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 OF

JAIPRAKASH SAINI Dr. N.S.BHOGAL
Enrollment No. 05/121540 Senior Scientist (soil science)

Work done at
 Tel: 05644-260495, 260379 (O), 260381 (R)
Fax: 05644- 260565
Email: arvind_mustard@rediffmail.com
dir_nrcrm@yahoo.co.in
Website : http://drmr.ernet.in

Dr. N.S.Bhogal
Senior 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)
 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)
 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)
 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)
 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 in
developing 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
77.100 E longitude and 27.150N latitude and is about 178.37 meters above mean sea.
DRMR
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.

 CONTENT

1.About DRMR
2.Micronutrient Status
a. Micronutrient Status by AAS(Atomic
Absorption Specto Photometer)
Inastrument
Principle
Method&Procedure
b.Micronutrient Status by Spectrophotometer
Principle
Method &Procedure
3.Analysis of fatty acid Composition by
GCMS(Gas Chromatography and Mass
Spectrophotometer
1. Instrument
Principle
Method&procedure
3.Effect of salinity
Na +k Ratio by flame Photometer
Instrument
Principle Method&Procedur

4.RESULT

5. REFERENCE
CHAPTER –1

INTRODUCTIO
N
 Introduction
India 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 seed
constitute 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 world’s 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
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 stored
material 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 and
soil 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 crop’s 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 plant’s 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
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

it’s required.
 ABOUT RAPESEED-MUSTARD

Among the oilseed crops, rapeseed and mustard (mostly Brassica species) are very important.
Family: Cruciferae
Genus: 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).
Rapeseed includes
1. Yellow sarson (Brassica rapa var. Yellow sarson)

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

3. Toria (Brassica compestries var. Toria)

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
This adventives or introduced plant is a summer or winter annual about 1-4' tall, branching
occasionally 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 ½" across, consisting of 4 yellow
petals, 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 ¾–
1¼" 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 resembles
Broccoli 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.
 (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 - 1.25

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

Absorption 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 the
principle that when a radiation from an external light source, emitting the
spectral line(s) that correspond to the energy required for an electronic transition
from the ground state to an excited state, is passed through the flame. The flame
gases are treated as a medium containing free, unexcited atoms capable of
absorbing radiation from an external source when the radiation corresponds
exactly to the energy required for a transition of the test element from the ground
electronic state to an upper excited level. Unabsorbed radiation then passes
through a monochromator that isolates the exciting spectral line of the light
source and into a detector. The absorption of radiation from the light source
depends on the population of the ground state, which is proportional to the
solution concentration sprayed into the flame. Absorption is measured by the
difference in transmitted signal in the presence and absence of the test element.
The layout of a basic flame atomic absorption spectrometer
 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 fine
mist of the sample solution. The region of spectrum to be measured is selected
by a monochromator. The isolated spectral line falls on the photomultiplier, the
detector 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 measured
twice, once with the sample in the flame and once without. The ratio of the two
readings is a measure of the amount of absorption, hence the sample.
Hollow Cathode Lamps
The hallow cathode lamp is stable, reliable, has a long operating life and is
the standard source in Atomic Absorption Spectrometry. Lamps may be
expected to run in excess of 5000mA hours without failure and many have been
known to run twice as long. The hollow cathode discharge lamp is known as a
fine 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 optical
window at one end made of glass or silica depending on the wavelength and
attached with a thermosetting resin or vacuum wax. The construction of a typical
lamp in use today is

Fig. Hallow cathode Lamp

 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 a
standard 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 mica
shield holds the structure rigid and helps to contain the discharge inside the
cathode cup. The cup usually has an internal diameter of about 2mm, to
concentrate the discharge into a small area and produce a high intensity line. The
available energy appears to dissipate in the metal resonance line rather than in
the 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 inert
gases are used and the glass is out-gassed at high temperature to remove
impurities absorbed onto the surface of the glass. The emission line of the inert
filler gas must not coincide with the resonance line of the emission line of the
inert filler gas and the line of the metal of interest must not coincide with the
resonance Neon has a higher ionization potential because it improves the
intensity of the discharge.
When a voltage of between 300-400 is applied between the anode and cathode
the discharge is set up and argon is ionized by the and becomes positive argon by
the mechanism of:

Ar +e- = Ar+ + 2e-

The positive Ar+ ion is attracted and accelerated toward the cathode where
it dislodges or “sputters” excited metal atoms into the metal cathode knocking
metallic atoms into the discharge, which improves the sensitivity of the
discharge. The excited atoms emit energy of their own characteristic wavelength
before returning to the ground state and the emitted light is used as the light
sources for the AAS system. After the atoms return to the ground state they form
a cloud of free atoms which return either to the walls of the glass lamp or to the
metal cathode.
 Lamp as uses to as

Atom Atomic no Atomic Primary Fuel flow Flame Type

mass wavelength(nm rate(L/min)
)
Mn 25 54.9380 279.5 0.9-.12 Air/acetylene
Fe 26 55.847 248.3 0.8-1.0 Air/acetylene
Cu 29 63.546 324.8 0.8-1.1 Air/acetylene
Zn 30 65.5 213.9 0.9-1.2 Air/acetylene

Double Beam Instruments

The 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 Instruments

At the detector end, the output signals which correspond to each are divided,
amplified separately and compared in a bridge circuit. The out-of-balance signal
is than compensated electronically and converted to absorbance.
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
 (i) Weigh 1.965 of DTPA.
(ii) Add 600 ml in 1 liter distilled water dissolve in the DTPA.
(iii) Weigh 1.470 gm CaCl2 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 NH4OH

(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.
 Sample reading by AAS concn mg / kg = M
µg / g or mg / kg of Zn / Cu / Fe / Mn = M x 2

Calculation for plant tissue:-

0.5 g plant tissue digested and volume made up 50 ml = X 100 Dilution
factor
Sample reading by AAS conc. In mg \ kg = P (ppm)
µg \ g or mg\kg of Zn\Cu\ Fe\ Mn = P X Dilution
factor (100)

Reading:-

1. Soil Sample
Sample Zn(ppm) Cu(ppm) Fe(ppm) Mn(ppm)
1 1.2089 1.0712 1.7176 1.8166
2 1.2785 1.0676 1.7411 1.2441
3 1.2082 1.0693 2.0504 2.0762
4 1.2012 1.0813 1.4002 1.3405
 5 0.8814 1.088 2.1152 1.5266
6 0.5973 1.0555 1.7631 1.8495
7 0.7228 1.0577 1.9081 1.2456
8 1.3261 1.0725 1.4352 1.0099
9 0.6257 1.0008 1.9527 0.8847
10 0.8206 1.0666 1.6584 1.2363
1.2785-0.5973 1.0888-1.0008 2.1152-1.4002 2.0762-0.8847
Max-min
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 55.51
2 2.313 152.09 323.89 52.6
3 1.333 133.58 508.82 27.55
4 2.281 281.97 294.6 25.55
5 2.507 260.65 321.54 29.33
6 2.411 253.15 320.87 31.81
7 2.165 257.25 555.39 27.85
8 1.097 236.58 530.73 30.97
9 2.46 254.14 628.6 23.64
10 2.452 217.13 199.96 55.51
2.507-1.097 299.19-133.58 628.6-199.96 55.51-23.64
Max-min
36.032
Mean 2.1363 234.573 407.954
Result
1. Critical limit in soil for of Zn, Cu, Fe, Mn are - 0.71, 0.60,7.0 and
3.0 respectively by Component the data in Cu table 1 all the soils are
deficient in Fe and Mn Where, they were sufficient in copper in Zn in
two soils table no 6 and 9 are deficient in zinc
 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.
 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
 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 the
solution 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 620mµ light maximum, read the %
transmittance and record the concentration of B in the test solution from the calibration curve,
 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 =Wg

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.

 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.

Reagents
Reagent 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.
 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 mµ
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)
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”
GAS CHROMATOGRAPHY
AND MASS SPECTROSCOPY
Composition of Fatty acid by
“GC-MS”
 FIG : “GC-MS”
(GAS CHROMATOGRAPHYAND MASS SPECTROSCOPY)
 ABOUT GC-MS INSTRUMENT:-

Gas chromatography

Gas liquid chromatography accomplish a separation by partitioning a sample

between a mobile gas phage and a tin layer of nonvolatile liquid held on a solid
support.
The sequence of a gas chromatography separation is as follows. A sample
containing the solute is injected in to a heating block where it is immediately
vaporized 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 phase
and then obsorbed by fresh carrier gas. This partitioning process occurs repeatedly
as the sample is moved towaed the outlet by the carrier gas. Each solute will travel
at its on rate through column, and consequently a band corresponding to each
solute will form. The band will separate to dwgree that is determine by the partition
ratios of the solute and the extent of the band spreading. The solute are eluted, one
after 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 as
a 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 the
concentration of the component of the mixture. Although the gas chromatography
method is limited to volatile material(about 15% of all organic compounds), the
availability of gas chromatography working at temperatures up to 450 c, pyrolytic
techniques, and the possibility of converting many materials into a volatile derivative
extend the applicability of the method.

Mass spectrometry:-

The first mass spectrometer dates back to the work in England of J.J.Thompson in
1912 and of F.W. Aston in 1919, but the instrument that served as a modle for more
recent once was constructed in 1932.
The mass spectrometer produces charged particles consisting of the parent ion and
ionic 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 information
concerning the structure of organic compounds and the elemental analysis of solid
state samples and for analyzing complex of organic mixture. A detailed
interpretation of the mass spectrum frequently makes it possible toplace functional
groups in to certain areas of the molecule and to see how they are connected to
one another. A mass spectrometer is an essential adjunct to the use of stable
isotopes in investigation reaction mechanisms and in tracer work. More over mass
spectrometry has contributed greatly to a more detailed understanding of kinetic
and mechanisms of unimolecular decomposition of molecules.
CHAPTER PARTICULARS PAGE
NO. NO.
1. INTRODUCTION

2. REVIEW OF LITERATURE

3. MATERIALS AND METHODS

4. RESULTS AND DISCUSSION

5. SUMMARY
6. CONCLUSION

7. BIBILIOGRAPHY

ABSTRACT
CHAPTER –1

INTRODUCTIO
N
CHAPTER –2

REVIEW
OF
LITERATURE
CHAPTER –3

MATERIALS
AND
METHODS
CHAPTER –4

RESULTS
AND
DISCUSSION
CHAPTER – 5

SUMMARY
CHAPTER –6

CONCLUSIO
N
 CHAPTER-
7

BIBILIOGRAP
HY