ijCEPr, Vol.1, No.1, April-August, 2010 by Sanjay K. Sharma

!!Om Sai Ram!!

Volume-1, Number- 1, May-August, 2010

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

Prof. (Dr.) Sanjay K. Sharma

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ijCEPr International Journal of Chemical,Environmental andPharmaceutical Research Volume-1, Number-1, May-August, 1-60 (2010) Contentsâ&#x20AC;Ś Kinetic Studies on Dairy Wastewater Using Immobilized Fixed Bed Anaerobic Digester Dwaraka Kothapalli and Meena Vangalapati

1-5

Fluoride and Flurosis Status in Groundwater of Todaraisingh Area of District Tonk (Rajasthan, India): A Case Study Ashok Kumar Yadav and Parveen Khan

6-11

Antimicrobial Activity of Leaf Extracts of Murraya Koenigii against Aerobic Bacteria Associated with Bovine Mastitis Abhishek Mathur, V. K. Dua and G. B. K. S. Prasad

12-16

Restoration of Oil Contaminated Soil by Bioremediation for Ground Water Management and Environment Protection Kiran H. Udiwal and V.M. Patel

17-26

Synthesis, Characterization and Spectral Studies of Heterobinuclear Complexes of Transition Metal ions and their Biological Activity Netra Pal Singh and Abhay Nanda Srivastava

27-31

Experimental and Modelling Studies of Andrographolide Extraction from Andrographis Paniculata Sumanjali Avanigadda and Meena Vangalapati

32-36

In vitro cytotoxicity of Argemone mexicana Against Different Human Cancer Cell Lines Satish Kumar Verma, Santosh Kumar Singh, Abhishek Mathur and Shivsharan Singh

37-39

Growth and Characterization of NLO Material: l-Alanine Sodium Chloride D.Prabha and S.Palaniswamy

40-46

Effect of Dimethyl Sulphoxide on the Conductance and Solvation Behaviour of Pyridinium Dichromate in Water V. Radhika ,N. Srinivas and P. Manikyamba

47-53

L-Alanine Sodium Nitrate (ASN), NLO Material: Growth and Characterization D.Prabha and S.Palaniswamy

54-60

INDEX of Contributors of this issue Detailed Guidelines to Authors for Manuscript Preparation Membership Form

ijCEPr International Journal of Chemical,Environmental andPharmaceutical Research Volume-1, Number-1, May- August, 1-60, (2010) AUTHOR INDEX OF THIS ISSUE

Abhay Nanda Srivastava,27 Abhishek Mathur,12,37 Ashok Kumar Yadav,6 D.Prabha,40,54 Dwaraka Kothapalli,1 G. B. K. S. Prasad,12 Kiran H. Udiwal,17 Meena Vangalapati,1,32 N. Srinivas,47 Netra Pal Singh,27 P. Manikyamba,47 Parveen Khan,6 S.Palaniswamy,40,54 Santosh Kumar Singh,37 Satish Kumar Verma,37 Shivsharan Singh,37 Sumanjali Avanigadda,32 V. K. Dua,12 V. Radhika,47 V.M. Patel,17 IJCEPR widely covers all fields of Chemical, Environmental and Pharmaceutical Research. Manuscript Categories: Full-length paper, Review Articles, Short/Rapid Communications. Manuscripts should be addressed to: Prof. (Dr.) Sanjay K. Sharma Editor-in-Chief 23, ‘Anukampa’,Janakpuri, Opp. Heerapura Power Station, Ajmer Road, Jaipur-302024 (India) E-mail: ijcepr@gmail.com Phone:0141-2810628(O), 09414202678(M)

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.1, 1-5 May-August, 2010

Kinetic Studies on Dairy Wastewater Using Immobilized Fixed Bed Anaerobic Digester Dwaraka Kothapalli* and Meena Vangalapati Department of Chemical Engineering, A.U.College of Engineering (A), Andhra University, Visakhapatnam. *E-mail : dwaraka.kothapalli@gmail.com Article history: Received: 23 April 2010 Accepted: 14 July 2010

ABSTRACT In any dairy plant, the quantity and characteristics of effluent is depending upon the extent of production activities, pasteurization to several milk products. The anaerobic digesters in the first phase of treatment, which is followed by high rate aerobic treatment, remain as the most common effluent treatment scheme for dairy plants. The Indian dairy industries is stated to have the growth at more than 15% and poised to cross the 150 million tones / annum. The requirement for milk and milk products is keep growing in steady pace, making a significant impact on the Indian agriculture domain. The dairy industries require large quantity of water for the purpose of washing of cans, machinery and floor, the liquid waste in a dairy originates from manufacturing process, utilities and service section. So there is every need to reuse the waste water generated with proper and efficient treatment methods. Here the source of waste generation is a mixed sludge from dairy processing unit. The present study, thus initiated, for evaluating a need based experimental work on anaerobic digester incorporated with immobilized poly urethane foams system for treating dairy effluent with four weeks of harvesting. The kinetic parameters are estimated using the experimental data to develop a model. Empirical relations were generated for the characteristics like COD, SCOD, BOD, TDS, and TSS using modeling equations. Key words: Kinetic parameters, COD (chemical oxygen demand), SCOD, BOD (biological oxygen demand), TDS (total dissolved solids), TSS (total suspended solids). ÂŠ 2010 ijCEPr. All rights reserved

INTRODUCTION Water management in the dairy industry is well documented, but effluent production and disposal remain a problematic issue for the dairy industry .To enable the dairy industry to contribute to water conservation, an efficient and cost-effective treatment technology has to be developed. To this effect anaerobic digestion offers a unique treatment option to dairy industry. Not only does anaerobic digestion reduce the COD of an effluent, but little microbial biomass is produced. The biggest advantage is energy recovery in the form of methane and up to 95% of the organic matter in a waste stream can be converted into biogas[13] .Many high-rate digester designs are currently available and some have successfully been used for the treatment of dairy effluents. A full-scale up flow anaerobic sludge blanket digesters in uses world-wide [12]. The fixed-bed digester is another high-rate digester that has been used for the treatment of dairy effluents. A high-rate combination design, using the up flow anaerobic sludge blanket (UASB) and the fixed-bed digester types, was developed [1]. This design was successfully used to treat landfill leachate and baker's yeast factory effluent. Landfill leachate and yeast effluent both are having high COD concentrations and both are difficult to degrade biologically. On the other hand, dairy effluents are fairly easily biodegradable, since they consist mainly of diluted dairy products. Thus, the aim of this study was to evaluate the use of anaerobic digester (fixed film fixed bed) in the treatment of a dairy effluent and to study the kinetics of other parameters [10] .The diary waste is collected from the final milk processing unit. Nearly 30-40m3 of waste water is produced daily in this dairy industry. A graphical Model was also developed to predict total COD level in dairy wastewater, providing an important design parameter for implementation of fixed-film anaerobic digestion systems [5]. The high strength industrial waste stream can be treated in such anaerobic system for system efficiency of 8090% COD reduction. The incorporation of immobilized microbial support systems in the reactors to have attachedgrowth systems of microorganisms will enable anaerobic systems to perform well with much more process stability.

Dwaraka Kothapalli and Meena Vangalapati .

Vol.1, No.1, 1-5 (2010)

MATERIALS AND METHODS The experimental setup consists of Immobilized Fixed Bed Anaerobic digester having effective reactor volume of 2.0 lit. The experimental model is of 1.5 lit effective volumes Immobilized digester system is fed by diluted Dairy wastewater [6]. Biomass sludge was activated by aerating the organisms which was fed in to anaerobic digester and the harvesting was carried up to 28 days. Mixed vegetable waste is used as a nutrient for the development of micro organisms [2]. Samples were collected from a local market and macerated using a domestic food blender so that the wastes had been reduced to the smallest possible sizes(125-250Âľm ) and stored in a refrigerator at 4Â°C.1 gram of mixed vegetable waste (on wet basis) is added per 1 liter of the waste water to be treated. The type of samples collected were Brinjal, cabbage, carrot, potato, pumpkin, tomato in their rotten form [8]. Each of these vegetables is added in equal quantities and grinded [1]. The reactor was observed to attain the steady state conditions after four weeks with an average COD removal of 80% to 90%.After the Inoculum development step, the influent was fed by the upper part of the immobilized digester at six different theoretical hydraulic retention times (HRT) in a decreasing order of 6, 5, 4, 3, 2 and 1 days, which corresponded to average organic volumetric loading rates (Bv) of 26,36,40,74,152,201,226 mg/L of COD .The experiment was run for five batches[7]. The operating conditions are interpreted for the parameters of organic loading rate (Bv, mg/L) COD, BOD and SCOD (filtered COD). Also the parameters like TDS and TSS for every operating batch were observed. Values are averages of 3 determinations taken over 3 weeks after the steady-state conditions had been reached [4]. The differences between the observed values were less than 3% in all cases. The features and characteristics of influent used are shown in Table1 which lists the average values and standard deviations of the separate analysis carried out.

RESULTS AND DISCUSSIONS Mathematical Model: The success of any biological treatment plant lies in the kinetics of the process as they determine the dimensions of the unit operation and dictates the control parameters and operating values. The experimental observations and their kinetic interpretation are used to evaluate the substrate utilization (COD removal) kinetics of the anaerobic process of treatment having attached growth system [11]. The removal of COD is envisaged for the maximum percentage, with necessary operating variables of influent COD, SCOD, TDS, TSS, and HRT. The loading rate of organics on the biological system, the composition of biological systems and the active status of the biological systems are correlated to explain the process of COD removal or in terms of (substrate) utilization [9]. Better the utilization of organics by the biological system for their energy requirement (during which they also stabilize most of the unstabilized waste constituents) better the COD removal. According to the results obtained by regression analysis, logarithmic type functions appear to describe the effect of Bv on the fractional removal efficiency [14]. The general mathematical expression that relates Bv and the fractional removal efficiency is given by the following equation-

Ef = K1[ln(1 / Bv)] + K 2(1)

(1) Where EF is the fractional removal efficiency at a given value of Bv, K1 is a dimensionless empirical constant and K2 is another empirical constant equivalent to the EF value obtained when Bv is equal to unity and, therefore, ln (1/Bv) is equal to zero. The values for the empirical constants K1 and K2 obtained in the experiment and the correlation factors are summarized in Table 2. Equation 1 is only valid within the experimental range of BV studied (226-26 mg/L COD dm).The effect of the organic volumetric loading rate on the effluent COD is illustrated in Fig 1. An increase of BV in the range from 26 to 226 mg/LCOD caused virtually a linear increase in the fractional removal efficiency of COD from 10 % to89%. When BV increased from 26 to around 226 mg/L COD, the effluent COD concentration increased moderately from 26 to 74 mg/L. Hence, the process was capable of assimilating a considerable increase of the organic loading without failure. The following empirical relationship was found between BV and effluent COD-

EfCOD = 0.0044 Bv + 1.0081

(2) Likewise, empirical relations were developed for TDS, TSS, BOD parameters whose fractional removal efficiency decreased with the increase of Bv .In TDS and TSS, the rate of removal efficiencies proceeded at a slower pace, particularly in case of TDS. Since the organic matter is the main substrate for anaerobes to degrade, no significant removal rates were seen in TDS and TSS .The BOD levels decreased at a satisfactory rate. The fractional removal efficiency equations are obtained as follows2

Dwaraka Kothapalli and Meena Vangalapati

Vol.1, No.1, 1-5 (2010)

EfSCOD = 0.0044 Bv + 0.9998 EfBOD = 0.0036 Bv + 0.8679 EfTDS = 0.0016 Bv + 0.4254 EfTSS = 0.0013Bv + 0.2813

(3) (4) (5)

(6) The plot of loading rate Bv versus the fractional efficiency is made to study the COD and as well independently for other parameters like SCOD, BOD, TDS, TSS. The plots of drawn curves are shown in the Fig. 2, 3, 4 and 5.

CONCLUSION According to the results obtained elimination of COD increases with the no. of days up to 45 mg/L .More than 85% COD removal efficiency was achieved in the reactor with influent COD concentration of 226mg/L. The empirical equations for removal efficiencies of other characteristics were developed. The results from this study proved the immobilized Fixed Bed anaerobic digester flexibility and excellent performance for treating domestic and easily biodegradable wastewater such as dairy wastewater.

Fig.-1: Fractional removal efficiency vs Bv.

Fig.-2: Fractional removal efficiency graph of Bv vs SCOD.

Dwaraka Kothapalli and Meena Vangalapati

Vol.1, No.1, 1-5 (2010)

Fig.- 3: Fractional removal efficiency graph of Bv vs BOD.

Fig.- 4: Fractional removal efficiency graph of Bv vs TDS.

Fig.-5: Fractional removal efficiency graph of Bv vs.TSS

Dwaraka Kothapalli and Meena Vangalapati

Vol.1, No.1, 1-5 (2010) Table-1: The influent characteristics of dairy waste water. Influent characteristics COD TS TDS TSS BOD pH

Values (mg/L) 304 1688 1393 295 81 7.6

Table-2: The empirical constants K1 and K2, and the regression coefficients of the parameters that were studied. Parameters

COD

0.0044

1.0081

0.9986

SCOD

0.0044

0.9998

0.9990

TDS

0.0016

0.4254

0.9233

TSS

0.0013

0.2813

0.9439

BOD

0.0036

0.8679

0.9698

REFERENCES Attilio C., Andriana Del Broghi., Journal of Chemical Technology Biotechnology 69 (1977) 231 Andreadakis A.D., Water Science Technology 25 (1992) 9. Berg Van den L., Kennedy K.J., Water Science Technology 15 (1983) 359. Bjornsson L., Murto M., Mattiosson B., Applied Microbiology Biotechnology 54 (2000) 844. Burak Demirel.,Orhan Yenigum.,Turgut T.,Ohay., Biochemistry 40 (2005) 2583. Cordoba P.,Sanchez R.,Sineriz F., Biotechnolology Letters 6 (1984) 753. Callaghan F.,Wase D.,Thayanithi K., Bioresource Technology 67 (1999) 117. Callaghan F.,Forster C., Biomass Bioenergy 27 (2002) 71. Ezeonu F.,Okaka A., Process Biochemistry 31 (1996) 7. Hamzawi N., Kennedy K., Mclean D., Water Science Technology 38 (1998) 127. Husain A., Biomass Bioenergy 14 (1998) 561. Lettinga G.,Hulshoff L.,Koster W.,Wiegant W.,Zeeuw J.,Rinzema A.,Grin D.,Roersma, R., Hobma S., Biotechnology Genetic engineering Reviews 2 (1984) 253. 13. Michal P.,Shlomo K.,Gedaliah S., Water Research 29 (1995) 1549. 14. Orhon D.,Gorgum E.,Germirli F.,Artan N., Water Reserach, 27 ( 1993) 635

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

Dwaraka Kothapalli and Meena Vangalapati

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.1, 6-11 May-August, 2010

Fluoride and Flurosis Status in Groundwater of Todaraisingh Area of District Tonk (Rajasthan, India): A Case Study Ashok Kumar Yadav* and Parveen Khan FIST sponsored Department of Chemistry, Govt. P.G. College, Tonk (Raj.) India E-mail: yadav_ashokyad@rediffmail.com Article history: Received: 23 July2010 Accepted: 14 August 2010

ABSTRACT The continuous uses of water carrying high amount of fluoride may prove toxic to human, animal and plants. Excessive fluoride concentrations have been reported in ground water of more than 20 developed and developing countries including India where 18 states are facing fluoride problem. In view of this an attempts were made to find out the fluoride content in groundwater of Todaraisingh Tehsil of Tonk (Rajasthan). Fluoride concentration over permissible limit (1.5mg/l) in drinking water lead to human health hazards such as dental fluorosis and skeletal fluorosis affecting millions of people. Preliminary investigation indicates that severe health disorders have been indentified in Todaraisingh area of Tonk district of Rajasthan due to excess intake of fluoride through drinking water. Most of people in this area suffer from dental & skeletal flurosis such as mottling of teeth, deformation of ligaments, bending of spinal column and ageing problem. Overall all water quality was found unsatisfactory for drinking purpose without any treatment. So an urgent need is to educate the people on the causes of flurosis, encouraging rain water harvesting and defluoridation technique for providing fluoride free water in the study area. Key words: Fluorosis, Fluoride, Ion-Selective Electrode ÂŠ 2010 ijCEPr. All rights reserved

INTRODUCTION Water is an essential natural resource for sustaining life and environment that we have always thought to be available in abundance and free gift of nature however chemical composition of surface or subsurface water is one of the prime factors on which the suitability of water for domestic, industrial and agriculture purpose depends. Fresh water occurs as surface water and ground water in this groundwater contributes only 0.6% of the total water resources on earth. It is major and preferred source of drinking water in rural and urban areas particularly in India. Water content many minerals like calcium, magnesium and fluoride etc. in this fluoride essential in minute quantity for normal mineralization of bone & teeth (for formation of dental enamel) [2] fluoride stimulate growth of many plant species [4] but on other hand when fluoride is taken up in excessive amount may prove toxic to plant and on feeding may toxic to animal & human as fluorosis. Fluorosis is now world wide problem not only India. the 20 developing countries like Argentina, U.S.A., Algeria, Libya, Turkey, Iran, China, Australia, south Africa, Kenya, Iraq, Srilanka, Canada, Thailand, Newzealand, Japan, and India etc[8]. But in the era of economical growth groundwater is getting polluted due to urbanization & industrialization. Presence of various hazardous contaminants like fluoride, nitrate, sulfate and other heavy metals etc. in underground water has been reported from different parts of India. It is well established that India has two acute public health problem induced by utilization of groundwater as a source of drinking water having excess fluoride and arsenic though the origin of these two hazardous elements is attributed to geological reasons. In India fluoride is major inorganic pollutant which natural origin in groundwater. Fluoride concentration is an important aspect of hydro geochemistry because of its impact on human health. Fluoride is a fairly common element that does not occur in the elemental state in nature because of its high reactivity. This is the 17th element in order of abundance of element in earthâ&#x20AC;&#x2122;s crust found as a complex fluoride. Fluoride is an ionic state of fluorine (the 9th element of Periodic Table). Fluorine is most electronegative element hence never found in nature as fluorine. Fluoride occurs in combined form of minerals as fluoride and represents .06 to .09% of the earth crust [13]. Fluorides frequently occurs in igneous as well as in metamorphic rocks, especially in alkali rocks, granite, basalt, shale, clays and calcium phosphate rocks are the main sources of fluoride. Table-1 represents various minerals having fluoride with their composition and the rocks in which they present. Minerals which have the greatest effect on the hydro geochemistry of fluoride are fluorite, apatite, mica, amphiboles, certain clays and villiamite. Fluoride occurs in almost all water from trace to high concentrations. Ashok Kumar Yadav and Parveen Khan

Vol.1, No.1, 6-11 (2010) Fluoride concentration in natural water depends on various factors such as temperature, pH, solubility of fluoride bearing minerals, anion exchange capacity of aquifer materials (OH for F) and nature of geological formation and contact time of water with particular formation. Fluoride is among the substances for which there are both lower (0.6 mg/l) and upper (1.2 mg/l) limits of concentration in drinking water, with identified health effect and benefits for human beings. Fluoride in minute quantity is an essential component for normal mineralization of bone, teeth and formation of dental enamel[2]. Very low doses of fluoride (<0.6 mg/l) in water promote tooth decay. However, when consumed in higher doses (>1.5 mg/l), it leads to dental fluorosis or mottled enamel and excessively high concentration (>3.0 mg/l) of fluoride may lead to skeletal fluorosis. In general, fluoride content in water between 1.5 and 2.0 mg/l may lead to dental mottling, which is characterized initially by opaque white patches on the teeth and in advanced stages leads to dental fluorosis (teeth display brown to black staining) followed by pitting of teeth surfaces. High manifestations of dental fluorosis are mostly found in children up to the age of 12 years, and skeletal fluorosis [1] may occur when fluoride concentrations in drinking water exceed 4–8 mg/l. The high fluoride concentration manifests as an increase in bone density leading to thickness of long bones and calcification of ligaments. The symptoms include mild rheumatic/arthritic pain in the joints and muscles to severe pain in the cervical spine region along with stiffness and rigidity of the joints. The disease may be present in an individual at sub-clinical, chronic or acute levels of manifestation. Crippling skeletal fluorosis can occur when the water supply contains more than 10 mg/l of fluoride [3,13]. The severity of fluorosis depends on the concentration of fluoride in the drinking water, daily intake, continuity and duration of exposure, and climatic conditions So it very necessary to understand the present contamination level, distribution and developing a methodology for safe drinking water source. The health problems arising as a result of fluoride contamination are more wide spread in India. The problem of excessive fluoride in ground water in India was first reported in 1937 in the state of Andhra Pradesh [10]. Today fluorosis is a major public health problem in 18 out of 32 constituent state of India [12]. Nearly 177 districts have been confirmed as fluoride affected area. The existence of fluorine as a fluoride in water was first reported in 1937 in India[10]. Recent studies show approximately 62 million People including 6 million children suffer from fluorosis because of consumption of water containing high concentration of fluoride [12]. In Rajasthan the existence of fluoride was first detected from jobner near Jaipur city [7] later during 1964 in the villages of nagour and in 1976 high fluoride content in drinking water were observed in bhilwara district and Mathur et al reported the prevalence of fluorosis in Ajmer district [9]. StudyArea: Tonk district is located in north eastern part of the state bordering jaipur in north. Swaimadhopur in the east Bundi & Bhilwara in the south & Ajmer in the west. Tonk is known for its unity among Hindus and Muslims for which it is same time called as “Hindus Muslims Ekta Ka Maskan”. The history of Tonk is Very old it was called as Nawabi Nagari “Tonk”. The Tonk is also known as the “Lucknow of Rajasthan” due to its elegance.Tonk is popular among tourist for its Magnificent Mosques, Mansion and Havelis. Climate conditions: Area = 7194 sq.km, Forests area = 27048 hectare, Latitude = 25.41’ and 26.24’ in north, Longitude = 75.19’&76.16 in east, Temperature = 26-45 oC in summer Temperature = 8- 20 oC in winter, Annual rainfall in Tonk = 62mm

MATERIALS AND METHODS Many methods have been suggested for the determination of fluoride ion in water given by official British and American compilation of Methods. The calorimetric & electrode method are the most satisfactory at the present time [11].10 Samples are collected in good quality polythene bottles of one liter capacity. Sampling has been carried out without adding any preservative in rinsed bottles directly for avoiding any contamination and brought to the laboratory. Fluoride concentration of sample was determined by ion electrode method. Fluoride Ion-Selective Electrode Method: Apparatus: Ion-Selective Meter, Fluoride Electrode, Magnetic Stirrer Reagent: Fluoride Standards of various ranges (0.2-20ppm) Fluoride Buffer (TISAB-Total ionic strength adjustment buffer) Procedure: Calibrate the instrument take 10ml sample in a beaker at 10ml buffer solution. Put stirring bar into the beaker immerse electrode & start the magnetic stirrer and wait until reading is constant withdrawal electrode rinse with distilled water. 7

Ashok Kumar Yadav and Parveen Khan

Vol.1, No.1, 6-11 (2010)

Fig.-1: Study Area- Tonk District (Rajasthan) Table-1: Minerals containing fluoride S.No. 1.

Mineral Fluorspar

Chemical Composition [CaF2.3Ca3(PO4)2]

Fluorite

CaF2

3. 4.

Lepidolite Tremolite Actinolite Rock Phosphate

K2(Li,Al)5(Si6Al2)O20(OHF)4 Ca2(MgFe+2)5(Si8O22)(OHF)2

Rocks Pegmatite Pneumatolitic deposits Pegmatite Metamorphosed limestone Gabbros, Dolerites Clay

NaCa2(MgFe+2)4(AlFe+3)(SiAl)8O22(OHF)2

Limestone, Fossils

RESULTS AND DISCUSSION In this study 32 sample are selects for fluoride analysis from different site and each direction of tehsil out of 32 samples 21 samples about 65.63% of the groundwater samples analysed in the study area exceeds the maximum permissible limits of fluoride (1.5 mg/l) set by the ISI [5] and WHO [13] In the study area fluoride contamination is mainly a natural process, i.e. leaching of fluorine-bearing minerals, since no man-made pollution has been noticed. Since fluorite, apatite, mica and various other minerals take part during rockâ&#x20AC;&#x201C;water interaction and liberate fluoride into the groundwater. Preliminary investigation indicates that severe health disorders have been indentified in Todaraisingh area of Tonk district of Rajasthan due to excess intake of fluoride through drinking water. Most of people in this area suffer from dental & skeletal flurosis such as mottling of teeth, deformation of ligaments, bending of spinal column and ageing problem. Since there are no published data available on the incidence of fluoride in the groundwater and its health hazards in the Todaraisingh area, Tonk District, we have carried out investigations on the fluoride content in groundwater of the villages affected with dental and skeletal fluorosis and also the probable source of fluoride in groundwater of the study area. Average high fluoride (>1.5 mg/l) distribution was found mainly 8

Ashok Kumar Yadav and Parveen Khan

Vol.1, No.1, 6-11 (2010) in the sample no.- 1, 2, 4, 5, 7, 9-12, 14-17, 19, 22, 23, 25-30, (Table-2). The highest fluoride concentration (9 mg/l) was recorded from sample no. 28. The people suffer from dental (Figure1, 2, 3) and skeletal fluorosis (Figure 4). In the fluoride-affected villages, both children and adults suffer from health disorders like mottling of teeth, deformation of ligaments, bending of spinal column and ageing problem. The rest of the village’s sample are not affected by fluoride disorders, because fluoride content in the groundwater is within the permissible limit (<1.5 mg/l). It is seen on observation of graph that in pre monsoon fluoride range from 0.5 mg/l to 10.7 mg/l and in post monsoon fluoride range from 0.2mg/l to 7.3mg/l. and fluoride has tends high in pre monsoon.

Fig.-2: Comparison of Fluoride Content in Pre Monsoon and Post monsoon session.

Comparison of fluoride content in the groundwater of the study area with drinking water standards Parameter ISI standards WHO standards NO of sample exceeding % of sample permissible limit permissible limit HDL MPL HDL MPL Fluoride

0.6–1.2

1.5

0.5

1.5

exceeding

65.63

CONCLUSION Fluoride in groundwater of this region is mainly due to dissolution from fluoride bearing minerals like Fluorspar, Fluorite etc. the present study was done at tehsil level in Tonk district. In this 65.63% samples are found exceeding permissible limit. A more detailed study is necessary for better understanding of the source and effects of fluoride problem in other tehsil of tonk district. In the study area local people ingesting the groundwater have not received medical attention till date since these people are dependent on the groundwater for domestic use. So remedial measures such as defluoridation techniques and rain water harvesting are needed Nutritional diet such as calcium and phosphorus rich food should be recommended to those affected with fluorosis as it decrease rate of accumulation of fluoride in the human body. Environmental awareness programme on “fluoride and fluorosis” urgent need is to educate the people for health.

Ashok Kumar Yadav and Parveen Khan

Vol.1, No.1, 6-11 (2010)

(1)

(2)

(3)

(4)

Fig.-3: Photographs of some flurosis affected persons Dental Fluorosis (Snaps 1, 2, 3) and Skeletal fluorosis ( Snap 4)

Table-2 : Fluoride level in Todaraisingh area of Tonk Fluoride status of Todaraisingh Tehsil S.NO

Pre monsoon F

Post monsoon F

S.NO

Pre monsoon F

Post monsoon F

1 2 3 4 5 6 7

5 3.1 1.4 5.6 3.2 0.66 2.5

2.17 2.3 1.2 4 2.8 0.5 1.3

17 18 19 20 21 22 23

4.5 1.5 5.4 0.47 0.5 4.5 6

3.5 0.6 2.6 0.3 0.2 2.3 4.4

Ashok Kumar Yadav and Parveen Khan

Vol.1, No.1, 6-11 (2010) 8 9 10 11 12 13 14 15 16

2.5 3.5 2 4.6 3.7 1.5 2 7 6.4

0.5 3.1 1.7 1.9 2.8 1.2 1.7 2.9 4.5

24 25 26 27 28 29 30 31 32

1.3 3.3 2.2 4.8 10.7 5 4.47 1.28 1.1

0.9 0.3 0.84 0.76 7.3 4.4 3.3 0.7 0.9

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13.

Apambire, W. B., Boyle, D. R. and Michel, F. A., Environ. Geol., 33(1997)13. Bell, M.C. and T.G. Ludwig, 1970, The supply of fluoride to man: ingestion from water, fluorides and human health, W.H.O. Monograph series 59, World Health Organization, Geneva Boyle, D. R. and Chagnon, M., Can. Environ. Geochem. Health,17(1995) 5 Daines, R.H., I.A.Leone and E.Brennan, 1952, Phytopathology.(Abstr).42:112 ISI, Drinking water standards, Table 1. Substance and characteristics affecting the acceptability of water for domestic use 18, 10500. Indian Standard Institution, New Delhi, 1983. JAOAC, Journal of Association of Analytical Chemist ,58(1975)477 Kalsiwal, R.M. and Soloman, S.K., J. Asso. Phys. India.,7(1959) 56. Mameri, N., Yeddou A.R., Lounici H., Grib H., Belhocine D., and Bariou B., Water Research, 32(5) (1998)1604. Mathur, G.M., Tamboli, B.I., Mathur, R.N., Ray, A.K., Mathur, G.L., and Goyal, O.P.; Preliminary Epidemiological Investigation of Fluorosis in Surajpura and Pratappura Village in Sarwar tehsil Ajmer District. I.J.P.S.M., 7(1976)90. Short, H.E., G.R.,Mcrobert, T.W., Dernard and Mannadinayar A.S. , Ind. J. Med. Res. ,25(1937) 553. Standard Methods for the Examination of water and Waste Water,Am. Pub. Health Assoc., New York, 15th Ed. (1981). Susheela A.K., Current Science,77(10) (1999)1250. World Health Organization, Fluorides and human health., Monogr. Ser. 59, 1970,World Health Organization Publ., WHO, Geneva.

Ashok Kumar Yadav and Parveen Khan

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.1, 12-16 May-August, 2010

Antimicrobial Activity of Leaf Extracts of Murraya Koenigii against Aerobic Bacteria Associated with Bovine Mastitis Abhishek Mathur*1, V. K. Dua2 and G. B. K. S. Prasad3 1

Department of Biochemistry, Sai Institute of Paramedical & Allied Sciences, Dehradun(U.K), India National Institute of Malaria Research, Hardwar (U.K),India 3 Department of Biochemistry, Jiwaji University,Gwalior(M.P),India. *E-mail:abhishekmthr@gmail.com 2

Article history: Received: 26 July 2010 Accepted: 18 August 2010

ABSTRACT The susceptibilities of some of the common aerobic bacterial isolates from 20 milk samples collected from different cows with udder inflammation were subjected to hexane and methanolic leaf extracts of Murraya koenigii was determined by the well diffusion method. The minimum inhibitory concentration (MIC) of test extracts that gave inhibition were determined using the tube dilution method. The phytochemicals present in the various leaf extracts were also qualitatively assayed using conventional techniques. The methanolic extract of leaves of Murraya koenigii inhibited Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus uberis, Pseudomonas aeruginosa, Escherichia coli, Corynebacterium gravis and Bacillus cereus. The hexane extract inhibited all microorganisms except Staphylococcus epidermidis, Streptococcus uberis and Bacillus cereus. The MIC values of the different methanolic extracts of leaves were found to vary greatly, and ranged from 8.25 mg/ml to 30mg/ml. Key words: Bovine mastitis, aerobic bacteria, Murraya koenigii,methanolic extracts. ÂŠ 2010 ijCEPr. All rights reserved

INTRODUCTION Bovine mastitis is the inflammation of the parenchyma of the mammary glands of cattle [7] associated with microbial infections [8] and physiological changes[9]. Mastitis is caused by a group of infective and potentially pathogenic bacteria [3], viruses [11], mycoplasma , fungi and algae. The most common bacteria causing bovine mastitis include; Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactia and Escherichia coli. In order to minimize the economic losses from bovine mastitis and dissipation of infection resulting from the consumption of contaminated milk and milk products, there is an urgent need to ascertain the current status and involvement of aerobic bacteria in bovine udder inflammation, their role as causative agents of bovine mastitis and their susceptibilities.

MATERIALS AND METHODS Collection of plant material Fresh leaves of Murraya Koenigii were collected from the local gardens and were rinsed properly in sterile distilled water, dried in shadow and then ground to powdered form. The leaf powder was then extracted using methanol and hexane as solvents with the soxhlet apparatus. The extracts so obtained were weighed and stored in sterile universal bottles at 4 0C in a refrigerator.

Fig.-1: Plant of Murraya Koenigii

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Vol.1, No.1, 12-16 (2010)

Phytochemical Screening The methanolic extract, aqueous extract and leaf powder of Murraya koenigii were used as samples for qualitative phytochemical screening for tannins, resins, alkaloids, saponins, tannins, glycosides and flavonoids following the standard procedures described by Trease and Evans and Faraz [6,10]. Test for resins: To 0.5g of each sample was added 5ml of boiling ethanol. This was filtered through Whatman No.1 filter paper and the filtrate diluted with 4ml of 1% aqueous HCl. The formation of a heavy resinous precipitate indicated the presence of resins [10]. This was filtered and 1ml of the filtrate tested with a few drops of Dragendorff's reagent and a second 1ml portion treated similarly with Wagners reagent. The formation of a precipitate was an indication of the presence of alkaloids [6]. Test For Saponins: 0.5g of each sample was stirred with water in a test tube. Frothing which persists on warming was taken as preliminary evidence for the presence of saponins [6]. Test For Tannins: 0.5g of each sample was stirred with 10ml of boiling distilled water. This was filtered and a few milliliters of 6% ferric chloride added to the filtrate. Appearance of deep green coloration indicated the presence of tannins. The second portion of the filtrate was treated with a few milliliters of iodine solution. Appearance of a faint bluish coloration confirmed the presence of tannins [10]. Test For Glycosides: 0.5g of each sample was stirred with 10ml of boiling distilled water. This was filtered and 2ml of the filtrate hydrolized with a few drops of concentrated HCl and the solution rendered alkaline with a few drops of ammonia solution. 5 drops of this solution was added to 2ml of Benedict`s qualitative reagent and boiled. Appearance of reddish brown precipitate showed the presence of glycosides [10] Test For Flavonoids: 0.5g of each sample was dissolved in 2ml dilute NaOH solution. A few drops of concentrated sulphuric acid was then added. The presence of flavonoids was indicated by the disappearance of color [10]. Isolation and Identification of Bacteria 20 samples (5ml each) of cow milk was aseptically collected from each quarter of the udders of 20 lactating cows into sterile sample bottles labeled with the number of the animal following the method of Schalm et al [12]. Each sample was examined macroscopically for discoloration clots and flakes. Somatic cell counts were done using the manual method. Each sample was then inoculated in duplicate onto 10% sheep blood agar plates and were incubated aerobically at 370C for 48 hours and were examined for growth over a period of 24 and 48 hours respectively. Cultures were then identified using conventional techniques [2]. Sensitivity Test Colonies of fresh cultures were suspended in 20 ml of nutrient broth in different sterile universal bottles and were incubated at 370C over night. The concentration of organisms in the broth was determined and was diluted down to 10-6 Macfarlandâ&#x20AC;&#x2122;s standard. One ml of this was used in flooding over nutrient agar plates in the well diffusion method of the in vitro antimicrobial sensitivity test. The plates were left for 5mins after which they were dried at 37 0 Cfor 1hour. Four wells, equally distant, were bored round the plate using a sterile cork borer. Various concentrations of the diluted extracts were put inside the wells. Solvents such as Methanol and Hexane were put inside the well in separate petriplates to serve as negative control while Chloramphenicol(1mg\ml) was used as positive control in the separate petriplates. The plates were left free for 1 hour after which there were incubated at 37 0C for 24 hours and were examined for zones of inhibition. Minimum Inhibitory Concentration (MIC)

The broth dilution assay was carried for different dilutions of the extracts using susceptible bacteria as described by Muray et al.[13]. 13

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Vol.1, No.1, 12-16 (2010)

RESULTS AND DISCUSSION

The phytochemical analysis of the leaf powder and various extracts gave the results as depicted in Table-1. Out of the 20 samples analysed, 18 had somatic cell counts (SCC) greater than 5x105 cells/ml. Sixteen out of these 18 samples gave positive cultures. Hence, 16 samples out of 20 were positive. Staphylococcus aureus was the predominant organism isolated with 5 isolates. Others were Streptococcus uberis and Corynebacterium gravis with 3 isolates each; Staphylococcus epidermidis with 2 isolates; Escherichia coli, Bacillus cereus and Pseudomonas aeruginosa with 1 isolate each. 2 samples gave a mixed culture of Staphylococcus aureus and Corynebacterium gravis(Table-2). Results obtained from the susceptibility testing of the organisms with the various extracts showed that the methanolic extract was the potent antimicrobial agent than hexane extract. Methanolic extract inhibited Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus uberis, Pseudomonas aeruginosa, Escherichia coli, Corynebacterium gravis and Bacillus cereus, while the hexane extract only inhibited Pseudomonas aeruginosa, Escherichia coli and Corynebacterium gravis (Table-3). Methanolic extract was found to be most potent antimicrobial agent against Staphylococcus epidermidis while hexane extract showed maximum antimicrobial activity against E.coli amongst all other microorganisms(Table-3). According to results obtained from MIC, the methanolic extract gave a MIC of 8.25mg/ml for Escherichia coli and Staphylococcus epidermidis and Bacillus cereus while 12.5 mg/ml for Staphylococcus aureus and Streptococcus uberis. Methanolic extract gave MIC of 30 mg\ml for Pseudomonas aeruginosa and Corynebacterium gravis(Table-4). Table-1: Phytochemical analysis of Murraya koenigii leaves extracts (+ means Present, ++ means Prominent, - means Absent) Test

Hexane extracts

Methanolic extracts

Steroids

Alkaloids

Saponins

Cardiac Glycosides

Reducing sugars

Flavonoides

Tannins

Table-2: Bacterial Isolates Number of Cattle selected

Milk samples

Sample with SCC 18

Positive Samples

Bacteria isolated

Number of Pure isolates

Staphylococcus aureus Staphylococcus epidermidis Streptococcus uberis Escherichia coli Pseudomonas aeruginosa Corynebacterium gravis Bacillus cereus

5 2

3 1 1 3 16

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Vol.1, No.1, 12-16 (2010)

Normal milk should contain less than 500,000 cells/ml and an elevated somatic cell count is an indication of inflammation in the udder [8]. As such elevated somatic cell counts in the present work was an indication of inflammation. Institute for Animal Health (2003) identifies all the organisms isolated during present work as causative agents of bovine mastitis and have produced experimental infections in cattle with these organisms. The inhibition of Staphylococcus epidermidis, Streptococcus uberis, Pseudomonas aeruginosa, Escherichia coli, Corynebacterium gravis and Bacillus cereus with an MIC range of 8.25 to 30 mg/ml suggest that Murraya koenigii stands as a promising alternate source of antibacterial agents for the management of diseases of animals however, further work is needed to refine the technique. Table-3: Antibacterial Activity of Leaf extracts of Murraya koenigii and diameter of zone of inhibition (mm) Bacteria Staphylococcus aureus Staphylococcus epidermidis Streptococcus uberis Pseudomonas aeruginosa Escherichia coli Corynebacterium gravis Bacillus cereus

Methanolic extract (mg/ml) 200 100 50 20.00 18.00 12.00 30.00 25.50 19.00 20.50 15.30 12.00 16.00 14.00 12.00 26.00 22.00 19.00 15.00 14.00 12.00 22.00 17.00 13.00

Hexane Extract (mg/ml) 200 100 50 18.00 15.00 12.00 0.00 0.00 0.00 0.00 0.00 0.00 13.00 11.00 9.00 14.00 12.00 11.00 13.00 12.00 12.00 0.00 0.00 0.00

Chloramphenicol (mg/ml) 25.00 23.00 26.00 26.00 25.00 28.00 20.00

Table-4: Minimum Inhibitory concentration(MIC) of Methanolic extract of Murraya koenigii Bacteria Staphylococcus aureus Staphylococcus epidermidis Streptococcus uberis Pseudomonas aeruginosa Escherichia coli Corynebacterium gravis Bacillus cereus

Methanolic extract 12.5 8.25 12.5 30 8.25 30 8.25

MIC(mg\ml)

AKNOWLEDGEMENTS The authors wish to acknowledge the local milk sellers for providing us the infected milk samples. Thanks are due to the Research staff of Sai Institute,Dehradun(U.K),India and National Institute of Malaria Research, Hardwar(U.K),India for providing us the adequate facilities of research.

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

Baron, P.R., Pfaller, E.J. , M. A., Tenover. F. C and Yolke, R. H, 1999, Manual of Clinical Microbiology, 7th edition, Washington: ASM. P 1527-1539 Barrow, G.I. and Feltham, P. K. A. , 1995, Cowan and Steelâ&#x20AC;&#x2122;s Manual for the identification of medical bacteria, 3rd Edition, Cambridge University Press, London. Bezek, D. M and Hall, B. L., Canidian Veterinary Journal 36(1995)106. FAO Food and Nutrition, 1984, Report of a I session of Codex committee on Residues of veterinary drugs in food. Paper No. 32,17. Institute for Animal Health (2003). Improving milk quality and reducing mastitis. http://www.iah.bbsrc.ac.uk/. Mojab Faraz, Kamalinejad Mohammed, Ghaderi Naysaneh, Reza Hamid Validipour, Iranian Journal of Pharmaceutical Research, 2(2003)77.

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Radostit. O. M, Blood, D.C, and Gray, C. C , 1996, Veterinary medicine. Text book of the Diseases of Cattle, Sheep, Goats and Horses, 8th edition, London Baillere Tindal 8. Schroeder, J. W, 1997, Mastitis control program. Bovine mastitis and milking management. AS1129.NDSU. Extension service. 9. Spronle, R., Surveillance, 122( 1995)16. 10. Trease, G.E. and Evans, W.C. ,1989, A Text book of Pharmacognosy, 13th edition., 83, 685. Bailliere Tindall Ltd., London, ISBN: 0702013617. 11. Wallenberg G.J., Van Der Poal, W. H. M and Vana Oirschot, J. T., J. Vet. Micro. 88(2003)27.

Abhishek Mathur et. al.

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.1, 17-26 May-August, 2010

Restoration of Oil Contaminated Soil by Bioremediation for Ground Water Management and Environment Protection Kiran H. Udiwal1* and V.M. Patel2 L.D. College of Engineering, Ahmedabad *E-mail: kiranudiwal@gmail.com Article history: Received: 22 May 2010 Accepted: 25 July 2010

ABSTRACT Water is most essential but scarce resource in our country. Total water resources include surface water and ground water. Groundwater which is the main source for irrigation and drinking in the most part of the world constitutes about 89% of the total fresh water resources in the planet. But in recent years, due to over exploitation of ground water and erratic nature of monsoon, there has been depletion of ground water across the world. Further the quality problems of drinking water – both due to geogenic factors leading to chemical contamination like excess fluoride, arsenic, iron, salinity, nitrate, etc. and anthropogenic factors resulting in bacteriological contamination, pose serious public health problems. The problem has been further aggravated by the Petroleum industries which generate huge amount of wastewater, solid waste and sludge, some of which may be considered hazardous because of the presence of toxic organics and heavy metals. Accidental discharges as a result of abnormal operations or leakages due to flow line rupture or effluent water carry over with storm water can be major environmental hazard. The article discusses the acute water shortage in the North Gujarat categorically in Mehsana where water pollution is increasing day by day due to accidental discharge of effluent water or crude oil during transportation through pipe lines and enters into lakes, streams, rivers, oceans, and other water bodies. They get dissolved or lie suspended in water or get deposited on the bed. This results in the pollution of water whereby the quality of the water deteriorates, affecting aquatic ecosystems. Pollutants can also seep down and affect the groundwater deposits and ground water recharge system. Against this context the present article attempts to analyze the need for sustainable ground water management in India and highlights the attempt made to restore oil contaminated soil by advance technology i.e. technique of bioremediation of crude oil/oily sludge using specialised bacteria These specialised bacteria have the capability to breakdown and digest crude oil/oily sludge and convert them into harmless products like carbon dioxide, water and metabolites. This article discusses the development of microbial consortium and a case study of implementation of bioremediation and result obtained through it at Oil and Natural Gas Corporation, Mehsana (Gujarat). Key words: Total petroleum hydrocarbons (TPH), atomic absorption spectrometer (AAS), Group Gathering Station (GGS), central tank farm (CTF), Gujarat Ecology Commission (GEC). © 2010 ijCEPr. All rights reserved

INTRODUCTION The petroleum industry is involved in the global processes of exploration, extraction, refining, transporting (often with oil tankers and pipelines), and marketing petroleum products. The largest volume products of the industry are fuel oil and gasoline (petrol). Petroleum is also the raw material for many chemical products, including pharmaceuticals, solvents, fertilizers, pesticides, and plastics. The industry is usually divided into three major components: upstream, midstream and downstream. Midstream operations are usually included in the downstream category. Petroleum is vital to many industries, and is of importance to the maintenance of industrialized civilization itself, and thus is critical concern to many nations. Oil accounts for a large percentage of the world's energy consumption, ranging from a low of 32% for Europe and Asia, up to a high of 53% for the Middle East. Other geographic regions' consumption patterns are as follows: South and Central America (44%), Africa (41%), and North America (40%). The world at large consumes 30 billion barrels (4.8 km³) of oil per year, and the top oil consumers largely consist of developed nations. While producing oil these industries also generate wastewater, oil sludge, drill cuttings, chemical and chemical mud / drilling mud which in turns contribute to water pollution[9]. Industrial wastewater usually contains specific and readily identifiable chemical compounds. During the last fifty years, the numbers of industries in India have grown rapidly. But water pollution is concentrated within a few subsectors, mainly in the form of toxic wastes and organic pollutants. When toxic substances produced from

Kiran H. Udiwal and V.M. Patel

Vol.1, No.1, 17-26 (2010) industries and enter lakes, streams, rivers, oceans, and other water bodies, they get dissolved or lie suspended in water or get deposited on the bed. The effects of wastewater discharged accidently by these industries are not only devastating to people but also to animals, fish, and birds and affecting aquatic ecosystems. Pollutants can also seep down and affect the groundwater deposits and it also form layer of suspended oil and grease at the top of the land, creates hindrance in ground water recharge system. The facilities to treat wastewater are not adequate in most of the industries in India. Presently, only about 10% of the waste water generated is treated; the rest is discharged as it is into our water bodies. Due to this, pollutants enter groundwater, rivers, and other water bodies[17]. To overcome the effect of water pollutants discharged from oil industries and to restore affected land, contaminated due to accidental discharge of effluent water or discharge of crude oil in transit from wells to GGS/CTF, the Mehsana Area of North Gujarat was considered. Brief about area of Study: Mehsana district is one of the districts of north Gujarat agro-climatic zone. The district is located on 200.02’’ to 240.42’’ north latitude and 680.08’’ to 740.48’’ east longitude with an elevation of 92.96 meter above sea level. This district is surrounded by the Banaskantha district in north, Ahmedabad district in south, Sabarkantha district in east and Patan district in west. Mehsana is district headquarter. The Oil and Natural Gas Corporation, a pioneer in oil industry, started its exploration activities in and around Mehsana city of Gujarat in the year 1964. The important commercially producing fields of the Asset are North Kadi, Santhal, Sobhasan, Balol, Jotana, Lanwa, Bechraji, Nandasan and Linch. Starting with meager production 26 TPD during 1968-69, this Asset is currently producing 6600 TPD Approximately[10].

Fig.-1: Site Map Ground water resources in North Gujarat: As per recent estimate by GEC, 2004 the State of Gujarat has total replenishable ground water resources of 15,811 MCM / yr, whereas utilizable ground water resources for irrigation are 15,020 MCM / yr. The draft from ground water structures is estimated to be 11,486 MCM / yr. The Level of development of ground water resources at present is 76.47%. This leaves a balance of 3,051 MCM / yr for future development of ground water resources[6]. Categorization of areas: The estimation of ground water resources has been carried out considering talukas as assessment units. Based on the level of ground water development the assessment units have been categorized as safe, semi critical, critical and over exploited. Out of 223 talukas in the state of Gujarat 31 units are over exploited (level of GW development > 100%), 12 are Critical (level of GW development between 90 & 100%) and 42 are Semi Critical (level of GW development between 75 & 90%). Over exploited talukas are mostly located in north Gujarat Alluvial plain area. The region wise status of talukas falling indifferent categories is shown in Table No. 1[6]. 18

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Vol.1, No.1, 17-26 (2010) Ground water scarcity areas: Ground water scarcity areas are mostly located in North Gujarat, Saurashtra and Kachchh regions of the state. In north Gujarat the ground water scarcity areas cover parts of Panchmahals, Banaskantha, Mehsana, Gandhinagar and Ahmedabad districts of Gujarat. The scarcity in these areas is faced on account of erratic and scanty rainfall, high level of irrigation development and partly due to inherently saline formations[3]. Beside this existing water scarcity, oily effluent generated by oil industry, spilled over on land, due to accidental leakage from pipe line during transportation. Waste disposal and treatment options: Many incident of ground water contamination from oil and gas producing industries have been seen across the world. ONGC, Mehsana, which is the largest on shore oil producing asset of Oil and Natural Gas Corporation Limited, India has also observed few such cases and have taken corrective measures to arrest them. Even though if soil becomes contaminated with spilled over hydrocarbons and other waste materials, it is to be disposed off or to be treated by applying various existing methods. The main disposal method is through burial, either on or off site. Treatment occurs via bioremediation (using microorganisms to convert toxic compounds into less toxic forms); or thermal technologies (using high temperatures to reclaim or destroy hydrocarbon-contaminated material). The following types of waste disposal and treatment sites are utilized for oil and gas wastes[9]1 Pits 2 Landfills 3. Land forms 4 Land Spreading 5. Thermal Technology 6. Underground Slurry Injection 7. Salt Caverns Soil degradation and Bioremediation of oil Contaminated land: Soil degradation is widespread in India, affecting, about 1880 lakh hectares or 57% of the total area of the country. Of this 1620 lakh hectares are affected by soil erosion and 250 lakh hectares are affected by in situ degradation (water logging, salinization, and nutrient depletion). Extending the limited data available indicates that area affected by soil degradation in 1947 was probably about 1100 lakh hectares[14]. It is estimated that the cumulative effects of degradation over time will lead to an economic loss of Rs. 8900 crore to Rs. 23200 crore in 1997, reflecting a loss of 11%-26% of annual agricultural output. Erosion has rendered 1180 lakh hectares or 36% of the total area of the country “uneconomical to cultivate because of a significant loss in productivity”, while a further 150 lakh hectares has been rendered unmanageable and uneconomical” to use. Erosion by water is the most significant contributor to the economic loss due to soil degradation, and accounts for about 87% of the total degraded area. This category alone accounts for around Rs. 6100 crore-Rs. 21600 crore of the total estimated losses. Salinity and water logging are the other major-kinds of degradation. Although salinization and water logging affect only 11 % of the total area affected by degradation, they account for up to 30% of the total estimated cost. The problems of water logging and salinity are altogether lead to an estimated loss of Rs. 1200 crore to Rs. 2700 crore annually. India, USA, Germany, France, Italy, UK, Japan, China, Russia and South Korea together constitute 68.6% of total oil consumed in the world. India is the sixth greatest energy consumer in the world and Gujarat ranks 1st. in on shore oil production 55.10% and 32.3% in gas production in India. The State has highest on shore and offshore oil and gas field (31.3%)[15]. India's crude output in the year to March 2010 is likely to rise 11 percent to 36.71 million tons, or about 734,000 barrels per day [12]. One of the Major contributors of oil and Natural Gas to the country is Oil and Natural Gas Corporation Limited, which is involved in exploration and production of crude oil and Natural Gas, is located in Gujarat. Oil and Natural Gas Corporation Limited, Mehsana is producing Heavy oil. Heavy crude oil or Extra Heavy Crude oil is any type of crude oil which does not flow easily. It is asphaltic and contains asphaltenes and resins. It is "heavy" (dense and viscous) due to the high ratio of aromatics and naphthenes to paraffin (linear alkanes) and high amounts of NSO's (nitrogen, sulfur, oxygen and heavy metals). Asphalt is a sticky, black and highly viscous liquid or semi-solid that is present in most crude petroleum and in some natural deposits. The primary use of asphalt is in road construction, where it is used as the glue or binder for the aggregate particles. So the effect of leakage of crude oil or effluent water on land is devastating and affecting ground water recharge system as well. While processing it at GGS/ CTF it also unavoidably generates enormous quantity of tank bottom crude oil/oily sludge as well as oil/oily effluent soaked soil and wastewater which constitutes a major challenge for hazardous waste management as well as environment management. Due to stringent norms by regulatory authority and corporate responsibility of oil industries to protect the environment, new techniques are continuously in demand in India to manage crude oil/oily sludge. However, in developed countries, new technologies are continuously being invented and implemented for treatment of crude 19

Kiran H. Udiwal and V.M. Patel

Vol.1, No.1, 17-26 (2010) oil/oily sludge. Several technologies, which were considered emerging a few years back, are now well accepted in the field of hazardous waste treatment. One of new technology is bioremediation of crude oil/oily sludge using specialised bacteria. These specialised bacteria have the capability to breakdown and digest crude oil/oily sludge and convert them into harmless products like carbon dioxide, water and metabolites. Selection of site for bioremediation: The bioremediation project at ONGC Mehsana was undertaken for a total quantity of 1500 tons of oil contaminated soil at the locations of Santhal – 1, near well no 205 and near Well no. 206. The treatment was carried out in three sites at the said locations. The area of the sites is as follows:

Site – I : Santhal- 1 scrap yard, South Santhal CTF : Approximate area = (87 m x 55 m) + (58 m x 20 m) = 5945 m 2 Site – II : Near Well no. 205 , South Santhal CTF : Approximate area = (40 m x 20 m) = 800 m 2 Site – III : Near Well no. 206 , South Santhal CTF : Approximate area = (40 m x 22 m) = 880 m 2 Total area of the bioremediation site at South Santhal CTF = 7625 m 2 approximately.

MATERIALS AND METHODS Site preparation for bioremediation job: Bioremediation of oil contaminated soil at ONGC, Mehsana, was undertaken to treat total 1500 tons of oil contaminated soil in all the three bioremediation sites. All the three sites were containing heaps of oil contaminated soil. The site preparation was done by spreading the oil contaminated soil on the whole site and leveling the same to a uniform height of 20 cm. The site was cleaned by removing stones, debris, iron scrap material and any other non biodegradables. This was done by using JCBs and tractors with the help of manual laborers. All the sites were properly segregated and identified. The site preparation job was completed by 25 days and then the actual bioremediation job was initiated by the application of Oil zapper on the bioremediation site. Development & Application of Oil zapper on the bioremediation site: Oil zapper is a new bacterial product developed for the purpose of bioremediation of contaminated soil[10]. This was developed by assemble of five bacterial species which could biodegrade aliphatic, aromatic, nitrogen, sulphur, oxygen containing compounds and asphaltene fractions of crude oil/oily sludge. Oil zapper (sludge degrading bacterial culture) was produced in a bioreactor at laboratory. The growth conditions in the bioreactor were as follows: temperature, 32°C; aeration, 0.75 volume of air/volume of medium/min; agitation, 250 rpm; pH 7.0 (adjusted with 1 N HCl-NaOH); and duration of growth, 15 h. Silicone oil was added to control excessive foaming in the bioreactor. After growth, the culture was immobilized onto the selected carrier material, corncob powder, a biodegradable agricultural residue, by simple mixing in a1:3 ratios of carrier material and culture. A total bacterial count (on Luria-Bertani agar [LA] plates) of 1010 CFU/g of carrier material with a moisture level of 70% was maintained while the bacterial consortium was immobilized. The carrier-based culture was dispensed into sterile reusable polyethylene bags (4 kg of culture immobilized onto carrier material in each 10-kg polyethylene bag) and stored at 4°C after the bag was aseptically sealed. Oil zapper culture (live sludge degrading bacteria) was applied on oil contaminated soil at the Site I, II & III of the bioremediation site. Nutrient mixture was also sprayed on oil contaminated soil. After application of Oil zapper, mixing of oil contaminated soil and Oil zapper bacteria was done by tilling of site using tractor with cultivator. Sampling at the bioremediation site: Total 1500 tonnes of crude oil contaminated soil was undertaken for the bioremediation at site I, II & III of South Santhal .Sampling was done in the sites of the bioremediation site at zero day i.e. before initiation of the bioremediation job and at regular intervals after application of Oil zapper on the bioremediation site till the completion of the bioremediation job. Samples of the oil contaminated soil was collected from the random points of the bioremediation site and collected in a plastic bag. The sampling point was considered by dividing the site in number of zones separated at 20 meter distance and from each zone samples were collected randomly. In Site – I samples were collected from 10 random sampling points whereas in Site – II & III, samples were collected from 5 random sampling points from each sites. Collected samples from bioremediation sites were mixed uniformly with 20

Kiran H. Udiwal and V.M. Patel

Vol.1, No.1, 17-26 (2010) equal quantity to get a composite homogenised mixture of the samples. These composite samples were analysed for related parameters for monitoring the bioremediation efficiency. Extraction of TPH: The moisture content was determined by heating the oil contaminated soil at 80 0 C, where the water layer will be separated from the oil contaminated soil. Total petroleum hydrocarbon (TPH) was extracted from the oil contaminated soil samples by using solvents (hexane, toluene, ethylene chloride and chloroform). Solvents were evaporated in a fume hood by gentle nitrogen stream. After solvent evaporation, the total petroleum hydrocarbon (TPH) in each sludge samples was quantified. TPH extracted from crude oil/crude oil contaminated soil samples were fractionated into alkane fraction, aromatic fraction, NSO fraction and asphaltene fractions by silica gel column. After TPH extraction the residue was further analysed. The residue was taken in crucibles and heated at 600°C in a Muffle furnace for 5-6 hours. After cooling, the amount of ash was quantified [4]. Analysis of selected heavy metals in crude oil contaminated soil: Selected heavy metals were analysed in composite samples of crude oil contaminated soil undertaken for the bioremediation collected at zero day and after completion of bioremediation. The sludge sample was digested in nitric acid. A known weight (approximately 1 gm) of sludge sample was taken in a clean and dry container (normally microwave digestion Teflon tube or 100 ml glass beaker). Concentrated nitric acid (approximately 15 ml.) was added to a container and the container was covered with a watch glass and heated at 140°C on a hot plate, in a fume hood, until most of the acid was evaporated. The step was repeated thrice in order to solubilise the metallic components. The solution was then filtered in another container through 0.45 micron Whatman filter paper number 42 and the insoluble residues on the filter paper were rinsed with 10% nitric acid. The residue was then discarded and the container was covered with a watch glass and heated at 140°C until complete evaporation of nitric acid. Now the container was heated to 400°C until barely dried and white ash appeared. The sample was not allowed to bake and the temperature was maintained at 400°C for six hours. The bottom of the watch glass was carefully rinsed into the container using 10% nitric acid. The sides of the container were also rinsed and solution was evaporated to dryness at 140°C. The filter paper blank and nitric acid blank were also prepared similarly. Each container was then cooled and the residue was dissolved in 1 ml of concentrated nitric acid. The clear solution was then quantitatively transferred into 50 ml volumetric flask and volume was made up to 50 ml by using 10% nitric acid. The selected heavy metals present in the extract were analyzed using Atomic Absorption Spectrophotometer (AAS) (AAS – TJA, SOLAAR M Series, Unicam, USA). Some metals such as Se, and as, were analyzed using AAS equipped with hydride generation system or cold vapour technique [10].

RESULTS AND DISCUSSION At zero days, the oil contaminated soil samples were collected from the bioremediation sites I, II & III of South Santhal CTF, ONGC, Mehsana Asset. The composition of the oil contaminated soil was estimated by the analysis methods mentioned above. As per the analysis it was found that the oil contaminated soil undertaken for bioremediation at the bioremediation site I contained 7.87 % solvent extractable total petroleum hydrocarbon (TPH), 18.51 % moisture / water content, 73.62 % organic heavy fraction of hydrocarbon and inorganic (Table 2). The site II of the bioremediation site contained 7.32 % solvent extractable total petroleum hydrocarbon (TPH), 19.42 % moisture / water content, 73.26 % organic heavy fraction of hydrocarbon and inorganic (Table 2). The site III of the bioremediation site contained 6.92 % solvent extractable total petroleum hydrocarbon (TPH), 18.25 % moisture / water content, 74.83 % organic heavy fraction of hydrocarbon and inorganic (Table 2). It was observed that the steam extractable TPH in the oil contaminated soil in all the three sites were below extraction level (Table 2). Total petroleum hydrocarbon extracted from the oil contaminated soil of the bioremediation site I contained 65% alkane fraction, 24% aromatic fraction, 11% NSO and asphaltene fraction as shown in Table 2. The same in site II contained 64% alkane fraction, 25% aromatic fraction, 11% NSO and asphaltene fraction as shown in Table 2. The same in Site III contained 66% alkane fraction, 24% aromatic fraction, 10% NSO and asphaltene fraction as shown in Table 2. Biodegradation of TPH in oil contaminated soil at the bioremediation sites ONGC Mehsana is given at Table 3 and at Figure - 2 GC chromatogram indicating the biodegradation of Alkane and Aromatic fractions of the TPH extracted from the samples collected from the bioremediation sites of Site – I are also taken and given at Figure – 3 and 4

Kiran H. Udiwal and V.M. Patel

Vol.1, No.1, 17-26 (2010) Reduction in TPH has taken place within a period of 135 days from 7.87% to 0.7% and biodegradation reached to 90.98% at site -I indicate successful in-situ bioremediation of the oil contaminated soil. The land has become cultivated. The physical result of the bioremediation on the affected oil contaminated sites can also be seen from the photographs taken. (Status Before and after bioremediation are given from Figure -5 to Figure -10).

CONCLUSION Bioremediation is a process that uses naturally occurring microorganisms to transform harmful substances to nontoxic compounds. Bioremediation exploits this natural process by promoting the growth of microbes that can effectively degrade specific contaminants. Thus Oil zapper technology utilizes the bioremediation potential of specific microbes that degrades the toxic hydrocarbon compounds leaving behind non hazardous end products or metabolites and hence no harmful effects. Not only this technique is environmental friendly but also highly cost effective when compared to storage of oily waste/oily sludge in sludge pits and removing and transporting oil contaminated soil from the affected site due to accidental leakage of effluent water or crude oil. Further, the technique is ecologically sound, natural process; existing microorganisms can increase in numbers when oily sludge and waste water effluent sludge (the contaminants) is present. When the contaminants are degraded, the microbial population naturally declines. The residues from the biological treatment are usually harmless products (such as carbon dioxide, water and fatty acids) and hence, bioremediation technique could greatly help in solving the problem of soil contamination, oil sludge and waste water management problems of oil industries and controlling environmental and ground water pollution and also providing access to recharge of ground water system to the great extent at places where layer of oil contaminated soil formed restricting water percolation. Table- 1: Region-wise status of categorization of Talukas. (Source – GEC 2004) Region

No. of safe Talukas

No. of Sami Critical Talukas

No. Of critical Talukas

No. of Over exploited talukas

Saline Talukas

Total No. of Talukas

North Gujarat Central Gujarat South Gujarat Saurashtra Kutchh Total

11 33 24 28 1 97

14 15 1 35 4 69

6 1 0 4 1 12

25 1 0 2 3 31

7 2 0 4 1 14

63 52 25 73 10 223

Table -2: Composition of oil extracted from contaminated soil undertaken for bioremediation at, ONGC, Mehsana Constituents of oil contaminated soil

Composition (%) in the oil contaminated soil collected from Site – I Site – II Site – III

Steam extractable total petroleum hydrocarbon (TPH) in oil contaminated soil Solvent extractable TPH in oil contaminated soil Water content in oil contaminated soil Heavy fraction of hydrocarbons & inorganics Constituents of TPH Alkane fraction Aromatic fraction NSO & Asphaltene fraction 22

Nil

7.87

7.32

6.92

18.51 73.76

19.42 73.26

18.25 74.83

65 24 11

64 25 11

66 24 10 Kiran H. Udiwal and V.M. Patel

Vol.1, No.1, 17-26 (2010)

Table- 3: Biodegradation of TPH in oil contaminated soil at the bioremediation sites ONGC Mehsana.

Site I Time period

Zero day After 15 days After 40 days After 75 days After 135 days

Site II

Site III )

TPH Biodegradatio TPH Biodegradatio TPH Biodegradation (%) n (%) n (%) (%) (%) (%) 7.87 -7.32 -6.92 -5.12 34.94 5.27 28.01 4.29 38.01 4.21 46.51 3.98 45.63 3.14 54.62 2.04 74.08 2.41 67.08 1.95 71.82 0.71 90.98 0.58 92.08 0.59 91.47

Fig. - 2: Bioremediation of oil contaminated soil at South Santhal CTF, ONGC Mehsana Asset.

Figure - 3: GC chromatogram indicating the biodegradation of Alkane fraction.

Kiran H. Udiwal and V.M. Patel

Vol.1, No.1, 17-26 (2010)

Figure - 4: GC chromatogram indicating the biodegradation of Aromatic fraction.

Fig.-5: Site â&#x20AC;&#x201C;I before Bioremediation

Fig.-6: Site â&#x20AC;&#x201C; I after Bioremediation

Kiran H. Udiwal and V.M. Patel

Vol.1, No.1, 17-26 (2010)

Fig.-7 Site – II before Bioremediation

Fig.-8: Site – II after Bioremediation

Fig.-9: Site – III before Bioremediation

Fig.-10: Site – III after Bioremediation

REFERENCES 1. 2.

CGWB- ministry of water resources (Guide on artificial recharge of ground water, May-2000) CGWB, 2004 – The Dynamic ground water resources of India.

Kiran H. Udiwal and V.M. Patel

Vol.1, No.1, 17-26 (2010) 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

District groundwater brochure, Central ground water board, Ministry of water resources, Government of India .Water year 2007. Govt of India, 1999, Report of the Commission on Integrated Water Resources Development. Gujarat ecology commission – Ecological restoration, GEC -2004. Gujarat- the growth engine of India- Report on vibrant Gujarat, 2009. http://wrmin.nic.in/ Crude Oil Degradation in Contaminated Soil Comparing Land-Farming, Surface Heating and Enhanced Biodegradation Ahmed Mahdia, Dar EI Tarbia American School, Cairo, Egypt. Notification from Ministry Of Environment and forests, New Delhi –Section 72.For oil exploration onshore/ offshore. ONGC and TERI Report No. 2006MB24, ONGC - HSE Manual. Planning Commission, 2000, Midterm Appraisal of 9th Five Year Plan. Planning Commission, 2002, 10th Five Year Plan. Report from Narmada water resources, water supply and Kalpsar Department, Govt. of Gujarat. (www.gujnwrws.gujarat.govt.in/water related_ issues in Gujarat. Soil Degradation/ Pollution “Times Of India 25 Augut 1997 Editorial Pages” The Risk of Groundwater Contamination from Infiltration of Storm water Runoff - By Robert Pitt, Associate Professor, University of Alabama-Birmingham. UNEP 2003, Groundwater its susceptibility to degradation. World Bank, 1999, “Groundwater regulation and Management in India”

Kiran H. Udiwal and V.M. Patel

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.1, 27-31 May-August, 2010

Synthesis, Characterization and Spectral Studies of Heterobinuclear Complexes of Transition Metal ions and their Biological Activity Netra Pal Singh* and Abhay Nanda Srivastava Depatrment of Chemistry,Meerurt College Meerut, U.P.(India). E-mail: netrapal_chem@yahoo.com Article history: Received: 15 July 2010 Accepted: 23 August 2010

ABSTRACT Heterobinuclear complexes of transition metal ions with bis(2-aminobenzaldehyde)malonyldihydrazone in the presence of 5nitroindazole Cu(II) / Ni(II)- chloride of the type [ML1M`L2Cl2]or [ML1FeL2Cl2]Cl, where M = Mn(II), Co(II), Ni(II), Cu(II) have been prepared. All the complexes have been characterized by IR, UV-vis.and EPR spectroscopy, elemental analysis, magnetic moment and molar conductance measurement. Spectral studies and magnetic moment measurement in DMF suggest the covalent nature of the complexes, except the [ML1FeL2Cl2]Cl complex which is 1:1 electrolyte. An octahedral geometry is proposed for M` and square planer for M for the heterobinuclear complexes. The low value of magnetic moment and overlapping EPR signals are due to spin cross over since both of the metal have unpaired electrons with same molecular symmetry. The lowering of the magnetic moment has been discussed. The biological activity (antifungal and antibacterial) of the represented compounds has been studied. Key words: Heterobinuclear complexes, malonyldihydrazone, 5-nitroindazole, biological activity. ÂŠ 2010 ijCEPr. All rights reserved

INTRODUCTION Many of the divalent metal ion are widely presented in vivo as trace elements and essential for the living organism to maintain and regulate biological activities[7,14].There has been a great interest in the synthesis of heterobinuclear complexes for their relevance as metal for interesting magnetic properties[10,15] and active sites of biomolecules[13]. Heterobinuclear bridged complexes can be formed in step wise fashion from a mononuclear compound which contains a dangling ligand. The first spin cross over complex were reported by Brewer[4]. These complexes are also of interest of bioinorganic chemistry due to the importance of the structurally similar porphyrin complexes with unsymmetrical axial ligation[2,11,16]. The aim of this work is preparation and characterization of heterobinuclear complexes of Fe(III),Co(II), Mn(II), Cu(II) and Ni(II). Many other works have been done earlier by various chemists which show current importance and interest of coordination chemistry of transition metal ions[1,12,17,21].

MATERIALS AND METHODS All the chemicals used in this work were analytical grade. Hydrated Mn(II), Co(II), Ni(II), Cu(II) and Fe(III) chloride (BDH), 5-nitroindazole(Fluka), DMSO, DMF, acetonitrile, malonyl dihydrazone, 2-aminobenzaldehydeand ethanol. Double distilled water was used. The transition metal complexesof 5-nitroindazole and bis(2aminobenzaldehyde)malonyl hydrazone were prepared the method reported earlier[3,18]. Preparation of [MnL1NiL2Cl2] A solution of MnL1Cl2 (0.462gm, 1mmol) in DMF (15 ml) was added to the solution of NiL2Cl2 (0.456gm, 1mmol) and refluxed for 15hr and then kept in refrigerator overnight. A light pink colour product was formed which was filtered and washed with ethanol, ether and dried in vacuo. Preparation of [MnL1CuL2Cl2] This compound was prepared by using same procedure as above. Preparation of [CoL1NiL2Cl2] A solution of CoL1Cl2 (0.466gm, 1mmol) in dry DMF (15ml) was refluxed with a methanolic solution (15 ml) of NiL2Cl2 (0.456gm, 1mmol). The purple colour solution of CoL1Cl2 turned blue on addition of the solution of

Netra Pal Singh and Abhay Nanda Srivastava

Vol.1, No.1, 27-31 (2010) NiL2Cl2. A light yellow coloured product was precipitated on refluxing for 6 h. The compound was filtered, washed with ethanol, ether and dried in vacuo. Preparation of [CoL1CuL2Cl2] This compound was prepared by using same procedure as above. Preparation of [NiL1FeL2Cl2]Cl A solution of NiL2Cl2 (0.456gm, 1mmol) in methanol (15 ml)was treated with a solution of [FeL1Cl2]Cl (0.488gm, 1mmol) in dry DMF (15 ml). The resultant solution was refluxed for 20 h. A brown product precipitated. The complex was filtered, washed with ethanol, ether and dried in vacuo. Preparation of [CuL1FeL2Cl2].Cl This complex was prepared by using same procedure as above.

RESULTS AND DISCUSSION The complexes were prepared according to the following chemical equations-

Where, L1 = bis(2-aminobenzaldehyde)malonyl hydrazone, L2 = 5-nitroindazole. Analytical data are given in Table 1. All the complexes are soluble in DMF and DMSO. (Fig. 1.) IR Spectra of the Heterobinuclear Complexes The relevant IR bands and their assignments are cited in Table 2. The IR spectra of the binuclear complexes under investigation show several bands belonging to ligands L1 and L2. They are considerably changed compared with the relevant bands of the ligands and monometallic complexes[22]. Results given in table are consistent with the some previous results[5,8,9,20]. Electronic Spectra and Magnetic Moments Electronic spectra (UV-vis.) and magnetic moment value of heterobinuclear complexes are given in Table 3. Magnetic moment values are measured in DMF solvent and show non- electrolyte nature of complexes, except [NiL1FeL2Cl2]Cl and [CuL1FeL2Cl2]Cl which are 1:1 electrolyte. The electronic spectra of metal complexes were recorded in DMF solvent and contain mixed transitions due to two different metal ions. The binuclear complexes possess antiferromagnetic properties at room temperature by intramolecular spin exchange interaction between M and M` metal ions. Results given in table are consistent with the heterobinuclear complexes[19]. EPR Spectra EPR spectra value of all metal complexes were given in Tabkle 3. the EPR spectra of hetrobinuclear complexes were recorded at room temperature. The spectra of [MnL1NiL2Cl2] show g = 1.91, g = 1.82 which show square planer Mn(II) complexes. The signals for two different metals are merdged together and new signals are obtained. Antimicrobial Activity In vitro antimicrobial activity of heterobinuclear metal complexes have been tested against the bacteria Bacillus subtilis and Escherichia coli and fungi Aspirgillus niger and Aspirgillus flavus and are summerised in Table 4. The values indicate that all complexes have higher antimicrobial activity than the free ligand. Such increased activity of the metal chelates can be explained on the basis of chelation theory. On chelation, the polarity of the metal ion will be reduced to a greater extent due to overlap of the ligand orbital and partial sharing of the positive charge of the metal ion with donor groups. Further, it increases the delocalization of Ď&#x20AC;-electrons over the whole chelate ring and enhance the penetration of the complexes into lipid membranes and blocking of the metal binding sites in enzymes of microorganism. These complexes also disturb the respiration process of the cell and thus block the synthesis of proteins, which restricts further growth of microorganism [6]. 28

Netra Pal Singh and Abhay Nanda Srivastava

Vol.1, No.1, 27-31 (2010)

Table-1: Analytical Data of Heterobinuclear Complexes Complexes

Molecular Formula (Formula weight)

Colour

M.P. ( )

Calcd. (found%)

Yield (%) C

[MnL1NiL2Cl2]

C31H26Cl2MnN12O6Ni (846.8)

Light pink

324

44.00 (43.92)

3.09 (3.04)

19.85 (19.65)

[MnL1CuL2Cl2]

C31H26Cl2MnN12O6Cu (852.07) C31 H26Cl2CoN12O6Ni (851.23) C31H26Cl2CoN12O6Cu (856.06) C31H24Cl3FeN12O6Ni (883.65) C31H24Cl3FeN12O6Cu (888.48)

Pink

328

Light Yellow Dirty Yellow Brown

336

318

342

Reddish Brown

346

43.70 (43.66) 43.74 (43.68) 43.49 (43.42) 42.13 (42.08) 41.90 (41.84)

3.07 (3.01) 3.08 (3.03) 3.06 (3.02) 3.00 (2.88) 2.95 (2.91)

19.73 (19.65) 19.75 (19.71) 19.63 (19.54) 19.02 (18.84) 18.91 (18.87)

[CoL1NiL2Cl2] [CoL1CuL2Cl2] [NiL1FeL2Cl2]Cl [CuL1FeL2Cl2]Cl

Table-2: IR Spectral data (cm-1) of the Heterobinuclear Complexes Complexes ν (C=O) ν (N-H) [MnL1NiL2Cl2] [MnL1CuL2Cl2] [CoL1NiL2Cl2] [CoL1CuL2Cl2] [NiL1FeL2Cl2]Cl [CuL1FeL2Cl2]Cl

1725 1718 1738 1735 1726 1730

Ring ν (NO2) ν (C=N) ν (M-N) ν (M-N) Stretching (Asym / Sym)

3318 3322 3328 3330 3324 3327

1615 1612 1618 1622 1632 1634

1528/1382 1530/1385 1538/1395 1536/1392 1564/1344 1558/1348

1615 1610 1628 1624 1570 1578

482 478 460 468 466 470

ν (M-Cl)

475 470 466 468 472 464

322 318 320 324 322 320

Table-3:Electronic Spectra, Magnetic Moment and EPR Data of Heterobinuclear Complexes.

Complexes

Transition(Cm-1) (values, cm-1M-1)

[MnL1NiL2Cl2]

20,020(302) 18,178(260) 12,502(45)

[MnL1CuL2Cl2]

38,320(56) 25,448(428) 20,408 16588(406)

[CoL1NiL2Cl2]

6,565(3.1) 14,415(5.3)

Assignments

A2g 4T1g(P) 4 A2g 4T1g 4 A2g 4T2g C.T. 4 A1g(G) 6A1g 4 T2g(G) 6A1g 2 Eg(G) 6B1g 4 T1g(G) 6A1g 4 T2g(F) 6T1g(F) 4 Ag(F) 4T1g(F) 29

µ eff (B.M.)

EPR Value

g∥

g⊥

1.91

1.82

3.90

5.10

1.93

Netra Pal Singh and Abhay Nanda Srivastava

Vol.1, No.1, 27-31 (2010) 21,268(3.4) 16,378(2.2) [CoL1CuL2Cl2]

6,565(3.4) 14,412(5.1) 18,230(5.2) 15,508(6.2) 20,302(2.7)

[NiL1FeL2Cl2]Cl

40,462 29,112(28,410) 19,542(26,457) 15,508(24,268) 21,266(3.4) 18,788(2.6)

[CuL1FeL2Cl2]Cl

40,460 29,111(28,409) 15,512(24,242) 19,541(26,458) 15,506(6.03) 18,305(8.2)

A1g 1B1g A1g 1B2g 4 T2g(F) 4T1g(F) 4 A2g(F) 4T1g(F) 4 4 T1g T1g(F) 2 1 A1g B1g 2 E1g 2B1g C.T. 4 E1g(G) 6A1g 4 6 T2g A1g 2 6 T1(G) A1g 1 A1g 1B1g 1 A1g 1A2g C.T. 4 A1g(G) 6A1g 4 6 T1g A1g 4 6 T2(G) A1g 1 B1g(G) 2A1g 2 B1g 2Eg 1

1.93

1.86

5.87

5.82

Table-4: Antibacterial and Antifungal Activity of Heterobinuclear Metal Complexes. Compounds

*Conc.

Bacterial Inhibition (%)

Antifungal Inhibition (%)

B. subtilis

E. coli

A. niger

A.flavus

[MnL1.NiL2Cl2]

100 500

40 48

51 58

62 74

65 78

[MnL1.CuL2Cl2]

100 500

48 53

51 55

71 83

72 85

[CoL1.NiL2Cl2]

100 500

52 61

73 82

82 91

80 86

[CoL1.CuL2Cl2]

100 500

42 58

62 74

68 77

70 79

[NiL1.FeL2Cl2].Cl

100 500

46 52

59 64

69 72

75 79

[CuL1.FeL2Cl2].Cl

100 500

48 55

54 68

65 81

78 84

* = (ŕŤ&#x201E;g moL-1), B. subtilis =Bacillus subtilis , E. coli = Escherichia coli, A. niger = Aspirgillus niger , A. flavus = Aspirgillus flavus.

Netra Pal Singh and Abhay Nanda Srivastava

Vol.1, No.1, 27-31 (2010)

O C C O

Cl NH

NO2 NH H N

N Cl

NO2

Fig.-1:Heterobinuclear complexes of the type [ML1.M`L2Cl2].

ACKNOWLEDGEMENTS The authors are thankful to ACBR, Delhi for providing spectral data and SAIF, CDRI, Lucknow for providing elemental analysis data. Authors are also thankful to SARC, Meerut for providing antimicrobial activity.

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

Arjmand Farukh et al., European Journal of Medicinal Chemistry,(2010), 1. Bag, N. et al., Inorganic Chemistry, 34, (1995), 753. Bandini, A. L. et al., Canadian Journal of Chemistry, 57, (1979), 3237. Brewer, C. T. et al., Journal of Chemical Society, Dalton Trans., 1, (1993), 1513. Campell, M.J.M. et al., Journal of Inorganic Nuclear Chemistry, 36, (1974), 2485. Dharmaraj, N. et al., Transition Metal Chemistry, 26, (2002), 105. Fenton, D. E. ,1995, Biocoordination Chemistry, First Ed., Oxford University . Ferraro, J.R. ,1971, Plenum Press, New York. Folgado, J.V. et al., Journal of Chemical Science. Dalton Trans, (1986), 1061. Gatteschi, D. et al.,1991, Magnetic Molecular Materials, Kluwev Academic Dordrecht. Kadish , K. M. et al., Inorganic Chemistry, 37, (1998), 2693. Kalam, A. et al., Turkish Journal of Chemistry, 34, (2010), 147. Karlin, K. D. , Tyeklav, Z.,1993, Bioinorganic Chemistry of Copper, Chapmanand Hall, London. Karlin, K. D., Zubieta, J.,1996, Biological and Inorganic Copper Chemistry, Academic Press, New York. Khan, O. ,1993, Molecular Magnetism, Wiley-VCH, New York. Kobayashi, K. et al., Journal of Biological Chemistry存255, (1980), 2239. Lashanizadegan, M. and Seraj, S., Turkish Journal of Chemistry, 34, (2010), 263. Rajavel, R. et al., E-Journal of Chemistry, 5(3), (2008), 620. Sakamoto, M., Synthesis Reactivity of Inorganic Metal Organic Chemistry, 27(4),( 1997), 567. Shah, N. , Kar, S.K. , Journal of Inorganic Nuclear Chemistry, 39, (1997), 1236. Travnicek Zdenek et al., Inorganica Chemica Acta, 363, (2010), 2071. Woo, L.K., Maurya, M.R. , Inorganic Chemistry, 30, (1991), 4671.

Netra Pal Singh and Abhay Nanda Srivastava

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.1, 32-36 May-August, 2010

Experimental and Modelling Studies of Andrographolide Extraction from Andrographis Paniculata Sumanjali Avanigadda and Meena Vangalapati* Centre for Biotechnology, Department of Chemical Engineering, AUCE (A), Andhra University, Visakhapatnam *E-mail: meena_sekhar09@yahoo.co.in Article history: Received: 24 April 2010 Accepted: 23 July 2010

ABSTRACT Andrographolide is the main diterpenoid lactone contained in the leaves of Andrographis paniculata. This bioactive component has multifunctional medicinal properties such as activity against fever, dysentery, diarrhoea, inflammation, and sore throat as well as immune disorder. The objectives of this work were to study the effect of polarity and Hildebrand solubility parameter of solvents in the extraction of andrographolide from A. paniculata and to develop a mathematical model to quantitatively describe the extraction phenomena. The extraction was carried out by employing various organic solvents and their mixtures with water as solvents using standard soxhlet method. Five grams of ground - dried A. paniculata leaves was extracted using 1.00 x 10-4 m3 of solvent for 80minutes. The standard soxhlet extraction method was conducted using methanol, ethanol, ethyl acetate and water at different extraction times to verify the mathematical model proposed in this work. Methanol was found to be the best solvent for the extraction of andrographolide from A. paniculata. The Hildebrand solubility parameter concept was not able to predict the extraction of andrographolide using polar organic solvents. The final form of the proposed model based on rapid mass transfer at the interphase of the solid-liquid surface and the introduction of volumetric mass transfer coefficient is Es = 0.8917t – 9.8114, where ,Es = total extract (g) and t = extraction time (in minutes). Keywords: Andrographolide, Andrographis Paniculata, Extraction, Modelling, Soxhlet extraction © 2010 ijCEPr. All rights reserved

INTRODUCTION Andrographis paniculata (Burm. f.) Nees (Acanthaceae) (AP) is an herb originated from India and widely distributed in southern China with annual growth of 0.30 - 0.70 m height. It has been used in traditional systems of medicine to treat a number of ailments including common cold, fever, diarrhoea, liver diseases, and inflammation [18]. Recent studies have revealed some cardiovascular effects of this herb [16,19]. It is also found to be a promising new way for the treatment of HIV, AIDS [1], and numerous symptoms associated with immune disorders [4],works effectively as a immunostimulant [11,13]. Three main diterpenoid lactones identified in the A. paniculata leaves were andrographolide, neo-andrographolide and deoxyandrographolide. The molecular formula of andrographolide is C20H30O5, while its molecular structure is shown in Figure 1. Andrographolide can be easily dissolved in methanol, ethanol, pyridine, acetic acid and acetone, but slightly dissolved in ether and water. The melting point of this compound is 228o – 230oC and the ultraviolet spectrum in ethanol, λ max is 223 nm. [17]. The analysis of andrographolide can be done by thin layer chromatography (TLC), high – performance liquid chromatography (HPLC) and crystallisation. The liquid solvent extraction is the most common method for separating bioactive components from their natural resources. The advantages of this method over other extraction methods are as follows [4]: Sample throughput can be increased by simultaneous extraction in parallel. It has the ability to extract more sample mass and it is non-matrix dependent.The sample is repeatedly brought into contact with the fresh portions of the solvent, thereby helping to displace the transfer equilibrium, The temperature of the system remains relatively high due to the heat applied to the distillation flask. However, for toxicological reason, drug and medicine producers are required to minimize the number and amount of solvents employed in pharmaceutical processes [19]. The presence of a solvent in the extract may also affect the kinetics of crystallisation and the crystal morphology of the product [3]. In order to optimise the utilisation of solvent in the extraction of bioactive components from natural resources, an estimation of the extract yield obtained is necessary. The objectives of this work were to study the effect of polarity and Hildebrand solubility parameter of solvents in

Sumanjali Avanigadda and Meena Vangalapati

Vol.1, No.1, 32-36 (2010) the extraction of andrographolide from A. paniculata and to develop a mathematical model to quantitatively describe the extraction phenomena.

MATERIALS AND METHODS Materials The leaves of A. paniculata were collected from chintapalli forest near Visakhapatnam. Information about its use as traditional Anticancer and other properties were collected from tribals and local Ayurvedic doctors. Various organic solvents were purchased from Qualigens; deionised water was generated in the Analytical Laboratory, Department of Chemical Engineering, Andhra University, and Visakhapatnam. The leaves are dried under sundry. Leaves were powdered using thimble and mixer. It is finely grounded to 80 mesh size (particle size 100micro meters). Prior to the solvent extraction study, 1 gram of dried - ground leaves of A. paniculata was placed in a cellulose thimble. Solvent extraction An amount of 1.00 x 10-4 m3 of solvent was used for the extraction using a standard soxhlet method for 80minutes in a soxhlet extraction system. The standard soxhlet extraction method [8]was conducted using methanol and other organic solvents at different extraction times, and different concentrations to verify the mathematical model proposed in this work[12,14]. The extracts were then concentrated using vacuum rotary evaporator and completely dried in an atmospheric oven. The crude extracts were then analysed for their andrographolide content using high performance liquid chromatography[15]. Modelling of extraction using soxhlet extractor In order to describe the andrographolide transfer from the leaf particles to the bulk of the solvent, the following hypotheses were used [6,7]. Every leaf particle is symmetrical. The mass transfer coefficient is constant. The solvent in the extractor is perfectly mixed, while the transfer resistance in the liquid phase is negligible and the andrographolide concentration in the solvent depends only on time, The transfer of the andrographolide is a diffusion phenomenon and independent of time, At the interface, the concentration of andrographolide in the solution between the internal liquid (in pores) and external to particles are equal. The final form of the equation obtained from this modelling is: Es = B (t) – D Where Es = total extract (g), t = extraction time (seconds), B, D is constants

RESULTS AND DISCUSSION In comparison to non – polar solvents, polar solvents could extract andrographolide at higher yield except water, where hydrolysis and thermal degradation might occur. Methanol was found to be the best solvent for the extraction of andrographolide[9,10].Ethanol and aqueous acetone extracted andrographolide at lower yield although their Hildebrand solubility parameters are closer to that of andrographolide. Solvents having moderate polarity extracted andrographolide much lower than ethanol did. Non - polar solvents were almost not able to extract andrographolide. The maximum androgapholide extracted at the concentration of 60% methanol. For the model development we considered 60% methanol. The generated model equation is Es = 0.8917t – 9.8114 The results are represented in Table 1, 2 and Figure 2, 3.The model showed a good agreement with the experimental data as shown in Figure 3.

CONCLUSION Methanol was found to be the best solvent for the extraction of andrographolide from Andrographis paniculata. However, the Hildebrand solubility parameter concept was not able to predict the extraction of andrographolide using polar organic solvents. Among the different concentrations, 60% methanol yield maximum. The final form of the proposed model is Es = 0.8917t – 9.8114.

Sumanjali Avanigadda and Meena Vangalapati

Vol.1, No.1, 32-36 (2010)

Table-1:Effect of solvent polarity and Hildebrand solubility parameters on Extraction yield. Solvent

Polarity

Extract Yield (%)

Extracted (g/1gm of dried leaves) andrographolide

6.6

Hildebrand solubility parameter 14.45

Methanol 100% Methanol 80% Methanol 60% Methanol 50% Methanol 40% Methanol 20% Ethanol 100% Ethanol 80% Ethanol 60% Ethanol 50% Ethanol 40% Ethanol 20% Ethyl acetate 100% Ethyl acetate 50% Water

15.518

0.131

7.08 7.56 7.8 8.04 8.28 5.2 5.96 6.72 7.1 7.48 8.06 4.3

16.242 18.034 18.93 19.826 21.618 12.90 15.016 17.132 18.19 19.248 21.364 9.04

20.91 27.068 23.75 14.619 11.92 18.49 20.06 24.34 22.39 18.61 17.62 14.619

0.18 0.266 0.531 0.29 0.0945 0.128 0.169 0.342 0.287 0.107 0.129 0.452

5.8

15.28

17.34

0.532

23.40

21.068

0.43

Table-2: Effect of Yield extract with Extraction time S. No

Time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Extract Yield (gm) 0 0 0 3.554 8.011 12.534 17.658 20.639 25.989 29.677 34.988 39.548 48.334 54.137 54.137 54.137 54.137

Sumanjali Avanigadda and Meena Vangalapati

Vol.1, No.1, 32-36 (2010)

E xtract (g m )

50 40 30 20 10 0 0

20 25

40 45

55 60

70 75

Time (min) Fig.-1: Effect of Yield extract with extraction time

y = 0.8917x - 9.8114 2 R = 0.9986

Extract (gm)

0 0

Time (min) Fig.-2: Comparison of extract weight calculated from the model and experimental data

Sumanjali Avanigadda and Meena Vangalapati

Vol.1, No.1, 32-36 (2010)

Fig.-3: Molecular structure of Andrographolide

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

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Calabrese, C. et al., Phytother. Res., 14(2000) 333 Choudhury, et al. Planta Med., 53(1987)135. Coon, J.T. and Ernst, E. , Planta Med 70(2004) 293. Dandu Anilkumar M. and Naseeruddin M. Inamdar, Pak. J. Pharm. Sci., 22(1)(January 2009)49. Kolar, et al. , Fluid Phase Equil. 194 -197(2002) 771-782. Kumoro, A.C., and Hasan Masitah ,Modelling of Andrographolide Extraction from Andrographis Paniculata Leaves in a Soxhlet Extractor Proceedings of the 1st International Conference on Natural Resources Engineering & Technology (2006) 664-670 Li, W. and Fitzloff ,J. F. ,J. Liq. Chromatogr. Relat.Technol., 27(15) (2004) 2407. Luque de Castro, M. D. and Garcia-Ayuso, L. E. , Analitica Chimica Acta, 369(1998)1 Misra Himanshu, et al. InPharm Communique (Suppl.) ,2(2), 51 Perry, et al. 1988. Perry’s Chemical Engineer’s Hand Book. New York: McGraw-Hill Book Company. Puri, et al, J. Nat. Prod. ,56(1993)995. Rajani, et al., Pharmaceutical Biology, 38(2000)204. Sediawan, B. S. and Prasetyo, A. , 1997. Pemodelan Matematis dan Penyelesaian Numeris dalam TeknikKimia dengan Pemrograman Bahasa BASICc dan FORTRAN. Yogyakarta: Andi Singh Pratibha ,et al, Journal of Medical Sciences., 114(2009)136 Tang, et al., Yaowu-Fenxi-Zazhi. , 20 (6) (2000) 420. T hi so d a, e t a l., E ur . J . P har maco l., 5 5 3 ( 2 0 0 6 ) 3 9 . Wongkittipong et al ., Separation and Purification Technology, 40(2000)147. World Health Organization (2002) WHO Monographs on Selected Medicinal Plants, Vol 2, pp 12–24, World Health Organization, Geneva, Switzerland. Zhang C.Y. and Tan B.K. , Clin. Exp. Pharmacol. Physiol., 23(1996) 675.

Sumanjali Avanigadda and Meena Vangalapati

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.1, 37-39 May-August, 2010

In vitro cytotoxicity of Argemone mexicana Against Different Human Cancer Cell Lines Satish Kumar Verma*1, Santosh Kumar Singh2, Abhishek Mathur1 and Shivsharan Singh3 1

Department of Botechnology, Sai Institute of Paramedical and Allied sciences, Dehradun (U.K.) India. Department of Microbiology, Gayatri College of Biomedical Sciences, Dehradun, Uttarakhand, India. 3 Doon (PG), Paramedical College and Hospital, Dehradun, Uttarakhand, India *E-mail: verma.satish@rediffmail.com 2

Article history: Received: 15 August 2010 Accepted: 31 August 2010

ABSTRACT Cancer is a public health problem all over the world. Large number of plants and their isolated constituents has been shown to possess potential anticancer activity. Whole plant ethanolic extract of Argemone mexicana showed in vitro cytotoxicity against different human cancer cell lines. There was no growth of inhibition recorded against liver cancer cell line. Sulforhodamine B dye (SRB) assay was done for in vitro cytotoxicity test assay. The in vitro cytotoxicity was performed against five human cancer cell lines namely of lung (A-549), liver (Hep-2) colon (502713 HT-29) and neuroblastima (IMR-32). Plant extract showed 83% growth of inhibition against lung (A-549) cell line. In case of liver (Hep-2), no activity was observed, where as plant extract showed maximum activity against colon 502713 cell line. It was found to be 99% and 96% respectively, in case of HT-29 liver human cancer line and IMR-32 neuroblastima cell lines. Keywords: Human cancer cell lines, in vitro, cytotoxicity test, SRB, Argemone mexicana © 2010 ijCEPr. All rights reserved

INTRODUCTION Argemone mexicana Linn. belongs to the family Papaveraceae. The plant is annually prickly herb. Its leaves are simple, alternate, cauline, sessile, exstipulate prickly deep cut, with spiny teeth and unicostate reticulate vanation. The flowers are large, yellow, hermaphrodite and actinomorphic [14]. It is widely used in folk medicines to alleviate several ailments especially for its analgesic effects [1]. Chemical investigations of this plant have revealed the presence of alkaloids [9,13], amino acids [5], phenolics [8] and fatty acids [7]. Thus the authors set forth the objective of evaluating the in vitro cytotoxicity activity of Argemone mexicana against different human cancer cell lines.

MATERIALS AND METHODS Plant material and Preparation of plant extracts Plants were collected in February to March 2008 from eastern regions of Uttar Pradesh, India. The whole plant ethanolic extract was used for in vitro cytotoxicity assays. Plant material was dried at 370C, powdered and extracted in ethanol. Extract was fine-filtered and freeze dried. For the preparation of the extracts, dried ground plant material was percolated with 95% ethanol and concentrated to dryness under reduced pressure. Extract was redissolved in Dimethylsulphoxide (DMSO) to form stock solutions, which were filter sterilized (0.2µm) before testing on cell lines. Human cell lines Human cancer cell lines namely of lung (A-549), liver (hep-2) colon (502713 HT-29) and neuroblastima (IMR-32) were grown in RPMI-1640 with 2 mM L-glutamine medium pH 7.2. Penicillin was dissolved in PBS and sterilized by filtering through 0.2µ filter in laminar air flow hood. The media was stored at low temperature (2-8oC). Complete growth medium contained 10% FCS. The medium for cryopreservation contained 20% FCS and 10% DMSO in growth medium. The cell lines were maintained at 370C in a 5% CO2 atmosphere with 95% humidity.

Satish Kumar Verma et. al

Vol.1, No.1, 37-39 (2010) In vitro assay for cytotoxic activity The anticancer activity was determined by evaluating the cytotoxic potential of the test material using human cancer cell lines that were allowed to grow on tissue culture plates in the presence of test material. The cell growth was measured using ELISA reader after staining with Sulforhodamine B dye (SRB) which binds to basic amino acid residues in the trichloroacetic acid (TCA) fixed cells. Preparation of Cell suspension for assay Human cancer cell lines were grown in multiple tri-conical flasks (TCFs) at 370C in an atmosphere of 5% CO2 and 90% relative humidity in complete growth medium to obtain enough number of cells. The flasks with cells at subconfluent stage were selected. Cells were harvested by treatment with Trypsin-EDTA. Cells were separated to single cell suspension by gentle pipetting action and the viable cells were counted in a hemocytometer using trypan blue. Cell viability at this stage should be >97%. Viable cell density was adjusted to 5,000 - 40,000 cells/100µl depending upon the cell line (Monks, 1991). Cell suspension (100µl) together with 100µl of complete growth medium was added into each well. The plates were incubated at 370C for 24 hours in an atmosphere of 5% CO2 and 90% relative humidity in a CO2 incubator. After 24 hours, the test material, DMSO (vehicle control) and positive control were added. Sulforhodamine B (SRB) assay The antiproliferative SRB assay was performed to assess growth inhibition. This is a colorimetric assay which estimates cell number indirectly by staining total cellular protein with the SRB dye Skehan [17]. The microtiter plates were taken out after 48 hours incubation of the cells with test materials and gently layered with chilled 50% TCA in all the wells to produce a final concentration of 10%. The tissue culture plates were incubated at 4°C for one hour to fix the cells attached to the bottom of the wells. The supernatant was then discarded. The plates were washed five times with distilled water to remove TCA, growth medium, low molecular weight metabolites, serum proteins etc. Plates were air dried and stored until further use. SRB solution was added to each well of the plates and incubated at room temperature for 30 minutes. The unbound SRB was removed quickly by washing the wells five times with 1 % acetic acid and then air dried. 100µl of Tris buffer (0.01 M, pH 10.4) was added and shaken gently for 5 minutes on a mechanical shaker. Optical density was recorded on ELISA reader at 515 nm.

RESULTS AND DISCUSSION Argemone mexicana is a storehouse of good variety of compounds. Chemically they could be alkaloids, flavonoids, coumarins, monoterpenes, sesquiterpenes, steroids, fatty acids, esters and phenolic acids etc. The flowers contain isorhamnein, isorham-nethin-3-glucoside and isorhamnetin-3, 7-diglucoside [4,16], Protopine, allocryptopine and berberine [15], Isoquinoline [2], argemexicaine A and argemexicaine, along with thirteen known alkaloids Chang et al.[3].Thus the plant Argemone mexicana is one of the most valuable medicinal plants and attracted the attention of many workers. Experimentally the test samples showing growth inhibition more than 70% at 100 µg/ml were considered to be active. The in vitro cytotoxicity was performed against lung (A-549) cell line showed 83% growth of inhibition. In case of liver (Hep-2), no activity was observed. Where as in case of colon 502713 cell line, it showed maximum growth inhibition. In case of HT-29 liver human cancer line and IMR-32 neuroblastima cell line plant extract showed 99% and 96% activity respectively. In the present study, we concluded that the plant extracts showed selective in vitro cytotoxity against some human cancer cell lines. The activity might be depended upon the morphology of cell lines and mechanism of action of the plant extract. Many plant extract kill cancer cell lines through activating apoptosis and effecting growth regulators. The plant is also shown to acclerate the growth of the Pestalotiopsis mangiferae that causes a serious leaf-spot disease of Mangifera indica. It is widely used in Sudanese traditional medicine for the treatment of trypanosomiasis [10]. The Ethanolic and aqueous extracts were also observed to show antibacterial properties and immunomodulatory properties [6,11]. It is not possible at this juncture to single out the most effective in vitro cytotoxicity constituent of Argemone mexicana. However, based on the published studies flavonoids, alkaloids seem to be most likely candidates eliciting in vitro cytotoxicity effect. Its reported in vitro cytotoxicity effects warrant further investigation for its use in the cases of clinical anticancer activity.

Satish Kumar Verma et. al

Vol.1, No.1, 37-39 (2010)

Fig.-1: In vitro cytotoxic activity of plant extracts

REFERENCES 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Capasso, A. , Piacente, S. , Pizza, C. , Tommasi, N. D. , Jativa, C. and Sorrentino, L. , Planta Medicine, 63 (1997) 326. Capasso,A. , Aquino, R. , Tommasi, N.D. , Piacente, S. , Rastrelli, L. and Pizza, C. , 2002, Neuropharmacology Activity of Alkaloids from South American Medicinal Plants Current Medicinal Chemistry-Central Nervous System Agents, Bentham Science Publisher 2, 1-15. Chang, Y.C. , Hsieh, P.W. , Chang, F.R. , Wu, R.R. , Liaw, C.C. , Lee, K.H. and Wu, Y.C. , Planta Medicine ,69 (2003) 148. Dalvi, R.R. , Experientia ,41 (1985) 77. Dinda, B. and Bandyopadhyay, M. J. , J. Indian Chemical Society, 63 (1986) 934. Farrukh , A. and Ahmad, I. , World J. of Microbio and Biotechnology, 19 (2003) 653. Gunstone, F. D., Holliday, J. A. and Scrimgeour, C. M. , Chem. Phys. Lipids ,20 ( 1977) 331. Harborne, J. B. and Williams, C. A., Phy-tochemistry, 22 (1983) 1520. Hussain, S. F , Nakkady, S., Khan, L. and Shamma, M. , Phytochemistry, 22 (1983) 319. Khalid, S.A. , Schmidt, T.J. and Brun, R. , Ethnopharmacology, 55 (1996) 1. Miguel, A. , Zavala, S. ,Salud, P.G. , Rosa, M. and Perez, G. , Phytother. Research, 11 (1997) 368. Monks, J. Natl. Cancer Institute, 83 (1991) 757. Nakkady, S. and Shamma, M. , Egypt. J. Pharmacy Science, 29 (1988) 53. Pandey B., P., 2003. Papaveraceae, Taxonomy of Angiosperm, pp. 329. Piacente, S. , Capasso, A. , Tommasi, N. , Jativa, C., Pizza, C. and Sorrentino, L. , Phytotherapy Research, 2 (1998) 155. Rai, M. K. , Pandey, A. K. and Acharya, D. , Journal of Non-timber Forest ,7( 2000) 237. Skehan, P. , Journal of National Cancer Institute, 82 (1990) 1107.

Satish Kumar Verma et. al

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.1, 40-46 May-August, 2010

Growth and Characterization of NLO Material: l-Alanine Sodium Chloride D.Prabha and S.Palaniswamy* Department of Physics, PSG College of Arts and Science, Coimbatore-641 014, India. *E-mail: drprabha76@rediffmail.com Article history: Received: 22 August 2010 Accepted: 2 September 2010

ABSTRACT Single crystal of alanine sodium chloride (ASC), a nonlinear optical material has been grown from solution by slow evaporation method. The iso-electric point of the alanine is 6 (1). So, the growth of crystals has been carried out at pH 6. The grown crystals have been subjected to powder X-ray diffraction studies to identify the crystalline nature. Single crystal X-ray diffractometer is utilized to measure the cell parameters and morphology of the grown crystals. The FTIR spectra taken for the crystals grown at a pH value of 6 show different peaks intensity which reveals the structure of the ASC crystal. The mechanical properties of the grown crystals are studied using Vickers micro hardness measurement. Surface morphology was studied by SEM analysis. Using Nd-YAG laser the NLO property of the crystal is studied. The transmittance and absorption of the crystal is studied by UVVisible spectrometer. Keywords: Characterization, X-ray diffraction, Slow evaporation, Sem analysis, Vickers micro hardness tester. © 2010 ijCEPr. All rights reserved

INTRODUCTION Non linear optical (NLO) crystals has emerged as one of the most attractive fields of current research in view of its vital applications in areas like optical modulation, optical switching, optical logic, frequency shifting and optical data storage for the developing technologies in telecommunications and in efficient signal processing[4]. Surface morphology was studied by using SEM studies. The development of highly efficient nonlinear optical (NLO) crystals for visible and ultraviolet region is extremely important for both laser spectroscopy and laser processing. MATERIALS AND METHODS Synthesis and crystal growth The solution is prepared using sodium chloride and alanine in the molar ratio of 1:1[7]. The pH value is low, so to increase the pH value, the pH of the solution is adjusted to 6 by adding 4 drops of NaOH. The above solution is filtered using the filter paper and transferred to a Petri dish. The Petri dish is covered with a filter paper with small hole, tied on top with rubber band to facilitate evaporation and crystal growth. Structure of L-Alanine molecule The α-carbon atom of L-alanine is bound with a methyl group (-CH3), making it one of the simplest α-amino acids with respect to molecular structure and also resulting in L-alanine being classified as an aliphatic amino acid. The methyl group of L-alanine is non-reactive and is thus almost never directly involved in protein function. The structure of L-alanine is –

L-alanine

D.Prabha and S.Palaniswamy

Vol.1, No.1, 40-46 (2010) Further its linear zwitter ionic structure is

The crystal structure of L-alanine is orthorhombic [10]. Its cell parameters are a = 6.032 A o, b= 12.343 A o, c = 5.784 A o. α = β = γ = 90o Characterization The crystals are characterized by FTIR spectroscopy, powder XRD, UV-Visible spectroscopy, Vickers micro hardness tester and SEM. The FTIR spectra are recorded on Shimadzu 8400s FTIR. Powder XRD is obtained by Philips X-ray generator mode 1pw 1390 with a nickel filter. The visible absorption data is obtained using Jaso corp v570. Powder XRD (Fig.1) X-ray diffraction technique is a powerful tool to analyze the crystalline nature of the materials. If the material to be investigated is crystalline, well defined peaks will be observed. The peaks in the diffractogram are indexed by using powder X-ray software [5]. FT-IR Analysis (Fig.2) The FTIR spectrum of alanine sodium chloride (ASC) is recorded using FTIR spectrometer in the region 2500500cm-1. T he region 3436cm-1 with strong intensity represents N-H stretching. 3085cm-1with medium intensity refers C-H asymmetric stretching. The region 2929cm-1 with weak (broad) represents OH stretching. 2112cm-1 with weak intensity refers C=C stretching.1506cm-1 weak intensity represents N-H in lane bending. The peak 1412cm-1 with weak band represents C=H bending.1235cm-1 with variable refers C-O-C stretching. 539cm-1 with weak intensity refers S-S stretching. The peak 486cm-1 with weak intensity represents S-S stretching [3]. Micro hardness (Fig.3) ASC crystal is subjected to Vickers micro hardness test with the load varying from 25 to 100g [1]. Hardness number of the crystal is calculated using the relation Hv = 1.8544 P/d 2 Kg/mm 2 Vickers micro hardness profile as a function of the applied test loads is illustrated by fig. It is evident from the plot that the micro hardness of the crystal increases with increasing the load. The value of the work hardening coefficient n is estimated from the plot of log p versus log d drawn by the least square fit method. It is observed that the Vickers hardness number of the crystal increases with increasing the load [11]. The value of the work hardening coefficient n is found to be 0.672. According to Onitsch, 1.0 ≤ n ≤ 0.672 for hard materials and n > 0.672 for soft materials [6]. Hence, it is concluded that ASC belongs to the soft materials. UV-Visible spectrometer analysis (Fig.4) The optical absorption spectra of alanine sodium chloride crystals (ASC) are recorded in the range 190-2500nm using JASCO corp V-570 spectrometer [8]. It is seen from the absorption spectrum the crystal is transparent in the range 900-2500nm without any absorption peak, which is an essential parameter of NLO crystals. Scanning electron microscope (SEM) (Fig.5) The quality of the grown crystals can be identified to some extent by identifying the surface morphology of crystal face is observed by optical microscope in reflection mode using JSM 6360 JEOL/EO. From the SEM photograph shown in figure, the following observations are evident: 1. at a magnification of 850 and at a scale of 20 micrometer we observe the crystals have smoothed, rectangular surfaces. 2. At a magnification of 15,000 and 1 micrometer scale we can observe that the crystals have an average thickness of 200µm [9].

D.Prabha and S.Palaniswamy

Vol.1, No.1, 40-46 (2010)

RESULTS AND DISCUSSION From the above experimental and characterization of alanine sodium chloride crystal the following results and the discussion are significant. 1. Single crystals of alanine sodium chloride (ASC) are successfully grown at a pH of 6.The grown crystals are larger in size having an average size of 3cmx4cm. 2. The grown crystals are characterized by using powder X-ray diffraction. 3. From the FTIR spectrum we can confirm the structure of the ASC to have both the alanine and sodium chloride molecules. These are arranged in alternate layers in the crystal. This is evident from the non damage of alanine structure. 4. From the SEM analysis we conclude that the crystal formation size in micro range is 200 micrometers. Further in the micro level the crystal surface is very smooth which shows that it can add more molecules to grow into a large crystal. 5. From the Vickersâ&#x20AC;&#x2122; micro hardness test we find the micro hardness number increases with load. Further the value of the work hardening coefficient is found to be 0.672. From this result we conclude that the crystal is soft. 6. From the UV visible spectrum we find that the crystal is transparent in the range 900-2500nm without any absorption peak. 7. The NLO test using Nd-YAG laser confirms that the crystal has NLO property.

Fig.-1: Powder XRD pattern of Alanine sodium chloride (ASC) REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Ambujam, K., Selvakumar, S., Prem Anand, D., Mohamed, G., and Sagayaraj, P., Cryst.Res.Technol.,41(7) (2006) 671. http://www.chemie.fu-berlin.de/chemistry/bio/aminoacid/ alanin_en.html Jag Mohan, 1992,Vol.II, Organic Chemistry,Himalaya publishing House. First Edition. Janarthanan, S., Kishore Kumar, T., Pandi, S. and Prem Anand, D., Indian Journal of Pure & Applied Physics, 47(May 2009)332. Milton Boaz, B., Samuel Selvaraj, R., Senthil Kumar, K. and Jerome, S., Das, Indian J. Phys. ,83(12) (2009) 1647. Onitsch, E.M., Mikroskopie, 95(1998)12. Palaniswamy, S. and Balasundaram, O.N., Rasayan J.Chem., 1(4) (2008), 782. Palaniswamy, S., Balasundaram, O.N., Rasayan J.Chem., 2(2) (2009), 386. 42

D.Prabha and S.Palaniswamy

Vol.1, No.1, 40-46 (2010) 9. Palaniswamy, S., Balasundaram, O.N., Rasayan J.Chem. , 2(1) (2009), 28. 10. Razzetti, C. , Ardoino, M. , Zanotti, L. , Zha, M. , Paorici, C. , Cryst. Res. Technol., 5(2002)456. 11. Vimalan, M., Ramanand, A. and Sagayaraj, P., Cryst. Res. Technol. , 42(11) (2007)1091.

Fig.-2: FTIR sopectrum of Alanine sodium chloride (ASC)

Fig.-3A: Variation of micro hardness number with load

D.Prabha and S.Palaniswamy

Vol.1, No.1, 40-46 (2010)

Fig.-3B: Variation of log(D) with log(P)

Fig.-4A: UV-Visible spectrum of Alanine sodium chloride (ASC)

D.Prabha and S.Palaniswamy

Vol.1, No.1, 40-46 (2010)

Fig.-4B: UV-Visible spectrum of Alanine sodium chloride (ASC)

Fig.-5A: SEM Photograph of Alanine sodium chloride (ASC)

D.Prabha and S.Palaniswamy

Vol.1, No.1, 40-46 (2010)

Fig.-5B: SEM Photograph of Alanine sodium chloride (ASC) Table-1: Charcteristic Absorption frequencies of various functional groups S. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Frequency Range 3436 3085 2929 2601 2112 1620 1591 1506 1455 1412 1361 1306 1235 1151 1113 1013 918 849 772 648 539 486

Intensity s m w (broad) w (broad) w s s w m w w w variable s m variable m (broad) w s s w w

Mode of vibration N-H stretching C-H asym.stretching OH stretching OH stretching Câ&#x2030;ĄO stretching C=O stretching C=O stretching N-H in plane bending C-H in plane bending C-H bending (in-Plane) C-H bending (in-Plane) C-H bending (in-Plane) Asym.C-O=C stretching Asym.C-O=C stretching Sym. C-O=C stretching C-CHO stretching OH bending (out of plane bending) C-H out of plane bending C-H out of plane bending C-H bending (out of plane) S-S stretching S-S stretching

D.Prabha and S.Palaniswamy

International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.1, 47-53 May-August, 2010

Effect of Dimethyl Sulphoxide on the Conductance and Solvation Behaviour of Pyridinium Dichromate in Water V. Radhika1 ,N. Srinivas and P. Manikyamba* Department of Chemistry, Kakatiya University, Warangal 506009. E-mail: mani_prerepa@yahoo.co.in Article history: Received: 17 June 2010 Accepted: 23 July 2010

ABSTRACT Conductance of pyridinium dichromate has been measured in dimethyl sulphoxide-water mixtures of different compositions in the temperature range 283-313K. The limiting molar conductance, Λo and the association constant of the ion pair, KA have been computed using Shedlovsky equation. Λo increases with increase in the proportion of water in the solvent mixture. KA values increases with increase in temperature. The effective ionic radii (ri) of (C5H5NH+)2 Cr2O7-2 have been determined from Λio values using Gill’s modification of the Stokes law. Walden product and thermodynamic parameters are also reported. The results of the study have been interpreted in terms of ion–solvent interactions and solvent properties. Keywords: Association constant, ion-pair, Walden product, Thermodynamic parameter, ion-solvent © 2010 ijCEPr. All rights reserved

INTRODUCTION Literature survey on behavior of different electrolytes in mixed solvent systems indicates that their conductance is influenced by number of factors like density, viscosity, dielectric constant of the medium, ion-solvent interactions and solvent-solvent interactions. Ion-solvent interactions stabilize the ion by solvating it. Such studies not only give an idea about ion-solvent and solvent-solvent interactions but also the preferential solvation of an ion. Though literature is replete with such type of information[6-9, 12-13,15-18,20-22,24], similar information on pyridinium dichromate is not available. Recently pyridinium dichromate has emerged as a very useful and versatile oxidant[1,3,11,14]. This is a stable oxidant which was prepared and analyzed by Corey E J and Schmidt[2].In the present communication the authors report their observations on the conductance behaviour of pyridinium dichromate in aqueous mixtures of dimethyl sulphoxide.

MATERIALS AND METHODS Deionised water was distilled and used. Dimethyl sulphoxide (s.d. fine-chem) was used as such. Pyridinium dichromate was prepared as reported in the literature[2]. A stock solution of this reagent was prepared by dissolving a known weight of the sample in water and standardized by iodometric method. A conductivity bridge (model M.180,ELICO) equipped with a glass conductivity cell of cell constant 1.103 cm-1 with smooth electrodes was used to measure the conductance of the solution. The conductance measurements were reproducible within ±5%. The conductivity values of the solvent systems used in the present study were less than 5 µS cm-1. The temperature was maintained constant using a thermostat (INSREF make) with an accuracy of ±0.20C. To maintain the temperature below room temperature an ice bath equipped with a stirrer was used. At each temperature the solution of pyridinium dichromate and the solvent mixture were thermally equilibrated. Conductance values of pyridinium dichromate at different concentrations were measured by diluting this solution using the thermally equilibrated solvent. The same procedure was followed at different temperatures in the range 283-313K. After making the solvent corrections the molar conductance values, Λm of pyridinium dichromate were evaluated at different compositions of dimethyl sulphoxide-water mixtures in the range 0-100% (v/v) of dimethyl sulphoxide. The molar conductance (Λm) values determined are analysed using Kraus-Bray equation [10]Eqn.(1) and Shedlovsky equation[10] Eqn.( 2).

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Vol.1, No.1, 47-53 (2010)

Λ C 1 1 = m 2 + 0 Λ m K c Λ0 Λ

(1)

S f ±2 K A C Λ m 1 1 = + 0 2 S Λm Λ Λ0

(2)

Λm is molar conductance at concentration C, Λo is the limiting molar conductance KA is the association constant of the ion pair, KC is the dissociation constant, f± is the mean ionic activity coefficient, S is a factor given by

 β CΛ β 2 CΛ   S =  + 1+ 03   4Λ0 3 / 2 4Λ  

 − 1.8246 × 10 6 (Cα )1 / 2 /(εT ) 3 / 2  log f ± =  8 1/ 2 1/ 2  1 + 50.24 × 10 R (Cα ) (εT )  SΛ α= 0 Λ 8.20 × 10 5 Λ0 82.5 + β = 3/ 2 (ε T ) η (ε T )1 / 2

(3) 2

(4)

(5) (6)

where R is ion-size parameter which is equal to the Bjerrum's critical distance q given by

R=q=

e2 2ε kT

(7)

k is the Boltzmann's constant and T is the temperature in degrees kelvin. S is calculated using Λo obtained from the Onsager model using the plot of Λm against √C. The least square analysis of the data (Λm and C) using the above two equations (1,2) is satisfactory with linear correlation coefficients in the range 0.92-0.97.Dichromate ion, Cr2O72- in the aqueous solution in the concentration range used in the present study exists in the monomeric form[23] as CrO42and ionizes as HCrO-4. Therefore the conductivity equations applicable to 1:1 electrolytes are used.

RESULTS AND DISCUSSIONS The limiting molar conductance values Λo thus obtained using both Kraus-Bray and Shedlovsky equations are presented in Table 1. The Λo values obtained from these two models are in good agreement (within 0-3% variation). These Λo values at each temperature, depend on the composition of the solvent system. Addition of dimethyl sulphoxide to water decreases the Λo values. This may be due to the change in the solvent molecular size and their viscosity of the medium and ion-solvent interactions. A decrease in mobility of the ion due to increases in solvation is also expected. Increase in the proportion of dimethyl sulphoxide in the solvent system increases the viscosity of the medium thus the mobilities of the ions decrease. The Λo values increase with increase in temperature. This variation may be due to increase in the mobility of the ions with increase in temperature. It is supposed that this variation has to follow Arrhenius relation, i.e Λ0=Ae-Ea/RT where A is a constant, Ea is activation energy of the conducting process, R is gas constant and T is the temperature on absolute scale. Ea values obtained from the slopes of the linear plot obtained when log Λ0 is plotted against 1/T. are presented in Table 2. Ea values are lower in pure water than in other solvent systems i.e aq DMSO. In solution the ions are in equilibrium with the ion-pairs. From the slopes of the linear least square analysis of the conductance data using Kraus-Bray and Shedlovsky models, the dissociation constant KC and the association constant KA of the ion - pair have been evaluated and presented in Table 3. These values increase with temperature. In general the association constant KA or dissociation constant KC depend on the viscosity, dielectric constant and temperature of the medium. At any given temperature the association constant, KA values are higher in binary solvent mixtures than in pure solvents. Change in enthalpy (∆H) determined from the slopes of the linear plots of log KA against T-1 are presented in Table 3.The free energy change accompanied by the ion-pair formation (∆GAo) is computed using the

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Vol.1, No.1, 47-53 (2010) relation ∆GAo = -RT lnKA. These values calculated at all temperatures are tabulated in Table 4. The differential free energy change in different solvents is computed , using the equations, ∆Gto = -RT ln(wKA/sKA) (8) and ∆Gto = ∆Go (s) - ∆Go (w) (9) Where wKA, sKA are association constant of the ion-pair in water and in solvent mixture respectively . ∆Gto is the free energy change accompanied by the addition of different amounts of dimethyl sulphoxide to water. The magnitude of ∆Gto depends on the relative magnitude of stabilization of the ion-pair and are presented in Table 4. Change in entropy ∆SAo are presented in Table 5 . These are all negative and are in the range 0.072-0.154. As suggested by Hammamy and coworkers[4] the ionic conductance

Λ0+ of the anion HCrO-4 is calculated from the

intercept ( Λ + η0) of the straight line obtained when the Walden product (Λo η0)of different salts with common anion 0

is correlated with reciprocal of the molecular weights of the salts. Using this Λ − the Λ + of the pyridinium ion is calculated applying Kohlrausch’s law. These ionic conductances computed in all the solvent systems used at 293K 0

and 303K are presented in Table 6. Λ − attains minimum value in 20%(v/v) DMSO-water mixture, while Λ + gradually increases due to the addition of water to DMSO and attains a maximum value at this composition. This variation in the ionic conductances suggests that the specific solvation of the anion is by water while DMSO from the solvent mixture solvates the cation. The effective ionic radii( ri) of the cation and anion in each solvent system used are calculated by using Stoke’s radius equation modified by Gill[5]0

ri =

0.820 Z

Λ + ηo

+ 0.0103∈ + ry

(8)

where ry is a parameter equal to 0.85 A for non associated solvents and 1.13 A for associated solvents. These values are tabulated in Table 6. These values which are radii of solvated ions vary with the solvent composition suggesting the operation of ion-solvent interactions. Λo, the limiting molar conductance values recorded in Table 1 indicate that these values decrease continuously due to the addition of DMSO to water. The Walden product Λ0η0, which is the limiting molar conductance of the electrolyte and viscosity of the solvent is calculated in each solvent system and presented in Table 7. At a given temperature this is expected to be constant if the sum of the effective radii of ions is same in all the solvent systems used. The variation in the Walden product as a function of the solvent compositionis generally regarded as an index of specific ion-solvent interaction including structural effects. In the present study the variable Walden product observed may be interpreted as due to variable ion-solvent interactions as the solvent composition is changed. The correlation of log Λ0 against 1/ε according to the equation 1

log Λ0 = log Λ0 −

Z AZ Be2 ∈ d AB k B T

(9)

resulted in a linear plot, from the slope of which dAB the distance between the centres of the two ions in the ion-pair 0

is calculated. This value is 11.32 and 11.77 A at 293 and 303K respectively which is higher than the sum of the ionic radii. This suggests the formation of a solvent separated ion-pair (SSIP). The solvation number is calculated using the relation

Sn =

d AB − (r+ + r− ) rsolvent

(10)

in each solvent, and are recorded in Table 8. These values increase as the proportion of water in the solvent mixture increases. This observation indicates that the ions are selectively sol vated with the water molecules. The change in the free energy accompanied by the solvation process of the ion ∆Gi-s is calculated using Born[19] equation at each composition of the solvent mixture at 293K and 303K . These values are shown in Table 9. These are all negative and change due to change in composition of the solvent mixture. ∆Gi-s is a measure of the stability of

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Vol.1, No.1, 47-53 (2010) the solvated system and larger the negative value higher will be its stability. The ∆Gi-s+ and ∆Gi-s- computed in the present system suggests that the solvated species is more stable in 100% DMSO system at which ion-solvent interactions are stronger than at other compositions.

CONCLUSIONS The presence of molecular interaction is further supported by the solvation number. Solvation is higher in water system compair to other organic solvent systems. Due to high polarity of water, solvent-solute interactions take place to greater extent there by increasing the solvation number at lower concentration of solute. Solvation number of PDC in water-dimethyl sulphoxide is better then compare to solvation number in BDC (Benzimidazolium dichromate)[15] in our earlier work. More polarity more-solvation number. The decrease in Solvation number might be due to disruption of solvent structure. The resultant values of Solvation number decided on the basis of dipole-dipole interactions.

ACKNOWLEDGEMETS The authors are thankful to the authorities of kakatiya University, Warangal for laboratory and instrumentation facilities respectively.

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

Coates, W. M. and Corrigan, Chem.and Ind., (1969),1594. Corey, E .J. , Schmidt, G., Tetrahedron Letters, 20 (1979) 399. Czerncki, S., Georgoulis, C., Stevens, C.L. and Vijayakumaran, Tetrahedron Letters, 26(1985),1699 El-Hammamy, N. H. , Amira, M F. , Abou El Enien, S. A. , El Halim, F. M. , Indian Journal Chem, 23(1984), 43. Gill, D. S. , Kumari , N., Chauhan, M. S. , Journal of Chem Soc Faraday Trans 1, 81(1985),687. Gill, D.S. , Rana, D., Gupta, R. , Journal of Thermochimica Acta ,478(2008)1. Gupta, R. , Chandra, A. , J. Chem. Phys,127 (2007)2043. Ishwara Bhat, J. , Sreelatha, T. N. , Indian Journal Chem., 45A (2006) 1165. Jalalif, A., Shaeghi Rad, Journal of the Iranian Society, 5(2) (2009)309. John, O. M., Bockris , Reddy, A. K. N. , Modern Electro Chemistry, 1970, Plenum, New York. Kabilan, S., et. al, J.Chem.Soc. Perkin Transactions, 2(2002) 1151. Kondo, K. , The Journal of Physical Chemistry B, 113 (35) ( 2009) 11988. Morteza, Jabbari, Farrokh, Gharib, Journal of Acta Chim. Slov, 57 (2010) 325. Parish, E. J. and Wei, T. Y., Synthetic Communications,17(10)(1987),1227. Radhika, V. , Manikyamba, P. , Indian Journal Chem, 47A(2008)1814. Radhika, V. , Manikyamba, P., , J Chem Eng Data, 53(12) (2008) 2766. Radhika, V. , Manikyamba, P., Proc. Nat. Acad. Sci.India , 79A,Pt.II(2009) 167. Rezaei Behbehani, G. , Waghorne, W. E. , Journal of Thermochemica Acta, 478(2008)1. Robinson , R. A., Stokes., R. H., 1959. Electrolyte Solutions, Butter worth’s Scientific, London. Sanjib, B. A. Maitra, Journal of Molecular Liquids, 137 (2008) 131. Senem, A. , Ksel Altun, Y., Nurullah, A. , Alsancak, G. L. , Beltran, L. , J. Chem. Eng. Data, 54 (11) (2009)3014. Victor, P. J. , Journal of Chemical & Engineering Data, 54 (10) (2008) 2902. Wiberg, K. B. Oxidations in Organic Chemistry ,1965. AP, New York. Yang, L. J. , Qing, X. , Yang, Huang, K. M. , Jia, G. Z. , Shang, H. , IInt. J. Mol. Sci., 10(3) (2009) 1261.

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Vol.1, No.1, 47-53 (2010)

Table-1: Limiting molar conductance ( Λo ) values in S cm2 mol-1 of pyridinium dichromate in aqueous mixtures of dimethyl sulphoxide at different temperatures. Dimethyl sulphoxide (%v/v) →

20%

T (K) ↓ 1 283 205.55 293 221.58 303 253.42 313 303.49 1 = Kraus-Bray Model

2 205.67 222.80 259.61 307.27

1 2 131.46 130.81 142.61 141.78 185.49 186.86 202.84 199.47 2 = Shedlovsky model

40%

1 78.05 100.15 129.10 154.85

60%

2 78.21 100.54 128.84 155.49

1 54.43 63.94 84.60 99.74

80%

2 53.75 62.81 85.86 103.85

1 39.38 45.09 59.92 65.97

100%

2 39.34 45.36 60.85 66.92

1 42.97 52.46 69.056 71.122

2 42.85 51.50 68.06 81.86

Table-2: Computed Values of Ea and ∆HA ( kJ mol-1) for pyridinium dichromate under varying compositions(v/v) of aqueous dimethyl sulphoxide mixtures .

Ea ∆HA

0%dimethyl sulphoxide 9.59 -31.43

20%dimethyl sulphoxide 11.40 -16.82

40% dimethyl sulphoxide 17.14 -18.72

60% dimethyl sulphoxide 15.75 -9.56

80% dimethyl sulphoxide 13.65 -46.26

100% dimethyl sulphoxide 11.31 -48.36

Table-3: KA and KC values of pyridinium dichromate in aqueous dimethyl sulphoxide mixtures 0% dimethyl 20% dimethyl sulphoxide sulphoxide KC KA KC KA 283 7.58 0.133 10.4 0.10 293 15.20 0.21 42.2 0.23 303 37.65 0.28 59.2 0.17 313 37.65 0.27 60.55 0.24 KA = Association constant from Shedlovsky equation T (K)

40% dimethyl 60% dimethyl 80% dimethyl sulphoxide sulphoxide sulphoxide KA KC KA KC KA KC 32.02 0.21 22.35 0.23 33.39 0.30 37.37 0.17 33.98 0.26 35.75 0.31 40.53 0.19 40.71 0.25 50.74 0.33 53.3 0.20 44.88 0.31 56.81 0.35 KC = Dissociation constant from Kraus-Bray equation

100% dimethyl sulphoxide KA KC 10.63 0.25 11.16 0.18 20.59 0.20 30.88 0.32

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Vol.1, No.1, 47-53 (2010)

Table-4: Computed change in free energy (∆Ga) and transfer(∆Gt) for pyridinium dichromate in aqueous dimethyl sulphoxide mixtures at all temperatures in kJ mol-1

T (K) 283 293 303 313

0% dimethyl sulphoxide ∆Ga ∆Gt -4.760 -4.032 -9.151 -9.513 -

20% dimethyl sulphoxide ∆Ga ∆Gt -5.52 -0.758 -9.13 -5.10 -10.25 -1.10 -9.63 -0.12

40% dimethyl sulphoxide ∆Ga ∆Gt -8.17 -2.65 -8.59 -4.56 -8.57 +0.58 -6.70 +2.81

60% dimethyl sulphoxide ∆Ga ∆Gt -8.24 -2.54 -8.51 -3.27 -9.33 -0.18 -9.21 +0.30

80% dimethyl sulphoxide ∆Ga ∆Gt -8.24 -3.48 -8.51 -4.48 -9.90 -0.75 -8.91 +0.60

100% dimethyl sulphoxide ∆Ga ∆Gt -5.57 -0.81 -5.88 -1.85 -5.91 +3.24 -8.91 +0.60

Table-5: Computed change in entropy (∆Sa) and transfer(∆St) for pyridinium dichromate in aqueous dimethyl sulphoxide mixtures at all temperatures in kJ mol-1

T (K) 283 293 303 313

0% dimethyl sulphoxide ∆Sa ∆St -0.154 -0.151 -0.129 -0.124 -

20% dimethyl sulphoxide ∆Sa ∆St -0.144 -0.168 -0.127 -0.159 -0.119 -0.170 -0.117 -0.156

40% dimethyl sulphoxide ∆Sa ∆St -0.005 -0.151 -0.003 -0.143 -0.003 -0.150 -0.009 -0.150

60% dimethyl sulphoxide ∆Sa ∆St -0.041 -0.025 -0.033 -0.022 -0.031 -0.031 -0.030 -0.032

80% dimethyl sulphoxide ∆Sa ∆St -0.030 -0.057 -0.028 -0.048 -0.023 -0.064 -0.025 -0.069

100% dimethyl sulphoxide ∆Sa ∆St -0.091 -0.570 -0.087 -0.400 -0.084 -0.052 -0.072 -0.053

Table-6: Ionic conductances and stoke’s radius of solvated ions of pyridinium dichromate in aqueous dimethyl sulphoxide mixtures at 293 and at 303K.

At 293K %DMSO.(%v/v) 0% 20% 40% 60% 80% 100%

Λ0+ (S cm2mol-1)

Λ0− (S cm2mol-1)

r+( A )

r-( A )

r++ r-( A )

174.35 114.90 61.32 23.70 23.27 10.37

48.54 26.88 39.22 40.29 21.0 31.63

2.42 2.31 2.09 4.66 4.61 5.37

3.60 4.55 3.50 3.35 1.58 2.66

6.02 6.86 5.59 8.01 6.19 8.03

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Vol.1, No.1, 47-53 (2010)

At 303K %DMSO(%v/v) 0% 20% 40% 60% 80% 100%

Λ0+ (S cm2mol-1) 169.15 107.56 50.11 35.64 20.84 19.8

Λ0− (S cm2mol-1)

r+( A )

84.27 77.93 78.10 49.03 39.09 29.26

2.50 2.14 3.12 3.69 5.12 5.14

r-( A ) 3.05 4.56 2.55 3.09 3.39 3.91

r+ + r-( A ) 5.55 6.7 5.67 6.78 8.51 9.05

Table-7: Walden product(Λ0η0),S cm2 m-1 poise of the conducting molecular species of pyridinium dichromate in aqueous dimethyl sulphoxide mixtures at 293,303K. T (K) 293 303

0%dimethyl sulphoxide 1.44 1.24

20% dimethyl sulphoxide 1.34 1.47

40% dimethyl sulphoxide 1.11 1.33

60% dimethyl sulphoxide 0.85 1.08

80% dimethyl sulphoxide 0.55 0.62

100% dimethyl sulphoxide 0.85 0.90

Table-8: Solvation number values for ion pair formation of pyridinium dichromate in aqueous dimethyl sulphoxide mixtures at 293,303K. T (K) 293 303

0%dimethyl sulphoxide 4.08 3.22

20% dimethyl sulphoxide 2.05 2.49

40% dimethyl sulphoxide 2.48 2.64

60% dimethyl sulphoxide 1.41 1.92

80% dimethyl sulphoxide 1.97 1.25

100% dimethyl sulphoxide 1.08 0.89

Table-9: Computed change in free energy of solvation (∆Gi-s+) and (∆Gi-s-) for pyridinium dichromate in aqueous dimethyl sulphoxide mixtures at 293,303K in kJ mol-1. 0% dimethyl 20% dimethyl 40% dimethyl 60% dimethyl 80% dimethyl 100% dimethyl sulphoxide sulphoxide sulphoxide sulphoxide sulphoxide sulphoxide T (K) ∆Gi-s+ ∆Gi-s∆Gi-s+ ∆Gi-s∆Gi-s+ ∆Gi-s∆Gi-s+ ∆Gi-s∆Gi-s+ ∆Gi-s∆Gi-s+ ∆Gi-s293 1.64 3.97 2.61 5.67 5.41 5.05 6.28 5.13 6.46 2.09 12.45 8.24 303 1.60 2.70 2.10 5.44 4.43 3.48 5.27 4.58 6.48 5.23 6.80 6.06

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International Journal of Chemical, Environmental and Pharmaceutical Research

Vol. 1, No.1, 54-60 May-August, 2010

L-Alanine Sodium Nitrate (ASN), NLO Material: Growth and Characterization D.Prabha and S.Palaniswamy* Department of Physics, PSG College of Arts and Science, Coimbatore-641 014, India. *E-mail: drprabha76@rediffmail.com Article history: Received: 22 August 2010 Accepted: 2 September 2010

ABSTRACT Single crystal of alanine sodium nitrate (ASN), a nonlinear optical material has been grown from solution by slow evaporation method. The isoelectric point of the alanine is 6(1). So, the growth of crystals has been carried out at pH 6. The grown crystals have been subjected to powder X-ray diffraction studies to identify the crystalline nature. Single crystal X-ray diffractometer was utilized to measure the cell parameters and morphology of the grown crystals. The FTIR spectra taken for the crystals grown at pH values show the peak intensity. The mechanical properties of the grown crystals are studied using Vickers micro hardness measurement. Surface morphology was studied by SEM analysis. Using Nd-YAG laser the NLO property of the crystal is studied. The transmittance and absorption of the crystal was studied by UV-Visible spectrometer. Keywords: Characterization, X-ray diffraction, Slow evaporation, SEM analysis, Vickers micro hardness tester. © 2010 ijCEPr. All rights reserved

INTRODUCTION Non-linear optics is a very useful technology because it extends the usefulness of lasers by increasing the number of wavelengths available both longer and shorter than the original can be produced by non-linear optics. Some materials change light passing through them, depending upon orientation, temperature, light wavelength etc.(red light, lower wavelength) releasing one photon of accumulated higher energy (blue and green light higher wavelength). The important nonlinear optical materials from the device point of view are generally in the form of single crystals and must need a wide variety of ancillary materials requirement for optical use. In general, they will require extra ordinary stability with regard to room temperature and high intensity light source.

MATERIALS AND METHODS SYNTHESIS AND CRYSTAL GROWTH The solution was prepared using 4.995gms of alanine and 4.765gms of sodium nitrate dissolved in 30ml of distilled water. The pH value is low, so to increase the pH value. The pH of the solution is adjusted to 6 by adding 10 drops of NAOH [5].The above solution is filtered in the filter paper and transferred to a petri dish. The petri dish is covered with a filter paper with small hole, tied on top with rubber band to facilitate evaporation and crystal growth. STRUCTURE OF L-ALANINE MOLECULE The α-carbon atom of L-alanine is bound with a methyl group (-CH3), making it one of the simplest α-amino acids with respect to molecular structure and also resulting in L-alanine being classified as an aliphatic amino acid. The methyl group of L-alanine is non-reactive and is thus almost never directly involved in protein function. The structure of L-alanine is

L-alanine Further its linear zwitter ionic structure is

D.Prabha and S.Palaniswamy

Vol.1, No.1, 54-60 (2010)

The crystal structure of L-alanine is orthorhombic [8]. Its cell parameters are a = 6.032 A o, b= 12.343 A o, c = 5.784 A o. α=β=γ=90o CHARACTERIZATION Powder XRD(Fig.1) The powder XRD of alanine sodium nitrate (ASN) is shown in the fig. The peaks in the fig show the crystalline nature of ASN. Further peaks are indexed [6]. The grown crystals have been characterized by powder X-ray diffractometer. The lattice parameter values of alanine sodium nitrate taken from the values area=10.83439 A o, b=15.39174 A o, c=6.19978 A o α=90.7995 o β =94.7954 o γ =76.8417 o Cell volume V=1003.19 A o3. The crystal crystal system is Triclinic. FT-IR Analysis (Fig.2) The FTIR spectrum of alanine sodium nitrate (ASN) was recorded using FTIR spectrometer in the region 2500500cm-1 by KBr technique to confirm the presence of different organic groups along with the inorganic materials presence in the table[1]. The region 3448cm-1 with strong intensity represents N-H stretching. 3212cm-1 with medium intensity refers N-H stretching. The region 3066cm-1 with weak (broad) represents OH stretching.2058cm-1 with medium intensity refers overtones & combination bands with prominent peaks near 2500 and 2000cm-1.The peak 1637cm-1 with strong band represents C=O stretching.829cm-1 with weak intensity refers C-H out of plane bending[3]. Micro Hardness(Fig.3) ASN crystal was subjected to Vickers micro hardness test with the load varying from 25 to 100g [1]. Hardness number of the crystal is calculated using the relationHv = 1.8544 P/d 2 Kg/mm 2 Vickers micro hardness profile as a function of the applied test loads is illustrated by fig. It is evident from the plot that the micro hardness of the crystal increases with increasing the load. The value of the work hardening coefficient n was estimated from the plot of log p versus log d drawn by the least square fit method. It is observed that the Vickers hardness number of the crystal increases with increasing the load [4]. The value of the work hardening coefficient n was found to be 0.08. According to Onitsch,1.0 ≤ n ≤ 0.08 for hard materials and n > 0.08 for soft materials [7]. Hence, it is concluded that ASN belongs to the soft materials. UV-Visible Spectrometer Analysis(Fig.4) The optical absorption spectra of alanine potassium chloride crystals (ASN) were recorded in the range 190-2500nm using JASCO corp V-570 spectrometer [6]. The UV-Visible transmission spectrum of alanine sodium nitrate crystal is shown in the Figure.. Crystal surface analysis by SEM(Fig.5) Surface analysis of alanine potassium chloride is carried out through JSM 6360 JEOL/EO make. The surface of the crystal was coated with a thin of carbon to make the sample conducting. From the figure, the following observations are evident: 1. At a magnification of 330 and at a scale of 50 micro meter we observe the crystals have smoothed needle surfaces. 2. At a magnification of 1500 and 1 micro meter scale can observe that the crystals have an average thickness of 421nm [7]. 55

D.Prabha and S.Palaniswamy

Vol.1, No.1, 54-60 (2010)

Fig.-1: Powder XRD pattern of Alanine Sodium Nitrate

Fig.-2:FTIR Spectrum of Alanine Sodium Nitrate

RESULTS AND DISCUSSION From the above experimental and characterization of alanine sodium nitrate crystal the following results and the discussion are significant. 1. Single crystals of ASN are successfully grown at a pH of 6. The grown crystals are larger in size having an average size of 1.5cmx11.5cm. 56

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Vol.1, No.1, 54-60 (2010)

2. The grown crystals are characterized by using powder X-ray diffraction. 3. From this we confirm the structure of the crystal to be triclinic and the cell parameters: a=10.83439 A o

6. 7.

A S N

7 0

MICROHARDNESS NUMBER

6 0

5 0

4 0

3 0

2 0 2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

1 1 0

L O A D

Fig.3A: variation of Micro hardness number with Load

A S N

-0 .1 2

-0 .1 4

-0 .1 6

Log(d)

b=15.39174 A o c=6.19978 A o From the FTIR spectrum we can confirm the structure of the ASN to have both the alanine and soiumnitrate molecules. These are arranged in alternate layers in the crystal. This is evident from the non damage of alanine structure. From the Vickersâ&#x20AC;&#x2122; micro hardness test we find the micro hardness number increases with increasing the load. Further the value of the work hardening coefficient is found to be 0.08. From this result we conclude that the crystal is soft. From the UV-Visible spectrum we find the crystal is transparent in the range 900-2500nm. From the SEM analysis we conclude that the crystal formation inmicro range is 421nm. Further in the micro level the crystal surface is very smooth which shows that it can add more molecules to into a large crystal.

-0 .1 8

-0 .2 0

-0 .2 2

1 .3

1 .4

1 .5

1 .6

1 .7

1 .8

1 .9

2 .0

L o g (P )

Fig.-3B: variation of log(D) with log(P)

D.Prabha and S.Palaniswamy

Vol.1, No.1, 54-60 (2010)

Fig.-4A: UV-Visible spectra of Alanine Sodium Nitrate

Fig.-4B: UV-Visible spectra of Alanine Sodium Nitrate

D.Prabha and S.Palaniswamy

Vol.1, No.1, 54-60 (2010)

Fig.-5A: SEM photograph of Alanine Sodium Nitrate

Fig.-5B: SEM photograph of Alanine Sodium Nitrate

REFERENCE 1. 2. 3.

Ambujam, K., Selvakumar, S., Prem Anand, D., Mohamed, G. and Sagayaraj, P., Cryst. Res. Technol., 41(7) (2006) 671 http://www.chemie.fu-berlin.de/chemistry/bio/aminoacid/ alanin_en.html Jag Mohan, 1992, Vol.II, Organic Chemistry, First Edition Himalaya publishing House. 59

D.Prabha and S.Palaniswamy

Vol.1, No.1, 54-60 (2010) 4. 5. 6. 7. 8. 9.

Onitsch, E.M., Mikroskopie, 95(1998)12 Palaniswamy, S. and Balasundaram, O.N., Rasayan J. Chem. ,1(4) (2008), 782. Palaniswamy, S. and Balasundaram, O.N., Rasayan J. Chem. ,2(1) (2009) 28. Palaniswamy, S. and Balasundaram, O.N., Rasayan J. Chem. ,2(2) (2009) 386 Razzetti, C. , Ardoino, M. , Zanotti, L. , Zha, M. , Paorici, C. , Cryst. Res. Technol, 37( 2002)456. Vimalan, M., Ramanand,. A. and Sagayaraj, P. , Cryst. Res. Technol. ,42(11) (2007)1091

Table-1: Characteristic Absorption Frequencies of Various Functional Groups S. No 1 2 3 4 5 6 7

Frequency Range 3448 3212 3066 2789 2606 2514 2058

Intensity

Mode of vibration

s m w (broad) w (broad) w (broad) w (broad) m

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1764 1637 1523 1384 1295 1216 1125 1102 1046 1004 922 844 829 721 677 531

s s s s s s variable variable variable variable w w w m m s

N-H stretching N-H stretching OH stretching OH stretching OH stretching OH stretching Overtones & combination bands with prominent peaks near 2500 and 2000 cm-1 C=O stretching C=O stretching N-H in plane bending CO-2 sym. stretching C-O stretching C-O stretching C-CHO stretching C-CHO stretching C-CHO stretching C-CHO stretching C-H out of plane bending C-H out of plane bending C-H out of plane bending N-H out of plane bending OCN deformation OCN deformation

Table-2: Crystalographic data of ASN Crystal ALANINE SODIUM NITRATE

CRYSTAL DATA a=10.83439 A o b=15.39174 A o c=6.19978 A o α=90.7995 o β =94.7954 o γ =76.8417 o

Lattice parameters

Cell volume V=1003.19 A o 3.

Cell Volume (V)

D.Prabha and S.Palaniswamy

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