PRACTICAL MANUAL

FOR

SOIL AND PLANT ANALYSIS

R.S. ANTIL
ANOOP SINGH
S.S. DAHIYA

2002
DEPARTMENT OF SOIL SCIENCE
CCS HARYANA AGRICULTURAL UNIVESITY
HISAR - 125004, INDIA
The Authors: DR. R.S. ANTIL, SOIL SCIENTIST
DEPARTMENT OF SOIL SCIENCE
CCS HARYANA AGRICULTURAL UNIVESITY
HISAR - 125004, INDIA

DR. ANOOP SINGH, PROFESSOR (SOIL SCIENCE)

DEPARTMENT OF SOIL SCIENCE
CCS HARYANA AGRICULTURAL UNIVESITY
HISAR - 125004, INDIA

DR. S.S. DAHIYA, PROFESSOR & HEAD

DEPARTMENT OF SOIL SCIENCE
CCS HARYANA AGRICULTURAL UNIVESITY
HISAR - 125004, INDIA

Correct citation : Antil, R.S., Singh, A. and Dahiya, S.S. 2002. Practical Mannual
for
Soil and Plant Analysis. Department of Soil Science, CCS
Haryana
Agricultural University, Hisar - 125004.
 FOREWARD
Rising population, persisting poverty, rapid urbanization,
industrialization, deforestation, intensification of agriculture, and lack of
appropriate policies are putting enormous pressure on our natural resource
base. This has led to both quantitative and qualitative degradation of land,
water, air and bioresources, endangering the ecological foundations that are
essential for sustainable advances in the productivity of major farming
systems. Food, nutrition and environmental secuirty are directly linked to
agriculture which play an important role in achieving sustainable food self-
sufficiency. Maintaining the soil health is essential for long-term sustainability
to meet the growing demand of food, fibre and fuel for increasing animal and
human population. The widespread use of high analysis nitrogenous fertilizers
without the application of organic manures resulted in deficiency of P, K, Zn, S
and Mn. Unless corrective measures and viable nutrient management
strategies are not taken into consideration, these may lead to further
deterioration in the soil health. Therefore, improvement in soil health is
utmost necessary to provide an optimum condition for plant growth. Thus,
Soil testing assumes considerable significance in the context of applying
need-based fertilizer doses for enhanced crop productivity. Enhancement in
productivity of soil through fertilizer and manures depends on the correct and
reliable estimation of nutrients in the soil.
Present practical manual is a timely contribution for quantifying
the chemical characteristics of soil and plant in relation to the fast changing
environment/scenario. I am very pleased to find that the authors have taken
pains to compile such a comprehensive practical manual convening the
principles and methods of analysis of soils and plants.
I am sure this manual would not only be useful to soil scientists
and agronomists but also to undergraduate and postgraduate students of
various disciplines interested in the analysis of soil and plant samples.
I appreciate the sincere efforts put by Dr(s). R.S.Antil, Anoop
Singh and S.S. Dahiya in bringing out this excellent practical manual entitled,
"Practical Manual for Soil and Plant Analysis".
 VINAY KUMAR
Vice-Chancellor

PREFACE
Intensive cultivation of the agricultural land is the only way to meet the
demands of growing human and animal population for food, fibre and fuel.
This is only possible at the cost of depleting soil nutrient reserves or with the
use of high inorganic fertilizer. Soil testing has been recognised long as a tool
for efficient fertilizer management.
The main aim of writing this manual is to provide an uptodate
knowledge of the methods of soil and plant analysis for quantifying the
chemical characteristics of soil and plants. A few practical manuals currently
available in the subject, are either very specific or highly advanced to a topic
and do not provide a complete required information. The present manual is an
effort in this direction. In fact, a need was felt by the ICAR and State
Agricultural Universities teachers to have a comprehensive manual for soil
and plant analysis at undergraduate level which motivated the authors to write
this practical manual.
The manual comprises six chapters, covering general considerations
for analysis, basic principles of the instruments used for soil and plant
analysis, soil and plant sampling techniques for analysis, methods of
analysing available as well as total nutrients in soil and plant samples. Each
chapter is presented in a concise and lucid manner, including reagent
preparation and stepwise procedure, outlining precautions, wherever
necessary. The principles, reactions involved and interpretation of the result
of analysis have been discussed in brief. It would suffice the purpose of
undergraduate and postgraduate students in their respective courses viz.
Soils 101 - Introduction to Soil Science, Soils 201 - Soil Fertility and Fertilizer
use, Soils 402 - Principles of Soil Fertility and Fertilizer use, Soil 406 - Soil and
Water Testing, Soils 502 - Soil Fertility, etc. The authors feel that the manual
will provide a very important tool to carry out soil and plant analysis teachers,
researchers, engineers, agricultural extension workers and other field
scientists engaged in agricultural research and teaching.
We are grateful to the Dean, College of Agriculture for generous
financial support under ICAR Development Assistance Scheme, which made
this publication possible. Our sincere thanks are due to Shri Vinay Kumar, Vice
-Chancellor, CCS Haryana Agricultural University, Hisar, for all his help
including writing a foreword for the manual. We are immensely thankful to Dr.
D.K. Bhandari, Sr. Soil Chemist cum-Incharge, Regional Soil Testing
Laboratory, Hisar for editing the manuscript.

R.S.ANTIL
ANOOP SINGH
S.S. DAHIYA
Hisar
March, 2002
5
 6

CONTENTS
_______________________________________________________________________
FOREWARD (I)
PREFACE (II)
1. General considerations of analytical determinations
2. Basic principles of the instruments used in soil and plant
analysis
2.1 pH meter
2.2 Conductivity meter
2.3 Colorimeter/Spectrophotometer
2.4 Flame photometer
2.5 Atomic Absorption Spectrophotometer (AAS)
2.6 Inductively Coupled Plasma Emission Spectroscopy (ICP-AES)
3. Collection and processing of soil samples for analysis
4. Soil test procedures
4.1 Soil reaction (pH)
4.2 Electrical Conductivity
4.3 Calcium carbonate
4.4 Cation Exchange Capacity (CEC)
4.5 Organic carbon
4.5.1 Wet digestion method
4.5.2 Colorimetric method
4.6 Available (mineralizable) nitrogen
4.7 Inorganic forms of nitrogen (ammonium, nitrite and nitrate)
4.8 Available phosphorous
4.8.1 Olsen's method
4.8.2 Bray's method
4.8.3 Ascorbic acid reductant method
4.9 Available potassium
4.10 Available sulphur
4.11 Available calcium and magnesium
 7

4.12 Available (DTPA extractable) zinc, manganese, copper and iron

4.13 Toxic heavy metals
4.14 Available boron
4.15 Available molybdenum
5 Plant analysis
5.1 Plant sampling and sample preparation
5.2 Total carbon by dry combustion method
5.3 Total nitrogen
5.3.1 Modified Kjeldahl method
5.3.2 Nessler's reagent (colorimetric) method
5.4 Total phosphorous
5.5 Total potassium
5.6 Total sulphur
5.7 Total calcium and magnesium
5.8 Total zinc, manganese, copper and iron
5.9 Total boron
5.10 Total molybdenum
6 Soil analysis for total nutrients
6.1 Total nitrogen except nitrogen
6.2 Total nutrients
References
 8

1. GENERAL CONSIDERATIONS OF ANALYTICAL DETERMINATION

1.1 Analytical reagents

Chemical reagents are supplied in different grades and each grade has a
distinct purpose and range of uses. The purest grade is analytical reagent (AR),
the second is laboratory reagent (LR), the third is guaranteed reagent (GR) and
fourth is technical grade. Second and third form of reagents, have a lot of
impurities of micronutrients. Therefore, for the determination of micronutrients,
AR grade reagent should be used.
1.2 Concentrated acids and bases
Strength of acids and bases are expressed on the basis of their Normality.
The strength of some concentrated acids and bases are presented in Table 1.
Table 1. Strength of concentrated acids and basis.

Reagent Concentration Approximate

Normality Per cent by weight specific gravity
HCl 11.6 37-38 1.19
H2SO4 35-36 97-100 1.84
HOAC 17.5 99.5 1.13
HNO3 16 70-71 1.42
HCLO4 9-11.6 60-70 1.51-1.67
H¬3PO4 45 85 1.71
NH4OH 15 28-29 0.90
HOH 55 100 1.00

1.3 Distilled Water

Distilled water is always used in chemical analysis. The quality of distilled
water varies from single distilled to double or triple depending upon the
requirement of analytical technique(s) e.g. double distilled water (glass distilled)
is always recommended for micronutrient and heavy metal analysis.
1.4 Filter paper
Several types of filter papers are available in the market depending on
their ash content, porosity and elemental composition. The commonly used filter
papers are Whatman, Munktells, Schleicher and Schuell. These filter papers are
available in different number(s) ranging from No.1 to 60. The pore size
decreases with their increasing numbers.
 9

1.5 Preparation of cleaning solution

1.5.1 Chromic acid cleaning solution
Chromic-sulphuric acid cleaning solution is very important for final
cleaning of glassware. Visible materials and organic solvents should be removed
with water before using cleaning solution. The cleaning solution can be prepared
either by dissolving 80 g of K2¬Cr2O7 or Na2¬Cr2O7 in about 300 ml of water (with
heating) in a 2000 ml of corning (Borosil) beaker. To the beaker, add one litre of
technical/commercial grade H2SO4. Considerable red chromic oxide (Cr2O3)
precipitates, or
a cleaning solution that does not involve Cr2O3 crystallization is made by
dissolving 5 g of K2¬Cr2O7 in a minimum of water and add one litre of technical/
commercial grade H2SO4 to this solution.
1.5.2 Aqua regia
Another cleaning solution used is aqua regia. The Aqua regia is
prepared by mixing concentrated HCl and H2SO4 in a ratio of 3:1.
1.6 Laboratory apparatus
1.6.1 Glass wares
Corning/Borosil glass that contains arsenic is preferred for most of the
laboratory analysis.
1.6.2 Sieve openings v/s meshes per inch
The size of sieve openings in mm approximately based on the assumption
that opening is 0.63 of the mesh interval, hence
16
mm per opening = -------------------
Meshes per inch

For example, a "100-mesh" screen has openings of 0.16 mm.

1.7 Preparation of standard solutions/expression of solution concentration

1.7.1 Molar solution
A one molar solution contains one mole or one gram molecular weight of
a chemical substance in one litre of the solution. This expression is written as:
 10

Molarity = No.of moles per litre

Wt. in g
No. of moles = -----------------------
Mol. wt.

1.7. 2 Molal solution

The molality of a solution is the number of moles per 1000 g of the solvent.
Molal solutions are prepared by dissolving gram mole(s) of chemical substance
in 1000 g of the solvent.
Molality = No. of moles per 1000 g of the solvent.

1.7. 3 Normal solution

The chemical reactions occur on chemical equivalent basis. Thus, one
gram equivalent weight of chemical compound is equivalent to one gram
equivalent weight of the other. When one gram equivalent weight is dissolved in
a solvent and volume made 1000 ml, the solution is referred to as Normal
Solution and concept is called Normality (N).
Mol.wt.
Normality = ----------
(Neutral salts) Valency
1.7.4 The milliequivalent weight
When equivalent weight is expressed in milligrams i.e.
Equivalent weight
Milliequivalent weight = ----------------------- x 1000
1000

Number of milliequivalents = Normality x 1000

ppm (parts per million)

Milliequivalent (meq./litre) = ------------------------------
Eq.wt. (Equivalent weight)

ppm = meq./litre x Eq.wt.

ppm
ppm to % = --------
10,000
 11

1.8 Standardization of solutions

The exact standardization of solution is done with primary standard
solution of approximate strength. Most commonly used primary standard
solutions are:
i) Acids: Potassium hydrogen phthalate
Benzoic acid
Constant boiling Hydrochloric Acid
Sulphamic acid
Potassium acid iodate
ii) Bases: Sodium carbonate
Mercuric oxide
Borax
iii) Oxidising agents: Potassium dichromate
Potassium bromate
Potassium iodate
iv) Reducing agents: Sodium Oxalate
Arsenious oxide
Iron metal
Potassium ferrocynide
v) Others: Sodium chloride
Potassium chloride

1.9 Preparation of standard solutions

1.9.1 Standard solutions of acids
The standard acid solutions can be prepared by using the equation given
below:
V1N1 = V2N2
where V1 = Volume of concentrated acid required
N1 = Normality of the concentrated acid
V2 = Total volume of the acid to be prepared
N2 = Normality of the acid to be prepared.
Example: Preparation of 100 ml of 4 N HCl solution, from concentrated HCl.
Calculations
V1N1 = V2N2
Where V1 = Volume of concentrated HCl (11.6 N)
N1 = Normality of concentrated HCl (11.6 N)
V2 = Volume of diluted acid (100 ml)
N2 = Normality of diluted acid to be prepared (4N)
 12

V1N1 = V2N2

V2 N2
Therefore, V1 = ----------
V1

100 (ml) x 4(N)

SoV1 = ------------------- = 34.5 ml
11.6 (N)

Thus, pipette 34.5 ml concentrated HCl (11.6N) and make volume 100 ml
with distilled water to obtain HCl of 4N strength.
Another method of preparing standard solutions of acids is based on the
specific gravity and percent purity of the acids.
Example: Prepare 1N 1000 ml H2SO4 from concentrated H2SO4 of the
specific gravity 1.84 and per cent purity 98%.
Calculations

i) Specific gravity of concentrated H2SO4 (D) = 1.84

ii) Per cent purity = 98

iii) Volume of 1N H2SO4 to be prepared = 1000

ml

Calculations:
M
D = ------
V

M 100
or V = ------ x ------------------ (ml)
D per cent purity

49 100
V = ------ x ------- = 27.17 ml
1.84 98

Pipette 27.17 ml of concentrated H2SO4 acid and dilute to 1000 ml with

distilled water.
 13

1.9.2 Standard solutions of bases

Example: Preparation of 1N 1000 ml NaOH solution.
Calculations:
1N NaOH solutions contains 1g equivalent weight of NaOH dissolved in
one litre water.

Mol. wt. of NaOH = 23+16+1= 40 g

or
Mol. wt
Eq. wt. of NaOH = ----------------
Valency

40
=-------- = 40 g
1
Dissolve 40 g of NaOH in distilled water and make volume to 1000 ml.
The strength of this solution is 1N.
Source: Extracted and modified from Jackson (1967) and Chopra and Kanwar

(1991).
 14

2. Basic Principles of the instruments used in soil and plant analysis

Most of the analytical methods used for soil and plant analysis involve
some kind of instrument. Therefore, it is very important to know about the basic
principles of the instruments in order to get the accurate results. The most
commonly used instruments in soil and plant analysis are given below:
1. pH meter
2. Conductivity meter
3. Colorimeter/Spectrophotometer
4. Flame photometer
5. Atomic Absorption Spectrophotometer (AAS)
6. Inductively Coupled Plasma Emission Spectroscopy (ICP-AES)
2.1 pH meter
pH meter is used for the measurement of the reaction of a solution. pH
determination with the help of a pH meter is based on the measurement of the
electromotive force (e.m.f.) of a pH cell consisting of a glass electrode, sensitive
+
to H ions and a reference electrode. The pH cell can be represented as given
below:
+
[H ] sensitive Reference Salt bridge Reference
electrode buffer or Test electrode
solution
The potential developed across in glass electrode on account of the
+
difference in activity of H ions in and out of the electrode. This electrical
potential, E, is defined by the Nernst Equation:
RT P
E = ----- Loge --------
nF Q

where R = Gas constant (8.313 Joules)

o o
T = Absolute temperature (298 K or 25 C)
+
n = Valency of the metal ion (1 for H )
F = Faraday Constant (96,500 coloumbs)
P = Pressure of the electrode
 15

Q = Osmotic pressure

RT 1
or E = ------- 2.303 log -------
+
nF H

By putting the value of R, T, n and F, we get

1
E = 0.058 log ------
+
H
or E = 0.058 pH
E
pH = --------
0.058

2.1 Conductivity meter

In soil and water, salinity is characterized by the total amount of the
dissolved inorganic salts. It is estimated by measuring the electrical conductivity.
The electrical conductivity is a measure of the ability of a salt solution to carry
electric current by the migration of ions under the influence of an electric field, as
ions are the carriers of electricity. Like metallic conductors, solutions also obey
Ohm's law. Increase in temperature promotes dissociation of the salts with
simultaneous increase in conductivity at the rate of approximately 2% of each
degree of Celsius rise in temperature. The unit of specific conductance is the
-1 -1
reciprocal of specific resistance in Ohms cm , i.e. mhos cm . If CS is the
-1
concentration of a solution in gram equivalent L , then the volume of solution in
ml per equivalent is 1000/CS, so the conductance will be 1000 K/CS, where K is
cell constant. At infinite dilution, the ions are theoretically independent of each
other and each ion has its contribution to the total conductance.
Thus,
+ -
λα = Σ (λ ) + Σ (λ )
where, λα is the total conductance
+
λ is the conductance of cations
-
λ is the conductance of anion at infinite dilution.
 16

2.3.Colorimeter/Spectrophotometer
Colorimeter/Spectrophotometer is used for the determination of the
concentration of a substance in a solution by measuring the relative absorption
or transmission of light w.r.t. a known concentration standard. Colorimetric
analysis is based on the measurement of intensity of radiant energy after it
passes through a sample solution. Spectrophotometer is generally controlled by
basic principles or laws of light. Spectrophotometer is based on the law of
Lambert, Beer and Bouger. When a beam of monochromatic light falls on a
homogeneous layer of a substance, part of the radiation is reflected, part is
absorbed, and part is transmitted. The substance can be in a solid, liquid, or gas
form. The incoming light is generally called the incident light. The relationship
between the intensity of incident light (Io) and the intensity of reflected light (Ir),
absorbed light (Ia), and transmitted light (It) can be expressed as follows:
Io = Ir + Ia + It
When comparing the intensity of the beams transmitted through the solution and
through the solvent, the effect of Ir is neglected in practice, and the relationship is
changed into:
Io = Ia + It
Law of Lambert and Bouger
This law indicates that transmission and absorption of light is a function
of thickness of the medium:
-1
It = Ia a
Where, a = Factor related to the fraction of incident light transmitted by a layer of
1 cm thickness. This factor is often called the transmission co-efficient.
Rearranging the above equation, we get
-1
It/Io = a =T
T is called transmittance.
By rearranging again and taking the log, the equation above changes into:
Io/It = 1/T
 17

Log(Io/It) = log(1/T) = D
Where D is the optical density (O.D.) or absorbance (A).

Beer's Law
This law indicates that transmission and absorption of light by a medium
is a function of concentration:
-c
It = Io a
Where a = transmission co-efficient, and c = concentration.
Law of Lambert and Beer
The combination of law of Lambert and Bouger and the Law of Beer gives
the law of Lambert and Beer's law the basis of absorption spectrophotometry:
-IC
It/Io = a
or in log form:
-IC
log(It/Io) = loga
log(It/Io) = (-Ic)log a or
log(It/Io) = (Ic)log a
Log a is also called "extinction coefficient" and is assigned the symbol ε.
The term "molar extinction" or molar absorption is used when the concentration
-1
is expressed in moles L . By using ε instead of log a, the above formula can be
changed into the fundamental equation of spectrometry:
log(It/Io) = ε Ic
which indicates that the optical density or absorbance is proportional to
concentration (C), and thickness of sample (I). If a cuvette or sample holder of 1
cm thickness is used, then I=1, and log(Io/It) = εC, in other words, absorbance is
directly proportional to concentration.
According to Lambert and Beer's law, a straight line is obtained if optical
density or absorbance is plotted against concentration on an ordinary graph
paper. However, if transmittance readings are plotted against concentration on a
semi log paper a curvilinear regression line is obtained, because T=(It/Io)
indicating that transmittance is not directly proportional to concentration as in
 18

the case of optical density.

2.4 Flame photometer

In flame photometry a flame is used for (1) converting the sample from
the liquid or solid state into the gas phase; (2) decomposing the sample into
atoms, and/or (3) exciting these atoms into light emission.
Flame photometer is based on the principle that when a solution of a salt
is sprayed into a flame, the salt breaks into the component atoms due to high
temperature. The atoms released of some specific element take energy from
flame and get excited to the higher orbit. Such atoms release energy of a
wavelength, which is specific for the element and is proportional to the
concentration of atoms of that element. However, since intensity of emitted light
is dependent on temperature, the sensitivity and reproducibility of the analysis
vary also with temperature.
2.5 Atomic Absorption Spectrophotometer
Atomic absorption spectrophotometer is based on the principle that when
a sample, in the form of a homogenous liquid, is aspirated into a flame where
"free" atoms of the element, to be analysed, are created. A light source (hollow
cathode lamp) is used to excite the free atoms formed in the flames by the
absorption of the electromagnetic radiation. The decrease in energy (absorption)
is then measured which follows the Lambert-Beer's law i.e. the absorbance is
proportional to the number of free atoms in the ground state.
2.6 Inductively coupled plasma-atomic emission spectroscopy (ICP-AES)
ICP-AES is based on the observation of atomic emission spectra, when
samples in the form of aerosol, thermally generated vapour or powder are
injected into an inductively coupled plasma atomization and excitation source. By
definition, plasma is simply a very hot gas in which a significant fraction of the
atoms is ionized. Plasma is electrically conducted and has been referred to as
electrical flames, as no combustion takes place. This is because inert argon gas
is used as a plasma source in such instruments.
 19

The ICP is produced by passing initially ionized argon gas through a quartz
torch located within an induction coil (Cu Coil), which is connected to a radio
frequency (RF) generator. The radio frequency generator produces 1.5 to 3 KW
power at a frequency of 27.1 mHz. an oscillating magnetic field is formed within
the quartz torch in response to the radio frequency energy passing through the
coil. Electrons and ions passing through the oscillating electromagnetic field flow
at a high acceleration rates within the quartz torch space. As argon gas enters
the magnetic field associated with the induction coil, its atoms collide with the
accelerated ions and electrons resulting in the ionization of the argon gas. These
collisions give rise to ohmic heating, which produces plasma with temperature
ranging from 600 to 1000 K. The resultant plasma is contained within a plasma
torch by means of argon flow.
The method of presenting the sample to the plasma is similar to that used
in flame atomic absorption. The liquid sample is aspirated into the plasma
through a nebulizer system by using argon carrier gas at a rate of about 10-12 L
-1
argon min . The prevailing high temperature in the plasma leads to complete
vaporization, atomization and excitation of the element to be analyzed. The
excited neutral atoms or ions of the sample emit radiation of characteristic
wavelengths within the intensity directly proportional to the element
concentration. Intensity of the emitted radiation is measured by
spectrophotometer component of ICP-AES instrument.
Source: Extracted and modified from Jackson (1967), Page et al. (1982), Singh et
al.
(1999).
 20

3. Collection and processing of soil samples for analysis

The chemical analysis becomes meaningless if there is an error in
collection and preparation of soil samples. Hence, the collection and preparation
of soil samples should be done with utmost perfection. The steps involved are:
3.1 Collection of soil samples
The soil sample collected should be representative of the area sampled. A
field can be treated as a single sampling unit if it is uniform in all respects.
Variation in slope, texture, color, crop(s) growth and management levels should
be taken into account for soil sampling. Separate sets of composite samples
need to be collected from areas differing in these characteristics. Recently
fertilized plots, bunds, channels, marshy tracts, area near trees, farm ways,
buildings, wells, compost piles or other non-representative locations must be
avoided during sampling. When crops are grown in rows, samples can be taken
in between the rows. Soil samples should be taken in zig zag pattern or randomly.
3.2 Depth of sampling
The plant roots penetration is very important for deciding the sampling
depth. The following parameters may be kept in consideration:
1. For field crops (cereals, vegetables and others seasonal crops) a sampling
depth of 0-15 cm (plough layer) is desired.
2. For deep-rooted crop like sugarcane or under dry farming conditions,
samples from different depths may be taken.
3. For horticultural crops, sampling is done at depths of 0-15, 15-30, 30-60,
60-90, 90-120, 12-150 and 150-180 cm.
4. In saline-alkali soils, salt crust (visible or suspected) should be sampled
separately and sampling depth be recorded.
5. Sampling should be done every year if the field is under intensive
cultivation. If one crop per year is grown, sampling once in three years is
sufficient. Soil sampling should be done at the same time in each year.
3.3 Preparation of composite sample
For making composite sample, mix the collected soil samples thoroughly
by hand on a clean thick paper or cloth or polythene sheet. Reduce the bulk
 21

sample to about 500 g by quartering process in which entire soil mass is spread,
divided into four quarters, two opposite samples are discarded and the remaining
two are remixed. Repeat this process until about 500 g soil is left.
3.4 Sampling tools
Soil sampling can be done with the help of following tools:
i) Tube auger
ii) Screw type auger
iii) Post-hole auger
iv) Spade or Khurpi.
For sampling soft and moist soil, a tube auger, spade or khurpi is quite
satisfactory. A screw type auger is more convenient on hard or dry soil, while the
post-hole auger is useful for sampling in excessively wet areas viz. rice fields.
Tube Auger is convenient for sampling from lower depths. If a spade or khurpi is
used, a V-shaped cut may be first made up to the plough layer and a uniform 2
cm thick slice is taken out.
3.5 Labeling of samples
For identification, label the soil samples. A label of thick paper with
identification mark along with the details of the sample should be put inside the
sample bag and another label carrying same details tied outside the bag. In
addition to location, field number, name of cultivator and relevant information
about slope, drainage, previous cropping history, irrigation, fertilizer, manure used
etc. must be recorded.
3.6 Processing of soil samples for analysis
Processing of soil samples involves several procedures in sequence as
follows:
- Drying
- Grinding
- Sieving
3.6.1 Drying
The soil samples should be dried in shade at room temperature.
3.6.2 Grinding
 22

Crush the soil clods lightly and grind with the help of wooden pestle and
mortar. Care is taken so that primary sand and gravel particles are not crushed.
3.6.3 Sieving
Sieve the entire quantity of soil through 2 mm stainless steel sieve.
Remove plant residues, gravel and organic material as much as possible by
retaining them on the sieve. For specific type of analysis (e.g. organic C) grind
the soil further and pass it through the 0.2 to 0.5 mm sieves. Remix the whole
quantity of sieved soil before a sample is weighed for analysis.
3.7 Precautions in collection and processing of soil samples
In order to avoid the contamination, special care is required in the
collection and processing of soil samples. Following precautions should be
taken to minimize error:

 Keep away the sample from chemicals, fertilizers or manure.

 For sampling of micronutrient analysis, always use auger made up of

stainless steel instead of rusted iron khurpi or spade.

 Do not use bags previously used for storing fertilizers or any chemical.

 Store soil samples in clean cloth or polythene bags.

 Use glass or polythene jars for storage of soil samples for a longer
duration.
 23

4. Soil test procedures

4.1 Soil reaction (pH)
The acidity, neutrality or alkalinity of a soil is measured in term of
hydrogen ion activity (active-concentration) of the soil-water system. The pH may
+
be defined as the negative logarithm of H activity, which is expressed in gram
+
ions per litre. The H activity in a very dilute solution can be expressed as
concentration in gram per litre. Thus,
a + +
pH = -log10 H or -log10[H ]

Principle
The pH is a measure of hydrogen or hydroxyl ion activity of soil-water
system. It is based on the measurement of potential measured across a glass
+
electrode, on account of the difference in activity of H ions in and out of
electrode. It indicates whether a soil is acidic, neutral or alkaline in reaction. For
+ -
pH below 7, the H concentration exceeds OH and the range is acidic. When the
- +
OH concentration is more than H , pH lies in between 7 and 14 and range is
alkaline.
Equipment and apparatus
pH meter, balance, beakers, measuring cylinder, spatula, glass rod.
Reagents

 0.1M CaCl2 solution: Dissolve 1.47 g of AR grade of CaCl2.2H2O in one litre

of distilled water.

 Standard buffer solution: Prepare buffer solutions of pH 4.0, 7.0 and 9.2 in
distilled water. Alternatively, dissolve one commercially available buffer
tablet in distilled water and make the volume to 100 ml.
Procedure
(a) pH of saturated soil paste
1. Transfer 200 g of soil sample in a 500 ml beaker for making a saturated
soil paste.
2. Add small amount of distilled water to the soil while working with a
spatula, time and again till soil paste glitters and flows slightly without
 24

sticking to spatula or flows slightly when the container is tilted.

3. Allow the paste to equilibrate for about an hour. In no case, free water
should appear on the soil surface nor should the paste stiffen or lose its
glistening characteristics on standing.
4. Switch on the pH meter; set the knob for the temperature. Calibrate with
two buffers, one in the acidic range and other in the alkaline range or
neutral pH.
5. Carefully insert the glass and calomel or combined electrode in the paste
and measure pH.
6. Move the electrode a little to ensure removal of water film around it and
read the pH again till a constant value is obtained.
(b) pH in soil-water suspension
1. Transfer 20 g of soil in a 100 ml beaker, and add 40 ml of distilled water in
it.
2. Stir the suspension with glass rod intermittently for 30 minutes.
3. Again stir just before immersing the electrodes and read the pH.
(c) pH in 0.01M CaCl2 solution
1. Transfer 20 g of soil in a 100 ml beaker and add 40 ml of 0.01 M CaCl2
solution
2. Stir the suspension well for 30 minutes and the record the pH of the
suspension on a pH meter.
Precautions

 The electrodes must be washed with a jet of distilled water and dried
gently with the help of filter or tissue paper before each measurement.

 The electrodes, while not in use, should be immersed in distilled water to

avoid their drying.
Interpretation of results
pH Soil reaction rating
<6.5 Acidic soil
6.5 - 7.5 Normal soil
>8.5 Alkali or sodic soil
 25

4.2 Electrical Conductivity

Principle
Electrical conductivity is a measurement of soluble salts in the soil-water
system. Ions, like metals, allow the electric current to pass through them. Hence,
electrical conductivity (EC) of soil-water system increases with increasing
content of soluble salts in the soil. Thus, the measurements of EC give the
concentration of soluble salts in the soil at any particular temperature. The
reverse of resistance is conductance.
Equipment and apparatus
Conductivity meter, balance, beakers, measuring cylinder, glass rod.
Reagents
0
 0.01N KCl Solution: Dissolve 0.7456 g of AR grade KCl (dried at 60 ) in
distilled water and make volume 1 litre. The electrical conductivity of this
-3 -1 o
solution is 1411.8 x 10 i.e. 1.41 dSm at 25 C.
Procedure
1. Transfer 20 g of soil sample in a 100 ml beaker and add 40 ml of distilled
water and stir the suspension intermittently for 30 minutes.
2. Allow to stand until clear supernatant liquid is obtained. The clear extract
after pH measurement can also be used for EC measurement.
3. Calibrate the conductivity bridge with the help of standard KCl solution
and determine cell constant.
1.4118 μhos/cm
Cell constant = ----------------------------------------------------------
Observed conductivity of KCl solution

4. Determine the conductivity of the supernatant liquid with the help of

Conductivity Bridge.
Calculations
-1
Electrical conductivity, dSm = Observed value of EC x cell constant x
temperature
o
factor (to express result at 25 C).
 26

Interpretation of results
-1
EC (dS m ) Soil rating
<4.0 Non-saline soil
>4.0 Saline soil

4.3 Calcium carbonate by Puri's method

Principle
Calcium carbonate of the soil can be determined by titrating the soil
suspension with 0.5N H2SO4 in the presence of bromothymol blue and
bromocresol green indicators. Calcium sulphate and aluminium chloride are also
added to make the appearance of color very distinct. Aluminium chloride is
added in order to lower the pH of the soil suspension, and brings the color
change just at the point, where no carbonates are present. Without its use, even a
soil free from CaCO3 gives green color. So, AlCl3 make sharp distinction between
the soils containing carbonates and without carbonates.
Reactions involved
CaCO3 + H2SO4 CaSO4 + H2O + CO2
Equipment and apparatus
Conical flask, pipette, burette, volumetric flask, hot plate.
Reagents

 0.5N H2SO4

 0.1N AlCl3.6H2O: Dissolve 8.05 g of aluminium chloride in a small quantity

of water and dilute to 1 litre with distilled water.

 Bromothymol blue (1% alcoholic solution): Dissolve 1 g of Bromothymol

blue in 100 ml of alcohol.

 Bromocresol green (1% alcoholic solution): Dissolve 1 g of Bromocresol

green in 100 ml of alcohol.
Procedure
1. Weigh 10 g of soil and stir it with 100 ml of distilled water in a conical
flask.
2. Add 0.2-0.5 g of CaSO4 and bring the suspension to boiling and add 10 ml
 27

of 0.1N AlCl3.6H2O solution.

3. Shake the suspension and add 10 drops of bromothymol blue and
bromocresol green indicators and note down the color.
4. Green color shows the amount of CaCO3 present, in more than 1% and a
golden yellow color is indicative of the absence of CaCO3.
If carbonate is present, follow the procedure given below:
5. Bring the contents of the flask to boiling point and titrate against 0.5N
H2SO4. Boil for a minute or two after each addition and allow to settle for a
couple of minutes. Continue the titration till golden yellow color persists
on boiling for a couple of minutes. Note down the volume of 0.5N H2SO4
used for titration.
Calculations
Weight of soil taken = 10 g
Amount of 0.5N H2SO4 used = Y ml
Amount of H2SO4 used is equivalent to total CaCO3 in soil.
1ml of 0.5 N H2SO4 = 1 ml of 0.5 N CaCO3 = 0.5 meq. of CaCO3
= 50/2 x 1/1000 = 0.025 g CaCO3
% CaCO3 in soil = 0.025 x Y x 100
10
= 0.25 Y
OR
% CaCO3 in soil = Volume of 0.5N H2SO4 used
4
Interpretation of results
----------------------------------------------------------------------------------------------------------
CaCO3 (%) Soil rating
----------------------------------------------------------------------------------------------------------
<5% CaCO3 Fit for all crop
5-10% CaCO3 Fit for all crops except citrus
>10% CaCO3 Unfit for all crops
---------------------------------------------------------------------------------------------------------
4.5 Cation Exchange capacity (CEC) by Hesse (1971)
 28

Principle
The soil is leached with sodium acetate solution (pH 8.2) for replacement
+
of exchangeable cations by Na ions. The excess of salt is washed down by
+ +
alcohol and adsorbed Na ions are replaced by NH4 , using neutral normal
+
NH4OAC solution. The Na ions released from the exchanged sites are measured
in NH4OAC leachate with the help of flame photometer.
Reactions involved

- +
CH3COONa CH3COO + Na

- +
X-Soil + CH3COO + Na Na-Soil + CH3COO-X

- +
CH3COONH4 CH3COO + NH4

+ + +
Na-Soil + NH4 NH4 - Soil + Na

Equipment and apparatus

Centrifuge, centrifuge tubes, volumetric flask, funnels, flame photometer.
Reagents

 Sodium acetate solution, 1N: Dissolve 136 g of sodium acetate trihydrate

in water and dilute to 1 litre and adjust pH to 8.2 by using NaOH and acetic
acid solution.

 Ammonium acetate solution, 1N: Dilute 57 ml of glacial acetic acid and 68

ml of ammonium hydroxide to 800 ml of water. Dilute to 1 litre and adjust
to pH 7.0 by addition of more ammonium hydroxide or acetic acid.
OR

 Dissolve 77 g NH4OAC in one litre distilled water and adjust pH to 7.0 by

addition of more ammonium hydroxide or acetic acid.

 Ethanol, 95%.

 Standard Na solution: Dissolve 152 g of reagent grade NaCl in one litre of

distilled water. This is 60 ppm stock solution of Na. Transfer 2.5, 5, 10, 25,
50 and 75 ml of the stock solution in a series of 100 ml volumetric flask to
obtain 1.5, 3, 6, 15, 30 and 45 ppm Na, respectively.
 29

 Preparation of standard curve for Na: Record the flame photometer

readings for each of the working standards of Na after adjusting blank to
zero and 100 at 60 ppm. Draw a standard curve by plotting the readings
against different concentrations of Na on ordinary graph paper.
Procedure
1. Transfer 5 g of soil in a 50 ml stoppered centrifuge tube.
2. Add 30 ml of sodium acetate solution and shake for 5 minutes.
3. Centrifuge for about 5 minutes at about 8000 rpm until the supernatant
liquid is clear.
4. Decant and discard the liquid and repeat the shaking and centrifuging
three times more with fresh sodium acetate solution.
5. Shake the soil with 30 ml of 95% ethanol for 5 minutes, centrifuge and
discard the supernatant.
6. Repeat the ethanol washing three times to remove excessive amounts of
sodium acetate.
7. Finally extract the soil with three lots of 30 ml of NH4OAC solution,
centrifuging and decanting each washing into a 100 ml volumetric flask.
Dilute the combined extracts with NH4OAC to 100 ml.
8. Determine Na concentration in the extract by using flame photometer.
Calculations
Weight of soil taken = 5g
Volume made after leaching of NH4OAC = 100 ml
Concentration of Na in the leachate against reading from standard curve = X
ppm
X
+ -1
CEC [meq./100 g soil or Cmol (P ) kg ] = ------ x 2
23

4.6 Organic C
The organic C content of soils is estimated by using the following
methods:
1. Wet digestion method (Walkley and Black, 1934).
 30

2. Colorimetric method (Datta et al. 1962).

4.6.1 Wet digestion method
Principle
Organic matter in the soil is oxidized with a mixture of potassium
dichromate and concentrated sulphuric acid, utilizing the heat of dilution of
sulphuric acid. The excess of potassium dichromate, not reduced by the organic
matter of the soil, is determined by titration using standard ferrous ammonium
sulphate solution in the presence of sodium fluoride or phosphoric acid using
diphenylamine as indicator.
Reactions involved
(i) The oxidation of carbon
-
K2Cr2O7 + 4H2SO4 K2SO4 + Cr2 (SO4)3 + 4 H2O + 3O [x 2]
-
3C + 6O 3 CO2
________________________________________________________________________
2K2Cr2O7 + 8H2SO4 + 3C 2K2SO4 + 2Cr2(SO4)3 + 8H2O + 3CO2
________________________________________________________________________
(ii) Reactions involved during titration

FeSO¬4(NH4)2SO4. 6H2O FeSO4+ (NH4)2SO4+6H2O [ x 2]

-
2 FeSO4 + H2SO4 + O Fe2(SO4)3 + H2O
Nascent
oxygen
________________________________________________________________________

-
2FeSO4(NH4)2SO4.12 H2O + H2SO4 + O 2(NH4)2SO4 + Fe2(SO4)3 + 13
H2O
________________________________________________________________________
(iii) The action of diphenylamine indicator
+0 +0
2C6H5NHC6H5 2(C6H5.NHC6H4) C6H5N-C6H4C6H4N-C6H5
diphenylamine -H2O -H2O diphenyl benzidine (violet)

Equipment and Apparatus

Conical flask, pipette, burette, measuring cylinder, balance
Reagents

 1N Potassium dichromate: Dissolve 49.04 g of AR grade K2Cr2O7 in about

500 ml of distilled water and make the volume to 1 litre.

 Concentrated H2SO4.
 31

 0.5N Ferrous ammonium sulphate (Mohr's salt): Dissolve 196.1 g of AR

grade FeSO4(NH4)2SO4.6H2O in about 400 ml of distilled water. Add 20 ml
of concentrated H2SO4 and make the volume to 1 litre with distilled water.

 Sodium fluoride and /or phosphoric acid (85%).

 Diphenylamine indicator: Dissolve 0.5 g of diphenylamine indicator in a

mixture of 20 ml of distilled water and 100 ml of concentrated H2SO4.
Procedure
1. Transfer 1 gm soil in a 500 ml conical flask.
2. Add 10 ml of 1N K2Cr2O7 and 20 ml of concentrated H2SO4.
3. Swirl the contents of the flask 2 or 3 times and allow the flask to stand for
30 minutes on asbestos sheet for the reaction to complete.
4. Add 200 ml of distilled water to the flask to dilute the suspension.
5. Add 10 ml of orthophosphoric acid or 0.5 g of NaF and 1 ml of
diphenylamine indicator. A deep violet color will appear.
6. Titrate it with 0.5N ferrous ammonium sulphate till the color changes from
violet to blue and finally bright green.
7. Note the volume of the ferrous ammonium sulphate used in titration.
8. Carry out a blank titration (without soil), the same way.
Calculations
(i) Weight of soil taken = W g
(ii) Volume of 0.5 N ferrous ammonium sulphate required for reducing 10 ml
K2C2O7 solution (blank reading) = X ml.
(iii) Volume of 0.5 N ferrous ammonium sulphate required for reducing the
excess of dichromate (sample reading) = Y ml
Difference = (X-Y) ml
(iv) 1 ml of 1N K2Cr2O7 (= 1 meq.) = 3(=12/4) mg of C or = 0.003 g of C

(X - Y) ml x 0.003 x 100
Organic carbon (%) in soil = ---------------------------------- = Z
2xW

(v) Organic carbon (%) in soil = Z x 1.3 = R

 32

There is incomplete oxidation of organic matter in this procedure. The

organic carbon is multiplied by 1.3 on the assumption that there is 77%
recovery.
(vi) Assume that organic matter contains 58% carbon, thus, the organic matter
content in the soil will be calculated as under:
Organic matter (%) in soil = R x 100
58
= R x 1.724
Precautions

 Don't use an iron or steel mortar to grind the soil samples, especially
coarse textured one, as ferrous ions result in positive error in the
estimation.

 Ferrous ammonium sulphate should be standardized daily because of

2+ 3+.
slow oxidation of Fe to Fe

 For quality control, a minimum of one reference sample should be

analyzed per batch of 20-25 samples.

 High chloride content, as in case of saline soils, interferes in the

estimation of carbon. It can be prevented by adding Ag2SO4 @ 1.25% to
the concentrated H2SO4.
Interpretation of results
_________________________________________________________________
Organic C (%) Soil rating
_________________________________________________________________
< 0.4 Low
0.40 - 0.75 Medium
>0.75 High
________________________________________________________________
4.6.2 Colorimetric method
Principle
The oxidation of soil organic matter is carried out by dichromate-sulphuric
acid mixture. The intensity of green color of the chromium sulphate formed is
measured to give directly the amount of carbon oxidized.
Equipment and Apparatus
Colorimeter/spectrophotometer, conical flask, volumetric flask, pipette.
 33

Reagents

 1N potassium dichromate: Dissolve 49.04 g of K2Cr2O7 in distilled water

make to one litre.

 Concentrated H2SO4.

 Anhydrous sucrose (AR grade).

 Preparation of standard solution and standard curve: Weigh 5, 10, 15, 20

and 25 g of anhydrous sucrose (AR) in separate 100 ml conical flasks,
develop the color as per the procedure explained and read transmittance
or absorbance on a colorimeter/spectrophotometer. Draw a curve by
plotting the concentrations of C v/s transmittance
Procedure
1. Transfer 1 g of soil sample in a 100 ml conical flask.
2. Add 10 ml of 1N K2Cr2O7 and 20 ml of concentrated H2SO4 and swirl the
content of the flask.
3. Keep the flask on asbestos sheet for 30 minutes.
4. Centrifuge the contents at 8000 rpm for about 5 minutes to ensure the
complete setting of soil particles.
5. Decant the clear liquid into a colorimeter/spectrophotometer cuvette and
measure the intensity of green color in transmittance or absorbance using
red filter at 660 nm wavelength.
6. Simultaneously also carryout a blank (without soil).
Calculations
Find out a factor to be multiplied by sample reading to get organic
carbon content (%) of soil. If the standard curve is prepared accurately, the
average values of this factor comes to be 0.0042 with little variation.
Organic C ( %) in soil = Colorimeter reading x 0.0042
OR
Weight of soil taken =1g
Volume of 1N K2Cr2O7 and concentrated H2SO4 solution added = (10+20)=
30 ml
Dilution factor = 30/1 = 30 times
 34

Transmittance (%) of the test solution = T

Concentration of C as read from standard curve against the observed T =
A ppm
Organic C (%) in soil = (30 x A x 100)/1000
=3xA
4.7 Available-Nitrogen (Subbiah and Asija, 1956)
Principle
A known weight of the soil is mixed with excess of alkaline KMnO4
solution and distilled. The organic matter present in the soil is oxidized by the
nascent oxygen, liberated by KMnO4, in the presence of NaOH and the released
ammonia is condensed and absorbed in a known volume of a standard acid, the
excess of which is titrated with a standard alkali, using methyl red as an indicator.
Reactions involved
(i) Distillation
alkaline
2KMnO4 + H2O 2MnO2 + 2KOH + 3O
medium Nascent oxygen

Oxidative
-
R.CHNH2COOH + O R.CO.COOH + NH3
Organic-N fraction deaminiation ammonia
distillation
NH3 + H2O NH4OH
absorption
2NH4OH + H2SO4 ( NH4)2SO4 + 2H2O
(ii) Titration
H2SO4 + 2 NaOH Na2SO4 + 2 H2O

Equipment and Apparatus

Kjeldahl distillation set, measuring cylinder, pipettes, burette, conical flask,
heater.
Reagents
 0.32% potassium permanganate solution: Dissolve 3.2 g reagent grade
KMnO4 in distilled water; make up the volume to 1 litre.

 2.5% sodium hydroxide solution: Dissolve 25 g of sodium hydroxide

pellets in distilled water and make up the volume to 1 litre.

 N/50 H2SO4: Prepare 0.1 N H2SO4 by adding 2.7 ml of Concentrated H2SO4

 35

to I litre of distilled water. From this, prepare N/50 H2SO4 by diluting a

suitable volume five times with distilled water. Standardize it against N/50
NaOH solution.

 Methyl red indicator (0.15%): Dissolve 0.15 g of methyl red powder in

alcohol and make volume to 100 ml.

 Liquid paraffin (extra pure)

Procedure
1. Transfer 20 g soil in a 800 ml Kjeldahl flask.
2. Add 20 ml of water and 100 ml of 0.32% KMnO¬4 solution.
3. Add 1 ml of liquid paraffin and a few glass beads or broken pieces of
glass to prevent frothing and bumping during distillation.
4. Pipette out 20 ml of N/50 H2SO4 in a conical flask. Add 2-3 drops of
methyl red indicator and dip the end of the delivery tube into it.
5. Run tap water in the condenser.
6. Add 100 ml of 2.5% NaOH solution into the flask and cork it immediately.
7. Switch the heater on.
8. Distill the ammonia gas from the distillation flask and absorb it in
standard H2SO4 solution (test by bringing a moist red litmus paper near
the outlet of the condenser, which will turn blue as long as ammonia is
being evolved) and collect approximately 40 ml distillate.
9. After the completion of distillation, first remove the conical flask
containing distillate and then switch off the heater to avoid back sucking.
10. Titrate the excess of H2SO4 against N/50 NaOH and note the volume of
NaOH used. The end-point is reached when the color changes from pink to
yellow.
11. Run a blank (without soil) with each set of 10-15 samples.
12. Carefully remove the Kjeldahl flask after cooling and drain the contents in
the sink.
Calculations
Weight of soil taken = 20 g
 36

Volume of N/50 H2SO4 taken = 20 ml

Volume of N/50 NaOH used in titration =Y ml
Volume of N/50 H2SO4 used for NH3 absorption = (20 - Y) ml
1 ml of N/50 H2SO4 = 0.02 meq. of N = 0.28 mg N = 0.00028 g
Available N (ppm) in soil
(20-Y) x 0. 28 1000
= x
20 100
= (20-Y) x 28

Available N (Kg/ha) in soil = 2 x [(20 - Y) x 28]

Precautions

 Check all the joints of the Kjeldahl apparatus to prevent any leakage and
loss of ammonia.

 Opening ammonia bottles in the laboratory should be prohibited during

distillation and titration.

 Prepare 2.5% NaOH solution fresh as NaOH absorbs CO2 and strength of
alkali comes down.

 Steam the apparatus with distilled water before use.

Interpretation of results
Available N (Kg/ha) Soil rating
<250 Low
250-500 Medium
>500 High

4.8 Inorganic forms of nitrogen (ammonium, nitrite and nitrate) by Keeney and
Nelson (1982)
Principle
The method involves equilibrium extraction of a soil sample with 2M KCl.
+ - -
The MgO-Devarda alloy methods of determining NH4 , NO2 and NO3 are based
+
on the finding that NH4 -N in solution containing glutamine and other alkali-labile
organic compounds can be determined quantitatively from the NH3-N liberated
 37

by steam distillation of these solutions with a small amount of MgO for 2 to 3

+ - -
minute and that (NH4 + NO3 + NO2 ) - N in such solutions can be determined
quantitatively by same method if finely divided Devarda alloy is added
+ -
immediately before distillation. The method for determination of NH4 and NO3 in
-
the presence of NO2 developed from the finding that sulfamic acid decomposes
- + -
NO2 rapidly but does not react with NH4 or NO3 and does not interfere with their
determination by steam distillation with MgO and Devarda alloy.
Equipment and Apparatus
Kjeldahl distillation set, measuring cylinder, pipettes, burette, conical flask,
heater.
Reagents

 2M potassium chloride solution: Dissolve 149 g of reagent-grade KCl in 1

litre of water.

 Magnesium oxide (MgO): Heat heavy MgO in an electric furnace at 600-

o
700 C for 2 hours. Cool the product in a desicator containing KOH pellets
and store it in a tightly stoppered bottle.

 Devarda alloy.

 Mixed indicator: Dissolve 0.07 g methyl red with 0.1 g bromocresol green
in 100 ml of 95% ethanol (ethyl alcohal).

 Boric acid-indicator solution: Dissolve 40 g of pure boric acid (H3BO3) in

about 700 ml of hot water. Transfer the cooled solution to a 1 litre
volumetric flask containing 200 ml of ethanol and 20 ml of mixed indicator
solution. After mixing the contents of the flask, add approximately 0.05N
NaOH cautiously until the color is reddish purple. Then dilute the solution
to volume with water and mix it thoroughly.

 Sulphuric acid, 0.02N standard.

+ -
 Preparation of standard (NH4 + NO3 )-N solution: Dissolve 0.236 g of
ammonium sulphate (NH4)2SO4) and 0.361 g of potassium nitrate (KNO3)
in water and dilute to a volume of 1 litre in a volumetric flask. This solution
+ -
contains 50 ppm of NH4 -N and 50 ppm of NO3 -N.
- - -
 Preparation of standard (NH4 +NO3 +NO2 )-N solution: Dissolve 0.236 g of
 38

(NH4)2SO4, 0.123 g of NaNO2, and 0.361 g of KNO3 in water, dilute the

solution to a volume of 1 litre in a volumetric flask. This solution contains
+ - -
50 ppm of NH4 -N, 25 ppm of NO2 - N, and 50 ppm of NO3 -N.
Procedure
i) Extraction of Exchangeable ammonium, nitrite and nitrate
1. Transfer 10 gm of soil in a 125 ml wide mouth bottle and add 100 ml of
2M KCl.
2. Shake the bottle on a mechanical shaker for 1 hour.
3. Filter the soil-KCl suspension through Whatman No.42 filter paper and
store the filtrate in a refrigerator until analysis can be performed.
ii) Steam distillation method for exchangeable ammonium, nitrite and nitrate
+ - -
Table 1. Steam distillation methods for determining NH4 , NO2 and NO3 -N.
Form of N Method*
+
NH4 Steam distillation with MgO
-
NO3 Steam distillation with MgO and Devarda alloy after
-
destruction of NO2 with sulfamic acid and removal of
+
NH4 by steam distillation with MgO**.
+ -
NH4 + NO3 Steam distillation with MgO and Devards alloy after
-
destruction with NO2 with sulfamic acid**.
- -
NO3 + NO2 Steam distillation with MgO and Devarda alloy after
+
removal of NH4 by steam distillation with MgO
+ - -
NH4 + NO3 + NO2 Steam distillation with MgO and Devarda alloy

* In each method the NH3 liberated by steam distillation is collected in boric acid-
indicator solution and determined by titration with standard (0.02N) H2SO4.
-
** If NO2 is absent, the treatment with sulfamic acid is omitted.
iii) Procedure in the presence of nitrite
(A) Ammonical-Nitrogen
1. Pipette an aliquot (normally 10-20 ml) of the soil extract into a
distillation flask and add 0.2 g of dried MgO.
2. Pipette 20 ml of 4% Boric acid containing mixed indicator in a 200 ml
conical flask and place it under the receiver tube. Dip the receiver tube
end in the boric acid.
3. Distill the contents in Kjeldahl assembly at a steady rate and continue
 39

distillation until about 100 ml of distillate is collected.

4. Titrate the distillate against 0.02N H2SO4 taken in burette until pink
color starts appearing (end point: green to pink).
(B) (Nitrate + Nitrite)- N
+
 After removal of NH4 -N from the extract, add 0.2 g of Devarda alloy to the
distillation flask, and determine the NH3-N liberated by steam distillation
as described in Section (A).
(C) (Ammonium + Nitrite +Nitrate)-N

 Proceed as describe in Section (A), but add 0.2 g of Devarda alloy to the
distillation flask immediately after addition of MgO before connecting the
flask to the distillation apparatus.
(D) (Ammonium + Nitrate)-N

 Proceed as described in Section (C), but treat the sample in the distillation
flask with 1 ml of sulfamic acid solution, and swirl the flask for a few
-
seconds to destroy NO2 before the addition of MgO and Devarda alloy.
(E) Nitrate-N
- -
 Follow the procedure described for the determination of (NO3 +NO2 )-N
(Section B) but before the analysis in a sample that has been treated with
-
sulfamic acid to destroy NO2 as described in Section (D).
iv) Procedure in the absence of nitrite
1. Ammonium-N: Proceed as described in Section (A).
2. Nitrate-N: Proceed as described in Section (B).
3. (Ammonium + Nitrate)-N: Proceed as described in Section (C).
+ -
Note Magnesium oxide - Devarda alloy methods for determining NH4 , NO2 and
NO3-N in soil can also be applied directly by placing 2 g of soil and 10 ml
of 2M KCl and do the distillation by adding MgO or Devarda alloy for
+ - -
determining the NH4 , NO2 and NO3 - N by following the procedures, given
above.
Calculations
1 ml of 0.02 N H2SO4 = 0.00028 g N

(20-X) x 0.00028 Volume of KCl (ml)

 40

N release (mg/kg) = ----------------------- x -----------------------------------

Weight of soil (g) Volume of filtrate distilled (ml)

= W

N release (kg/ha) = W x 2.0

4.9 Available phosphorus

For the determination of available phosphorus in soils, two methods are
commonly used. The Olsen's method (Olsen et al., 1954) is used for neutral -
alkaline soils while the Bray's method (Bray and Kurtz, 1945) is used for acid
soils.
4.9.1 Olsen's Method
Principle
The soil is extracted with 0.5M NaHCO3 pH 8.5 in the presence of Darco G-
60 (which adsorbs dispersed organic matter and helps in giving clear extract).
Phosphorus in the extract is treated with ammonium molybdate, which results in
the formation of heteropoly complexes (phosphomolybdate). The
phosphomolybdate is reduced by the use of Sncl2 (a reducing agent). Due to this
6+ 3+ 5+
reduction, some of MO is converted to Mo and/or Mo , and the complex
assumes the blue color. The intensity of blue color obtained can be measured at
an appropriate wavelength on a visible spectrophotometer.
In calcareous, alkaline or neutral soils, NaHCO3 buffered to pH of 8.5
controls the ionic activity of Ca through precipitation of Ca as CaCO3. As a result,
-
the concentration of H2PO4 in solution increases. In acid soils, Al and Fe
-
phosphates (the H2PO4 ) concentration in the solution increases as pH rises by
way of suppressing Al and Fe activities by adding NH4F. Again the secondary
precipitation reactions are kept at minimum because the concentration of Al, Ca
and Fe remains at a low level in the extractant.
Reactions involved
(i) Extraction
Ca3(PO4)2 + 6 NaHCO3 3 CaCO3 + 2H3PO4 + 3 Na2CO3
 41

(ii) Color development

(NH4)6 Mo7O24.4H2O + 6 HCl 7 H2MoO4 + 6 NH4Cl

(ammonium molybdate) (molybdenic acid)

H3PO4 + 12 H2MoO4 H3P (Mo3O10)4 + 12 H2O

(phosphoric acid) (phosphomolybdate)
Yellow - colored complex

reduction
Phosphomolybdate + Sncl2 Reduced phosphomolybdate
(light yellow color) (blue color complex)

Equipment and apparatus

Spectrophotometer, pipette, volumetric flasks, funnel, Whatman No.1
filter paper.
Reagents

 0.5M NaHCO3 : Dissolve 42 g of sodium bicarbonate in one litre of distilled

water and adjust pH to 8.5 with dilute NaOH or HCl.

 Darco-G60 (activated charcoal) should be made free from soluble P by

repeated leaching with NaHCO3.

 1.5% Ammonium molybdate solution: Dissolve 15 g of ammonium

molybdate in 300 ml warm water. Cool to room temperature and add 300
ml conc. HCl. Cool and make volume to 1 litre.

 40% stannous chloride stock solution: Weigh 10 g of SnCl2 in a 100 ml

glass beaker. Add 25 ml of concentrated HCl and dissolve by heating. Cool
and store in an amber colored bottle in dark, after adding a small piece of
Zn metal to prevent oxidation. From this, prepare a dilute Sncl2 solution
(0.5 ml diluted to 66 ml with distilled water) immediately before use.

 Preparation of standard phosphate solution: Weigh 0.439 g of AR grade of

O
KH2PO4 (dried in oven at 60 C for 1 hr) in a one litre beaker, add about 500
ml of distilled water to dissolve. Add to it 25 ml of 7N H2SO4 and make
volume to one litre. This gives 100 ppm stock solution of P. From this,
prepare a 2 ppm solution by 50 times dilution of the stock solution and
prepare standard curve by sequential dilution of 2 ppm P solution to get a
 42

final P concentration range varying from 0.1 to 0.8 ppm.

Procedure
1. Transfer 2 gm of soil in a 100 ml wide mouth plastic bottle or glass bottle.
2. Add a pinch of Darco G-60 and 40 ml of 0.5M NaHCO3 solution.
3. Shake for 30 minute on mechanical shaker and filter the suspension
through Whatman No.1 filter paper.
4. Transfer 5 ml of the filtrate in a 25 ml volumetric flask, and add 5 ml of
ammonium molybdate solution.
5. Shake slowly and carefully to drive out the CO2 evolved.
6. When frothing completely ceases, add distilled water, washing down the
sides, to bring the volume to about 20 ml.
7. Add 1 ml of freshly diluted Sncl2 solution, make up the volume with
distilled water and mix the contents of the flask.
8. Read the blue color intensity at a wavelength of 660 nm using a red filter
on a spectrophotometer.
9. Run a blank with all the reagents except soil.
Calculations
Weight of soil taken = 2g
Volume of 0.5M NaHCO3 solution added = 40 ml
First dilution = 40/2 = 20 times
Volume of filtrate (extract) taken = 5 ml
Final volume following color development = 25 ml
Second dilution = 25/5 = 5 times
Total dilutions made = 20 x 5 = 100 times
Transmittance (%) of the test solution = T
Conc. of P as read from the standard curve
against the observed Transmittance (T) = A ppm
Available P (ppm) in soil = A x 100
OR
Available P (kg/ha) in soil = A x100 x 2.00
OR
 43

Available P2O5 (kg/ha) in soil = A x 100 x 2.00 x

2.29
Precautions

 The filtrate should be colorless, otherwise, add more Darco G-60, shake
and filter again.

 The dilute Sncl2 should be prepared just before use.

 The blue color starts fainting after 15 minutes. Therefore, take readings
within 10-15 minutes.

 If the intensity of the color is too high, take a smaller volume of the aliquot
and redevelop the color.
Interpretation of results
Available P (Kg/ha) Available P2O5 (Kg/ha) Soil rating
<10 <23 Low
10-20 23-46 Medium
>20 >46 High

4.9.2 Bray's Method (Bray and Kurtz, 1945)

Principle
The combination of HCl and NH4F is designed to remove easily acid-
soluble P forms, largely calcium phosphates, and a portion of the so-called
aluminum and iron phosphates. The NH4F complexes, aluminum and iron in acid
medium thereby protecting reprecipitation of the extracted phosphate ions.
Reactions involved
3NH4F + 3HF + AlPO4 H3PO4 + (NH4)3AlF6

3 NH4F + 3 HF + FePO4 H3PO4 + (NH4)3FeF6

H3PO4 + 12H2MoO4 H3P (Mo3O10)4+12H2O

(phosphate) (molybdenic acid) (Phosphomolybdete)

(yellow-colored)

Equipment and Apparatus

Same as previously described in section 4.9.1.
 44

Reagents

 Ammonium Fluoride (NH4F), 1N: Dissolve 37 g of NH4F in distilled water,

and dilute the solution to one litre. Store this solution in a polyethylene
bottle.

 Hydrochloric acid, 0.5N: Dilute 20.2 ml concentrated HCl to a volume of

500 ml with distilled water.

 Extracting solution: Pipette 15 ml of 1N NH4F and 25 ml of 0.5N HCl and

dilute to 500 ml with distilled water. This gives a solution composition of
0.03 N NH4F in 0.025N HCl.

 1.5% Dickman and Bray's reagent: Dissolve 15 g of AR grade ammonium

molybdate in 300 ml of warm water, cool and add 350 ml of 10N HCl.
Make the volume to one litre.

 40% SnCl2 solution: Same as previously described in section 4.9.1.

 Preparation of standard solution of P: Same as previously described in

section 4.9.1.
Procedure
1. Transfer 5 g soil in a 100 ml conical flask.
2. Add 50 ml of the extracting solution to the soil and shake for 5 minutes.
3. Filter through Whatman No.1 filter paper quickly so as to collect the
filtrate within 10 minutes.
4. Transfer 5 ml aliquot into a 25 ml volumetric flask.
5. Add 5 ml of ammonium molybdate solution, shake a little and dilute to
about 20 ml.
6. Add 1 ml of diluted SnCl2 (freshly prepared) and make the volume up to
the mark with distilled water.
7. Measure the intensity of blue color developed, using a spectrophotometer
at 660 nm wavelength by using a red filter.
Calculations
Same as previously described in section 4.9.1.
Interpretation of results
Same as previously described in section 4.9.1.
 45

4.9.3 Ascorbic acid reductant method (Watanabe and Olesen, 1965) for color
development in the extracts of P
The ascorbic acid method has proved to be reliable and less subjected to
interferences in color development than SnCl2 method. The color is stable for 24
hrs. This method is common for both the extractants i.e. NaHCO3 and Bray's.
Principle
The principle is based on the reduction of the (NH4)3PO4.2MoO3 complex
by ascorbic acid, in the presence of antimony-potassium tartrate. The blue color
complex is produced which is stable for 24 hrs, and is less subjected to
interfering substances than methods involving reduction by stannous chloride.
Reactions involved
For color development

(NH4)6 Mo7O24.4H2O + 6 HCl 7 H2MoO4 + 6 NH4Cl

(ammonium molybdate) (molybdenic acid)

H3PO4 + 12 H2MoO4 H3P (Mo3O10)4 + 12 H2O

(phosphate) (phosphomolybdate)
Yellow - colored complex

reduction
Phosphomolybdate + Ascorbic acid Reduced
phosphomolybdate
(light yellow) (blue color complex)

Reagents

 Solution A: Dissolve 12 g of AR grade ammonium molybdate in 250 ml of

distilled water. In 100 ml of distilled water, dissolve 0.291 g of antimony-
potassium tartrate separately. Mix both of the solutions and add 148 ml of
conc. H2SO4 and make the final volume to 2 litres with distilled water.

 Ascorbic acid solution (Solution B): Dissolve 1.056 g of ascorbic acid in

200 ml of solution A. This should be prepared fresh as and when required.
Procedure
1. Transfer 5 ml of extract (extracted with NaHCO3 or Bray's extractents)
in a 25 ml volumetric flask. Add 0.5 ml of 5N H2SO4 to it and shake for
 46

a while till CO2 evolution disappears.

2. Add 4 ml of ascorbic acid solution (solution B) to it and make the
volume with distilled water
3. Wait for 10 minutes and read the intensity of blue color using
spectrophotometer at 760 nm wavelength by using red filter.
4. Run a blank without soil in identical manner.
Calculations
Same as previously described in section 4.9.1.
Interpretation of results
Same as previously described in section 4.9.1.
4.10 Available (exchangeable) potassium
Principle
The method is based on the principle of equilibrium of soils with an
exchanging cation made of the solution of neutral normal NH4OAC in a given soil:
solution ratio. During an equilibrium, ammonium ion exchange with
exchangeable K ions of the soil. The K content in the equilibrium solution is
estimated with a flame photometer.

Reactions involved

K NH4
K NH4
Clay K + 5 CH3COONH4 Clay NH4 + 5 CH3COOK
K NH4

Equipment and apparatus

Mechanical shaker, flame photometer, pH meter, funnels, beakers, pipette,
conical flask, volumetric flask.
Reagents

 Neutral N NH4OAC solution: Dissolve 77.08 g of NH4OAC in distilled water

and make volume to 1 litre. Adjust the solution pH to 7.0 with glacial
acetic acid or NH¬4OH.

 Preparation of standard K solution: Make a stock solution of 1000 ppm K

o
by dissolving 1.908 g of AR grade potassium chloride (dried at 60 C for 1
 47

hr) in distilled water and diluting to 1 litre. Dilute suitable volumes of stock
solution to get 100 ml of working standard containing 5,10,15,20,25 and
40 ppm K.

 Preparation of standard curve for K: Record the flame meter readings for
each of the working standards of K after adjusting blank to zero and 100
at 40 ppm K solution. Draw a standard curve by plotting the readings
against different concentrations of K on ordinary graph paper.
Procedure
1. Transfer 5 g soil in a 100 ml conical flask.
2. Add to it 25 ml of neutral N NH4OAC solution and shake for 5 minutes and
filter through Whatman No.1 filter paper.
3. Measure K concentration in the filtrate using flame photometer.
Precautions
1. The filtrate should be clear.
2
2. The air pressure should be between 0.4 to 0.6 kg/cm .
3. The gas inlet should be opened after opening air inlet, and closed before
shutting off the air supply.
4. The flame should be soot-free and blue with all the burner cones visible
clearly.
5. The flame photometer should be warmed up for 10 to 15 minutes before
use with continuous distilled water feeding.
Calculations
Weight of the soil taken = 5g
Volume of the neutral N NH4OAC added = 25 ml
Dilution factor = 25/5 = 5 times
Reading of the flame photometer for the test sample = R
Concentration of K in the sample from standard
curve against the reading R = C

Available K (ppm) in soil = Cx5

Available K (kg/ha) in soil = C x 5 x 2.0 = C x 10
 48

Available K2O (kg/ha) in soil = C x 10 x 1.21

Interpretation of results
Available K2O (kg/ha) Soil rating

<125 Low
125- 300 Medium
>300 High

4.10 Available sulphur (Chesnin and Yien, 1950)

Principle
Soil is extracted with a solution of calcium chloride. Soluble sulphate
estimated in an aliquot of the extract, using barium chloride in the presence of
2-
gum acacia solution. The turbidity produced by the precipitation of SO4 as
barium sulphate is measured on spectrophotometer at 420 nm wavelength by
using blue filter.
Reactions involved
++ -
CaCl2.2H2O Ca + 2 Cl + 2H2O

- 2-
Soil Colloid SO4 + 2Cl Soil Colloid Cl + SO4

2+ -
BaCl2 Ba + 2 Cl

2+ 2-
Ba + SO4 BaSO4
(precipitate)

Equipment and Apparatus

Conical flask, volumetric flask, pipette and Whatman No. 42 filter paper,
spectrophotometer.
Reagents

 0.15% Calcium Chloride dihydrate (CaCl2.2H2O): Dissolve 1.5 g of

CaCl2.2H2O in about 700 ml of distilled water and make volume to 1 litre
with distilled water.

 Gum acacia solution, 0.25%: Dissolve 0.25 g of gum acacia in distilled

water, and dilute to 100 ml.

 Barium Chloride: Grind BaCl2.2H2O crystals in a mortar, until they pass

 49

through a 20 to 30 mesh, but retained on a 60 mesh sieve.

 Standard sulphate Solution: Dissolve 0.543 g of reagent grade K2SO4 in

one litre of distilled water. This is 100 ppm stock solution of S. Transfer
0.25, 0.50, 0.75, 1.0, 2.5 and 7.5 ml of the 100 ppm stock solution in a
series of 25 ml volumetric flask to obtain 1, 2, 4, 10, 20 and 30 ppm S,
respectively. Prepare a standard curve by plotting concentration vs
transmittance on a semi-log graph paper.
Procedure
1. Transfer 5 g of soil in a 100 ml conical flask and add 25 ml of 0.15% CaCl2
solution to it.
2. Shake for 30 minutes on a shaker and filter the suspension through
Whatman No.42 filter paper.
3. Transfer 10 ml of the aliquot to a 25 ml volumetric flask.
4. Add 1.0 g of the sieved BaCl2 crystals and shake for 1 minute.
5. Add 1 ml of gum acacia solution, make the volume to mark and shake for
1 minute.
6. Measure the turbidity, 25 to 30 minute after the precipitation, on
spectrophotometer, using a blue filter at a wavelength of 420 nm.
7. Run a blank as per the procedure given above but without soil.
Calculations
Weight of the soil taken = 5g
Volume of extractant added = 25 ml
Volume of aliquot of the extract taken
for developing turbidity = V ml
Final volume made = 25 ml
Transmittance (%T) as read from
spectrophotometer = T
ppm of S from the standard curve against T = C
First dilution = 25/5 = 5 times
Second dilution = 25/V times
Total dilution = 5 x (25/V) = 125/V times
 50

Available S (ppm) in soil = C x (125/V)

Available S (kg/ha) in soil = C x (125/V) x 2.0
Interpretation of results
Soils, having <10 ppm S is classified as S deficient.
4.11 Calcium and Magnesium (Cheng and Bray, 1951)
Principle
For the determination of Ca and Mg simultaneously or individually, the
versenate titration method is used in which EDTA, disodium salt, solution is used
to chelate them. A known volume of the solution is titrated with standard EDTA
using Calcon indicator in the presence of NaOH solution. The end point is a
change of color from pink to blue at pH 12 when whole of calcium forms a
complex with EDTA. For calcium plus magnesium, stable complex with EDTA is
formed at pH 10 using Erichrome Black T indicator.
Reactions involved

EDTA - Na2 H2Y.2H2O, where Y is tetravalent anion of EDTA

(General formula)
2+ -2 -2 +
Ca +HY CaY +2H
- -2 -2 -2 +
Ca + MgI n + H2Y Ca + MgY + HI n +H
Wine red Blue or green
(Ca + Mg present) (Ca + Mg absent)

-2 2++ -2 +
H2Y + Ca CaY + 2 H
Pink Blue
(Ca-present) (Ca-absent)

Equipment and Apparatus

Buckner funnel, vacuum pump, micro burette, pipette, magnetic needles,
stirring plate, china clay dish.
Reagents

 Neutral N NH4OAC solution: Dissolve 77.09 g of NH4OAC in distilled water

and make volume to 1 litre. Adjust solution pH to 7.0 by adding acetic acid
or NH4OH solution.

 Ammonium chloride-ammonium hydroxide buffer solution: Dissolve 67.5 g

 51

of NH4Cl in 570 ml of concentrated NH4OH and dilute to 1 litre with

distilled water.

 Sodium hydroxide, 4N: Dissolve 160 g of NaOH in I litre of water. Store in

plastic bottle.
O
 Standard CaCl2 solution, 0.01N: Dissolve 0.500 g of CaCO3, dried at 150 C,
in 10 ml of approximately 3N (1+3) HCl and dilute to a volume of exactly 1
litre.

 Erichrome Black T indicator: Dissolve 0.5 g of Erichrome Black T and 4.5 g

of hydroxylamine hydrochloride in 100 ml of 95 per cent ethanol.

 Calcon indicator : Dissolve 0.5 g of calcon in 100 ml of 95 per cent ethanol.

 EDTA (Di-sodium salt), 0.01N: Dissolve 2.0 g of ethylene diamine-tetra-

acetic acid-disodium salt and 0.05 g MgCl2.6H2O in distilled water, and
dilute the solution to 1 litre. Standardize it by titrating against 0.01 N CaCl2
solution.

 Polyvinyl alcohol: 1 % solution.

 Triethanolamine.
Procedure
i) Extraction of Ca and Mg
1. Transfer 10 g of soil in a 250 ml conical flask, and add 100 ml of 1N
NH4OAC (pH = 7.0).
2. The flask is stoppered and shake for several times and allow to stand for
overnight.
3. Transfer the content of the flask to a 5.5 cm-Buchner funnel in which
moist Whatman No. 42 filter paper is placed by gentle suction.
4. The soil is leached with an additional 400 ml of NH4OAC and determine Ca
and Mg in the extract.
Determination of Ca in NH4OAC extract
1. Pipette 5 to 25 ml aliquot of the extract (usually 5 ml) containing not more
than 0.1 meq. of Ca in to a china clay dish and dilute to a volume of
approximately 25 ml.
2. Add 0.25 ml (5 drops) of reagent 4N NaOH and 4-5 drops of Calcon
 52

indicator. Titrate with EDTA, using 10 ml micro burette. The color changes
from pink to blue. When close to the end point, EDTA should be added at
the rate of about a drop every 5 to 10 seconds, as the color change is not
instantaneous.
3. Carryout a blank (without Ca extract) in the same manner.

Determination of Ca + Mg
1. Pipette 5 to 25 ml ml aliquot of the extract (usually 5 ml) containing not
more than 0.1 meq. of Ca plus magnesium in a china clay dish and dilute
to approximately 25 ml with distilled water. Add 0.5 ml (10 drops) of
ammonium chloride-ammonium hydroxide buffer solution and 3 or 4
drops of erichrome black indicator.
2. Titrate with EDTA, using 10-ml micro burette. The color changes from
wine red to blue or green. No tinge of the wine red color should be
remained at the end point. Carryout a blank (without Ca + Mg extract) in
the same manner.

Determination of Mg

Mg is calculated from the difference between the (Ca+Mg) and the Ca

determination.

Calculations
T x normality of EDTA solution x 1000
Milliequivalent per litre Ca or (Ca + Mg) =
Aliquot (ml) taken

Where T = Volume in (ml) of standard EDTA used in titration.

Milliequivalent Ca or (Ca + Mg) per 100 gm soil

100 Extract volume (ml)

= ------------------------ x ------------------------ x meq. of Ca or (Ca + Mg)/litre
soil weight (g) 1000

or

ml. of EDTA solution used x normality of EDTA used x vol. of extract made
 53

x100
= ----------------------------------------------------------------------------------------------------
Vol. of aliquot taken for analysis x weight of soil

4.12 Available (DTPA extractable) Zinc, Manganese, Copper and Iron (Lindsay
and Norvell, 1956)
Principle
DTPA, a chelating agent, extracts the easily soluble zinc, iron, copper and
manganese by forming soluble complexes. The extracting solution is buffered at
pH 7.3 by triethanolamine (TEA) and also includes CaCl2 to prevent dissolution of
CaCO3. These conditions permit the right amount of Zn, Fe, Cu and Mn to be
extracted and CaCl2 to stabilize the pH of the extractant. DTPA extractant has the
++ ++.
ability to chelate Zn, Cu, Fe and Mn in competition with Ca and Mg The
elements in the DTPA extract are determined by atomic absorption
spectrophotometer.
Equipment and apparatus
Conical flasks, volumetric flasks, pipettes, pH meter, mechanical shaker,
Whatman No.42 filter paper, Atomic absorption spectrophotometer.
Reagents

 DTPA extracting solution: To prepare 1 litre of DTPA extracting solution,

dissolve 13.1 ml reagent grade TEA, 1.967 g of AR grade DTPA and 1.47 g
of CaCl2 in 1 litre of double distilled water. Allow sufficient time for the
DTPA to dissolve, and dilute to 900ml. Adjust the pH to 7.3 with dilute HCl
(1:1) while stirring and dilute to 1 litre. Addition of approximately 4 ml of
1N HCl will bring the pH of the solution to 7.3. This solution is stable for
several months. This reagent has 0.005 M DTPA, 0.1 M TEA and 0.01M
CaCl2.H2O.

 Preparation of zinc standard solution: Dissolve 0.439 g of AR grade

ZnSO4.7H2O in 200 ml of double distilled water in a 500 ml beaker. Add 5
ml of dilute H2SO4 (1:5) and make up the volume to 1 litre. This is a
standard solution of 100 ppm Zn. Dilute 10 ml of this standard solution to
100 ml in a volumetric flask to have a stock solution of 10 ppm. Dilute
 54

1,2,4 and 6 ml of stock solution (10 ppm) to a 100 ml volumetric flask to

get working standards containing 0.1, 0.2, 0.4 and 0.6 ppm Zn ,
respectively.

 Preparation of manganese Standard Solution: Dissolve 0.288 g potassium

permanganate (KMnO4) AR grade in 300 ml of double distilled water in a
0
500 ml beaker. Add 20 ml concentrated H2SO4, warm to about 60 C and
add oxalic acid solution drop wise to make the solution colorless. Cool
and transfer to a 1 litre measuring flask and make volume to the mark.
This is 100 ppm stock solution of Mn. Make use of this solution for the
preparation of working standards (0, 0.2, 0.4, 0.8, 1, 2, 5 and 10 ppm).

 Preparation of copper Standard Solution: Dissolve 0.392 g copper

sulphate (CuSO4.5H2O) of AR grade in 300 ml of double distilled water, and
make up volume to 1 litre. This is standard solution of 100 ppm Cu. To
prepare working standards dilute 100 ppm Cu stock solution to obtain
1,2,4,6 and 8 ppm Cu.

 Preparation of iron Standard Solution: Dissolve 0.702 g of AR grade

ammonium ferrous sulphate (NH4)2SO4 FeSO4.6H2O in 300 ml of double
distilled water in a 500 ml beaker. Add 5 ml of dilute H2SO4 (1:5). Transfer
to a 1 litre measuring flask and make volume up to the mark. This is a
standard solution of 100 ppm Fe. Finally prepare working standards
containing 1,2,4 and 6 ppm Fe by diluting appropriate volumes of 100 ppm
Fe. Feed the working standard solutions and prepare a standard curve by
plotting AAS readings against Zn, Mn, Cu and Fe concentrations.
Procedure
1. Transfer 10 g of soil in a 125 ml polyethylene bottle and add 20 ml of
DTPA extracting solution and close the mouth with stopper.
2. Shake continuously for 2 hrs on a shaker and filter through Whatman
No.42 filter paper.
3. Determine the content of these elements (Zn, Mn, Cu and Fe) in the DTPA
extract of the soil by Atomic Absorption Spectrophotometer using
respective cathode lamps.
 55

4. A blank solution (without soil) should also be run.

Calculations
Weight of soil taken = 10 g
Volume of DTPA extracting solution added = 20 ml
Dilution factor = 20/10 = 2 times
Available (DTPA-extractable) Zn/Mn/Cu/ Fe (ppm) = A x 2
Where A stands for the Zn/Mn/Cu/Fe concentration in aliquot as
read from standard curve against the sample readings.
Interpretation of results
Element Available micronutrient (ppm) Soil rating
Zn <0.6 Deficient
Mn <1.0 Deficient
Fe <4.5 Deficient
Cu <0.2 Deficient

Precautions
 For micronutrient analysis, soil sample must be taken preferably with a
stainless steel auger or khurpi from a desired depth.

 Soil sampling grinding should be done in wooden pestle and mortar and
sieved through a synthetic wire sieve.

 Only double distilled water should be used.

 Use only corning /borosil glass apparatus and AR grade chemicals.

4.13 Toxic Heavy Metals
Like micronutrients heavy metals such as Pb, Cd, Ni and Cr etc. can also
be extracted by DTPA extractant can be analysed by Atomic Absorption
Spectrophotometer. Standard curves are to be prepared separately for each
metal as per the requirement of the Atomic Absorption Spectrophotometer
method. The concentration limits of metals for preparing their standard curves to
obtain maximum sensitivity are given below:
Metal AAS Method Standard curve limits
(ppm)
 56

Pb Flame 10
Cd Flame 2
Ni Flame 8
Cr Flame 10
Co Flame 8
If a sample contains metal more than these concentrations, then dilution
of the sample is recommended to bring the concentration within the range of the
curve.
4.14 Available Boron
Out of several methods devised to assess the level of available B in soil,
hot water soluble B method of Berger and Truog (1939) has been most widely
accepted. Recent description of method includes some changes (Keren and
Bigham, 1985; Sippola and Ervco, 1987), but the basic procedure remains the
same. This method is rapid, reliable and more convenient to use than traditional
procedure employing carmine, curcumin or quinalizarin.
Principle
Boron is extracted from the soil with hot water and is subjected to
colorimetric estimation, following reaction with azomethine-H reagent.
Azomethine-H reagent form a stable colored complex with H3BO3 at pH 5.1, in
aqueous media.
Equipment and apparatus
Spectrophotometer, boron-free glassware, viz., conical flasks, volumetric
flasks, pipette, a dispenser, heater or water bath with a reflex system.
Reagents

 Azomethine-H reagent: Dissolve 0.45 g of azomethine-H in 100 ml of 1% L-

ascorbic acid solution in polypropylene reagent bottle. Fresh reagent
should be prepared each week and stored in a refrigerator.

 Buffer solution: Dissolve 250 g of ammonium acetate and 15 g of EDTA-

disodium salt in 400 ml of distilled water and slowly add 125 ml of glacial
acetic acid and mix.

 Boron standard solution: Dissolve 0.570 g boric acid (H¬3BO3) in distilled

 57

water and dilute to one litre to obtain a stock solution of 100 ppm B. Dilute
5 ml of the stock solution to 100 ml with distilled water which will give a B
concentration of 5 ppm. Pipette 0, 0.25, 0.50, 1.0, 2.0 and 4.0 ml of 5 ppm
solution to a 25 ml volumetric flask to have working standards of 0,0.05,
0.10, 0.20, 0.40 and 0.80 ppm B, respectively, for preparing a standard
curve.
Procedure
1. Transfer 25 g soil in a 250 ml B-Free (quartz) conical flask and add 50 ml
of water and about 0.5 g of activated charcoal.
2. Heat the flask until first sign of boiling and then reflux the contents for
exactly 5 minutes.
3. Allow to cool and filter through Whatman No. 42 filter paper.
4. Transfer 5 ml of the extract in a 25 ml volumetric flask and add 4 ml of
buffer solution and mix well.
5. Add 4 ml of azomethine-H reagent and mix the contents. The color is
allowed to develop for 1-hr, and the volume is made up to the mark.
6. Intensity of the color is measured on a spectrophotometer at 420 nm.
Read the concentration of B against the reading from the standard curve.
Precautions
Throughout the boron analysis, use of borosilicate glassware should be
avoided even for storage of chemicals. Plastic containers or corning/borosil
glassware should be used.
Calculations
Weight of the soil taken = 25 g
Volume of the extract (water) = V ml
Volume of aliquot taken = V1 ml
Final volume made up to = 25 ml
Transmittance (%) as read from spectrophotometer = T
Concentration of B (ppm) in the solution
against the transmittance T = C
First dilution = (V/25) times
 58

Second dilution = (25/V1) times

Total dilution = (V/25) x (25/VI) times = Z
Available B (ppm) in soil = CxZ
Available B (kg/ha) in soil = C x Z x 2.0
Interpretation of results
Soils having < 0.5 ppm hot water soluble boron is classified as boron deficient.
4.15 Available molybdenum
Principle
Molybdenum is extracted from the soil with a solution of ammonium
oxalate-oxalic acid in 1:10 soil: solution ratio. The extractant is advantageous as
it is easy to prepare, does not allow a significant change in pH during shaking
due to buffering action and molybdenum form stable complex with oxalic acid.
-
The extraction is based on the replacement of MoO4 ions from exchange sites,
which is irreversible due to formation of strong Mo-oxalic acid complexes.
Equipment and apparatus
Spectrophotometer, hot plate, refrigerator, water bath, muffle furnace,
separating funnel, glass stoppered flasks, volumetric flasks, pipettes.
Reagents

 50% Potassium iodide solution: Dissolve 50 g in 100 ml of double distilled

water (DDW).

 50% Ascorbic acid solution: Dissolve 50 g in 100 ml of DDW.

 10% Tartaric acid: Dissolve 10 g in 100 ml of DDW.

 10% Thiourea solution: Dissolve 10g in 100 ml of DDW and filter. Always
prepare a fresh just before use.
o
 Dithiol solution (Toluene-3,4-dithiol): One g of dithiol (51 C) is added in
o
100 ml of 10% NaOH solution. The contents are warmed up to 51 C with
frequent stirring for 15 minutes on a hot plate. Then 1.8 ml of thioglycolic
acid is added and stored in refrigerator.

 Isoamyl acetate solution.

 Ferrous ammonium sulphate solution: Dissolve 63 g of ferrous

 59

ammonium sulphate in distilled water, add 10 ml concentrated H2SO4 and

make the volume to 1 litre.

 Ethyl alcohol (Ethanol).

 Extracting solution: Dissolve 24.9 g of AR grade ammonium oxalate and

12.6 g oxalic acid in water, and dilute the solution to a volume of 1 litre.
Adjust the pH to 3.3 with dilute HCl or dilute ammonia solution.

 Standard Mo solution: Dissolve 0.150 g of AR grade ammonium

molybdate in 100 ml of 0.1 N NaOH, make it slightly acidic with dilute HCl
and make volume to 1 litre to obtain a stock solution of 100 ppm. Dilute 1,
2, 3, 4, 5, 6, 8,10 ml of 100 ppm standard Mo solution to 1000 ml with
water in volumetric flask to obtain respective working standards 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.8, 1.0 ppm of Mo in order to prepare standard curve.
Procedure
i) Soil Extraction
1. Weigh 25 g of soil in a 500 ml conical flask. Add 250 ml of the
extracting solution (1.10 ratio) and shake for 10 hours.
2. Filter the suspension through Whatman No. 42 filter paper. Collect 200
ml of the clear filtrate in a 250 ml beaker and evaporated to dryness on
a water bath.
o
3. Heat the contents in the beaker to dryness and then ignite at 500 C for
5 hours in a muffle furnace to destroy organic matter and oxalates.
4. Digest the contents of the beaker with 5 ml of HNO3-HClO4 mixture
(4:1), then with 10 ml of 4N H2SO4 and H2O2, each time bringing to
dryness.
5. Add 10 ml of 0.1N HCl and filter. Wash the filter paper with separate 10
ml of 0.1N HCl and double distilled water until the volume of filtrate is
100 ml.
6. Run a blank side by side without soil.
ii) Color Development
1. Transfer 50 ml of the filtrate in 250 ml separatory funnels and add 0.25
ml of ferrous ammonium sulphate solution and 20 ml of double
 60

distilled water and shake vigorously the funnel, up and down.

2. Add excess of KI solution and clear the liberated iodine by adding
ascorbic acid drop by drop on continuous stirring/shaking.
3. Add one ml of tartaric acid and 2 ml of thiourea solution and again
shake vigoursly.
4. Add 5 drops of dithiol solution and allow the mixture to stand for 30
minutes.
5. Add 10 ml of isoamyl acetate and separate out the contents (green
color) in glass cuvettes to read the colour intensity (% T) at 680 nm on
a spectrophotometer by using red filter.
Preparation of Standard Curve
Develop colour in working standard solutions by following the procedure
described above and read the colour intensity (% T) on a spectrophotometer at
680 nm and prepare standard curve by plotting concentration on X-axis and
readings (% transmittance) on Y-axis on a semi-log graph paper.
Precautions

 Keep separating funnel on a revolving type separatory funnel stand.

 Wash all glassware with alcohol before use.

 Dry separatory funnels before use.

 Separate out aqueous phase first and take out organic phase (green
coloured complex).

 Don't store dithiol solution for more than one week.

 Remove stopper from separating funnel before taking out the two phases.
Calculations
1. Weight of soil taken = 25 g
2. Volume of extracting solution added = 250 ml
3. Volume of filtrate taken for color development = 200 ml
250 1 C
Available Mo in Soil (ppm) = C x ------- x ------ = ------
200 25 20
Where C stands for Mo concentration obtained from standard curve against the
reading (%T)
 61

C
Available Mo (kg/ha) in soil = -------- x 2.0
20
Interpretation of results
A soil is classified deficient in Mo if its Mo content is less than 0.2 ppm.
 62

5. Plant Analysis
Knowledge of nutrient concentration in growing plants can serve as a tool
for correcting any deficiencies. Plant analysis is also an aid to soil testing. Error
in plant sampling may results in wrong interpretation of the results for making
recommendations. Therefore, selection of right plant part, stage of growth and
time of sampling are very important for plant analysis.
5.1 Plant sampling and sample preparation
For a meaningful plant analysis, collection of particular plant part and
stage of growth is very important. It would be wrong and wasteful to pluck any of
the growing plant part at any time and send to laboratory for analysis. Thus,
depending on the purpose of analysis, plant sampling needs to be planned. Plant
parts (index tissue) recommended to be sampled for analysis are given in Table
2. The index tissue sampling is designed for nutrient diagnosis, monitoring and
efficient nutrient management for optimum yield and excellent quality.
After collection of plant samples, fresh tissues should be free from dust
and other foreign material by washing with detergent solution followed by 0.1 N
HCl and deionized water. The 0.2% liquid detergent solution will remove waxy
coating on the leaf surface. The 0.1N HCl will remove metallic contaminants and
deionized water will wash the previous two solutions. Place the washed samples
on filter paper sheets and air dried for 24 hrs. Then put the plant samples in new
o o
paper bags to dry in a forced air oven at 65 C + 2 C for 48 hrs. Grind the samples
in an electric stainless steel grinder using 0.5 mm sieve. Put each sample in
oven and dry again for few hours more for constant weight. Store in paper bags
for further analysis.
Table 2. Plant parts recommended to be sampled for analysis
________________________________________________________________________
Category Crop Part to be sampled with stage/age
________________________________________________________________________
Grain, Wheat Flag-leaf, before head emergence
rd
Pulses, Rice 3 leaf from apex, at tillering
Oilseeds, Maize Ear-leaf, before tasseling
Fibre and Barley Flag-leaf at head emergence
Commercial Oat Flat-leaf, before inflorescence emergence
Crops Pulses Recently matured leaf, at bloom initiation
 63

Groundnut Recently matured leaflets, at maximum tillering

Sunflower Youngest mature leaf blade, at initiation of flowering
Mustard Recently matured leaf, at bloom initiation
rd
Soybean 3 leaf from top, after 2 months of planting
th
Cotton Petiole, 4 leaf from the apex, at initiation of
flowering.
Jute Recently matured leaf, at 60 days age
rd
Sugarcane 3 leaf from top after 3-5 months of planting
Sugar beet Petiole of youngest mature leaf at 50-80 days age
rd
Tea 3 leaf from tip of young shoots
rd th
Coffee 3 or 4 pair of leaf from apex of lateral shoots, at
bloom

Vegetables Potato Most recent, fully developed leaf (half-grown)

Tomato Leaves adjacent to influorescence (mid-bloom)
Onion Top non-white portion (1/3 to 1/2 grown)
Brinjal Blade of most recent fully developed leaves
Beans Uppermost, fully developed leaves
Cauliflower Most recent, fully matured leaf, at heading
Cabbage Wrapper leaf at 2-3 months age
Pea Leaflets from most recent, fully developed leaves at
first
bloom
Carrot Most recent, fully matured leaf, at mid-growth
Radish Most recent, fully developed leaf
Turnip Most recent, fully developed leaf
Beetroot At 30-50 days age.
th
Spinach 5 leaf from tip (omit unfurled) at the stage of bud
Starting to small fruits.
Cucumber Most recent, fully developed leaf.

Ornamental Bougainvillea Most recent, fully developed leaves at the stage of

plants Jasmine, flower bud of pea size.
Croton, Fern,
Ficus, Geranium
Gerbera,Fladiolus
Lilly, Orchid,
Hibiscus and Rose

rd
Fruit crops Almond 3 leaf from top, at the beginning of bloom
Apple, Pear Leaves from middle of terminal shoot growth,
th
8-12 weeks after full bloom, 2-4 weeks after
formation of terminal buds in bearing trees.
Blackberry Latest matured leaf from non-tipped canes,
th
4-6 weeks after peak bloom
Cherry Fully expanded leaves, mid shoot current growth in
 64

July-
August
Peach Mid-shoot leaves, fruiting or non-fruiting spurs, mid-
summer leaves, fruiting
Strawberry Youngest, fully expanded matured leaf without petiole,
at
peak or harvest period
Plum Leaves from middle of current season's extension
growth,
in January-February.
rd
Banana Petiole of 3 open leaf from apex, 4 months
after planting.
th
Cashew 4 leaf from tip of matured branches, at beginning of
flowering
th
Grapes 5 petiole from base at bud differentiation for yield,
and petiole opposite to bloom for quality
th
Citrus fruits 3-5 month old leaves from new flush,
st
1 leaf of the shoot, in June.
rd
Guava 3 pair of recently matured leaves, at bloom (August
or December)
Mango Leaf with petiole (4-7 month old) from middle of
shoot.
th
Papaya 6 petiole from apex, six months after planting
rd th
Pineapple Middle 1/3 portion of white basal portion of 4 leaf
from apex, at 4-6 months stage.
th
Pomegranate 8 leaf from apex at bud differentiation, in April
and August.
th
Falsa 4 leaf from apex, one month after pruning
th
Ber 6 leaf from apex from secondary or tertiary shoot, 2
months after pruning.
________________________________________________________________________
Sources: Extracted and modified from Kensworthy (1964), Tandon (1984),
Reuter and
Robinson (1986), Jones et al. (1991), Bhargav and Raghupati (1993),
Singh et
al. (1999).

5.2 Total Carbon by dry combustion method (Nelson and Sommer, 1982)
Principle
The dry combustion method is based on oxidation of C and thermal
decomposition of carbonate materials in a medium-temperature resistance
furnace. Loss of weight is determined gravimetrically.
Equipment and apparatus
 65

China crucible, balance, electric furnace.

Procedure
Weigh 5 g of the well-mixed plant/organic material in a preweighted china
o
crucible and put in furnace for 1 hr at 550 C. After an hour the furnace is allowed
to cool and the ash produced is weighed.
Calculations
Weight of crucible = W1
Weight of crucible + plant/organic material = W2
Weight of crucible + plant/organic material after ignition = W3
Weight of ash = W 3 - W1
W 3 - W1
% of ash in sample = ------------ x 100 = Z
W 2 - W1

% C in sample = (100 - Ash % + % total N and S) / 1.724

5.3 Total nitrogen

Total N in plant samples is determined commonly by two methods:
i) Modified Kjeldahl method (Bremner and Mulvaney, 1982)
ii) Colorimetric method (Lindner, 1944)
5.3.1 Modified Kjeldahl method to include nitrite and nitrate
Principle
Plant samples are digested in sulphuric acid and salicylic acid containing
substances that promote oxidation of organic matter and conversion of organic
+
N to NH4 - N, the substances generally favoured being salts such as K2SO4,
which increase the temperature of digestion, and catalysts such as Cu or Se,
which increase the rate of oxidation of organic matter by H2SO4. Salicyclic acid
forms nitro compound, which is reduced by Na2S2O3 to amino compound. The
+
NH4 -N in the digest is determined by collection of NH3 liberated by distillation of
digest with alkali. The distilled ammonia is quantitatively absorbed in boric acid
and titrated against standard alkali.
Equipment and Apparatus
Kjeldahl digestion assembly, ammonia distillation assembly conical flask,
 66

pipette, Kjeldahl flask, balance.

Reagents

 Sulphuric acid - salicylic acid mixture: Dissolve 25 g of salicyclic acid in 1

litre of concentrated H2SO4.

 Digestion mixture: Mix 10 parts of K2SO4, 1 part CuSO4.5H2O and 1 part

selenium powder.

 Sodium thiosulphate pentahydrate (Na2S2O3.5H2O): Powder crystals of

sodium thiosulphate pentahydrate to pass through a 60-mesh sieve.

 40% sodium hydroxide solution.

 0.1N Hydrochloric acid

 Mixed indicator: Dissolve 0.07 g methyl red with 0.1 g bromocresol green
in 100 ml of 95% ethanol (ethyl alcohal).

 Boric acid-indicator solution: Dissolve 40 g of pure boric acid (H3BO3) in

about 700 ml of hot water. Transfer the cooled solution to a 1 litre
volumetric flask containing 200 ml of ethanol and 20 ml of mixed indicator
solution. After mixing the contents of the flask, add approximately 0.05N
NaOH cautiously until the color is reddish purple. Then dilute the solution
to volume with water and mix it thoroughly.
Procedure
1. Weigh 0.5 g of plant material and transfer to a 300 ml Kjeldahl flask.
Moisten with a little water and then add 25 ml of sulphuric acid-
salicyclic acid mixture rotating the flask to ensure complete contact of
the acid with the material.
2. Shake it for some time and add 5 g of Na2S2O3.5H2O. Heat it gently for
5 minutes and cool.
3. Add 10 g of digestion mixture and heat it strongly until the solution
become clear. Continue heating for another 2 hours to complete the
digestion. Temperature during the digestion should be maintained in
o
between 360 to 400 C. At lower temperature, digestion is not
o
complete, whereas at temperature higher than 400 C, some ammonia
is lost due to decomposition of ammonium sulphate.
 67

4. Now pipette out 25 ml of 4% boric acid containing mixed indicator in a

250 ml conical flask. Place this flask in the distillation apparatus and
dip the end of the delivery tube in the boric acid.
5. Dilute the digested plant material with about 150 ml water and transfer
it to a 800 ml Kjeldahl flask and add few pieces of glass beads to avoid
bumping.
6. Add 100 ml of 40% NaOH solution in such a way that it runs down to
the bottom without mixing.
7. Fit it immediately in the distillation apparatus and ignite the burner.
8. Mix the solution thoroughly by swirling and continue distillation till
about 100 ml of the distillate is collected.
9. Titrate the boric acid solution with 0.1N HCl till blue color just
disappears and turns to pink.
10. Also carry out a blank (without plant material) in the same way.

Calculations

Blank reading = X ml of 0.1 N HCl

Amount of 0.1 N HCl used for neutralization of NH3 in boric acid = Y ml

Amount of 0.1 N HCl used for ammonia titration = (X - Y) ml

Weight of N = (X- Y) x 0.0014

(X - Y) x 0.0014 x 100
% of N in plant sample = -------------------------------
Weight of plant material taken

5.3.2 Colorimetric (Nessler's reagent) method (Lindner, 1944)

Principle
Plant samples are digested in diacid mixture of H2SO4 and HClO4 in the
ratio of 9:1. Colorimetric procedure, uses Nessler's reagent-potassium
tetraiodomercuriate (I4HgK2), from which an orange color complex of
oxidimercurammonium iodide, HgOHg(NH2)I is obtained. Intensity of coloured
complex is measured on a spectrophotometer at 440 nm wavelength by using
 68

blue filter.
Reactions involved

HgI2 + 2KI K2HgI4

NH3+ 2K2HgI4 + 3 KOH Hg2O (NH2I) + 7 KI + 2 H2O

Orange colour complex

Equipment and apparatus

Spectrophotometer, conical flasks, volumetric flask, wash bottle, pipette,
hot plate.
Reagents

 10% sodium hydroxide solution: Dissolve 100 g of AR grade NaOH in 1 litre

distilled water.

 10% sodium silicate solution: Dissolve 100 g of AR grade sodium silicate

in 1 litre of distilled water.

 Nessler's reagent: Weigh 45.5 g of mercuric iodide (HgI2) and place it in an

1 litre volumetric flask. Dissolve 35 g of KI in about 400 ml of distilled
water in a 500 ml beaker. Add KI solution to the flask containing HgI2
slowly by rotating the flask, so that all HgI2 is dissolved. Take another 500
ml beaker and dissolve 112 g of KOH in about 400 ml distilled water. Cool
it and add to HgI2 solution and make the volume up to 1 litre with distilled
water. Keep it for overnight, because some precipitation is there which
gets settled down. Next day, use the superanent liquid in analysis. The
solution is called Nessler's reagent.

 Preparation of standard solution and standard curve: Prepare a stock

solution of 50 ppm N from any nitrogenous compound viz. EDTA. From
the stock solution, take the appropriate quantity to prepare the working
standard solutions of 0.5, 1, 1.5, 2, 2.5 and 3 ppm N. Add all the reagents
except NaOH (because here, there is no acidity). Prepare standard curve
by plotting % transmittance (T) on Y-axis and concentration on X-axis on a
semi-log graph paper.
Procedure
 69

i) Digestion of sample
Weigh 0.2 to 0.5 g plant material depending upon the N content in the
plant in a 50 or 100 ml conical flask. Add 10 ml of diacid mixture of H¬2SO4 and
HClO4 in a ratio of 9:1. Keep it for overnight. Keep on a hot plate and heat gently
at first. Then heat more vigoursly until a clear colorless solution results or till
while fumes cease to come out. Do not take it to dryness. Discontinue heating,
when the volume is reduced to 3-4 ml. Cool it and transfer to 50 ml volumetric
flask and make volume up to the mark by adding distilled water. Filter it through
Whatman No. 42 filter paper and used for further analysis.
ii) Color development
1. Take 0.2-1 ml of digested plant material (depending upon the N content in
plant) in a 25 ml of volumetric flask and add about 5 ml of distilled water.
2. Add approximately 1-2 ml (depending upon the acidity of digested sample)
of 10% NaOH to neutralize the acidity of solution.
3. Add 1 ml of 10% sodium silicate (to avoid the turbidity) and then make the
volume to about 20 ml with distilled water.
4. Add 2 ml of Nessler's reagent, it will give orange color complex slowly and
slowly and make the volume up to the mark with the help of distilled water.
5. Read the intensity of color on spectrophotometer by using blue filter at
440 nm wavelength.
6. Calculate N content of sample using the standard curve.
Calculations
Weight of plant material taken = 0.5 g
Volume of the digested material made = 50 ml
Volume of digested plant material taken for
Color development = 0.2 ml
Final volume made = 25 ml
First dilution = 50/0.5 = 100 times
Second dilution = 25/0.2 = 125 times
Total dilution = 100 x 125 = 12500 times
 70

Transmittance (%) of the test solution = T

Concentration of N as read from standard
curve against transmittance T = A ppm
Total N (ppm) in plant sample = A x 12500

A x 12500
Total N (%) in plant sample = -----------
10,000
= A x 1.25
Precautions
 Flask should be neat and clean.
 Wash the neck of the volumetric flask after addition of each reagent.

 Always add calculated amount of NaOH to neutralize the acidity of the

plant digest.
5.4 Total P by Vanadomolybdophosphoric yellow color method (Koenig and
Johnson, 1942)
Principle
Phosphorus in the acid digest of plant samples can be determined by
following the method based on vanadomolybdo-phosphoric acid yellow color
developed by the reduction of heteropoly complex. Heteropoly complexes are
thought to be formed by co-ordination of molybdate ion with phosphorous as a
central co-coordinating atom, oxygen of molybdate is substituted for that of
phosphate.

Reaction involved
H3PO4 + 12H2MoO4 H3P (Mo3O10)4 + 12H2O
Heteropoly complex
Equipment and apparatus
Same as previously described in section 5.3.2.

Reagents

 Vanadate-molybdate reagent: Prepare solution 'A' by dissolving 25 g of

ammonium molybdate in about 400 ml of warm water. Prepare solution
 71

'B' separately by dissolving 1.25 g of ammonium metavanadate in about

o
300 ml of warm (70 C) water, cool it to room temperature and add 250 ml
of concentrated HNO3 and cool again at room temperature. Now add
solution 'A' to solution 'B' slowly with continuous stirring and dilute to one
litre with distilled water.

 6N HCl solution: Dilute 517 ml of concentrated HCl to one litre.

 2,4 di-nitrophenol indicator, 2.5%: Dissolve 2.5 g of 2,4 di-nitrophenol in

100 ml of 95 per cent ethanol.

 Ammonia solution.

 Preparation of standard curve: Dissolve 0.2195 g of dried KH2PO4 in water,

acidifying with 25 ml of 7N H2SO4 and dilute to 1 litre. This solution
contains 50 ppm P. From 50 ppm standard P solution, take appropriate
volumes to prepare working solutions of 0.05, 0.1, 0.2, 0.3, 0.4, 0.8 and 1.0
ppm. Develop the color in identical manner. Prepare standard curve by
plotting P concentrations on X-axis and per cent transmission on Y-axis
on a semi log graph paper.
Procedure
i) Digestion of sample
Weigh 0.5 g plant material in a 50 or 100 ml conical flask. Add 10 ml of
diacid mixture of HNO3 and HClO4 in a ratio of 4:1. Keep it for overnight.
Keep on a hot plate and heat gently at first. Then heat more vigoursly until
a clear colorless solution results or till while fumes cease to come out. Do
not take it to dryness. Discontinue heating, when the volume is reduced to
3-4 ml. Cool it and transfer to 50 ml volumetric flask and make volume up
to the mark by adding distilled water. Filter it through Whatman No.42
filter paper and used for further analysis.
ii) Color development
1. Transfer 1-5 ml of aliquot (digested plant material) in a 25 ml
volumetric flask.
2. Add 2-3 drops of 2,4 di-nitrophenol indicator.
3. Add ammonia solution till yellow color appears and now add 6 N HCl
 72

(drop wise) till it become colorless.

4. Add 5 ml of vanadomolybdate solution and dilute to 25 ml with distilled
water.
5. Mix well and read the intensity of yellow color on spectrophotometer
by using blue filter at 440 nm wavelength.
6. Run a blank without P solution simultaneously.
7. Calculate P content of sample using the standard curve against the
measured transmittance.
Calculations
Weight of plant material taken = 0.5 g
Volume of the digested material made = 50 ml
Volume of digested plant material taken for
color development. = 5.0 ml
Final volume made = 25 ml
First dilution = 50/0.5 = 100 times
Second dilution = 25/5.0 = 5 times
Total dilution = 100 x 5 = 500 times

Transmittance (%) of the test solution = T

Concentration of P as read from standard
curve against transmittance T = A ppm
Total P (ppm) in plant sample = A x 500

A x 500
Total P (%) in plant sample = -----------
10,000
= A x 0.05
5.5 Total potassium
Principle
Potassium in the acid digest of plant samples can be determined by using
flame photometer. It is based on the principle that atoms of some specific
element absorb energy from flame and get excited to the higher orbit. Such
 73

atoms release energy of a characteristic wavelength, which is specific for that

element and is proportional to the concentration of atoms of that element in a
sample.
Equipment and apparatus
Same as previously described in section 4.9.
Reagents

 Concentrated HNO3

 HClO4

 Standard K solution: Same as described in section 4.9.

Procedure
1. Digestion of sample: Same as previously described in section 5.4.
2. Potassium in the acid digest of plant samples can be determined using
flame photometer, as previously described in section 4.9. Depending upon
the concentration of K, the digest of plant samples can be used either
directly or after dilution for flame photometric determination of K.
Calculate K content of sample using the standard curve.
Calculations
Weight of the plant material taken = 0.5 g
Volume made after digestion = 50 ml
Dilution factor = 50/0.5 = 100 times
Flame photometer reading = 20 (say)
Potassium concentration (ppm) from
standard curve against reading =Y
Total K (ppm) in plant sample = Y x 100
Y x 100
Total K (%) in plant sample = -----------
10,000
= Y x 0.01

5.6 Total sulphur

Principle
Sulphur in the HNO3-HClO4 digest of the plant can be determined by
precipitation of sulphate as barium sulphate with the addition of BaCl2 salt
 74

(turbidimetric method).
Reactions involved
X.SO4 + BaCl2 BaSO4 + X.Cl2
precipitation
Equipment and apparatus
Same as previously described in section 4.10.
Reagents

 Concentrated HNO3 and HClO4

 Same as described in section 4.10.

Procedure
1. Digestion of sample: Same as previously described in section 5.4.
2. Follow the same procedure previously described (Section 4.10) for
available sulphur after extraction of soil.
Calculations
Weight of plant material taken = 0.5 g
Volume of the digested material made = 50 ml
Volume of digested plant material taken for
color development = 5.0 ml
Final volume made = 25 ml
First dilution = 50/0.5 = 100 times
Second dilution = 25/5.0 = 5 times
Total dilution = 100 x 5 = 500 times
Transmittance (%) of the test solution = T
Concentration of S as read from standard
curve against transmittance T = A ppm
Total S (ppm) in plant sample = A x 500
A x 500
Total S (%) in plant sample = -----------
10,000
= A x 0.05
5.6 Total calcium and magnesium
Principle
 75

A known volume of the acid digest of plant samples is titrated with

standard EDTA using calcon indicator in the presence of NaOH solution. The end
point is a change of color from pink to blue at pH 12 when whole of calcium
forms a complex with EDTA. For calcium plus magnesium, stable complex with
EDTA is formed at pH 10.
Equipment and apparatus
Same as previously described in section 4.11.
Reagents

 Concentrated HNO3 and HClO4.

 Standard stock solution of Ca: To 2.247 g of primary standard calcium

carbonate (CaCO3), add 5 ml of distilled water. Add drop wise a minimum
volume of HCl (approximately 10 ml), to effect complete dissolution of
CaCO3. Dilute to 1 litre with distilled water. This will give 1000 ppm Ca. 10
ml of this solution is diluted to 100 ml to get 100 ppm Ca concentration.

 Standard stock solution of Mg: Dissolve 10.141 g of MgSO4.7H2O in 200

ml of distilled water and make up the volume to 1 litre. This will give 1000
ppm Mg. Dilute 10 ml of 1000 ppm Mg to 100 ml in order to get a 100 ppm
Mg. Prepare a standard curve by plotting concentrations of Ca and Mg on
X-axis and % transmittance on Y-axis on an ordinary graph paper.
Procedure
1. Digestion of sample: Same as previously described in section 5.4.
2. Follow the same procedure as previously described (Section 4.11) for available
calcium and magnesium after extraction of soil.
Calculations
Weight of plant material taken = 0.5 g
Volume made after acid digestion = 50 ml
Dilution factor = 50/0.5 = 100
times
Ca or (Ca + Mg) content in plant sample (meq.) per 100 g plant material

ml of EDTA solution used x normality of EDTA used x 100

= -------------------------------------------------------------------------- x 100
 76

Volume of aliquot taken for analysis

5.8 Total Zinc, iron, manganese and copper

Principle
The Zn, Mn, Fe and Cu in acid digest of plant samples can be determined
with the help of Atomic Absorption Spectrophotometer (AAS).
The AAS is based on the principle that atoms of metallic elements (Zn, Mn,
Fe, Cu etc.), which normally remain in ground state, under flame conditions,
absorb energy when subjected to radiation of specific wavelength. The
absorption of radiation is proportional to the concentration of atoms of that
element. The absorption of radiation by the atoms is independent of the
wavelength of absorption and temperature.
Equipment and apparatus
Same as previously described in section 4.12.
Reagents

 Concentrated HNO3 and HClO4

 Preparation of standard solutions of Zn, Mn, Fe and Cu: Same as

previously described in section 4.12.
Procedure
1. Digestion of sample: Same as previously described in section 5.4.
2. Above-mentioned metallic cations can be determined in diacid digest
of plant samples using AAS.
Calculations
Weight of plant material taken = 0.5 g
Volume made after acid digestion = 50 ml
Dilution factor = 50/0.5 = 100
times
Concentration of Zn/Mn/Fe/Cu (ppm) in plant sample = M x 100
Where M stands for the Zn/Mn/Fe/Cu concentration in aliquot as
read from standard curve against the sample readings

5.9 Total boron

Principle
 77

Boron in the acid digest of plant sample can be determined by a

spectrophotometer using azomethine-H reagent.
Equipment and apparatus
Same as previously described in section 4.14.
Reagents

 Concentrated HNO3 and HClO4

 Same as previously described in section 4.14.

Procedure
1. Digestion of sample: Same as previously described in section 5.4.
2. Color development: Pipette 1 ml aliquot from the acid digested
solution and proceed for B determination by azomethine-H method as
previously described in section 4.14.
Calculations
Weight of the plant material taken = 0.5 g
Volume made after acid digestion = V ml
Volume of aliquot taken = V1 ml
Final volume made = 25 ml
Transmittance (%) as read from spectrophotometer = T
Concentration of B in the solution (ppm)
against the transmittance T =C

First dilution = (V/0.5) times

Second dilution = (25/V1) times
Total dilution = (V/0.5) x (25/VI) times = Z
Content of B (ppm) in plant sample = CxZ
5.10 Total Molybdenum
Principle
Molybdenum in the plant samples can be determined by ashing and fusion
of the sample.
Equipment and Apparatus
Same as previously described in section 4.15.
 78

Reagents

 Anhydrous Na2CO3.

 Ethanol

 All the reagents previously described in section 4.15.

Procedure
1. Transfer suitable quantity of plant material (1 to 10 g) in a platinum
dish.
o
2. Ash in an electric furnace at 500 C.
o
3. After ashing, fuse the ash with 2 g of anhydrous Na2CO3 at 1000 C in
the electric furnace, ensuring complete contact of the entire ash with
the flux.
4. Cool the dish and drop the cake in a 250 ml beaker by inverting the
dish.
5. If cake does not detach, place the dish in beaker and disintegrate the
cake in 100 ml water containing 2% ethanol (by volume).
6. Measure the suspension in a cylinder and filter through Whatman
No.42 filter paper in a 250 ml beaker.
7. Measure the undiluted clear filtrate and use for Mo determination as
previously described in section 4.15.
Calculations
Weight of the plant material taken = 0.5 g
Volume made after acid digestion = 50 ml
Volume of aliquot taken for color development = 20 ml
50 1
Mo content (ppm) in plant sample = C x ------- x ------ = C x 5
20 0.5
Where C stands for Mo concentration obtained from standard curve
against the reading (%T).
Interpretation of plant test results for macro and micronutrients
The optimum range (critical concentration) of macro and micronutrients in plants
is given in Table 3.
-------------------------------------------------------------------------
 79

Nutrients Optimum range

(Critical concentration)
------------------------------------------------------------------------
Macronutrients (%)
N 2.0-5.0
P 0.2-0.5
K 1.0-5.0
Ca 0.1-1.0
Mg 0.1-0.4
S 0.1-0.3
Micronutrients (ppm)
Zn 20-100
Mn 50-250
Cu 20-300
B 10-100
Mo 0.1-0.5
------------------------------------------------------------------------
Source: Extracted and modified from Singh et al. (1999).
 80

6. Soil analysis for total nutrients

6.1 Total N by modified Kjeldahl method to include Nitrate and Nitrite (Bremner
and Mulvaney, 1982)
Of the total amount of N present in soils, nearly 95-99% is in organic forms
and 1-5% in the inorganic form as ammonium and nitrate. Bremner and Shaw
(1955) gave salicyclic acid-thiosulphate modification of Kjeldahl method to get a
- -
quantitative recovery of NO2 and NO3 -N.
Principle
Soil is digested with sulphuric-salicyclic acid mixture in presence of
certain catalysts (Cu or Se) and salts (K2SO4), which raises the digestion
temperature. From the ammonium salts thus formed, ammonia is distilled with
sodium hydroxide and absorbed in boric acid - indicator solution. The excess of
boric acid left unneutralized can be determined by titration with standard HCl.
Reactions involved
i) Digestion
NaNO3 + H2SO4 Na2SO4 + 2 HNO3

C6H4(OH)COOH + HNO3 C6H3(OH)NO2.COOH + H2O

Salicylic acid Nitrosalicyclic acid

Na2S2O3 + H2SO4 Na2SO4 + H2S2O3

Sodium thiosulphate

H2S2O3 H2SO3 + S

C6H3(OH)NO2.COOH + 3 H2SO3 + H2O C6H3(OH)(NH2).COOH + 3H2SO4

(Amino salicylic acid)

2C6H3(OH)(NH2).COOH + 27 H2SO4 (NH4)2SO4+ 6SO2+ 14CO2 +

30H2O

ii) Distillation

(NH4)2SO4 + 2 NaOH Na2SO4 + 2NH3 + 2H2O

Distillation flask

+ -
NH3 + H3BO3 NH4 + H2BO3
 81

+ -
H + H2BO3 H3BO3

Ammonium borate formed by the reaction of NH3 with H3BO¬3 is converted

back to H3BO3, when distillate is titrated with H2SO4 or HCl.
Equipment and apparatus
Kjeldahl digestion assembly, ammonia distillation assembly, conical flask,
pipette, Kjeldahl flask, balance.
Reagents

 Sulphuric acid - salicylic acid mixture: Dissolve 25 g of salicyclic acid in 1

litre of conc. H2SO4.

 Digestion mixture: Mix 10 parts of K2SO4, 1 part CuSO4.5H2O and 1 part

selenium powder.

 Sodium thiosulphate pentahydrate (Na2S2O3.5H2O): Powder crystals of

sodium thiosulphate pentahydrate to pass through a 60-mesh sieve.

 40% sodium hydroxide solution.

 0.1N Hydrochloric acid.

 Mixed indicator: Dissolve 0.07 g methyl red with 0.1 g bromocresol green
in 100 ml of 95% ethanol (ethyl alcohal).

 Boric acid-indicator solution: Dissolve 40 g of pure boric acid (H3BO3) in

about 700 ml of hot water. Transfer the cooled solution to a 1 litre
volumetric flask containing 200 ml of ethanol and 20 ml of mixed indicator
solution. After mixing the contents of the flask, add approximately 0.05N
NaOH cautiously until the color is reddish purple. Then dilute the solution
to volume with water and mix it thoroughly.
Procedure
1. Weigh 5 g of processed soil sample in a 500 ml Kjeldahl flask and moisten
it with about 10 ml of distilled water.
2. Add 30 ml of sulphuric acid-salicyclic acid mixture and 10 g of digestion
mixture.
3. Shake the contents of the flask until thoroughly mixed, and allow to stand
for 30 minutes with frequent shaking.
 82

4. Add 5 g of sodium thiosulphate, heat the solution for 5 minutes and cool.
5. Heat first at low flame and then gradually raises the flame. Continue
digestion, until the mixture is colourless. Cool and add about 50 ml water.
Swril well and cool under water tap.
6. Transfer the solution by decantation to a 80 ml distillation flask. Wash the
contents of the digestion flask with water till free from acid. Do not
attempt to transfer the sandy material to the distillation flask.
7. Make up volume to about 400 ml and add a few glass beads to prevent
bumping.
8. Run a blank (without sample) under identical conditions.
9. Transfer 25 ml of 4% boric acid containing mixed indicator in a 250 ml of
conical flask and put it under the ammonia-receiving tube of distillation
assembly.
10. Add about 100 ml of 40% NaOH solution and immediately attach to the
alkali trap of the distillation unit.
11. Continue distillation for about half an hour collecting about 100 ml of
distillate and remove the conical flask.
12. Titrate it against 0.1 N HCl until a purple color appears to start.
Calculations
Blank reading = X ml of 0.1 N HCl
Amount of 0.1 N HCl used for neutralization of NH3 in boric acid = Y ml
Amount of 0.1 N HCl used for ammonia = (X - Y) ml
Weight of nitrogen = (X - Y) x 0.0014

(X - Y) x 0.0014
% N in soil = --------------------- x 100
Weight of soil taken

Interpretation of results

Total N (%) Soil rating

< 0.03 Low
0.03 - 0.06 Medium
 83

>0.06 High

6.2 Total nutrients except nitrogen

Principle
For the release of mineral elements from soil, wet oxidation method is
widely used. This method is easier, less-time consuming and convenient. Wet
oxidation employs oxidizing acids like HNO3- HClO4 diacid or HNO3-H2SO4-HClO4
tri-acid mixture.
Equipment and apparatus
Same as previously described in section 5.3.2.
Reagents

 Concentrated HNO3 (AR grade)

 HClO4 (AR grade)

 All reagents previously described in section 5.3.1.

Procedure
1. Transfer 2 g of soil in a 250 ml conical flask.
2. Add 10 ml of diacid mixture of HNO3 and HClO4 (4:1) and keep it for
overnight.
3. Put the flask on a hot plate in acid-proof digestion chamber having fume
o
exhaust system and heat at about 100 C for first one hour and then raise
o
the temperature to about 200 C.
4. Continue digestion until the contents become colorless and only white
dense fumes appear.
5. Reduce the acid contents to about 2-3 ml by continuous heating at the
same temperature. Do not allow the content to dry up.
6. Remove from hot plate, cool and add 10 ml of water.
7. Make the volume to 100 ml in volumetric flask with distilled water and
filter through Whatman No.42 filter paper and use for further analysis.
8. Total nutrients except N in the soil digest can be determined by following
the procedures previously described in section 5.4, 5.5, 5.6, 5.7, 5.8, 5.9
and 5.10 for determination of total P, K, S, Ca and Mg, Zn/Mn/Fe/Cu,
 84

boron and molybdenum, respectively.

Calculations
Follow the same method as previously described in section 5.4, 5.5, 5.6,
5.7, 5.8, 5.9 and 5.10 for determination of total P, K, S, Ca and Mg,
Zn/Mn/Fe/Cu, boron and molybdenum, respectively.
 85

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