Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 5(7), 20-28, July (2015) Res. J. Chem. Sci. International Science Congress Association 20 Synthesis, Characterization and Antimicrobial screening of some Azo compounds derived from Ethyl vanillinPagariya S.K., Pathade R.M. and Bodkhe P.S. Department of Chemistry, Vidyabharati Mahavidyalaya, Amravati, 444602, INDIAAvailable online at: www.isca.in, www.isca.me Received 19th June 2015, revised 30th June 2015, accepted 17th July 2015 AbstractAzo compounds were synthesized in excellent yield by diazotization of some substituted aromatic amines using NaNO and concentrated HCl followed by coupling with ethyl vanillin in alkaline medium. These azo compounds were characterized by FTIR and H1 NMR spectroscopic technique and have been tested against the growth of five gram positive and negative microorganisms in order to assess their antimicrobial activity. Keywords: Azo compounds, ethylvanillin, diazotization, antimicrobial activity. Introduction Azo compounds are mostly used as dyes due to its various applications in the fields such as textile fibres, colouring of different materials, biomedical studies and organic synthesis1-2. The azo dyes containing azo linkages have advanced applications in high technology areas like lasers, LCD color filters. In addition to this, azo dyes were reported to have variety of biological applications like antineoplastics, antidiabetics, antiseptics, anti-inflammatory and other useful chemotherapeutic agents5-8. Scarlet red and diamazon are the most commonly used azo dyes which are antiseptics. Several azo compounds derived from thymol, aspirin10, paracetamol11, m-cresol12, resorcinol13 and vanillin14 moieties have been frequently reported and exhibit excellent biological properties. In the present work, we have synthesized four azo compounds derived from ethyl vanillin and characterized by FTIR and H1 NMR spectral technique. The antimicrobial potential of synthesized azo compounds of ethyl vanillin has been tested against the growth of five gram positive and negative microorganisms using agar well diffusion method. Material and Methods In the present synthesis, chemicals and reagents used were of analytical grade, Merck and Alfa Aesar Company Ltd. The azo compounds were characterized by FTIR and H NMR spectroscopic techniques. The Perkin-Elmer spectrum One FTIR instrument was used for characterization of IR spectra in the form of KBr pallet. Bruker Avance II 400 MHz NMR spectrometer was used for characterization of H NMR spectra of azo compounds using CDCl3 as a solvent and TMS as an internal standard. The purity of all the compounds was checked by thin layer chromatography and were recrystalised from hot ethanol. The melting points were measured by open capillary method and they are uncorrected. Procedure for the synthesis of azo compounds9-10: Aromatic amines (0.01 mole) were mixed with 2.5 ml of concentrated HCl. To this resultant solution, 25 gm of crushed ice and 2.5 ml of NaNO (4N) was added with constant stirring. The temperature of the reaction mixture was kept constant up to 0-C. The diazonium salt solution prepared was added to the alkaline solution of ethyl vanillin drop by drop with constant stirring for 10-25 minutes at constant temperature of 5-10C. The colored precipitate given out was filtered and washed with water number of times. The colored product was recrystallised from hot ethanol. The general reaction scheme for synthesis of azo compounds of ethyl vanillin is shown in figure-1. Antimicrobial Activity: The azo compounds 1a-d were analysed for their antibacterial activity against five gram positive and negative pathogens viz. Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Bacillus subtilis and Staphylococcus aureus by using agar well diffusion method15-16. These compounds were mixed in DMSO to form solutions of concentration 1mg/ml. Sterile discs were dipped in this solutions, dried it and placed on nutrient agar plates spreaded with the bacteria. The plates were further incubated for 24 to 48 hours at 37C and the diameters of zones of inhibition were measured in millimeter. Results and Discussion The data of synthesized azo compounds of ethyl vanillin (i.e. symbols, compounds name, molecular formulae, molecular weights, melting points and percentage yield) are given in table-1. The FTIR and H NMR spectroscopic data of synthesized azo compounds are illustrated in table-2 and are shown in figure-6 to 13. A total four azo compounds of ethyl vanillin have been synthesized, recrystalised and used separately to study its antimicrobial activity against five gram positive and negative microorganism’s viz. Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Bacillus subtilis and Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 20-28, July (2015) Res. J. Chem. Sci. International Science Congress Association 21 Staphylococcus aureus. The data on antimicrobial activity of azo compounds of ethyl vanillin 1a-d against five pathogens are presented in table-3. From the results it was observed that the azo compounds of ethyl vanillin have showed miraculous antibacterial potential against all five pathogens. The compound 1a showed 10.4 and 9.6 mm zones of inhibition against the test pathogens Escherichia coli and Staphylococcus aureusrespectively as shown in figure-2 and do not showed any inhibitory action against Salmonella typhi Pseudomonas aeroginosa and Bacillus subtilis. The azo compound 1b showed 10.6, 12.7, 11.8 and 9.8 mm zones of inhibition against the test pathogens Escherichia coli, Salmonella typhi, Bacillus subtilis and Staphylococcus aureus respectively shown in figure-3 but do not showed any inhibitory action against Pseudomonas aeroginosa. The azo compound 1c showed 10.3, 13.1, 13.5 and 12.8 mm zones of inhibition against the pathogens Escherichia coli, Salmonella typhi, Bacillus subtilis and Staphylococcus aureus respectively as shown in figure-4 but do not showed any inhibitory action against Pseudomonas aeroginosa. The azo compound 1d showed 13.6, 12.8, 15.6 and 14.4 mm zones of inhibition against the test pathogens Escherichia coli, Salmonella typhi, Pseudomonas aeroginosa and Staphylococcus aureus asshown in figure-5. Compound 1d showed 11.3 mm zone of inhibition against the pathogen Bacillus subtilis. R NH NaNO+HCl0-5 N=N Cl 1 C2H5O 2R C2H5O 5-10N=N Ar 1a-dOH CHO OH CHO Ar O2N CH abcd Figure-1 The general reaction scheme for synthesis of azo compounds of ethyl vanillinTable-1 The symbols, compounds name, molecular formulae, molecular weights, melting points and percentage yield of synthesized azo compounds of ethyl vanillin Symbols for antimicrobial activity Symbols for FTIR and NMR Compounds Name Molecular Formulae Molecular Weights Melting points C) Yield (%) 1a 4P 3-ethoxy-4-hydroxy-5-(phenyldiazenyl)benzaldehyde 1514 270.28 67 67 1b 4Q 3-ethoxy-4-hydroxy-5-((2-nitrophenyl)diazenyl)benzaldehyde 1513 315.28 128-130 65 1c 4R 3-ethoxy-4-hydroxy-5-(p-tolyldiazenyl)benzaldehyde 1616 284.31 64 72 1d 4S 3-ethoxy-4-hydroxy-5-(napthalen-1-yldiazenyl)benzaldehyde 1916 320.34 127-129 80 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 20-28, July (2015) Res. J. Chem. Sci. International Science Congress Association 22 Table-2 FTIR and H NMR data of synthesized azo compounds of ethyl vanillin Symbols for compounds Types of spectra FTIR and H NMR spectral data 4P FTIR (KBr,cm-1) 3362 (OH of Phenol), 2829 (C-H of H-C=O), 1686 (C=O), 1516 (C=C), 1255 (C-O), 1280 (C-N), 1579 (N=N). NMR ( ppm) 1.4 (t, 3H of -CH), 4.2 (q, 2H of -OCH), 6.2 (s, 1H of Phenolic -OH), 9.8 (s, 1H of -CHO), 7.0 to 7.5 (m, 7H of Aromatic-H). 4Q FTIR (KBr,cm-1) 3398 (OH of Phenol), 2882 (C-H of H-C=O), 1673 (C=O), 1519 (C=C), 1262 (C-O), 1232 (C-N), 1590 (N=N). NMR ( ppm) 1.5 (t, 3H of -CH), 4.4 (q, 2H of -OCH), 4.2 (s, 1H of Phenolic -OH), 9.8 (s, 1H of -CHO), 7.0 to 8.5 (m, 6H of Aromatic-H), 9.9 due to moisture. 4R FTIR (KBr,cm-1) 3363 (OH of Phenol), 2828 (C-H of H-C=O), 1681 (C=O), 1514 (C=C), 1281 (C-O), 1168 (C-N), 1580 (N=N). NMR ( ppm) 1.5 (t, 3H of -CH), 4.3 (q, 2H of -OCH), 6.3 (s, 1H of Phenolic -OH), 9.8 (s, 1H of -CHO), 7.0 to 7.4 (m, 6H of Aromatic-H). 4S FTIR (KBr,cm-1) 3333 (OH of Phenol), 2842 (C-H of H-C=O), 1682 (C=O), 1515 (C=C), 1261 (C-O), 1231 (C-N), 1592 (N=N). NMR ( ppm) 1.5 (t, 3H of -CH), 4.1 (q, 2H of -OCH), 4.3 (s, 1H of Phenolic -OH), 9.8 (s, 1H of -CHO), 7.0 to 8.5 (m, 9H of Aromatic-H). Table-3 Antimicrobial activities of the synthesized azo compounds of ethyl vanillin Symbols for compounds Microbial species and diameter of zone of inhibition in mm Escherichia coli Salmonella typhi Pseudomonas aeroginosa Bacillus subtilis Staphylococcus aureus 1a 10.4 No inhibition No inhibition No inhibition 9.6 1b 10.6 12.7 No inhibition 11.8 9.8 1c 10.3 13.1 No inhibition 13.5 12.8 1d 13.6 12.8 15.6 11.3 14.4 E. coli S. aureus Figure-2 Zone of inhibition of azo compound 1a E. coli S. typhi B. subtilis S. aureusFigure-3 Zone of inhibition of azo compound 1b Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 20-28, July (2015) Res. J. Chem. Sci. International Science Congress Association 23 E. coli S. typhi B. subtilis S.aureus Figure-4 Zone of inhibition of azo compound 1c E. coli S. typhi P. aeroginosa S.aureus Figure-5 Zone of inhibition of azo compound 1d Pagariya-21.sp - 4/21/2014 - 4P 4000.03600320028002400200018001600140012001000800600400.00.010152025303540455055.4cm-1%T 3729,53 3362,13 2979,30 2934,29 2887,34 2829,29 2797,32 2722,35 2681,40 2539,42 2331,45 2097,49 1908,49 1782,52 1686,7 1674,8 1604,18 1579,5 1516,7 1477,29 1439,17 1398,26 1380,27 1280,4 1255,9 1197,14 1168,4 1115,10 1104,10 1041,15 970,23 890,37 852,27 839,20 802,27 786,21 734,40 653,27 633,25 585,28 524,26 416,47 Figure-6 FTIR spectrum of azo compound 1a Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 20-28, July (2015) Res. J. Chem. Sci. International Science Congress Association 24 Pagariya-22.sp - 4/21/2014 - 4Q 4000.03600320028002400200018001600140012001000800600400.031.535404550556065.4cm-1%T 3398,57 2982,56 2926,58 2882,59 2843,58 2757,60 1673,37 1590,41 1519,38 1441,41 1335,48 1308,41 1262,34 1232,42 1126,43 1041,48 878,45 851,45 829,45 742,42 639,45 590,51 530,54 Figure-7 FTIR spectrum of azo compound 1b S K Pagariya-1.sp - 5/12/2015 - 4R 4000.03600320028002400200018001600140012001000800600400.07.010121416182022242628303233.5cm-1%T 3778,31 3363,17 2980,20 2934,19 2887,20 2828,19 2796,20 2724,20 2540,23 2321,24 2096,27 1907,28 1681,11 1580,11 1514,11 1439,14 1396,17 1382,17 1281,10 1257,11 1168,10 1107,12 1040,13 969,16 889,21 837,16 801,18 783,16 633,16 584,18 522,19 415,30 Figure-8 FTIR spectrum of azo compound 1c Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 20-28, July (2015) Res. J. Chem. Sci. International Science Congress Association 25 S K Pagariya-2.sp - 5/12/2015 - 4S 4000.03600320028002400200018001600140012001000800600400.02.0101214161818.7cm-1%T 3333,14 3054,12 2981,10 2927,11 2882,12 2842,11 2758,13 2351,16 2007,18 1938,18 1871,17 1682,4 1592,5 1515,6 1487,6 1442,5 1401,6 1308,5 1283,5 1261,4 1231,5 1165,7 1136,5 1041,8 1003,8 878,6 860,6 828,6 795,6 767,5 741,6 716,5 638,6 590,8 574,8 545,9 489,11 424,14 Figure-9 FTIR spectrum of azo compound 1d 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm 0.0000 1.4622 1.4797 1.4971 4.1735 4.1910 4.2085 4.2260 6.3361 7.0335 7.0538 7.2680 7.4013 7.4200 7.4245 9.8170 3.35 2.20 1.04 1.01 2.06 1.00 Current Data Parameters NAME May13-2014-Administrator EXPNO 50PROCNO 1F2 - Acquisition ParametersDate_ 20140513Time 17.32INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT CDCl3NS 8DS 2SWH 12019.230 HzFIDRES 0.183399 HzAQ 2.7263477 secRG 287DW 41.600 usecDE 6.00 usecTE 296.7 KD1 1.00000000 secTD0 1======== CHANNEL f1 ========NUC1 1HP1 10.90 usecPL1 -3.00 dBSFO1 400.1324710 MHzF2 - Processing parametersSI 32768SF 400.1300064 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.004PBRUKERAVANCE II 400 NMRSpectrometerSAIFPanjab UniversityChandigarh avtar_saifpu@yahoo.co.in Figure-10 NMR spectrum of azo compound 1a Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 20-28, July (2015) Res. J. Chem. Sci. International Science Congress Association 26 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm 1.2431 1.2542 1.4019 1.4666 1.4743 1.4841 1.4916 1.5016 1.5091 1.5321 1.6176 1.6380 4.0919 4.1092 4.1662 4.1787 4.1831 4.1963 4.2137 4.2310 7.0325 7.0520 7.2635 7.3995 7.4190 7.4232 7.4318 7.4470 7.4673 7.5105 7.5176 7.5474 7.5574 7.5760 7.6299 7.6450 7.6590 7.6634 7.6724 7.6995 7.7180 7.7311 7.7855 8.0220 8.0542 8.0701 8.1306 8.1637 9.8171 9.9354 13.7555 14.5427 2.21 6.97 2.16 1.04 3.57 0.68 1.00 3.45 5.47 4.50 0.56 1.05 0.38 0.36 Current Data ParametersNAME May13-2014-AdministratorEXPNO 60PROCNO 1F2 - Acquisition ParametersDate_ 20140513Time 17.37INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT CDCl3NS 8DS 2SWH 12019.230 HzFIDRES 0.183399 HzAQ 2.7263477 secRG 912DW 41.600 usecDE 6.00 usecTE 296.7 KD1 1.00000000 secTD0 1======== CHANNEL f1 ========NUC1 1HP1 10.90 usecPL1 -3.00 dBSFO1 400.1324710 MHzF2 - Processing parametersSI 32768SF 400.1300083 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.004QBRUKERAVANCE II 400 NMRSpectrometerSAIFPanjab UniversityChandigarh avtar_saifpu@yahoo.co.in Figure-11 NMR spectrum of azo compound 1b 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm 0.0000 1.4595 1.4770 1.4945 4.1702 4.1877 4.2052 4.2226 6.3759 7.0331 7.0528 7.2702 7.4008 7.4196 7.4239 9.8151 3.31 2.15 1.00 1.07 2.03 1.01 Current Data ParametersNAME May13-2014-AdministratorEXPNO 70PROCNO 1F2 - Acquisition ParametersDate_ 20140513Time 17.42INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT CDCl3NS 8DS 2SWH 12019.230 HzFIDRES 0.183399 HzAQ 2.7263477 secRG 512DW 41.600 usecDE 6.00 usecTE 296.8 KD1 1.00000000 secTD0 1======== CHANNEL f1 ========NUC1 1HP1 10.90 usecPL1 -3.00 dBSFO1 400.1324710 MHzF2 - Processing parametersSI 32768SF 400.1300055 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.004RBRUKERAVANCE II 400 NMRSpectrometerSAIFPanjab UniversityChandigarhavtar_saifpu@yahoo.co.inFigure-12 NMR spectrum of azo compound 1c Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 20-28, July (2015) Res. J. Chem. Sci. International Science Congress Association 27 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm 1.5105 1.5283 1.5551 1.5725 1.5899 1.6178 1.6350 1.6524 4.1694 4.1869 4.2044 4.2239 4.2417 4.2592 4.2766 4.2870 4.3362 4.3539 7.0285 7.0480 7.2588 7.3936 7.4127 7.4987 7.5020 7.5276 7.5420 7.5508 7.5764 7.6001 7.6197 7.6355 7.6403 7.6640 7.6810 7.7272 7.7743 7.7797 7.8555 7.9336 7.9517 7.9716 7.9963 8.0301 8.0430 8.0498 8.0617 8.1352 8.1390 8.4134 8.4831 8.5038 8.5623 9.8137 9.9637 14.8291 1.30 4.64 2.94 0.49 0.70 0.81 1.55 4.88 1.99 2.32 1.17 1.00 0.98 1.01 Current Data ParametersNAME May13-2014-AdministratorEXPNO 80PROCNO 1F2 - Acquisition ParametersDate_ 20140513Time 17.49INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT CDCl3NS 8DS 2SWH 12019.230 HzFIDRES 0.183399 HzAQ 2.7263477 secRG 812DW 41.600 usecDE 6.00 usecTE 297.0 KD1 1.00000000 secTD0 1======== CHANNEL f1 ========NUC1 1HP1 10.90 usecPL1 -3.00 dBSFO1 400.1324710 MHzF2 - Processing parametersSI 32768SF 400.1300103 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.004SBRUKERAVANCE II 400 NMRSpectrometerSAIFPanjab UniversityChandigarh avtar_saifpu@yahoo.co.in Figure-13 NMR spectrum of azo compound 1dConclusion The current investigation reveals that, there was a miraculous inhibition of compound 1d shows antibacterial activity against all the tested organisms while the compound 1a was found to be most resistant by the pathogens. The highest zone of inhibition was obtained for 1d against Pseudomonas aeroginosa (15.6 mm) while lowest zone was recorded for 1a against Staphylococcus aureus (9.6 mm). The compounds 1b and 1c shows moderate inhibition of growth against tested pathogens. References1.Towns A.D., Developments in azo disperse dyes derived from heterocyclic diazo compounds, Dyes and Pigments, 42, 3-28 (1999)2.Zollinger H., Synthesis, properties and applications of organic dyes and pigments, Color Chemistry, third revised edition,Wiley-VCH.,Weinheim (2003)3.Gayatri C. and Ramalingam A.,Z-scan determination of the third order optical nonlinerities of an azo dye using dipole: pumped Nd:Yag laser, Optik, 119(9), 409-414 (2008)4.Sakong C., Kim Y. D., Choi J. H., Yoon C. and Kim J.P., The synthesis of thermally stable red dyes for LCD color filters and analysis of their aggregation and spectral properties, Dyes and Pigments, 88(2),166-173 (2011)5.Bae J.S., Freeman H.S. and El-Shafei A., Metallization of non-genotoxic direct dyes, Dyes and Pigments, 57(2), 121-129 (2003)6.Sanjay F.T., Dinesh M.P., Manish P.P. and Ranjan G.P., Synthesis and antibacterial activity of novel pyraazolo [3,4-b] quinoline base heterocyclic azo compounds and their dyeing performance, Saudi Pharm. Journal, 15(1), 48-54 (2007)7.Child R.G., Wilkinson R.G. and Tomcu-Fucik A., Effect of substrate orientation of adhesion of polymer joints, Chem. Abstr., 87, 6031 (1977) 8.Garg H.G. and Prakash C.J., Preparation of 4-arylazo-3,5-disubstituted-(2H)-1,2,6-thiadizine1,1-dioxides, Med. Chem.,15(4), 435-436 (1972)9.Koshti S.M., Sonar J.P., Sonawane A.E., Pawar Y.A., Nagle P.S., Mahulikar P.P. and More D.H., Synthesis of azo compounds containing thymol moiety, Indian J. Chem., 47B, 329-331 (2008) 10.Pathan R.U. and Borul S.B., Synthesis and antimicrobial activity of azo compounds containing drug moiety,Oriental J. Chem., 24(3), 1147-1148 (2008) Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 5(7), 20-28, July (2015) Res. J. Chem. Sci. International Science Congress Association 28 11.Rathod K.M., Synthesis and antimicrobial activity of azo compounds containing paracetamol moiety, Oriental J. Chem., 26(3), 1163-1166 (2010)12.Rathod K.M. and Thakare N.S., Synthesis and antimicrobial activity of azo compounds containing m-cresol moiety, Chem. Sci.Trans., 2 (1), 25-28 (2013)13.Rathod K.M., Synthesis and antimicrobial activity of azo compounds containing resorcinol moiety, Asian J. Research Chem., 4(5), 734-736 (2011)14.Pagariya R.F., Rathod K.M. and Thakare N.S., Synthesis, characterization and antibacterial screening of azo compounds containing vanillin moiety, Asian J. Research Chem., 6(9), 824-827 (2013)15.Dahake P.R. and Kamble S.I., Investigatory study on antimicrobial activity and phytochemical screening of Butea monosperma Linn, BBRA, 11(3), 1695-1698 (2014)16.Alzoreky N.S. and Nakahara K., Antimicrobial activity of extracts from some edible plants commonly consumed in Asia, Int. J. Food Microbial, 80, 223-230 (2003)