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Micro Nutrient status, Analysis of Fatty acid Composition and

Effect of Salinity on plant by Rapeseed mustard

DISSERTATION

SUBMITTED TO

LORDS INTERNATIONAL COLLEGE CHIKANI(ALWAR)RAJASTHAN UNIVERSITY(JAIPUR)

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

BACHLOR OF SCIENCE IN BIOTECHNOLOGY

2009-10

SUBMITTED BY UNDER THE SUPERVISION OFJAIPRAKASH SAINI Dr. N.S.BHOGALEnrollment No. 05/121540 Senior Scientist (soil science)

Work done at
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Tel: 05644-260495, 260379 (O), 260381 (R)Fax: 05644- 260565

Email: [email protected][email protected]

Website : http://drmr.ernet.in

Dr. N.S.BhogalSenior Scientist (soil science) DATE: 2dec. 2009

Certificate

It is to certify that the thesis entitled micronutrient status ,analysis of fatty acid Composition

and Effect of Salinity on Plant by Rapeseed mustard is an original piece of done by Mr.Jaiprakash

Saini, s/o Jaiprakash Saini under my supervision for partial fulfillment of the requirement for the degree

of Bachelor of Science in Biotechnology to the Institute of lords international collage

alwar,University of rajasthan,Japur. I further certify that:

It embodies the original work of candidate himself and no part of work has been submitted for

any other degree or diploma.

It is up to the required standard both the respect of its contents and literary presentation for being

referred to the examiners.The candidate has worked under my supervision for the required period at Directorate of

Rapeseed-Mustard Research Sewer, Bharatpur (Raj.), India . The assistance and help received

during the course of this investigation and sources of literature have been duly acknowledged.

(N.S.BHOGAL)
mailto:[email protected]:[email protected]


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DEPARTMENT OF BITECHNOLOGY

LORDS INTERNATIONAL COLLEGE CHIKANI (ALWAR)

INSTITUTE OF UNIVERSITY OF RAJASTHAN,JAIPUR

INDIA

Certificate

This is to certify that dissertation entitled micronutrient status, analysis of fatty

acid composition and effect of salinity in plant by repeseed mustard has been carried

out at Directorate of Rapeseed-Mustard Research, Sewer, Bharatpur (Raj.) under the

supervision of Dr.N.S.Bhogal, Senior Scientist (soil science), by Mr. Jaiprakash s/o

Prabhudayal student of lords international collage chikani ,alwar(university of

rajasthan ,jaipur) and submitted in partial fulfillment of the requirements for the degree of

Bachlor of Science in Biotechnology. It is further certified that the candidate has put in

the necessary stay in department during the thesis period as per university rules.

. (Teena Agariwal)

Co-Ordinator(External Examiner)


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DECLARATION

Certificate

I hereby declare that the thesis entitled Micronutrient status ,analysis of fatty

acid composition and effect of salinity in plant by rapeseed mustard being submitted

as the partial fulfillment of the requirement for the degree of Bachlor of Science in

Biotechnogy. to the Institute of lords international collage chikani ,alwar(University of

rajasthan.jaipur). Campus is a record of bonafied research carried out by me under the

supervision of Dr. N.S.Bhogal, Senior Scientist (Soil science), Directorate of Rapeseed-

Mustard Research, Sewer, Bharatpur (Raj.) To the best of my knowledge any part or the

whole of thesis has not being submitted elsewhere for the award of any other degree or

any qualification of any university or examining body.

(Jaiprakash Saini)


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AKNOWLEDGEMENTS

First of All I Thanks To God, Whose showers His Kindness and blessings to

Maintain me all cheerful throughout the Course of this thesis work / research

programme.

I acknowledge my deepest sense of debt and gratitude to my esteemed

advisor Dr.N.S.Bhogal Senior Scientist (Soil science), Directorate of Rapeseed-

Mustard Research Sewer, Bharatpur (Raj.) India , under whose valuable supervision

and guidance this work was competed. I have no words to express my heartiest

gratitude for the undoubting support, constant encouragement, painstaking efforts

and motivation provided by him at every stage of my present work and during the

preparation of this manuscript.

Words can hardly acknowledge the help made by Dr. Arvind Kumar, Director

DRMR, Bharatpur, for providing necessary facilities and benevolent patronage.

I also express my great pleasure and deep sense of gratitude to Shri.

H.P.Meena (technical Officer) for his valuable advice and help in analysis.

I feel honored to record my highest regard to my reverend parents and all

the family members for untiring help, love, affection and encouragement without I

would not have reached up to this level of stage in life.

Last but not the least, I express my sincere thanks to all beloved and respected people who helped

me but could not find a separate mention.

PLACE: DRMR, BHARATPUR

DATE:

(JAIPRAKASH SAINI )


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ABOUT DRMR

The Indian Council of Agriculture Research (ICAR) established the Directorate Research on

Rapeseed- Mustard (DRMR) on October 20, 1993 to carry out basic, strategic and applied research

on rapeseed mustard. Besides, generating basic knowledge and material, it also engages indeveloping ecologically sound and economically viable agro production and protection technology.

The All India Coordinate Research Project on Oilseed (AICRPO) was established in April

1967 for the improvement of oil seeds in country. Separate Project Coordinating Units were

established in the fifth plan for further strengthening of the research programmes. The Project

Coordinators (Rapeseed-Mustard) unit was accordingly, established on January 28, 1981 at the

campus of the Haryana Agricultural University, Hissar. The Indian Council of Agriculture

Research (ICAR) established National Research Centre on Rapeseed-Mustard at Sewar, Bharatpur

(Rajasthan) during eighth plan in 1993 on the recommendation of the task force constituted in 1990

to select a suitable site. The centre is located on Agra-Jaipur National highway and is 7 km far from

Bharatpur railway station and 3 km from Rajasthan Roadways bus station. Internationally known

for Keoladeo National Bird Sanctuary, Bharatpur is on Delhi-Bombay main railway track just 35

km ahead of Mathura. Bharatpur is well connected with Jaipur, Delhi and Agra by road. The

campus of the centre is spread over an area of 44.21 hectares of which about 80% is experimental

and rest is covered by residential and Laboratory-cum-Administrative building. It is situated at

77.10 0 E longitude and 27.15 0N latitude and is about 178.37 meters above mean sea.


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DRMR


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Mandate of Institute: National repository for rapeseed-mustard genetic resources.

Basic, strategic and applied research to improve the productivity, quality of oil and seed

meal.

Development of ecologically sound and economically viable agro-production and protection

technologies for different situations.

Generation of location specific, interdisciplinary information based on multi-location testing

and coordination.

Establishment of linkages and promotion of cooperation with national and international

agencies objectives envisaged.

To extent technical expertise and consultancies.


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CONTENT

1.About DRMR2.Micronutrient Statusa. Micronutrient Status by AAS(Atomic

Absorption Specto Photometer)InastrumentPrincipleMethod&Procedure

b.Micronutrient Status by Spectrophotometer PrincipleMethod &Procedure

3.Analysis of fatty acid Composition byGCMS(Gas Chromatography and MassSpectrophotometer 1. Instrument

PrincipleMethod&procedure

3. Effect of salinityNa +k Ratio by flame Photometer InstrumentPrinciple Method&Procedur

4.RESULT

5. REFERENCE


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CHAPTER 1

INTRODUCTION


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IntroductionIndia is largest vegetable oil economics in the world next only to USA and China. In the

agriculture economy of India, Oil seed crops are important next only to food grains in terms of

hectares, production and value. In India, mustard is known both as oil seed as well as spice.

Internationally, however, it is more popular as a spice. In India Indian mustard is the main crop of the

group rapeseed mustard is cultivate in the state of Assam, Bihar, Gujarat, Haryana, Himachal Pradesh,

Jammu and Kashmir, M.P., Maharashtra, North Eastern states, Orissa, Punjab, Rajasthan, Uttar

Pradesh and West Bengal. Indian mustard contributes more than 75 % of the total rapeseed mustard

area. Haryana, Madhya Pradesh, Rajasthan and Uttar Pradesh are the major rapeseed-mustard

production states in India. They are representing over 80% of total hetarage and production as well.

Rajasthan ranked first, contributing around 40% of the total hetarage and production.

India accounts for 7 % global output, 7% of global oil meal production, 6 % of global oil meal

exports, 6 % of global vegetable oil production, 1.4 % of global vegetable oil imports and 10 % global

edible oil. The total market size of the Indian oil seed sector is about Rs. 600 billion. (U.S. $ 13.4

billion). Oil seed cultivation in the countries takes place on about 26 million hectares of land. Oil seedconstitute the 2nd largest agriculture commodity in India after cereals accounting for nearly 5 % of the

Grass National Product (GNP) & 10 % of the value of all agricultural products. These crops hold a

sizable share (14.4 %) of the countries gross cropped area contributes around 5 % of its GNP and

about 10 % of the volume of agricultural. Along the oil seed, a rapeseed mustard group of crops

occupies prominent position in India as well as in the world. Usually India accounts for 20.3 % and

11.6 % of the total acreage and production of rapeseed mustard (USDA 2008). Asia contributes

around 59 % of hectares and 49 % of the world production. India has a premier position for global oil

seed production contributing 9 % to the worlds oilseed. The production has been estimated to be at

6.43 m tones only during 2007 08 from an area of 5.9 m ha. Compared to 6.79 m ha, 7.43 m tones

during 2006-07 (DRMR, 2008).

The genus Brassica consists of over 150 species of annual or biennial herbs several of which are

cultivated as oil seed crops like mustard. Other oil seed crops in genus are toria and rapeseed. There


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are many others, which are cultivated mainly as vegetable like cabbage, turnip etc. There are many

others, which are being grown as fodder.

The normal seed contains materials which it utilizes during process of its germination. These

substances are frequently found in the endosperm. Thus endosperm may contain variety of storedmaterial such as starch, oil and proteins etc. In some plants however the reserve food material is

present in cotyledons. Plants usually yield to any stress conditions and its reaction may be elastic or

plastic. In the formal the reaction is temporary and the plant reverts back to its original states however

in the later state the plants are deformed and the change are not reversible. Seed biotech describe the

generation of seed. Seed is defined as a ripened ovule consisting of embryo and its coats.

Anatomically a seed consists of some old or parental saprophyte tissue viz. the seeds coats which are

derived from the integuments and nucleus. Some endosperm which may be gametophytic tissue or

fertilized tissue and the embryo, the new young saprophytes.

The oil yielding Brassicas that are predominantly cross pollinated, constitute a group about

which considerable confusion exists, regarding their identification and nomenclature. Black mustard is

ground with white mustard for preparing table mustard and also various medicinal preparations, such

as bath mustard, mustard bran and mustard flour. The seeds of black mustard are used in pickles and

curries. In India mustard is mainly cultivated in the semi-arid to arid tropical zones nearly 33% rainfed

where scarcity of water (drought) severely affects crop growth and development, consequently seed

yield. Besides this, the crop in these regions is mainly sown at the time when the plant nutrients andsoil that adversely affects not only the germination of the crop but also the early seedling

establishment.

Lot of work is going on how to make crop tolerant to high concentration minerals and drought

stress. Due to these minerals, crops growth and development were affected and caused toxicity

symptoms therefore they play an important role in environmental factors for realization of optimum

growth and economic produce in arid and semiarid regions.

Here sixteen chemical elements are known to be important to crops growth and survival. These

are divided into two main groups Mineral and Non-Mineral. The Non-Mineral Nutrients are hydrogen

(H), oxygen (O) and carbon (C). These nutrients are found in the air and water. These are used in

photosynthesis. The 13 mineral nutrients, which come from the soil, are dissolved in water and

absorbed through a plants roots. These are divided into two group macronutrients and micronutrients.

Macronutrients are nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg)

and sulfur (S). These major nutrients are present in sufficient amount in the soil but sometimes these


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are lacking from the soil because plants use large amounts for their growth and survival. This is why

many farmers and gardeners use fertilizers to add the nutrients to the soil.

Micronutrients are those elements essential for plant growth which are needed in only very small

quantities. These elements are boron (B), copper (Cu), iron (Fe), chloride (Cl), manganese (Mn),

molybdenum (Mo) and zinc (Zn). These are also called as trace elements.Soils vary widely in their micronutrient content and in their ability to supply micronutrients in

quantities sufficient for optimal crop growth. Soils deficient in their ability to supply micronutrients to

crops are alarmingly widespread across the globe, and this problem is aggravated by the fact that

many modern cultivars of major crops are highly sensitive to low micronutrient levels. Original

geologic substrate and subsequent geochemical and pedogenic regimes determine total levels of

micronutrients in soils. Total levels are rarely indicative of plant availability, however, because

availability depends on soil pH, organic matter content, adsorptive surfaces, and other physical,

chemical, and biological conditions in the rhizosphere.

Micronutrient availability to plants can be measured in direct uptake experiments, or estimated

with techniques that correlate quantities of micronutrients extracted chemically from soils to plant

uptake and response to micronutrient fertilization. Rational management of micronutrient fertility and

toxicity requires an understanding of how total and plant-available soil micronutrients vary across the

land.

Although in the past, several efforts have been made to evaluate Brassica germplasm for

identification of resistant sources against the minerals in soil, the degree of tolerance in identified

genotypes is quite low and thus requires continuous efforts in this regard. The present studies were,

therefore, undertaken to evaluate diverse Brassica germplasm in the field to tap better sources of

minerals tolerant. The study was undertaken with the following objectives:

1. To identify the macro elements and micro elements which are present in the soil.

2. To identify that how much nutrient elements were uptake by the plant and how much

its required.


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ABOUT RAPESEED-MUSTARD

Among the oilseed crops, rapeseed and mustard (mostly Brassica species) are very important.

Family : CruciferaeGenus : Brassica sp.

Local Hindi name is Sarson but it varies from place to place. Most of the edible oils are

obtained from Rapeseed and Mustard. The crops play an important role in oilseed economy in India

and are considered as cash crop.

This group of crops includes rai or raya (B.juncea), toria and Sarson (B.rapa), gobhi Sarson

(B.napus) and taramira or rocket salad (Eruca sativa).


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Rapeseed includes

1. Yellow sarson (Brassica rapa var. Yellow sarson)

2. Brown sarson (Brassica rapa var. Brown sarson)

3. Toria (Brassica compestries var. Toria)
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4. Taramira (Eruca sativa)

5. Gobhi sarson (B. napus)

Mustard also includes

1. Indian mustard or rai (Brassica juncea)

2. Karan rai (Brassica carcinata)

ABOUT INDIAN-MUSTARD


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T his adventives or introduced plant is a summer or winter annual about 1-4' tall, branchingoccasionally in the upper half. Initially, there is a rosette of basal leaves, but during warm weather this

plant has a tendency to bolt and develops flowering stems. These stems are round and hairless. The

alternate leaves are up to 12" long and 4" across. The typical leaf is pinnatifid, tapering gradually to a

stout petiole and becoming broader toward the large terminal lobe. There is a stout central vein along

its length. A few of the upper leaves may be unlobed. These leaves are bluish green (usually),

glabrous, and glaucous, while their margins are undulate or dentate.

The

upper

stems

terminate in narrow racemes of yellow flowers. Each flower is about " across, consisting of 4 yellowpetals, 4 yellowish green sepals, a short green pistil with a knobby stigma, and several stamens with

yellow anthers. The rounded petals are slightly notched at their tips, and have faint veins running

across their length. The pedicel of each flower is about 1/3" long or longer. The blooming period

usually occurs from late spring to mid-summer, but some plants bloom during the late summer or

early fall. Individual plants remain in bloom for about a month. Each flower is replaced by a hairless

silique (i.e., a seedpod) that is cylindrical and held more or less erect. A mature silique is about


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1" long, and has a conspicuous beak at its tip. There are 2 faint lines running along its length. The

small round seeds are arranged in a single row within each silique. The root system consists of a

taproot. This plant spreads by reseeding itself.

This species is the source of many cultivated forms of Indian and Chinese mustard. It resemblesBroccoli or Collards in appearance, but these cultivated plants are apparently different forms of

Brassica olearacea (Wild Cabbage). Indian mustard differs from Wild Cabbage by the absence of

leaves that clasp the central stem. It differs from other Brassica spp. (Mustards) in the wild by the lack

of hairs on the foliage, seedpods, or stems. While some cultivated forms of Indian mustard have leaves

that are incredibly hot and spicy, the wild form of this plant has leaves with a mild flavor. They are

edible and can be used as a potherb. This plant typically grows in full sun under mesic to dry

conditions. It is not fussy about the characteristics of the soil, and can often be found in clay-loam or

gravelly sites. However, fertile soil will produce larger plants. Disease rarely bothers this plant in the

wild, although various insects often chomp holes in the foliage

ECONOMIC IMPORTANCE

The leaves, seed, oil and oil cake are economically useful in one way or other. Rapeseed and

mustard yield the most important edible oil. The oil content seeds of different ranges from 10 to 46

percent. The leaves of young plants are used as green and plants are used as green fodder for cattle.

Oil

(A) Edible oil: The Rapeseed and mustard oil is the most important edible oil in the northern and

eastern India. Oils are used for cooking and for the preparation of various products. The seed and

oil are used as condiment in the preparation of pickles and for flavoring curies and vegetable. The

oil is also used as medicine.


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(B) Industrial uses: The mustard oil is used in the preparation of hair oil, soap and in the

manufacture of mineral oil for lubrication. It is also used in bakery, tea industry and in the

preparation of vanaspati ghee. Rapeseed oil is used in the manufacture of grease, tanning industry

and mustard oil is used for softening leather. But the use of mustard oil for industrial purpose is

rather limited on account of its high cost.

Oil cake

The oil cake is obtained as by product after extraction of oil. The oil cake is mainly used as cattle

feed. A part of oil cake product in India is also used as organic manure in the field. The mustard

and rapeseed contain on an average of 10-24 percent protein. The mustard oil cake contains 5.1-

5.2 % of nitrogen, 1.8-1.9 % of phosphorous and 1.1-1.2 % potassium.

Table: Specification of Rapeseed and mustard oil

Physio-chemical properties Indian standards (IS : 546-1975)

Moisture (%) 0.1 - 1.25Specific gravity at 10/10 c -Refractive index at 40 c 1.4646-1.4646Saponification value 169.0 -177.0Iodine value 98.01 110.0Acid value 0.5 40.0Bellier turbidity temperature c 27.5Unsaponifiable matter (%) 1.2 2..0


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Methods and Materials

Micronutrient availability to plants can be measured in direct uptake experiments, or estimated

with techniques that correlate quantities of micronutrients extracted chemically from soils to plant

uptake and response to micronutrient fertilization. Rational management of micronutrient fertility and

toxicity requires an understanding of how total and plant-available soil micronutrients vary across the

land.

Digestion of plant sample for estimation of nutrients

It is a method in which the harvested plant material is converted into solution form, so that the

estimation of nutrients could be done.

1- The harvested plant material is first surface cleaned using with a mild acidic detergent

like 3N HCl.

2- Then it is rinsed with distilled water.

3- This washed material is air dried.

4- Once the plant material is dried completely it is ready for grinding.

5- After the material is grinded it is placed in a suitable air tight container.

By above methods the husk, straw, seed and root are digested and the procedure is as

follows;

Digestion

Take 0.5g of sample in a conical flask of 150 ml.

Add 10ml of conc. Nitric acid.

Leave overnight for pre-digestion.

After pre-digestion add 20ml in ratio of 9:3 of Nitric acid and perchloric acid.


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Then heat the conical flask using hot plate in the presence of fume extraction hood.

After some time the plant material would appear in a form of a light colored paste.

Now allow the conical flask to cool down.

Then make up the volume up to 50 ml using volumetric flask.

FIG: DIGESTION INSTRUMENT

Precautions:


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Gloves should always be used during working with acid.

Borosil flask should not be used.

Sulphuric acid could be used if we are not interested in estimating sulphur.

Estimation of Micronutrients by AtomicAbsorption System

About AAS instrument

The Atomic Absorption Spectrophotometer (AAS) used in the study was of Thermo make model M5 having Graphite and hydride unit. AAS is based on theprinciple that when a radiation from an external light source, emitting thespectral line(s) that correspond to the energy required for an electronic transitionfrom the ground state to an excited state, is passed through the flame. The flamegases are treated as a medium containing free, unexcited atoms capable of absorbing radiation from an external source when the radiation correspondsexactly to the energy required for a transition of the test element from the groundelectronic state to an upper excited level. Unabsorbed radiation then passesthrough a monochromator that isolates the exciting spectral line of the lightsource and into a detector. The absorption of radiation from the light sourcedepends on the population of the ground state, which is proportional to thesolution concentration sprayed into the flame. Absorption is measured by thedifference in transmitted signal in the presence and absence of the test element.The layout of a basic flame atomic absorption spectrometer


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Fig. Atomic Absorption Spectrophotometer

Light from a line source of characteristic wavelength for the element being

determined passes through the flame into which it has been sprayed as a finemist of the sample solution. The region of spectrum to be measured is selectedby a monochromator. The isolated spectral line falls on the photomultiplier, thedetector and the output is amplified and to a readout device meter, digital or analogue, to a chart recorder or through a computer data processing system,printer or digital display unit. The intensity of the resonance line is measuredtwice, once with the sample in the flame and once without. The ratio of the tworeadings is a measure of the amount of absorption, hence the sample.


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Hollow Cathode Lamps

The hallow cathode lamp is stable, reliable, has a long operating life and isthe standard source in Atomic Absorption Spectrometry. Lamps may beexpected to run in excess of 5000mA hours without failure and many have beenknown to run twice as long. The hollow cathode discharge lamp is known as afine line sources capable of producing spectra where fine structure could be

studied. It consist of a glass tube with the electrode sealed inside with an opticalwindow at one end made of glass or silica depending on the wavelength andattached with a thermosetting resin or vacuum wax. The construction of a typicallamp in use today is

Fig. Hallow cathode Lamp


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The two electrodes are sealed in the glass envelope and the window located

at the opposite end to the cathode. Electrical connections are made through astandard octal plug at the base of the lamp. The cathode is shaped in the form of a hallow cup inside which the discharge takes place. This cup is constructed of or contains the element of interest of interest whose spectra is required. A micashield holds the structure rigid and helps to contain the discharge inside thecathode cup. The cup usually has an internal diameter of about 2mm, toconcentrate the discharge into a small area and produce a high intensity line. Theavailable energy appears to dissipate in the metal resonance line rather than inthe filler gas in this type of construction. The lamp is usually filled to pressure of 4-10 Torr with an inert gas such as helium, argon or neon. Highly purified inertgases are used and the glass is out-gassed at high temperature to removeimpurities absorbed onto the surface of the glass. The emission line of the inertfiller gas must not coincide with the resonance line of the emission line of theinert filler gas and the line of the metal of interest must not coincide with theresonance Neon has a higher ionization potential because it improves theintensity of the discharge.When a voltage of between 300-400 is applied between the anode and cathodethe discharge is set up and argon is ionized by the and becomes positive argon bythe mechanism of:

Ar +e - = Ar + + 2e -

The positive Ar +

ion is attracted and accelerated toward the cathode whereit dislodges or sputters excited metal atoms into the metal cathode knockingmetallic atoms into the discharge, which improves the sensitivity of thedischarge. The excited atoms emit energy of their own characteristic wavelengthbefore returning to the ground state and the emitted light is used as the lightsources for the AAS system. After the atoms return to the ground state they forma cloud of free atoms which return either to the walls of the glass lamp or to themetal cathode.


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Lamp as uses to as

Atom Atomic no Atomicmass

Primarywavelength(nm

)

Fuel flowrate(L/min)

Flame Type

Mn 25 54.9380 279.5 0.9-.12 Air/acetyleneFe 26 55.847 248.3 0.8-1.0 Air/acetyleneCu 29 63.546 324.8 0.8-1.1 Air/acetyleneZn 30 65.5 213.9 0.9-1.2 Air/acetylene

Double Beam InstrumentsThe light from the sources is split into two beams by means of a rotating

half-silvered mirror, or by a beam splitter which is a 50% transmitting mirror.This directs beam alternatively through the flame and along a path which by-passes the flame at a frequency usually at 50Hz or higher. Once past the flame,the beams are recombined with a half a half-silvered mirror as shown below:

Fig. Double Beam InstrumentsAt the detector end, the output signals which correspond to each are divided,amplified separately and compared in a bridge circuit. The out-of-balance signalis than compensated electronically and converted to absorbance.


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Estimation of micronutrients

Analyses of different trace element from soils are considered as a

diagnostic tool for identifying nutrient deficiency or excess in soils. The soil test

method in practical/realistic till the nutrient question extracted by the chemical

extractants from the soil shown a high degree of correlation with crop yield.

Plant analysis of as compared to soil analysis can evaluate better considerable

from nutrient deficiency. Mostly deficiency of micronutrient like Zn, Cu, Fe,Mn, B, Mo etc. appears in the early growing stage and by usual symptom could

identify the micronutrient deficiency the time the usual symptom appear the

deficiency of nutrient have all ready done the damage to the crop. It in hear that

soil analysis has the advantage over that of plant analysis as well as symptom in

indicating the extent of tract element deficiency of their requirement for crops to

seeding. However, despite number of demerits soil analysis has covered itself as

a diagnostic tool in monitoring nutrient status of soil. For the estimation

available micronutrient are in use still most acceptable method as 0.005 m DTPA

extractable method of at pH 7.3 (Lindsay and Norvell 1978).

Apparatus

Atomic Absorption Spectrophotometer

Reagents


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(i) Weigh 1.965 of DTPA.

(ii) Add 600 ml in 1 liter distilled water dissolve in the DTPA.

(iii) Weigh 1.470 gm CaCl 2 and then dissolve in the DTPA solution.

(iv) Then add 13.29 ml of Triethanolamine (TEA) to this solution.

(v) Add 300 ml distilled water and then the solution was adjusted at pH 7.3 by

adding dilute. HCl or NH 4OH

(vi) After pH was adjusted at pH 7.3 the volume was made up

to 1000 ml.

Procedure

(i) 10 gm of soil is taken in 250 gm plastic shaking bottle.

(ii) Add 20 ml of DTPA solution Adjusted at pH 7.3 in the plastic

shaking bottle.

(iii) The plastic bottle is placed on mechanical shaker for 2 hours.(iv) After 2 hour of shaking the soil solution is filtered with Whatman no

of 42.

(v) The filtrate in used for the analysis of micronutrient and pollutant

element.

(vi) Atomic Absorption Spectrophotometer is use to analysis the

micronutrient and pollutant element of different optimal wavelength,

current and slit width after it is calibrated for the specific element.

Calculation for soil

10 gm + 20 ml extracted solution = x 2 dilution factor.


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5 0.8814 1.088 2.1152 1.52666 0.5973 1.0555 1.7631 1.84957 0.7228 1.0577 1.9081 1.24568 1.3261 1.0725 1.4352 1.00999 0.6257 1.0008 1.9527 0.8847

10 0.8206 1.0666 1.6584 1.2363Max-min 1.2785-0.5973 1.0888-1.0008 2.1152-1.4002 2.0762-0.8847

Mean 0.98707 1.06305 1.7742 1.423

Table.1 Soil sample

2.Plant sample

Sample Zn (ppm) Cu(ppm) Fe(ppm) Mn(ppm)1 2.344 299.19 395.14 55.512 2.313 152.09 323.89 52.63 1.333 133.58 508.82 27.554 2.281 281.97 294.6 25.555 2.507 260.65 321.54 29.336 2.411 253.15 320.87 31.817 2.165 257.25 555.39 27.858 1.097 236.58 530.73 30.979 2.46 254.14 628.6 23.64

10 2.452 217.13 199.96 55.51Max-min 2.507-1.097 299.19-133.58 628.6-199.96 55.51-23.64

Mean 2.1363 234.573 407.95436.032


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Result

1. Critical limit in soil for of Zn, Cu, Fe, Mn are - 0.71, 0.60,7.0 and3.0 respectively by Component the data in Cu table 1 all the soils aredeficient in Fe and Mn Where, they were sufficient in copper in Zn intwo soils table no 6 and 9 are deficient in zinc


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Determination of available Boron in soil

Introduction:-

Boron (B) is unique in soils by its narrow range between deficiency for (plant growth) and

toxicity. Less than 1ppm of B may lead to a deficiency, yet 3ppm may be toxic. It is associated with

calcium uptake by plants, and, in fertility studies, it is, often, useful to measure boron-calcium ratio.

The most useful measure of available B is the water-soluble form. The range of water soluble B, in

mineral soils of the humid region, is generally, from 0.2 to 1.5ppm onto 2ppm or more in muck soils,

and down to 0.2ppm in fairly fertile sandy soils. The content of water-soluble B in soils is influenced

by pH, organic matter and amount of colloids.

Importance:-

Knowledge of water-soluble B in soils is of considerable agricultural significance in the context

of its narrow limits between deficiency and sufficiency, its interaction with calcium and its

precipitation as calcium metaborate in extreme cases.

Extraction of Boron from soil:-

Take a 250 ml of low boron conical flask and weigh 20 g of air dried soil.

Add 400 mg of activated charcoal to deactivate the carbon content.

Add 40 ml 0.01M CaCl2 and then attach a reflux condenser.

Heat the flask for 5 min from the first sign of boiling appears.

Allow the flask to cool without removing the condenser till all the drops of condenser land

back into the conical flask.

Filter the suspension with Buchner funnel and suction pump or centrifuge until the supernatant is

clear.


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Store the sample in the plastic ware for analysis.

Methods of Determination:-

Once extracted from the soil, B can be analysed by colorimetric methods, using different

reagents. Of the numerous reagents, used for colorimetric determination of B, Quinalizarine,

Curcumin and Azomethine-H are the most popular. Other methods include Inductively Coupled (ICP)

and atomic emission spectrometry.

Quinalizarine method:-

Principle:-

Boron is extracted from the soil with hot water and is subjected to colorimetric estimation,

following reaction with quinalizarine (1, 2, 5, 8-tetrahydroxyanthraquinone). The reaction of

quinaliarine, with soluble B (boric acid), forms a chelate ring and gives a blue-coloured complex in

strong acid medium, the precise strength of the acid, influencing the intensity of the colour developed.

Nitrates are removed by treatment with hyposulphite in the presence of hydrochloric acid.

Instrument and Apparatus:-

Spectrothotometer boron-free glassware, viz., conical flasks, volumetric flasks, pipette, a

dispenser or a fine pipette, heater or water bath.

Reagent:-

Copper sulphate, CuSO4.5H2O, hydrogen peroxide, 30%, conc. H2SO4, NaOH, 0.5N


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Quinalizarine in H2SO4 0.01%: Dissolve 0.01g of quinalizarine in a mixture of 90ml of H2SO4

and 10ml of water.

Standard boric acid solution, 100ppm of B:

Dissolve 0.5716g of boric acid (H3BO3) in distilled water, and dilute to one litre. One ml of this

solution contains 0.1mg of boron. Dilute 50ml of this solution to 500 ml with distilled water, which

will give a B concentration of 10mg per ml (10ppm).

Procedure:-

* Place 10 g of soil (or 5 g peat) in a 100ml conical flask, and treat with 30 ml hot distilled

water.

* Add 0.5ml of CuSO4. 5H2O solution, and boil it for 5 minutes. Then, agitate and filter.

* Take 10ml of the filtrate in a 50ml volumetric flask, and add 0.5ml of 30% H2O2 Boil thesolution gently for about 1 minute until the solution loses its colour.

*Add 1ml of 0.5N NaOH solution, and heat the solution gently until evaporation. Then, add to it

0.5ml of 30% H2O2, and very slowly evaporate until dry, to avoid loss of boron.

*Treat the residue with 4.5ml of conc. H2SO4 until the salts have completely dissolved. If the

solution is brown in colour, another heating is required.

*Add to the solution 0.4ml of potassium hyposulphite (a reducing agent) in HCl system for

elimination of nitrates.

*Following the disappearance of gas bubbles, add 0.5ml of quinalizarine solution in H2SO4,

agitate the contents, and make up the volume to the mark with distilled water.

*Stopper the flask and allow it to stand for 25 minutes.

*Measure the blue color intensity on the colorimeter, using 620m light maximum, read the %

transmittance and record the concentration of B in the test solution from the calibration curve,


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prepared from the working B standards (using 10ppm stock solution) over a range of 0-10 ppm

as per the procedure outlined above.

Observation:-

a. Weight of the soil = W g

b. Volume of the extract (hot water) made up to = V ml

c. Volume of aliquot taken (test solution) = 10 ml

d. Final Volume made up to = 50 ml

e. Transmittance (%) as read from the colorimeter = T (say)

f. Concentration (ppm) of B in the solution = C (say)

Calculations:-

g. First dilution: = (v/w) times

h. Second dilution: (50/10) = 5 times

i. Total dilution = (v/w) x 5 times

Now, available B in the soil (ppm) = Cx [(v/w) x 5]

Thus, available B in the soil (Kg/ha) = Cx [(v/w) x5] x2.24

Where Cx is the conc. of the unknown sample in O.D.


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Estimation of P by Ascorbic acid reductant method

The ascorbic acid method has clean edge over the method described above, as the reduced blue

coloured complex, due to ascorbic acid, is stable for 24 hours, and is not affected by moderate

variations in acidity, temperature and reducing agent.

Principle

The underlying principle of the method is based on the reduction of the (NH4)3PO 4. 12MoO3

complex by ascorbic acid, in the presence of antimony- potassium tartarate. The blue colour is

produced which is stable for 24 hours, and is less subject to interfering substances than methods

involving reduction by stannous chloride.

ReagentsReagent A:

Dissolve 12gm of ammonium molybdate (AR) in 250 ml of distilled water. In 100ml of

distilled

Water, dissolve 0.291 gram of antimony potassium tartarate. Add both of these solutions to

100ml of approximately 5 N H2SO4; mix thoroughly and make up to 2 liters with distilled

water.

Reagent B:

Dissolve 1.056gm of ascorbic acid in 200ml of reagent A and mix well. This should be

prepared fresh as and when required.


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p-nitrophenol indicator .

Procedure

Pipette out 5ml of extracts in to a 25ml volumetric flask and acidify with 5 N H2SO4 to pH 5.

This can be easily done by taking 5ml of 0.5 M NaHCO3 in a separate 25ml flask and determining the

volume of acid required to bring the solution to pH 5, using p-nitrophenol indicator, the yellow colour

of which disappears at this pH.

After having the pH adjusted to 5, dilute to 20ml with distilled water, and add 4ml of reagent

B.

Wait for 10 minutes and read the blue colour intensity on a colorimeter using 730 840 m

filter.

Run a blank (without soil) along with.

Observations and calculations

a. Weight of the soil taken = 1gm

b. Volume of 0.5 M NaHCO3 solution added = 20ml c. First

dilution = 20/1= 20 times

d. Volume of the extract taken = 5ml

e. Final volume following colour development = 25ml

f. Second dilution = 25/5 = 5 times

g. Total dilution = 20X5 =100 times

h. Transmittance (%) of the test solution = T (say)


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i. Concentration of P as read from the standard curve = A ppm (say)

j. Available P in the soil = A X 100 ppm

k. Available P in the soil = A X 100 X 2.24 kg/ha.

l. Available P2O5 in the soil = A X 100 X 2.24 X

GC-MS


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GAS CHROMATOGRAPHYAND MASS SPECTROSCOPY


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Composition of Fatty acid by

GC-MS


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FIG : GC-MS

(GAS CHROMATOGRAPHYAND MASS SPECTROSCOPY)


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ABOUT GC-MS INSTRUMENT:-

Gas chromatography

Gas liquid chromatography accomplish a separation by partitioning a samplebetween a mobile gas phage and a tin layer of nonvolatile liquid held on a solidsupport.The sequence of a gas chromatography separation is as follows. A samplecontaining the solute is injected in to a heating block where it is immediatelyvaporized and swept as a plug of vapour by the carrier gas streem in to the column

inlet. The solutes are absorbed at the head of the column by the stationary phaseand then obsorbed by fresh carrier gas. This partitioning process occurs repeatedlyas the sample is moved towaed the outlet by the carrier gas. Each solute will travelat its on rate through column, and consequently a band corresponding to eachsolute will form. The band will separate to dwgree that is determine by the partitionratios of the solute and the extent of the band spreading. The solute are eluted, oneafter another, in the increasing order of their partition ratios and enter a detector attached to the column exit. If a recorder is used, the single appear on the chart asa plot of time versus the composition of thr carrier gas stream. The time of emergence of a peak identifies the component, and the peak area reveals theconcentration of the component of the mixture. Although the gas chromatographymethod is limited to volatile material(about 15% of all organic compounds), theavailability of gas chromatography working at temperatures up to 450 c, pyrolytictechniques, and the possibility of converting many materials into a volatile derivativeextend the applicability of the method.

Mass spectrometry:-

The first mass spectrometer dates back to the work in England of J.J.Thompson in1912 and of F.W. Aston in 1919, but the instrument that served as a modle for morerecent once was constructed in 1932.The mass spectrometer produces charged particles consisting of the parent ion andionic fragments of the original molecule,and soet these ion according to their mass/charge ratio.Mass spectrometer are a powerful tool for extracting a wealth of informationconcerning the structure of organic compounds and the elemental analysis of solidstate samples and for analyzing complex of organic mixture. A detailed


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interpretation of the mass spectrum frequently makes it possible toplace functionalgroups in to certain areas of the molecule and to see how they are connected toone another. A mass spectrometer is an essential adjunct to the use of stableisotopes in investigation reaction mechanisms and in tracer work. More over massspectrometry has contributed greatly to a more detailed understanding of kinetic

and mechanisms of unimolecular decomposition of molecules.


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CHAPTER

NO.

PARTICULARS PAGE

NO.1. INTRODUCTION

2. REVIEW OF LITERATURE

3. MATERIALS AND METHODS

4. RESULTS AND DISCUSSION

5. SUMMARY


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6. CONCLUSION

7. BIBILIOGRAPHY

ABSTRACT


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CHAPTER 1

INTRODUCTION


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CHAPTER 2

REVIEW OF

LITERATURE


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CHAPTER 3

MATERIALSAND

METHODS


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CHAPTER 4

RESULTSAND

DISCUSSION


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CHAPTER 5

SUMMARY


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CHAPTER 6

CONCLUSION


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