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Volume-1, Number- 2, September-December, 2010

ijCEPr International Journal of Chemical, Environmental and Pharmaceutical Research Editor-in-Chief

Prof. (Dr.) Sanjay K. Sharma

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ijCEPr International Journal of Chemical, Environmental and Pharmaceutical Research Volume-1, Number-2, September- December, 61-122 (2010) Contents‌ Ultrasonic Studies of Binary Liquid Mixtures: Ethyl Acetate + 2-Butanone S. Anbarasu, K. Kaviyarasu, T. Kishore Kumar, S. Selvakumar, A.J. Clement Lourdhu Raj and Prem Anand Devarajan

61-70

Polarographic Study of La(III)- 3-Hydroxy-3-p-Tolyl-1-p-Sulphonato(Sodium Salt) Phenyltriazene Complex Dipen Upadhyay , Pooja Joshi , Neelam Pareek , Girdharpal Singh, Amit Bhandari, Rekha Dashora, A. K. Goswami and R. S. Chauhan

71-73

Alterations in the Activity of Enzymes as a Method to Characterize Herbicide Tolerance Santosh Kumar Singh, Satish Kumar Verma, Md. Aslam Siddiqui and Sachin Chauhan

74-79

Synthesis, Sectral Characterization, Thermal and Anti-microbial Studies of New Binuclear Metal Complexes Containing Tetradentate Schiff Base Ligand P. Jayaseelan, S. Prasad, S.Vedanayaki and R. Rajavel

80-88

Gas-Phase Structure and Rotational Barrier of Hydroxyphosphinecarbothialdehyde: A Computational Study Abdulhakim A. Ahmed

89-94

RP-HPLC Method for the Estimation of Eletriptan in Pharmaceutical Dosage Forms D. Suneetha1 and A. Lakshmana Rao

95-99

Polarographic Studies on Interaction of 3-Hydroxy-3-Phenyl-1-p-Sulfonato (Sodium Salt) Phenyltriazene with Ni (II) in Aqueous Medium Neelam Pareek, Pooja Joshi, Dipen Upadhyay, G.P.Singh, Amit Bhandari, Anita Mehta, R. S. Chauhan and A. K. Goswami

100-102

Application of 2-Hydroxyethyl Methacrylate Polymer in Controlled Release of 4Aminosalicylic Acid: A Colon Targeted Prodrug Approach RajeshYadav ,O.P.Mahatma and D.S.Rathore

103-110

Physico-Chemical Analysis of Some Groundwater Samples of Kotputli Town Jaipur, Rajasthan Ranjana Agrawal

111-113

Novel Treatment Process for Dyeing Industries Waste Water and Recycling: A Green Approach to Treat Effluents Ashok Patni INDEX of Contributors of this issue Authors Guidelines: for RASAYAN J. Chem. SUBSCRIPTION Form

114-122


ii

ijCEPr International Journal of Chemical, Environmental and Pharmaceutical Research Volume-1, Number-2, September- December, 61-122 (2010) AUTHOR INDEX OF THIS ISSUE A. K. Goswami , 71, 100 A. Lakshmana Rao, 95 A.J. Clement Lourdhu Raj, 61 Abdulhakim A. Ahmed, 89 Amit Bhandari, 71, 100 Anita Mehta, 100 Ashok Patni, 114 D. Suneetha, 95 D.S.Rathore, 103 Dipen Upadhyay , 71, 100 Girdharpal Singh, 71, 100 K. Kaviyarasu, 61 Md. Aslam Siddiqui, 74 Neelam Pareek , 71, 100 O.P.Mahatma, 103 P. Jayaseelan, 80

Pooja Joshi , 71, 100 Prem Anand Devarajan, 61 R. Rajavel, 80 R. S. Chauhan, 71, 100 RajeshYadav, 103 Ranjana Agrawal, 111 Rekha Dashora, 71, 100 S. Anbarasu, 61 S. Prasad, 80 S. Selvakumar, 61 S.Vedanayaki , 80 Sachin Chauhan, 74 Santosh Kumar Singh, 74 Satish Kumar Verma, 74 T. Kishore Kumar, 61

IJCEPR widely covers all fields of Chemical, Environmental and Pharmaceutical Research. Manuscript Categories: Full-length paper, Review Articles, Short/Rapid Communications. Manuscripts should be addressed to: Prof. (Dr.) Sanjay K. Sharma Editor-in-Chief 23, ‘Anukampa’,Janakpuri, Opp. Heerapura Power Station, Ajmer Road, Jaipur-302024 (India) E-mail: ijcepr@gmail.com Phone:0141-2810628(O), 09414202678(M)

Adopt GREEN CHEMISTRY Save Our Planet. We publish papers of Green Chemistry on priority. If you think that you may be a potential reviewer in field of your interest, write us at ijcepr@gmail.com with your detailed resume and recent color photograph.


International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.2, 61-70 September-December, 2010

Ultrasonic Studies of Binary Liquid Mixtures: Ethyl Acetate + 2-Butanone S. Anbarasu1, K. Kaviyarasu1, T. Kishore Kumar2, S. Selvakumar3, A.J. Clement Lourdhu Raj4 and Prem Anand Devarajan1* 1*

Department of Physics, St. Xavier’s College, Palayamkottai. Department of Physics, Presidency College, Chennai 3 Department of Physics, L.N. Govt. College of Arts & Science, Ponneri. 4 Department of Physics, St. Josephs College, Trichy *E-mail: dpremanand@yahoo.co.in 2

Article History: Received:20 November 2010 Accepted:14 December 2010

ABSTRACT Binary liquid mixtures of Ethyl acetate + 2-Butanone at various mole fractions were prepared. The molecular interactions between the binary mixtures were analyzed by ultrasonic measurements using interferometer method. The densities of pure liquid mixtures were elucidated by relative measurement method. The mole fractions of Ethyl acetate and 2-Butanone were found to be 98.50 and 98.06 respectively. The FTIR spectrum shows a drastic change in the frequency for 0.6 mole fraction of Ethyl acetate and 0.4 mole fraction of 2-Butanone. The shift in the frequency values might be due to interstitial accommodation or induced dipole interaction. Keywords: Ethyl acetate, 2-Butanone, FTIR, ultrasonic measurements. ©2010 ijCEPr. All rights reserved

INTRODUCTION Ion-solvent or solvent-solvent interaction involved in a binary mixture system can be studied by various methods. The principle of acoustics is one among them [1,2]. Studies on acoustic parameters have become an emerging hid in recent years [3,4]. To understand solution chemistry, it is essential to know the salvation behavior of binary mixture system. Acoustic parameters are sensitive to changes and are useful in elucidating the solvent-solvent interaction. Moreover the ultrasonic velocity measurements have been successfully employed to detect and assess weak and strong molecular interactions, present in binary and ternary. In prevailing literature, many contributions have been made in the strong of liquid mixtures [7-13]. Literature does not show any report on the ultrasonic behavior of Ethyl acetate + 2-Butonone. In the present paper, an attempt has been made to determine the densities and ultrasonic velocities of the above said title binary mixtures have been reported.

MATERIALS AND METHODS Commercially available AR grade Ethyl acetate (E-merk) and 2-Butanone (E-merk) were used as such. Densities were measured with the help of bicapillary pyknometer. All the weighings were made using single pan digital balance. The binary mixtures were prepared by volume, by mixing selected volumes of liquid components in airtight glass bottles. In all the property measurements, an INSREF thermostat was used at a constant digital temperature display accrete to ( ± 0.1mg) and the measurement of mass were made using an electronic balance. Accuracy of density measurement was 0.0001 gcm-3. A set of eleven compositions was prepared for each system and their physical properties were measured on the same day. A 10 ml. specific gravity bottle and electronic balance were used for the determination of density measurements. Speed of sound was determined using constant frequency (2 MHz) variable path ultrasonic interferometer (Model F- 81, Mittal Enterprises, New Delhi) with an accuracy of

± 2 ms-1 and was calibrated using water and benzene. RESULTS AND DISCUSSION Ultrasonic Velocity Measurement The velocity of ultrasonic waves in the mixture has been measured by Interferometer method. The interferometer consists of two parts namely high frequency generator and the measuring cell. The interferometer generates

S. Anbarasu et al.


Vol.1, No.2, 61-70 (2010) alternating field for variable frequencies. The frequency of the alternating field in the interferometer can be selected by changing the selector available on the front panel. Thus, alternating field of a fixed frequency is generated by the interferometer. The measuring cell is a double walled vessel with a provision to circulate water from the water bath between the inner and outer walls. Thus the temperature of the mixture (taken in the inner cell) can be kept constant. At the top of the cell, a fine micrometer screw is fitted with a (metal) reflector, which is immersed in the mixture. The reflector plate in the mixture can be raised or lowered through a know distance using a micrometer screw. The least count of the micrometer screw is 0.001 mm. A quartz crystal is mounted at the bottom of the cell. The reflector plate and the quartz crystal are parallel to each other. The alternating field from the generator is applied to the quartz crystal. Therefore, quartz crystal gets into resonant vibrations and hence generates longitudinal ultrasonic waves. The longitudinal ultrasonic waves generated by the crystal pass through the mixture and get reflected at the surface of the parallel reflector place. If the distance between the plate and the crystal is exactly an integral multiple of half wavelength, standing waves are formed within the medium. This leads to acoustic resonance, resulting in a change of potential difference at the generator, which excites the quartz crystal. Thus, the anode current of the generator becomes maximum. The change in the anode current can be measured from the micro-ammeter fitted with the frequency generator. The distance‘d’ between the plate and crystal is slowly varied using the micrometer screw, resulting in a decrease in anode current. The micrometer screw is adjusted such that the anode current increases up to a maximum once again i.e., the needle in the ammeter complete one oscillation. By noting the initial and final position of the micrometer for n complete movements (maxima-minima-maxima) of the micro-ammeter needle, one can determine the distance (d) moved by the parallel reflector. The wavelength is calculated as, 2 d λ = n (1) Therefore, the velocity of ultrasonic longitudinal waves in the mixture is given by,

U =λ f

(2)

Where, f is the frequency of the generator, which is used to excite the crystal.

Error Analysis Let Uexpt and Ucal be the experimental and theoretically calculated values of ultrasonic velocities Percentage Deviation The percentage deviation in the values of ultrasonic velocity is given by, P e r c e n t a g e d e v i a t io n =

U

ex p t

U

−U

ca l

X 100

exp t

(3)

Molecular Interaction Parameter The Molecular Interaction Parameter (MIP) is given by,

U M IP =  U 

cal exp

  

2

−1 (4)

This MIP is multiplied by 100 for convenience of presentation of values.

Chi- Square value Chi- square test of goodness of fit enables us to find whether the deviations of the theoretical values from the experimental ones are due to chance or really due to the inadequacy of the theory to fit the experimental data.

χ2

Value is given by,

χ

2

n

∑ i=1

(U

exp t

U

−U

cal

)2 (5)

cal

Determination of Mole fraction The Mole Fractions for Liquid-1 [Ethyl Acetate] and Liquid-2 [2-Butanone] are calculated as following as 62

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Vol.1, No.2, 61-70 (2010)

Step 1: To find the Mass of the water Mass of the empty R.D bottle (m1) Mass of the water +R.D bottle (m2) Therefore, Mass of the water alone (M) is M= (m2-m1)

= 18.8701g = 28.7041g = 9.8340g

Step 2: To find the Mass of the Liquid 1 Mass of the empty R.D bottle (l1) Mass of the Liquid 1 + R.D bottle (l2) Therefore, Mass of the Liquid 1 alone (L1) is L1= (l2-l1)

= 18.8701g = 27.6804g = 8.8103g

Step 3: To find the Mass of the Liquid 2 Mass of the empty R.D bottle (l’1) = 15.1564g Mass of the Liquid 1 + R.D bottle (l’2) = 22.3990g Therefore, Mass of the Liquid 1 alone (L2) is L2 = (l’2-l’1) = 7.2426g Relative Density Formulae: R.D.= [ Mass of the liquid/ Mass of the water]X ρw Where, ρw is density of water.

(6)

Note: Density of water(ρw) = 1000 Kg/m3 but, we take the approximate value of 0.9984 Kg/m3.

Step 4: To find the Density of the Liquid 1 D1= [ Mass of the liquid 1/ Mass of the water]X ρw Therefore, D1= [ 8.8103/ 9.8340]X 0.9984 D1 = 0.8944 Kg/m3

Step5: To find the Density of the Liquid 2 D2= [ Mass of the liquid 2/ Mass of the water]X ρw Therefore, D2= [ 7.2426/ 9.8340]X 0.9984 D2 = 0.7353Kg/m3

Step 6: To find the Molecular weight of the liquids Molecular weight of the Liquid 1 (MW1) = 88.10 g/mol Molecular weight of the Liquid 2 (MW2) = 72.11 g/mol Step 7: To find the Mole Fraction The Mole Fraction for Liquid1 (MF1) is calculated asMF1= [MW1/D1]= [88.10/0.8944] = 98.5017 The Mole Fraction for Liquid2 (MF2) is calculated asMF2= [MW2/D2]= [72.11/0.7353] = 98.0688

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Vol.1, No.2, 61-70 (2010) The result of MF1 and MF2 are divided by 2 and 3

FTIR Spectrum Analysis The FTIR spectrum of the binary mixtures was recorded in the frequency range 400-4000 cm-1 employing Broker model IFS 66V FTIR spectrometer. The spectra are shown in Fig. (1-11). The various frequency assignments pertaining to different ratios are tabulated in Table1.

Fig.1

Fig.2

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Vol.1, No.2, 61-70 (2010)

Fig.3

Fig.4

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Vol.1, No.2, 61-70 (2010)

Fig.5

Fig.6 66

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Vol.1, No.2, 61-70 (2010)

Fig.7

Fig.8

67

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Fig.9

Fig.10 68

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Vol.1, No.2, 61-70 (2010)

Fig.11

Observations of FTIR Spectrum Table-1 1 (EA) + 0.0 (2BUT) 3449.55 2991.58 2489.19 2362.08 2088.87 1763.76 1637.15 1378.64 1242.81 1056.51 928.00 622.08

0.9 (EA) + 0.1 (2BUT) 3449.85 2369.39 2089.05 1739.40 1637.90 1409.82 1244.60 1110.28 1049.78 574.78

0.8 (EA) + 0.2 (2BUT) 3450.45 2990.51 2488.21 2365.16 2089.01 1762.32 1637.33 1376.73 1242.82 1164.40 1056.35 931.02 623.89

0.7 (EA) + 0.3 (2BUT) 3755.77 3450.69 2990.49 2489.94 2375.18 2090.50 1761.48 1637.41 1375.96 1242.79 1165.69 1056.63 932.09 622.77

0.6 (EA) + 0.4 (2BUT) 3778.13 3447.05 2928.83 2376.34 2093.87 1636.17 1418.32 1245.49 1113.89 1021.27 577.76

0.5 (EA) + 0.5 (2BUT) 3768.93 3450.23 2990.28 2378.86 2091.95 1757.15 1637.35 1373.67 1242.87 1168.70 1056.34 935.24 595.26

69

0.4 (EA) + 0.6 (2BUT) 3775.07 3448.38 2989.86 2370.49 2092.69 1757.83 1635.10 1373.15 1242.82 1164.26 1109.66 1056.57 1005.30 938.26 594.26

0.3 (EA) + 0.7 (2BUT) 3765.76 3448.32 2988.64 2368.72 2094.28 1746.98 1635.26 1370.94 1242.92 1114.23 595.48

0.2 (EA) + 0.8 (2BUT) 3906.73 3769.93 3449.06 2375.62 2089.51 1636.11 1405.90 1243.50 1116.30 1008.25 670.17

0.1 (EA) + 0.9 (2BUT) 3920.45 3772.35 3449.47 2988.97 2380.41 2104.31 1740.66 1628.07 1367.68 1243.00 1167.31 1008.88 586.32

0 (EA) + 1 (2BUT) 3921.99 3778.25 3445.42 2930.86 2087.47 1815.01 1626.89 1409.75 1247.00 1117.88 1011.07 563.25

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Vol.1, No.2, 61-70 (2010)

CONCLUSION In the case of Ethyl acetate + 2-Butanone mixture, the VE is maximum negative for 0.6 mole fraction of Ethyl acetate which shows the presence of interstitial accommodation of one type of molecule into other and dipole – induced dipole interaction. The FTIR spectrum taken shows a drastic change in the frequency values for 0.6 mole fraction of Ethyl Acetate (0.6 of Ethyl Acetate and 0.4 of 2-Butanone). So, the observation from the VE study has been confirmed by the FTIR spectrum measurement.The shift in the frequency values of FTIR spectrum measurement is generally due to following observed factors: 1. Interstitial accommodation. 2. Strong interactions (H- bond type, or dipole – dipole or dipole – induced dipole interactions).

ACKNOWLEDGEMENTS One of the authors, S. Anbarasu would like to thank Prof. I.Sebasdiyar, Head of the Department of Physics for his constant support, help and encouragement.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Everest F.A., Master handbook of acoustics (Mc Graw Hill, New York), 2000. David N.J., Fundemendals and applications of ultrasonic waves (RC press, New York), 2002. Ishwara Bhat.J and Shivakumar H.R., Indian J. Chem A.,37 (1998) 252 Ishwara Bhat.J and Shivakumar H.R., Indian J. Pure and Applied Physics, 38 (2000) 306. Jeyakumar S, Karunanithi N and Kannappan V, Indian J. Pure and Applied Physics, 34 (1996) 761. Prasad N, Singh R, Prakash O and Prakash S, Indian J. Pure and Applied Physics, 14 (1976) 676 Marewein B.L and Bhat S.N., Acustica, 58 (1985). Carter S, J. Chem. Soc A, 404 (1968). Sheshagiri Rao M.G., Indian J. Pure and Applied Physics, 9 (1971) 169. Varma R.P. and Surendrakumar, Indian J. Pure and Applied Physics, 38 (2000) 96. Yadav S.S., Singh Y.P.and Rajkumar, J. Indian Chem., 16 (1999) 20. Sheshagiri K and Reddy K.C., Acoustica, 29 (1973) 59. Ali A, Tiwari K, Nair A.K. and Chakravarthy V, Indian J. Physics B, 74 (2000) 351. Upadhayay S.K., Indian J. Chemistry, 39 (2000) 537. [ijCEPr-125/2010]

RASĀYAN Journal of Chemistry http:// www.rasayanjournal.com ISSN: 0974-1496 (Print); ISSN: 0976-0083(Online)

Highlights of RASĀYAN • • • • • .

It is a full text open access international journal of Chemical Sciences. Covers all fields related to Chemistry. Research papers will be published on the website and also in the printed version simultaneously. Manuscript is reviewed and published very quickly. Full text of the article is available on the site http://www.rasayanjournal.com all over the world. Reprints may be downloaded directly from the website. Papers can be submitted through e-mail to rasayanjournal@gmail.com

70

S. Anbarasu et al.


International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.2, 71-73 September-December, 2010

Polarographic Study of La(III)- 3-Hydroxy-3-p-Tolyl-1-p-Sulphonato(Sodium Salt) Phenyltriazene Complex Dipen Upadhyay , Pooja Joshi , Neelam Pareek , Girdharpal Singh, Amit Bhandari, Rekha Dashora, A. K. Goswami* and R. S. Chauhan Department of Chemistry, M.L Sukhadia University.Udaipur -313001 (Raj.), *E-mail : akumargoswami@rediffmail.com Article History: Received:24 November 2010 Accepted:8 December 2010

ABSTRACT The electrochemical behaviour of complex of La (III) with 3- hydroxy-3-p-tolyl-1-p-sulphonato (sodium salt) phenyltriazene (HPST) was studied . It was observed that HPST forms 1:1 complex with La(III) in Citric acid and Na2HPO4 buffer solution between pH 6.0 to 7.5. It was found that the reduction process of La (III) - HPST complex is two electron reversible reduction process. The stability constant of the hydroxy-3-p-tolyl-1-p-sulphonato(sodium salt) phenyltriazene complex was evaluated with the Lingane method at different ligand concentrations.The logarithm value of stability constant of 1:1 La(III)-3-hydroxy-3-p-tolyl-1-psulphonato(sodium salt) phenyltriazene complex is 10.05. Keywords : Hyroxytriazene, Polarography, La (III)- HPST complex. ©2010 ijCEPr. All rights reserved

INTRODUCTION Hydroxytriazenes are well established chelating agents as revealed by reviews appearing on them during last few years[1,2,6,7]. These compounds have been used as spectrophotometric and complexometric reagents for determination of transition and non-transition elements[3,5,8]. In the pesent work complex formation of La (III) with HPST at D.M.E in aqueous and alcoholic medium has been studied polarographically. Overall stability constant of La(III)-HPST has been determined .

MATERIALS AND METHODS Synthesis of 3-hydroxy-3-p-tolyl-1-p-sulphonato (sodium salt) phenyltriazene (HPST) In a one litre beaker (0.1mol) of p-nitrotoluene, 5 gm of NH4Cl 50 ml water and 50 ml C2H5OH were mixed, stirred mechanically and cooled to 0° C. 20 gm Zn dust was added in small lots such that the temperature of reaction mixture remained between 50-60°C. The reaction mixture was stirred mechanically for another 15 min. The solution of p-tolylhydroxylamine was obtained after filteration Thus, kept in freezer and used as such for coupling with diazotized product. In a 500 ml beaker (0.1 mol) of sulphanilic acid was dissolved in 20 ml Na2CO3 solution and then NaNO2 (6.9gm) was added to sulphanilic acid and dissolved this mixture in 20 ml HCl and 100 ml water in small lots at 0 to 5°C under constant mechanical stirring. The diazotized product so obtained was directly used for coupling. The p-tolylhydroxylamine was coupled with the diazotized product at 0 to 5°C under mechanical stirring with occasional addition of sodium acetate solution for maintaining the pH close to 5 during coupling process, Now sodium chloride (50 gm) was added to the reaction mixture. The compound of 3-hydroxy-3-p-tolyl-1-psulphonato(sodiumsalt) phenyltriazene was obtained as yellowish brown micro crystals after crystallization from double distilled water. C H N analysis corroborated the purity of compound. The compound was subjected to IR spectral analysis and following bands are given as: IR (KBr) cm-1: 3249 (O-H str.), 3078 (C-H str. Ar), 2981 (C-H str., CH3), 1632 (N=N str.), 1419 (N-N str.).The spectra showed the compound to be in pure state. IR spectra (KBr) were recorded on FT IR RX1 Perkin Elmer Spectrometer. A systronics polarograph 1632 was used for obtaining C.V. curves. Physical and analytical data are given in Table-1. Polarographic study of La(III)-HPST complex

Dipen Upadhyay et al.


Vol.1, No.2, 71-73 (2010) Metal solution(1mM) was prepared using La(NO3)3 and ligand solution was prepared by dissolving requisite quantity of HPST(.01 M) in double distilled water. Citric acid and Na2HPO4 solution were used as buffer to maintain pH. Ionic strength was kept constant by using KCl as supporting electrolyte, gelatin (.002%)was use as maximum suppressor. The capillary had following characteristics t=1 drop/sec .IR drop correction were applied. The polarographic study of La(III)-HPST has been done at D.M.E in aqueous medium. Solution was deareated by purging of oxygen free nitrogen through the polarographic cell. A 1×10-3 M Cu(II) solution in N/10 KCl has been used to obtain polarograms of La(III). This showed an E1/2 at 1.9 Vs SCE. Polarographic study was done on La(III) with various concentration of HPST. The polarogram showed the half wave potentials shifted towards more negative value with increasing concentration of ligand indicating complex formation and the diffusion current was found to decrease regularly with increase of HPST concentration.

RESULTS AND DISCUSSION A single well defined wave was obtained for La(III)- HPST system between pH 6.0-7.5. Diffusion controlled nature of each wave was verified from id Vs C and id Vs √h plots where id =diffusion current in µA; C=conc. In m mole lit1- , h=height of mercury column. Slope of the linear plots of log (i/id-i) Vs Ede was found to be in the range of 30-32 mV, thereby showing the reversible nature of reduction process involving two electrons. The plot of half wave potential E1/2 Vs log Cx (where Cx = concentration of complex in m mole lit1- ) have been found to be a straight line showing the formation of most stable complex. The coordination no. (j) of the metal complex is obtained from the slope of this plot, as may be expressed by: d(E1/2 ) /d log Cx = -j.0591/n where n = no. of electrons involved (here n =3).The value of j was found to be 2.This shows that composition of the complex is 1:1 (metal: ligand). Determination of stability constant The stability constant of the La(III)--HPST complex has been determined by classical method of Lingane[4], as the method is applicable for maximum coordination number and for the stability constant of highest complex formed. The E1/2 has a linear correlation with ligand concentration; which shows that there is only one complex formed. The following equation has been used to calculate the stability constant of the complex studied. ∆ (E1/2) = 0.0591/n log β + j 0.0591/n log Cx Here, ∆ (E1/2) =Difference of half wave potentials of simple metal ion and complexed ion, n =number of transfered electron, log β = Stability constant of complex formed, j = Coordination number, Cx = concentration of ligand. Thus the value of log β has been found to be 10.05. Polarographic data of Cu (II)- 3-hydroxy-3-m-tolyl-1-psulphonato (sodium salt) phenyltriazene are given in Table-2.

CONCLUSION The present work has opened up possibility of studying La(III)--HPST complexes by D.C polarographic method. Stability constant ( log β) was obtained with polarography .This proves the validity of polarographic techniques for studies of hydroxytriazenes metal complexes.

Table-1:Elemental analysis of 3-hydroxy-3-p-tolyl-1-p-sulphonato (sodium salt) phenyltriazene Molecular formula Melting %C %N %H point (C12H10N3O4.S.Na) H2O 180° C (d) Th. 43.2 12.6 3.6 Exp. 42.4 12.4 3.6

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Vol.1, No.2, 71-73 (2010) Table-2: Polarographic characterstics of La (III)- 3-hydroxy-3-p-tolyl-1-p-sulphonato (sodium salt) phenyltriazene . S.No Cx Log Cx E1/2 Log β 1 2 3 4 5 6 7 8 9

0.00 0.01 0.015 0.020 0.025 0.030 0.035 0.04 0.045

0.00 -2 -1.8239 -1.6987 -1.6020 -1.5228 -1.4559 -1.3979 -1.3467

1.900 1.950 1.970 1.985 2.005 2.020 2.040 2.055 2.070

12.16 11.15 10.47 10.32 9.54 9.20 8.91 8.65

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Chakrovorty D., Majumdar A. K., J. Indian Chem.Soc., 54 (1977) 258. Dutta R. L., Sharma R. S., J.Sci.Industr.Res.India, 40 (1981) 715. Gorgi D. K., Chauhan R. S., et.al., Revs. Anal. Chem., 17(4) (1998) 223. J. Lingane., J.Chem.Rev., 24 (1941) 1. Kumar S., Goswami A. K., et.al Revs. Anal. Chem., 22 (2003) 1. Purohit D. N., Talanta., 14 (1967) 207. Purohit D. N., Nizammudin et.al.,Revs. Anal. Chem., 8 (1985) 76. Purohit D. N., Tyagi M. P., et.al., Revs. Anal. Chem., 11 (1992) 269.

[ijCEPr-126/2010] __________________________________________________________________________________________

http:// www.rasayanjournal.com ISSN: 0974-1496 (Print); ISSN: 0976-0083(Online)

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It is a full text open access international journal of Chemical Sciences. Covers all fields related to Chemistry. Research papers will be published on the website and also in the printed version simultaneously. Manuscript is reviewed and published very quickly. Full text of the article is available on the site http://www.rasayanjournal.com all over the world. Reprints may be downloaded directly from the website. Papers can be submitted through e-mail to rasayanjournal@gmail.com. 1. 2.

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73

Dipen Upadhyay et al.


International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.2, 74-79 September-December, 2010

Alterations in the Activity of Enzymes as a Method to Characterize Herbicide Tolerance Santosh Kumar Singh*1, Satish Kumar Verma2, Md. Aslam Siddiqui3 and Sachin Chauhan4 *1Department of Microbiology, Gayatri College of Biomedical Sciences, Dehradun(U.K.)India 2 Department of Biotechnology, Sai Institute of Paramedical and Allied Sciences, Dehradun (U.K.) India. 3 Department of Life Sciences, BFIT, Dehradun(U.K.) India. 4 Department of Biotechnology, GCBMS, Dehradun(U.K.) India. *E-mail: res_mol_bio@sify.com Article History: Received:19 September 2010 Accepted:6 December 2010

ABSTRACT Exposure of Ocimum gratissimum seeds to Oxyfluorfen showed a varied response. At low concentration of herbicide shoot length and its fresh and dry weight mass was observed to be stimulated though it showed an remarkable decrease in root length and its mass. Chlorophyll and carotenoid contents also showed slight enhancement in values at low doses, in comparison to untreated controls. But they decreased gradually at higher concentration of herbicide. The growth in terms of total protein contents decreased progressively at high concentration (4 ppm), though at low doses (0.5 ppm), it showed an enhancement. It might be due to the increased enzymatic activities to overcome stress. Enhanced generation of active oxygen species, increased the level of total MDA contents showing high degree of lipid peroxidation, at high dose of Oxyfluorfen (% control increase = 15-40 %). Enhanced exposure of herbicide to the seedlings stimulated the antioxidant enzymes. Superoxide dismutase and catalase activity were enhanced over controls (15% - 98%) but Peroxidase activity was observed to be decreased at 4 ppm concentration (21% as compared to the untreated samples). IAA oxidase activity assays showed its greater sensitivity towards the herbicide Oxyfluorfen. A considerable oxidative damage was observed due to the treatments of herbicide in seedlings. Keywords: Lipid peroxidation, oxidative stress, photosynthetic pigments, catalase, SOD, Peroxidase, IAA oxidase ©2010 ijCEPr. All rights reserved

INTRODUCTION Since India is an agriculture based country and it is a key factor in ‘Indian Economy’, about 64% of the population is dependant on agriculture for their livelihood. Peoples are diverting their attention towards various applied techniques to achieve the target of fulfilling the nutritional requirements of growing population. It might help not only to increase the food productivity, but also to prevent the losses of grains and vegetables by different invader pests and herbs. The application of insecticides and herbicides, the groups of pesticides, in crop fields for selective control of pests in modern age led to serious environmental contamination resulting in greater loss of crop productivity and growth of many microorganisms [4, 23]. Applications of herbicides are favored due to their low cost, easy availability and lack of regulatory implementation. The removal of these insecticides from soil and aquatic ecosystems has become a difficult problem and as a result of this they persist in the ecosystem for longer duration of time [24] and might harm lower and higher photosynthetic non target plants. Oxyfluorfen (2-chloro-1-(3ethoxy-4-nitrophenyl)-4-trifluoromethyl) benzene) belongs to the chemical family of diphenyl ether herbicides. This is used to control broadleaf and grassy weeds in the culture of a variety of fields, fruits and vegetables crops, ornamentals as well as non crops sites. Because Oxyfluorfen has been identified as being persistent in water and mobile in soils, there is concern for ground water contamination and harm to some non target plants. Ocimum gratissimum, the Ban Tulsi is a part of important group of aromatic and medicinal plants that yield many essential oils and aroma chemicals and find diverse uses in the perfumery and cosmetic industries as well as in indigenous systems of medicine. It belongs to the family Labiatae (Lamiaceae). It is well known for its antioxidant potentials [10, 26]. Since activities of enzymatic antioxidants that help plants in recovering from oxidative stress such as catalases, superoxide dismutase and peroxidase are previously reported to be changed under different stresses [17, 21], authors wished to screen the effects of herbicide Oxyfluorfen on modulation of their activity, in Ocimum gratissimum. The authors have set forth the objective of evaluating the alterations in growth behavior and photosynthetic pigment contents, analysis of the level of lipid peroxidation and modulations in enzymatic antioxidants in plants exposed to the herbicide Oxyfluorfen.

Santosh Kumar Singh et al.


Vol.1, No.2, 74-79 (2010)

MATERIALS AND METHODS Selection of Experimental plants and treatment of herbicide Seeds of the wild Ocimum plants were surface sterilized in 5% Sodium hypochlorite solution. Seedlings (2 weeks old) were selected for the pretreatment of herbicide- Oxyfluorfen. On 10th day, plantlets of each set were harvested and various parameters were analyzed with respective to the control plantlets (untreated). Measurement of growth, photosynthetic pigment and Lipid Peroxidation levels Length and fresh mass of 10 seedlings were recorded separately and then dried in an Oven at 60-700C for 4 days to determine dry mass. Fresh leaves (0.02 g) from different Ocimum species were taken and cut into small pieces and photosynthetic pigments were extracted in 80% (v/v) acetone [2]. The quantification of pigments was done by standard methods [15]. Extraction of protein from plant leaf samples were done by boiling them in 0.5 N NaOH for 4 minutes. Samples were centrifuged at 5000 rpm and supernatant was used for protein estimation using lysozyme as the standard [16]. The level of lipid peroxidation was measured in terms of total MDA contents and the reaction reagent consisted of 0.4 N TCA + 19.68 ml of distilled water + 0.4 ml of HCl + 100mg TBA [12]. Prepared leaf extract (in phosphate buffer) was added to the reaction reagent and absorbance was taken at 532 nm. MDA content was calculated as underConcentration of MDA = Absorbance x 6.45/ml/mg fresh wt.

(1)

Estimation of Catalase (EC 1.11.10.6) and Superoxide dismutase (EC 1.15.1.1) activities In vivo catalase activity was determined by making homogenates of leaves in fresh 50 mM of phosphate buffer (pH 7.0). In each samples catalase activity was determined by recording O2 evolution for 1 min after the addition of 5 ml of 50 mM phosphate buffer (pH 7.0) containing 50 mM H2O2 [7]. Further 1 ml of cell suspension was added and O2 evolution was monitored in darkness. For the measurement of SOD activity the reaction mixture contained 1.3 µM riboflavin, 13 mM L- methionine, 0.05 M Na2CO3, (pH 10.2), 63 µM p– nitroblue tetrazolium chloride (NBT) and crude plant extract [9]. Reaction was carried out under illumination (75 µmol photon m-2 s-1) from fluorescent lamp at 25oC. The initial rate of reaction as measured by the difference in increase in absorbance at 560 nm in the presence and absence of extract was proportional to the amount of enzyme. Estimation of Peroxidase and IAA oxidase activities Peroxidase (EC 1.11.1.7) was estimated by adding 0.1 M Phosphate buffer (pH 7.0) to homogenized leaf samples. The enzyme reaction mixture consisted of 0.1 M Phosphate buffer + 20 mM guaiacol + 12.5 mM H2O2 and plant extract. Optical density was measured at 436 nm [20]. IAA oxidase activity was assayed using the enzyme reaction mixture 0.071 M Phosphate buffer + 0.5 mM MnCl2 + 0.05% paracoumaric acid + enzyme extract [3]. After ½ hour incubation in dark 5 M perchloric acid and 0.1 M ferric nitrate solution was added. After incubation for 60 minutes in dark, optical density was measured at 535 nm.

RESULTS AND DISCUSSIONS It was observed that the exposure of Oxyfluorfen herbicide to Ocimum gratissimum for 7 days resulted decrease in root length as compared to untreated seedlings (33% decrease at 0.5 ppm and 88% at 4.0 ppm of Oxyfluorfen as compared to the control). Progressive inhibition in root fresh mass and dry mass was found out. Though the exposure of Oxyfluorfen showed varied results with shoot length (% control increase = 8% at 0.5 ppm and 17% at 1.0 ppm; % control decrease at high concentration of Oxyfluorfen was observed: % control inhibition= 53%). Shoot fresh and dry mass showed same variations as compared to the untreated cultures (Table 1). Similar findings were reported by other authors in seedlings of Triticum aestivum [18], Barley and maize [25] exposed to cobalt stress. It was seen that initially chlorophyll contents increased up to 78-88% while the high dose of Oxyfluorfen decreased the % control value up to 27%. Carotenoid contents showed the increase in values at 0.5 ppm to 1.0 ppm of Oxyfluorfen but high doses decreased the value up to 62% at 4 ppm concentration respectively (Table 1). Total protein contents showed initially low doses of herbicide induced the high rate of increase in protein contents that showed a decrease with time duration (days). But the high dose of Oxyfluorfen (4 ppm) was found to reduce the total protein contents speedily with increasing days (13% at 4 ppm as compared to the control) (Figure 1a). The declining trend in pigment and protein contents continued with rising concentration of the herbicide while low doses showed their recovery. The growth of photosynthesizes reflects the status of key physiological processes such as photosynthesis. Thus to understand the impact of herbicides on photosynthetic pigments and protein contents might 75

Santosh Kumar Singh et al.


Vol.1, No.2, 74-79 (2010) give us clues for its impacts on total biomass yield. Initial increase in total protein contents might be due to the increase in pool of enzymatic antioxidants to overcome the stress produced by the herbicide Oxyfluorfen and other herbicides as suggested by other authors [5, 19]. Oxyfluorfen induced lipid peroxidation of the cellular components in Ocimum gratissimum was studied by estimating the level of MDA in treated and untreated plantlets and the related data are depicted in the figure 1b. The lipid peroxidation in non- stressed Ocimum gratissimum was observed as 1.60 nmol MDA (mg fresh mass)-1. Treated plantlets showed 15-40 % increase in total Malondialdehyde contents as compared to the untreated plants. Since MDA is an intermediate compound produced due to lipid peroxidation, the measurements of its contents can be used as an index for the injury caused by free radicals produced during oxidative stress. The results obtained here are in agreement with other authors [1, 6, 11], who reported the increase in MDA content with the exposure to other stresses in Oryza sativa, Cassia sp. and Ulva fasciata, respectively. It was observed that the catalase activity showed an enhancement in herbicide treated plantlets (% control induction = 15% - 97% at 0.5 ppm to 4 ppm doses). The values ranged from 0.65 to 1.11 units minute-1 mg protein-1 as compared to the control (0.562 units minute-1 mg protein-1). Lower dose of Oxyfluorfen 0.5 ppm stimulated catalase activity a little (15%) but higher concentration increased the enzymatic activity rapidly (figure 2a). The increase in the activity of catalase might be due to the need to decompose H2O2 and to protect membranes. The activity of the superoxide dismutase in non-stressed plants was 5.89 Units g-1 minute-1 which indicated that plant samples appeared to be more resistant against superoxide radicals produced due to various kinds of stresses. When plantlets were treated with Oxyfluorfen, there was remarkable increase in the activity of the enzyme at high concentration of herbicide, respective to the control (figure 2b). The lower dose treatment (0.5 ppm) enhanced the SOD activity only by 11%. The enhancement in the activity of SOD may be as a consequence of increased production of O2.- radicals. SOD converts relatively less toxic O2. - radicals to more toxic H2O2. Thus H2O2 scavenging activity is increased [22]. The Peroxidase activity showed varied responses with herbicide stress. Peroxidase activity was increased with the low dose of Oxyfluorfen (0.5 ppm) by 10% and this was continued linearly with 1 and 2 ppm of the herbicide doses (6.2 and 7.1 units minute-1 mg protein-1 as compared to the control (5.15 units minute-1 mg protein-1). but at the higher dose activity decreased by 21% as compared to the untreated samples (figure 2c). Increased activity of Peroxidase indicates more powerful mechanism of detoxification of overproduced H2O2. It can be depicted from the figure 2d that IAA oxidase activity increased initially showing an enhancement in the enzyme activity by 15% at 0.5 ppm Oxyfluorfen but it showed remarkable reduction in values at higher doses (10% to 73% reduction as compared to the untreated seedlings). The results were in accordance with the studies done previously in Vigna radiata [8]. Exposure of stresses like heavy metals, insecticides, pesticides, ultraviolet radiations etc are reported to induce production of active oxygen species that might trigger the responses of antioxidative defense systems [14]. The present piece of work has proved the Oxyfluorfen induced increase in the activity of enzymatic antioxidants like SOD, Catalase, Peroxidase etc. However the high doses affected the defenses adversely proving the loss and damage to recovery system. Initial increases in the enzymatic activities proved the extent of antioxidant potential of plants against free radical induced damage. It might provide suitable keys to assess the antioxidant potential of plants growing against various stresses. It can be said that the increase in the values of enzymatic antioxidants at high concentrations of herbicide might be due to their successful recovery [13]. The study also helps us to encourage the proper evaluation of the toxicity of pesticides before their uses in agricultural fields so that they might not contaminate our water and soil reservoirs and non-target organisms.

CONCLUSION According to the results obtained, it may be concluded that the herbicide affected the enzymatic antioxidants of the non-target plants (Ocimum species) severely. Initial increase in the enzymatic activities might be due to the increased activities of stress relief genes and their gene products. The results also indicated that the proper estimation and evaluation of the lethal doses of pesticides must be done prior to their use in agricultural lands to avoid any damage to non target organisms.

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Santosh Kumar Singh et al.


Vol.1, No.2, 74-79 (2010)

(a)

(b)

Fig.-1: Alterations in protein contents (a) and the level of lipid peroxidation (b) with enhanced exposure of Oxyfluorfen, herbicide. The values are means +SE and significantly different from control (p<0.05).

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Santosh Kumar Singh et al.


Vol.1, No.2, 74-79 (2010)

a

b

c

d

Fig.-2: Effect of increased exposure of Oxyfluorfen on Catalase (a), Superoxide dismutase (b), Peroxidase (c) and IAA oxidase activities; in Ocimum gratissimum plantlets. All the values were significantly different from their respective controls (p<0.05). Table-1: Effect of Oxyfluorfen on different growth parameters of Ocimum gratissimum. Parameters

Root length (cm)

Control (untreated) 3.89+0.80

0.5 ppm

Concentration of Oxyfluorfen 1.0 ppm 2.0 ppm

2.60+0.34

2.45+0.14 78

2.13+0.13

4.0 ppm 0.45+0.06 Santosh Kumar Singh et al.


Vol.1, No.2, 74-79 (2010) Shoot length 12.35+0.45 13.5+0.30 (cm) Root fresh mass 0.190+0.05 0.125+0.08 (g/root) Root dry mass 0.03+0.04 0.020+0.001 (g/root) Shoot fresh mass 0.98+0.36 1.01+0.036 (g/shoot) Shoot dry mass 0.098+0.28 0.110+0.040 (g/shoot) Chlorophyll 0.118+0.056 0.210+0.045 contents Carotenoid 0.054+0.008 0.068+0.04 contents *Values are means+ SE of triplicate samples

14.56+0.26

11.20+0.19

5.80+0.16

0.10+0.006

0.078+0.001

0.035+0.004

0.016+0.004

0.010+0.002

0.008+0.002

1.060+0.04

0.85+0.04

0.450+0.05

0.125+0.024

0.080+0.02

0.045+0.004

0.280+0.14

0.10+0.003

0.085+0.002

0.090+

0.044+0.03

0.020+0.006

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Agarwal S., Pandey V., Indian Journal of Plant Physiology 8:3 (2003) 264. Arnon D.I., Plant Physiology 24 (1949) 1â&#x20AC;&#x201C;13. Byrant S.D., Lane F.E., Plant Physiology 63 (1979) 696. Cedergreen N., Streibig J.C., Pest Management Science 61 (2005) 1152. Christopher D.N. et al., International Journal of Environmental Research and Public Health 7 (2010) 3298. Dai Q. et al., Physiol Plant 101 (1997) 301. Egashira T. et al., Plant Cell Physiol 30 (1989) 1171. Garg N. et al., Res Bull Punjab University Sci 39 (1988) 196. Giannopolitis C.N., Ries S.K., Plant Physiology 59(1977) 309. Gupta S.K. et al., Indian Journal of Experimental Biology 40 (2002) 765. Hasanuzzaman M. et al., American Journal of Plant Physiology 5 (2010) 295. Heath R.L., Packer L., Arch Biochem Biophysics 125 (1968) 189. Holt J.S., Plant Physiol Plant Mol Biol 44 (1980): 203. Kondo N., Kawashima M., Journal of Plant Research 113 (2000) 311. Lichtenthaler H.K., Welburn W.R., Biochem Soc Trans 11 (1983) 591. Lowry O.H. et al., J Biol Chem 193 (1951) 265. Meriles J.M. et al., Journal of Phytopathology 154 (2006) 309. Prasad S.M. et al., Biochem Cell Arch 2 (2002) 29. Prasad S.M. et al., Photosynthetica 43:2 (2005) 177. Prasad S.M., Zeeshan M., Environment and Experimental Botany 52 (2004) 175. Rao M.V., Paliyath G., Osmrod D.P., Plant Physiology 110 (1996) 125. Ratcliff A.W., Busse M.D., Shestak C.J., Applied Soil Ecology 34 (2006) 114. Shetty P.K. et al., Current Science 79 (2000) 1381. Singh PK., Arch Microbiology 89 (1973) 317. Vergaro O., Hunter J.G., Annals of Botany 17 (1952) 317. Young I.S., Woodside J.V., Journal of Clinical Pathology 54 (2001) 174. [ijCEPr-122/2010]

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79

Santosh Kumar Singh et al.


International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.2, 80-88 September-December, 2010

Synthesis, Sectral Characterization, Thermal and Anti-microbial Studies of New Binuclear Metal Complexes Containing Tetradentate Schiff Base Ligand P. Jayaseelan, S. Prasad, S.Vedanayaki and R. Rajavel* Department of Chemistry, Periyar University, Salem-636 011, Tamilnadu, India. *E-mail:drrajavel@rediffmail.com Article History: Received:26 November 2010 Accepted:11 December 2010

ABSTRACT A novel binuclear Schiff base ligand was prepared by the reaction between 3,3’diaminobenzidine with o-hydroxyacetophenone. The ligand and metal complexes have been characterized by elemental analysis,UV,IR,1H,magneticsucceptibility,conductivity measurements and EPR. The molar conductance studies of Cu(II),Co(II) and Mn(II) complexes showed non-electrolyte in nature where as Ni(II) complex showed electrolytic in nature. The spectroscopic data of metal complexes indicated that the metal ions are complexed with azomethine nitrogen and phenolic oxygen atoms. The binuclear metal complexes exhibit different geometrical arrangements such as square planar and octahedral arrangements. The microbial activities and thermal studies have also been studied. In microbial activity all complexes showed good microbial activity higher than ligand against gram positive,gram negative bacteria and fungus. Keywords: Schiff base, epr (electron spin resonance), o-hydroxyacetophenone, microbial activity ©2010 ijCEPr. All rights reserved

INTRODUCTION Schiff base complexes have been extensively investigated in recent and past years and have been employed in areas of catalysis[6], material chemistry[11], and magneto chemistry[9]. Binuclear Schiff base complexes have been of continuing interest because of their roles as biological models, catalyst for organic reaction as components in the formation of new materials[12].Copper complexes are considerably interesting due to their variety in coordination chemistry, technical application, catalysis, spectroscopic properties, anion selectivity, and their biological significance.[2,19]. A wide variety of cobalt(II) complexes are known to bind dioxygen more or less reversibly and are therefore frequently studied as model compounds for natural oxygen carriers and for their use in O2 storage, as well as in organic synthesis due to their catalytic properties under mild conditions [14]. For these applications, we are extending this field in synthesis of novel binuclear Schiff base complexes. In this paper the novel complexes derived from o-hydraxyacetophenone with 3,3’ diaminobenzidine were synthesized and characterized by elemental analysis, UV, IR, NMR, EPR and molar conductance. Thermal study has also been studied. The Schiff base ligand and its complexes were investigated for their anti-bacterial and anti-fungal properties. One gram-positive bacteria (Staphylococcus aureus), one gram-negative bacteria (Escherichia coli) and one fungus ( Aspergillus fumigatus) were used in this study to assess their antimicrobial properties.

MATERIALS AND METHODS Chemicals and Physical measurements All the chemicals used were of analytical reagent grade and the solvents were dried and distilled before use according to standard procedure [23]. O-hydroxyacetophenone and 3,3’-diaminobenzidine were purchased from Aldrich and were used as received.. Physical measurements (Apparatus and experimental condition) C,H and N contents were determined by Perkin Elmer CHN 2400 elemental analyzer, and IR Spectra was recorded in the range 4000 cm−1 to 100 cm−1 with a Bruker IFS66V in KBr and polyethylene medium for manganese complex and other complexes recorded in the range 4000 cm−1 to 400 cm−1. The molar conductance of the complexes in DMF (10−3 M) solution was measured at 27±3 °C with an Elico model conductivity meter. UV-visible spectra were recorded in DMF with Elico spectrophotometer 164 in the range of 200-800 nm. H1NMR spectra was recorded on Bruker 300 spectrophotometer using DMSOd6 as solvent. Chemical shifts are reported in ppm relative P. Jayaseelan et al.


Vol.1, No.2, 80-88 (2010) to tetramethylsilane, using the solvent signal as internal reference. EPR spectra were recorded at room temperature on JEOL JESTE100 ESR spectrometer. The spectrometer was operated at X-band (8-12 Ghz) with microwave power of 1mW. The room temperature magnetic moments were measured on a PAR vibrating sample magnetometer (Model-155). The TGA and DTA curves of the complexes were recorded on NETZSCH-STA 409PC thermal analyzer in heating rate of 10°K/min with the range of 50 °C to 900 °C. Anti-Microbial activity The Schiff base ligand and its complexes were investigated for anti-bacterial and anti-fungal properties. One Grampositive bacteria (Staphylococcus aureus), one Gram-negative bacteria (Escherichia coli) and one fungus (Aspergillus fumigatus) were used in this study to assess their antimicrobial properties. All complexes exhibit antibacterial and antifungal activities against these organisms and are found to be more effective than the free ligand. The antimicrobial activity was carried out at Progen Lab at Salem, Tamilnadu (India). The standard disc-agar diffusion method [1] was followed to determine the activity of he synthesized compounds against the sensitive organism S.sureus as gram positive bacteria and E.coli as Gram-negative and the fungus A.fumigatus. The antibiotic chloramphenicol was used as standard reference in the case of Gram-negative bacteria, Tetracycline was used as standard reference in case of gram-positive bacteria and clotrimazole was used as standard anti-fungal reference. The tested compounds were dissolved in DMF (Which have no inhibition activity), to get concentration of 50,100,150 µg/mL. The test was performed on medium potato dextrose agar contains infusion of 200 g potatoes, 6 g dextrose and 15g agar [7]. Uniform size filter paper disks (3 disks per compound) were impregnated by equal volume from the specific concentration of dissolved tested compounds and carefully placed on incubated agar surface. After incubation for 36 h at 27 °C in the case of bacteria and for 48h at 24 °C in the case of fungus, inhibition of the organism which evidenced by clear zone surround each disk was measured and used to calculate mean of inhibition zones. Synthesis of Ligand O-hydroxyacetophenone 4mmol was dissolved in methanol, and 3,3’diaminobenzedine 1mmol dissolved in methanol. Both were mixed together and reflux for 2 h at 90 °C. The resulting dark brown color solution was allowed to cool. The dark brown color product was obtained. This product was filtered and dried in air.Yield-85 %. M.p 220 °C Synthesis of complexes The metal complexes were prepared by reacting Copper(II)nitrate, Cobalt(II)nitrate, Nickel(II) acetate and manganese(II) chloride (2 mmol) and ligand (1 mmol) in acetonitrile were mixed separately and refluxed for about 2 h at 90 °C. The resulting product was filtered and dried over anhydrous P2O5. Color, yield, melting point were shown in the Table1.

RESULT AND DISCUSSION The color, melting point, elemental analysis and empirical formulae of the prepared complexes are listed in Table1. The results of the elemental analysis are in good agreement with the calculated values. The metal contents of the complexes were determined according to literature methods [3]. The binuclear complexes are stable in air, nonhygroscopic, insoluble in water and most organic solvents, but are easily soluble in DMF & DMSO. The electrolytic nature of the complexes is measured in DMF at 10−3M. The conductivity Λm lies between 13 to 8 Ω−1 cm2 mol−1 for copper, cobalt and manganese complexes. This result shows that the complexes were non-electrolyte in nature, and anions were coordinated inside the coordination sphere [16]. For nickel complex the conductivity lies in 100 Ω−1 cm2 mol−1. This is due to the presence of anion which is present in the outside of coordination sphere. The IR spectra of metal complexes and ligand were recorded in the range of 400 cm−1 to 4000 cm−1and for the manganese complex in the range of 100 cm−1 to 4000 cm−1. The azomethine group (C=N) stretching frequency of free ligand appears around 1604 cm−1. The frequency have been shifted to lower number in the range of 1590 to 1575 cm−1 is accordance with the coordination of the azomethine function to the metal ion for all the complexes. The lowering wave number is due to decrease in electron density of the azomethine group. In IR spectra of ligand OH was band observed at 3370 cm−1. A band observed at 1300 cm−1 was assigned at phenolic oxygen for free ligand. On complexation this band is shifted to higher frequency in the range 1308 to 1315 cm−1 and it is further supported by the disappearance of OH frequency at 3370 cm−1in all complexes. The absorption of the co-ordinated 81 P. Jayaseelan et al.


Vol.1, No.2, 80-88 (2010) ions at 1460-1450, 1300-1310 and 1040-1455 cm−1 suggest the presence of the co-ordinated nitrate groups[20]. The bands in the region 500 to 550 cm−1and 400 to 480 cm−1 were due to the formation of M-O and M-N bands[4]. The absorption at 1350 cm−1was assigned to uncoordinated acetate ion. The bands at 315 cm−1are due to the M-Cl[13]. Electronic spectra of all the complexes were recorded in DMF medium. The data are listed in the Table 3.The bands observed in 240 to 260 nm are due to π→π* transition of benzene ring and azomethine group [18]. The bands were shifted to higher range, which is due nitrogen and oxygen that involved in coordination with metal ion. The absorption bands are observed in the range of 320 to 370 nm due to n→π* transition from imine group corresponding to the ligand or metal complexes. The copper(II) binuclear complex shows a broad absorption peak at 642 nm and arises due to the d-d transition 2Eg→ 2T2g, of Cu(II) ion suggest that the copper ion exhibits a octahedral geometry [8]. Electronic spectra of the nickel(II) binuclear complex shows bands at 520, 635 nm which are assigned to 1Ag→1B1g and 1A1g→1A2g transitions, respectively suggesting an square planner arrangement around the nickel(II) complex [8]. The electronic spectra of binuclear cobalt(II) complexes exhibit absorption at 520, 616 nm are assigned to 4T1g (F) →4T1g(P), 4T1g → 4A2g transitions, respectively corresponding to cobalt(II) octahedral complex [8]. The Mn(II) binuclear complex shows bands at 540,584 nm, respectively are corresponding to 6A1g → 4 Eg(4D), 6A1g → 4T2g(4G) transitions which are compatible to an octahedral geometry around manganese(II) ion [8]. The structure of ligand was confirmed by H1 NMR. The triplet observed at 2.44 to 2.43 ppm was attributed to methyl group. The multiplet observed 6.61 to 7.83 ppm were due to aromatic system. The singlet at 14.95 ppm was assigned to proton of Ar-OH. EPR and magnetic studies EPR measurement has been made for copper complex using powder sample at room temperature, which could provide only value of giso and does not give g parallel and g perpendicular values. The giso value of the complex is 2.095. The value of giso shows that the copper (II) complex is in octahedral environment. The magnetic moments of copper(II), Cobalt(II) and Manganese(II) are 1.81, 4.14, 5.85 B.M respectively which are almost equal to the total spin only value. This indicates that the two paramagnetic centers are equivalent and there is no interaction between the metal centers. The pairing of electron is prevented by greater distance between two metal centers [21]. Thermal study In copper(II) binuclear complex one endothermic peak was observed at 120 °C which is assigned to the elimination of 4NO3- molecule at 30-210°C 23(23.46) %. Two exothermic peak and two endothermic peak were observed at 240,515 °C and 315,670 °C respectively which is attributed to loss of aromatic ligand group from 211 to 760°C 29.00(28.76) %. After 760 °C the decomposition was not completed. In nickel(II) binuclear complex one endothermic peak at 70 °C and another exothermic peak observed at 105 °C were assigned to the loss of four acetate ions at 30 to 200°C 22.00(22.80) %. One exothermic peak at 315 °C and another exothermic peak at 435 °C were due to loss of aromatic ligand groups from 201 to 615°C 30.00(29.37) %. One endothermic peak at 670 °C and one exothermic peak at 825 °C were assigned to loss of four CH3CN at 616-900°C 16.5(15.84) %. After 900 °C the decomposition was not completed (Table 4). Anti-microbial assay Biological activity of the ligand and a series of its metal complexes [Cu(II), Ni(II), Co(II) and Mn(II)] were screened for antibacterial activity against S.sureus as gram positive bacteria and E.coli as Gram-negative and the fungi A.fumigatus by using broth micro dilution procedures. From table(5), the Gram positive bacteria on all metal complexes were found to inhibit all tested bacteria at different rates and the activity as following order Co > Ni > Cu > Mn. In Gram negative bacteria also follows the same order and all complexes have higher bacterial activity than ligand. In fungal activity, the ligand showed activity against Aspergillus fumigatus and metal complexes show activity in the following order Cu > Co > Ni >Mn. It is known that chelation tends to make the ligand to act as more powerful and potent bacterial agent. A possible explanation for this increase in the activity upon chelation is that, in chelated complex, positive charge of the metal is partially shared with donor atoms present on ligands and there is an electron delocalization over the whole chelating ring. This, in turn, increases the lipid layers of the bacterial membranes. Generally, it is suggested that the chelated complexes deactivate various cellular enzymes, which play a vital role in various metabolic pathways of these microorganisms [5,10,15,17,22].

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Vol.1, No.2, 80-88 (2010)

CONCLUSION The ligand and its metal complexes were prepared and characterized by physio-chemical methods. In molar conductance of binuclear copper(II), cobalt(II) and manganese(II)complexes were non-electrolytic in nature whereas nickel(II) binuclear complex showed electrolyte in nature. The spectroscopic data of metal complex indicated that the metal ions are complexed with nitrogen of the imine and phenolic oxygen atoms. In magnetic moments studies complexes showed that there is no interaction between two metal centers. The TGA showed that in nickel complex anion coordinated outside the coordination sphere. Hence the copper(II),cobalt(II) and manganese(II) complexes have been octahedral structures. Nickel(II) binuclear complex has been in square planar structure. In antibacterial studies cobalt(II) binuclear complex showed good activity and in antifungal studies of copper(II) binuclear complex showed good activity. Table-1: Physical data and elemental analysis.

Complex

Color

Molecular weight g

Yield in %

Ligand

Dark brown Deep green Yellowish green Brown

686

Dark red

[Cu2(L)(NO3)4] [Ni2(L)]4+4Ac[Co2(L) (NO3)4] [Mn2(L)Cl4]

C% Found(Cal)

H% Found(Cal)

N% Found(Cal)

Metal % Found(Cal)

85

m.p. in °C 220

76.5(76.9)

5.4(5.5)

8.2(8.1)

-

1057

80

>270

49.4(49.9)

3.1(3.2)

10.3(10.6)

12.2(12.0)

1035

75

>270

60.4(60.2)

4.5(4.4)

5.2(5.4)

11.1(11.3)

1048

83

>270

50.1(50.3)

3.2(3.2)

10.3(10.6)

11.4(11.2)

933

77

>270

56.9(56.5)

3.5(3.6)

6.1(6.0)

11.2(11.7)

Table-2: IR Spectral Studies of Ligand and Metal Complexes (in cm−1)

Complex

O–H

C=N

M–O

M–N

Ligand

3370

1604

-

-

[Cu2(L)(NO3)4]

-

1579

525

442

[Ni2(L)]4+4Ac-

-

1575

533

450

[Co2(L) (NO3)4] [Mn2(L)Cl4]

-

1585

543

448

-

1590

537

456

No3-

M–Cl

CH3COO-

C-O

-

-

-

1300

1450,1310 ,1040 -

-

-

1315

-

1350

1308

-

-

1311

315

-

1309

1455,1302 1036 -

Table-3: UV Spectral and Magnetic Studies

Ligand

-

Λm Ω cm2 mol−1 -

[Cu2(L)

1.81

13

Complex

µ eff (B.M)

λmax in nm

−1

π→π*

83

n→π*

dd

240

320

-

255

353

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Vol.1, No.2, 80-88 (2010) (NO3)4] [Ni2(L)]4+4Ac-

-

100

260

365

520,635

[Co2(L) (NO3)4] [Mn2(L)Cl4]

4.41

11

258

350

520,616

5.85

08

252

370

540,584

REFERENCES 1. Anbu S., Kandasamy M.,Sudakaran P., Velmuragan V., Varghese J. J., Inorganic Biochemistry, 103 (2009) 401.

2. Chattopadhyay S., Drew M.G.B., Ghosh A., Inorganic Chimica Acta A, 359 (2006) 4519. 3. Feffery G.H., Basset J., Mendhan J., Denny R.J., Vogel’s quantitative chemical analysis, fifth ed., Longman Science and tech, Sussex UK (1989) 449.

4. Ferraro J.R., 1971 Low frequency vibrations of inorganic and coordination compounds 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

(New York:Plenumpress). Franklin T.J., Snow G.A., 1971 Biochemistry of Antimicrobial Action, 2nd ed.Chapman And Hall, London. Gianneshi N.C., Ngugen S.T., Mirkin C.A., Journal of American Chemical Society, 127(2005) 1644. Gross D.C., De Vay S.C., Physiology Plant Pathology, 11 (1977) 13. Lever A.B.P., 1984 Inorganic electronic spectroscopy Amsterdam, The Netherlands Elsevier. Lu.J.W., Huang Y.H., Lo S.I., Wei H.H., Inorganic Chemistry Communications (2007) 10. Mehmet So¨nmez .; Metin Celebi.; Ismet Berber.; European Journal of Medicinal Chemistry, 45 (2010) 1935. Morris G.A., Zhou H., Stern C.L., Nguyen S.T., Inorganic Chemistry, 40 (2001) 3222. Morris G.A., Ngugen S.T., Happ S.T., Journal of molecular catalysis A, 174 (2001) 15 Murphy B., Nelson J., Nelson S.M., Drew M.G.B., Yates P.C., J Chemical Society Dalton Transistion, 123 (1987) 127. Niederhoffer E.C., Timmons J.H., Martell A.E., Chemical Reviews, 84 (1984) 137. Prasad S., Jayaseelan P., Rajavel R., International Journal of Pharmacy and technology, 2 (2010) 694 Refat M.S., El-Korashy S.A., Kumar DN., Ahmad A.S., Spectrochimica Acta A, 70 (4) (2008) 898 Sellappan R., S.Prasad S., Jayaseelan P., Rajavel R., Rasayan Journal of Chemistry, 3 (2010) 556 Serbest K., Karabocek S., Degirmencioglu I., Guner S., Transition Metal Chemistry, 26 (2001) 375. Sharma V.B., Jain S L., Sain B., Journal of Molecular Catalysis A: Chemical, 219 (2004) 61-64 Uc¸an S.Y.. Mercimek B., Synthesis Reactivity Inorganic Metal-Organic Nano-Metal Chemistry, 35 (2005) 197. Upadhyay M.J.,Bhattacharya P.K.,Ganeshpure P.A.,Satish S.,Journal of Molecular Catalysis,73(1992) 277. Vedanayaki S., Jayaseelan P., Sandanamalar D., Rajavel R., Asian Journal of Chemistry, 23 (2011) 407 Vogel A.I., 1989 Text Book of Practical organic chemistry 5th ed. Longman London. [ijCEPr-128/2010]

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Vol.1, No.2, 80-88 (2010)

M=Copper(II) ,Cobalt(II) X=Nitrate M=Manganese(II) X=Chloride

M=Nickel X=Acetate

Fig.-1: Synthesis of Ligand and its metal complexes

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Vol.1, No.2, 80-88 (2010)

Fig.-2: TG/DTA of [Cu2(L) (NO3)4] complex

Fig.-3: TG/DTA of [Ni2(L)]4+4Ac- complex

Fig.-4: EPR Spectrum of [Cu2(L) (NO3)4] Complex

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Vol.1, No.2, 80-88 (2010)

Fig.-5a: Anti-bacterial studies (S.aureus gram positive) of Schiff base ligand and its metal Complexes.

Fig.-5b: Anti-bacterial studies (E.coli gram negative)) of Schiff base ligand and its metal complexes

Fig.-5c: Anti-fungal studies of (A.fumigatus) Schiff base ligand and its metal complexes 1=Ligand 2=copper complex, 3=nickel complex, 4=cobalt complex and 4=manganese complex Inhibition zone in cm.

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Vol.1, No.2, 80-88 (2010)

Table-4: Thermal Studies

Complex [Cu2(L) (NO3)4]

[Ni2(L)]4+4Ac-

Range °C 30-210 °C 211-760 °C >760 °C

DTA°C Endo-120 °C Endo-315,670 °C Exo240,515 °C

30-200 °C 201-615 °C 616-900 °C >900 °C

Endo-70 °C Exo-105 °C Exo-315,435 °C Endo-670,Exo825 °C

Estimated loss(Cal)% Mass loss % Total loss % 23.00(23.46) 52.00(52.22) 29.00(28.76)

22.00(22.80)

68.5(68.01)

30.00(29.37) 16.50(15.84)

Assignments 1. Elimination of 4NO3ions. 2. Elimination of aromatic ligand groups. 3. Decomposition in progress. 1. Elimination of four acetate ions. 2. Elimination of aromatic ligand groups. 3. Elimination of 4CH3CN groups. 4. Decomposition in progress.

Table-5: Anti-microbial Activities of Ligand and Metal Complexes Fungi

Bacteria

Sample

Gram-positive S.aureus 50 100 150 µg/mL µg/mL µg/mL

Gram-negative E.Coli 50 100 150 µg/mL µg/mL µg/mL

A.Fumigatus 50 100 150 µg/mL µg/mL µg/mL

Ligand

4

10

13

5

9

12

4

11

13

[Cu2(L) (NO3)4]

10

13

18

11

15

17

12

15

19

[Ni2(L)]4+4Ac-

9

11

16

10

14

15

12

14

17

[Co2(L) (NO3)4]

11

13

19

13

16

19

11

14

18

[Mn2(L)Cl4]

8

10

15

9

14

15

10

15

19

Inhibition zone in cms.

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International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.2, 89-94 September-December, 2010

Gas-Phase Structure and Rotational Barrier of Hydroxyphosphinecarbothialdehyde: A Computational Study Abdulhakim A. Ahmed Chemistry Departments, Faculty of Science, University of Garyounis, Benghazi, Libya *E-mail : dr_hakeem@garyounis.edu Article History: Received:19 November 2010. Accepted:20 December 2010

ABSTRACT The molecular structure of hydroxyphosphinecarbothialdehyde been studied in the gas phase. In addition, the interconversion of few isomeric tautomers of hydroxyphosphinecarbothialdehyde via intramolecular hydrogen transfer has been investigated by density functional calculations. The global isomeric structures, the transfer potential surfaces, rotational barrier, the harmonic frequency and transition states were calculated at the B3LYP/6-31++G(dp) // B3LYP/6-31G(d) levels of theory. Excluding the thiol forms with charge separating structures (CS1) and (CS2), the order of stability of these tautomers was 1b> 4b> 1a> 5b> 2b>> 3b, calculated at the single point level. Besides the hypervalent molecules 1b and 3b which was containing P=O bond character. The 1a, 2b and 5b are the thione forms, whereas 4b is the thiol form. The energy difference among the structures is no greater than 6.60 kcal mol-1. The reaction pathway for the interconversion between tautomers was through the transition structures TS1 toTS7. TS3 was involved in the rate-determining step. Apart from the TS3, the ring strain was clearly affecting the activation barrier; in addition the calculations revealed that the bond lengths and the atomic charges have a direct role in the stability of the structures. Keywords: Transition state, Activation barrier, Harmonic frequency and stability. ©2010 ijCEPr. All rights reserved

INTRODUCTION The organophosphorus compounds have been potent inhibitors of cholinesterase, their action is non-competitive and not readily reversible, and furthermore many applications of organophosphorus compounds were investigated previously [1-3]. The structures of hydroxyphosphinecarbothialdehyde which was the phosphorus analogues of thioformohydroxamic acid was constructed by replacement of nitrogen atoms by phosphorus atoms. Hydroxamic acids like their thiohydroxamic acid counterparts play important roles in analytical and biological chemistry [4]. The structure and the deprotonation of the derivatives of these compounds have been the subject of several theoretical investigations [5-7]. Many hydroxamates exhibit metalloproteinase inhibition activity [8,9]. The existence of the phosphorus analogues of thiohydroxamic acids has not been proved experimentally and therefore no structural details are available, furthermore, no theoretical calculation has been carried out on this compound. Earlier theoretical calculations have been shown that the substitution of the central carbon atom with the silicon in formohydroxamic acid significantly influences the structure and acidity by comparison with parent molecule [10]. The aim of this work is to provide a consistent and reliable set of gas-phase structures for hydroxyphosphinecarbothialdehyde using high level theoretical calculations. Additional interests are the molecular geometries, activation barrier and how these properties change upon isosteric substitution of nitrogen atom in thioformohydroxamic acid molecule by phosphorus.

MATERIALS AND METHODS The calculations were investigated the relative stabilities of the various tautomeric forms of hydroxyphosphinecarbothialdehyde, and then studied the reaction path leading from one to the other. The DFT calculations were performed with the B3LYP three parameter density functional, which includes Becke’s gradient exchange correction [11] and the Lee–Yang–Parr correlation functional.[12,13] The geometries of all conformers, products and transition states were fully optimized at the B3LYP/6-31G(d) level of theory. This was followed by harmonic frequency calculations at this level; the optimized structures were confirmed to be real minima by frequency calculation (no imaginary frequency). The vibrational frequencies were scaled by a factor of 0.9614 [14]. The

Abdulhakim A. Ahmed


Vol.1, No.2, 89-94 (2010) zero-point vibrational energy contribution is also considered. Single point calculations were then performed at the B3LYP/6-31++G(d,p) level for the geometries optimized at the B3LYP/6-31G(d) level. The SCF = Tight option was used in these calculations, performed using Gaussian 03 Revision C.02 [15].

RESULTS AND DISCUSSION The optimized eight local minimum structures, including the thiol forms with charge separating species (CS1), (CS2) and seven corresponding transition structures TS of intramolecular hydrogen transfer are shown in Figure 1. The full optimized geometry of the structures and the barrier height in the processes are given in Table 1. The calculations indicates that the main structures should be represented by three resonance structures, of which the later two are of major importance for the rotation barrier and charged isomer is suitable for the formation of metal complexes as in the scheme below. Consequently the reaction path produced three isomeric tautomers of organophosphours compounds namely hydroxyphosphinecarbothialdehyde (I), (hydroxyphosphoranylidene)methanethiol (II) and (mercaptomethylene)phosphine (III) structures. The 1b structure showed three-member ring involving CSP atoms, the structure has the longest r(C-S) bond length 1.90Ǻ and the lowest non-bonded distance between r(S---P) 2.08Ǻ. CS1 and CS2 have the shortest r(C-P) bond length which is equal to 1.66 Ǻ, in addition they have the highest sulfur charge +0.08 and +0.17 associated with CS1 and CS2 respectively. The seven transition structures (TS1 to TS7) was found on the potential energy surface of the reaction. TS3 is located on the reaction coordinate for 1b and 2b conversion; it is clear that it’s the transition state of highest energy in the path and is involved in the rate-determining step. The other transition structures are located for proton transfer between pair of structures. Most of the optimized structures were found to be non-planar. The only planar structure was 4b with SCPO angle been the highest (180°), and thus in this structure the phosphorus adopted a pyramidal orientation. The relative energies are listed in Table 2 and the schematic potential energy profile for the proton transfer is given in Figure 2. At the calculated level the 1b structure is calculated to be the most stable, and the energy values reported related to 1b in the hydroxyphosphinecarbothialdehyde. As expected the CS1 and CS2 have the highest energies which was 19.45 and 19.36 kcal mol-1 above the global minimum 1b respectively. The stability order for the local minimum structures are 4b>1a>5b>2b>3b. The energy analysis indicated that the difference in energies among the structures is no greater than 6.60 kcal mol-1. If the transformation of CS2 to 5b were to take place in one step, the only possible path would be the direct transfer of proton attached to sulfur to oxygen atom. It seems very difficult since the distance between the hydrogen and the oxygen is calculated to be 4.98 Å in the trans position and therefore, there is no sufficient kinetic energy to initiate such direct transfer. Thus interconversion between the CS2 and 5b form occur via a path (CS2→TS6→4b→TS7→5b) that has an overall activation barrier of 51.38 kcal mol-1. This result is in excellent agreement with the activation barrier of thiohydroxamic acid which has been reported as 52.20 kcal mol-1 [16]. Excluding TS3, the difference in transition states energies are clearly related to the ring strain of the structure, therefore three-member ring TS4, TS6 and TS2 have higher energy thanTS5 and TS7. The TS3 has a unique structure since the oxygen atom lie above the SCP plane almost by right angle 92.98°. The computed vibration frequencies are listed in Table 3. The computed infrared spectra of 2b and 4b tautomers are given in Figure 3. The calculated vibration frequencies are in conformity with the assignments of the experimental infrared spectra for related structures [16-18]. The computed vibrational frequencies for 4b structure showed a υ(SH) at 2561 cm-1 which in conformity with previously reported results [16]. The vibration stretching frequencies for (O-H), (C-H) and (P-O) are observed at 3629 and 3064 and 756 cm-1 respectively. On the other hand the 2b structure showed a very characteristic bands which are not observed for the thiol 4b structure. Thus the computed spectra had the υ(PH) and υ(S=O) at 2286 and 1020 cm-1 respectively. The DFT calculations showed that the νO-H of 2b is shifted to lower frequency in comparison with 4b form which has been attributed to intramolecular hydrogen bonding. The results for the other structures showed no different trend.

ACKNOWLEDGMENT I am grateful to Dr. M. C. Sameera at Glasgow University Scotland UK for his help with the Gaussian package.

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Vol.1, No.2, 89-94 (2010)

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17. 18.

A.F. Childs, D. R. Davies, A. L. Green and J. P. Rutland, Brit. J. Pharmacol., 10(1955)463. V. I. Yudelevich, E. V. Komarow and B. I. Ionin., Pharmace. Chem. J., 19(1986)382. M. Eddleston, L Szinicz, P. Eyer and N. Buckley., 95(2002)275. A. Chimiak, W. Przychodzen and J. Rachon, Heteoat. Chem., 13(2002)69. S. Bohm and O. Exner,Org. Biomol. Chem., 1(2003) 1176. S. Yen, C. Lin and J. Ho, J. Phys. Chem. A., 104(2000) 11771. D. Wu and J. Ho, J. Phys. Chem. A., 102(1998) 3582. M. Whittaker, D. C. Floyd, P. Brown and H. J. A. Gearing, Chem. Rev., 99(1999) 2735. H. Nagase and J. F. Jr. Woessner, 274(1999) 21491. M. Remko and J. Sefcikova, J. Mol. Struct., 258(2000) 287. A. D, Becke., Phys. Rev. A. 38(1988) 3098 C. Lee, W. Yang and R. G. Parr, Phys. Rev. B. 37(1988) 785. B. Miehich, A. Savin, H. Stoll and H. Preuss, Chem. Physi. Lett., 157(1989) 200. A. P. Scott and L. Radom; J. Phys. Chem.; 100(1996) 16502. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgomery, T. Jr. Vreven, K. N. Kudin, J. C. Burant,J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G.. Scalmani, N. Rega, G.. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W Chen, M. W. Wong, C. Gonzalez and J. A. Pople, Gaussian 03, Revision C.02.Gaussian, Inc., Wallingford CT, (2004). R. Kakkar, A. Dua, and S. Zaidi, Org. Biomol. Chem., 5(2007) 547. L. K. Ashrafullina, N. I. Monakhova and R. R. Shagidullin, J. Appl. Spectros., 51(1989) 690. T. C. Stringfellow, J. D. Trudeau and T. C. Farrar, 97(1993) 3985. [ijCEPr-134/2010] H

S

H

H

S

S H

H H

P H

P

P+ H

O I

O

-

II

H

O

III

Table 1: Optimized geometries of the structures (bond length in Angstroms). System 1a TS1 CS1 TS2 1b TS3 2b TS4 3b

C-S 1.64 1.68 1.76 1.82 1.90 1.69 1.63 1.64 1.62

C-P 1.83 1.78 1.66 1.71 1.79 1.87 1.83 1.80 1.85

P-O 1.64 1.55 1.50 1.49 1.49 1.59 1.66 1.60 1.49

S-H 1.35 -

91

r(S--P) 3.07 2.90 3.08 3.18 2.08 2.95 3.07 3.02 3.05

SCPO 10.2 12.8 10.4 20.6 64.1 75.6 26.3 158.7 141.0

â&#x2C6;&#x2020;Ea 22.61 40.22 80.17 63.79

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Vol.1, No.2, 89-94 (2010) TS5 CS2 TS6 4b TS7 5b

1.66 1.77 1.74 1.76 1.70 1.63

1a TS1 CS1 TS2 1b TS3 2b TS4 3b TS5 CS2 TS6 4b TS7 5b

1.87 1.66 1.73 1.68 1.73 1.83

1.50 1.45 1.60 1.68 1.67 1.68

1.36 1.35 1.35 -

2.83 2.95 3.11 3.10 2.75 3.06

Table 2: The energies of the structures in (kcal mol-1). ENERGY Single energy ZPE 0.040151 -854.647307 -854.747197 0.036136 -854.6112721 -854.7111624 0.038057 -854.6220243 -854.7226941 0.035200 -854.5574993 -854.6585888 0.042513 -854.6610503 -854.7536863 0.034134 -854.5270196 -854.625923 0.040139 -854.6409908 -854.7417737 0.034926 -854.540366 -854.6401057 0.039602 -854.6346042 -854.7299134 0.034256 -854.5669258 -854.666309 0.038470 -854.6239928 -854.7228383 0.034272 -854.5388451 -854.6409539 0.039542 -854.6445617 -854.7491448 0.036640 -854.587249 -854.6908118 0.040119 -854.642145 -854.7431635

154.0 172.1 171.2 180 32.7 21.1

39.91 51.38 36.60

Relative E 4.07 26.68 19.45 59.67 0.00 80.17 7.48 71.27 14.92 54.83 19.36 70.74 2.85 39.45 6.60

Table-3: Calculated vibrational frequencies in cm-1 for 2b and 4b structures.

2b 98 186 301 417 598 771 807 873 940 1061 1105 1283 2378 3074 3728

factorized 94 179 289 401 575 741 776 839 904 1020 1062 1234 2286 2929 3584

4b 90 186 209 301 337 710 774 786 956 1004 1093 1284 2664 3187 3775

92

factorized 87 179 201 289 324 683 744 756 919 965 1051 1234 2561 3064 3629

Abdulhakim A. Ahmed


Vol.1, No.2, 89-94 (2010)

H

H

S

H

S

S H

H

P

H

P

H

H

H

P + O

O

O

1a

TS1

-

CS1

H H

S

H

H

H

S P

H

+P

P

O

O

H

TS3

H

H

S

H

S

P O

H

2b

H

S

H

P

O

P

O

H

H

3b

TS4

H

S

H

S

H

H P +

S

H

O

P

O

H

H

H

H

P +

-

O

-

TS2

H

H

O

1b

H

-

S

CS2

TS5

TS6

H

S

H

S

H

S

H H P

P

O H

P

O

4b

O

H

TS7

H

H

5b

Fig.-1: The compound structures and the transition states interconnecting them.

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Vol.1, No.2, 89-94 (2010)

Fig.-2: Schematic potential energy profile for the proton transfer in the compound.

Fig.-3(a): The Computed infrared spectrum of the 4b structure.

Fig.-3(b): The Computed infrared spectrum of the 2b structure.

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International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.2, 95-99 September-December, 2010

RP-HPLC Method for the Estimation of Eletriptan in Pharmaceutical Dosage Forms D. Suneetha1 and A. Lakshmana Rao*2 1

A.K.R.G. College of Pharmacy, Nallajerla, A.P., India. V.V. Institute of Pharmaceutical Sciences, Gudlavalleru, A.P., India. * E-mail: dralrao@gmail.com 2

Article History: Received:20 November 2010 Accepted:21December 2010

ABSTRACT A reverse phase high performance liquid chromatographic method has been described for the estimation of eletriptan in its pharmaceutical formulations using an inertsil ODS C-18, 5 µm column having 250 mm × 4.6 mm I.D., in isocratic mode using acetonitrile:methanol:0.01M phosphate buffer in the ratio of 40:40:20 v/v/v. The detection was carried out using UV detector at 251 nm. Linearity of eletriptan was found to be in the concentration range of 200 to 1000 µg/ mL. The flow rate was 1.0 mL/min and the run time was 10 min. The developed method was validated with respect to linearity, precision, accuracy and specificity as per the International Conference on Harmonisation (ICH) guidelines. The mean recoveries were found to be with in the limits. The developed method was simple, fast, accurate and precise and has been successfully applied for the analysis of eletriptan in bulk sample and in pharmaceutical dosage forms. Keywords: Eletriptan, HPLC, Estimation, Linearity. ©2010 ijCEPr. All rights reserved

INTRODUCTION Eletriptan hydrobromide is a novel, orally active, selective serotonin 5-HT1B/1D receptor agonist and is second generation anti-migraine drug [1]. Eletriptan hydrobromide is chemically designated as (R)-3-[(1-methyl-2pyrrolidinyl)methyl]-5-[2-(phenylsulfonyl)ethyl]-1H-indole monohydrobromide (Fig. 1). Eletriptan hydrobromide used for the treatment of acute migraine headaches. Its pharmacological effects include the constriction of cerebral blood vessels and neuropeptides secretion blockade which eventually relieves the pain [2]. The pharmacokinetics and metabolism of eletriptan have been investigated in the rat, dog and human. In all three species, eletriptan was rapidly absorbed and extensively cleared by metabolism. The pathways of eletriptan metabolism are similar in the rat, dog and human and principal routes include pyrrolidine N-demethylation to N-desmethyl eletriptan, together with N-oxidation, oxidation of the pyrrolidine ring and formation of tetracyclic quaternary ammonium metabolites [3].

Fig.-1: Structure of Eletriptan hydrobromide

Literature survey revealed that very few analytical methods have been reported for the determination of eletriptan in pure drug, pharmaceutical dosage forms and in biological samples using HPLC [4,5] and LC-MS [6] techniques. The aim of the present work is to develop and validate a simple, fast, reliable and appropriate chromatographic method with UV detection for the determination of eletriptan in bulk drug and in pharmaceutical formulations. Confirmation of the applicability of the developed method was validated according to the International Conference on Harmonization (ICH) guidelines [7] for the determination of eletriptan in bulk sample and in tablet dosage forms.

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Vol.1, No.2, 95-99 (2010)

MATERIALS AND METHODS Drugs and Chemicals Acetonitrile and methanol (HPLC grade) were purchased from Merck Specialities Pvt. Ltd, Mumbai, India. Water (HPLC grade) was purchased from Loba Chemie, Mumbai, India. All other reagents used in the study were of AR grade. Eletriptan hydrobromide was kindly supplied by R.V. Labs, Guntur, India. Instruments A high performance liquid chromatograph (Shimadzu HPLC class VP series) with binary LC-20 AT VP pumps, variable wave length detector SPD-20 A VP, SCL-20 A VP system controller (Shimadzu) and a reverse phase inertsil ODS C-18 column (250 mm × 4.6 mm I.D., 5 µm particle size) was used for the estimation. The HPLC system was equipped with the software Class VP series version 5.03 (Shimadzu). All weighings were done on electronic balance (Shimadzu AY-120). Chromatographic conditions The mobile phase consisting of acetonitrile, methanol and 0.01M phosphate buffer (KH2PO4, pH adjusted to 4.4 with orthophosphoric acid) were filtered through 0.45 µ membrane filter, degassed and were pumped from the solvent reservoir in the ratio of 40:40:20 % v/v/v and was pumped into the column. The flow rate of mobile phase was maintained at 1.0 mL/min and detection was done by using UV detector at 251 nm with a run time of 10 min. The volume of injection loop was 20 µL prior to injection of the drug solution the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The column and the HPLC system were kept in ambient temperature. Procedure About 1 gm of eletriptan hydrobromide was accurately weighed and dissolved in methanol and finally makes up the volume up to 100 mL in volumetric flask with methanol so as to give 10 mg/mL solution. Subsequent dilution of this solution was made to obtain 100 µg/ mL. Linearity solutions containing 200, 400, 600, 800 and 1000 µg/mL were prepared from the above stock solution. Initially the mobile phase was pumped for 30 min to saturate the column there by to get the baseline corrected. Then solutions prepared as above filtered through 0.45 µ membrane filter and then 20 µL of the filtrate was injected each time in to the column at a flow rate of 1.0 mL/min. Evaluation of the drug was performed with UV detector at 251 nm. The peak area for each of the drug concentrations was calculated. The plot of peak area vs the respective drug concentration gives the calibration curve. The recovery studies were carried out by adding a known amount of eletriptan hydrobromide to the pre analyzed samples and subjecting them to proposed HPLC method. Estimation of eletriptan hydrobromide in pharmaceutical dosage forms Commercial formulations of eletriptan hydrobromide were not available in local market. Two formulations of eletriptan hydrobromide were prepared in-house. Twenty tablets each containing 40 mg of eletriptan hydrobromide were accurately weighed and powdered. From this powder mixture, an amount of the tablet powder equivalent to 40 mg of eletriptan hydrobromide was transferred to a 100 mL standard volumetric flask. A small amount of methanol was added and sonicated to dissolve. The volume was made up with methanol, filtered with a 0.45 µ membrane filter and the above filtrate solution was diluted to 100 mL with methanol and 20 µL of tablet sample solution was injected each time into the HPLC system and a chromatogram was obtained at a flow rate of 1.0 mL/min. The injections were repeated six times and the peak areas were recorded. The mean value of the peak area was calculated and the drug content in each tablet was quantified using the regression equation. The same procedure was followed for the estimation of eletriptan hydrobromide for both in-house formulations of tablet dosage forms.

RESULTS AND DISCUSSION The typical chromatogram for the proposed method is shown in Fig. 2. Linearity A good linear relationship (r=0.9998) was observed with a concentration range of 200-1000 µg/mL. The regression equation was constructed by linear regression fitting and its mathematical expression was y=1219.9+199.19x, where y is the peak area and x is the concentration of eletriptan (µg/mL). It was found that correlation coefficient and 96

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Vol.1, No.2, 95-99 (2010) regression analysis are within the limits. The peak areas of different concentrations are shown in Table 1. The peak areas of eletriptan hydrobromide were reproducible, as indicated by a low coefficient of variation.

Fig.-2: RP-HPLC chromatogram for eletriptan hydrobromide

Table-1: Standard graph for the estimation of eletriptan hydrobromide Concentration of eletriptan hydrobromide (µg/mL) Peak area 200 41003 400 81120 600 119227 800 163151 1000 199182 Specificity Placebo, blank and sample run were carried out to determine the specificity of the chromatographic method developed for eletriptan. The chromatograms indicate that the placebo (which had the excipients of the tablet formulation but not the drug) did not show any peak, indicating that there was no interference with or suppression of the peak at the retention time of eletriptan due to the commonly used tablet excipients. Ruggedness Ruggedness was established by determining eletriptan in the tablet formulation using two different chromatographic systems (Shimadzu, HPLC binary LC-20 AT VP pumps with SPD-20 A VP UV detector) and two different analysts. The RSD for analyst and inter-system variations were 0.63-1.22 % (limit < 2.0 %) and 0.84-1.38 % (limit < 2.0 %), respectively. This indicates that the method was rugged. Robustness Robustness of a method is its ability to remain unaffected by small deliberate variations in the method parameters. The following changes in the optimum parameter values were examined, the flow rate of the mobile phase (adjusted by ± 0.02 mL/min) and the detection wave length (adjusted by ± 1 nm).

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Vol.1, No.2, 95-99 (2010) System suitability For the determination of eletriptan, different compositions of mobile phases were employed. Finally the ratio was fixed to acetonitrile, methanol and 0.01M phosphate buffer (KH2PO4, pH adjusted to 4.4 with orthophosphoric acid) in the ratio of 40:40:20 % v/v/v, where eletriptan was eluted at 3.07 minutes with symmetric peak shape and shorter retention time. The system suitability parameters were given in Table 2. Table-2: Summary of validation parameters System suitability Results Theoretical plates (N) 8284 Linearity range (µg/mL) 200-1000 Retention time (min) 3.07 Tailing factor 1.68 Correlation coefficient 0.9998 LOD (µg/mL) 0.080 LOQ (µg/mL) 0.120

Intra-day and inter-day precision The precision was determined in terms of intra-day and inter-day precision. For intra-day precision evaluation, a standard solution of fixed concentration was injected at various time intervals and RSD was 1.14 % (limit RSD < 2.0 %). In addition, the day-to-day (inter-day) precision was studied by injecting the same concentration of standard solution on consecutive days and the RSD was 1.14 % (limit RSD < 2.0 %). The results are provided in Table 3. Table-3: Intra- and inter-day precision

Concentration of eletriptan (1000 µg/mL) Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Average Standard Deviation % RSD * RSD= relative standard deviation

Peak area Intra-day

Inter-day

183705 184352 186732 184869 184640 184859 1134.1 1.14

183891 184635 186947 184963 184831 185053 1136.8 1.14

Accuracy The accuracy of the method was assessed by recovery of eletriptan in the dosage formulation at three different concentration levels (50, 100 and 150 %) with reference to label claim of tablet. The recovery studies were replicated 3 times. The accuracy was expressed in terms of recovery and calculated by multiplying the ratio of measured drug concentration to the expected drug concentration with 100 so as to give the percentage recovery. Recoveries ranged from 98.36 to 99.55 %. The results are furnished in Table 4. Table-4: Accuracy data Concentration (Spike level) 50 % 100 % 150 %

Amount added (mg) 300 600 900

Amount found (mg) 305 602 904 98

% Recovery

Mean recovery

98.36 99.66 99.55

99.29 %

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Vol.1, No.2, 95-99 (2010)

Limit of detection (LOD) and limit of quantification (LOQ) The limit of detection (LOD) for eletriptan was 0.080 µg/mL while the limit of quantification (LOQ) for eletriptan was 0.120 µg/mL. Analysis of in-house formulations The amount of the drug present in the tablet dosage forms were calculated by using the regression equation obtained for the pure drug. The relevant results are furnished in Table 5. Table-5: Assay of tablet formulations Formulation Formulation-1 Formulation-2

Label claim (mg) 40 40

Amount found (mg) 40.12 39.86

% Amount found 99.70 100.35

In the proposed method, the retention time for eletriptan hydrobromide was found to be 3.07 minutes. Quantification was linear in the concentration range of 200-1000 µg/mL. The regression equation of the linearity plot of concentration of eletriptan over its peak area was found to be y=1219.9+199.19x, where y is the peak area and x is the concentration of eletriptan (µg/mL). The number of theoretical plates calculated was 8284, which indicates efficient performance of the column. The limit of detection and limit of quantification were found to be 0.080 µg/mL and 0.120 µg/mL respectively, which indicate the sensitivity of the method. The use of acetonitrile, methanol and 0.01M phosphate buffer pH 4.4 in the ratio of 40:40:20 % v/v/v resulted in peak with good shape and resolution. The high percentage of recovery indicates that the proposed method is highly accurate. The absence of additional peaks indicates no interference of the excipients used in the tablets.

CONCLUSION The validated RP-HPLC method employed here proved to be simple, fast, accurate, precise and sensitive. This developed method can be used for routine analysis of drug in bulk sample as well as in tablet dosage forms.

ACKNOWLEDGEMENTS The authors are grateful to R.V. Labs, Guntur, India, for providing the gift sample of eletriptan hydrobromide.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Milton K.A., Scott N.R., Allen M.J., Abel S., Jenkins V.C., James G.C., Rance D.J., Eve M.D., J Clin Pharmacol, 42(2002)528. Evans D.C., O’Connor D., Lake B.G., Evers R., Allen C., Hargreaves R., Drug Metab Dispos., 31(2003)861 Morgan P., Rance D., James C.G., Milton K.A. ,Headache, 37(1997)324 Zecevic M., Jocic B., Agatovonic-Kustrin S., Zivanovic L., J. Serb. Chem. Soc.,71(2006) 1195. Cooper J.D.H., Muirhead D.C., Taylor J.E., J. Pharm. Biomed. Anal.,21(1999) 787. Jocic B., Zecevic M., Zivanovic L., Protic A., Jadranin M., Vajs V., J. Pharm. Biomed. Anal.,50(2009) 622 ICH topic Q2 (R1), Validation of analytical procedures: text and methodology ,62(1997)27463. [ijCEPr-136/2010]

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International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.2, 100-102 September-December, 2010

Polarographic Studies on Interaction of 3-Hydroxy-3-Phenyl-1-p-Sulfonato (Sodium Salt) Phenyltriazene with Ni (II) in Aqueous Medium Neelam Pareek, Pooja Joshi, Dipen Upadhyay, G.P.Singh, Amit Bhandari, Anita Mehta, R. S. Chauhan and A. K. Goswami* Coordination Chemistry Lab, Department of Chemistry, M.L. Sukhadia University, Udaipur – 313001 (Raj.), India *E-mail : akumargoswami@rediffmail.com Article History: Received:26 November 2010 Accepted:8 December 2010

ABSTRACT Polarographic studies of Ni (II) - 3-hydroxy-3-phenyl-1-p-sulfonato (sodium salt) phenyltriazene (HPST) have been done in aqueous medium. Ni (II) forms 1:2 complex with HPST and the electrochemical reduction of the complex is diffusion controlled in nature between the pH 7.5 to 8.5. Well defined waves are obtained and the E1/2 shifts to more negative side with the addition of HPST. The reduction mechanism indicates two electron reversible reduction process and the stability constant Log β value found is 10.88. Keyword: 3-hydroxy-3-phenyl-1-p-sulfonato (sodium salt) phenyltriazene, Polarographic study. ©2010 ijCEPr. All rights reserved

INTRODUCTION In the present work HPST – Ni (II) complexes have been examined polarographically and stability constants calculated are in very good agreement with results of spectrophotometric studies of this system. The structure of 3hydroxy-3-phenyl-1-p-sulfonato (sodium salt) phenyltriazene (HPST) is given below. C 6H 5 N HO

N N SO 3 Na

Fig.-1: HPST

MATERIALS AND METHOD Apparatus and solutions A Systronics polarograph 1632 was used for obtaining current- voltage curves. Metal solution (1mM) was prepared using nickel sulphate heptahydrate and ligand solution was prepared by dissolving HPST (.01M) in double distilled water. Citric acid and Na2HPO4 solutions were used as buffer to maintain pH of test solution. Ionic strength was kept constant by using KCl as supporting electrolyte. Gelatin (.002%) was used as maximum suppressor. The D.M.E. had the following characteristicsm = 1.35 mg/sec. t = 1 sec per drop. The electrochemical behaviour of Ni (II) – HPST has been studied at d.m.e. in aqueous medium. Solution was deaerated by purging of oxygen free nitrogen through the polarographic cell. Temperature was maintained at 298 K. Synthesis 3-hydroxy-3-phenyl-1-p-sulfonato (sodium salt) phenyltriazene has been synthesized as per reported method[3]. In this method nitrobenzene (12.3 ml) was reduced with Zn dust (20 g) in the presence of NH4Cl (5.3 g) at 40-60°C to obtain phenyl hydroxylamine. The diazotized product was obtained by adding sodium nitrite (6.9 g) to sulphanilic acid (17.3 g) dissolved in 20 ml HCl and 100 ml water in small lots at 0-5°C under constant mechanical stirring. The diazonium compound was coupled with phenyl hydroxylamine at 0-5°C under mechanical stirring with occasional addition of sodium acetate solution for maintaining pH close to 5 during coupling process. After complete addition of diazonoum salt NaCl was added in sufficient quantity for salting out. The hydroxytriazene was obtained as

Neelam Pareek et al.


Vol.1, No.2, 100-102 (2010) yellowish brown micro crystals after crystallization from double distilled water. Its purity was checked by m.p. determination and CHN analysis. M.P. was found 157°C. The theoretical and experimental values of %C, %N and %H were obtained 43.2, 12.6, 3.6 and 42.4, 12.6, 3.6 respectively. Further the compound was subjected to IR spectral analysis which yielded the characteristics bands reported for hydroxytriazenes[4] and their values for νO-H, νN-H, δN-H and δN-OH are 3450, 3190, 1590 and 940 respectively. The IR spectra confirmed their presence establishing purity of compound. Determination of half wave potential of Ni (II) with supporting electrolyte 1 × 10-3 M Ni (II) solution in N/10 KCl has been used to obtain polarogram of Ni (II). This showed an E½ at -1.100V vs. SCE. Study of Ni (II) – HPST System Solution of Ni (II) 1 mM at various concentration of HPST was prepared from the stock solution and polarographed. The shift of half-wave potentials towards a more negative value with increasing concentration of ligand indicated complex formation and the diffusion current was found to decrease regularly with increase of HPST concentration. To avoid any precipitation, the polarographic behaviour of Ni (II) – HPST system has been studied in a solution containing a ten fold excess of ligand.

RESULTS AND DISCUSSION A single well defined wave was obtained for Ni (II) – HPST system between pH 7.5 to 8.5. Diffusion controlled nature of each wave was verified by id Vs C and id Vs

h plots. The slope value of linear plots of

 i   Vs Ede was found to be in the range of 30-32 mV, thereby showing the reversible nature of reduction log  id − i   process involving two electrons. Determination of coordination number The plots of half wave potential (E1/2 ) Vs log Cx (where Cx is equal to concentration of complex in mole/lit) have been found to be a straight line showing the formation of most stable complex. The value of j (coordination number) as determined by slope is 4. This shows that the complex composition is in 1:2 (M: L) ratio.

  d  E 1  .0591  2 c =− J d log C x n

(1)

Determination of stability constant The stability constant log β of the Ni (II) – HPST complex has been determined by classical method of Lingane[2], as this method is applicable for maximum coordination number and for the stability constant of highest complex formed. The ∆E1/2 has a linear correlation with ligand concentrations; which shows that there is only one complex formed in the solution. The following equation has been used to calculate the stability constant of the complex studied.

∆E 1 = 2

Here, ∆E 1

.0591 .0591 log β + j log C x n n =

(2)

Difference of half wave potentials of simple metal ion and complexed ion.

2

n Log β j Cx

= Number of transferred electrons = Stability constant of complex formed. = Coordination number = Concentration of ligand

Thus, the value of log β has been found to be 10.88. The value of log β fall in good agreement with results of hydroxytriazene - Ni (II) complexes studied spectrophotometrically[1,5]. The data are presented in Table I. 101

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CONCLUSION The results of Polarographic study of Ni (II) – HPST system is similar to the results found in spectrophotometric study of the same system.

Table-1: Polarographic study of Ni (II)–HPST system in aqueous medium Ni (II):1 mM S.No. 1 2 3 4 5 6 7 8 9

Cx (mol/lit) 00.00 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045

log (Cx ) -2.0000 -1.8239 -1.6980 -1.6020 -1.5228 -1.4559 -1.3979 -1.3467

E1/2 (-V vs SCE) 1.150 1.250 1.255 1.260 1.265 1.278 1.285 1.325 1.335

log β 11.38 10.85 10.52 10.30 10.42 10.40 11.52 11.65

REFERENCES Bhatt R., Rezaii B., Chauhan R. S., Goswami A. K., and Purohit D. N., J. Indian Chem. Soc. 73 (1996) 89. Lingane, J. J., Chem. Rev., 21 (1941) 1. Mehta C., Ph.D Thesis, “3-Hydroxy-3-alkyl or aryl-1-phenyltriazenes in copper determinations.” M. L. Sukhadia University, Udaipur (1992). 4. Purohit D. N., Spectro Chimica Acta, 41(A) (1985) 873. 5. Shekhawat R. S., Shekhawat R. S., Singh R., Chauhan R. S., Goswami A. K., and Purohit D. N., Asian J. Chem., 13(2) (2001) 783. [ijCEPr-127/2010] __________________________________________________________________________________________

1. 2. 3.

Highlights of RASĀYAN J. Chem. • • • • • Note:

It is a full text open access international journal of Chemical Sciences. Covers all fields related to Chemistry. Research papers will be published on the website and also in the printed version simultaneously. Manuscript is reviewed and published very quickly. Full text of the article is available on the site http://www.rasayanjournal.com all over the world. Reprints may be downloaded directly from the website. Papers can be submitted through e-mail to rasayanjournal@gmail.com. 1. Authors are requested to prepare the manuscript strictly according to RJC guidelines. 2. All contacts shall be by e-mail. All the authors are advised to have an email id. Manuscripts should be addressed to: Prof. (Dr.) Sanjay K. Sharma, Editor-in-Chief 23, ‘Anukampa’,Janakpuri, Opp. Heerapura Power Station, Ajmer Road, Jaipur-302024 (India) E-mail: rasayanjournal@gmail.com, drsanjay1973@gmail.com Phone: 0141-2810628(O), 09414202678(M)

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International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.2, 103-110 September-December, 2010

Application of 2-Hydroxyethyl Methacrylate Polymer in Controlled Release of 4-Aminosalicylic Acid: A Colon Targeted Prodrug Approach RajeshYadav*1, O.P.Mahatma1 and D.S.Rathore2 1

Department of Pharmaceutics, B.N Girls College of Pharmacy, Udaipur. Rajasthan Pharmacy College, Jaipur *E-mail:rajpharma21@gmail.com

2

Article History: Received:20 December 2010 Accepted:31 December 2010

ABSTRACT Acrylic type polymeric systems having degradable ester bonds linked to the 4-aminosalicylic acid (4-ASA) was synthesized and evaluated for colon targeted drug delivery. The obtained prodrug was characterized by FTIR,1HNMR, Melting point and Rf value. In vitro drug release study was conducted at pH 1.2, pH 7.4 and in rat fecal matter (pH7.4). Drug release in rat fecal matter at pH 7.4 was found to be most satisfactory. A burst release of 40.55% was observed in the first two hours followed by a sustained release over a period of 12 hours. A maximum of 91.64% of the drug was released from the prodrug and the time taken for 50% drug release (t50) was found to be nearly 3.5 hours. The best linearity for the prodrug was found in Higuchi’s equation, where r2 value was 0.9927, indicating the release of drug from prodrug as square root of time dependent process based on Fickian diffusion. The result suggest that the studied polymers in the present investigation can be used in the achievement of controlled drug release or slow release, prolongation of transit time and are useful as drug carriers for development of colon targeted delivery. Key words: 4-aminosalicylic acid, 2-hydroxy ethyl methacrylate, IBD, polymeric prodrug. ©2010 ijCEPr. All rights reserved

INTRODUCTION Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the mucosal membrane of the large intestine. Although many treatments have been recommended for IBD, they do not treat the cause but are effective only in reducing the inflammation and accompanying symptoms in up to 80% of patients. The primary goal of drug therapy is to reduce inflammation in the colon that requires frequent intake of anti-inflammatory drugs at higher doses. Sulfasalazine (5-ASA) is well known drug used in the treatment of IBD, [1]. In this study a prodrug of 4-aminosalicylic acid was synthesized, as 4-Aminosalicylic acid (4-ASA) differs from its 5-ASA counterpart by the position of the NH2 group and is considered as a second line anti-tuberculosis agent in the treatment of drug-resistant tuberculosis caused by Mycobacterium tuberculosis [2]. 4-ASA has been used in the treatment of mild to moderate ulcerative colitis in patients who are intolerant of sulfasalazine and in the treatment of Crohn’s disease; the drug is designated as an orphan drug by the FDA for use in mild to moderate ulcerative colitis [3-4]. 4-ASA has been claimed to be beneficial in the topical treatment of ulcerative colitis and in contrast to 5ASA, has no effect on arachidonic acid metabolism in human neutrophils or on the free radical 1,1-diphenyl-2picrylhydrazyl [5]. 4-ASA has been suggested as an effective treatment for both active and quiescent ulcerative colitis with lesser side effects [6]. The present work describes concept-based mutual prodrug design and synthesis of ester conjugates of 4-ASA with 2-hydroxy ethyl methacrylate (HEMA), for its colon-targeted delivery. This would facilitate delivery of intact prodrug to colon. Microbial degradation of ester prodrug by hydrolytic action of esterase secreted by the colonic microflora would further ensure the release of 4-ASA only in colon. This novel approach of polymeric drug derivatives, where the drug molecules are covalently linked to the polymeric backbone and linkages with limited stability in the physiological environment can be used. This approach should modify the pharmacokinetics of the drug and also obtain preferential localization. If a polymeric pro-drug wherein, the drug is covalently attached to the polymeric backbone is synthesized, and if it is capable of cleaving itself and release the drug in the alkaline environment (lower GIT) rather than the acidic environment (upper GIT), this should avoid direct contact with the gastroduodenal mucosa and thus prevent local imitation. Such a system would also prolong the pharmacological response of the drug thus leading to a good sustained release system. Dose dumping, associated with other conventional reservoir systems, can also be avoided here.

RajeshYadav et al.


Vol.1, No.2, 103-110 (2010) The objective of the present work is, therefore, to synthesize and evaluate polymeric pro-drug containing a nonsteroidal anti-inflammatory drug, namely 4-Aminosalicylic acid, for sustained and site- specific delivery and evaluate their in vitro release behaviour. For the synthesis of the polymeric pro-drug a polymeric drug carriers, namely, poly (hydroxyethyl methacrylate); [poly (HEMA)] was chosen because this carrier has a poor tendency to absorb biological species as a result of which they show good biocompatibility. They also have low interfacial energies in aqueous solutions. Moreover, they are also expected to be excreted as such since they are not absorbed by mucosal surfaces [7]. Among the several linkages that were proposed, the ester linkage was proposed to be used because it is perhaps the most appropriate covalent linkage for attaching the anti-inflammatory drugs (4-ASA) to the polymeric carriers. This is because, the ester linkage not only shows relatively stability in the acidic environment but also hydrolyses easily in physiological basic medium. Thus, the amount of drug released is more in the lower GI tract. Although hydrolysis of the pro-drug in the upper GI tract takes place, it is relatively much less since the residence time of the drugs in the upper GI tract is about two hours whereas, in the lower GI tract it is much higher[8]. The pro-drug was proposed to be synthesized by initially obtaining the monomeric drug derivatives, characterizing them and then polymerizing them by suitable polymerization techniques. This procedure was considered suitable for the preparation of the pro-drugs since it would result in polymeric pro-drugs with 100% degree of substitution which is required for higher yields of drug release[9]. 4-Aminosalicylic acid was proposed to be studied in the synthesis of the pro-drug because it is weak carboxylic acid with a pKa value of 3-4 range. In the gastric pH this is present as an unionized molecule. It is known that cell membranes are more permeable to unionized molecules than the ionized ones because of greater lipid solubility. The drug is, therefore, absorbed predominantly in the upper GI tract. Local irritation to the mucosal surfaces is more likely. The pro-drug was proposed to be synthesized with the aim that it would convey the release of the drug in the lower GI tract, in a site-specific manner thus avoiding local side effects[10]. The pro-drug has been evaluated for their in vitro drug release behaviour at pH 1.2 ,7.4 and in rat fecal matter at pH 7.4, stimulating the upper and lower GI tract to assess their capability to release the drug largely in the alkaline environment of the lower GI tract. Prodrug was fitted to various models such as zero‐order, first‐order, Higuchi, Hixcon‐crowel, Korsmeyer and peppas to ascertain the kinetic modeling of drug release. Hydrolysis & stability studies of the prodrug were also conducted to analyze the same.

MATRIALS AND METHODS 4-Aminosaliculic Acid was purchased from Acros Organics (Thermo Fisher Scientifics), 2-Hydroxy ethyl metha acrylate (HEMA) was obtained from Sigma Aldrich Life Sciences, New Delhi, Thionyl chloride was obtained from S.D fine chemicals, DMSO was purchased from Ranbaxy fine chemicals, Benzoyl Peroxide from Spectrochem Pvt. Ltd, Mumbai; all other chemicals were reagent grade or purer. Preparation and Characterization of 4-Aminosalicylic prodrug: - 2-Hydroxyethyl methacrylate (HEMA) [12.2 ml] was taken in a 250ml double necked round bottom flask fitted with a stirrer and condenser. The flask containing HEMA was then heated in an oil bath to 40°C. Thionyl chloride (7.2 ml] was added dropwise to the reaction flask using a dropping funnel. When the addition was complete, the temperature of the flask was raised to 700C and the flask was stirred at this temperature for 3-4 hours. The excess of thionyl chloride was removed by distillation and the chloro ethyl methacrylate derivative obtained as a liquid was distilled and used for further experiments. FTIR spectrum was done to analyze the chloro derivative of HEMA. The monomeric drug derivative was prepared by reacting the sodium salt of 4-Aminosalicylic Acid with Chloroethyl methacrylate. The 4-ASA (5.25gm) was taken in a double necked round bottom flask fitted with a stirrer and condenser, now Dimethyl sulphoxide (50ml.) was added to the flask and stirred until the drug was completely dissolved. Chloroethyl methacrylate (4.17ml) was then added and the flask was heated to 1200C and maintained at this temperature with constant stirring for 10 hours. After the reaction period the contents in the flask were poured into 500ml. of distilled water with vigorous stirring. A light brown precipitate was obtained which was allowed to settle overnight. The precipitate was filtered and dried. The monomeric drug derivative was purified by dissolving in 20ml acetone and reprecipitating it by pouring into 200ml distilled water. It was purified by  odeling ed on . The monomeric drug derivatives were  odeling ed o by FTIR spectra, thin layer chromatography, physical appearance and melting point. The monomeric dug derivative of hydroxyethyl methacrylate (HEMA) [5gm.] was taken in a double necked round bottom flask fitted with a stirrer and condenser. Dimethyl sulphoxide (50 ml.) was added and stirred to dissolve the monomeric derivative. Nitrogen gas was allowed to bubble through the reaction mixture throughout the reaction 104

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Vol.1, No.2, 103-110 (2010) period. The flask was heated in a water bath to 700 C. Benzoyl peroxide (0.1 g.) was then added to initiate  odeling ed on. The reaction was stirred for 8 hours. After the reaction period, the flask was cooled to room temperature and the contents were poured into 500 ml. of distilled water. A light brown precipitate was obtained. The precipitate was filtered and dried. The polymeric pro-drug thus obtained was purified by dissolving it in 20 ml. of acetone, reprecipitating it from distilled water and drying at reduced pressure to constant weight. The absence of HEMA was confirmed by a single spot in thin layer chromatography. Polymeric prodrug was confirmed by 1HNMR spectra, thin layer chromatography, physical appearance and melting point. Estimation of drug content Polymeric pro-drugs (100 mg.) synthesized was dissolved separately in 100 ml of 0.1 M. sodium hydroxide solution containing 2% w/v rat fecal material which were then kept overnight for complete release of the drug from its prodrugs by hydrolysis. From this 5 ml each were transferred separately into a 100 ml. volumetric flask and diluted upto the mark with acetonitrile and the absorbance was measured at their respective λmax (298nm). [11] In vitro drug release study In vitro drug release study was carried out by placing 100 mg prodrug containing a known amount of drug into a hard gelatin capsule. The study was carried out in different pH levels i.e. pH 1.2, and pH 7.4. Samples of 5 ml. were withdrawn at time intervals of 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 hours. The absorbances of the samples withdrawn after suitable dilution were measured against the reagent blank at their respective λmax (276 nm for pH 1.2 and 290 for pH 7.4) of the drug determined[12]. Release study in rat fecal matter (pH 7.4) The prodrug was dissolved separately in phosphate buffer (pH7.4) so that final concentration of the solution was 250µg/ml. Fresh fecal material of rats was weighed (about 1g) and placed in different sets of test tubes. To each test tube, 1ml of the prodrug solution was added, and diluted to 5ml with phosphate buffer (50 µg/ml). The test tubes were incubated at 37ºc, for different time intervals (0.5, 1, 2, 3, 4, 6, 8, 10, and 12 hours). For analysis, the concentration of released drug from prodrug was estimated on a double beam UV Spectrophotometer at 290nm of λmax through their calibration curve[13]. Kinetic modeling of drug release The dissolution profile of the prodrug was fitted to various models such as zero‐order, first‐order, Higuchi, Hixcon‐crowel, Korsmeyer and peppas to ascertain the kinetic modeling of drug release[14]. Characterization of hydrolysis product Twenty milligram of the polymer-drug conjugates was dispersed into 20 ml of buffered solution (pH 7.4) and maintained at 37 °C. After 24 h, the hydrolysis solution was sampled, neutralized with 1 N HCl and the solvent was removed in vacuum. The resulting crude product was treated with 10 ml of acetone and heated. The suspension was then filtered and the acetone solution was evaporated under reduced pressure. The residue was characterized by melting point measurement, Rf value (TLC method) and FTIR spectroscopy [15]. Stability studies The stability of the prodrug at room temperature (20oC-25 oC) was carried out over a period of 3 months. Sample was withdrawn at the end of 30, 60, and 90 days and analyzed for physical appearance, melting point, FTIR spectra and drug content while in vitro drug release study was carried out directly after 90 days to analyze the f1 (difference factor) & f2 (similarity factor) factors in release pattern[16].

RESULTS AND DISCUSSIONS The synthesis scheme for the polymeric prodrug is given in Fig.[1]. The FTIR spectrum of the chloro derivative of HEMA) shows the absence of any signals in the region of 3000 to 3500 cm-1 and the presence of characteristic bands at 1722 cm-1 (C=O), 1636 cm-1 (C=C) and 759 cm-1 (C-Cl), thus indicating the replacement of the hydroxyl group by chlorine. The monomeric drug derivative has a brown color monomeric drug derivative with melting point 1870C (while m.p of 4-ASA is 150.50C) and Rf value 0.71 (while Rf value of 4-ASA is 0.49), indicating the formation of new product. Prodrug which is synthesized from 4-ASA and HEMA, it shows all the peaks for 4-ASA & polymer, in FTIR spectra (Fig. 41) like 3386.77 cm-1 for O-H stretching,3226 cm-1for NH stretching, 3028.03 cm1 for C-H aromatic stretching, 2918.10 for C-H aliphatic stretching. A new peak that was absent in case of FTIR of pure drug was at 1714.60 cm-1 for C=O stretching of ester confirming the formation of ester bond. HEMA shows its intense bands at 1568 cm-1 for C=C, 875.62 cm-1for C-H stretching. Polymerisation of the monomeric drug derivative was characterized by a light brown color drug derivative with melting point 2120C (while m.p of monomeric drug derivative and 4-ASA are 1870C and 150.50C respectively) and Rf value 0.65 (while Rf values of monomeric drug derivative and 4-ASA are 0.71 and 0.49 respectively), indicating 105

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Vol.1, No.2, 103-110 (2010)

CH3 C ), 2.720 (S, 2H, -CH2=), the formation of new product. 1HNMR d DMSOd6, 300 MHz) δ 2.504 (S, 3H, 4.428 (t, 2H, OCH2), 5.69 (t, 2H, COOCH2), 6.29-7.803 (m, 3H, Ar ring), 7.860 (S, 1H, Ar-OH) Fig[2]. Estimation of drug content from the prodrug was done and each of 100 mg prodrug was contained 54.13 of drug content. While in vitro release profile is given in Fig.[3].The polymeric produg shows sustained & targeted drug release behaviour over a period of 12 hrs where a maximum of 16.51% of the drug was released over a period of 12 hrs while the drug release in the first two hours was only 5.86%. Drug release at pH 7.4 was found to be significantly more compared to pH 1.2. A maximum of 32.52% of the drug was released over a period of 12 hours while the drug release in the first two hours was only 11.70%. Drug release in rat fecal matter at pH 7.4 was found to be most satisfactory. A burst release of 40.55% was observed in the first two hours followed by a sustained release over a period of 12 hours. A maximum of 91.64% of the drug was released from the prodrug and the time taken for 50% drug release (t50) was found to be nearly 3.5 hours. In order to explain the in vitro drug release data and the sustained and site-specific nature of drug release envisaged in the present study, an understanding of the drug release mechanism is essential. Drug release in the case of the polymeric pro-drug  odeling ed should depend on the nature of the functional group undergoing hydrolysis and steric hinderence. There are two hydrolysable ester groups, one adjacent to the polymeric backbone and the other relegated to the pendant chain by a spacer group. It is obvious that hydrolysis of the latter ester group is much more facile than the one adjacent to the polymeric backbone because of steric reasons. In the alkaline environment of the lower GI tract, however, hydrolysis is mainly take place by microfloral enzymes. Where esterase enzyme released by microbes is expected to hydrolyse the ester linkage and releasing the free drug. In other words base hydrolysis reactions proceed to completion Even allowing for a certain amount of hydrolysis of the pro-drug in the acidic environment of the upper GI tract, the amount of drug released here should be much less because the residence time in the upper GI tract (stomach and duodenum) is less than two hours .As well as a certain amount of hydrolysis of the pro-drug taken place even in absence of microfloral enzymes due to a nucleophilic attack of the hydroxyl group on the electron deficient carbonyl carbon in the lower GI tract, but the most of drug release takes place predominantly in the lower GI tract only in presence of microfloral enzymes especially esterase which is expected to hydrolyse the ester linkage and releasing the free drug thus allowing for site-specific delivery . Kinetic  odeling of drug release was fitted to various models .The best linearity for the prodrug was found in Higuchi’s equation plot Fig. [4], where r2 value was 0.9927 that is close to one, indicating the release of drug from prodrug as square root of time dependent process based on Fickian diffusion. After hydrolysis, the residue was characterized & confirmed as 4-ASA Fig. [5], because its melting point and Rf value was found 150.50C and 0.49 respectively. While FTIR spectra shows 3396.41cm-1(OH stretching), -1 cm-1 3361.69cm (NH stretching), and 3332 (CH aromatic stretching), 1637.45 cm-1 (C=O stretching), 860cm-1(CH out cm-1 of plane bending), 1595 (C=C aromatic stretching) .These all are the characteristics of 4-ASA. Stability studies showed no significant change in the physical appearance, melting point, and its FTIR spectra. While in vitro drug release behaviour when compared by mean of similarity & difference factors ,the result indicated that their similarity factor was found to be more than 98 that was in the range between 50-100 (according to FDA), ensuring the sameness of products. While the difference factor was found to be less than .2 that was in the range between 0-15 (according to FDA), ensuring minor difference between two products. Therefore there is no significant change in the release pattern, indicating no changes occurred during storage.

CONCLUSION In this work, HEMA polymeric prodrug containing 4-ASA was synthesized. The structure of the obtained prodrug was characterized 1HNMR, FTIR, Melting point and Rf value. Release studies confirmed that the prodrug was stable and did not release (significantly lower) 4-ASA in aqueous buffers of pH 1.2 and 7.4. Thus, the objective of by passing the upper GIT without any free drug release was achieved. The hydrolysis was further studied in rat fecal matter [17] to confirm the colonic hydrolysis of ester prodrug, over a period of 12 hrs. A maximum of 92.46% of the drug was released from the prodrug and the time taken for 50% drug release (t50) was found to be nearly 3 hours. However, a certain amount of 4-ASA can be released by hydrolysis of the polymeric produg in small intestine (pH 5–7), but the amount of released 4-ASA in colon (pH 7.4) is very high. Therefore, the studied polymers in the present investigation can be used in the achievement of controlled drug release or slow release, prolongation of transit time and are useful as drug carriers for development of colon targeted delivery.

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Vol.1, No.2, 103-110 (2010) The prodrug therefore is expected to reduce the frequency of administration and avoid the gastrointestinal adverse effects associated with 4-ASA as a drug. Future aspect: In vivo work is in progress with respect to safety profile and in the management of ulcerative colitis that will be available in next issue. Step-1 CH3 H 2C

SOCl2

+

COO(CH 2)2OH

C

700C O

CH3 H 2C

C

COO(CH 2)2Cl

NaO

+

C

NH2

HO

1200C

DMSO CH3 H2 C

O

C

COO(CH 2)2

O

C

NH2

HO

Monomeric drug derivative Step-2 CH3 H2C

CH3

C

C H2

CO

C

n

CO

Redical Polymerisation O

O

(CH2)2

(CH2)2

O

O

CO

CO

R

R

NH2

R=

HO Polymerisation of monomeric drug derivative Fig.-1:Synthesis of the polymeric prodrug

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Vol.1, No.2, 103-110 (2010)

Fig.-2: 1HNMR spectra the polymeric drug derivative

HCLbuffer PH 1.2

Phosphate buffer of PH 7.4

Phosphate buffer of PH 7.4+ Rat fecal matter

100.00 90.00

Cumulative % Release

80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 0

2

4

6

8

10

12

Time (hrs)

Fig.-3:Graph showing a comparison of in vitro drug release profile of polymeric prodrug at pH 1.2, pH7.4 and in rat fecal matter

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Vol.1, No.2, 103-110 (2010)

100.00

y = 25.608x + 1.5819 R2 = 0.9927

Cumulative % Release

90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 0

0.5

1

1.5

2

2.5

3

3.5

4

Square root of time

Fig.-4: Higuchi release model of polymeric prodrug of 4-ASA

Fig.-5: FTIR Spectrum of 4-ASA (Product after hydrolysis of prodrug)

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Vol.1, No.2, 103-110 (2010)

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Nagpal Deepika., Singh R., Gairola Neha., Bodhankar, S.L., Dhaneshwar, S.,Indian Journal of Pharmaceutical Sciences, 68 (2006)171 Hassan M.M.A., Jado A.I., Zubair M.U., Analytical Profiles of Drug Substances, 10(1981) 20. Food and Drug Administration, Orphan designations pursuant to section 526 of the Federal Food and Cosmetic Act as amended by the Drug Act 28 (1996). D. Fady P., Seksik C., Wulfran R., Jian P., Marteau, Inflamm. Bowel Dis.,10 (2004)258. Neilson O.H., Ahenfelt R.I., Pharmacol. Toxicol.,62 (1988) 223. Schreiber S., Howaldt S., Raedler A., Gut ,35 (1994) 1081 Sjoergren J., Churchill Livingstone, Edinberg., 38 (1985) 45

Chourasia M.K., Jain S.K., J Pharm Pharm Sci., 6 (2003) 33

Babazadeh M,. Int Jour of Pharm. ,316 (2006) 68. Kane S.V., Bjorkman D.J., Rev Gastroenterol Disord, 3 (2003) 210. Chandrasekar M.J.N., Nanjan M.J., Suresh B. Indian.J.Pharma.Sci., 66 (2004) 66. Deelip V,.Mrudula Bele., Kashlival Nkihil,. Asian J.Pharmaceutics., 2 (2008) 30. Sunil K,. Jain Gopal., Rai D., Saraf K., Agrawal G.P. Pharmaceutical Technology.,1 (2005) 44 Chau DM,. Sylvestri M. F., Snyder S., Banker U. V., Makoid M. C., Drug Development and Industrial Pharmacy, 17 (1991) 1279 15. Mirzaagha Babazadeh.,Ladan Edjlali., Lida Rashidian.,J Polym Res., 14 (2007) 207 16. Yuksel N., Kanik AE., Baykara T., Int J Pharm. ,209 (2000) 57. 17. Rubinstein A., Nakar D., Sintov A., Pharm. Res., 9 (1992) 276. [ijCEPr-138/2010]

___________________________________________________________________________

Highlights of RASĀYAN J. Chem. • • • •

It is a full text open access international journal of Chemical Sciences. Covers all fields related to Chemistry. Research papers will be published on the website and also in the printed version simultaneously. Manuscript is reviewed and published very quickly. Full text of the article is available on the site http://www.rasayanjournal.com all over the world. Reprints may be downloaded directly from the website. Papers can be submitted through e-mail to rasayanjournal@gmail.com.

• Note: • Authors are requested to prepare the manuscript strictly according to RJC guidelines. • All contacts shall be by e-mail. All the authors are advised to have an email id. All correspondences should be addressed to: Prof. (Dr.) Sanjay K. Sharma

Editor-in-Chief 23, ‘Anukampa’,Janakpuri, Opp. Heerapura Power Station, Ajmer Road, Jaipur-302024 (India) Phone: 0141-2810628, 09414202678, 07597925412 E-mail: rasayanjournal@gmail.com, drsanjay1973@gmail.com

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International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.2, 111-113 September-December, 2010

Physico-Chemical Analysis of Some Groundwater Samples of Kotputli Town Jaipur, Rajasthan Ranjana Agrawal* Department of Chemistry, Birla Institute of Technology, MESRA(Ranchi) Extension Centre, Jaipur(Rajasthan)-302017, India *E-mail: ranjana1904@rediffmail.com Article History: Received:15 December 2010. Accepted:21December 2010

ABSTRACT Groundwater samples were taken from different locations of Kotputli town. Studies of Physico-chemical characteristics of groundwater quality based on Physic-chemical parameters have been taken up to evaluate its suitability for different purposes. Total 13 samples were collected. The quality analysis has been made through the pH, EC, TDS, Total Hardness, Sodium, Potassium, Calcium, Magnesium, Chloride, Sulphate, Nitrate, Fluoride and Alkalinity. Comparative studies of samples in different seasons were conducted. It was found that there is no appreciable change in the different parameters during rainy season. It was also analyzed that Electrical Conductivity and Total Dissolved Solids (TDS) were decreased in the rainy season, and Alkalinity, Total Hardness were increased after the rainfall. The results were compared with standards prescribed by WHO and ISI. A systematic calculation of the correlation coefficient has also been carried out between different analyzed parameters. Keywords: Drinking water, Groundwater, Physico-Chemical characteristics, Seasonal Variations. Š2010 ijCEPr. All rights reserved

INTRODUCTION Groundwater plays a vital role in the development of arid and semi-arid zones. Water is extremely essential for survival of all living organisms. The quality of water is vital concern for mankind since it is directly linked with human welfare. The quality of public health depends to a greater extent on the quality of ground water, which should be clean and fresh. In India, most of the population is dependent on groundwater as it is the only source of drinking water supply. The groundwater is believed to be comparatively cleaner and free from pollution than surface water. The modern civilization,  rbanization tion,  rbanization and prolonged discharge of industrial effluents, domestic sewage and solid waste dump cause the groundwater to become polluted and created health problems1. As the water is the most important component of eco-system, any imbalance created in term of amount, which is presence of impurities added to it can harm the whole eco-system2-4. Hence, there is always a need for and concern over the protection and management of groundwater quality5. Any imbalance in its physical or chemical properties beyond permissible limit would be harmful for the whole eco-system. Looking to the above aspects of groundwater contamination, the present study was undertaken to investigate the groundwater quality.

MATERIALS AND METHODS Water samples were collected in Polythene bottles of 2.5 and 2.0 liters from different locations of the town. The samples were collected from bore wells as well as from deep hand pumps. The samples were also collected in different seasons from same bore wells and tube wells. It was ensured that the concentrations of various water quality parameters do not changes in time that elapses between drawing of samples and the analysis in the laboratory. The bottles were thoroughly cleaned with Hydrochloric acid and then washed with tape water rendered free of acid and than washed with distilled water twice and again rinsed with the water sample to be collected and then filled up the bottle with the sample leaving only a small air gap at the top, stopper and sealed the bottle with paraffin wax. Some samples which were turbid or containing suspended matter were filtered at the time of collection6. All the glassware, casserole and other pipettes were first cleaned with tape water thoroughly and finally with de-ionized distilled water. The pipettes and burette were rinsed with solution before final use.

Ranjana Agrawal


Vol.1, No.2, 111-113(2010) The chemicals and reagent were used for analysis were of analar grade. The pH meter, conductivity meter, spectrophotometer, flame photometer instruments were used to analyze these parameters. The procedure for calculating the different parameters were conducted in the laboratory. The samples collected from Kotputli town were analyzed and results presented in Table-1 and correlation coefficient depicted in Table-2.

RESULTS AND DISCUSSION The value of pH was within maximum permissible limit in 10 samples and in 3 samples it was more than 9. It was ranging from 7.8 to 9.3. The Electrical conductivity was ranging from 1080 to 4637 Âľm/cm and in 76.9% samples the E.C. was out of maximum permissible limit. The Total Hardness (TH) of samples was ranging from 80 to 710. 15.4% samples were out of maximum permissible limit. Total Dissolved Solids (T.D.S.) value were ranging from 700 to 3200 and 7.7 % of the samples were out of maximum permissible limit. Calcium values were ranging from 16 to 96 and Sulphate values were ranging from 6 to 126. In Calcium and Sulphate both all the samples were within maximum permissible limit. Value of Potassium were ranging from 2 to 78 and 38.5% samples were out of maximum permissible limit. Fluoride contents were ranging from 0.50 to 8 and in 23.1 % samples it was more than maximum permissible limit. Nitrate value was ranging from 2 to 60 and 61.5% samples were having value more than maximum permissible limit. Alkalinity was ranging from 300 to 1243 and in 7.7% samples it was more than maximum permissible limit. Chlorine content was ranging from 15 to 1190 and in 7.7% samples it was more than maximum permissible limit. Magnesium was more than 100 in 4(30.8%) samples. Pre-monsoon and post monsoon samples were collected from different locations. It was found that there are no major changes in chemical properties of the samples. It was due to the fact that the rainfall in the state was below average. Ground water recharge was very less. Although in summer seasonal concentration of solids were higher than rainy season and at the same time Alkalinity, EC, Total Hardness and TDS of the samples shown down trend from summer season to rainy season.

CONCLUSION The study carried out in the Kotputli town on ground water samples conform that the pH level of ground water was within limit. In 10 samples were having Electrical Conductivity more than Maximum Permissible Limit. The value of T.D.S. were more than maximum permissible limit in 1 samples, these sample water are not suitable for drinking but samples which are having TDS more that 3000 water cannot be used even for irrigation purposes, only 1 samples were found which are having TDS more than 3000. Nitrate concentration was higher in 8 samples. Excess fluoride may lead to tooth decay and kidney disease. In 3 samples the fluoride was found more than maximum permissible limit and it is very high. The need for new institutional economics approach to deal with current and emerging problems has become very crucial. These problems have been addressed by various agencies in different states. The values of correlation coefficients will help in selecting proper treatment to minimize groundwater pollution. Table-1: Reading of Ground Water Samples Collected from Kotputli Town

pH EC Alkalinity TH Chloride Sulphate Nitrate Fluoride TDS Calcium Magnesium Sodium Potasium

S1 8.2 1296 390 250 150 10 60 0.8 840 16 30 204 2

S2 8.2 1296 390 250 150 10 60 0.8 840 80 44 110 15

S3 7.8 1188 380 300 140 26 30 0.8 770 62 20 144 10

S4 8 1080 330 240 125 6 32 0.5 700 35 68 42 32

S5 8 1080 350 260 140 6 50 0.5 700 34 70 48 12

S6 8.2 1392 470 320 146 18 16 2 924 23 38 210 4

112

S7 7.9 1948 530 290 315 50 6 0.7 1232 35 10 382 6.2

S8 8.3 3298 570 710 690 66 31.4 0.5 2240 56 150 400 4

S9 9.1 2345 540 130 430 48 2 1.2 1520 72 164 125 42

S10 8.8 4637 536 620 190 80 21.8 1.1 3200 96 152 632 6

S11 9.1 2022 519 80 330 20 40 8.4 1440 23 92 245 7

S12 8.6 3842 582 500 880 126 32.8 1.5 2720 46 96 570 78

S13 9.3 3246 1243 170 305 48 2 2 2160 24 136 410 4

Ranjana Agrawal


Vol.1, No.2, 111-113(2010) Table-2: Correlation Matrix for Different water Quality Parameters Cl-

SO4-2

NO3-

0.455 0.617 0.019

1.000 0.820 -0.198

1.000 -0.377

1.000

0.151

-0.406

0.064

-0.086

0.028

1.000

0.998

0.515

0.620

0.624

0.876

-0.360

0.069

1.000

0.067

0.407

-0.167

0.434

0,071

0.337

-0.049

-0.318

0.397

1.000

Magnesiu m Sodium

0.759

0.732

0.482

0.296

0.469

0.495

-0.372

0.105

0.724

0.387

1.000

0.392

0.916

0.487

0.638

0.563

0.862

-0.341

0.050

0.918

0.225

0.415

1.000

Potasium

0.148

0.243

-0.069

0.061

0.616

0.561

-0.064

-0.103

0.261

0.163

0.208

0.137

pH

EC

Alkalinity

TH

pH EC Alkalinity Total hardness Chloride Sulphate Nitrate

1.000 0.595 0.683 -0.185

1.000 0.535 0.616

1.000 -0.058

1.000

0.323 0.357 -0.388

0.613 0.872 -0.392

0.287 0.368 -0.574

Fluoride

0.532

0.039

TDS

0.594

Calcium

F-

TDS

Ca+2

Mg+2

Na+

K+

1.000

REFERENCES 1. 2. 3. 4. 5. 6.

Raja R E, Lydia Sharmila, J.Princy Merlin, Chritopher G, Indian J Environ Prot., 22(2)(2002) 137. Hem, J.D., Reort A. Taft Sanitary Engr. Centre, Report WEI-5,(1961). Kannan Krishnan, Fundamental of Environmental Pollution, S. Chand & Co. Ltd., New Delhi(1991). De. A.K., Environmental Chemistry, 4th Edn., New Age International Publishers(P) Ltd., New Delhi(2000). Patil P R, Badgujar S R. and Warke A M, Oriental J Chem.,17 (2) (2001)283. American Public Health Association American Water Works. Association & Water pollution Control Federation 1962 Standard Methods for Examination of water and waste water. 12th ed. APHA. New York(1962) [ijCEPr-135/2010]

__________________________________________________________________ Highlights of RASĀYAN J.Chem. • • • • • Note:

It is a full text open access international journal of Chemical Sciences. Covers all fields related to Chemistry. Research papers will be published on the website and also in the printed version simultaneously. Manuscript is reviewed and published very quickly. Full text of the article is available on the site http://www.rasayanjournal.com all over the world. Reprints may be downloaded directly from the website. Papers can be submitted through e-mail to rasayanjournal@gmail.com. 1. 2.

Authors are requested to prepare the manuscript strictly according to RJC guidelines. All contacts shall be by e-mail. All the authors are advised to have an email id. Manuscripts should be addressed to: Prof. (Dr.) Sanjay K. Sharma, Editor-in-Chief 23, ‘Anukampa’,Janakpuri, Opp. Heerapura Power Station, Ajmer Road, Jaipur-302024 (India) E-mail: rasayanjournal@gmail.com, drsanjay1973@gmail.com Phone: 0141-2810628(O), 09414202678(M)

113

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International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.2, 114-122 September-December, 2010

Novel Treatment Process for Dyeing Industries Waste Water and Recycling: A Green Approach to Treat Effluents Ashok Patni M/s. C&I Systems, Kota – 324 005 (Rajasthan) E-mail: ashokpatnijain@gmail.com Article History: Received: 10 December 2010 Accepted:27December 2010

ABSTRACT A ‘Batch Process’ for Quick treatment of dyehouse waste water (Fibre, Flock, Yarn, Fabric dyeing industries) containing left over dye liquor having disperse, acid, basic, vat, direct dyes etc. leveling agents, cationic, anonic, amphoric dyeing assistants for reclaimation and reuse for consecutive dyeing and/ or rinsing or the like in which oxidation process using ‘bleaching powder’ to decolourise the chromopheric functional group of the dye stuffs, is used to complete the process with in one hour time. Thus, treated decolourised reclaimed water at desired pH is then recycled back to the dye house for reuse. Keywords: Dyes, Textiles, Oxidation, pH, Antichlor, Chlorination. ©2010 ijCEPr. All rights reserved

INTRODUTION For Textile world, dyeing process is essentially desired for production of varieties of products as per market requirements. With dyeing process, use of dyes, dyeing assistance with water is must and there by generation of unreacted dyes in waste water causing disposal problems due to pollution and environment factors. The present article shows and describes very practical solution to the problem of disposal of waste water not only with• Short time (ony 1 hr treatment time instead of minimum 8 hrs) • Low cost (chlorination with 1/3rd cost of normal chemicals used for effluent treatment) • Energy saving ( only mixing with aeration for oxidation reaction during chlorination processas co mpared to process of primary, secondary clarifiers & disposal of solid mass). But also with Batch Process decided norms of input and out put of reactants, total solids, pH for recycling of treated water for dye house are easy to maintion and repeat time and again. Reduction of 70 to 80% in dye waste water discharge is possible with this process thus saving not only fresh water cost, cost of auxillaries to be added in each batch for dyeing process and last but not the least pollution problems arising with dyed effluent discharge for human being, penetraction to ground water sources for pollution further. Accordingly, it would be highly deisirable, both from econimic stand point and from an ecological stand point to provide of treating such waste waters so as to reduce or eliminate the necessity of their disposal.

MATERIALS AND METHODS For the proposed Novel treatment for ETP which is simple, quick, time and energy saving as compared to conventional effluent treatment the material required (Schamatic diagram No.1 and 2 ) are as under1.

Equalisation Tank pump capacityof 10,000 Ltrs/hr

2.

pH adjustment system

3.

Preparation Tank1000 Ltrs capacity for (Bleaching Powder) with Agitator

Desired as per Batch size of dyeing (50 KL capacity) with discharge waste water to be treated . (i) Hydrochloric acid to adjust pH 6.0-6.5 (ii) pH meter or pH paper strips for evaluation and checking. (i) Provision of drain valve to discharge settled material at bottom (ii) Feeding valve at bottom by gravity flow for prepared

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Vol.1, No.2, 114-122 (2010)

4. 5. 6. 7. 8. 9.

Aeration system suitable for Air diffusion Oxidation Tank with transfer pumps cap. 10,000 Litres/hr Anticlor Preparation/ Feed Tank (200 Ltrs cap) Casustic soda addition Tank (100 Ltrs cap) Sand & activated carbon filters capacity 10M³ cap each. Storage Tank 100 KL with transfer pump.

solution. Provided with Air diffusion caps for homogenisation and mixing of effluent for treatment and oxidising solution. Capacity 60 KL for treatment and oxidation. For removal of excess chlorine after oxidation process For pH adijustment of finally treated effluent For filtration of inpuritics & colour removal of treated effluent for recycling back to process. Treated effluent storage for dye house use.

Chemicals required for the oxidation and total effluent treatment process are(i) Bleaching powder. (ii) Anti chlor (iii) Hydrochloric acid (iv) Caustic soda lye. (v) Activated carbon.

- Stable Grade (Free chlorine Mnimum 33%) - Sodium sulphite or sodium bisulphite commercial grade) - Commercial grade 35% w/v. - 40% Solution - Granular 0.5—1.0 mm dia – M.B. value – 200

Preparation of solution Bleaching Powder: Solution is prepared by mixing 100 Kg stable bleaching powder in 1000 Ltrs fresh water and allowed to settle for 30 mts. Super- natant solution is used for oxidation process. Process Description From equalization tank, sample of dye house waste water is checked for pH (using pH strip). If pH of the sample is above pH 7.0, using commercial Hydrochloric acid the same is adjusted to pH 6.0 to 6.5 [ Quantity of Hydrochloric acid required is to be calculated from Laboratory experiment using 100c.c. waste water sample & addition of Hydrochloric acid from 0.1 ml graduated pipette of 5 ml capacity]. pH adjusted waste water is pumped in to Oxidation Tank through pump (discharge capacity 10,000 ltrs/hr) and bleaching powder solution prepared Supernatant solution is added in mixing tank with agitator and mixture is collected in the Oxidation Tank. During the Oxidation process following reaction takes placeCl2 + H2O Chlorine from bleaching powder

HCl + HOCl Hydrochloric acid Hypo chlorous Acid

The Hydrochloric acid (strong acid)  ecolon and suppresses ionization of the hypoclorous acid (weak acid). The latter is most effective as a  ecolonizati agent when it is in the unionized from. Such agent  ecolonizat the dyestuff while keeping it otherwise intact. The oxidation process is effective at pH 6.0—6.5 and typical treatment times are about 10- 40 mts. Amount of bleaching powder required to generate required chlorine necessary to declorise the dye-stuff can be calculated or adjusted to provide slightly in excess for complete oxidation process based on laboratory experimental calculations using 100 c.c pH adjusted waste water and bleaching powder super natent solution prepared as above]. It is essential to maintain desired pH since as the pH increases above about neutral pH, the  ecolonization reaction slows down. At pH 9.0 and above, no or little depolarization occurs. In oxidation tank Total waste water from equalisation Tank is I though mixing tank, bleaching powder solution addition and aeration/ mixing is continued in oxidation tank to complete decolourisation process in 10 to 40 mts. It may be noted that acid dyes require only 2 to 7 minutes to decolurise where as disperse dyes take 25 to 35 mts to decolourse . Fibre reactive dyes decolourise about as rapidly as acid dyes, but requires a higher doses of chlorine and higher reaction temperature. Direct dyes like disperse dyes require longer contact time for decolourisation. (Schematic diagram No.2 ) 115

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Vol.1, No.2, 114-122 (2010)

Dechlorination When oxidation process is complete the pale-yellow decolourised treated waste water checking for excess chlorine if present, may be done by Starch iodide strip (if turned violet shows presense of excess chlorine). Sodium bi sulphate or sodium sulphite solution is used as ‘antichlor’ to neutralise or destroy excess chlorine remaining after decolourisation. Estimation of antichlor to be added for neutralisation is done in the laboratory as per process given belowEstimation of excess chloride Reagent required 1. 0.1 N sodium thio sulphate: Dissolve 25gm Na2S2O3 , 5 H2O & dilute to 1000 Freshly boiled & cooled distilled water, add 5ml. chloroform as preservative. 2. Starch indicator: 1.0 gm starch powder is added to small quantity of water to prepare paste. Add 100ml boiling water and continue to boil for 2-3 mints,cool and use. 3. Solid potassium Iodide crystal. 4. Acetic acid. Procedure Pipette out 25 ml sample (to be tested for free chlorine) in conical flask and i. Add. Pinch of KI & 100 ml distilled water. ii. Add 10 ml. acetic acid and allow reaction to complete iii. Titrate free Iodine liberated with 0.1N Thio sulphate and record consumed (A) iv. Carry out blank using distilled water as above (B) Calculations Available Chlorine mg/litre = (A-B)*0.1*35450 Available Chlorine (ppm) = (A-B)*141.8 Note: 1. For free chlorine quick estimation “lovibond” comparator with chlorine disc of 1.0 to10 ppm range is desired for plant operations control. 2. Free chlorine is detected by Immersion of potassium –Iodide indicator Strip for quick evaluation. Final pH adjustment After dechlorination pH of treated water is checked for final adjustment with caustic-soda to 7.0-8.0 as per requirement of dye house. Filteration and activated carbon treatment Treated water with ph adjustment is now passes through sand filter and activated carbon treatment process and transferred to storage tank of 100 KL capacity for feeding to dye house for re-use in the process Re-claimed waste water The reclaimed waste water can be used to dye leather goods, cellulosic material, synthetic and natural fibre and like, in each instance reducing the over all cost of the dyeing process by reduction of elimination of the cost of a destroying dye molecules for decontimination puposes before passage of waste dye treating water to external sources. RESULTS AND DISCUSSIONS Bleaching powder was used for oxidation process to declorise the dye house effluent in five different industries process houses. 1. Nylon-66 Flocks dye house effluent quantity 100 KL/day (Brown – Black effluent discharge) pH 6.0 –6.5 temperature 50 -55°C. 2. Cotton/ viscose / Polyester staple fibre dye house effluent quantity 250 KL/day (Navy blue- Balck effluent discharge pH 8.5 –9.0) temperature 50 -55°C. 3. Polyester / cotton/ viscose shirting – suiting fabric dye house effluent quantity 1200 KL/day (Navy bluebroun effluent discharge pH 9.0 –10.0) temperature45 – 50°C

116

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Vol.1, No.2, 114-122 (2010) 4. 5.

Viscose/ cotton/ Polyester shirting – suiting fabric dye house effluent quantity 700 KL/day (Black – brown –Red effluent discharge pH 9.0 –9.5) tempoerature 45 – 50°C. Cotton /Polyester/ viscose shirting – suiting fabric dye house effluent quantity 1500 KL/day. (Navy Blueblack effluent discharge pH – 9.0 – 9.5) temperature 50˚- 55˚C.

Convventional process of ETP In all above 5 Industries dye houses (Flock, staple fibre, fabric) input effluent was having different pH (6.0 to 9.5 depending on dyes & process). After equalisation tank addition of lime slurry, Ferrous sulphate, Polyelectrolyte, decolourant gives alkaline pH 8.5 – 9.5 and floculation process In “Primary clarifier” sludge formation takes place which is removed through filter press. Over flow from Primary clarifier is passed to “secondary clarifier” where after aeration and desired pH adjustment treated effluent is passed through sand filter, activated carbon filter, R.O. process depending on need of Industry (Figure-3). Novel Oxidation Process Experiment and Observation For all five industries, using bleaching powder for oxidation, decided steps were used as under1. pH adjustmenty to 6.0 – 6.5 using Hydrocloric acid. 2. Supernatent solution of bleaching powder slurry was added to pH adjusted waste water in steps with continuous agitation and in 10 to 50 mts the waster water was declorised to pale yellow colour with out sediments. 3. Excess chlorine was checked with starch – iodide strip and in two cases neutralised with sodium sulphite solution based on lab. Expts calculation. 4. Treated effluent of (iii) was passed through sand and activated carbon filters and clear treated water of pH 6.5 7.0 was given for dyeig experimentation and assessments.. Dyeing Observation All five industries compared dyeing of flock, fibre, fabrics with different receipes with normal water (being used for dyeing by industry) and Novel oxidation process treated water. Recorded observation were as underi. Slight ‘tone variation’ was observed in dyed samples with Normal & oxidation process treated waters. ii. Dyed colours & samples were very much comparative with both types of water used. iii. No variation in dyed samples (stripes or uneven dyeing) was noticed. Novel oxidation process used for dyed liquor effluent is successful, cost benefit and environment friendly with less energy and time consumptions and that’s why it is truly a Green Approach to address the problem under consideration.(Table-1). Most of the dyeing house are using conventional effluent treatment process which has following critical stepsi. Transfering of dye liquor with various chemicals, (such as Ferous sulphate, lime, polyelectrolyte, decolourant etc) ii. Removal of sludge residue. iii. Continuous process of treatment with longer time. iv. Treated effluent is not colourless & further passed through Reverse-osmosis (R.O.) treatment process. v. Effluent Treatment cost is very high for recycling of waste water. The proposed Novel oxidation process is having One step effluent treatment for dye houses explained in details as above and definitely has following advantages over previous conventional processes being used. (a) Simple batch process which is having reproducible results of effluent treatment. (b) Economical with almost 1/3rd cost of chemicals used for treatment. (c) Energy conservation and operational cost is less since total treatment time is 50% of conventional process (Table-2). (d) Removal of solid residue is not involved in the process. (e) Treated effluent is pale-yellow in colour which can be processed through sand filter and activated carbon filter before using in dye house as recyled water. Addition of dyeing assistant will be less due to their presence in the treated effluent since dye molecules are only decolourised by oxidation process. Typical and Critical Issues

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Vol.1, No.2, 114-122 (2010) 1. 2. 3.

For light shades dyeing requirements, above treated water may give different tone of dyed shades which needs to be adjusted with receipes. Total dissolved solides from batch to batch could vary, adjustment of the same needed to be done for consistancy of results with addition of fresh water. For initial pH adjustments to 6.0 – 6.5, addition of hydrochloric acid will increase cost & increase in total dissolved solids (TDS).

CONCLUSION A Novel Process for treating dye house waste water for reclaimation and reuse of there of, said dyeing process normally in co-operating a levelling dyeing assistant and dye in a dye step there of, said waste water comprising an aqueous solution of dye stuffs selected from the group consisting essential acid and direct dyes, which process comprisesPlacing said waste water in to a equalisation tank,adding hydrochloric acid to adjust pH 6.0 –6.5 adding bleaching powder sulution equivalent to 1.0 to 1.5 Kg per 10,000 Lits of said waste water sufficient to decolourise the chromophoric functional group of said dye stuff retaining the site seeking substituents there of and keeping said dye stuff otherwise intact and then destroying any excess chlorine, to form a reclaimed solution containing decolourised but otherwise intact dye stuff, and using said decolorised dyestuff contained in said reclaimed solution in an acid or direct dye bath as a levelling assistant. A Novel process where in said halogen is chlorine using bleaching powder as oxidant, where in by oxidation with chlorine excess chlorine is destroyed by addting anticlor (sodium sulphite),the process is a batch process and used in flock, fibre, yarn, fabric dye houses,.Quick effluent treatment process with 1/3rd cost and energy saving as compared to conventional ETP process with environment friendly applcations and recylisation of waste water treated as above.

ACKNOWLEDGEMENTS We acknowledge with thanks experts of various fields including Dr. N.D. Sharma(M/S. H.B.N. Chemicals, Kota), Dr. M.L. Mittal(M/S. Chlorochem Ind, Kota), M/S. Sangam (India) Ltd., M/S. Sangam Bhilwara, Mr. Longinas Lasa(Los Angeles, U.S.A.) Mr. D.S. Bhadoria(M/S. RSWM Gulabpura, Rajasthan), Mr. Rajeev Saxena( M/S. Anant Syntex Ltd Bhilwara, Rajasthan), Mr. C.S. Sharma M/S. BSL Process, Bhilwara, Rajasthan), Mr. Sushil (M/S. Sona Processors, Bhilwara, Rajasthan), Mr. I.D. Khemchandani(M/S. Shriram Rayons, Kota), who helped us to carry out experimentation with encouraging guidelines and support to publish above article. Table-1: Comparision between Two Processes Conventional Process Oxidation process (Cost / day in Rs. ) (Cost / day in Rs. ) Chemicals Chemicals 1. Lime 700/1. HCl 13/2. Ferrous Sulphate 1100/2. Bleaching Powder 650/3. Poly 83/3. Sodoum Sulphite 100/4. D.C. 471/4. Caustic Soda 200/Total 2354/963/Man Power Man Power Three Operators per Day( Three shifts working) One Operators per Day( One shift working) Energy Consumption 1/3rd Energy consumption since entire Quantity of effluent is treated in one shift instead of three shifts. Concluding Remark: Using Bleaching powder for oxidation process,total cost of effluent Treatment is approximately 1/3rd as compared to conventional ETP Treatment.

REFERENCES 1.

U.S Deptt. Of Commerce, National Technical information Services (NTIS).Vol – 1, Technical Report EPA – 600/2 840709 March 1984, Jon F. Bergentha 118

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Vol.1, No.2, 114-122 (2010) 2. 3. 4. 5. 6. 7. 8. 9.

Groves, G.R., Bakley C.A. and Turrbull R.H., Journal Water Pollution Control Federation.,51(3)(1979)499. Fox, Edward. “Salem Installs Water Savings System,” Carpet and Rug Industry, July 1976 Montgomery, Vera, Modern Textiles, 57(8) (1976)36. Perkins, W.S.J.F. Judkins, Jr. W.D. Perry,Textile Chemist and Colorist, 12(8-10)(1980)182 Textile Systems Incorporated. It’s water reclamation system, “manufacturer’s brochure. United States Patent 3,807,947. “ Leveling Acid or Direct Dyes with chlorin Decolored Acid or Direct Dye Wastes,” April 30, 1974. United States Patent 3,927,965. “Direct and Acid Dye with the same Dye Chlorine Decolored as leveller”, December 23, 1975. Water Control Systems, Inc. “Water Reclamation system,”unpublished paper. [ijCEPr-132/2010]

Schematic Diagram No.1: Oxidation Process

119

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Vol.1, No.2, 114-122 (2010)

PH Adjustment by HCl to 6.0 – 6.5 EQUALIZATION TANK

DYEHOUSE WASTEWATER

BATCH TREATMENT TANKS Bleaching Powder Solution

OXIDATION TANK 1

Caustic Soda

OXIDATION TANK 2

Sodium Bisulphite Solution OXIDATION TANK 3

ANTICHLOR

STORAGE TANK

FILTER Sand + Activated

RECYCLE TO DYEHOUSE

Carbon

OXIDATION TREATMET SYSTEM SCHEMATIC

Schematic Diagram No.2

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Vol.1, No.2, 114-122 (2010)

CONVENTIONAL EFFULENT TREATMENT FLOW CHART EQUALISATION TANK

FLASH MIXER

DOSING OF LIME

FLOCUATOR DOSING OF Ferous sulphate poly electrolyte and De-colouring agent PRIMARY SETTLING TANK

SOLID SLUDGE REMOVAL

SECONDARY SETTLING TANK

HOLDING SUMP

PRESSURE SAND FILTER

ACTIVATED CARBON FILTER

TREATED WATER HOLDING SUMP

Dosing of chemicals (a)Lime 90% (b)Ferous sulphate © De-colouring agent (d) Poly electrolyte

400kg/day 400kg/day 300kg/day 0.5kg/day

Total treated water

80 KL

Raw effulent parameters (1) Ph (2) Temp (3) TSS (4) Hardness as ca & mg (5) TDS (6) COD (7) BOD

5 to 6 38°C 1200ppm 350ppm 2200ppm 1000ppm 600ppm

Fig.-3

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Vol.1, No.2, 114-122 (2010) Table-2: Comparision of Effluent Treatment Process S.No. 1. 2.

Process Involved Equalisation Tank Aearation Aearation of chemicals

-

3.

Sludge Removal

4.

Secondary clarifier

5.

Sand/ Activated Carbon filter

6.

Recycling back to dye house

Conventional ETP

Innovation ETP

Yes

No

Lime Alum/ Ferrous Sulphate Poly electrolyte Decolourant

Only bleaching Powder solution for Oxidation

Yes

No

Aearation

Addition of “Antichlor”

Yes

Yes

Not before sofner/ R.O.

Yes

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