PROJECT

REPORT
Analysis of Fatty Acids by GC-
MS,

Estimation of Micronutrient by
Atomic Adsorption

At

Directarate of Rapeseed
Mustard Research,
Sewar, Bharatpur (Raj.)

During

Sept 10, 2009 – Nov 10, 2009

 Under Supervision
Submitted By
Dr. N.s. Bhogal
Jaiprakash saini
(Senior scientist)
B.sc Biotech(Int.)3ed year

Submitted to
Lords International collage
Chakani, Alwar
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

upto 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)
 CONTENTS

1. Analysis of Fatty acid by GC-Ms

a. Introduction 1

b. Estimation of Fatty acid 2

c. Tic graph 7

2. Estimation of Micronutrients by
Atomic Absorption System

a. About AAS instrument

13

b. Hollow Cathode Lamps

15

c. Double Beam Instruments

17
d. Estimation of micronutrients
18

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 Atomic Primary Fuel flow Flame Type

no 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
Max-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 55.51
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
Max-min 2.507-1.097 299.19-133.58 628.6-199.96 55.51-23.64
Mean 2.1363 234.573 407.954 36.032
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