ijCEPr, Vol.1(2), Sept.-Dec., 2010

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

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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 4-Aminosalicylic 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

114-122

INDEX of Contributors of this issue Authors Guidelines: for RASAYAN J. Chem. SUBSCRIPTION Form

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ijCEPr

International Journal of Chemical,Chemical,Chemical,Chemical, Environmental andEnvironmental andEnvironmental andEnvironmental and Pharmaceutical ResearchPharmaceutical ResearchPharmaceutical ResearchPharmaceutical Research

Volume-1, Number-2, September- December, 61-122 (2010) AUTHOR INDEX OF THIS ISSUE

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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

International Journal of Chemical, Environmental and

Pharmaceutical Research

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

S. Anbarasu et al.

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. Xaviers College, Palayamkottai. 2Department of Physics, Presidency College, Chennai 3Department of Physics, L.N. Govt. College of Arts & Science, Ponneri. 4Department of Physics, St. Josephs College, Trichy *E-mail: [email protected]

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

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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 distanced 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 dn

= (1)

Therefore, the velocity of ultrasonic longitudinal waves in the mixture is given by, U 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,

e x p

e x p1 0 0t c a l

t

U UP e r c e n ta g e d e v ia t io n X

U

=

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

2

e x p1c a lUM IP

U

=

(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.

2 Value is given by,

2e x p2

1

( )n t c a li c a l

U UU

=

(5)

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

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Step 1: To find the Mass of the water

Mass of the empty R.D bottle (m1) = 18.8701g Mass of the water +R.D bottle (m2) = 28.7041g Therefore, Mass of the water alone (M) is M= (m2-m1) = 9.8340g

Step 2: To find the Mass of the Liquid 1

Mass of the empty R.D bottle (l1) = 18.8701g Mass of the Liquid 1 + R.D bottle (l2) = 27.6804g Therefore, Mass of the Liquid 1 alone (L1) is L1= (l2-l1) = 8.8103g

Step 3: To find the Mass of the Liquid 2

Mass of the empty R.D bottle (l1) = 15.1564g Mass of the Liquid 1 + R.D bottle (l2) = 22.3990g Therefore, Mass of the Liquid 1 alone (L2) is L2 = (l2-l1) = 7.2426g

Relative Density Formulae:

R.D.= [ Mass of the liquid/ Mass of the water]X w (6) Where, w is density of water.

Note: Density of water(w) = 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 w

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 w

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 as-

MF1= [MW1/D1]= [88.10/0.8944] = 98.5017

The Mole Fraction for Liquid2 (MF2) is calculated as- MF2= [MW2/D2]= [72.11/0.7353] = 98.0688

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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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Fig.3

Fig.4

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Fig.5

Fig.6

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Fig.7

Fig.8

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Fig.9

Fig.10

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Fig.11

Observations of FTIR Spectrum Table-1

1 (EA) + 0.0 (2BUT)

0.9 (EA)

+ 0.1 (2BUT)

0.8 (EA)

+ 0.2 (2BUT)

0.7 (EA)

+ 0.3 (2BUT)

0.6 (EA)

+ 0.4 (2BUT)

0.5 (EA)

+ 0.5 (2BUT)

0.4 (EA)

+ 0.6 (2BUT)

0.3 (EA)

+ 0.7 (2BUT)

0.2 (EA)

+ 0.8 (2BUT)

0.1 (EA)

+ 0.9 (2BUT)

0 (EA)

+ 1 (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

3449.85 2369.39 2089.05 1739.40 1637.90 1409.82 1244.60 1110.28 1049.78 574.78

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

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

3778.13 3447.05 2928.83 2376.34 2093.87 1636.17 1418.32 1245.49 1113.89 1021.27 577.76

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

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

3765.76 3448.32 2988.64 2368.72 2094.28 1746.98 1635.26 1370.94 1242.92 1114.23 595.48

3906.73 3769.93 3449.06 2375.62 2089.51 1636.11 1405.90 1243.50 1116.30 1008.25 670.17

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

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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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. Everest F.A., Master handbook of acoustics (Mc Graw Hill, New York), 2000. 2. David N.J., Fundemendals and applications of ultrasonic waves (RC press, New York), 2002. 3. Ishwara Bhat.J and Shivakumar H.R., Indian J. Chem A.,37 (1998) 252 4. Ishwara Bhat.J and Shivakumar H.R., Indian J. Pure and Applied Physics, 38 (2000) 306. 5. Jeyakumar S, Karunanithi N and Kannappan V, Indian J. Pure and Applied Physics, 34 (1996) 761. 6. Prasad N, Singh R, Prakash O and Prakash S, Indian J. Pure and Applied Physics, 14 (1976) 676 7. Marewein B.L and Bhat S.N., Acustica, 58 (1985). 8. Carter S, J. Chem. Soc A, 404 (1968). 9. Sheshagiri Rao M.G., Indian J. Pure and Applied Physics, 9 (1971) 169. 10. Varma R.P. and Surendrakumar, Indian J. Pure and Applied Physics, 38 (2000) 96. 11. Yadav S.S., Singh Y.P.and Rajkumar, J. Indian Chem., 16 (1999) 20. 12. Sheshagiri K and Reddy K.C., Acoustica, 29 (1973) 59. 13. Ali A, Tiwari K, Nair A.K. and Chakravarthy V, Indian J. Physics B, 74 (2000) 351. 14. Upadhayay S.K., Indian J. Chemistry, 39 (2000) 537.

[ijCEPr-125/2010]

RASYAN Journal of Chemistry http:// www.rasayanjournal.com

ISSN: 0974-1496 (Print); ISSN: 0976-0083(Online)

Highlights of RASYAN 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 [email protected].




Dipen Upadhyay et al.

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 : [email protected]


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-p-sulphonato(sodium salt) phenyltriazene complex is 10.05. Keywords : Hyroxytriazene, Polarography, La (III)- HPST complex.


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 50-60C. 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 5C 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 5C 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-p-sulphonato(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

Vol.1, No.2, 71-73 (2010)


72

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 110-3 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.


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 A; 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 + 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 = Stability constant of complex formed, j = Coordination number, Cx = concentration of ligand.

Thus the value of log has been found to be 10.05. Polarographic data of Cu (II)- 3-hydroxy-3-m-tolyl-1-p-sulphonato (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 ) 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 point

%C %N %H

Th. 43.2 12.6 3.6 (C12H10N3O4.S.Na) H2O 180 C (d) Exp. 42.4 12.4 3.6

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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

1 0.00 0.00 1.900 - 2 0.01 -2 1.950 12.16 3 0.015 -1.8239 1.970 11.15 4 0.020 -1.6987 1.985 10.47 5 0.025 -1.6020 2.005 10.32 6 0.030 -1.5228 2.020 9.54 7 0.035 -1.4559 2.040 9.20 8 0.04 -1.3979 2.055 8.91 9 0.045 -1.3467 2.070 8.65

REFERENCES 1. Chakrovorty D., Majumdar A. K., J. Indian Chem.Soc., 54 (1977) 258. 2. Dutta R. L., Sharma R. S., J.Sci.Industr.Res.India, 40 (1981) 715. 3. Gorgi D. K., Chauhan R. S., et.al., Revs. Anal. Chem., 17(4) (1998) 223. 4. J. Lingane., J.Chem.Rev., 24 (1941) 1. 5. Kumar S., Goswami A. K., et.al Revs. Anal. Chem., 22 (2003) 1. 6. Purohit D. N., Talanta., 14 (1967) 207. 7. Purohit D. N., Nizammudin et.al.,Revs. Anal. Chem., 8 (1985) 76. 8. 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)

Highlights of RASYAN 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

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Santosh Kumar Singh et al.

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 2Department of Biotechnology, Sai Institute of Paramedical and Allied Sciences, Dehradun (U.K.) India. 3Department of Life Sciences, BFIT, Dehradun(U.K.) India. 4Department of Biotechnology, GCBMS, Dehradun(U.K.) India. *E-mail: [email protected]

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


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-(3-ethoxy-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.

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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 under-

Concentration 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 M riboflavin, 13 mM L- methionine, 0.05 M Na2CO3, (pH 10.2), 63 M p nitroblue tetrazolium chloride (NBT) and crude plant extract [9]. Reaction was carried out under illumination (75 mol 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 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

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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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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

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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

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Shoot length (cm)

12.35+0.45 13.5+0.30 14.56+0.26 11.20+0.19 5.80+0.16

Root fresh mass (g/root)

0.190+0.05 0.125+0.08 0.10+0.006 0.078+0.001 0.035+0.004

Root dry mass (g/root)

0.03+0.04 0.020+0.001 0.016+0.004 0.010+0.002 0.008+0.002

Shoot fresh mass (g/shoot)

0.98+0.36 1.01+0.036 1.060+0.04 0.85+0.04 0.450+0.05

Shoot dry mass (g/shoot)

0.098+0.28 0.110+0.040 0.125+0.024 0.080+0.02 0.045+0.004

Chlorophyll contents

0.118+0.056 0.210+0.045 0.280+0.14 0.10+0.003 0.085+0.002

Carotenoid contents

0.054+0.008 0.068+0.04 0.090+ 0.044+0.03 0.020+0.006

*Values are means+ SE of triplicate samples

REFERENCES 1. Agarwal S., Pandey V., Indian Journal of Plant Physiology 8:3 (2003) 264. 2. Arnon D.I., Plant Physiology 24 (1949) 113. 3. Byrant S.D., Lane F.E., Plant Physiology 63 (1979) 696. 4. Cedergreen N., Streibig J.C., Pest Management Science 61 (2005) 1152. 5. Christopher D.N. et al., International Journal of Environmental Research and Public Health 7 (2010) 3298. 6. Dai Q. et al., Physiol Plant 101 (1997) 301. 7. Egashira T. et al., Plant Cell Physiol 30 (1989) 1171. 8. Garg N. et al., Res Bull Punjab University Sci 39 (1988) 196. 9. Giannopolitis C.N., Ries S.K., Plant Physiology 59(1977) 309. 10. Gupta S.K. et al., Indian Journal of Experimental Biology 40 (2002) 765. 11. Hasanuzzaman M. et al., American Journal of Plant Physiology 5 (2010) 295. 12. Heath R.L., Packer L., Arch Biochem Biophysics 125 (1968) 189. 13. Holt J.S., Plant Physiol Plant Mol Biol 44 (1980): 203. 14. Kondo N., Kawashima M., Journal of Plant Research 113 (2000) 311. 15. Lichtenthaler H.K., Welburn W.R., Biochem Soc Trans 11 (1983) 591. 16. Lowry O.H. et al., J Biol Chem 193 (1951) 265. 17. Meriles J.M. et al., Journal of Phytopathology 154 (2006) 309. 18. Prasad S.M. et al., Biochem Cell Arch 2 (2002) 29. 19. Prasad S.M. et al., Photosynthetica 43:2 (2005) 177. 20. Prasad S.M., Zeeshan M., Environment and Experimental Botany 52 (2004) 175. 21. Rao M.V., Paliyath G., Osmrod D.P., Plant Physiology 110 (1996) 125. 22. Ratcliff A.W., Busse M.D., Shestak C.J., Applied Soil Ecology 34 (2006) 114. 23. Shetty P.K. et al., Current Science 79 (2000) 1381. 24. Singh PK., Arch Microbiology 89 (1973) 317. 25. Vergaro O., Hunter J.G., Annals of Botany 17 (1952) 317. 26. Young I.S., Woodside J.V., Journal of Clinical Pathology 54 (2001) 174.

[ijCEPr-122/2010]







P. Jayaseelan et al.

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:[email protected]


ABSTRACT A novel binuclear Schiff base ligand was prepared by the reaction between 3,3diaminobenzidine 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


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.


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 cm1 to 100 cm1 with a Bruker IFS66V in KBr and polyethylene medium for manganese complex and other complexes recorded in the range 4000 cm1 to 400 cm1. The molar conductance of the complexes in DMF (103 M) solution was measured at 273 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

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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 10K/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 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. 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 g/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 3,3diaminobenzedine 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, non-hygroscopic, 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 103M. The conductivity m lies between 13 to 8 1 cm2 mol1 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 1 cm2 mol1. 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 cm1 to 4000 cm1and for the manganese complex in the range of 100 cm1 to 4000 cm1. The azomethine group (C=N) stretching frequency of free ligand appears around 1604 cm1. The frequency have been shifted to lower number in the range of 1590 to 1575 cm1 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 cm1. A band observed at 1300 cm1 was assigned at phenolic oxygen for free ligand. On complexation this band is shifted to higher frequency in the range 1308 to 1315 cm1 and it is further supported by the disappearance of OH frequency at 3370 cm1in all complexes. The absorption of the co-ordinated

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ions at 1460-1450, 1300-1310 and 1040-1455 cm1 suggest the presence of the co-ordinated nitrate groups[20]. The bands in the region 500 to 550 cm1and 400 to 480 cm1 were due to the formation of M-O and M-N bands[4]. The absorption at 1350 cm1was assigned to uncoordinated acetate ion. The bands at 315 cm1are 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 pipi* 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 npi* 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 1Ag1B1g and 1A1g1A2g 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 4Eg(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 30-210C 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 760C 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 200C 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 615C 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 616-900C 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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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 %

m.p. in C

C % Found(Cal)

H % Found(Cal)

N % Found(Cal)

Metal % Found(Cal)

Ligand Dark brown

686 85 220 76.5(76.9) 5.4(5.5) 8.2(8.1) -

[Cu2(L)(NO3)4] Deep green

1057 80 >270 49.4(49.9) 3.1(3.2) 10.3(10.6) 12.2(12.0)

[Ni2(L)]4+4Ac- Yellowish green

1035

75 >270 60.4(60.2) 4.5(4.4) 5.2(5.4) 11.1(11.3)

[Co2(L) (NO3)4]

Brown 1048 83 >270 50.1(50.3) 3.2(3.2) 10.3(10.6) 11.4(11.2)

[Mn2(L)Cl4] Dark red 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 cm1)

Complex OH C=N MO MN No3- MCl CH3COO- C-O

Ligand 3370 1604 - -

- - - 1300

[Cu2(L)(NO3)4] - 1579 525 442 1450,1310,1040

- - 1315

[Ni2(L)]4+4Ac-

- 1575 533 450 - - 1350 1308

[Co2(L) (NO3)4]

- 1585 543 448 1455,1302 1036

- - 1311

[Mn2(L)Cl4] - 1590 537 456 - 315 - 1309

Table-3: UV Spectral and Magnetic Studies

max in nm Complex

eff (B.M)

m 1 cm2

mol1 pipi* npi* d d

Ligand - - 240 320 -

[Cu2(L) 1.81 13 255 353 642

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84

(NO3)4] [Ni2(L)]4+4Ac- - 100 260 365 520,635

[Co2(L) (NO3)4]

4.41 11 258 350 520,616

[Mn2(L)Cl4] 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., Vogels 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 (New

York:Plenumpress). 5. Franklin T.J., Snow G.A., 1971 Biochemistry of Antimicrobial Action, 2nd ed.Chapman And Hall,

London. 6. Gianneshi N.C., Ngugen S.T., Mirkin C.A., Journal of American Chemical Society, 127(2005) 1644. 7. Gross D.C., De Vay S.C., Physiology Plant Pathology, 11 (1977) 13. 8. Lever A.B.P., 1984 Inorganic electronic spectroscopy Amsterdam, The Netherlands Elsevier. 9. Lu.J.W., Huang Y.H., Lo S.I., Wei H.H., Inorganic Chemistry Communications (2007) 10. 10. Mehmet Sonmez .; Metin Celebi.; Ismet Berber.; European Journal of Medicinal Chemistry, 45 (2010)

1935. 11. Morris G.A., Zhou H., Stern C.L., Nguyen S.T., Inorganic Chemistry, 40 (2001) 3222. 12. Morris G.A., Ngugen S.T., Happ S.T., Journal of molecular catalysis A, 174 (2001) 15 13. Murphy B., Nelson J., Nelson S.M., Drew M.G.B., Yates P.C., J Chemical Society Dalton Transistion, 123

(1987) 127. 14. Niederhoffer E.C., Timmons J.H., Martell A.E., Chemical Reviews, 84 (1984) 137. 15. Prasad S., Jayaseelan P., Rajavel R., International Journal of Pharmacy and technology, 2 (2010) 694 16. Refat M.S., El-Korashy S.A., Kumar DN., Ahmad A.S., Spectrochimica Acta A, 70 (4) (2008) 898 17. Sellappan R., S.Prasad S., Jayaseelan P., Rajavel R., Rasayan Journal of Chemistry, 3 (2010) 556 18. Serbest K., Karabocek S., Degirmencioglu I., Guner S., Transition Metal Chemistry, 26 (2001) 375. 19. Sharma V.B., Jain S L., Sain B., Journal of Molecular Catalysis A: Chemical, 219 (2004) 61-64 20. Ucan S.Y.. Mercimek B., Synthesis Reactivity Inorganic Metal-Organic Nano-Metal Chemistry, 35 (2005)

197. 21. Upadhyay M.J.,Bhattacharya P.K.,Ganeshpure P.A.,Satish S.,Journal of Molecular Catalysis,73(1992) 277. 22. Vedanayaki S., Jayaseelan P., Sandanamalar D., Rajavel R., Asian Journal of Chemistry, 23 (2011) 407 23. Vogel A.I., 1989 Text Book of Practical organic chemistry 5th ed. Longman London.

[ijCEPr-128/2010]

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85

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

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

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86

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

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


87

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.

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


88

Table-4: Thermal Studies

Estimated loss(Cal)%

Complex

Range C

DTAC Mass loss % Total loss %

Assignments [Cu2(L) (NO3)4]

30-210 C 211-760 C

>760 C

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

23.00(23.46) 29.00(28.76)

52.00(52.22) 1. Elimination of 4NO3- ions. 2. Elimination of aromatic ligand groups. 3. Decomposition in progress.

[Ni2(L)]4+4Ac- 30-200 C

201-615 C 616-900 C

>900 C

Endo-70 C Exo-105 C

Exo-315,435 C Endo-670,Exo-

825 C

22.00(22.80)

30.00(29.37) 16.50(15.84)

68.5(68.01) 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

Bacteria

Gram-positive Gram-negative

Fungi

S.aureus E.Coli A.Fumigatus

Sample 50

g/mL 100

g/mL

150 g/mL

50 g/mL

100 g/mL

150 g/mL

50 g/mL

100 g/mL

150 g/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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Abdulhakim A. Ahmed

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 : [email protected]

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.


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.


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 Beckes gradient exchange correction [11] and the LeeYangParr 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

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

Abdulhakim A. Ahmed 90

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].


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 1.90 and the lowest non-bonded distance between r(S---P) 2.08. CS1 and CS2 have the shortest r(C-P) bond length which is equal to 1.66 , 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 its 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 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 (CS2TS64bTS75b) 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 (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 (PH) and (S=O) at 2286 and 1020 cm-1 respectively. The DFT calculations showed that the O-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.

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


REFERENCES

1. A.F. Childs, D. R. Davies, A. L. Green and J. P. Rutland, Brit. J. Pharmacol., 10(1955)463. 2. V. I. Yudelevich, E. V. Komarow and B. I. Ionin., Pharmace. Chem. J., 19(1986)382. 3. M. Eddleston, L Szinicz, P. Eyer and N. Buckley., 95(2002)275. 4. A. Chimiak, W. Przychodzen and J. Rachon, Heteoat. Chem., 13(2002)69. 5. S. Bohm and O. Exner,Org. Biomol. Chem., 1(2003) 1176. 6. S. Yen, C. Lin and J. Ho, J. Phys. Chem. A., 104(2000) 11771. 7. D. Wu and J. Ho, J. Phys. Chem. A., 102(1998) 3582. 8. M. Whittaker, D. C. Floyd, P. Brown and H. J. A. Gearing, Chem. Rev., 99(1999) 2735. 9. H. Nagase and J. F. Jr. Woessner, 274(1999) 21491. 10. M. Remko and J. Sefcikova, J. Mol. Struct., 258(2000) 287. 11. A. D, Becke., Phys. Rev. A. 38(1988) 3098 12. C. Lee, W. Yang and R. G. Parr, Phys. Rev. B. 37(1988) 785. 13. B. Miehich, A. Savin, H. Stoll and H. Preuss, Chem. Physi. Lett., 157(1989) 200. 14. A. P. Scott and L. Radom; J. Phys. Chem.; 100(1996) 16502. 15. 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).

16. R. Kakkar, A. Dua, and S. Zaidi, Org. Biomol. Chem., 5(2007) 547. 17. L. K. Ashrafullina, N. I. Monakhova and R. R. Shagidullin, J. Appl. Spectros., 51(1989) 690. 18. T. C. Stringfellow, J. D. Trudeau and T. C. Farrar, 97(1993) 3985.

[ijCEPr-134/2010]

P

SH

OHH P

SH

OH

H

P

SH

H

H

O+

-

I II III

Table 1: Optimized geometries of the structures (bond length in Angstroms). System C-S C-P P-O S-H r(S--P) SCPO Ea

1a 1.64 1.83 1.64 - 3.07 10.2 TS1 1.68 1.78 1.55 - 2.90 12.8 22.61 CS1 1.76 1.66 1.50 1.35 3.08 10.4 TS2 1.82 1.71 1.49 - 3.18 20.6 40.22 1b 1.90 1.79 1.49 - 2.08 64.1

TS3 1.69 1.87 1.59 - 2.95 75.6 80.17 2b 1.63 1.83 1.66 - 3.07 26.3

TS4 1.64 1.80 1.60 - 3.02 158.7 63.79 3b 1.62 1.85 1.49 - 3.05 141.0

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


TS5 1.66 1.87 1.50 - 2.83 154.0 39.91 CS2 1.77 1.66 1.45 1.36 2.95 172.1 TS6 1.74 1.73 1.60 1.35 3.11 171.2 51.38 4b 1.76 1.68 1.68 1.35 3.10 180

TS7 1.70 1.73 1.67 - 2.75 32.7 36.60 5b 1.63 1.83 1.68 - 3.06 21.1

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

Table 2: The energies of the structures in (kcal mol-1). ENERGY Single energy ZPE Relative E

1a -854.647307 -854.747197 0.040151 4.07 TS1 -854.6112721 -854.7111624 0.036136 26.68 CS1 -854.6220243 -854.7226941 0.038057 19.45 TS2 -854.5574993 -854.6585888 0.035200 59.67 1b -854.6610503 -854.7536863 0.042513 0.00

TS3 -854.5270196 -854.625923 0.034134 80.17 2b -854.6409908 -854.7417737 0.040139 7.48

TS4 -854.540366 -854.6401057 0.034926 71.27 3b -854.6346042 -854.7299134 0.039602 14.92

TS5 -854.5669258 -854.666309 0.034256 54.83 CS2 -854.6239928 -854.7228383 0.038470 19.36 TS6 -854.5388451 -854.6409539 0.034272 70.74 4b -854.6445617 -854.7491448 0.039542 2.85

TS7 -854.587249 -854.6908118 0.036640 39.45 5b -854.642145 -854.7431635 0.040119 6.60

2b factorized 4b factorized 98 94 90 87 186 179 186 179 301 289 209 201 417 401 301 289 598 575 337 324 771 741 710 683 807 776 774 744 873 839 786 756 940 904 956 919 1061 1020 1004 965 1105 1062 1093 1051 1283 1234 1284 1234 2378 2286 2664 2561 3074 2929 3187 3064 3728 3584 3775 3629

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


P

SH

OHHP

SH

OHH P

SH

OH

H

SH

HP

O

H

H

OP

H

S

H

SH

OP

H

H

SH

OP

H

H

SH

OP

H

H

SH

OP

H

H

H

OP

H H

S

P

SH

HO

H

P

SH

O

H

H

SH

OP

H

H

SH

OP

H

H

SH

OP

H

H

TS1 1a CS1

+

-

TS2

2b

1bTS3

-

+

TS4 3b

+-

TS5CS2

+

TS6

4b TS7 5b

-

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

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.




D. Suneetha and A. Lakshmana Rao

RP-HPLC Method for the Estimation of Eletriptan in Pharmaceutical Dosage Forms

D. Suneetha1 and A. Lakshmana Rao*2 1A.K.R.G. College of Pharmacy, Nallajerla, A.P., India. 2V.V. Institute of Pharmaceutical Sciences, Gudlavalleru, A.P., India. *E-mail: [email protected]

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 m 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 g/ 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.


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-2-pyrrolidinyl)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.

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

D. Suneetha and A. Lakshmana Rao

96


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 m particle size) was used for the estimation. The HPLC system was equi

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