IJCEPR, Vol.2, No.1, , 1-66, Jan.-April, 2011 by Sanjay K. Sharma

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Volume-2, Number- 1, January-April, 2011

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International Journal of Chemical, Environmental and Pharmaceutical Research Volume-2, Number-1, January-April, 1-66 (2011)

Contentsâ&#x20AC;Ś CHEMICAL RESEARCH Synthesis of 2-Substituted-1,3,4-Oxadiazole Derivatives Vijay V. Dabholkar and Nitin V. Bhusari

1-4

Molecular Interaction Studies between H-Bonded Ternary Mixtures of p-Cresol with simple Aldehydes in Cyclohexane at Different Temperatures M. Aravinthraj, S. Venkatesan and M. Kamaraj

5-11

Chemical Analysis on Different Oils Use in Tyre Tread Cap Compound N. Kumar, R.K. Khandelwal, P.L. Meena, K. S. Meena, T.K. Chaki, D.K. Mahla and S. Dasgupta

12-19

Infra Red Spectral and X-ray Diffraction Study of Fe (II), Co(II), Cu (II), Metal Chelates with N1-(5, 6-dimethoxypyrimidin-4-yl) Sulphanilamide Jitendra H. Deshmukh and M. N. Deshpande

20-25

Comparative Study between soda Lignin and soda Anthraquinone lignin in terms of Physiochemical Properties of Ipomoea carena Preeti Nandkumar

26-29

ENVIRONMENTAL RESEARCH Use of Millet Husk as a Biosorbent for the Removal of chromium and Manganese Ions from the Aqueous Solutions. Manju Chaudhary

30-33

Study Regarding Lake Water Pollution with Heavy Metals in Nagpur City (India) P.J. Puri, M.K.N. Yenkie, S. P. Sangal, N.V. Gandhare, G. B. Sarote

34-39

Knowledge, Attitude and Practices regarding Waste Management in Selected Hostel Students of University of Rajasthan, Jaipur Lalita Arora and Sunita Agarwal

40-43

Utilisation of Thiocyanate (SCN-) by a Metabolically Active Bacterial Consortium as the Sole Source of Cellular Nitrogen Yogesh B. Patil

44-48

ii Construction of an open loop temperature control system for thin film fabrication in PC based instrumentation. K.Tamilselvan* , K.Anuradha, S.Deepa , O.N.Balasundaram and S.Palaniswamy

49-51

PHARMACEUTICAL RESEARCH Accumulation of Natural Antioxidants in Ferns Exposed to Mutagenic Stress Alok Kr. Singh, Santosh Kr. Singh, Satish K. Verma, H.V. Singh, A.K. Mishra, Pavan K. Agrawal, Abhishek Mathur and Md. Aslam Siddiqui

52-55

Reversed Phase HPLC Analysis of Valsartan in Pharmaceutical Dosage Forms V. Bhaskara Raju and A. Lakshmana Rao

56-60

A Review on Fibrinolytic Enzyme: Nattokinase Haritha Meruvu and Meena Vangalapati

61-66

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

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.

iii

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International Journal of Chemical, Environmental and Pharmaceutical Research Volume-2, Number-1, January-April, 1-66 (2011) AUTHOR INDEX OF THIS ISSUE A. Lakshmana Rao,56 A.K. Mishra,52 Abhishek Mathur,52 Alok Kr. Singh,52 D.K. Mahla,12 G. B. Sarote,34 H.V. Singh,52 Haritha Meruvu,61 Jitendra H. Deshmukh,20 K. S. Meena,12 K.Anuradha,49 K.Tamilselvan,49 Lalita Arora,40 M. Aravinthraj,5 M. Kamaraj,5 M. N. Deshpande,20 M.K.N. Yenkie,34 Manju Chaudhary,30 Md. Aslam Siddiqui,52 Meena Vangalapati,61 N. Kumar,12

N.V. Gandhare,34 Nitin V. Bhusari,1 O.N.Balasundaram,49 P.J. Puri,34 P.L. Meena,12 Pavan K. Agrawal,52 Preeti Nandkumar,26 R.K. Khandelwal,12 S. Dasgupta,12 S. P. Sangal,34 S. Venkatesan,5 S.Deepa,49 S.Palaniswamy,49 Santosh Kr. Singh,52 Satish K. Verma,52 Sunita Agarwal,40 T.K. Chaki,12 V. Bhaskara Raju,56 Vijay V. Dabholkar,1 Yogesh B. Patil,44

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International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 1-4 January-April, 2011

Synthesis of 2-Substituted-1,3,4-Oxadiazole Derivatives Vijay V. Dabholkar* and Nitin V. Bhusari Organic Research Laboratory, Department of Chemistry, KC College, Churchgate, Mumbai-20 1 Mumbai University, Maharashtra, Mumbai (India) *E-mail: nitin.bhusari7@gmail.com Article History: Received: 30 December 2010 Accepted: 20 January 2011

ABSTRACT Diethyladipate on reaction with Hydrazine hydrate gave Succinohydrazide (1) which on further treatment with Carbon disulfide, Aromatic aldehydes and Cynogen bromide yielded 1,2[di-(2-Mercapto-1,3,4-oxadiazole-5yl)] ethane (2), 1,2[di-(2-Phenyl-1,3,4oxadiazole-5yl)] ethane (3a-f) and 1,2[di-(2-Amino-1,3,4-oxadiazole-5 yl)] ethane (4) respectively. The structures of the compounds have been elucidated on the basis of spectral analysis. Keywords: Diethyladipate, Succinohydrazide, Carbon disulfide and Cynogen bromide. ÂŠ2011 ijCEPr. All rights reserved

INTRODUCTION The chemistry of heterocyclic compounds has been an interesting field of study for a long time. The synthesis of novel Oxadiazole derivatives and investigation of their chemical and biological behavior have gained more importance in recent decades for biological, medical and agricultural reasons. Different classes of Oxadiazole compounds possess an extensive spectrum of pharmacological activities. In particular, compounds bearing 1,3,4Oxadiazole nucleus are known to exhibit unique anti-edema and anti-inflammatory activity [1-4,9]. Differently substituted Oxadiazole moiety has also been found to have other important activities such as analgesic [3,4] antimicrobial [5,6], antimycobacterial [7], anticonvulsant [8], antitumor [9], antimalarial [10] and anti-hepatitis B viral activities [11]. Substituted 1,3,4-Oxadiazoles exhibit antibacterial [12], Pesticidal [13] and antifungal [14] activities. 1,3,4-oxadiazoles are biologically active [15], synthetically useful and important heterocyclic compounds. for these reasons the chemistry of 1,3,4-oxadiazoles have been the subject of many investigations [16-19].One pot synthesis of 1,3,4-oxadiazoles by the reaction of appropriate hydrazide and carboxylic acid has been reported [20]. Cerric ammonium nitrate has received considerable attention as an inexpensive and easily available catalyst for various organic reactions such as Oxidation, Oxidative addition, Nitration, Photo-oxidation, Polymerization etc. In recent report Cerric ammonium mediated synthesis of 1,3,4-Oxadiazoles has also been described [21,22].

MATERIALS AND METHODS Melting points of all synthesized compounds were determined in open capillary tubes on an electrothermal apparatus and are uncorrected. The progress of reaction was monitored by thin layer chromatography on silica gel coated aluminium plates (Merck) as adsorbent and UV light as visualizing agent. IR spectra (KBr in cm-1) were recorded on a Perkin-Elmer spectrophotometer in the range of 4000-400 cm-1. 1H NMR spectra were recorded on a Varian 500 MHz NMR spectrometer using CDCl3/DMSO-d6 as solvent and TMS as an internal standard (chemical shifts in Î´ppm). Succinohydrazide (1): General procedure A mixture of Diethyladipate (0.08 mole), Hydrazine hydrate (3.85 ml, 0.08 mole), and ethanol (10 ml) was refluxed on water bath for 4-5 hrs. The mixture becomes almost solid. This reaction mixture is allowed to cool at room temperature. The white solid so obtained is then filtered and washed with cold ethanol and finally recrystallized from hot water to yield (1). 1,2[di-(2-Mercapto-1,3,4-oxadiazole-5yl)] ethane (2) A mixture of Succinohydrazide (1.0 gm, 0.0068 mole), Carbon disulfide (0.82 ml, 0.0136 mole), and Potassium hydroxide (0.768 gm, 0.014 mole) was refluxed in ethanol (10 ml) on water bath for 5-6 hrs. The reaction is then allowed to cool. The red colored solid so obtained is then filtered and washed with ethanol and finally recrystallized from hot dichloromethane to yield (2). 1,2[di-(2-Phenyl-1,3,4-oxadiazole-5yl)] ethane (3a-f)

Vijay V. Dabholkar and Nitin V. Bhusari

Vol.2, No.1, 1-4 (2011) A mixture of Succinohydrazide (1.0 gm, 0.0068 mole), aromatic aldehydes (0.0136 mole), and Cerric ammonium sulphate (0.5 gm, 0.0008 mole), in Dichloromethane (10 ml) as a solvent was taken in 100 ml round bottom flask and the mixture was refluxed on water bath for 4-5 hrs. After monitoring the reaction on TLC, the reaction mixture was cooled and dumped on to the ice, filtered and recrystallized from ethanol. 1,2[di-(2-Amino-1,3,4-oxadiazole-5 yl)] ethane (4) A mixture of Succinohydrazide (1.0 gm, 0.0068 mole), Cynogen bromide (0.0136 mole), and Sodium bicarbonate (1 g, 0.012 mole), in ethanol (10 ml) as a solvent was taken in 100 mL round bottom flask and the mixture was refluxed on water bath for 4-5 hrs. After monitoring the reaction on TLC, the reaction mixture was cooled and dumped on to the ice, filtered and recrystallized from dimethylformamide to yield (4). The schematic data of the compound 2, 3(a-f) and 4 are listed in the Table-1. Antimicrobial evaluation Representative samples were screened for their antimicrobial and antifungal activity against gram-negative bacteria, E coli and P aeruginosa and gram-positive bacteria, S aureus, and C diphtheriae using disc diffusion method [23,24]. The zone of inhibition was measured in mm and the activity was compared with standard drug. The results of antibacterial screening studies are reported in Table-2.

RESULTS AND DISCUSSION Diethyladipate on reaction with Hydrazine hydrate gave Succinohydrazide (1) which on further treatment with Carbon disulfide, Aromatic aldehydes and Cynogen bromide yielded 1,2[di-(2-Mercapto-1,3,4-oxadiazole-5yl)] ethane (2), 1,2[di-(2-Phenyl-1,3,4-oxadiazole-5yl)] ethane (3a-f) and 1,2[di-(2-Amino-1,3,4-oxadiazole-5 yl)] ethane (4) respectively with good yield. Further, the representative compounds were screened for their antimicrobial activity against gram negative as well as gram positive bacteria, which shows convincing activity.

ACKNOWLEDGEMENTS The authors are grateful to the Principal Ms. Manju J. Nichani and Management of K.C. College, Mumbai for providing necessary facilities. Authors are also thankful to the Director, Institute of Science, Mumbai for providing spectral analyses. Table-1: Characterization of synthesized compounds 2, 3(a-f) and 4. Compd.

Mol. Formula

Yield (%)

m.p. (째C )

Spectral data IR (KBr cm-1)/1H NMR/13C NMR (ppm) in DMSO-d6

C6H6N4O2S2

62-64

C6H5

C18H14N4O2

114-115

p-OCH3-C6H5

C20H18N4O4

97-99

p-Cl-C6H5

C18H12N4O2Cl2

90-94

o-OH-C6H5

C18H14N4O4

124-127

IR (KBr): 2581 (S-H), 1363 (C=N). 1H NMR: 2.7 (t, 4H, CH2), 9.7 (s, 2H, SH). [Found: C,35.27, H,2.57, N,24.35, S,27.85%. Required: C,31.30, H,2.61, N,24.35, S,27.83%.] IR (KBr): 1621-1432 (Ar), 1377 (C=N), 1H NMR: 2.9 (t, 4H, CH2),7.4 (m,10H,ArH) [Found: C,67.27, H,3.87, N,17.35%. Required: C,67.92, H,4.4, N,17.61%.] IR (KBr): 1373 (C=N), 1142 (-OCH3). 1 H NMR: 2.9 (t, 4H, CH2),3.7 (s, 6H, OCH3), 6.9 (d, 4H), 7.5 (d,4H).13C NMR: 55.3 (OCH3), 72.4 (CH2),114.3-128.5 (Ar-C), 152.3 (C=N) [Found: C,63.29, H,4.38, N,14.42%. Required: C,63.49, H,4.76, N,14.81%.] IR (KBr): 1353 (C=N), 781 (C-Cl). [Found: C,55.31, H,3.04, N,14.21%. Required: C,55.81, H,3.1, N,14.47%]. IR (KBr): 3302 (-OH), 1308 (C=N). [Found: C,61.65, H,4.06, N,16.07%. Required: C,61.71, H,4.00, N,16.00%].

Vijay V. Dabholkar and Nitin V. Bhusari

Vol.2, No.1, 1-4 (2011) 3e

CH=CH-C6H5

C22H18N4O2

112-114

p-OCH3-m-OHC6H5

C20H20N4O6

93-95

C18H16N6O2

86-89

IR (KBr): 3021 (CH=CH), 1328 (C=N). [Found: C,70.99, H,4.53, N,15.01%. Required: C,71.35, H,4.86, N,15.14%.] IR (KBr): 3311 (-OH), 1331 (C=N), [Found: C,58.02, H,4.77, N,13.52%. Required: C,58.25, H,4.85, N,13.59%.]. IR (KBr): 1381 (C=N), 1267 (NH2). 1 H NMR: 2.9 (t, 4H, CH2), 8.9 (s, 4H, NH2). [Found: C,62.10 H,4.48, N,24.06%. Required: C,62.06, H,4.59, N,24.14%].

Table-2: Antibacterial Activity of compound 2, 3(a-f) and 4 Zone of inhibition (in mm) Comp.

Gram Positive S.aureus 22

Gram negative

C.diphtheria 20

P.aeruginosa 21

E.coli 19

Amphicilin trihydrate

DMSO

* Diameter of the disc was 6mm, concentration of the compounds taken was about 100 Âľg/mL. O O

C2 H5

C2H5

O O C2H5OH

NH2.NH2.H2O O

H N

NH2

H2N (1)

N H

O CS2

CNBr Ar-CHO

NaHCO3/C2H5OH

alc.KOH CAS/MDC

O SH

(2)

NH2 O

NH2

R N

(3)

N (4)

Scheme-1 3

Vijay V. Dabholkar and Nitin V. Bhusari

Vol.2, No.1, 1-4 (2011)

REFERENCES Omar F. A., Mahfouz M.N., Rahman A.M., Eur. J Med. Chem., 31 (1996) 819 Franski R., Asian J. Chem., 17 (2005)2063. Narayana B., Vijayraj K. K., Ashalatha B. V., Kumari N. S., Arch. Pharm. (Weinheim) ,338 (2005) 373. Amir M., Kumar S., Acta Pharm., 31 (2007) 57 Gaonkar L. S., Rai M. K., Eur. J. Med. Chem., 41 (2006)841. Mishra P., Rajak H., Mehta A., J. Gen. Appl. Microbiol., 51 (2005) 133. Ali M. A.,Yar M. S., Bioorg. Med. Chem. Lett., 17 (2007) 3314. Zargahi A., Tabalabai S. A., Faizi M., Ahadian A., Navabi P., Zanganeh V., Shafiee A., Bioorg. Med. Chem. Lett., 15 (2005) 1863. 9 Bezerra N. M., De-Oliveira S. P., Srivastava R. M., Da Silva, J. R. Farmaco., 60 (2005) 955. 10 Zareef M., Iqbal R., De Dominquez N. G., Rodrigues J., Zaidi J. H., Arfan M., Supuran C. T., J. Enzyme Inhib. Med. Chem., 22 (2007)301. 11 Tan T. M., Chen Y., Kang K. H., Bai Li Y., Lim S. G., Ang T. H., Lam Y., Antiviral Res., 71, (2006)7. 12 Hui P. X., Chu H. C., Zhang Y. Z., Wang Q. and Zhang Q., Indian J. Chem., 41B (2002), 2176. 13 Khanum A. S., Shashikanth S., Sudha S. B., Deepak A. S. and Shetty S. H., Pest Manag. Sci., 60 (2004), 1119. 14 Palaska E., Sahin G., Kelicen P., Durlu T. N. and Altinok G., J.R. Farmaco., 57 (2002) 101. 15 Perez S., Lasheral B., Oset C., Carmen A., J. Heterocycl. Chem., 34 (1997) 1527. 16 Hutt M. P., Elslanger E. F., Werbet M. L., J. Heterocycl. Chem., 8 (1970) 511. 17 Baltazzi E., Wysocki A., J. Chem. Ind. , 31 (1963) 1080. 18 Chiba T., Mitsuhiro O., J Org. Chem., 57 (1992) 1375. 19 Shah V. R., Vadodaria M., Parikh A. R., Ind. J Chem., 36B (1997) 101. 20 Bentiss F. and Laqrenee M., J. Heteocycl. Chem., 36 (1999), 1029. 21 Minoo D., Peyman S., Mostafa B. and Mahboobeh B., Tetrahedron Lett., 47 (2006), 6983. 22 Kalluraya B., Jyothi N. Rao and Sujith V. K., Indian J Heterocycl. Chem. ,17 (2008), 359. 23 Cruickshank R., Duguid J. P. and Marmion B. P., Medicinal Microbiology,12th edn, Vol 11, (1975), (Churchill Livingstone, London). 24 Arthington- Skaggs B. A., Motley M. and Morrison C. J., J. Clin. Microbiology, 38, (2000) 2254. [IJCEPR-139/2011] __________________________________________________________________________________________ 1 2 3 4 5 6 7 8

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Vijay V. Dabholkar and Nitin V. Bhusari

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 5-11 January-April, 2011

Molecular Interaction Studies between H-Bonded Ternary Mixtures of p-Cresol with simple Aldehydes in Cyclohexane at Different Temperatures M. Aravinthraj1,*, S. Venkatesan2 and M. Kamaraj1 1

Department of Physics, Sacred Heart College, Tirupattur, Tamilnadu, India. Department of Chemistry, Sacred Heart College, Tirupattur, Tamilnadu, India. *E-mail: mar.rebo84@gmail.com 2

Article History: Received:12 February 2011 Accepted:27 February 2011

ABSTRACT The ultrasonic velocity (U), Density (ρ) and Viscosity (η) have been measured for three ternary liquid mixtures of P-Cresol + Cyclohexane + Formaldehyde, P-Cresol + Cyclohexane + Acetaldehyde and P-Cresol + Cyclohexane + Benzaldehyde at 303K, 313K and 323K. The experimental data have been used to calculate the acoustical parameters such as adiabatic compressibility (β), free length (Lf), free volume (Vf), internal pressure (πi), viscous relaxation time (τ) and Gibb’s free energy (∆G) were evaluated. The obtained results support the occurrence of dipole-dipole interactions and molecular association through intermolecular hydrogen bonding in these ternary liquid mixtures. Key words: Ternary liquid mixture, Acoustical parameter, Molecular association, dipole-dipole interactions. ©2011 ijCEPr. All rights reserved

INTRODUCTION The studies of multi-component (Binary and ternary liquid) mixtures and solutions have found wide applications in chemical, textile, leather and nuclear industries [1-3]. The study and understanding of thermo dynamical and transport properties of liquid mixtures and solutions are more essential for their application in these industries. It increases interest among several workers for the study of molecular interaction in binary [4, 5] and ternary [6, 7] liquid mixtures in recent past employing ultrasonic velocity measurement. This precisely helps to understanding the molecular interactions and structural behavior of molecules and their mixture. In this paper, we report on the ultrasonic study of four ternary liquid mixtures (p-Cresol + Cyclohexane + Formaldehyde, p-Cresol + Cyclohexane + Acetaldehyde and p-Cresol + Cyclohexane + Benzaldehyde). The cyclohexane is a non-polar unassociated, inert hydrocarbons possesses globular structure [8], it has ring structure as benzene without any S electron and serves as reference point for comparison of the molecular interactions. Cresols are organic compounds which are methyl phenols. It has isomeric and methyl group substituted onto the benzene ring of a phenol molecule. It has three forms (ortho, meta and para), one of the o-,m- or p- positions relative to the OH group. The compounds are highly flammable moderately soluble in water and soluble in ethanol, ether, acetone cyclohexane and alkalies. Chemically these alkyl phenols undergo electro-philic substitution reactions at the condensation reaction with aldehydes, ketones [9]. The interactions between cresols and aldehydes may probably be dipole-dipole or may be due to intra molecular hydrogen bonding [10]. EXPERIMENTAL The chemicals used in the present work were Analytical reagent (AR) grades with a minimum assay of 99.9%, obtained from E-Merck (Germany) and Loba chemicals; they are used without further purification. In all the systems, the various concentrations of the ternary liquid mixtures were prepared in terms of mole fraction, out of which the mole fraction of the second compound, cyclohexane (X2=0.3) was kept fixed while the mole fraction of the remaining two (X1 and X3) were varied from 0.0 to 0.7. The ultrasonic velocity was measured by a single crystal interferometer with a high degree of accuracy operating frequency of 2MHz supplied by M/s. Mittal Enterprises, New Delhi. Water was circulated around the double walled sample holder to maintain the experimental temperatures. (Say 303K, 313K and 323K) The density of all compounds was measured by a 10 ml specific gravity bottle calibrated with double distilled water and acetone. An Ostwald’s viscometer with 10ml capacity was used for the viscosity measurements of all the compounds. The viscometer was immersed in fresh conductivity water bath that can be operated at desired temperatures. The flow time of water (tw) and the flow time of solution (ts) were measured with a digital stop clock with an accuracy of 0.01s (RACER HS-10w).

M. Aravinthraj et al.

Vol.2, No.1, 5-11 (2011) Theory and Calculations The longitudinal ultrasonic velocity (U), density (ρ) and viscosity (η) of unknown liquid mixtures at any experimental temperatures are calculated using the following relations. And the density, viscosity of water at different temperatures was taken from literature [11, 12]. The accuracy in the measurement of density in this method depends on the accuracy of the weight. The accuracy in the measurement of density is of the order of ± 0.1 kgm-3. And the accuracy in the measurement of viscosity depends on the accuracy in the determination of time and density. The overall accuracy of the measurement of viscosity in this method is ± 0.001Nsm-1. Ultrasonic velocity (U) (1) Where is the frequency of Ultrasonic wave in Hz, and λ is the wavelength of the Ultrasonic wave in solution under study in meter. Viscosity (ηs) (2) Where

is the co-efficient of viscosity of water in Nsm-1,

time for water in seconds, Density (ρ2)

is the density of water in Kg/m3, 3

is the density of solution in Kg/m , and

is the flow of

is the flow of time solution in seconds. (3)

are weights of distilled water and experimental liquid and are the densities of water and Where experimental liquid. Using experimentally determined values of ultrasonic velocity (U), density (ρ) and viscosity (η), the following acoustic and thermodynamic parameters are evaluated. Adiabatic compressibility (β) (4) 3 Where U is the velocity measured in meter/second and ρ is density measured in Kg/m . Free Length (Lf) (5) -1

Where KT is Jacob’s constant and β is the adiabatic compressibility of a liquid mixtures measured in N m . Free Volume (Vf) Suranarayana et al [13] obtained a formula for free volume in term of the ultrasonic velocity (V) and the viscosity of the liquid (η) as, (6) Where Meff is the effective molecular weight (

in which mi and xi are the molecular weight and the

mole fraction of the individual constituents respectively) and K is a temperature independent constant equal to 4.28×109 for all liquids. Internal Pressure (πi) On the basis of statistical thermodynamics, Surayanarayana [14] derived an expression for the determination of internal pressure through the use of the concept of free volume (7) Where T is absolute temperature in Kelvin, R is the universal gas constant and b is the cubic packing fraction factor is assumed to be ‘2’ for all liquid systems. Viscous relaxation time (ττ) (8) Where η is the viscosity of the solution in Nsm-2 and β is adiabatic compressibility in N-1m2

M. Aravinthraj et al.

Vol.2, No.1, 5-11 (2011) Gibb’s free energy (∆G) The Gibb’s free energy of activation flow in the mixtures can be obtained on the basis of Erying rate process theory and it can be able to calculate from the relation, (9) Where τ is the viscous relaxation time measured in meters, K is the Boltzmann’s constant, h is a Plank’s constant and T is the temperature measured in kelvin.

RESULT AND DISCUSSION The experimentally measured values of density, viscosity and ultrasonic velocity of the three ternary systems of pCresol + Cyclohexane + Formaldehyde, p-Cresol + Cyclohexane + Acetaldehyde and p-Cresol + Cyclohexane + Benzaldehyde at 303K, 313K and 323K are presented in Table-1. The systems taken for the present study is basically an H-Bonded complexes of phenol (p-cresol) with aldehydes in cyclohexane. Here mole fraction of cyclohexane is fixed as 0.3 and the mole fraction of the other two components are varied from 0.0 to 0.7. Acoustical parameters such as adiabatic compressibility (β), intermolecular free length (Lf), free volume (Vf), internal pressure (πi), kinetic parameter like viscous relaxation time (τ) and thermo dynamical parameter like Gibb’s Free Energy (∆G) were calculated from the measured ultrasonic velocity, density and viscosity using standard equations given in theory and calculations. From the measured data, it is observed that there is a sudden fall of density in all the systems for second molar concentration, and from the second concentration onwards p-cresol is made to dissolve in cyclohexane for which aldehydes are used as doped. When aldehyde and cyclohexane are taken without p-cresol, the density is found to be higher. This serves the possibility of greater interaction between the aldehydes and solvent due to dipole-dipole interaction as well as possibility of weak H-bond shown in Figure (a). The solvent has hydrogens placed at the 1,3-diaxial position, such that any of these hydrogens are free to interact with non-bonding electrons (or) n-electron from the carbonyl oxygen of aldehydes Figure (b). This serves to the increasing density with the increasing of aldehydes concentration. From the second molar concentration onwards a gradual increasing trend in density is observed. Figure (a) shows the interaction between Oxygen of the p-cresol and flagpole Hydrogen of the Cyclohexane. This dipole-dipole interaction serves for the gradual increase in density.

Fig. (a)

Fig. (b)

From the second concentration onwards the influence of non bonding electron is frankly reduced when p-cresol is began to introduce. Another kind of factor is observed when the mole fraction of aldehydes is taken as 0(zero), and the mole fraction of p-cresol is taken as 0.7, here also the density is greater because of the possible interaction between the non bonding electrons of oxygen and the cyclohexane. The mole fraction of cyclohexane is taken as 0.3 M, which is constant for the overall experiment. From the second concentration onwards p-cresol and aldehydes are mixed with different molar concentration in cyclohexane, and then increasing trend is observed in density, viscosity and velocity. This is because of more hydrogen bonding and dipole-dipole interaction observed as shown in Fig (c) and Fig (d) respectively. This behavior at such concentrations is same as the ideal mixtures behavior can be attributed to intermolecular interactions in the systems studied [16]. It is evident from table-1.

M. Aravinthraj et al.

Vol.2, No.1, 5-11 (2011)

Fig. (c)

Fig. (d)

It is found that from the table-2, the adiabatic compressibility and intermolecular free length decreases with increase in concentration of p-cresol in all the three systems. This shows an inverse behavior as compared to ultrasonic velocity. This indicates that there is a significant interaction between solute and solvent molecules. It can be taken as the indication of complex formation. The addition of interacting molecules breaks up the molecular clustering of the other, releasing several interaction, because p-cresol has the possibility of forming intermolecular H-bonding with such carbonyl oxygen. However free volume increases with increase in molar concentration of p-cresol, the p-cresol system has the lone pair of electron in oxygen of p-cresol system interact with Hydrogen in hydroxyl group of another p-cresol system. This is clearly observed in the increasing trend in free volume with increasing molar concentration of p-cresol. At the same time the mole fraction of doped is purposely decreased, because this factor holds well only from the introduction of pcresol. From Table-3 it is found that, internal pressure decreases with increase in concentration of p-cresol, because the hydrogen releasing ability of p-cresol is becoming low. The decreasing density with the increase in concentration of p-cresol and the decreasing concentration of aldehydes is contributed from the above explanation. Also a decrease in internal pressure with the decrease in size of the alkyl group of aldehyde is observed. This is naturally due to the increasing available space with the decreasing size of alkyl group. However, the viscous relaxation time increases with increasing molar concentration of p-cresol as shown in Table -3 and a decrease in all these values is also noted with the increasing temperature. This is clearly due to the increasing free space between the molecules and the weakening of intermolecular forces. Generally, viscous relaxation time is of the order of 10-12 s, for identifying structural relaxation between the component molecules. This shows the presence of molecular interactions [17], Gibb’s free energy confirms the same, which indicates the need for smaller time for the cooperative process or the rearrangement of the molecules in the mixtures. Table -1: Density (ρ), Viscosity (η) and Velocity (U) of p-Cresol + Cyclohexane + Formaldehyde, p-Cresol + Cyclohexane + Acetaldehyde and p-Cresol + Cyclohexane + Benzaldehyde at 303K, 313K and 323K Density (ρ)

Mole fraction

Viscosity (η)

-3

(Kg/m )

Velocity (U)

-2

(ms-1)

(10 Nsm )

303K

313K

323K

303K

313K

0.000

0.705

887.7

883.8

880.3

1.704

1.339

0.100

0.606

846.2

841.1

837.0

2.890

0.197

0.498

870.6

866.1

861.2

0.301

0.403

871.3

867.9

0.399

0.297

873.0

0.497

0.195

0.607 0.705

323K

303K

313K

323K

1.106

1310.1

1306.5

1298.1

2.344

1.829

1323.9

1318.7

1309.9

2.955

2.389

1.853

1338.1

1330.0

1318.9

863.3

3.112

2.398

1.899

1345.9

1339.2

1323.2

867.9

864.0

3.075

2.435

1.911

1357.3

1348.9

1336.8

875.1

870.3

866.8

3.232

2.458

2.023

1364.5

1353.1

1349.3

0.104

877.0

873.5

869.8

3.345

2.701

1.985

1374.7

1363.4

1354.8

0.000

879.0

875.2

870.6

3.538

2.901

2.189

1361.0

1357.3

1349.3

1309.3

1300.5

1295.1

p-Cresol + Cyclohexane + Formaldehyde

p-Cresol + Cyclohexane + Acetaldehyde 0.000

0.701

871.2

868.1

863.9

1.415

1.050

0.880

M. Aravinthraj et al.

Vol.2, No.1, 5-11 (2011) 0.102

0.604

842.1

837.5

830.3

2.190

1.845

1.407

1319.8

1310.1

1301.9

0.199

0.501

867.1

852.8

847.7

2.503

2.106

1.524

1326.9

1320.1

1312.1

0.305

0.398

868.3

863.3

859.5

2.621

2.161

1.577

1333.8

1328.6

1322.4

0.398

0.301

870.3

866.8

862.3

2.859

2.223

1.590

1349.7

1337.5

1331.6

0.504

0.200

874.4

869.3

865.8

3.105

2.236

1.777

1356.4

1350.0

1339.6

0.601

0.098

875.0

872.5

867.2

3.256

2.388

1.954

1360.0

1354.0

1347.8

0.700

0.000

879.0

875.5

869.6

3.506

2.896

2.185

1360.1

1355.5

1350.9

0.000

0.700

870.2

868.5

867.3

1.024

0.885

0.773

1307.9

1304.5

1300.3

0.101

0.603

838.2

835.2

832.3

1.216

1.004

0.890

1310.7

1296.0

1289.1

0.203

0.501

858.8

855.2

851.8

1.426

1.193

1.046

1321.7

1305.8

1295.0

0.299

0.404

860.1

859.8

857.4

1.711

1.362

1.199

1329.7

1310.7

1302.2

0.403

0.308

869.3

867.2

865.9

1.950

1.604

1.315

1338.4

1318.8

1309.4

0.503

0.199

870.0

868.9

864.6

2.427

2.025

1.560

1345.2

1331.9

1320.5

0.599

0.104

874.4

872.9

871.8

2.879

2.463

1.758

1350.4

1333.6

1326.0

0.703

0.000

878.0

876.9

872.0

4.096

2.850

2.223

1359.5

1351.4

1328.6

p-Cresol + Cyclohexane + Benzaldehyde

Table -2: Adiabatic compressibility (β), Free Volume (Vf), and Free Length (Lf) of p-Cresol + Cyclohexane + Formaldehyde, p-Cresol + Cyclohexane + Acetaldehyde and p-Cresol + Cyclohexane + Benzaldehyde at 303K, 313K and 323K

Mole fraction X1

Adiabatic compressibility (β) -10 (10 N-1m2) 303K

313K

323K

Free length (Lf) (10-10m) 303K

313K

Free Volume (Vf) -7 (10 m3 mol-1) 323K

303K

313K

323K

p-Cresol + Cyclohexane + Formaldehyde 0.000

0.705

6.5630

6.6280

6.7406

5.0564

5.0814

5.1243

0.1719

0.2457

0.3241

0.100

0.606

6.7417

6.8365

6.9626

5.1248

5.1607

5.2080

0.0908

0.1236

0.1776

0.197

0.498

6.4143

6.5264

6.6750

4.9988

5.0423

5.0993

0.1045

0.1424

0.2059

0.301

0.403

6.3355

6.4244

6.6158

4.9680

5.0027

5.0767

0.1158

0.1700

0.2368

0.399

0.297

6.2174

6.3316

6.4759

4.9215

4.9665

5.0227

0.1461

0.2054

0.2915

0.497

0.195

6.1368

6.2751

6.3367

4.8895

4.9443

4.9684

0.1719

0.2561

0.3414

0.607

0.104

6.0332

6.1580

6.2630

4.8480

4.8979

4.9395

0.2121

0.2886

0.4538

0.705

0.000

6.1412

6.2017

6.3090

4.8912

4.9153

4.9576

0.2670

0.3584

0.5419

p-Cresol + Cyclohexane + Acetaldehyde 0.000

0.701

6.6950

6.8109

6.9006

5.1070

5.1510

5.1848

0.3690

0.5713

0.7399

0.102

0.604

6.8164

6.9559

7.1049

5.1531

5.2055

5.2610

0.2160

0.2763

0.4108

0.199

0.501

6.5500

6.7283

6.8512

5.0514

5.1197

5.1662

0.1998

0.2569

0.4137

0.305

0.398

6.4735

6.5618

6.6524

5.0218

5.0559

5.0907

0.2143

0.2846

0.4533

0.398

0.301

6.3074

6.4486

6.5395

4.9570

5.0121

5.0473

0.2182

0.3139

0.5154

0.504

0.200

6.2175

6.3114

6.4360

4.9215

4.9585

5.0072

0.2265

0.3683

0.5137

0.601

0.098

6.1789

6.2513

6.3472

4.9062

4.9349

4.9726

0.2509

0.3968

0.5324

M. Aravinthraj et al.

Vol.2, No.1, 5-11 (2011) 0.700

0.000

6.1495

6.2163

6.3011

4.8945

4.9210

4.9545

0.2703

0.3583

0.5438

p-Cresol + Cyclohexane + Benzaldehyde 0.000

0.700

6.7172

6.7653

6.8191

5.1155

5.1337

5.1541

1.5849

1.9652

2.3944

0.101

0.603

6.9437

7.1278

7.2292

5.2010

5.2695

5.3068

1.2331

1.6165

1.9208

0.203

0.501

6.6652

6.8571

6.9996

5.0956

5.1685

5.2219

0.9860

1.2654

1.5216

0.299

0.404

6.5750

6.7692

6.8774

5.0610

5.1352

5.1761

0.7586

1.0451

1.2539

0.403

0.308

6.4210

6.6292

6.7353

5.0014

5.0818

5.1223

0.6322

0.8285

1.1041

0.503

0.199

6.3511

6.4868

6.6325

4.9741

5.0270

5.0831

0.4594

0.5938

0.8670

0.599

0.104

6.2709

6.4413

6.5228

4.9426

5.0093

5.0409

0.3587

0.4447

0.7310

0.703

0.000

6.1616

6.2441

5.3932

4.8993

4.9320

4.5837

0.2141

0.3655

0.5946

Table -3: Internal Pressure (πi), Viscous Relaxation Time (τ) and Gibb’s free energy (∆G) of p-Cresol + Cyclohexane + Formaldehyde, p-Cresol + Cyclohexane + Acetaldehyde and p-Cresol + Cyclohexane + Benzaldehyde at 303K, 313K and 323K

Mole fraction X1

Internal pressure (πi) (10-6 Pa) 303K

313K

323K

Viscous Relaxation Time (τ) (10-12s) 303K

313K

323K

Gibb’s Free Energy (∆G) (10-20 KJ mol-1) 303K

313K

323K

p-Cresol + Cyclohexane + Formaldehyde 0.000

0.705

14.090

12.472

11.342

1.4911

1.1836

0.9943

0.4077

0.3898

0.3686

0.100

0.606

15.867

14.397

12.878

2.5982

2.1373

1.6985

0.5086

0.4948

0.4722

0.197

0.498

14.769

13.754

11.907

2.5280

2.0793

1.6497

0.5036

0.4897

0.4666

0.301

0.403

12.561

11.314

9.971

2.6295

2.0545

1.6758

0.5017

0.4874

0.4696

0.399

0.297

10.921

9.529

8.429

2.5493

2.0563

1.6505

0.5051

0.4876

0.4667

0.497

0.195

9.357

8.127

6.977

2.6448

2.0565

1.7094

0.5118

0.4857

0.4735

0.607

0.104

7.818

6.712

6.034

2.6908

2.2184

1.6581

0.5149

0.5018

0.4676

0.705

0.000

6.829

5.661

4.915

2.8985

2.3989

1.8416

0.5284

0.5165

0.4879

p-Cresol + Cyclohexane + Acetaldehyde 0.000

0.701

8.691

7.495

6.854

1.2634

0.9539

0.8101

0.3776

0.3435

0.3289

0.102

0.604

9.684

8.887

7.742

1.9907

1.7115

1.3336

0.4602

0.4531

0.4254

0.199

0.501

9.630

8.997

7.603

2.1867

1.8898

1.3923

0.4773

0.4717

0.4337

0.305

0.398

8.883

7.948

6.873

2.2624

1.8907

1.3992

0.4834

0.4718

0.4347

0.398

0.301

8.345

7.268

6.096

2.4048

1.9118

1.3870

0.4945

0.4739

0.4330

0.504

0.200

7.719

6.539

5.837

2.5746

1.8818

1.5252

0.5069

0.4709

0.4514

0.601

0.098

6.924

5.931

5.356

2.6831

1.9910

1.6542

0.5144

0.4815

0.4671

0.700

0.000

6.782

5.664

4.907

2.8753

2.4005

1.8364

0.5269

0.5166

0.4873

p-Cresol + Cyclohexane + Benzaldehyde 0.000

0.700

3.464

3.220

3.012

0.9178

0.7988

0.7035

0.3196

0.3102

0.3016

0.101

0.603

3.669

3.344

3.150

1.1262

0.9543

0.8582

0.3568

0.3436

0.3401

0.203

0.501

4.013

3.682

3.453

1.2676

1.0910

0.9767

0.3782

0.3687

0.3651

0.299

0.404

4.379

3.605

3.362

1.5007

1.2301

1.0996

0.4089

0.3912

0.3881

M. Aravinthraj et al.

Vol.2, No.1, 5-11 (2011) 0.403

0.308

4.679

4.269

3.875

1.6696

1.4183

1.1814

0.4283

0.4179

0.4019

0.503

0.199

5.203

4.773

4.193

2.0560

1.7520

1.3800

0.4661

0.4575

0.4320

0.599

0.104

5.663

5.265

4.458

2.4073

2.1157

1.5297

0.4947

0.4929

0.4519

0.703

0.000

6.737

5.632

4.771

3.3652

2.3731

1.5989

0.5555

0.5145

0.4605

CONCLUSION From the experimental and calculated parameters, there is a significant interactions observed only after introducing p-Cresol. This increases density, viscosity and velocity of all the liquid mixtures. This increasing trend is due to the intermolecular hydrogen bonding between carbonyl oxygen of aldehydes with hydroxyl hydrogen in p-cresol and dipole- dipole interaction between the same compounds with carbonyl carbon of aldehydes and hydroxyl oxygen. Hence this investigation provides comprehensive idea about the molecular interactions between solute and solvent. The order of interactions is found to be Formaldehyde > Acetaldehyde > Benzaldehyde.

REFERENCE Rowlinson J.S., and Swinton P. L., 1982. Liquids and liquid mixtures. 3rd edition, Butterworth scientific, London. 2. Acree W.E., 1984. Thermodynamic properties of Non-electrolytic solutions, academic press, New York. 3. Prausnitz J.M., Linchenthalr and Azevedo E.G., 1986. Molecular thermodynamics of fluid phase equlibria. 2nd edition, Engle wood cliffs, Prentic Hall Inc. 4. Sastry N.V., and Patel S.R., Int. J. Thermo phys, 21(5)(2000). 5. Prabakar S., and Rajagopal., Ind J pure & appli ultra, 27, (2005), 4. 6. Sharma V.K., Kalra., Romi and Kotach., Ind J Chem., 42A, 292. 7. Asghar J., Liakath Ali Khan F and Subramani K., Rasaya J Chem, 3(4)( 2010), 697. 8. Ali, A.S. Adida Hydar and A.K Nain., Ind J Phys, 76B (15), (2002), 661. 9. Thirumaran S and Deepesh George., ARPN, 4(4)(2009), 1. 10. Jayakumar S., Karunanithi S and Kannappan V., Ind J pure & Appli Phys, 34(1996) 761. 11. Riddick J.A., Bunger W.B and Sanano T.K., 1984. Techniques in Chemistry, Vol. II, Organic Solvent. 4th edition, John Willey, New York. 12. Hand book of chemistry and physics, 1984-85, 65th edition, the chemical rubber company, Cleveland, Ohio, USA. 13. Surayanarayana. C.V. and kuppusamy.T , J.Acou.soc.Ind, 4, (1976), 75. 14. Surayanarayana. C.V. ,J.Acou.soc.Ind, 7, (1979), 131. 15. Baldev Raj., Rajendran V and Planichami P ., 2004. Science and technology of ultrasonics., 3rd edition, Narosa publication, India. 16. Guptha A K, Krishnakumar and Birendra Kumar Karn, J.Ind. Council.Chem, 26(1)(2009), 77. 17. Kannappan A N, Keasvasamy R and Ponnuswamy V, Am.J Eng and Appli.Sci, , 1(2)( 2008), 95. [IJCEPR-151/2011] __________________________________________________________________________________________ 1.

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M. Aravinthraj et al.

International Journal of Chemical, Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 12-19 January-April, 2011

Chemical Analysis on Different Oils Use in Tyre Tread Cap Compound N. Kumar*, R.K. Khandelwal1, P.L. Meena1, K. S. Meena1, T.K. Chaki2 , D.K. Mahla2 and S. Dasgupta3 1

P.G.Department of Chemistry, M.L.V.Govt.P.G.College, Bhilwara-311001 Rajasthan Indian Institute of Technology Kharagpur West Bengal-721302, India 3 J K Tyre HASETRI Kankroli, Rajasthan *E-mail: nitinkumariitkgp@gmail.com 2

Article History: Received: 17 March 2011 Accepted:23 March 2011

ABSTRACT The global market place is increasingly demanding safe process oils to reduce the environmental impact of tires. The replacement of classified distillate aromatic extracts by non-carcinogenic MES, TDAE, or naphthenic process oils will reduce the PAH emissions. . In the present work three types of low PCA and one regular high PCA Petroleum oils were chemically analyzed. The oils were characterized for different chemical analysis . These low PCA oils can act as the best alternative processing aids for rubber industry. The rheological, properties of SSBR loaded with different LPCA & HPCA oils have been studied. in order to obtain similar properties. The data show that the best results are obtained using LPCA. A comparative study has been carried out on SSBR filled with various oils. Key words: low PCA oils, Polycyclic aromatics, carcinogenesis, PAH, risk assessment. ©2011 ijCEPr. All rights reserved

INTRODUCTION In compounding rubber and rubber composition for use in pneumatic tyre, it is common to utilize processing oils to soften and extend the rubber[7]. Typical, aromatic processing oils, having a certain content of poly aromatic compound or polyaromatic hydrocarbons ,have bee used .recently , regulatory concerns have necessisted the use of processing oils having alower PCA content. Rubber formulations used in various tyre components previously have been designed using conventional processing oils .How ever, in changing to the use of the lower PCA content oils ,some lose in rubber compound performance is noted. It is , there for necessary to develop new rubber compounds that provide desirable performance levels wile in corporating the use low PCA oils[8].

EXPERIMENTAL Materials studied are given in Table 1. Physicochemical characterization The oils were characterized for Specific gravity by hydrometer (ASTM D 1298) , flash and fire point (ASTM D92), pour point (ASTM D97), specific gravity (ASTM D1298), saybolt viscosity (ASTM D88), Fourier transform infrared (FTIR) spectroscopic study of the petroleum oils was performed in a FTIR System from PERKIN ELMER, USA for checking functional groups present[1-3]. Compound mixing Mixing of rubber compound was carried out using a two-wing rotor laboratory Banbury mixer (Stewart Bolling, USA) in three stages (master batch remill and final batch) and the formulations are given in Table Master batch mixing was done setting the temperature control unit (TCU) at 90°C and rotor speed at 60 rpm.. After the power integrator (PI) indicated achievement of 0.32 kWh, the master batch was dumped. The dump temperature of the master batches was found to be within 140 - 150°C. The master batches were sheeted out in a laboratory tworoll mill. Further mixing of the master batches were carried out after a maturing period of 8 hours[8]. For final batch mixing, the TCU was kept at 600C and rotor speed at 30 rpm. The earlier prepared master batch was mixed with sulfur, accelerator and scorch inhibitor. The batch was dumped at a PI reading of 0.12 kWh. The dump temperature of the batches was found to be within 95 – 105°C. The final batches were also sheeted out on a laboratory two-roll mill8-12 Formulation is according to Table 2.

N. Kumar et al.

Vol.2, No.1, 12-19 (2011) Table-1: Material Required 1 2 3 4 5 6 7 8 9 10 11 12 13

S SBR having regular aromatic oil S SBR having low PCA oil (3830) Regular oil and LPCA OIL No.1,2,3, Filler N339 black ZnO Stearic Acid 6PPD MC Wax MS 40 S TBBS DCBS PVI

RESULTS AND DISCUSSION 1. Flash and Fire Point The flash point of a volatile liquid is the lowest temperature at which it can vaporize to form an ignitable mixture in air. Measuring a liquid's flash point requires an ignition source. At the flash point, the vapor may cease to burn when the source of ignition is removed. The flash point is not to be confused with the autoignition temperature, which does not require an ignition source. The fire point, a higher temperature, is defined as the temperature at which the vapor continues to burn after being ignited. Neither the flash point nor the fire point is related to the temperature of the ignition source or of the burning liquid, which are much higher. The flash point is often used as a descriptive characteristic of liquid fuel, and it is also used to help characterize the fire hazards of liquids. “Flash point” refers to both flammable liquids and combustible liquids. There are various standards for defining each term. The flash and fire point results are shown in Table 3. Flash and fire point is one of the important criteria for determining the process safety while handling the rubber compound during mixing, calendaring, extrusion etc. Higher flash and fire point of oils always indicates good process safety. High flash and fire point of oils may be due to presence of carbonyl groups, alkaloids groups etc[11]. 2. Pour Point The pour point of a liquid is the lowest temperature at which it will pour or flow under prescribed conditions. It is a rough indication of the lowest temperature at which oil is readily pumpable. Also, the pour point can be defined as the minimum temperature of a liquid, particularly a lubricant, after which, on decreasing the temperature, the liquid ceases to flow.The pour point results are shown in Table 4. All the Low PCA oils show pour point less than 0°C except aromatic oil. Lower pour point improves the handling of oils during winter season. Surrounding temperature during winter season reduces drastically when some processing oils require heating arrangement for ease of flow to the Banbury chamber for mixing. Additional energy consumption is required for such heating process. With natural oils, such additional processing costs can be eliminated. 3. Saybolt viscosity Viscosity describes a fluid's internal resistance to flow and may be thought of as a measure of fluid friction. For example, high-viscosity felsic magma will create a tall, steep stratovolcano, because it cannot flow far before it cools, while low-viscosity mafic lava will create a wide, shallow-sloped shield volcano. All real fluids (except superfluids) have some resistance to stress and therefore are viscous, but a fluid which has no resistance to shear stress is known as an ideal fluid or inviscid fluid. The study of flowing matter is known as rheology, which includes viscosity and related concepts.The saybolt viscosity results are shown in Table 5. High Saybolt viscosity indicates higher aromaticity. 4. Aniline Point Aniline point is defined as the temperature at which equal volumes of aniline(C6H5NH2) and diesel oil are completely miscible. The value gives an indication of the aromatic content of diesel oil, since aniline is an aromatic compound which is dissolved on heating by the aromatics in diesel oil. The greater the aniline point, the lower the aromatics in diesel oil. A higher aniline point also indicates a higher proportion of paraffin. The diesel index is directly related to aniline point as:

N. Kumar et al.

Vol.2, No.1, 12-19 (2011) DIESEL INDEX = ((ANILINE POINT(DEG F))(API GRAVITY))/100 A higher aniline point (and therefore a lower aromatic content) in diesel oil is desirable, in order to prevent autoignition in diesel engines.In cases where the Aromatic content in the oil is very high, in such cases "Mixed Aniline Point" needs to me measured to determine the approximate content of Aromatic in the oil.The aniline point results are shown in Table 6. All the Low PCA oils show higher values whereas High PCA oils show lower values. Aniline point indicates the presence of aromatic ring in the oils. Higher the aromatic groups lower the aniline point. 5. Specific gravity Specific gravity is the heaviness of a substance compared to that of water, and it is expressed without units. In the metric system specific gravity is the same as in the English system[10]. In relationship to liquids, the term specific gravity is used to describe the weight or density of a liquid compared to an equal volume of fresh water at 4°C (39° F). If the liquid you are comparing will float on this water it has a specific gravity of less than one. If it sinks into the fresh water the specific gravity is more than one. As you have already guessed fresh water at 4°C (39° F) has been assigned a value of one. The specific gravity results are shown in Table 7. 6. Aromatic content (CA) The aromatic content results are shown in Table 8.Higher aromatic content is basically the presence of polycyclic group in the oils. 7. FTIR Study for surface group IR bands of aromatic C-H stretching at 3010 and 3080 cm-1 and overtone and combination band due to C-H out-ofplane at 1600-2000 cm-1 were observed. Also, IR band of aromatic ring CC stretching and aromatic C-H in-plane appeared in 1000-1200 cm-1 region[9].The FTIR results are shown in Table 9.

CONCLUSION The recent change in world scenario in shifting towards low PCA oils and restriction on high PCA oils. Present study is focused on chemical, analytical and compound characterization of petroleum oils. These oils were found to be suitable on the basis of low PCA content.. All non-carcinogenic process oils contain very low concentrations of polycyclic aromatic hydrocarbons and meet the 1 mg/kg benzo[a]pyrene limit set by the VDA. Hence, the replacement of HPCA by non-carcinogenic process oils in oil extended natural or synthetic rubber and therefore in finished tires will reduce the PAH emission from tire wear by more than 98 %. Test results are intended to support the rubber and tire industries in their environmental challenge to replace the classified aromatic oils. Further extensive compounding and evaluation work will be required by each company using its proprietary tire formulation technology. Demand for these oils is expected to rise as car manufacturers realise that carcinogenic emissions from tires can hereby be greatly reduced. It has demonstrated on a commercial scale that this challenge can be met by a change to safer alternatives such as LPCA . The production LPCA oil are already on the market[12].

ACKNOWLEDGEMENTS The author would like to thank IIT Kharagpur (W.B.) & J K Tyre HASETRI Kankroli, Rajasthan for excellent cooperation, extensive evaluations and discussion. Flash and fire point (°C) 270 260 250 240

Flash and fire point (°C)

230 220 210 Aro.oil LP Reg. CA-1

LP CA-2

LP CA-3

Fig.-1: Flash and fire point 14

N. Kumar et al.

Vol.2, No.1, 12-19 (2011) Table-2: Formulation Ingredients

HPCA

LPCA-1

LPCA-2

LPCA-3

RMA4

VSL5525

Tufden3830

N339

Reg Ar. Oil

LPCA-1

LPCA-2

LPCA-3

ZnO(WS)

2.25

St Acid

0.5

6PPD

1.9

MC Wax

2.4

MS 40

S(108)

2.2

TBBS

1.2

DCBS

0.6

PVI

0.15

Batch weight

191.2

Table-3: : Flash and fire point Name of oils Aromatic oil regular Low PCA oil No.1 Low PCA oil No.2 Low PCA oil No.3

Flash and fire point(째C) 235 240 230 267

Table-4: Pour point Name of oils Aromatic oil regular Low PCA oil No.1 Low PCA oil No.2 Low PCA oil No.3

Pour point (째C) 7 -8 4 -4

Table-5: Saybolt viscosity Name of oils Aromatic oil regular Low PCA oil No.1 Low PCA oil No.2 Low PCA oil No.3

Saybolt viscosity(sec) 130 105 65 120

N. Kumar et al.

Vol.2, No.1, 12-19 (2011) Table-6: Aniline point Name of oils Aromatic oil regular Low PCA oil No.1 Low PCA oil No.2 Low PCA oil No.3

Aniline point (°C) 46.5 100 30 105

Table -7: Specific gravity Name of oils Aromatic oil regular Low PCA oil No.1 Low PCA oil No.2 Low PCA oil No.3

Specific gravity 1.001 0.916 0.938 0.921

Table -8: Aromatic content S.No.

Carbon type analysis (%) 1 2 3

CA CP CN

Low PCA 1 19.8 59.8 20.4

Low PCA 2

Low PCA 3 -

Aromatic oil

16.1 68.4 15.5

36.8 58.7 4.5

Table-9: FTIR study Name of oils Aromatic oil regular Low PCA oil No.1 Low PCA oil No.2 Low PCA oil No.3

Surface group present Alkyl group –CH2-R St. Aromatic substituent C-H St. Alkyl group CH2-R St. Alkyl group CH2-R St. Aromatic substituent C-H St. Aliphatic hydrocarbon C-H St. Aliphatic hydrocarbon –CH2-R St. Aromatic substituent C-H St Aliphatic hydrocarbon St. Aliphatic hydrocarbon (Short chain compound or substituent)

Flash and fire point (°C) 270 260 250 240

Flash and fire point (°C)

230 220 210 Aro.oil Reg.

LP CA-1

LP CA-2

LP CA-3

Fig.-2: Flash and fire point 16

N. Kumar et al.

Vol.2, No.1, 12-19 (2011)

Pour point

(째C)

8 6 4 2 0

Pour point (째C)

-2 -4 -6 -8 Aro.oil LPCA- LPCA- LPCAReg. 1 2 3

Fig.-3: Pour point

Saybolt viscosity (sec) 140 120 100 80 Saybolt viscosity (sec)

60 40 20 0 Aro.oil LPCA- LPCA- LPCAReg. 1 2 3

Fig.-4: Saybolt viscosity Aniline point (째C) 120 100 80 60 Aniline point (째C)

40 20 0 Aro.oil LPCA- LPCA- LPCAReg. 1 2 3

Fig.-5: Aniline point 17

N. Kumar et al.

Vol.2, No.1, 12-19 (2011)

Specific gravity 1.02 1 0.98 0.96 0.94 Specific gravity

0.92 0.9 0.88 0.86 Aro.oil LPCA- LPCA- LPCAReg. 1 2 3

Fig.-6: The specific gravity 86.1

564.38 576.98

75 2360.15

1907.41

463.88

3482.62 2360.48 577.79

428.26

60 963.58 965.14 1031.86

1031.85 1603.27

869.22 867.54

1168.61

960.56 2670.21

1605.36

1156.33

2728.51

2729.56

814.48

2727.09

813.52 1060.17

35 1166.60

1032.83

722.55

1306.59

1601.68 1306.43

864.49 726.71 728.31

749.81

15 1376.91

810.24

1376.76

1460.29 1377.05 2906.97

1460.29 1460.26

2870.61

0.0 4000.0

3600

3200

2800

2400

2000

1800

1600

1400

1200

1000

800

600

cm-1

Fig.-7: FTIR of oils: Black color high PCA/ Blue color Oil No1/ Red color Oil 2 Table-10: Rheometric Properties @ 1930C/2.5min TEST

HPCA

LPCA-1

LPCA-2

LPCA-3

MIN TQ. (lb-in)

0.23

0.22

MAX.TQ.(lb-in)

1.19

1.84

1.87

1.82

Final TQIlb-in)

13.61

13.18

13.06

13.26

tS1 (min)

0.49

0.5

0.48

tS2 (min)

0.66

0.64

0.63

N. Kumar et al.

400.0

Vol.2, No.1, 12-19 (2011) tC10 (min)

0.53

0.51

0.52

0.5

tC40 (min)

0.83

0.8

0.81

0.8

tC50 (min)

0.88

0.84

0.86

0.84

tC90 (min)

1.19

1.15

1.17

1.14

Table-11: Rheometric Properties @ 160 C/30min (Final) TEST

HPCA

LPCA-1

LPCA-2

LPCA-3

MIN TQ. (lb-in)

2.45

2.67

2.64

2.76

MAX.TQ.(lb-in)

15.84

15.76

15.4

16.09

Final TQ.(Ilb-in)

14.56

14.21

13.88

14.57

tS1 (min)

4.1

4.09

4.22

tS2 (min)

5.07

4.94

4.74

5.08

tC10 (min)

4.59

4.47

4.32

4.64

tC40 (min)

5.69

5.58

5.38

5.77

tC50 (min)

5.87

5.77

5.59

5.97

tC90 (min)

7.79

7.66

7.57

7.85

Max-Min Tq.(lb-in)

13.39

13.09

12.76

13.33

REFERENCES 1. 2. 3.

Internet “Definitions of terms relating to oil”. ASTM D1566-06, “Standard Terminology Relating to Rubber”. Encyclopedia of Polymer Science and Engineering, “Cellular Materials to Composites”, IInd Edition, A Wiley-Interscience Publication, 3, 619 (1985). 4. ASTM D2230-96 (Reapproved 2002), “Rubber property-Extrudability of Unvulcanised Compounds”. 5. J. S. Dick and H. Pawlowski, Rubber World, 211(1995) 20. 6. A.Y. Coran and J. B. Donnet, Rubber Chem. Tech., 65(1992) 973. 7. H. D. Luginsland, J. Frohlich and A. Wehmeier, Rubber Chem. Tech.75(2002) 563. 8. S. Das Gupta, presented at 19th Indian Rubber Manufacturers Research Association (IRMRA) Conference”, p. 187, Bombay, India, December 2005. 9. S. Das Gupta, S. L. Agrawal and R. Mukhopadhyay, “Proceedings of the 19th Indian Rubber Manufacturers Research Association (IRMRA) Conference, Bombay, India, December 2005. 10. en.wikipedia.org/wiki/Oil 11. United State patent No. US6,984,687,B2 Jan.10,2006. 12. J. Fraser Stoddart a,*, Howard M. Colquhoun, Tetrahedron 64 (2008) 8231. [IJCEPR-153/2011] ___________________________________________________________________________________________

Highlights of RASĀYAN • • • • • Note:

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.

N. Kumar et al.

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 20-25 January-April, 2011

Infra Red Spectral and X-ray Diffraction Study of Fe (II), Co(II), Cu (II), Metal Chelates with N1-(5, 6-dimethoxypyrimidin-4-yl) Sulphanilamide Jitendra H. Deshmukh*1 and M. N. Deshpande2 1

Department of chemistry, Yeshwant Mahavidyalaya, Nanded-431602 Head P.G. Department of chemistry, NES, Science College, Nanded-4316053 *E-mail: jitendra_deshmukh25@rediffmail.com 2

Article History: Received: 14 April 2011 Accepted:25 April 2011

ABSTRACT N1-(5, 6-dimethoxypyrimidin-4-yl) sulphanilamide (DMPS)forms chelates having general formula [Fe (DMPS)2 Cl2] H2O, [Co (DMPS)Cl22H2O] and [Cu(DMPS)ClH2O]Cl .Infra red ,electronic and X-ray diffraction study of Fe(II), Co(II), Cu(II) metal chelates. Keywords: N1-(5,6-dimethoxypyrimidin-4-yl)sulphanilamide, Infra red, electronic and X-ray diffraction study ©2011 ijCEPr. All rights reserved

INTRODUCTION Literature survey reveals that complexes of metal salts are more potent and less toxic in many cases as compared to the parent drug[1]. These complexes are found to be interesting due to their biological application like antifungal[2], antibacterial[3] activity. Large number of drugs has been used to synthesize the complexes with many metals with a view to enhance their therapeutic action[4-6]. In continuation of our studies on the metal complexes of N1-(5,6 dimethoxypyrimidin-4-yl) sulphanilamide we report here Infra red ,electronic and X-ray diffraction study of Fe(II), Co(II), Cu(II) metal chelates.

EXPERIMENTAL All the chemicals used in the present study were from BDH grade. Metal salts and solvents were used of reagent grade. Metal ion solution and ligand solution in appropriate as desired and pH of resulting mixture were maintained about 6.8 to 7.1 by putting alcoholic ammonia solution. The reaction mixture was refluxed for three hours by keeping the round bottom flask on steam bath, The solid thus separated on cooling was filtered ,washed and dried in vacuum desiccators over anhydrous CaCl2.The detailed preparation of complexes were discussed in earlier papers.

RESULTS AND DISCUSSION Infrared Spectral study Important absorption frequencies of ligand and complexes along with their assignment are presented in the table-1. The assignments are well supported by literature survey. The comparision of IR spectrum data of Fe (II), Co (II) and Cu (II) complexes with ligand N1_ (5, 6-dimethoxypyrimidin-4-yl) sulphanilamide helps in determining bonding pattern in the complexes. Table-1: Infrared spectral data of the ligand (DMPS) and their metal complexes Compound DMPS [Fe(DMPS)2 Cl2] H2O [Co(DMPS) Cl2 2H2O] [Cu (DMPS) Cl H2O] Cl

ν(NH2) 3238 3296 3334 32003449

ν(NH) 3216 3256 3248 32003449

ν(OCH3) 1010 1010 1010

ν(M-Cl) 340 317

ν(M-N) 459 417

ν(M-O) 553 519

1010

299

432

502

IR spectra of ligand shows strong bond in the region 3238 cm-1 which is assigned to ν(NH2) Stretching vibration[7]. Ligand shows band at 3116 cm-1 assigned to ν(NH) stretching[8]. Band at 1010 cm-1 is due presence of (OCH3) group in ligand[9].

Jitendra H. Deshmukh and M. N. Deshpande

Vol.2, No.1, 20-25 (2011) The IR of Fe (II) complex shows strong band at 3406 cm-1 assigned to presence of lattice water in the complex[10].In the IR Spectra of Fe (II) complex, ν(NH2) band shifted and observed at 3296 cm-1 indicating formation of co-ordinate bond with metal. Similarly ν(NH) band in ligand is at 3216 cm-1 which shift to higher frequency region 3256 cm-1 in complex. The additional bands around 459 cm-1 and 553 cm-1 are assigned to ν(M-N) and ν(M-0) Stretching vibration, respectively. These bands were not observed in ligand. Co-ordination with the chlorine atom is supported by the appearance of band in the far IR region at 340 cm-1 which may be assigned to ν(M-Cl) linkage[11]. IR spectra of Co (II) complex shows intense bond at 3334cm-1 due to ν(NH2) band. This band in ligand observed at 3338 cm-1. This band shifting indicates coordination of nitrogen with metal ion. In ligand ν(NH) observed at 3216 cm-1 but Co(II) complex it is shifted towards higher frequency and appears at 3248 cm-1. In the far IR spectra region of the complex, the bands at 417cm-1 and 519cm-1 observed can be assigned to ν(M-N) and ν(M-O) stretching vibration respectively. Strong band at 3465 cm-1 indicates the presence of lattice water and ν(M-Cl) stretching vibration is observed at 317cm-1. In the IR spectra Cu (II) Complex band at 3475 cm-1 is observed indicating the presence of coordinated water. ν(NH2) and ν(NH) bands merge in the complex and appeared 3200-3449 cm-1. Similarly low intense band is observed at 432 cm-1 and 502 cm-1 due to formation of ν(M-N) and ν(M-O) bond. Band at 299 cm-1 indicates formation of coordinate bond of chlorine with metal ν(M-Cl). Electronic spectra The electronic spectrum deals with transitions in UV and Visible region. The electronic spectra of the complexing agent N1_ (5, 6-dimethoxypyrimidin-4-yl) sulphanilamide and their metal complexes were obtained from SAIF, IIT Chennai. The ligand exhibit strong bands around 36000 cm-1 with a shoulder at 33,000cm-1 assigned to π→π* and n→π* transitions respectively[12]. Different absorption bands and corresponding transitions are given in the table 2. Table-2: Electronic spectral data in cm-1 and magnetic moment values of Fe (II), Co (II) and Cu (II) complexes with DMPS Compound [Fe (DMPS)2Cl2] H2O [Co(DMPS)Cl2 2H2O]

[Cu (DMPS) Cl H2O] Cl

Absorbance band cm-1 23774, 21505, 16606 22779, 16339, 14925 22727, 14513

17921,

Transitions No specific assignment are made 4 T1g(F)→4T1g(P) 4 T1g(F)→4A2g(F), 4T1g(F) → 4 T2g, 4

A2→ 4T1g charge transfer

Magnetic Moment (BM) 5.05 3.85

2.08

Electronic spectra of Fe (II) complex have different transitions and different absorbance peaks due to presence of the metal ion in the complex. Fe (II) complex is d6 system having four unpaired electrons. Therefore complex is paramagnetic. Due to presence of the d-d transitions some different peaks are observed in electronic spectra of the complex than the electronic spectra of the ligand. In electronic spectra of Fe(II)complex low intensity bands 23774 cm-1, 21505 cm-1 and 16606 cm-1 no specific assignments are made[13].Complex may acquire octahedral geometry outer hybrid orbital are used to form co-ordinate bond between donor atom of ligand and the central metal ion. The magnetic moment shown by metal complex is 5.05 BM. Electronic spectra of Co (II) complex exhibit three absorbance peaks at 22779 cm-1, 16339 cm-1 and 14925 cm-1. These absorbance maxima due to 4T1g(F)→4T1g(P), 4 T1g(F)→4A2g(F) and 4T1g(F) → 4T2g, transitions respectively characteristics of the octahedral geometry around Co(II)metal ion[14]. Magnetic moment is found to be 3.85 BM. Electronic spectra of Cu (II) complex have three bands at 22727 cm-1, 17921cm-1 and 14513cm-1 indicating the transition between the ligand to copper metal ion. The geometry of the complex is tetrahedral. Magnetic susceptibility indicates the presence of one unpaired electron. The presence of above bands in electronic spectra of Cu (II) complex indicates 4A2→4T1g transition and also the transitions due to charge transfer. Magnetic moment for the complexes is found to be 2.08 BM. Electron Spin Resonance study

Jitendra H. Deshmukh and M. N. Deshpande

Vol.2, No.1, 20-25 (2011) The ESR spectrum of the Fe (II), Co (II) and Cu (II) complexes was recorded at room temperature using tetracyanoethylene radical as ‘g’ marker. The H|| and H⊥ values were measured from the spectrum and used to calculate the g|| and g⊥ values by using the formula are given in the table-3.

g av =

1 ( g || + 2 g ⊥ ) 3

ESR spectra of Fe (II) complex in polycrystalline state shows two peaks. From the observed ESR of Fe (II) complex, the ‘g’ value is 2.203 which is less than 2.3 indicating the coordinate bond between donors atoms of ligand with metal ion have partial covalent character.From ESR spectra of Co (II) complex, g|| obtained is 1.84 and g⊥ is 2.36. gav value for Co (II) complex is found to be 2.18 which is less than 2.3 indicating covalent character of the metal ligand bond[15]. ESR spectra of Cu (II) complex show two peaks. One of intense absorption peak at high field and the other of less intensity peak at low field. From these two peaks, g|| and g⊥ have been calculated. The gav value of Cu (II) is 2.34 which are more than 2.3 indicating the presence of ionic character in the complex. Table-3: ESR spectral data of Fe(II), Co(II) and Cu(II) complexes with DMPS ESR spectral parameters g|| g⊥ gav

[Co(DMPS)Cl2 2H2O] 1.84 2.36 2.18

[Fe (DMPS)2Cl2] H2O 1.78 2.40 2.203

[Cu (DMPS) Cl H2O] Cl 1.91 2.55 2.34

X-ray diffraction Study The crystal lattice parameters of Fe (II), Co (II), Cu (II) complexes with DMPS were found out by X-ray diffraction powder method. The X-ray diffraction of complexes was recorded in the range 200 to 800 on 2θ value. The major refluxes were measured and the corresponding d-values were obtained. An independent indexing for each of these refluxes was carried out by least square method[16]. The miller indices (h k l) were calculated and refined using Back-cal program by computational method and data has been summarized in the following tables. Table-4:Cell data and crystal lattice parameters for [Fe (DMPS)2Cl2] H2O complex Volume (A0)3 = 19578.25 a (A0) = 30.052210 ± 0.035028 b (A0) = 30.052210 ± 0.032651 Dcal = 1.0387 g/cm3 0 Dobs = 1.2985 g/cm3 c (A ) = 25.031710 ± 0.093737 Standard deviation=0.049395 Z = 16 = 4.9% Crystal system = Hexagonal α=90° β=90° γ=120° Porosity (%) = 20 I/Io Dobs Dcal h k l 11 4.362115 4.378966 6 -1 2 49 3.902708 3.919956 7 -1 1 65 3.789483 3.783258 7 -1 2 38 3.682723 3.677650 8 -3 1 32 3.533657 3.532010 7 -3 4 72 3.486692 3.467847 8 -2 2 33 3.115265 3.110134 7 -1 5 16 2.847374 2.851161 10 -3 2 51 2.455439 2.451257 9 2 3 100 2.196959 2.198960 10 3 1 28 2.049400 2.051326 6 6 7 27 1.990465 1.990256 9 6 0 19 1.882965 1.885334 9 2 9 17 1.798814 1.798980 8 8 4

Jitendra H. Deshmukh and M. N. Deshpande

Vol.2, No.1, 20-25 (2011) 16 1.662780 1.662464 10 8 1 The cell data and crystal parameters of Fe (II) complex is given in the table indicates that the complex have hexagonal crystal system. Table-5: Cell data and crystal lattice parameters for [Co (DMPS)Cl2 2H2O] complex a (A0) = 30.059050 ± 0.047643 Volume (A0)3 = 19552.39 0 b (A ) = 30.0659050 ± 0.045038 Dcal = 1.2940 g/cm3 0 c (A ) = 24.970650 ± 0.053964 Dobs = 1.3988 g/cm3 Standard deviation = 0.0050235 Z = 32 = 0.50% Crystal system = Hexagonal α= 900 β= 900 γ =1200 Porosity (%) = 7.49 Dcal h k l I/Io Dobs 20 3.902708 3.921863 7 -1 1 9 3.646795 3.641020 4 -2 6 7 3.631565 3.611176 8 -2 0 25 2.675749 2.677321 5 0 8 14 2.478043 2.479616 8 -1 7 23 2.411530 2.414165 8 4 2 14 2.308712 2.309016 10 0 5 13 2.214781 2.215209 10 1 5 9 2.019460 2.022433 10 4 3 16 1.935065 1.936715 9 6 3 39 1.870411 1.871525 10 2 8 16 1.702111 1.700870 9 8 4 19 1.572922 1.569306 10 9 2 Cell data and crystal lattice parameters of Co (II) complex attributed to hexagonal crystal system. Table-6: Cell data and crystal lattice parameters for [Cu (DMPS) Cl H2O] Cl Complex a (A0) = 21.878380 ± 0.015787 Volume (A0)3 = 14362.44 0 b (A ) = 23.412220 ± 0.041224 Dcal = 0.8558 g/cm3 0 Dobs = 1.0963 g/cm3 c (A ) = 28.039510 ± 0.054712 Standard deviation = 0.0030459 Z = 16 = 0.3% Crystal system = Orthorhombic α= 900 β= 900 γ = 900 Porosity (%) = 24.05 I/Io Dobs Dcal h k l 8 4.087049 4.098696 5 2 0 27 3.789483 3.793231 3 5 2 17 3.533657 3.528988 6 0 2 12 3.440960 3.434774 3 5 4 11 3.396491 3.397027 6 0 3 100 2.878237 2.873455 7 2 3 7 2.675844 2.680874 7 3 4 10 2.524552 2.526895 6 0 8 11 2.478043 2.477680 8 4 0 10 2.369269 2.368666 6 0 9 11 2.232888 2.232415 8 6 1 7 2.128633 2.125735 10 2 2 10 1.935064 1.935126 9 3 8 Cell data and crystal lattice parameters of Cu (II) complex indicates that complex have orthorhombic crystal system.

Jitendra H. Deshmukh and M. N. Deshpande

Vol.2, No.1, 20-25 (2011) O

H 3C

N N

H 3C

O S

H Fe H 2O H H

O O

CH3

N N

CH3

Mol. formula C24H30N8O9S2Cl2Fe; Mol.wt. = 765.64 Dichloro bis N1(5, 6-dimethoxypyrimidin-4-yl) sulphanilamide Fe (II) complex. H

O H Co H

H N

S O O

CH3

N N

CH3

Mol. Formula: C12H18N4O6SCl2Co; Mol. wt. = 476.32 Dichloro N1â&#x20AC;&#x201C;(5, 6-dimethoxypyrimidin-4-yl) sulphanilamide diaquo Co (II) Complex H

H O

Cu Cl N

Cl O H H 3C

S O

O N

H3C

Molecular Formula: C12H16N4O5S Cl2Cu ; Mol. wt. = 462.82 Monochloro N1-(5,6-dimethoxypyrimidin-4-yl) sulphanilamide aquo Cu(II) Chloride Complex.

CONCLUSION All the complexes are paramagnetic. Electronic spectrum of each metal complexes produce intense peak at higher wave number. From the foregoing observations, the suggested chemical structures for the prepared complexes under investigation are as follows.

Jitendra H. Deshmukh and M. N. Deshpande

Vol.2, No.1, 20-25 (2011)

ACKNOWLEDGEMENTS The author would like to thanks the Principal and Head Department of Chemistry, Science College, Nanded for providing all necessary facilities. In addition, the author wishes to acknowledge Principal Yeshwant College, Nanded for encouragement.

REFERENCES 1. 2. 3.

Singh A. and Singh P., Indian J. Chem.,39A (2000)874 Sharma R.C. and Parashar R.K., J. Inorg. BioChem. ,32(1998) 163. Adbel-Waheb Z.H., Mashaly M.M., Salman A.A., El-Shetary B.A. and Faheim A.A. Spectrachim Acta., 60 (2004)2861. 4. Reedijk J., Pure Appl.Chem. ,59(1987) 181. 5. Lochrer P.J. and Einhorn L.H., Ann.inten, Med .,100(1984) 704. 6. Bell R.A., Lock C.J.L., Scholten C. and Villiant J.F., Inorg. Chem. Acta, 274 (1998) 137. 7. Farinan J.A., Patel K.S. and Nelson L.O.; J. Inorg. Nucl. Chem., 38(1976) 77. 8. Mitu L. and Kriza Angela, Asian J. of Chem. , 19(1)(2007) 658. 9. Bellamy L.J., The infrared organic molecules,1958, John Wiley Sons, Inc. New York. 10. Nakamoto K., 1963, IR spectra Wiley New York . 11. Goldstein M. and Unworth D., Inorg. Chem. Acta., 4(1970)37. 12. Seetharama Rao T., Laxma Reddy K. and Lingaih Ind. Acad Sci. (Chem.Sci.) , 100(5)(1988) 333. 13. Anuradha G.H. and Chandrapal A.V., Ori. J. Chem., 23 (1) (2007) 287. 14. Mathew and Watson (1971). 15. Kivelson D. and Meinan R., J. Chem.Phys., 35 (1961) 149. 16. Stout G.H. and . Jensen L.H., 1968, X-ray structure determination a practical guide, MacMillan; New York. [IJCEPR-161/2011] __________________________________________________________________________________________

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_____________________________________________________________________________ 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, â&#x20AC;&#x2DC;Anukampaâ&#x20AC;&#x2122;,Janakpuri, Opp. Heerapura Power Station, Ajmer Road, Jaipur-302024 (India) E-mail: ijcepr@gmail.com Phone:0141-2810628(O), 09414202678, 07597925412(M)

Jitendra H. Deshmukh and M. N. Deshpande

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 26-29 January-April, 2011

Comparative Study between soda Lignin and soda Anthraquinone lignin in terms of Physiochemical Properties of Ipomoea carena Preeti Nandkumar Department of Applied Chemistry, M. P. Christian College of Engineering & Technology, Bhilai – 490026. E-mail: nandupreeti@yahoo.co.in Article History: Received: 17 March 2011 Accepted: 27 April 2011

ABSTRACT Non wood plants are more common as raw material where wood is scarce. In view of its easy availability of ipomoea carnea was utilized as raw material.In this study soda lignin and soda anthraquinone lignin were studied.A comparision has been made between the physico chemical properties and structural features of isolated lignin.These characterisation was done by Fourier Transform Infrared specrometry(FTIR),Ultraviolet(UV)and High Perormance Liquid chromatography(HPLC).Nitrobenzene oxidation was performed with the two types of lignin especially soda lignin and soda anthrquinone lignin.According to the FTIR report, there is no significant difference in terms of functional groups that exists in both the lignin. HPLC results however identified that in both the lignin samples the presence of vanillin and syringaldehyde was found. Key words: Ipomoea carnea, lignin, anthraquinone, black liquor. ©2011 ijCEPr. All rights reserved

INTRODUCTION Ipomoea carnea is a common weed and locally known as BESHRAM. It is also known as bush morning glory which is fast growing and attains optimum size in about a year’s time. Due to its high adaptability and resistance towards adverse climatic conditions, it may grow in all types of climate and soils, marshy as well as dry. A large diffused or straggling shrub with milky juice native of South America, the plant was originally used for making fence for the road side fields, but due to its massive growth and rapid propagation it has grown rapidly in barren waste lands. Plantations of ipomoea carnea may be undertaken in the month of June, July with the onset of monsoon[1]. Ipomoea carnea plant is poisonous to animals. Its leaves contain a polysaccharide-ipomus, one glucosideanthracene a gum-gelapin and saponin .Out of the two materials, one is soluble in water and the other is in ether. Both polysaccharide and anthracene present in ipomoea carnea are water soluble poisons when enters into the central nervous system it damages the respiratory track[2], the scarcity and the restricted supply of high quality pulp and the rising price of utilities will force paper mills to adopt new technologies to conserve energy, minimum inputs, keeping environmental aspects in view, much efforts have been directed towards finding a chemical pulping process giving higher pulp yield coupled with economic and environmental considerations[3]. The process of producing cellulosic pulp from ipomoea carnea jacq requires delignification with sodium hydroxide under pressure.This process frees the cellulosic fiber from ipomoea carnea and produces a large quantity of black liquor that is discharged into surface water without effective treatment[4].Based on the study of ipomoea carnea as lignocellulosic raw material for the pulp and paper industry, sodium hydroxide lignin extracted from soda pulping has been compared with soda anthraquinone (AQ) lignin extracted from soda AQ pulping in this study. Lignin extracted during the pulping process has so far not being investigated for its usefulness. Before its application can be considered, knowledge of its structural characterization is required; this study represents such an effort. Lignin is an amorphous polyphenolic material arising from an enzyme mediated dehydrogenates polymerization of three major phenyl propanoid monomer which is coniferyl, sinapyl and p-coumaryl alcohol. The lignin structural elements are linked by carbon-carbon and ether bond to form tridimensional network associated with the hemicelluloses polysaccharide inside the cell wall. Lignin is usually insoluble in all solvents and can only be degraded by physical or chemical treatment. During the chemical pulping process at high temperature and high pressures degradation of lignin occurs and dissolves into the spent liquor. The delignification reactions involved the cleavage of non phenolic β-O-4 linkage, phenolic α-O-4 linkage and releasing from the associated by the polysaccharide. Addition of small quantity of anthraquinone to the alkaline pulping process increases lignin removal by promoting cleavage of interunit bonds in the lignin molecules that are not cleaved in the absence of

Preeti Nandkumar

Vol.2, No.1, 26-29 (2011) anthraquinone. Anthraquinone helps to minimize recondensations reactions by reacting with the carbohydrates to increase lignin removal during pulping process[5,6]. This study was conducted to characterize soda lignin and soda anthraquinone lignin in terms of their physicochemical properties and their structural features. The objective is to determine that adding anthraquinone to the pulping process changes the properties of the lignin produced. Complimentary destructive nitrobenzene oxidation and non destructive Infrared (IR), Ultraviolet (UV) and high performance liquid chromatography (HPLC) to evaluate the cross linked lignins and their linkages to cell wall polysaccharide.

MATERIAL AND METHODS Raw material used for the laboratory experiment is ipomoea carnea jacq. The sample were collected, cleaned, chipped and screened. The screened chips were used for the experiment. About 500 gms of screened chips of ipomoea carnea jacq was pulped by soda pulping and soda anthraquinone pulping in a 20 liter stainless steel rotatory digester unit with 25% NaOH (cooking liquor) in 3hrs at a maximum cooking temperature of 170oC at a pressure of 10psi with a cooking liquor to ipomoea carnea ratio of 10:1 by weight. For soda AQ pulping 0.1% anthraquinone was added to the soda pulping system. The soda and soda anthraquinone lignin were precipitated from the black liquor by acidifying it to pH 50mg of dry soda lignin or soda anthraquinone lignin was added to 7ml of 2M, NaOH and 0.4ml of nitrobenzene in a 15 ml steel autoclave. The autoclave was sealed tightly with a screw cap fitted with Teflon gasket and heated to 165oC for 3hrs in an oil bath. After heating the autoclave was cooled quickly by immersion in ice water. The soda lignin mixture was transferred to a liquid-liquid extractor for continuous extraction with 10ml chloroform to remove any remaining nitrobenzene reduction products and excess nitrobenzene. The oxidized mixture was acidified with conc.HCl to pH 3-4 and then extracted with 20ml chloroform. The chloroform was removed by using a rotatory evaporator at 40oC under reduced pressure to obtain nitrobenzene oxidation mixture which was used with a stock solution for further analysis. Table-1:Yield and molar ratio of degradation products of soda lignin and soda AQ lignin by nitrobenzene oxidation Oxidation peak

Oxidation product

A B C D E F G Molar Ratio

p-hydroxy benzoic acid(H1) Vanillic Acid (V1) Syringic Acid (S1) p-hydroxy benzaldehyde(H2) Vanillin (V2) p-coumaric acid(B) Syringaldehyde (S2) S/S V/S H/S S=S1+S2, V=V1+V2, H=H1+H2

Soda retention time

Liquid yield %

Soda AQ retention time

Lignin yield %

4.29 5.23 5.55 6.53 8.44 10.13 12.38

4.98 3.98 4.74 26.54 30.33 26.54 2.84 1 5 4

4.3 5.28 5.6 6.58 8.51 10.17 12.42

0.64 5.65 4.92 15.97 36.86 31.95 3.69 1 5 2

Table-2: IR Stretching frequencies S.No. 1.

Type of Bond O-H Bond

Stretching frequencies 3430-3400cm-1

2. 3. 4. 5.

C-H Bond( in methyl group) C-O ( in carbonyl compounds) C-O (in conjugated carbonyl compounds with aromatic ring Aromatic ring

6. 7.

C-H Bond (bending vibrations from aromatic group ) C-O ( in syringyl group )

2940-2930cm-1 1720-1660cm-1 1712-1702cm-1 1609-1604cm-1 1516-1510cm-1 1426-1422cm-1 1470-1460cm-1 1330-1325cm-1 1117-1115cm-1

Intensity Strong, broad Medium Strong Medium Strong

Medium Weak Medium Preeti Nandkumar

Vol.2, No.1, 26-29 (2011) 8. 9.

C-O(in syringyl and guaiacyl group) C-O(in guaiacyl group)

10.

Bending vibrations inside aromatic plane for guaiacyl ring

11.

1220-1215cm-1 1158-1155cm-1 1038-1030cm-1 1038-1030cm-1 -1

C-H deformation and ring vibration

838-834 cm

Strong Strong Strong Medium

High performance liquid chromatography (HPLC) was used to analyze the nitrobenzene mixture. Stock solution(0.25ml) was pipette into 25ml volumetric flask and made up to volume with acetonitrile:water (1:2 v/v).forty micro liter of the filtrate was injected into an HPLC system equipped with hypersil bond C18 column to identify oxidation product. A 1:8 mixture of acetonitrile: water containing 1% acetic acid was used as an eluent with a flow rate of 2ml min-1.the eluent was monitored with an UV (ultraviolet) detector at 280nm.IR spectra were recorded with a Perkin Elmer spectrophotometer for each sample. KBr pellets were prepared containing 1% finely ground sample. For UV spectra-A Hitachi spectrophotometer was used to obtain the results. Prior to the analysis,5mg samples were dissolved in 10ml 90% (v/v) dioxane:water.The sample was then measured its absorbance for range of 210 to 350 nm.

Fig.-1

RESULTS AND DISCUSSION Yield of soda anthraquinone lignin was much higher as compared with the yield of soda lignin. It was found that anthraquinone lignin was 9.6% high than soda lignin. The amount of solubilized lignin in soda AQ black liquor is higher because anthraquinone serves as a catalyst for the soda pulping process.AQ has a marked catalytic effect on the delignification.AQ acts in a redox sequence and cycles between its oxidized and reduced forms. The oxidized AQ form reacts with quinine methide segments of the lignin polymer to increase the rate of delignification. Nitrobenzene oxidation is one of the standard methods for analyzing lignin by chemical degradation technique in order to gain information about the composition of the original polymer. The production of aromatic aldehyde upon oxidation of lignin with alkaline nitrobenzene takes place. Three monomeric lignin unitsâ&#x20AC;&#x2122; i.e. p-hydroxyphenyl (H), guaicyl (V) and syringyl (S) based on the amount of their degradation product. The degradation product of syringaldehyde and vanillin were analysed.Syringaldehyde was found to be predominant followed by vanillin as a second major degradation product. HPLC chromatogram for soda lignin and soda AQ lignin are similar. In general the S: V: H ratio for both syringaldehyde and vanillin. The ligninâ&#x20AC;&#x2122;s are about the same which is 1:5:4 for soda lignin and 1:5:2 for soda AQ lignin. IR spectra (Fig.-1) of soda lignin and soda AQ lignin precipitates have a strong and broad band at 3406cm-1 which is a characteristic of OH group or phenolic compound from soda AQ lignin. The band width and strength could be due to moisture in the sample, since the OH vibration of water usually is very broad. The clear peak at 2934-2844 cm-1 for the soda AQ lignin is attributed to the vibration of a Methoxyl (-OCH3) group while slightly different values were observed for soda lignin (2936-2844 cm-1).the band at 1462 cm-1 is assigned to CH stretching of methyl or methylene groups and the broad medium band at 1712 cm-1 is due to conjugated carbonyl stretching. The three bands at 1606, 1515 and 1425 cm-1 are characteristics of aromatic rings due to aromatic vibrations and the band at 832cm-1 indicates CH deformation and ring vibrations. The bands at 1329 cm-1 for soda AQ lignin and 1328 cm-1 for soda lignin may be due to the vibration of C aryl-O in syringyl derivatives. The bands at 1328-1329cm-1 and 1216

Preeti Nandkumar

Vol.2, No.1, 26-29 (2011) cm-1 corresponds to a syringyl units and the small bands at 1033-041cm-1 are assigned to guaiacyl units of lignin molecules

CONCLUSION Addition of anthraquinone to the pulping process does not affect the quality of lignin precipitated from soda black liquor; even though it nearly doubles the amount of lignin precipitated from black liquor. Rate of delignification was higher with 0.1% addition of anthraquinone. The production of aromatic aldehyde upon oxidation of lignin with alkaline nitrobenzene takes place the product yielded are vanillin and syringaldehyde. Molar ratio of syringyl and guaicyl unit varies from species to species even in the same genus and exerts its influence on rate of delignification. Higher the ratio better is the material from delignification and from processing lignin containing spent liquor points of view.

REFERENCES Ipomoea Carnea; The Wealth of India CSIR publication, raw material, 5 (1950) 58. Nair Preeti , Shukla R.N., Indian Journal of Applied and Pure Bio., 19(2) (2004)189. Venica, A., C.L.Chen and J.S.Gratzl, Delignification of hardwoods during alkaline pulping; reactions, mechanisms and characteristics of dissolved lignin’s during soda aqueous pulping of poplar, Tappi proceeding, pp.503, 1989. 4. Sucking I.D., The role of anthraquinone in sulphite pulping TAPPI wood and pulping chemistry Tappi proceedings, pp. 503(1989). 5. Lin, S.Y., and C.W.Dence, Method in Lignin Chemistry, Springer pp 65-67, 71-73, 75-80.,(1992). 6. Sun, R.C., J. Tompkinson and G.L.Jones ,Fractional Characterization of Ash by Successive Extraction with Organic Solvents.,68 (2000)111. [IJCEPR-154/2011] __________________________________________________________________________________________

1. 2. 3.

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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) Adopt GREEN CHEMISTRY Save Our Planet. We publish papers of Green Chemistry on priority.

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Preeti Nandkumar

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 30-33 January-April, 2011

Use of Millet Husk as a Biosorbent for the Removal of chromium and Manganese Ions from the Aqueous Solutions. Manju Chaudhary Department of chemistry, Siddhi Vinayak College of Science and Higher Education, Alwar, Rajasthan. E-mail: m75anju@yahoo.co.in Article History: Received:28 January 2011 Accepted:5 February 2011

ABSTRACT Movement of heavy metal ions into the water sources has made it unfit for consumption. Rural areas situated near the industrial area are facing the problem of contaminated water. Present work is an effort to develop a self sufficient, easy to assemble and eco friendly system for the removal of heavy metal ions from the water. Millet husk readily available in the rural areas of eastern Rajasthan has been tried and tested for the removal of chromium and manganese ions from the aqueous system. The results are exiting and indicate that it could be used for the removal of heavy metal ions from the water. A column of 12 inches height and 2 inches diameter of pretreated millet husk was used as a biosorbent. This column is sufficient to treat a 50 ml solution (0.012g/L) of chromium ions and 65ml solution (0.001g/L) of manganese ions. The rate of flow of the solution was kept 2ml/min at room temperature and Ph3-4. Key words: biosorbent , millet husk, biosorption, Chromium ions, manganese ions. ÂŠ2011 ijCEPr. All rights reserved

INTRODUCTION Growing industries gave good job opportunities and pace to the economic growth of India and at the same time pose serious threat to the environment. Water being most important part of industries and environment has been affected most. Quality of water in the nearby areas of the industrial growth is facing a serious problem of heavy metal contamination. Surface water as well as underground water is equally affected. About 20 metals have been identified as toxic to human health and out of this half are emitted into the environment in quantities that pose risk to human health. A number of chemical and physical processes are available for the removal of these toxic ions from the water samples. Of all the available processes which can remove these toxic ions from water have some or the other technical or economical problem. A study of such systems and their difficulties has been reported [1]. Therefore some methods which are environment friendly and low cost are desirable. A study on the different types of biosorbents and their probable use has been presented by Volesky et al [2]. Here is a useful method which in recent years has gained a lot of attention. Biosorption can be defined as a â&#x20AC;&#x2DC;non directed physico-chemical interaction that may occur between metal/radionuclide species and microbial cells [3].A variety of micro organisms as biosorbents have been identified and reported[4-7] for the removal of heavy metal ions. Some plant wastes like obtained from agriculture farms have also been reported to be efficient biosorbents[8-15].Removal of chromium[16-18], iron[16,19], lead[19,20] and copper [19-21])have also been studied and reported . In the present paper the biosorption of lead, copper and cadmium on the two biosorbents that are available in plenty in southern Rajasthan are reported.

MATERIALS AND METHOD Preparation of biosorbent The biowaste that is the husk of millet and oat have been procured from the nearby village. The husk was washed thoroughly with water to remove sand etc. Then it was treated with dilute HCl to remove natural colour of the husk and then again washed with demineralised water. Thoroughly washed husk have dried in an hot air oven at about 60 0 C for 24 hours. The properly dried husk is now ground to a fine powder and kept in air tight jars and taken as and when required. A steel column of 20 inches height and 2inches diameter has been taken. The biosorbent was filled into the column till a height of 12 inches only. All the experiments have been carried out using this column only.

Manju Chaudhary

Vol.1, No.2, 30-33 (2011) Preparation of the solutions: Standard solutions (1000mg/L) of chromium (VI) and manganese (VII) were prepared in the laboratory by dissolving potassiumdichromate and potassium permanganate in doubly distilled water. Stock solutions were kept in covered and labeled bottles. These stock solutions were used after further dilution according to the requirement. The pH of the solutions was adjusted using NaOH and HCl. Method The test solution containing metal ions was allowed to flow through the prepared column of 12 inches height and 2 inches diameter. The flow of test solution was maintained by using a burette. The flow rate was maintained 3 ml/min for all the experiments except when effect of variation of the rate of flow was studied. A clean tumbler properly washed with deionised water was used to collect the percolated water. All the test solutions were examined before and after the treatment using atomic absorption spectrophotometer. Sorption efficiency (%) of the biosorbent was calculated in terms of amount of metal ion adsorbed per gram of the biomass using following formula. Q = (C0-C)/M X V Sorption efficiency (%) = C0 â&#x20AC;&#x201C; C /C0 X 100 Where Q is the amount of metal ion biosorbed per gram of the biomasss mg/g.C0 and C are the initial and final concentrations of the test solution. V is the volume of the test solution; M is the mass of the biomass (g).

RESULTS AND DISCUSSIONS Effect of pH on the sorption capacity of the biosorbent Biosorption of metal ions on to the surface of the biosorbent is greatly influenced by the pH of the solution. It is expected the sorption of the metal ions increase with increase in the pH of the solution. The possible reason may be that at lower pH, H+ and H3O+ ions compete with the metal ions for adsorption onto the surface of biosorbent. At higher pH less no of H+ ions are there to occupy the adsorbent sites are available for the metal cations. The pH of the solution was varied from 2 to7. The pH of the normal water is around 7 therefore the pH 7 is chosen to conclude the adsorption capacities of the biosorbent. The case is different with the chromium ions.Chromium ions are available as anions CrO32- or HCrO3- in the solution therefore they showed a different character. It has been found that biosorption of chromium decrease with increase in the pH of the solution. Probable reason may the presence more number of positive sites on the biosorbent at lower pH which can adsorbe chromate ions. Similar is the case with the manganese ions. It is clear from the following table that the extent of adsorption decreases with increase in the pH of the solution for manganese ions and chromate ions. The results are shown in table1. Table-1 pH 1 2 3 4 5 6 7

Percentage removal of Cr(VII) 78% 88% 65% 49% 18% 8% 2%

Percentage removal of Mn(VII) 75% 90% 82% 32% 12% 2% 0

Effect of Rate of flow of solution through column Rate of flow of solution through the biosorbent column effect the net percentage removal of metal ions from the solution. If the solution travels quickly down the column then the metal ions get less time to adhere to the surface of the biosorbent. Similarly if the rate of flow through the column is slow than the metal ions will get sufficient time to get adhere to the surface of the biosorbent. Therefore the effect of rate of flow on the percentage removal of the ions from the test solutions must be considered and studied. Following figures illustrates that with the increase in the contact time between the adsorbent and the metal ions from test solutions percentage removal of the ions is also increased. The results are shown as below in table 2.

Manju Chaudhary

Vol.1, No.2, 30-33 (2011) Table-2 Percentage removal of Cr(VII) 88% 72% 66% 62% 53%

Rate of flow of solution (mL/ min) 2 4 6 8 10

Percentage removal of Mn(VII) 90% 53% 48% 34% 20%

Effect of height of column Keeping the diameter of the column fix and varying the height of the column it is seen that the sorption of ions is greatly influenced. Increase in the height of the column means increase in the number of adsorption sites. Therefore the extent of sorption is to increase with the increase in the height of the column. Height of the column is varied from 8inches to 14 inches. Results are shown below in Table 3. Table-3 Height of column in inches (diameter 2 inches in all the cases) 2 4 6 8 10 12

Percentage removal of Cr(VI)

Percentage removal of Mn(VII)

23% 35% 50% 64% 76% 88%

18% 34% 52% 68% 83% 90%

Effect of initial concentration of the metal ion solution Initial concentration of metal ions in the solutions affects the rate sorption of ions on the sorbent surface. Initially the rate of adsorption increase with increase in initial concentration of the ions in solution. This is due to large number of adsorption sites available for few metal ions in the solution. On further increase in the concentration the rate of adsorption nearly become stable when number of metal ions and number of adsorption sites become equal. Beyond this concentration there is no increase in the rate of adsorption. The initial concentration of metal ions taken for the study is 20 mg/L each. The equilibrium is reached at about 80 mg/L and then after no further increase in the rate of adsorption is noticed. The results are shown below in table 4. Table-4 Initial concentration of the metal ions(mg/L) 20 40 60 80 100

Metal uptake of Cr(VII) (mg/g)

Metal uptake of Mn(VII) mg/g

2.5 4.8 5.4 6.8 7.2

1.5 3.6 5.2 6.8 6.8

CONCLUSION Biosorption capacities of Pennisetum typhoideum (millet husk) are found enough for the removal of chromium and mangenese ions from the waste water. The optimum ph range for the purpose lies in the acidic range. There is a need to study the system in more detail and find the practical utility. Widely available and ecofriendly biowastes like husk of millet and oat can serve the purpose of water purifier in the rural areas.

ACKNOWLEDGEMENTS I am thankful to Ms Seema Bhardwaj and Ms Akansha Arora for helping me in editing and formatting of the paper. My sincere thanks are due to the college and chemistry department who provided me the laboratory facility.

Manju Chaudhary

Vol.1, No.2, 30-33 (2011)

REFERENCES Alhalya N., Ramchandra T.V., Kannamadi R. D., Res.J.Chem.Environ., 7 (2003) 71. Volesky B. and Holan Z. R., Biotechnol Prog., II (1995) 235. Shumate S. E., Strankberg G. W., Comprehensive Biotechnology; Pergamon Press, New York (1985) 235. Kapoor A., Virarangahavan T., Bioresour.Technol, 63(1998) 109. Kar R. N., Sahoo B. N., Shukla C. B., Pollut.Res.,11 (1992) 1. Kolishka T., Galin P., Z.Naluforsch, 57 (200) 629. Loderro P., Cordero B., Grille Z., Herror R.,Sastre de Vicente ME, Biotechnol.Bioeng., 88(2004) 237. Sarin V. and Pant K.K., Bioresour.Technol., 97 (2006) 15. Ricordel S., Taha S., Cisse I. and Dorange G., Sep.Purf.Technol., 24 (2001) 389. Selwakumari S., Murugan M., Pattabi S., Sathish Kumar M., Bull.Environ. Cont. Toxicol., 69(2001) 195. Gangsun and Weixing Shi, Ind. Eng.Chem.Res., 37(4) (1998) 1324. Espinola A., Adamiuan R. and Gomes L.M.D., Waste Treat Clean Technol.Proc., 3(1999) 2057. Iqbal M., Saeed A. and Akhtar N., Biores. Technol., 81(2002) 153. Tee T. W. and Khan R.A.M., Environ.Technol. Lett., 9(1988) 1123. Orhan Y. and Buyukgungar H., Water Sci.Technol., 28(1993) 247. Ahalya N., Kanamadi R.D. and Ramachandra T. V., J.Environmental Biology 28(4) (2007) 765. Vinodini V., Anabarasu and Nilanjana Das, Int. J.of natural products and resources, 1(2)(2009) 174. Saifuddin M., Nomanbhay and Kumaran Paanisamy, Environmental Biotechnology, 8(1) (2005). Fan Z., Xiaotao J., Int. J. Chem., 4(2002) 34. Haluk C., Ulki Y., Water S. A., 27 (2001) 15. Muraleedharan T.R., Venkobachr C., Biotechnol.Bioeng, 35(1990) 320. [IJCEPR-144/2011] __________________________________________________________________________________________

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International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 34-39 January-April, 2011

Study Regarding Lake Water Pollution with Heavy Metals in Nagpur City (India) P.J. Puri*,1, M.K.N. Yenkie1, S. P. Sangal2, N.V. Gandhare2 and G. B. Sarote3 *,1

Department of Chemistry, LIT, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440 001, Department of Chemistry, LIT, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440 001, 2 Department of Chemistry, Nabira Mahavidyalaya, RTM, Nagpur University, Katol - 66302 3 Regional Forensic Science Laboratory, Dhantoli, Nagpur – 440 012 E-mail: puripj@rediffmail.com 1

Article History: Received:6 January 2011 Accepted:20 January 2011

ABSTRACT This paper is intended to be a study concerning water pollution with heavy metals in Nagpur City, Maharashtra, India. The levels of the occurrence of heavy metals like cadmium (cd), iron (Fe), zinc (Zn), arsenic (As), mercury (Hg), lead (Pb) and chromium (Cr) were estimated in Futala, Ambazari, Gandhisagar and Gorewada lake, within Nagpur city, for the session January to December 2008. Sampling points were selected on the basis of their importance. The monitoring was made over a period of one year comprising of three seasons; summer, winter and rainy season respectively. The study demon started gradual increase in pollution input of heavy metals in studied lakes. The yearly variation in the concentration of heavy metals had definite upward trends. Present study revealed that dissolved constituents of Fe, Pb, Zn and Cr were above ranges of unpolluted water indicating their contamination throughout the season in cases of Pb, Fe and Zn and occasional for As and Hg. The metals Zn, Fe, Cd, Ni almost remained in natural level while arsenic (As) was always below the detection limit of 0.0001ppm. The Futala, Ambazari and Gandhisagar except Gorewada lake could be identified as probable area of contamination of these metals. The average levels of metals in studied lakes followed the order Zn > Cr > Fe > Cd > Pb > Hg > As. Keywords: Heavy metals, pollution, Lakes, Water Quality. ©2011 ijCEPr. All rights reserved

INTRODUCTION Heavy metals are important environmental pollutants and their toxicity is a problem of increasing significance for ecological, evolutionary, and environmental reasons[1]. Heavy metals have played great roles in genesis of present day civilization. In ancient times, the wealth of Emperors and Kings was attributed to the possession of metals like iron, gold, silver copper etc. in different forms. Still today, the dependence of heavy metals has not decreased as these are very commonly used in agriculture, medicine, engineering etc. The magnitude of danger of environmental pollution by heavy metals was probably for first time realized with the Minimata disaster in Japan, where thousand of peoples suffered with mercury poisoning after consuming the fish caught in Minimata Bay. The bay got contaminated with mercury released from vinyl chloride plant between 1953 and 1960[2]. Similarly, it was also reported in Japan in 1955 that cadmium caused itai-itai Byo’ disease in human beings, mainly in women over forty. This was due to high level of cadmium in local foodstuffs attributable to irrigation water from soil heaps of an abandoned mine. Minimata raised its ugly head once again, not in Japan this time, but in fishing communities of Amazon rain forest. Heavy metal pollution has been worked out in recent days[3-13]. Thus in view of this widely used practice; it was of interest to undertake further investigation on these lines. The purpose of study is to promoting and coordinating activities in the field of environmental chemistry as well as health related water microbiology and hygiene.

MATERIALS AND METHOD Physicochemical characteristics (only pH, conductivity and turbidity), of water samples were determined within twenty four hours of the collection of water samples using standard methods[14]. Total trace metals were determined in acidified water samples after pre concentration by atomic absorption spectrophotometric methods. The metals Fe, Pb, Hg, Cu, Zn, and As were estimated using Atomic Absorption Spectrometer (Make - GBC Australia, Model GBC 932). Air-acetylene flame technique was used for all these metals and hydride generator technique was used for arsenic. For dissolved metals, water samples were preserved by adding HNO3 and filtered through Millipore filtering unit. One liter of the filtered sample was evaporated to dryness and digested with HNO3,

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Vol.2, No.1, 34-39 (2011) HCIO4 mixture. The digested samples were made up to the volume and dissolved metals were analyzed by AAS. Iron, manganese and zinc were subjected to ten-fold concentration by evaporation. Cadmium, chromium, copper and lead were concentrated by complexing with ammonium pyrrolidine dithiocarbamate (APDC) and subsequently extracting the complex in methyl isobutyl ketone (MIBK). Iron, manganese and zinc in aquatic solution and cadmium, chromium, copper and lead in organic solution, were determined by atomizing respective solutions in Atomic Absorption Spectrometer. The instrument was operated in flame mode using air as oxidant and acetylene as fuel Operation parameters were optimized for maximum response. Background correction was made for lead and cadmium and flame rich in acetylene was used for chromium determination. Water samples were analyzed by both classical and automated instrumental methods as appropriated in standard methods for analysis of water and waste water[14]. All reagents used were of analytical grade and instruments pre-calibrated appropriately prior to measurement. Replicate analyses were carried out for each determination to ascertain reproducibility and quality assurance

RESULTS AND DISCUSSION Seasonal variations were noted in the physicochemical properties of studied lake water Different properties like pH, EC, Temperature, HCO3- and conductivity showed maximum values during summer, while minimum values were recorded during autumn season. The observed trend could be attributed to the evaporation of water from studied lake (Gandhisagar, Ambazari, Futala and Gorewada Lake) during summer and subsequent due to precipitation and runoff from catchment area during rainy season[15-16]. High pH values in all studied lakes during summer could be ascribed to increased photosynthetic assimilation of dissolved inorganic carbon by planktons[17]. A similar effect could also be produced by water evaporation through loss of half bound CO2 and precipitation of monocarbonate[18]. The alkaline pH and high alkalinity of Futala, Ambazari and Gandhisagar lake water might be due to use of detergents by neighboring population for washing of cloths, vehicles, and utensils. Higher alkalinity in Futala, Ambazari and Gandhisagar indicated the potential susceptibility of these water bodies for eutrophication. Lake water bodies with alkalinity values above 100 mgL-1 is considered nutritionally rich [19] and on the basis of this observation most of lakes in Nagpur city could be considered prone to eutrophication problems. Seasonal variation in different heavy metal concentration in water from Futala, Ambazari, Gandhisagar and Gorewada lake are presented in Table 1-4. Graphical representation of seasonal variation in different heavy metal concentration in water from Futala, Ambazari, Gandhisagar and Gorewada lake are presented in Figure1-4. Figure 5 represents map showing Gandhisagar, Ambazari, Gorewada and Futala lake, Nagpur (MS) India. The concentration of heavy meals in water of studied lakes remained below toxic limits with few exceptions. A remarkable high concentration of (Fe) iron, ranged from 0.022 mgL-1 to 0.035 mgL-1 , 0.014 mgL-1 to 0.031 mgL-1 , 0.025 mgL-1 to 0.031 mgL-1 and 0.012 mgL-1 to 0.016 mgL-1 in Futala, Gandhisagar, Ambazari and Gorewada lake respectively. The Fe content indicated that this metal was abundant in soil and rocks of catchment area from where the water reaches to these lakes. As regards the effect of season on heavy metals concentration in water of Futala, Ambazari and Gorewada lakes, concentration of metals like Cd, Cr, Fe, Ni, Pb, Zn, Hg were maximum during summer and rainy season while minimum concentrations were observed during autumn season. This trend could be attributed to the evaporation of water from lakes during summer and subsequent dilution due to precipitation and run off from catchment area during rainy season[20]. The variation of level of occurrence of heavy metal in Ambazari, Futala and Gandhisagar lake were found different from each other due to the variation of the solubility of the existing forms of metal in water as well as their availability in the immediate environment. Among metals the level of Zinc ranged from 0.025 mgL-1 to 0.0.048 mgL-1 , 0.016 mgL-1 to 0.041 mgL-1 , 0.021 mgL-1 to 0.032 mgL-1 and 0.012 mgL-1 to 0.021 mgL-1 in Futala, Gandhisagar, Ambazari and Gorewada lake respectively. Arsenic (As) level was always less than 0.1ppb in Ambazari, Futala, Gorewada and Gandhisagar respectively. The average level of metals (in ppm) followed the similar order Zn > Cr > Fe > Cd > Pb > Hg > As for both Futala and Gandhisagar lake respectively. The ranges of variation in present study revealed that the dissolved constitutes of Pb, Cd, Zn, Cr and Zn was above the ranges of unpolluted water indicating their contamination in water. Cadmium was detected in traces and ranged from 0.010 mgL-1 to 0.012 mgL-1 , 0.004 mgL-1 to 0.008 mgL-1 , 0.003 mgL-1 to 0.016 mgL-1 and 0.001 mgL-1 to 0.002 mgL-1 in Futala, Gandhisagar, Ambazari and Gorewada lake respectively. Cadmium is a non essential metal that is toxic even when present in very low concentration. The toxic effect of cadmium is exacerbated by the fact that it has an extremely long biological half â&#x20AC;&#x201C; life and is therefore retained for long periods of time in organisms after bioaccumulation. Cadmium is a respiratory poison and may contribute to high blood pressure and heart diseases[21]. Cadmium has been found to be toxic to fish and other aquatic organisms. Its effect on man includes kidney damage and serves pain in bones (itai â&#x20AC;&#x201C; itai in Japan). The level of lead (Pb) was higher during summer and rainy season as compared to autumn season in all studied lakes. Variations in lead (Pb) level 35

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Vol.2, No.1, 34-39 (2011) varied from 0.014 mgL-1 to 0.026 mgL-1 , 0.018 mgL-1 to 0.022 mgL-1 , 0.025 mgL-1 to 0.034 mgL-1 and 0.011 mgL-1 to 0.021 mgL-1 in Futala, Gandhisagar, Ambazari and Gorewada lake respectively. Adverse Chronic effects may occur at 0.5 mgL-1 to 1.0 mgL-1 (Pb). At levels greater than 0.1 mgL-1 possible neurological damage in fetuses and children may possible. The possible source of Pb in studied lakes could be from domestic sewage, immersion of idols of God and Goddess during festival season and effluent discharge from waste disposal sites as well as geology of catchments. Chromium was detected with lower levels in autumn and higher throughout monitoring period during summer and rainy season respectively. The heavy metals concentration in all studied lakes showed distinct temporal and spatial variations. Among metals, concentration of arsenic (As) remained always below the detection level throughout study period in entire stretch except Futala and Ambazari lake. Thus it could be presumed that levels of (As) arsenic in Gorewada lake remained almost in natural level and there was probably no anthropogenic input in Gorewada lake for its enrichment. Since pH of studied lake water lies in the range of neutral to alkaline the levels of studied metals could not rise so much as there are natural mechanism to remove these metals from aqueous solution and prevent from enrichment. A high degree of yearly variation was observed in zinc concentration. Its yearly variation showed an upward trend. In natural water system Zn remains as either hydroxide or carbonate form with having almost same solubility which is higher than solubility of existing forms of other metals. This could be the reason for comparatively higher values of Zn in studied lake water. The average level of metals followed the order Zn > Cr > Fe > Cd > Pb > Hg > As for both Futala and Gorewada lake respectively. Higher values of metals in all studied lakes are due to washing activities, recreational activities, immersion of idols of God & Goddess during and after festival seasons, vehicle washing, farming (agricultural) activities, weathering of minerals and soils, atmospheric deposition, storm water run off resulting from rainfall, and (poor) sewage. Thus, water system with enriched toxic metals can serve as reservoirs and may becomes a potential source to supply toxic metals in the environment. chromium content in studied lakes ranges from 0.028 mgL-1 to 0.042 mgL-1 , 0.028 mgL-1 to 0.036 mgL-1 , 0.014 mgL-1 to 0.028 mgL-1 and 0.016 mgL-1 to 0.018 mgL-1 in Futala, Gandhisagar, Ambazari and Gorewada lake respectively. Chromium ingestion over admissible limits leads to allergic phenomena and lung cancer. Mercury is considered to be the most toxic metal. In organic form it enters the human through fish. Fishes being one of main aquatic organism in food chain may often accumulate large amount of certain metals[22]. Highly significant difference was noticed in case of mercury (Hg) in water samples collected from Futala, Ambazari and Gandhisagar lake. The concentration of mercury in water varied from 0.005 mgL-1 to 0.018 mgL-1 , 0.008 mgL-1 to 0.018 mgL-1 , 0.010 mgL-1 to 0.021 mgL-1 and 0.001 mgL-1 to 0.003 mgL-1 in Futala, Gandhisagar, Ambazari and Gorewada lake respectively. The continuous increase in heavy metal contamination in studied lake is a cause of concern as these metals have ability to bioaccumulate in tissues of various biotaâ&#x20AC;&#x2122;s and may also affect distribution and density of benthic organisms as well as composition and diversity of faunal communities. Since pH and temperature affects solubility and toxicity of metals in aquatic ecosystem, this pH and temperature ranges were also used to access the metal toxicities in studied lakes. Metals such as Cd, Pb and Zn are most likely to have increased detrimental environmental effects as a result of lowered pH.. Table-1: Heavy metal content (ppm) during different seasons of the year at Futala lake. Heavy Metals (mgL-1 )

Season Zn

Summer

0.048

0.042

0.035

0.012

0.026

BDL

0.018

Winter

0.025

0.039

0.022

0.010

0.014

BDL

0.005

Rainy

0.036

0.028

0.024

0.011

0.021

BDL

0.016

BDL :Below Detectable Limit Table-2: Heavy metal content (ppm) during different seasons of the year at Gandhisagar lake. Heavy Metals (mgL-1 )

Season

Summer

0.041

0.036

0.031

0.008

0.022

BDL

0.012

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Vol.2, No.1, 34-39 (2011) Winter

0.018

0.028

0.014

0.004

0.018

BDL

0.008

Rainy

0.016

0.030

0.018

0.006

0.020

BDL

0.018

BDL :Below Detectable Limit Table-3: Heavy metal content (ppm) during different seasons of the year at Ambazari lake. Heavy Metals (mgL-1 )

Season Zn

Summer

0.032

0.028

0.025

0.005

0.031

BDL

0.016

Winter

0.025

0.016

0.029

0.003

0.025

BDL

0.010

Rainy

0.021

0.014

0.031

0.016

0.034

BDL

0.021

BDL :Below Detectable Limit Table-4: Heavy metal content (ppm) during different seasons of the year at Gorewada lake. Heavy Metals (mgL-1 )

Season Zn

Summer

0.018

0.020

0.011

0.002

0.016

BDL

0.001

Winter

0.012

0.018

0.012

0.001

0.014

BDL

0.001

Rainy

0.020

0.013

0.001

0.010

0.002

BDL

0.003

BDL :Below Detectable Limit

Concentration (ppm)

Fig-1: Heavy metal content (ppm) during different seasons of the year at Futala lake.

0.06 0.04 Summer

0.02

Winter

0 Zn

Rainy

Heavy Metals

Fig.-2: Heavy metal content (ppm) during different seasons of the year at Gandhisagar lake.

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Vol.2, No.1, 34-39 (2011)

Fig.-3: Heavy metal content (ppm) during different seasons of the year at Ambazari lake.

Fig.-4: Heavy metal content (ppm) during different seasons of the year at Gorewada lake.

CONCLUSION The heavy metal concentration in studied lake showed distinct temporal and spatial variations. There was significant seasonal variation in metal concentration within the study period. The dry season registered elevated levels of metals as compared to wet season. Dilution effect of rainy season due to storm run off into receiving lakes and excessive evaporation of surface water with its attendant pre-concentration of most of metals may be responsible for observed trend. The results of study have indicated gross pollution of lakes especially regards heavy metals. This poses a healthy risk to several communities in catchment who rely on these lakes primarily for their domestic sources without treatment. An elevated level of heavy metals in water is a good indication of man â&#x20AC;&#x201C; induced pollution as a result of (poor) sewage, domestic waste and immersion of idols of God and Goddess during and after festival season into studied lakes. There were definite upward yearly trends in the concentration of chromium, iron, lead and zinc in studied lakes which indicated increased input of their pollution load. The levels of mercury (Hg) and arsenic (As) in studied lake water were comparatively lower. The average level of metals followed the order Zn > Cr > Fe > Cd > Pb > Hg > As and for all studied lakes. Through some of detected heavy metals are beneficial for human and plants up to a certain limit; it may be harmful beyond that. Adoption of adequate measures to remove heavy metals load from industrial waste water, prevention of immersion of idols of God and Goddess along with fruits, flowers and worship materials along with washing activities are suggested to avoid further deterioration of lake water quality.

ACKNOWLEDGMENTS The authors hereby acknowledge the kind and wholehearted support of the Dr. S. B. Gholse Director, LIT, RTM, Nagpur University, Nagpur.

REFERENCE 1. 2. 3. 4. 5.

Nagajyoti, P.C., Dinakar, N., Prasad, T.N.V.K.V., Journal of Applied Sciences Research, 4(1) (2008) 110. Friedman, M., Environ. Sci. Technol., 6 (5) (1972) 457. Akeson, M. and D. Munns, Journal Plant Nutri., 13 (1990) 467. Anderson, A. and K. O. Nilsson, Sewish Jounal Agricult. Res., 6 (1976) 15. Antonovics, J., A. D. Bradshw and B.G. Turner : Adv. Ecol. Res., 7 (1975) 1. 38

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Asami Teuro: Sci. Res. Fac. Agric,. Iberaki Univ ., 23 (1975) 43. Azad, A.S., B.R. Arora, B. Singh and G.S. Sekhon: Ind. J. Ecol., II (1) (1984) 1. Borovik, A.S., CRC Press Boca Raton. Florida, 3-5 (1990). Bowen, J. E. : Plant Physiol, 44 (1973) 255. Dutta, I. and Mookerjee, A., Indian J. Environ. Health, 22(3) (1980) 220. Fytianos, K., V. Samanidou and T. Agelidis, Ambia 15 (1) (1986) 42. Mc. Calla, T.M., J.R. Perterson and C.I., Lue ; Ed. Elliot, L.F. and F.J. Stevensn, Sashngton, 28-36 (1977). Vander Werff, M. and J. P. Margaret : Chemosphere. 11(8). (1982). APHA; Standards Methods for the examination of water and waste water, 18th edition, (1998). Wiklander, L. and Kantol Vahtras : Geoderma 19 (2) (1977) 123. Radhika, C. G. Mini, J., & Gangaderr, T.: Pollution Research, 23, (2004) 49. King, D.L., Journal for the Water Pollution Control Federation , 42(1970) 2035. Khan, M.A. G., & Choudhary, S.H., Tropical Ecology, 35, (1994) 35. Patil, P.R., Chaudhari, D.N. & Kinage, M.S. Environemental Ecology, 22, (2004) 65. Bhatt, L.R. Lacoul, P., Lekhak, H.D., & Jha, P.K. : Pollution Research, 18 (1999) 353. Friberg, L. and C. J. Elinder; Health and Diseases, 18 (1988) 559. Purandara, B.K., Vararajan, N. and Jayashree K. Poll., Res, 22 (2) (2003) 189.

Fig. -5: Map showing Gandhisagar, Ambazari, Gorewada and Futala Lake, Nagpur (MS) India. [IJCEPR-142/2011]

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International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 40-43 January-April, 2011

Knowledge, Attitude and Practices regarding Waste Management in Selected Hostel Students of University of Rajasthan, Jaipur Lalita Arora* and Sunita Agarwal Department of Home Science, University of Rajasthan, Jaipur, India *E mail: guddu.arora20@gmail.com Article History: Received: 7 February 2011 Accepted: 27 February 2011

ABSTRACT The risk of unhealthy disposal of solid waste is one of the important problems in many societies. Environmental knowledge attitude practices of young people (like students) appears to be crucial as their point of view ultimately plays an important role in providing solution to future environmental problems. The study was conducted aiming to find knowledge attitude and practices of University students with respect to waste management. Total 300 students were included in this study. Data collected by self administered questionnaire and analyzed, using‘t’ test. It was found that knowledge attitude and practices of University students regarding waste management was low, less favorable and moderate respectively and correlation between knowledge and attitude, attitude and practices was not found, but significant correlation was found between knowledge and practices. Keywords: Knowledge, attitude, practices, waste. ©2011 ijCEPr. All rights reserved

INTRODUCTION With the development of civilization and globalization drastic changes have come in our life style and in every activity like education, recreation, traveling, feeding, clothing and housing, we are generating lots of wastes. The modern 'culture of consumerism' has aggravated the waste problem. To this has added the culture of 'disposable' where large number of goods in the society is being manufactured for 'one time use' and to be discarded as waste after use. These wastes products create particularly serious problems for the municipalities and its safe disposal is becoming a serious environmental problem and an ecological crisis is slowly brewing up, threatening to choke the earth and its life supporting systems. A number of studies have been carried out by various organizations which provide an estimate about the quantity of waste generation in various cities. According to Asokan et al [1] about 960 million tones of solid waste is being generated in India annually, as byproducts during industrial mining municipal, agricultural and other processes. Of this 350 million tones are organic waste from agricultural sources, 290 million tones are inorganic waste of industrial and mining sectors and 4.5 million tones are hazardous in nature. Unhealthy disposal of solid waste is considered as one of the most important problems in many societies. The problem of waste management has arisen recently in developing countries where there is little history of the implementation of formal and informal community environmental education awareness program. Environmental attitude of young people appears to be crucial as they ultimately play a direct role in providing knowledge based solutions to in coming environmental problems[3,5]. The few studies conducted regarding children and young people, show that the level of environmental awareness is relatively low [6]. The information acquired is mostly factual in nature and is not systematized. Begum, R. et al [2] found that majority of the doctors, nurses, and housekeepers have unsatisfactory knowledge and inadequate practice related to health care waste management. Keeping all this in view, the present study was planned to analyze their knowledge attitude and practices of hostel students regarding waste management.

MATERIALS AND METHODS This study attempts to identify the knowledge attitude and practices of University hostel students regarding waste management. Total 807 students were residing in selected girl’s hostels out of which 300 students were selected for study. The selection of the respondents was done by stratified sampling method. Students were divided into strata according to their level of education (PG and UG). 150 students from UG hostels and 150 students from PG hostels

Lalita Arora and Sunita Agarwal

Vol.2, No.1, 40-43 (2011) were selected by selecting every 3rd number from the list of students. Then each of these strata was further subdivided according to their stream of education viz Sc and NonSc and 75 students from each stratum were selected. The self administered questionnaires were used to identify knowledge attitude and practices of University students in the study area. Before it was used, the questionnaire was pretested in the pilot study. Split half method was used to calculate the reliability. Reliability of the questionnaire was .96, .94 and .96 for knowledge attitude and practices questionnaire respectively. Information collected through questionnaire included (1) General information on respondents including age, education, family type and size etc. (2) knowledge regarding waste management (3) attitude regarding waste management (4) practices regarding waste management. The respondents were well informed about the purpose of the study and about the questionnaire by the research investigator prior to data collection. After collecting data, data were edited and tabulated before data analysis. Descriptive statistics i.e. percentage, mean and standard deviation were used to describe studied variables. ‘t’ test and correlation tests were used according to the objective of this study.

RESULTS AND DISCUSSION Knowledge regarding waste management Knowledge about waste management was enquired using (300) questionnaire. The responses were given scores and thus the students were categorized as possessing low, medium and high level of knowledge. It was found that 162(54%) of the respondents could be classified as possessing low knowledge, whilst 138(46%) students were having medium level of knowledge regarding waste management (Table-1). Attitude regarding waste management The responses on attitude were classified into less favorable, favorable and most favorable. It was highly striking to note that majority of hostel students (64.33%) had less favourable attitude towards waste management and only 6.10% (Table-1) were found to have most favourable attitude. Practices regarding waste management The responses to practices by respondents are shown in Table-1. Those who had good practices were assumed to be managing the waste in proper manner and be able protect themselves and environment from negative impact of waste. From the results of this study it was found that only 1.33% of the respondents could be classified as having good practices, whilst more than half of the respondents had moderate practices and nearly half of the respondents 140 (46.66%) were found to have poor practices towards waste management. This indicates that they need to improve their practices regarding waste management. Hebel-Ulrich, Maja[8] has found that many responses regarding knowledge indicate that the awareness about hygiene exists, but is not being practiced. Also the observation of several risk behaviors, such as open defecation, lack of personal hygiene and irresponsible waste management suggests the need for hygiene educational program. Factors influencing knowledge of the respondents Factors influencing included in this part of the study were level of education and stream of education. ’t’ test was used to find out the difference in knowledge scores according to their level of education and stream of education. It can be observed from the Table-2 that there was a significant difference in the knowledge regarding waste management base on educational levels of the respondents. It means that PG students have higher scores of knowledge as compared to UG students. Saini, S. et al [11] measured the knowledge regarding biomedical waste management. Their results show that consultants, residents, and scientists respectively have 85%, 81%, and 86% knowledge about the bio medical waste management. Nurses and sanitary staff, operation theatre and laboratory staff have respectively 60%, 14%, 14%, and 12% awareness of the subject. This shows that the people with higher education have more awareness about the waste management issues. A significant difference was also observed between Sc and NonSc students which signify that stream of education makes an impact on knowledge regarding waste management. According to Ehrampoush, M.H. et.al. [4] the knowledge of the students regarding waste management was not appropriate. About 66% of students did not participate in segregation and recycling of solid waste. Factors influencing attitude of the respondents It was found that level of education did not make any impact on attitude of the respondents regarding waste management as no significant difference was observed between UG and PG students regarding attitude as shown in Table-3. Paengkaew, W. et.al [9] observed that majority of Asian students appeared to have lack of environmental consciousness and attitude needed to protect their environment. Therefore it is important to develop skills, awareness, and attitude and put in to practice. But stream of education is showing a significant difference on attitude. This may be due to that Sc students have some chapters on environmental pollution and waste management in their course and therefore they are little aware 41

Lalita Arora and Sunita Agarwal

Vol.2, No.1, 40-43 (2011) regarding waste management. As per the study done by Saini, S. et al [11] measured the attitude regarding biomedical waste management of doctors, nurses, and other support staff. They found that the people with higher education and knowledge have better attitudes towards the subject. Factors influencing practices of the respondents Practices of students were affected by both the variables i.e. level of education and stream of education. To find out the difference both the variables ‘t’ test was performed and ‘t’ values were found to be 3.86 and 4.14 (Table-4) for level of education and stream of education respectively. From ‘t’ values a significant difference between UG V/s PG and between Sc V/s NonSc was found suggesting that level and stream of the respondents affect the practices regarding waste management. Pothimamaka, J. (2008) found that more than half of the house holds had no waste separation practices and they concluded that their practices were not appropriate towards solid waste management and people must be taught to deal with solid waste by separating it in their homes, schools and work places. Association between knowledge, attitude and practices regarding waste management. Pearson ‘r’ correlation test was used to find out the association between knowledge, attitude and practices regarding waste management. As shown in Table-5 it was observed that there was a significant association between knowledge and practices with the correlation coefficient of 0.167 at 0.01 levels. It means those who possess good knowledge also have good level of practices, thus are able to manage the waste in proper manner. Grodzinska Jurczak,M.S and Friedlin, K. [6] also found that a correlation between the level of students’ knowledge and their activities was found regarding waste management. According to the Table-5 no significant association between knowledge and attitude with correlation coefficient of 0.04 and attitude and practices with correlation coefficient of 0.003 was found for waste management. Same results were found from Wai, S. Tantrakarnapa, K and Huangprasert, S. [12] that there was a significant association between knowledge and practices with correlation coefficient of 0.39 and knowledge and attitude with correlation coefficient of 0.289. But there was no significant association between attitude and practices for environmental sanitation.

CONCLUSION The majority of the respondents have unsatisfactory knowledge attitude and inadequate practices related to waste management. This study has shown a need to improve the knowledge about waste management to protect environment from negative impact of waste. It is recommended to implement the need based training programme for students at their school hostels and work places.

S.No. 1.

Table-1: Knowledge Attitude and Practices regarding waste management Category Number Low 162 Knowledge Medium 138 High Less favourable 193 Attitude Favourable 89 Most favourable 18 Poor 140 Practices Moderate 156 Good 4 variables

(%) 54 46 64.33 29.66 6.10 46.66 52 1.33

Table-2: Factors influencing Knowledge of the respondents S.No. 1.

Factors Level of education UG PG 2. Stream of education Sc. NonSc * Significant ** Non Significant

Mean

S.D.

‘t’ value

10.21 10.87

2.73 2.95

2.00*

11.09 9.99

2.86 2.76

3.38*

Lalita Arora and Sunita Agarwal

Vol.2, No.1, 40-43 (2011) Table-3: Factors influencing Attitude of the respondents Factors Mean S.D. Level of education UG 62.61 22.02 PG 66.31 23.44 2. Stream of education Sc. 67.17 24.15 NonSc 61.75 21.05 * Significant ** NonSignificant S.No. 1.

Table-4: Factors influencing Practices of the respondents Factors Mean S.D. Level of education UG 20.09 8.00 PG 23.18 5.03 2. Stream of education Sc. 23.29 7.38 NonSc 19.99 6.37 * Significant ** NonSignificant S.No. 1.

‘t’ value

1.40** 2.06*

‘t’ value

3.86* 4.14*

Table-5: Association between variables S.No. Variables 1. Knowledge and Practices 2. Knowledge and Attitude 3. Attitude and practices * Significant ** Nonsignificant

Co-efficient of reliability 0.16* 0.04** -.003**

REFERENCES Asokan, P., Sexena, M. and Asolekar, S., Building and Environment., 42 (2007) 2311. Begum. Ara, Rawshan and Pereira, Jacqueline, Joy., Asian Journal of Water, Environment and Pollution., 5(3) (2008) 15. 3. Bradley, C. J., Waliczek. T. M. and Zajicek, J. M., Journal of Environmental Education., 30(3) (1999) 17. 4. Ehrampoush, M.H., Baghiani Moghadam, M.H., Iranian Journal of Environmental Health Science Engineering., 2(2) (2005) 26. 5. Eagles, P.F.J. and Demare, R., Journal of Environmental Education., 30(4) (1999) 33. 6. Grodzinska-Jurczak, M.S., Resource Conversion and Recycling., 32(2) (2001) 85. 7. Grodzinska-Jurczak, M.S. and Friedlein, K., Environmental Science and Pollution Res., 3(3) (2002) 215. 8. Hebel-Ulrich, Maja., Danish Committee for Aid to Afghan Refugees (DACAAR) (2005) www.dacaar.org. 9. Paengkaew, W., Roongtawanreongsri, S. and Kittitoronkool, J., Paper presented at SEAGA Conference., 28-30 November (2006) Singapore. 10. Pothimamaka,J.,Environment Asia., (Available online at www.tshe.org/EA) (2008) 43 11. Saini, S., Nagarajan, S.S. and Sharma, R. A., Journal of the Academy of the Hospital Administration., 17(2) (2005). 12. Wai,S.,Tantrakarnapa, K. and Huangprasert, S., Thai environmental Engineering Journal., 19(2) (2005) 19. [IJCEPR-149/2011]

1. 2.

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International Journal of Chemical, Environmental and Pharmaceutical Research Research

Vol. 2, No.1, 44-48 January-April, 2011

Utilisation of Thiocyanate (SCN-) by a Metabolically Active Bacterial Consortium as the Sole Source of Cellular Nitrogen Yogesh B. Patil Department of Energy and Environment, Symbiosis Institute of International Business (SIIB) [A Constituent of Symbiosis International Institute (SIU)], G. No. 174/1, Rajiv Gandhi Infotech Park, Hinjewadi, Pune – 411 057, Maharashtra, India. E-mail: dr_ybpatil@rediffmail.com Article History: Received:26 April 2011 Accepted:28 April 2011

ABSTRACT Thiocyanate (SCN-), a toxic chemical species from cyanide family, consists of both carbon (C) and nitrogen (N) in equimolar ratio and is being emanated through liquid effluents by several industrial processes. Since bioremediation technologies for waste treatment are gaining enormous importance in the recent times; microbial treatment of SCN- is therefore being researched worldwide. However, utilisation of SCN- by microbes as a suitable growth substrate (C and/or N source) is poorly understood and presents the problem in waste treatment systems. A heterotrophic bacterial consortium comprising of three Pseudomonas sp. isolated from activated sludge and having potentials for environmental clean-up, was capable of utilising thiocyanate (SCN-) from aqueous solutions as the sole source of cellular nitrogen (N) in the presence of carbon (C) source viz. glucose. The consortium ceased to grow and degrade SCN- when supplemented with C and N sources alone. Diauxie pattern was observed when the consortium culture was supplied with two N sources (NH4Cl and SCN-) in the presence of glucose as a source of carbon (C). NH4Cl was the preferred growth substrate utilised by the bacterial consortium followed by SCN-. Keywords: Biodegradation, Bacterial consortium, Diauxie, Nitrogen source, Thiocyanate. ©2011 ijCEPr. All rights reserved

INTRODUCTION Industrial processes like metal extraction, dyeing, photo-finishing, thiourea, pesticide production and electroplating industries produce large quantities of thiocyanate (SCN-) bearing effluents [6]. The concentration of SCN- in these effluents is in the range of 5 to 100 mg/L. Since SCN- is toxic to all living cells [14], it is imperative for the industries to detoxify the effluents prior to their discharge in environment. Several physical-chemical technologies have been reported for the SCN- removal [4]; the most widely being used is chlorination. However, conventional methods are beset with problems that are environmentally hazardous and fail to bring SCN- level within safe limits. Moreover, SCN- content in the waste inhibits the degradation of other pollutants (like cyanide and metal-cyanides) present in the waste, and therefore, has detrimental impact on aquatic flora and fauna. It is, thus, necessary to develop an alternative treatment process capable of achieving high degradation efficiency at low-cost. Bioremediation technologies using microorganism for detoxification of waste chemical is an eco-friendly alternative. Considerable literature is available on the removal of toxic C-1 compounds like free cyanide and metal cyanides by metabolically active [5,9] and metabolically inactive (passive) microorganisms [8]. In the recent times Thakur and Patil (2009) had reported the removal of SCN- from solutions using low-cost waste biomass [13]. Although several reports are available on microbial SCN- degradation [4,11,12], utilisation of it by microbes as a suitable growth substrate (C and/or N source) is poorly understood. Lack of scientific knowledge in this regard may pose problems in the biological treatment systems. The present paper highlights some key laboratory experiments that confirm the degradation of SCN- by a heterotrophic bacterial consortium utilising it as the sole source of cellular N in the presence of external carbon (C).

MATERIALS AND METHODS A heterotrophic bacterial consortium comprising of three Pseudomonas species and capable of utilising SCN- was isolated from activated sludge by an enrichment culture technique [11]. The consortium was grown for 24 - 48 h in M-9 minimal salts medium – MSM [7] with 1 ml/L micronutrient solution [3], which contained SCN- (50 mg/L ≅ 1 mM) and glucose (10 mM) as the sole N and C & energy sources, respectively. The medium was totally free of synthetic organics like peptone, beef extract and yeast extract. Bacterial cell suspension (0.1 ml) containing 108 cells/ml was used as an inoculum. Batch culture experiments on SCN- biodegradation were performed under aseptic

Yogesh B. Patil

Vol.2, No.1, 44-48 (2011) and aerated conditions in 250 ml Erlenmeyer flasks with 100 ml M-9 MSM. The medium was supplemented with C and N sources with various permutations and combinations as shown in Table 1. Table-1: Experimental manipulation of carbon and nitrogen sources in various proportions for bacterial growth Combinations

Carbon Source

Nitrogen Source

KSCN (50 mg/L)

B C

Potassium thiocyanate i.e. KSCN (50 mg/L ≅ 1 mM) Glucose (10 mM) KSCN (50 mg/L)

Glucose (10 mM)

KSCN (50 mg/L) Ammonium chloride i.e. NH4Cl (1 mM) KSCN (50 mg/L) + NH4Cl (1 mM)

Overall C/N Molar Ratio in M-9 MSM 1 11 0.5 5.5

The pH of medium before starting the experiment was adjusted to 7.0. All the flasks were incubated at 30°C in a rotary shaker incubator (Remi, CIS-24 BL) at 150 rpm for 48-72 h. Suitable controls were run simultaneously along with experimental flasks to detect air stripping or auto-oxidation of thiocyanate, if any. Experiments were repeated twice to confirm the results. Analytical grade chemicals were used in all the experiments. Reagents were prepared in RO (Sartorius, Arium-61315) water (conductivity <5 µS) and refrigerated (4°C). SCN- content was measured spectrophotometrically (Spectronic-20D) as per the Standard Methods [1]. pH was determined using pH meter (Elico, Ll-120). Bacterial population was checked microscopically (Metzer, 778A) using Neubauer’s chamber (FeinOptik, Blankenburg) and by total viable count (TVC) procedure.

RESULTS AND DISCUSSION The data in Fig. 1 shows the growth of bacterial consortium when SCN- was supplemented in the medium as both C and N source. The results clearly indicated that the consortium failed to utilise SCN- as either C or N source. The SCN- concentration and bacterial population remained constant throughout the experimental period of 45 h. The data in Fig. 2 depicts that the bacterial consortium was capable of utilising SCN- (with an efficiency of > 99%) as the sole source of cellular N in the presence external C source like glucose, wherein increase in bacterial cell density from 105 to >108 cells/ml was observed with simultaneous decrease in SCN- level from 50 to < 0.1 mg/L. In uninoculated control, the SCN- level remained unaltered (Fig. 2). SCN- when supplemented with NH4Cl, inhibited growth of bacterial consortium (Fig. 3). Fig. 4 shows diauxic growth (diauxie) pattern of the bacterial consortium when SCNwas supplied along with C and N sources (Glucose and NH4Cl). It was observed that when SCN- and NH4Cl were supplied, the consortium did not utilise both N sources simultaneously. The consortium initially utilised NH4Cl and then SCN- as N source. The growth of consortium in the first 25-30 h and the unchanged levels of SCN- in the same period confirmed utilisation of NH4Cl during first phase and later the SCN-. This resulted in a biphasic (two phase) growth pattern known as diauxie / diauxic growth (Fig. 4). The prime objective of the present work was to elucidate the potentials of isolated bacterial consortium for its utilisation of SCN- from aqueous solution as (i) both C and N source or (ii) only C source or (iii) only N source for growth. Revealing this fact is important in biological treatment of wastewater because if toxic chemical like SCN- is used by the consortium as both C and N source, then at practical scale, external supplementation of nutrients wouldn’t be required, which in author’s opinion, would be beneficial from the economic point of view to the user industries. This holds true even for the wastewaters containing other toxic compounds like cyanide and metalcyanides. However, in the present study, the bacterial consortium failed to utilise SCN- as both C and N source (C/N molar ratio=1). This fact clearly indicates that toxic SCN- compound presents problem to the consortium for growth utilising it as suitable substrate (Fig. 1). Thus, a sufficiently high concentration of SCN- to support appreciable growth might prove to be too toxic to allow growth to occur. Since the concentration of N required for a given amount of growth is less than the requirement for C, it might be easier for bacterial consortium to utilise SCN- as the source of N in the presence of a separate source of C and energy. Therefore, microorganisms capable of degrading SCN- as the source of N were isolated by an enrichment technique [11]. The consortium in the present study clearly showed (Fig. 2) the utilisation of SCN- as the sole N source in the presence of external C source like glucose (C/N molar ratio=11). Therefore, from the process development point of view, it is essential to supplement some cheaper

Yogesh B. Patil

Vol.2, No.1, 44-48 (2011) source of C like molasses, which is readily available in developing country like India at cheaper rate. In the earlier studies, Patil (1999) had successfully demonstrated the use of molasses as the source of C to develop a microbial technology for metal cyanides biodegradation/removal from wastes utilising it as the sole source of N [10]. Uninoculated controls run simultaneously along with the experiments did not show any decrease in SCN- levels (Fig. 2) confirmed that biodegradation of SCN- was the predominant reaction taking place by the bacterial consortium. There are a few reports, which describe microbial SCN- degradation utilising it as the sole N source [4, 12]. When SCN- was supplied as the sole C source in the presence of external N (viz. NH4Cl), the consortium ceased to grow keeping the 50 mg/L of SCN- amount unaltered (Fig. 3). This might be due to the higher amount of available N (2 mM) compared to the C (1 mM) source (C/N molar ratio=0.5). Obviously, the consortium culture will find it more difficult to obtain the energy from low amount of C and utilise the toxic SCN-. Diauxic (Biphasic) growth pattern was observed (Fig. 4) when two N salts (i.e. SCN- and NH4Cl) along with one C source (glucose) were supplied to the consortium. NH4Cl was the preferred growth substrate utilised by the bacterial consortium followed by SCN- degradation suggests that SCN- utilisation by consortium is inducible. Diauxie pattern in Escherichia coli (and many other microorganisms) in the presence of two C sources viz. glucose and lactose is a well known example [2]. However, there are no reports on the diauxie pattern when two N sources like SCN- (a toxic compound) and NH4Cl are supplemented and therefore, this paper may be considered as the first report. In the present study, the diauxic growth experiments (Fig. 4) showed rapid removal/decrease of toxic SCN- from the medium within 25 h. This was because in the first phase of diauxie, the consortium grew to a substantial level (cell density from initial 105 to final >108 cells/ml) utilising glucose and NH4Cl. After the exhaustion of NH4Cl from the medium, the consortium in the second phase (after a lag of 10-12 h) degraded toxic SCN- rapidly utilising it as the sole N source in the presence of glucose (10 mM) as C. It has not escaped through authorâ&#x20AC;&#x2122;s notice that high initial cell density leading to rapid SCN- biodegradation in diauxie experiment, immediately suggests its possible application in wastewater treatment reactor vessels capable of retaining high microbial biomass by way of immobilisation, which is the key to hasten biodegradation of SCN-.

8 40

7.5 7

30 6.5 20

6 5.5

Log (N o. of bacterial cells)

Thiocyanate C onc. (m g/L)

8.5 50

5 0

4.5 0

Time (h) Fig.-1: Supplementation of SCN- as both C and N source. Cessation of bacterial growth ( ) and unaltered SCNconcentration (â&#x2013;˛)

Yogesh B. Patil

Vol.2, No.1, 44-48 (2011)

9.5

Thiocyanate Conc. (mg/L)

8.5 8

7.5 30

7 6.5

6 5.5

Log (No. of bacterial cells)

5 0

4.5 0

Time (h) Fig.-2: Utilisation/Degradation of SCN- by bacterial consortium as the sole source of N in presence of external C source. Growth of bacterial consortium (▲) with simultaneous decrease in SCN- concentration (●); SCNconcentration in the absence of consortium ( )

Thiocyanate Conc. (mg/L)

8 40

7.5 7

30 6.5 20

6 5.5

Log (No. of bacterial cells)

8.5 50

5 0

4.5 0

Time (h) Fig.-3: SCN- as the sole C source with external supplementation of N (NH4Cl). Growth of bacterial consortium (▲); SCN- concentration in presence (●) and absence of consortium ( )

Yogesh B. Patil

Vol.2, No.1, 44-48 (2011)

12.5

10.5 40 9.5 30

8.5 7.5

20 6.5 10

Log (No. of bacterail cells)

Thiocyanate Conc. (mg/L)

11.5 50

5.5 0

4.5 0

Time (h) Fig.-4: Diauxic growth pattern exhibited by bacterial consortium in the presence two N sources (SCN- and NH4Cl) in presence of glucose as C source. Growth of consortium ( ) and SCN- degradation (â&#x2013;˛) in the presence of two N sources; SCN- concentration in absence of consortium (â&#x2014;?); Cessation of bacterial growth in absence of either N or C source ( )

CONCLUSION Foregoing experimental results and discussion conclude that the bacterial consortium isolated from activated sludge is capable of utilising/degrading SCN- as the sole source of cellular N from aqueous solutions in the presence of external C source.

ACKNOWLEDGEMENT This research was supported by University Grants Commission (UGC), WRO, Pune through a grant to YBP.

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

APHA, AWWA, WPCF, Standard Methods for the Examination of Water and Wastewater, Washington, DC, 18th Edn. (1998). Atlas, R.M., Principles of Microbiology, McGraw-Hill, New York, (1997). Bauchop, T., Elsden, S.R., General Microbiology, 23 (1960) 457. Bipinraj, N.K., Joshi, N.R., Paknikar, K.M. Biohydrometallurgy: A Sustainable Technology in Evolution, In: International Biohydrometallurgy Symposium, Athens, (2003) 491. Gurbuz, F., Ciftci, H., Akcil, A., Karahan, A.G. Hydrometallurgy, 72 (2004) 167. Hughes, M.N. General Chemistry, In: Newmann, A.A., (Ed.), Academic Press, London (1975). Millar, J.H., Experiments in Molecular Genetics, Cold Spring Harbour, NY: Cold Spring Harbour Laboratory (1972). Patil, Y.B., Paknikar, K.M., Biotechnology Letters, 21 (1999) 913. Patil, Y.B., Paknikar, K.M., Process Biochemistry, 35 (2000) 1139. Patil, Y.B., Ph.D. Thesis, University of Pune, Pune, India (1999). Patil, Yogesh B., Research Journal of Chemistry and Environment, 12(1) (2008) 69. Sorokin, Dimitry Y., Tourova, Tatyana P., Lysenko, Anatoly M., Kuenan, Gigs J., Applied and Environmental Microbiology, 67(2) (2001) 528. Thakur, Ravindra Y., Patil, Yogesh B., South Asian Journal of Management Research, 1(2) (2009) 85. Westley, J. Cyanide and Sulfane Sulfur. In: Vennesland, B., (ed.), Academic Press, London (1981) 201. [IJCEPR-163/2011]

Yogesh B. Patil

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 49-51 January-April, 2011

Construction of An Open Loop Temperature Control System for Thin Film Fabrication in PC Based Instrumentation K.Tamilselvan*,1 , K.Anuradha2, S.Deepa2 , O.N.Balasundaram2 and S.Palaniswamy2 *,1

Department of Electronics, PSG College of Arts and Science, Coimbatore-641 014. Department of Physics, PSG College of Arts and Science, Coimbatore-641 014. E-mail: tamilselvan.psgarts@gmail.com 2

Article History: Received: 4 April2011 Accepted: 27 April 2011

Abstract The paper describes an open loop control for controlling the chemical hot bath temperature in PC based dip coating system for thin film fabrication. The control of temperature is achieved by changing the duty cycle and supply voltage. The function of the duty cycle at each supply voltage in obtaining maximum temperature is studied. The open loop control of temperature is implemented using PC based instrumentation. The temperature control system is optimized using relation between the duty cycle and temperature. The performance of this system is compared to that of a conventional close loop controller on a laboratory test. Results are presented that shows a good control of the hot bath temperature. Keywords: open loop control, PC based dip control system, duty cycle. ÂŠ2011 ijCEPr. All rights reserved

INTRODUCTION The use of personal computers in automation systems is widespread. The realization of PC-based automation systems has been allowed by several factors, among them are1. The impressive grown of the PCâ&#x20AC;&#x2122;s performances which ensures the possibility of locating and executing, inside the same machine, both the types of tasks involved: real-time control and monitoring /supervision. 2. The use of high level language allows for an efficient scheduling of the tasks assigning higher priorities to the most critical ones. 3. Increasing availability of devices equipped with bus interfaces which makes possible, the realization of an automation system using hardware/software products of different manufacturers. In the first part of this paper, we describe the general structure of the PC-based dip coating unit with open loop temperature control and an experimental evaluation of its performance using PWM technique.

MATERIALS AND METHOD A Personal Computer with Pentium processor, 256 MB RAM and 80 GB Hard disk drive along with external driver circuits are used. A small mechanical arrangement driven by stepping motor is used to move the substrate upward / downward. The stepper motor drives the screw rod, moving pulley and substrate holder. The proper gear reduction mechanism and well polished gear teeth wheels are employed to avoid mechanical slip and jerk. The screw rod is precisely machined to give smooth upward and downward movement. The control signals are taken from printer port or Programmable Peripheral Interface ( PPI ) card. Transistor driver controls the voltage applied to heater of hot bath. The programs written in C++ are executed. The instrumental setup is shown in figure.

Fig.-1: Instrumental Setup

K.Tamilselvan et al.

Vol.1, No.2, 49-51 (2011) 100V

150V

100

70 80

6s 8s

10s 12s 14s

ON 2s 4s 6s 8s

TEMP(celcius)

TEMP(CELSIUS)

ON 2s 4s

10s 12s 14s 16s 18s

16s 18s

30 10

20 1

106 113 120

10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 TIME(s)

TIME(S)

Graph-ii: Variation of temperature with time for 150V and .05Hz for various duty cycles.

Graph- i: Variation of temperature with time for 100V and .05Hz for various duty cycles. 200V

VOLTAGE VS TEMP

120

100

ON 2s 4s 6s 8s 10s 12s 14s

80 TEMP(CELSIUS)

TEMP(celcius)

16s 18s

2S 4S 6S 8S 10S 12S 14S

16S 18S

0 1

0 100

120

140

160

TIME(s)

180

200

220

VOLTAGE(V)

Graph-iii: Variation of temperature with time for 200V and .05Hz for various duty cycles.

Graph-iv: Plot with voltage and temperature for various duty cycles.

DUTYCYCLE VS TEMP 120

100

TEMP(celcius)

100V 150V 200V

0 0

DUTYCYCLE(s)

Graph-v: Plot with duty cycle and temperature for fixed voltages. Fig.-3: Different Graphs

RESULTS AND DISCUSSION Temperature control The temperature of the hot bath can be controlled by two methods namely, i. Closed loop control and ii. Open loop control Closed loop control Closed loop control system requires a sensor, a feedback circuit and a complex controller circuit, which is a drawback of the closed loop control. On the other hand the temperature control in this system can be done very precisely with a variation of 1ยบC or 2ยบC. Open loop contropl 50

K.Tamilselvan et al.

Vol.1, No.2, 49-51 (2011) An open loop control is simple.It does not require a sensor, a feedback circuit and also a complex circuit, which adds to the advantages of a open loop control. It requires only a driver circuit to control the temperature of the hot bath. In this system, temperature control can be done by using PWM technique , ie i. by varying the duty cycle ii. by varying the frequency where, Duty cycle = ON time/ Total Time when the duty cycle decreases the output voltage also decreases and when the duty cycle increases the output voltage also increases.

Fig.-2: Driver circuit Optimization The temperature of the hot bath varies accordingly with duty cycle, frequency and the supply voltage.The change in temperature of hot bath for fixed interval of time is studied until the temperature of the hot bath reaches the saturation temperature for fixed frequency and supply voltage . The change in temperature is plotted, graphically. For a supply voltage of 100V the saturation temperature is minimum and varies according to the duty cycle. Similarly for 150V and 200V the saturation temperature varies according to the duty cycle. From these observations we can fix the required temperature of the hot bath by fixing the corresponding duty cycle and the voltage.

CONCLUSIONS This newly designed simple instrumental setup is cost effective and convenient for preparation of Thin Films. The well known technique PWM is used to control the temperature. By changing the ON time and keeping the TOTAL time unchanged, the temperature can be increased or decreased. The proper mechanical setup and programming techniques improves the system performance.

REFERENCES 1. 2. 3. 4.

Maissel, L.I. , Glang R. , Handbook of Thin Films Technology, McGraw- Hill, New York, 1970. Ohring M., The Materials Science of Thin Solid Films, Academic Press, New York, 1992. George W. Gorsline ,Computer organisation, hardware/software, PHI. Stephen J. Bigelow ,Trouble shooting Maintaining & Repairing PCâ&#x20AC;&#x2122;s, Tata McGraw Hill Co, New Delhi 1999. [IJCEPR-159/2011]

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K.Tamilselvan et al.

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 52-55 January-April, 2011

Accumulation of Natural Antioxidants in Ferns Exposed to Mutagenic Stress Alok Kr. Singh1, Santosh Kr. Singh2*, Satish K. Verma3, H.V. Singh1, A.K. Mishra4, Pavan K. Agrawal3, Abhishek Mathur3 and Md. Aslam Siddiqui5 1

Department of Botany, S.G.R. P.G. College, Dobhi, Jaunpur Department of Biotechnology, S.B.S. (P.G.) Institute of Biomedical Sciences and Research, Balawala, Dehradun, Uttarakhand 3 Sai Institute of paramedical and allied Sciences, Dehradun 4 Kashi Naresh Rajkiya Snatkottar Mahavidhyala, Gyanpur, Bhadohi 5 Baba Farid Institute of Technology, Dehradun. *E-mail: res_mol_bio@sify.com *,2

Article History: Received:3 April 2011 Accepted:10 April 2011

ABSTRACT Different species of ferns were analyzed for the modulations in the pool of non-enzymatic antioxidants in response to the maleic hydrazide treatments. Treatments at very low doses were found to trigger the accumulation of both ascorbate and proline contents. Total amount of protein and chlorophyll contents showed varying degree of sensitivity in all cultivars of ferns. Proline accumulation was found to be high in treated plants compared with control. Proline, ascorbate and flavonoid contents were found to be accumulated in all plants exposed to high doses of maleic hydrazide. All the three species showed high proneness towards the mutagen. Improved tolerance in treated plants might be explained on the basis of the elevated level of enzymatic and nonenzymatic antioxidants. Key Words: Mutagen, Ascorbate, Proline, protein, Ferns ©2011 ijCEPr. All rights reserved

INTRODUCTION Ferns are found abundantly in many different habitats of the world. They were the dominant part of the vegetation during the Carboniferous Period which is called as ‘Age of Ferns’. Most of the ferns of the Carboniferous became extinct but some later evolved into our modern ferns. There are about 12,000 species in the world today [2]. Three fern species were selected for the present study: Cheilanthes farinose, Lygodium scandens and Adiantum caudatum. The plants prefer light (sandy), medium (loamy) and heavy (clay) soils [12, 13]. The plants are dominant in soils of acidic, neutral and alkaline nature. Plants possess many antioxidants, usually classified in two broader categories. They are: enzymatic antioxidants and non-enzymatic antioxidants. The alterations in the activities of antioxidants is observed in the plants exposed to different environmental stresses such as drought, heavy metals, pesticides, ultraviolet radiations etc. Since human activities are increasing the level of pollutants in the environment day by day, it has become an interesting area of research to observe their effects on plant communities (Producers of the Ecosystems). The damage to the biological ecosystems may be measured in terms of the morphological and biochemical alterations in primary producers. Numerous studies have been conducted on photosynthetic enzymes, pigments, proteins, seed patterns and antioxidant compound contents in plants. Maleic hydrazide is one of the agents that have been found to bring heritable alterations in the genes, chemical mutagens are undoubtedly very potent ones which can induce genetic or physical alterations in dormant seed or spore. This is a strong mutagen that induces mitotic inhibition and cytological abnormalities in a number of higher plants [4, 8, 16]. It possesses growth regulating properties [19, 22]. It is a known depressor of auxin transport in plant. Stem growth, root growth and seed germination can be regulated by its treatment. The presence/absence and increasing/decreasing property of antioxidant compounds, production of free radicals and the amount of lipid peroxidation in terms of elevated level of antioxidants might provide significant clues to assess and evaluate the antioxidant potential of various Cheilanthes species against environmental stresses. Plants are able to develop special mechanisms for adjusting the changed environment. Many groups of stresses like heavy metals, ultraviolet radiations etc are shown to generate singlet oxygen and other active oxygen species at various sites of photosynthetic electron transport chain [9] and affect the growth of plant. Many studies have been done with emphasis on morphological, biochemical and genetic characteristics of Cheilanthes rufa, Lygodium scandens and Adiantum caudatum with respect to ultraviolet radiations, gamma radiations and various light qualities like red light,

Alok Kr. Singh et al.

Vol.1, No.2, 52-55 (2011) blue light etc. The present study was done with setting forth the objective of studying the effect of Maleic hydrazide on the accumulation of non-enzymatic antioxidants in different fern species.

MATERIALS AND METHODS Organisms and culture conditions Spores of Cheilanthes rufa, Lygodium scandens and Adiantum caudatum were collected from plants growing in the kushmi forest of Gorakhpur (a tarai area of north India). The spores were surface sterilized with 2% sodium hypochlorite solution and then sown uniformly on 25 ml of autoclave sterilized (15 1b/in2) inorganic medium at pH 5.4 in petri dishes [20]. Sowing of spores was done in an inoculation chamber fitted with germicidal UV lamp (USA). Then the plates were maintained at 24 ± 20c under continuous white fluorescent illumination at the intensity of 2700 lux. Spores for treatments were placed in liquid nutrient media for 72 hours and then subjected to various concentrations of maleic hydrazide. Each experiment was conducted in triplicates. Extraction and estimation of total Protein, Malondialdehyde and peroxide contents Growth determination was done by estimating total protein contents after 12 days of treatments. Protein contents were determined using Folins-Lowry method with lysozyme as the standard [14]. Total amount of hydrogen peroxide radicals was estimated by using ferrithiocyanate method as described by Sagisaka (1976) [18]. Standardization of H2O2 was performed to minimize the interference of catalase. The level of lipid peroxidation was measured in terms of total malondialdehyde (MDA) contents. The reaction reagent consisted of 0.4 N TCA + 19.68 ml of distilled water + 0.4 ml of HCl + 100mg TBA [11]. Prepared leaf extract (in phosphate buffer) was added to the reaction reagent and absorbance was taken at 532 nm. Flavonoids: Extraction and Estimation Flavonoids were extracted in mutagen-treated and untreated fern leaflets by using the method of Mirecki and Teramura (1984). Extraction mixture consisted of acidified methanol (methanol: water: HCl, 78: 20: 2, v/v) + leaflets, incubated for for 24h at 40C. The filtered extract was then used for measuring the absorbance at 320 nm, which is indicative of relative concentration of UVB absorbing pigments [15]. Flavonoid contents were expressed as absorbance g-1 fresh mass of tissue at 320 nm. Extraction and estimation of Proline contents Proline contents in leaf homogenate of mutagen- treated and untreated cultures were estimated according to the standard method [3]. Proline contents in unknown samples were calculated by comparing with standard curve of Lproline. Amount of proline is represented in terms of µg g-1 FW. Ascorbic acid estimation Ascorbic acid was extracted by dehydrating ascorbic acid by shaking it with acid washed NORIT* in the presence of acetic acid. After coupling with 2, 4-Dinitrophenyl hydrazine, the solution is treated with sulfuric acid to produce the red color whose absorbance was measured at 540 nm. *Acid washed NORIT preparation: 200 gram NORIT (charcoal) is suspended in 1000 ml of 10% HCl, heated upto boiling point and filtered under suction. The cake is removed and stirred with 1000 ml water and filtered. This procedure is repeated until the washing give a negative test for Fe3+ ions. The NORIT is then dried overnight at 110-1200C.

RESULTS AND DISCUSSIONS Results observed were found to be variable with different fern species. Overall growth and accumulation of ascorbate, proline and flavonoid contents showed modulations in vales in response to the mutagenic chemical– Maleic hydrazide. Growth measured in terms of protein contents showed initial increase at low doses of mutagen (% control increase = 16-22% in all three ferns). But the high doses were found to reduce the total protein contents speedily (5-60% at 125 ppm as compared to the control) (Figure 1a). The declining trend in protein contents continued with rising concentration of maleic hydrazide. Initial recovery in protein contents might be explanined on the basis of increase in the pool of enzymatic antioxidants that help plants in overcoming the oxidative stress [6, 7] The present study might give us clues for the impacts of mutagens on total biomass yield. Cells contain important non-enzymatic antioxidants such as carotenoids, ascorbic acid, proline, glutathione, αtocopherol etc., for mitigating the toxic effects of free radicals and AOS (active oxygen species) under oxidative stress. In the present work, ascorbate contents showed increase in the amounts (% control increase= 10-45% in all plants) with mutagen exposure up to 16-62 ppm and the decrease at high dose (6-15% after 125 ppm exposure of maleic hydrazide), compared to untreated samples. (figure 1 b). There are two possibilities regarding increase in ascorbic acid contents; either its synthesis has increased or its regeneration rate through the Asada-Halliwell pathway has increased (as observed in Ulva fasciata) [21]. 53

Alok Kr. Singh et al.

Vol.1, No.2, 52-55 (2011) The chemical evolution and significance of flavonoids has been assumed to play an important role in overcoming the oxidative stress in cells [17]. Evidences suggest that the presence of flavonoids in UV-B irradiated leaves could alter the perception or response of other defense mechanisms. Presently, flavonoid contents showed enhanced synthesis in maleic hydrazide treated fern species- C. rufa, Lygodium scandens and Adiantum caudatum. A % control increase of 5-40% was observed at the doses of 16-62 ppm but at the high dose (125 ppm), the values decreased to 25%, 20% and 30% in C. rufa, Lygodium scandens and Adiantum caudatum, respectively (Figure 1 b). Earlier findings showed remarkable increase in the Flavonoids contents of the stressed soybean cultivars. Since flavonoids inhibit the enzymes responsible for superoxide anion production thus the increase in their values may be attributed to the protection from free radical induced damage. Similarly proline contents were found to be accumulated at all the doses of maleic hydrazide in all the fern cultivars- C. rufa, Lygodium scandens and Adiantum caudatum. A high accumulation (10-60%) of proline contents were observed at the highest dose of mutagen (125 ppm). The accumulation and protective effect of proline has been observed in many higher plants and bacteria as well as protozoa, algae, and marine invertebrates [5]. Mutagen induced lipid peroxidation of the cellular components in plants were studied by estimating the level of MDA in treated and untreated plantlets and the related data are depicted in the figure 2 a. The lipid peroxidation in non- stressed C. Rufa was observed as 1.4609 nmol MDA (mg fresh mass)-1, whereas it was found to be 1.351 and 1.6125 nmol MDA (mg fresh mass)-1. Treated plantlets showed 10-45% increase in total Malondialdehyde contents as compared to the untreated plants showing high level of lipid peroxidation in maleic hydrazide treated plants. Similarly the increase in peroxide radical contents was observed to be linearly related with the level of lipid peroxidation (Figure 2 b). 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, 10].

CONCLUSION According to the results obtained, it may be concluded that the mutagen-maleic hydrazide affected the overall growth of all fern species, severely. Decrease in total protein contents and high increase in the level of lipid peroxidation proved the oxidative damage caused by free radicals formed in response to the mutagen. Increase in non-enzymatic antioxidants may be attributed to the elevation of natural antioxidant defense system. Initial increase in the enzymatic activities might be due to the increased activities of stress relief genes and their gene products. Ferns are good Phytoremediator and can be used to remove heavy metals and other pollutants from the polluted soil. The study might provide suitable keys for studying the interrelationships of the chemical treatments and plant defense systems.

(b)

(a)

Fig.-1: Effect of Maleic hydrazide on protein (a) and non-enzymatic antioxidants (b) in ferns.

Alok Kr. Singh et al.

Vol.1, No.2, 52-55 (2011)

(b)

(a)

Fig.-2: Effect of Maleic hydrazide on lipid peroxidation (a) and hydrogen peroxide radicals (b) in ferns.

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

Agarwal S., Pandey V., Indian Journal of Plant Physiology, 8(3) (2003) 264. Antony R.S., Khan A.E., Thomas, J., Journal of Economic & Taxonomic Botany, 24 (2000) 413. Bates L.S., Waldren R.P., Teare I.D., Plant and Soil, 39(1) (1973) 205. Carrier H.B., Day B.E., Crafts A.S., Bot. Gaz., 112 (1950) 272. Cechin I., Rossi S.C., Oliveira V.C., Fumis T.F., Photosyn., 44(1) (2006) 143. 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. Darlington and McLeish J., Nature, (1951) 167. Halliwell B., and Gutteridge J.M.C., Lancet, 1 (1994) 1396. Hasanuzzaman M. et al., American Journal of Plant Physiology, 5 (2010) 295. Heath R.L., Packer L., Arch Biochem Biophysics, 125 (1968) 189. Hevly, R.H. et al., J Ariz. Acad. Sci., 2 (1963) 164. Hitchcock C.L., Cronquist A., Ownbey M., Thompson J.W., University of Washington Press, Seattle, WA. (1969) Pp. 914. Lowry O.H., Rosenbrough N.J., Farr A.L., Randall R.J., J. Biol. Chem., 193 (1951) 265. Mirecki R.M., Teramura A.H., Plant Physiol., 74 (1984) 475. Naylor A.W., Davis E.A., Bot. Gaz. ,112 (1950) 112. Rozema J., Boelen P., Blokker P., Environ. Pollut., 137(3) (2005) 428. Sagisaka, S., Plant Physiol ,57 (1976) 308. Schoene D.L., Hoffman O.L., Science, 109 (1949) 588. Sheffield E., Douglas, G.E., Hearned S.J., Huxham S., Wynn, J.M., Amer. Fern J., 91 (2001) 179. Shiu, C.T., and Lee T.M., J. Exp. Bot., 56(421) (2005) 2851. White D.G., Maleic hydrozide. Discovery, II. (1950) 379. [IJCEPR-158/2011]

Alok Kr. Singh et al.

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 56-60 January-April, 2011

Reversed Phase HPLC Analysis of Valsartan in Pharmaceutical Dosage Forms V. Bhaskara Raju1 and A. Lakshmana Rao2* 1

Sri Vasavi Institute of Pharmaceutical Sciences, Tadepalligudem- 534 101, A.P., India. V.V. Institute of Pharmaceutical Sciences, Gudlavalleru- 521 356, A.P., India. * E-mail: dralrao@gmail.com 2

Article History: Received:8 February 2011 Accepted:27 February 2011

ABSTRACT A rapid, precise, accurate, specific and sensitive reverse phase liquid chromatographic method has been developed for the estimation of valsartan in pure and tablet formulation. The chromatographic method was standardized using a Xterra C18 column (100×4.6 mm I.D., 5 µm particle size) with UV detection at 210 nm and flow rate of 1 ml/min. The mobile phase consisting of a mixture of phosphate buffer pH 3 and acetonitrile in the ratio of 50:50 v/v was selected. The proposed method was validated for its sensitivity, linearity, accuracy and precision. The retention time for valsartan was 4.450 min. The % recovery was within the range between 98.6 % and 101.2 %. The percentage RSD for precision and accuracy of the method was found to be less than 2 %. This method can be employed for routine quality control analysis of valsartan in tablet dosage forms. Keywords: Valsartan, Estimation, Validation, Tablets, RP-HPLC. ©2011 ijCEPr. All rights reserved

INTRODUCTION Valsartan [1] is a nonpeptide, orally active and specific angiotensin II receptor blocker acting on the AT1 receptor subtype. Valsartan is chemically described as N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4yl]methyl]-L-valine (Fig. 1) [2]. Angiotensin II is formed from angiotensin I in a reaction catalyzed by angiotensinconverting enzyme (ACE II). Angiotensin II is the principal pressor agent of the renin-angiotensin system, with effects that include vasoconstriction, stimulation of synthesis and release of aldosterone, cardiac stimulation and renal reabsorption of sodium. Valsartan blocks the vasoconstrictor and aldosterone-secreting effects of angiotensin II by selectively blocking the binding of angiotensin II to the AT1 receptor in many tissues, such as vascular smooth muscle and the adrenal gland. Its action is therefore independent of the pathways for angiotensin II synthesis. A few spectrophotometric [3-7], HPLC [8-14], UPLC [15] and LC-MS [16-19] methods were reported earlier for the determination of valsartan in bulk and pharmaceutical dosage forms. In the present study the authors report a rapid, sensitive, accurate and precise HPLC method for the estimation of valsartan in bulk samples and in tablet dosage forms.

Fig.-1: Chemical structure of valsartan

MATERIALS AND METHOD Chromatographic conditions The analysis of the drug was carried out on a Waters HPLC system equipped with a reverse phase Xterra C18 column (100x4.6 mm., 5 µm), a 2695 binary pump, a 20 µl injection loop, a 2487 dual absorbance detector and

V. Bhaskara Raju and A. Lakshmana Rao

Vol.1, No.2, 56-60 (2011) running on Waters Empower software. The UV spectrum of valsartan was taken using a Elico SL-159 UV-Visible spectrophotometer. Chemicals and solvents The reference sample of valsartan was supplied by Lupin Pharmaceutical Industries Ltd., Ahmadabad. HPLC grade water and acetonitrile were purchased from E. Merck (India) Ltd., Mumbai. Potassium dihydrogen phosphate and orthophosphoric acid of AR grade were obtained from S.D. Fine Chemicals Ltd., Mumbai. Preparation of pH 3.0 phosphate buffer Seven grams of KH2PO4 was weighed into a 1000 ml beaker, dissolved and diluted to 1000 ml with HPLC water. 2 ml of triethyl amine was added and pH adjusted to 3.0 with orthophosporic acid. Preparation of mobile phase and diluents 500 ml of the phosphate buffer was mixed with 500 ml of acetonitrile. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 µ filter under vacuum. Procedure A mixture of phosphate buffer and acetonitrile in the ratio of 50:50 v/v was found to be the most suitable mobile phase for ideal separation of valsartan. The solvent mixture was filtered through a 0.45 µ membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1.0 ml/min. The column was maintained at ambient temperature. The pump pressure was set at 800 psi. The column was equilibrated by pumping the mobile phase through the column for atleast 30 min prior to the injection of the drug solution. The detection of the drug was monitored at 210 nm. The run time was set at 7 min. Under these optimized chromatographic conditions the retention time obtained for the drug was 4.450 min. A typical chromatogram showing the separation of the drug is given in Fig. 2.

Fig.-2: Typical chromatogram of valsartan Calibration plot About 10 mg of valsartan was weighed accurately, transferred into a 100 ml volumetric flask and dissolved in 25 ml of a 50:50 v/v mixture of phosphate buffer and acetonitrile. The solution was sonicated for 15 min and the volume made up to the mark with a further quantity of the diluent to get a 100 µg/ml solution. From this, a working standard solution of the drug (10µg/ml) was prepared by diluting 1 ml of the above solution to 10 ml in a volumetric flask. Further dilutions ranging from 5-25 µg/ml were prepared from the solution in 10 ml volumetric flasks using the above diluent. 20 µl of each dilution was injected six times into the column at a flow rate of 1.0 ml/min and the corresponding chromatograms were obtained. From these chromatograms, the average area under the peak of each dilution was computed. The calibration graph constructed by plotting concentration of the drug against peak area was found to be linear in the concentration range of 5-25 µg/ml of the drug. The relevant data are furnished in Table-1. The regression equation of this curve was computed. This regression equation was later used to estimate the amount of valsartan in tablet dosage forms.

V. Bhaskara Raju and A. Lakshmana Rao

Vol.1, No.2, 56-60 (2011) Table-1: Calibration data of the method Mean peak area (n=5) Concentration (µg/ml) 5 605930 10 1244187 15 1865633 20 2468676 25 3062337 Validation of the proposed method The objective of the method validation is to demonstrate that the method is suitable for its intended purpose as it is stated in ICH guidelines [20]. The method was validated for linearity, precision, accuracy, specificity, stability and system suitability. Standard plots were constructed with five concentrations in the range of 5-25 µg/ml prepared in triplicates to test linearity. The peak area of valsartan was plotted against the concentration to obtain the calibration graph. The linearity was evaluated by linear regression analysis that was calculated by the least square regression method. The precision of the assay was studied with respect to both repeatability and intermediate precision. Repeatability was calculated from five replicate injections of freshly prepared valsartan test solution in the same equipment at a concentration value of 100 % (10 µg/ml) of the intended test concentration value on the same day. The experiment was repeated by assaying freshly prepared solution at the same concentration additionally on two consecutive days to determine intermediate precision. Peak area of valsartan was determined and precision was reported as % RSD and the results are furnished in Table-2. Table-2: Precision of the proposed HPLC method Concentration of valsartan (10 µg/ml)

Peak area Intra-day

Inter-day

Injection-1

1237412

1241721

Injection-2

1238580

1241059

Injection-3

1239480

1242984

Injection-4

1241807

1244489

Injection-5

1244696

1247070

Average

1240395

1243465

Standard Deviation

2895.0

2403.4

% RSD

0.23

0.19

The accuracy of the HPLC method was assessed by analyzing solutions of valsartan at 50, 100 and 150 % concentrated levels by the proposed method. The results are furnished in Table-3. The system suitability parameters are given in Table-4. Table-3: Accuracy studies Concentration Amount added (mg) Amount found (mg) % Recovery % Mean recovery 50 % 5.02 4.95 98.6 % 100.2 % 100 % 10.1 10.17 100.7 % 150 % 15.1 15.28 101.2 % Table-4: System suitability parameters Parameter Result 5-25 Linearity (µg/ml) 0.9998 Correlation coefficient Theoretical plates (N) 4547 1.20 Tailing factor V. Bhaskara Raju and A. Lakshmana Rao 58

Vol.1, No.2, 56-60 (2011) 0.012 0.040

LOD (µg/ml) LOQ (µg/ml)

Estimation of valsartan in tablet dosage forms Two commercial brands of tablets were chosen for testing the suitability of the proposed method to estimate valsartan in tablet formulations. Twenty tablets were weighed and powdered. An accurately weighed portion of this powder equivalent to 25 mg of valsartan was transferred into a 100 ml volumetric flask and dissolved in 25 ml of a 50:50 v/v mixture of phosphate buffer and acetonitrile. The contents of the flask were sonicated for 15 min and a further 25 ml of the diluent was added, the flask was shaken continuously for 15 min to ensure complete solubility of the drug. The volume was made up with the diluent and the solution was filtered through a 0.45 µ membrane filter. This solution was injected into the column six times. The average peak area of the drug was computed from the chromatograms and the amount of the drug present in the tablet dosage form was calculated by using the regression equation obtained for the pure drug. The relevant results are furnished in Table-5.

Formulation Formulation 1 Formulation 2

Table-5: Assay and recovery studies Label claim (mg) Amount found (mg) 40 40.12 40 39.86

% Amount found 99.70 100.35

RESULTS AND DISCUSSION Selection of the detection wavelength The UV spectra of valsartan in 50:50 v/v mixture of phosphate buffer and acetonitrile was scanned in the region between 200 and 400 nm and shows λmax at 210 nm. Optimization of the chromatographic conditions Proper selection of the stationary phase depends upon the nature of the sample, molecular weight and solubility. Mixture of phosphate buffer and acetonitrile was selected as mobile phase and the effect of composition of mobile phase on the retention time of valsartan was thoroughly investigated. The concentration of phosphate buffer and acetonitrile were optimized to give symmetric peak with short run time. A short run time and the stability of peak asymmetry were observed in the ratio of 50:50 % v/v of phosphate buffer and acetonitrile. It was found to be optimum mobile phase concentration. In the proposed method, the retention time of valsartan was found to be 4.45 min. Quantification was linear in the concentration range of 5-25 µg/ml. The regression equation of the linearity plot of concentration of valsartan over its peak area was found to be Y=8161.7+122746.06X (r2=0.9998), where X is the concentration of valsartan (µg/ml) and Y is the corresponding peak area. The number of theoretical plates calculated was 4547, which indicates efficient performance of the column. The limit of detection and limit of quantification were found to be 0.012 µg/ml and 0.040 µg/ml respectively, which indicate the sensitivity of the method. The use of phosphate buffer and acetonitrile in the ratio of 50:50 v/v resulted in peak with good shape and resolution. The high percentage of recovery indicates that the proposed method is highly accurate. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in tablet formulations did not interfere with the estimation of the drug by the proposed HPLC method.

CONCLUSION The proposed HPLC method is rapid, sensitive, precise and accurate for the determination of valsartan and can be reliably adopted for routine quality control analysis of valsartan in its tablet dosage form.

ACKNOWLEDGEMENTS The authors are thankful to M/s Lupin Pharmaceutical Industries Ltd., Ahmadabad, for providing a reference sample of valsartan.

REFERENCES 1. 2. 3. 4.

Budavari S. The Merck index, Merck and Co. Press: Whitehouse Station, NJ, 12th Edn, 1997. www.rxlist.com Gupta K.R., Wadodkar A.R., Wadodkar S.G., International Journal of Chem Tech Research, 2 (2010) 985. Gupta K.R., Mahapatra A.D., Wadodkar A.R., Wadodkar S.G., International Journal of Chem Tech Research, 2 (2010) 551. 59

V. Bhaskara Raju and A. Lakshmana Rao

Vol.1, No.2, 56-60 (2011) Tatar S., Saglik S., Journal of Pharmaceutical and Biomedical Analysis, 30 (2002) 371. Nevin E., Analytical Letters, 35 (2002) 283. Satana E., Altınay S., Goger N.G., Sibel A., Ozkan S.A., Senturk Z., Journal of Pharmaceutical and Biomedical Analysis, 25 (2001) 1009. 8. Kul D., Dogan-Topal B., Kutucu T., Uslu B., Ozkan S.A., Journal of AOAC International, 93 (2010) 882. 9. Patro S.K., Kanungo S.K., Patro V.J., Choudhury N.S.K., E-Journal of Chemistry, 7 (2010) 246. 10. Brunetto M.R., Contreras Y., Clavijo S., Torres D., Delgado Y., Ovalles F., Ayala C., Gallignani M., Estela J.M., Martin V.C., Journal of Pharmaceutical and Biomedical Analysis, 50 (2009) 194. 11. Chitlange S.S., Bagri1 K., Sakarkar D.M., Asian Journal of Research in Chemistry, 1 (2008) 15. 12. Kocyigit-Kaymakcoglu B., Unsalan S., Rollas S., Pharmazie, 61 (2006) 586. 13. Macek J., Klima J., Ptacek P., Journal of Chromatography, B 832 (2006) 169. 14. Daneshtalab N., Lewanczuk R.Z., Jamali F., Journal of Chromatography, B 766 (2002) 345. 15. Krishnaiah CH., Raghupathi Reddy A., Ramesh Kumar., Mukkanti K., Journal of Pharmaceutical and Biomedical Analysis, 53 (2010) 483. 16. Sampath A., Raghupathi Reddy A., Yakambaram B., Thirupathi A., Prabhakar M., Pratap Reddy P., Prabhakar Reddy V., Journal of Pharmaceutical and Biomedical Analysis, 50 (2009) 405. 17. Selvan P.S., Gowda K.V., Mandal U., Solomon W.D.S., Pal T.K., Journal of Chromatography, B 858 (2007) 143. 18. Li H., Wang Y., Jiang Y., Tang Y., Wang J., Zhao L., Gu J., Journal of Chromatography, B 852 (2007) 436. 19. Koseki N., Kawashita H., Hara H., Niina M., Tanaka M., Kawai R., Nagae Y., Masuda N., Journal of Pharmaceutical and Biomedical Analysis, 43 (2007) 1769. 20. ICH, Q2B. Validation of analytical procedures methodology, In Proceedings of The International Conference on Harmonization, Geneva 1993. [IJCEPR-150/2011] _____________________________________________________________________________________________

5. 6. 7.

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V. Bhaskara Raju and A. Lakshmana Rao

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 2, No.1, 61-66 January-April, 2011

Nattokinase :A Review on Fibrinolytic Enzyme Haritha Meruvu , Meena Vangalapati* Center for Biotechnology, Department of Chemical Engineering, College of Engineering, Andhra University, Visakhapatnam–530 003, Andhra Pradesh, INDIA. Email: meena_sekhar09@yahoo.co.in Article History: Received:26 April 2011 Accepted:28 April 2011

ABSTRACT Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. It is extracted and highly purified from a traditional Japanese food called Natto which is made from fermented soybeans, Glycine max (L.) Merr.Natto is produced by a fermentation process by adding Bacillus subtilis, subsp. Natto to boiled soybeans. The ensuing Nattokinase enzyme is produced when Bacillus subtilis acts on the soybeans. Nattokinase has caused natto wide attention around the world Natto is not only unique flavor and rich nutrition, but also due to a variety of health functions, known as "super health food”. The present review attempts to encompass the up-to-date comprehensive literature analysis on Nattokinase with respect to its properties, source and its various medical uses. Keywords: Nattokinase, Natto, Glycine max (L.), Bacillus subtilis subsp. Natto. ©2011 ijCEPr. All rights reserved

INTRODUCTION Nattokinase was discovered in 1980 by Dr Hiroyuki Sumi, researcher at Chicago University after testing over 173 natural foods as potential thrombolytic agents, searching for a natural agent that could effectively dissolve thrombus allied with cardiac and cerebral infarction [3-5]. Nattokinase was discovered in Natto, a fermented cheese-like food that has been used in Japan for over 1000 year. Natto is a traditional Japanese food made of soybeans . To prepare the beans are cooked and then by the action of the bacterium Bacillus subtilis ssp.natto fermented [8,27] .During this process is formed a slimy, stringy substance to the beans. In the traditional method of preparation are the bacteria from rice straw , into which the beans are wrapped [3] . In the modern manufacturing process, the bacteria cultures inoculated with beans, so that the use of rice straw is no longer necessary [21]. The botanical source for Nattokinase is Glycine max(L. )Merr. It appears as a yellow-white fine powder [22].

Fig.-1: Natto, traditionally wrapped in rice straw Natto is considered a very healthy food; a health product in the fermentation is some evidence for emerging substances [46]. Nattokinase is used for cardiovascular diseases including heart disease, high blood pressure, stroke, chest pain (angina), deep vein thrombosis,, “hardening of the arteries” (atherosclerosis), hemorrhoids, varicose veins, poor circulation,and peripheral artery disease[43-45]. It is also used for pain, fibromyalgia, chronic fatigue syndrome, endometriosis, uterine fibroids, muscle spasms, infertility, cancer, and a vitamin-deficiency disease called beriberi [40]. Properties Nattokinase is a fibrinolytic enzyme, meaning that it breaks down fibrin, an insoluble white protein produced by the conversion of fibrinogen (a protein in the plasma of blood for clotting) by thrombin (a blood clotting enzyme). Nattokinase is a serine protease with 275 amino acid residues and a molecular weight of 27,728 Daltons.

Haritha Meruvu , Meena Vangalapati

Vol.1, No.2, 61-66 (2011) Nattokinase has a high homology with the subtilisin enzymes and DNA sequencing shows 99.5 and 99.3% homology to subtilisin E and amylosacchariticus respectively [36, 38-39]. Nattokinase degrades fibrin clots both directly and indirectly. Nattokinase degrades fibrin directly in clot lysis assays with activity comparable toplasmin. Kinetic assays suggest that it is 6 times more active than plasmin in degrading cross-linked fibrin. Nattokinase degrades fibrin indirectly by affecting plasminogen activator activity. The other names of nattokinase are BSP, Natto Extract, Nattokinasa, NK, Fermented Soybeans, Soy Natto and Subtilisin NAT. Below, is the chemical structure of nattokinase [26-28].

Fig.-2: Chemical structure of nattokinase

Protien(Enyme) names Gene names Organism Taxonomic identifier Taxonomic lineage Protein attributes Sequence length Sequence status Sequence processing Protein existence General Annotation Function Catalytic activity Subunit structure Subcellular location Sequence similarities Biophysicochemical properties

Characteristics of nattokinase [15, 17, 19-21] Recommended name: Subtilisin NAT, EC=3.4.21.62 Alternative name(s):Nattokinase,cardiokinase, Name: aprN Bacillus subtilis subsp. natto 86029 [NCBI] Bacteria › Firmicutes › Bacillales › Bacillaceae › Bacillus 381 AA. Complete. The displayed sequence is further processed into a mature form. Evidence at protein level. Subtilisin is an extracellular alkaline serine protease, it catalyzes the hydrolysis of proteins and peptide amides. Subtilisin NAT also has fibrinolytic activity. Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides. Monomer. Secreted. Belongs to the peptidase S8 family. Kinetic parameters: KM=0.48 mM for Suc-Ala-Ala-Pro-Phe-pNA

Researches suggest that Nattokinase may promote normal blood pressure, reduce whole blood viscosity and increase circulation being an effective supplement to support cardiovascular health[19].Its strong thrombolytic activity promotes arterial health both directly, dissolving existing thrombus, and indirectly, enhancing body’s production of plasmin and urokinase by a direct cleavage of plasminogen activator inhibitor [32-35]]. The human body produces several types of enzymes for making thrombus, but only one main enzyme for breaking it down and dissolving it - plasmin. Nattokinase has plasmin-like bio-characteristic that lyses fibrin directly or indirectly in three different pathways [9, 10, 27]: Haritha Meruvu , Meena Vangalapati 62

Vol.1, No.2, 61-66 (2011) 1. 2. 3.

Nattokinase lyses fibrin directly. Nattokinase enhances plasmin through active pro-urokinase (endogenous). t-PA (Tissue Plasminogen Activators) is like urokinase and active plasmin,Nattokinase increases the concentration of t-PA.

Fig.-3: The physiological effects of nattokinase on fibrin

Source The botanical source for Nattokinase extraction is Glycine max(L. )Merr. It is a dicotyledonous annual herb belonging to fabaceae with common names wild soybean and reseeding soybean; with synonyms Dolichos soja L., Glycine gracilis Skvortzov, Glycine hispida (Moench) Maxim., Glycine soja (L.) Merr., nom. illeg., non Glycine soja Siebold & Zucc., Glycine ussuriensis Regel & Maack, Phaseolus max L., Soja hispida Moench, Soja max (L.) Piper [22-26 ].

Fig.-4: Images for Glycine max (L.) Merr Classification: Glycine max (L.) Merr. Kingdom Subkingdom Superdivision Division Class Subclass Order Family Genus

Plantae – Plants Tracheobionta – Vascular plants Spermatophyta – Seed plants Magnoliophyta – Flowering plants Magnoliopsida – Dicotyledons Rosidae Fabales Fabaceae – Pea family Glycine Wild. – soybean 63

Haritha Meruvu , Meena Vangalapati

Vol.1, No.2, 61-66 (2011) Species

Glycine max (L.) Merr. – soybean

Table-1: Compositional analysis of soybean [4] Raw soybean Boiled soybean pH 6.4 6.9 Moisture Ash Crude fibre

10.49 4.22 14.22

68.42 1.06 0.08

Protien

32.92

12.15

Fat

8.19

1.01

Reducing sugar(g/l) Ammonia(g/100ml)

0.29 0

0.22 0

The microbial source for nattokinase extraction is Bacillus subtilis subsp. Natto. The other name is Bacillus subtilis var. natto. This bacterium is used to produce natto by fermentation. Cooked soy beans are inoculated with the bacterial starter culture. The fermentation process takes at room temperature in a day; this time may be reduced to eight hours to six, if the temperature is increased from 40° C to 43 ° C. The maximum temperature reached during the fermentation process should be is 50 ° C; above which the fermentation stops as the bacteria die [11, 16, 17].

Fig.-5: Natto: Fermented soybean Bacterial Classification › Firmicutes › Bacilli › Bacillales › Bacillaceae › Bacillus › Bacillus subtilis group › Bacillus subtilis

Fig.-6: Bacillus subtilis Natto observed under microscope 64

Haritha Meruvu , Meena Vangalapati

Vol.1, No.2, 61-66 (2011)

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

The various strains of Bacillus subtilis subsp. Natto are CCRC 14716 IAM 1028 IAM 1163 IAM 1232 NC2-1 NR-1 OK2 Microbial fermentation is carried out using substrates like soybean, wheat bran, shrimp shell. These are the three substrates that are efficient in production of nattokinase enzyme upon fermentation. The essential components for fermentation are listed in comparison with three substrates [6-11]. Table-2: Compositional analysis of various substrates Constituents (%)

Soybean meal

Wheat bran

Shrimp shell meal

Fibre Moisture Protein nitrogen lipid carbohydrate

7 8 43 7 7 27

30 14 16 14 4 6

19 14 35 8 9 3

Medical uses In the main nattokinase works to support healthy blood circulation in two different ways. First off, nattokinase resembles plasmin, so it can break down fibrin directly. Secondly, nattokinase enhances the body’s natural production of plasmin, which also helps to break down fibrin. [2,11] In a nutshell, Nattokinase: • Supports normal circulation, blood flow, and blood viscosity (thickness) • Supports the body’s normal blood-clotting mechanism • Supports the body’s production of plasmin, which reduces fibrin • Helps to maintain normal blood pressure levels Nattokinase is used for cardiovascular diseases including heart disease, high blood pressure, stroke, chest pain (angina), deep vein thrombosis, hardening of the arteries (atherosclerosis), hemorrhoids, varicose veins, poor circulation, and peripheral artery disease stroke, venous stasis, thrombosis, emboli, fibromyalgia/chronic fatigue, claudication, retinal pathology, hemorrhoid, varicose veins, soft tissue rheumatisms, muscle spasm, poor healing[31-35]. . It is also used for pain, fibromyalgia, chronic fatigue syndrome, endometriosis, uterine fibroids, muscle spasms, tissue oxygen deprivation, infertility and cancer. Because nattokinase is an edible enzyme and is been used as nutrient supplement, it can be used to digest amyloids in body. Nattokinase can be used to remove infectious prion from animal feed, surgical instrument, and blood product [3-4, 44].

CONCLUSION Nattokinase is a potent fibrinolytic enzyme discovered in the extract of natto and produced via fermentation of Bacillus subtilis natto from boiled soybean.The safety record of its potent fibrinolytic enzyme, Nattokinase, is based upon the long term traditional use of the food, and recent scientific studies. Nattokinase has many benefits including its prolonged effects, cost effectiveness, and its ability to be used preventatively. It is a naturally occurring, food-based dietary supplement that has demonstrated stability in the gastrointestinal tract, as well as to changes in pH and temperature. Stressful era of modernization has led to high rates of cardiovascular diseases; thence it would then seem prudent to add this effective natural product to our heart health preventive arsenal as more recently, both clinical and non-clinical studies have demonstrated that Nattokinase supports heart health and promotes healthy circulation. Hereby, this paper paraphrases the properties, biological activity and the botanical and microbial sources of nattokinase. Moreover, the assorted therapeutic and medicinal uses are also summed in herewith. 65

Haritha Meruvu , Meena Vangalapati

Vol.1, No.2, 61-66 (2011)

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