viz sodium nitrate concentration, hydrochloric acid concentration, time of diazotization reaction, sulfamic acid concentration, chromotropic acid concentration, sodium hydroxide concentration, and time of coupling reaction) were first optimized, for the development of color dye, univariatly by systematic study of the effects of each parameters in 283 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 the development of color product. The optimization steps were carried out by varying the parameters each one at a time and controlling all others fixed. Effect of diazotization reaction time It was found that the diazotization reaction was taking place instantaneous when the time required for diazotization reaction was studied in the range (0-20) minute at 0 ºC. The diazotized product was found to be decomposed with time therefore, diazotization was carried out instantaneously. Effect of sodium nitrite Nitrite concentration for diazotization was optimized in the range of (0.025-0.500 %) (Figure 3). Maximum absorbance was observed when 1.0 mL of 0.050 % sodium nitrite was added and the absorption of dye product was measured at 511.5 nm. Below and above this concentration the absorbance was decreased. Effect of different acidic solutions Effect of many acids on diazotization reaction was studied such as H2SO4, HCl, HNO3, CH3COOH with 1 M of concentration. HCl was responded to the stable diazotized product thus it was used for the following experiments (Table1). Effect of acidity It was found that the absorbance of the formed azo dye product was enhanced as the concentration hydrochloric acid was decreased in the studied range of the acid (0.025–2.000 M), Figure 4. Maximum absorption intensity was achieved when 1.0 mL of 0.500 M HCl was used, and this amount was used for the subsequent work. Effect of sulfamic acid concentration According to the results obtained upon studying the effect of the addition of 1.5 mL of (0.25– 2.00 %) of sulfamic acid on the reaction product, the optimum amount was found to be 1.5 mL of 0.50 % solution (Figure5). Effect of chromotropic acid concentration The effect of coupling reagent concentration on the intensity of the color dye development was tested by using different concentrations of CTA in the range (0.1–2.0 %), Figure 6. The result shows that 1.0 mL of 0.7 % of CTA solution was optimum for this method. Effect of different bases In order to develop intensity and color of dye product, different bases νiz sodium hydroxide, sodium carbonate, potassium hydroxide and ammonium hydroxide were studied. The results indicate that the sodium hydroxide was suitable alkaline medium to give a maximum absorption as shown in (Table 2). Effect of alkalinity The stability and formed azo dye product depends upon the nature of reaction medium. The formed azo dye was found to have a reasonable stability when the reaction medium render alkaline via addition of 2.0 mL of 0.1 M sodium hydroxide. Figure 7 illustrates the results of the study. Effect of coupling reaction time The maximum time required for coupling reaction to be completed was found to be 10 min. at room temperature. After that period, the absorbance remained constant for at least 3.5 hours as presented in (Table 3). On the other hand, simplex program was employed to find the optimum experimental conditions of three important parameters that considerably affect the colored product formation (νiz the concentration of sulfamic acid, the concentration of CTA, and the concentration of sodium hydroxide) for the determination of (SFA). The boundary conditions for the studied parameters delineated above, were first set as depicted in (Table 4) together with the step size values. 284 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Four (numbers of studied parameters +1) arbitrary experimental conditions were chosen, by selecting the values of these parameters within specified boundaries for each, at which they affected the measured absorptions signal of the colored products (experiment 1-4 in Table 5), the absorbencies of these four experiments together with the applied experimental conditions were fed into a multi simplex computer program. The program run the simplex system to produce a new set of experimental conditions. An experiment is carried out under the new generated conditions and the measured absorption signal was fed again to the program and so on. Only 22 experiments were enough to evaluate the proper conditions that yield the maximum response. Results given in Table 5, and Figure 8 show the progress of the simplex, at step 20 ( i.e. 0.25 % sulfamic acid, 0.5 % CTA and 0.075 M sodium hydroxide) the highest response function value was obtained, which was taken to be the optimum conditions for the determination of (SFA). Calibration curves and analytical data І- For univariate According to the optimum experimental conditions, obtained νia univariable method, linear calibration graph for sulfacetamide was obtained, Figure 9, which shows that Beer’s law is obeyed in the concentration range of (0.5-15.0 µg.mL-1). The regression equation, correlation coefficient, molar absorptivity, Sandell's sensitivity and detection limit are given in Table 6. ІІ- For simplex Table 7 and Figure10 show the results for the calibration curve and statistical data. Better optical characteristics for calibration curve and statistical data were obtained under optimum conditions obtained by multi simplex optimization, in comparison with those obtained via univariate method. Precision and accuracy The accuracy (in term of relative error percent) and the precision (in term of coefficient of variation) of the proposed method under univariate and multivariate conditions were evaluated by doing five replicate analyses of SFA at three different levels of concentration within Beer’s law range (Table 8). The results indicate good accuracy and precision of the proposed method at the studied concentration levels. Interference studies The effect of various foreign species, which may be present in pharmaceutical products and affecting the reaction between the sulfacetamide and CTA, were studied. Optimum experimental conditions, for simplex optimization, were employed to determine 10 μg.ml-1 concentration of SFA. Table 9 shows that the presence of 1000 µg.mL-1 of the studied interfering excipient cause errors less than ± 3 %. Application on synthetic sample The developed method was applied for the determination of the amount of SFA in its synthetic sample. The results of the application of the proposed method given in Table 10 were satisfactory. The recovery was ranged from (98.9-101.2 %) and the coefficient of variation range was (0.4326-2.0458 %). Application on spiked urine sample The proposed method was also applied for the determination of SFA in spiked human urine samples.The results, listed in Table 11; indicate that the proposed method could successfully be applied. The recovery values were ranged (98.4-104.0 %) with the coefficient of variation of (0.4157-1.5678 %). Application on spiked urine by standard addition method 285 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 To increase the insurance, the proposed spectrophotometric method was applied for the determination of SFA in spiked urine sample following the standard addition technique. Good recoveries of the drug present in studied sample indicate that non-interference from urine matrix. Figure 11 shows the standard addition plot and Table 12 shows the result of recovery and coefficient of variation for the method. Conclusions Diazotization reaction of primary amine group followed by coupling with chromotropic acid in alkaline medium was found to be a simple, sensitive, accurate and economic spectrophotometric method for quantitative determination of (SFA) in pure form and synthetic samples. The classical univariate and modified simplex method have been used for optimizing the different variable affecting the completion of the reaction. The proposed method offers good linearity and precision. References 1- www.micromedex.com, (2012) Food and Drugs Administration (FDA)-labelled indication 2- CataláIcardoa, M.; Garc´ıa Mateo, J. V.; Fernández Lozano, M. and Mart´ınez Calatayud, J. (2003) "Enhanced flow-injection chemiluminometric determination of sulphonamides by on-line photochemical reaction" Anal. Chimi. Acta 499 : 57–69. 3- Azeez, Y. J. (2009) "Flow- Injection Spectrophotometric Determination of some Sulpha Drugs via Oxidative Coupling with 4-Amino-N, N-diethylaniline. Application to Various Samples" Journal of ZankoySulaimani 12(1): 67-76. 4- Paseková, H. ; Polášek, M.; Cigarro, J. F. and Dolejšová, J. (2001) "Determination of some sulphonamides by sequential injection analysis with chemiluminescence detection" AnalyticaChimicaActa 438: 165–173. 5- Go´ recki, H. Sh. T. (2012) "Optimization and validation of a fast ultrahigh-pressure liquid chromatographic method for simultaneous determination of selected sulphonamides in water samples using a fully porous sub-2 lm column at elevated temperature" J. Sep. Sci. 35: 216– 224. 6- Bonta, V.; Marghitas, L. A.; Dezmirean, D. and Bobis, O. (2009) "Determination of Six Sulfonamide Residues in Honey by HPLC with Fluorescence Detection" Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Animal Science and Biotechnologies 66 (1-2). 7- Tolika, E. P.; Samanidou, V. F. and Papadoyannis, I. N. (July 2011) "Development and validation of an HPLC method for the determination of ten sulfonamide residues in milk according to 2002/657/EC" j. Sep. Sci. 34: 1627–1635. 8- RadeInjac; Kočevar, N. and Štrukelj, B. (2009) "Optimized Method for Determination of Amoxicillin, Ampicillin, Sulfamethoxazole, and Sulfacetamide in Animal Feed by MicellarElectrokinetic Capillary Chromatography and Comparison with High-Performance Liquid Chromatography" Croati. Chemi. Acta 82 (3): 685–694. 9- Gallego, L. and Arroyo, P. (2003) "Determination of prednisolone acetate, sulfacetamide and phenylefrine in local pharmaceutical preparations by micellar electro kinetic chromatography" J. Pharm. Biomedi. Anal. 31(5): 873-884. 10- Nagaraja, P.; Naik, Sh. D.; Shrestha, A. K. and Shivakumar, A. (2007) "A sensitive spectrophotometric method for the determination of sulfonamides in pharmaceutical preparations" Acta Pharm. 57: 333–342. 11- Chaluvaraju, K. C.; Ishwar Bhat, K. and Zaranappa (2010) "Quantitative spectrophotometric determination of sulphacetamide sodium in bulk and in pharmaceutical dosage form" J. Pharm. Res. 3(1): 47-48. 286 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 12- Al-NuriIsraa, I. J. and Al-Obaydi, A. (2009) "Direct Determination of Sulfacetamide Sodium by Derivative UV Spectrophotometry" J. Raf. Sci. 20(4): 17-26. 13- Nagaraja, P.; K. R. Sunitha; R. A. Vasantha and H. S. Yathirajan (2002) " Iminodibenzyl as a novel coupling agent for the spectrophotometric determination of sulfonamide derivatives" Euro. J. Pharm. Biopharm. 53: 187–192. 14- Nagaraja, P.; Sunitha, K. R.; Vasantha, R. A. and Yathirajan H. S. (2002) "Rapid Spectophotometric Determination Of Sulphonamide Derivatives With Resorcinol" Ind. J Pharm. Sci. 64: 391-393. 15- Connors, K. A. (1973) "Reaction Mechanisms in Organic Analytical Chemistry" John Wiley & Sons: New York,. Table (1): Effect of different acids on diazotization of 10 µg.mL-1 of SFA. Acidic solution (1M) Absorbance H2SO4 0.200 HCl 0.253 HNO3 0.178 CH3COOH 0.081 Table (2): Effect of different bases. Base (4M) Absorbance sodium hydroxide 0.854 sodium carbonate 0.687 potassium hydroxide 0.609 ammonium hydroxide 0.268 Table (3): Effect of coupling reaction time. Time (min) Absorption 0 0.982 5 0.985 10 1.047 15 1.029 20 1.012 210 1.012 Table (4): Boundary of simplex for the studied variables. Variable Minimum boundary maximum boundary Step size Conc. of sulfamic acid (%) 0.25 2.0 0.25 Conc. of CTA (%) 0.1 2.0 0.1 Conc. of NaOH (M) 0.025 2 0.025 287 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Table (5): Multivariate experiments (Simplex) for the determination of (10 μg.mL-1) SFA. Exp. No. Conc. of sulfamic acid (%) Conc. of chromotropic acid (%) Conc. of sodium hydroxide (M) Abs. 1 0.25 0.2 0.125 1.019 2 0.5 0.7 0.05 1.024 3 1.5 1.2 0.025 0.681 4 2.0 0.1 2.0 0.096 5 0.25 1.3 0.025 1.234 6 0.25 0.6 0.075 1.244 7 0.25 1.4 0.025 1.212 8 0.25 1.2 0.05 1.223 9 0.25 0.4 0.1 1.263 10 0.25 0.1 0.125 0.830 11 0.25 0.1 0.1 0.893 12 0.25 0.9 0.075 1.242 13 0.25 1.0 0.05 1.238 14 0.25 0.1 0.15 0.813 15 0.25 0.9 0.05 1.233 16 0.25 0.4 0.075 1.250 17 0.25 0.1 0.1 0.893 18 0.25 0.7 0.075 1.243 19 0.25 0.3 0.1 1.186 20 0.25 0.5 0.075 1.273 21 0.25 0.3 0.075 1.195 22 0.25 0.6 0.05 1.237 Table (6): Optical characteristics and statistical data for the determination of SFA by univariable method. Parameter Value λmax (nm) 511.5 Color Orange Linearity range (µg.mL-1) 0.5 – 15.0 Regression equation A= 0.1255 [SFA. µg.mL-1] - 0.0198 Calibration sensitivity 0.1255 Correlation coefficient (R) 0.9997 Correlation of linearity (R2) 0.9994 Molar absorptivity (L.mol-1.cm -1) 3.1907*104 Sandell's sensitivity (µg.cm-2) 7.9681 Detection limit (µg.mL-1) 0.071 288 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Table (7): Optical characteristics and statistical data for the determination of SFA by simplex method. Parameter Value λmax (nm) 511.5 Color Orange Linearity range (µg.mL-1) 0.5-20.0 Regression equation A= 0.1266 [SMX. µg.mL-1] - 0.0282 Calibration sensitivity 0.1266 Correlation coefficient (R) 0.9998 Correlation of linearity (R2) 0.9996 Molar absorptivity (L.mol-1.cm -1) 3.2186*104 Sandell's sensitivity (µg.cm-2) 7.8989 Detection limit (µg.mL-1) 0.054 Table (8): Accuracy and precision of the proposed method. Conc. of SFA (µg.mL-1) Relative Error % C.V Taken Found* For univariate conditions 2.00 2.05 2.500 1.395 7.00 6.97 -0.428 0.536 15.00 15.06 0.400 0.208 For simplex conditions 2.00 1.95 -2.500 0.834 7.00 7.04 0.571 0.391 15.00 14.99 -0.066 0.228 *mean of five replicate. Table (9): Percent recovery for 10 µg.mL-1 of sulfacetamide in the presence of 1000 µg.mL-1 of excipients. Sulfacetamide Conc. Taken 10 µg.mL-1 Concentration of Excipient µg.mL-1 Excipients % Recovery Conc. Found µg.mL-1 97.00 9.70 1000 Vanillin 101.44 10.14 Glucose 101.12 10.11 Sucrose 102.20 10.22 Starch Table (10): Application of the proposed method to the SFA concentration measurements in synthetic sample. Weight of SFA in 25 mg of sample *Weight (mg) of SFA found in 25 mg of sample Conc. taken µg.mL-1 *Conc. found µg/mL Recovery % C.V* 20 20.24 2 2.024 101.2 2.0458 20.20 5 5.050 101.0 0.4992 19.78 10 9.890 98.9 0.4326 *Average of three determinations. 289 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Table (11): Application of the proposed method to the SFA concentration measurements in spiked urine. Sample Conc. taken µg.mL-1 Conc.* found µg.mL-1 Recovery % C.V* SFA in urine 2.00 2.08 104.0 1.5678 5.00 4.92 98.4 0.5891 10.00 9.87 98.7 0.4157 *Average of three determinations. Table (12):Application of the proposed method to the SFA concentration measurements in spiked urine by standard addition method. Sample Conc. taken µg.mL-1 Conc.* found µg.mL-1 Recovery % C.V* SFA in urine 200.00 202.52 101.26 0.8266 *Average of three determinations. Scheme (1): The proposed reaction mechanism for diazotization and reaction between SFA and CTA. H2N S H N O O O ++ N S H N O O O N Cl OH OH S S OH O O HO O O OH OH S S -O O- O OO O Na+ Na+ N S H N O O O N NaOH + HClNaNO2 (Diazonium ion) (Azo dye) (SFA) (CTA) 290 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Figure (1): The chemical structure of sulfacetamide. Figure (2): Absorption spectrum of (A) chromogen against (B) reagent blank solution. Figure (3): Effect of sodium nitrite on the color development of dye in the determination of chromogen from 10 µg.mL-1 of SFA. H2N S H N O O O 0.23 0.24 0.25 0.26 0 0.1 0.2 0.3 0.4 0.5 0.6 A bs ob na nc e Concentration of NaNO2 (M) Figure (4): Effect of HCl concentration on the color development of dye in the determination of chromogen from 10 µg.mL-1 of SFA. Figure (5): Effect of sulfamic acid concentration on the determination of chromogen from 10 µg.mL-1 of SFA. Figure (6): Effect of CTA concentration on the determination of chromogen from 10 µg.mL-1 of SFA. 0.22 0.24 0.26 0.28 0.30 0.0 0.5 1.0 1.5 2.0 2.5 A bs or ba nc e Concentration of HCl (M) 0.26 0.27 0.28 0.29 0.30 0.31 0.32 0.0 0.5 1.0 1.5 2.0 2.5 A bs or ba nc e Concentration of sulfamic acid (wt/v) % 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.4 0.8 1.2 1.6 2.0 A bs or ba nc e Concentration of sulfamic acid (wt/v) % 292 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Figure (7): Effect of NaOH concentration on the determination of chromogen from 10 µg.mL-1 of SFA. Figure (8): Response function progress for simplex. Figure (9): Calibration curve for the determination of SFA under univariate optimal conditions. 0.8 0.9 1 1.1 1.2 1.3 0 0.4 0.8 1.2 1.6 2 A bs or ba nc e Concentration of NaOH (M) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 5 10 15 20 25 A bs or ba nc e Experiment number y = 0.1255x - 0.0198 R² = 0.999 0.0 0.4 0.8 1.2 1.6 2.0 0 2 4 6 8 10 12 14 16 A bs or ba nc e Concentation of sulfacetamide (µg.mL-1) 293 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Figure (10): Calibration curve for the determination of SFA under simplex optimal conditions. Figure (11): Determination of SFA in spiked urine sample by standard addition method. y = 0.1266x - 0.0282 R² = 0.999 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 0 5 10 15 20 25 A bs or ba nc e Concentation of sulfacetamide (µg.mL-1) y = 0.9175x + 1.1613 R² = 0.998 0.0 0.5 1.0 1.5 2.0 2.5 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 A bs or ba nc e Volume of standard solution (mL) 294 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 التقدیرات الطیفیة للسلفاسیتماید المتضمنة األزوتة البسیطة واألزدواج مع حامض الكروموتروبیك سماعیلإ سمر أحمد درویش محسن الحیدري عبدالمحسن عبد الحمید عالء كریم محمد سلطان سرمد بھجت دیكران اوانیس بن الھیثم)/ جامعة بغداد.إكلیة التربیة للعلوم الصرفة (قسم الكیمیاء/ .2013أیلول 24، قبل في: 2013یار آ 27أستلم في: الخالصة وفي نماذج بشكلھ النقي (SFA)طریقة طیفیة بسیطة وحساسة ودقیقة وغیر مكلفة للتقدیر الكمي للسلفاسیتاماید طورت أزوتة مجموعة األمین األولي في السلفاسیتاماید بمعاملتھ مع نتریت الصودیوم . تعتمد الطریقة على مصنعة وفي األدرار وحامض الھیدروكلوریك ثم اجراء تفاعل االزدواج مع حامض الكروموتروبیك في وسط قلوي للحصول على كروموجین إتمام فيالتي تؤثرنانومتر. وقد تمت دراسة العوامل 511,5 عند (λmax)لون برتقالي یظھر أعظم امتصاص يمستقر ذ التفاعل بعنایة للحصول على الظروف الفضلى وذلك باتباع نمط المتغیر الواحد وباالعتماد على طریقة السمبلكس المحورة. مع قیمة (µg.mL-1 20.0 –0.5)عند الظروف الفضلى، وجد أن قانون بیر ینطبق على مدى من التراكیز یتراوح بین داللةوµg.mL-1 0.054 وكان حد الكشف یساوي L.mol-1.cm-1 104*3.2186 ة لـ مساوی ریةالموال یةاالمتصاص . لقد أمكن تطبیق الطریقة المقترحة بنجاح لتقدیر السلفاسیتاماید في نماذج مصنعة وكذلك µg.cm2 7.8989ساندل یساوي فى االدرار. .الكلمات المفتاحیة: التقدیر الطیفي، سلفاسیتماید، تفاعل األزوتة، تفاعل األزدواج، حامض الكروموتروبیك 295 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013