Determination of soil pH

Principle

The soil pH or soil reaction is meant to measure acidity or alkalinity of soil and it gives the measure
of activity of H+ ions in the soil solution. The pH of a solution is measured by pH meter which consists
of a calomel-glass electrode assembly connected to a voltmeter. The voltmeter measures potential
difference between two electrodes which share a common electrolyte. The two electrodes are standard
reference electrode (of which potential is known-Calomel electrode) and a measuring electrode (Glass
electrode). Glass electrode used for pH measurement employs a glass membrane of special chemically
pure soft glass, which is sensitive to H+ ions. When the electrode assembly is immersed in a solution,
there develops a potential, which is based on Nernst equation and this is amplified electronically and
recorded on a millivolt meter calibrated in pH units. In order to measure the H + ion activity
potentiometrically, one more electrode is needed as a reference. A calomel electrode is used for this
purpose.

Apparatus required
• pH meter with glass and reference electrodes or with a
combined electrode.
• Glass wares: 50 ml beaker and glass rod
• Wash bottle, tissue paper etc.

Reagents- Buffer solutions- pH 4, 7, 9.2

Preparation of buffer solutions from tablets:

Dissolve one buffer tablets each (4, 7 and 9.2) in distilled water and make up to 100 ml to get the
desired pH.

Procedure

A. Caliberation of pH meter
1. Switch ON the instrument 10 minutes before to warm up.
2. Take approx. 30 ml of buffer solution (pH 4) in 50 ml beaker
3. Introduce electrode in the buffer solution
4. Adjust the standardization knobe to get a meter reading 4
5. Remove the electrode from the buffer solution
6. Wash the electrodes with distilled water, wipe clean and dry with tissue paper
7. Keep the electrodes back in the distilled water
8. Repeat the process with other buffer solutions
B. Preparation of soil water suspension
 1. Take 10 g air dried 2mm sieved soil sample into a 50 ml beaker
2. Add 25 ml distilled water
3. Stir it in shaker for 30 minutes
4. Stir the suspension with glass rod just before taking the reading
C. Determination of pH of soil samples
1. Stir the soil water suspension and introduce the glass electrode in it and note the reading
of the samples
2. Wash the electrodes with distilled water, wipe clean and dry with tissue paper (it should
be done after each samples)
3. Keep the electrodes back in the distilled water and switch OFF the instrument.

*if the electrode gets dry, fill with saturated KCl solution and allow it to rest for 24 hour

Soil reaction (pH) and Lime requirement

Sl. pH range Class Lime requirement

No. (Kg CaCO3 ha-1)
1. < 3.5 Ultra acid 1000
2. 3.5 – 4.5 Extremely acid 850
3. 4.5 – 5.0 Very strongly acid 600
4. 5.0 – 5.5 Strongly acid 350
5. 5.5 – 6.0 Moderately acid 250
6. 6.0 – 6.5 Slightly acid 100
7. 6.6 – 7.3 Neutral Not applicable
8. 7.4 – 7.8 Slightly alkaline Not applicable
9. 7.9 – 8.4 Moderately alkaline Not applicable
10. 8.5 – 9.0 Strongly alkaline Not applicable
11. > 9.0 Very strongly alkaline Not applicable
Determination of electrical conductivity of soil

Principle
Electrical conductivity is a measure of the ability of a solution to carry electric current. It is often made
use for salinity measurements. Electrical Conductivity can be measured in the sample that is prepared
in soil: water ratio of 1:2.5 which is used for pH determination. EC of soil samples is detected using
EC meter which is measured in terms of resistance to the flow of current and expressed in milli
mhos/cm or deci seimen/m(dS/m).As the amount of soluble salts increases, conductivity also
increases.

Apparatus required
• Conductivity meter
• Glasswares:50 ml beaker and glass rod
• Wash bottle, tissue paper etc.

Reagents (either of the following)

1) 0.01 N KCl – 0.7456 g KCl dissolved in 1L of distilled
water will have EC of 1.41 mmhos/cm at 250C
OR
2) Saturated calcium sulphate solution in distilled water gives an EC of 2.2 mmhos/cm

Procedure
1. Switch on the conductivity bridge and wait for 10 minutes.
2. Calibrate the instrument with standards (0.01 N KCl or standard calcium sulphate) and adjust
the knob to get reading of 1.41 and 2.2 mmhos/cm)
3. Take 10 g of soil in a beaker
4. Add 25 ml distilled water (1:2.5 ratio)
5. Shake well and allow to settle down
6. Supernatant of the suspension can be used for measurement of EC.
7. Immerse the electrode in soil solution (supernatant).
8. Note the reading in mmhos/cm

Rating of TSS (Conductivity in mmhos/cm)

TSS Remarks

Below 1 Normal, suitable for all crops

1-2 Suitable for semi tolerant crops

>3 Suitable for tolerant crops only

SOIL SAMPLING AND SOIL SAMPLE PREPARATION
Soil sampling is the process of extracting a small volume of soil for subsequent analysis at a soil
testing laboratory. The primary objective of soil sampling is to provide a representative sample of
the fertility status of the field or area. Utility of the results obtained from the laboratory depends on
the sampling precision.

Requirements : Sampling tools, Cloth/polythene bags, paper, pestle & mortar, sieve, etc.

Collection of soil sample

• This is one of the most important steps in soil testing programme.
• Sample must properly represent the area.
• A field can be treated as a single sample unit only if it is greatly uniform in all respects.
• Variation in colour, texture, crop growth and management would be taken into account and
separate sets of composites samples should be collected.
• Recently fertilized plots, channels, marshy areas, areas under trees, fences, roads, wells,
compost pits, area near water taps and other non representative locations must be avoided during
sampling.
• Once the fertilizer is applied, soil sample is taken only after 3 months.

Depth of Sampling
• Root penetration is an important factor to be considered for ascertaining the depth of sampling.

Particulars Sampling depth or Remarks

Routine soil testing Upto plough depth (0-15 cm)
Salt problem in Fixed depth (0-10 cm)
large areas
Reclamation Upto the plough layer; but the salt crusts (visible or suspected) on the
purpose soil surface should be sampled separately.
Seasonal crops & Upto 15 cm
cereals
Deep rooted crops Upto 30 cm
Horticultural plants Different depths depending on the root penetration of plants.
Sampling tools Screw-type Auger Post hole type Auger
1. Soft and moist soil - soil tube,
spade or Khurpi.
2. Hard and dry soil - Screw type
auger.
3. Excessively wet areas like rice
fields - Post hole auger.

Time of sampling
Summer is the good time to collect
soil sample because by this time
manures and fertilizer applied to the
soil would have equilibrated with the
soil. It is good to sample at least after 3 crops in order to maintain a good fertility status.

Information sheets
These should contain details of location, field number, name & address of the farmer/owner, name of
crop/cultivar/variety, irrigation practices, previous cropping history, fertilizers applied, the crops to be
raised in the next season, etc. This has to be sent alongwith the soil samples to soil testing laboratories.

Sampling procedure (Soil sample preparation)

For routine testing
• First traverse the field to be sampled and note the variation in slope, colour, texture etc.
• Demarcate the field into a uniform portion, each of which is to be sampled separately, use proper
sampling tools.
• Take composite samples from each uniform area.
• Scrape away the surface litter and then take the sample from surface to plough depth (0-15 cm).
• Take samples from 10-20 spots in the field depending upon the area in the field. Sampling is
done between plants or rows, with spade / Khurpi. When crops are planted in rows, samples are
drawn in between the lines.
• Dig a ‘V’ shaped hole and then cut out a uniformly thick (about 2 inches) slice of soil from the

exposed soil phase (cut ends) so as to get soil from top to bottom of the pit.
• Collect the sample in a clean bucket. Collect all the soil samples from uniform area in the same
bucket.
• Pour the soil from the bucket into a clean paper, mix thoroughly, spread the homogenized
sample in square share in uniform thickness in a level surface and divide the sample into 4 equal
parts.
 • Reject 2 opposite quarters. Mix remaining quarters and repeat the procedure to get approximate
500 g of soil.
• Sample is air dried in shade and stored in cloth or polythene bags with suitable identification.
Soil samples should be dried only under shade and not in the bright sun or in an oven.
Precaution in collection and storage of soil samples
● Care in handling soil sample against contamination is extremely important - Any contact with
chemicals, fertilizer and manures must be avoided.
● Cotton, jute or plastic bag which have been previously containing fertilizers, salts /lime should
not be used.
● Samples should be preferably stored in polythene bag.

For soil reclamation

On saline and alkali soils, samples can be taken by either using a soil auger or by digging a 90 cm
deep pit. In case a pit is dug, soil samples should be collected as follows:
1) Make one side of the pit vertical and put mark on it as 15, 30, 60 and 90 cm depth from the
surface.
2) Hold a suitable container at 15 cm markand scrap off a uniform of soil from the surface down
to this mark and collect about 500 g of the soil sample.Transfer the soil sample to a cloth bag
and mark it as 0-15 cm.
3) Similarly, coect 500 g soil sample from each layer, 15-30, 30-60, and 60-90 cm and put them
separately in 3 cloth bags after drying in shade.
4) Take a separate sample of the surface crust also, if any.
5) Prepare 2 label for each sample showing the depth from which sample has been taken, name of
the farmer, name of the village, exact location of the field, condition/growth of crop if any.
6) Put up one label inside the bag and the other on the bag. Label should be weitten with a copying
pencil.
7) Information sheet may also be prepared if necessary.
8) Send the sample alongwith the information sheet to the nearest soil testing laboratory.
Precautions
• Sampling should be done from a uniform piece of land.
• Each hectare land should be represented by at least 2-3 pits.
• If there is hard pan in the pit, it should be sampled separately andalso note its depth and
thickness.

Sampling for Garden plantation

The success of fruit tree plantation depends upon the physico-chemicalcharacteristics and fertility
status of the sub-soil layers. There fore it is necessary totest soil before tree plantation.Soil.sample for
plantation are taken as follows:
1) Dig a pit of 1.80 m depth and make its one side vertical.Put mark on it as 15, 30, 60, 90, 120,
150 and 180 cm depth from the surface.
2) Collect samples separately from 0-15, 15-30, 30-60, 60-90, 90-120, 120-150, and 150-180 cm
depths in the same way as that of the saline-alkali soils.
3) In case there is a hard pan in the pit, sample it separately and note down its depth and thickness.
4) Pack the soil sample in depth-wise in separate cloth bags.
5) Put up label indicating the depth, name of the farmer, name of village, location of the field, etc.
6) Send the samples to nearest soil testing laboratory, keeping record of the sample with you.
Precautions:
• Soil samples should be dried only under shade and not in the bright sun or in an oven.
• All materials used for collection and processing of samples should preferably be made-up of
stainless steel, plastic or wood in order to avoid contamination due to undesirable materials.
• During processing of samples efforts should be made to repeatedly crush and pass the whole of
the soil sample through the polyethylene sieve and contamination through carryover from one
sample to the other should be avoided.

Processing of soil samples in laboratory

Soil samples received in the laboratory should be alloted laboratory numbers.These should be air-dried
if moist and then clouds should be ground using a pestle and mortar (wooden, porcelain or stainless
steel), and sieved using polyethylene, iron or stainless steel sieve. Air-dried soil passed thorough.
Before sieving samples should be dried and clods are crushed with a pestle and mortar (made of wood,
porcelain or stainless steel). Plant residues, gravels and other foreign materials retained in the sieve
are discarded.

• Routine : 0.5 mm polyethylene sieve

• 2 mm sieve (made of iron or stainless steel)
• For analysis of organic carbon - sieve of fine mesh size (0.2 – 0.5 mm).
• The sieve made of brass or copper should be avoided if the sample is used for estimation for
micronutrients.

It is important that the samples are processed in a separate room away from the instrument room and
working laboratory. The samples, thus prepared should then be transferred to the soil samples storage
room.
Collection and preparation of plant samples for analysis
Plant analysis is done to supplement soil analysis and is used as a diagnostic tool for making
recommendations for fertilizer application to soil. Soil testing gives a measure of the potential
availability of nutrients to crops. Plant analysis indicate the actual removal of the nutrients by crops
from the soil. Plant analysis indicate the accessibility of nutrients from the soil. A knowledge of
nutrient concentration in the growing plant can serve as a tool for correcting any nutrient deficiency if
carried out early enough and can thus safe guard yield. It can also be used to evaluate the efficiency
of recently applied fertilizer. The information provided by analysis of mature or harvested plants can
help in planning nutrient application for the subsequent crop or for subsequent years.
Plant analysis is based on the assumption that the amount of a given element in plant is the
indication of supply that element in soil. But all the element do not enter the plant at a constant rate.
The movements of nutrients within plant will also show variation. Under normal growing condition,
nutrient absorption and plant growth run at parallel rates during the period of vegetable growth. Any
hindrance to normal growth rate varies the elemental composition within plant. Mobile nutrients move
from older parts to newly developing parts. Considerable movements also occur during seed
formation. Thus the nature of plant and stage of growth assume importance in plant analysis. Whole
plant analysis and Tissue analysis are the two types of techniques generally employed for plant
analysis.
Whole plant analysis
Whole plant analysis overrides the problem of uneven distribution of elements. But this is more
tedious as the volume of tissue to be handled is quite large & sometimes impossible & subsampling is
also difficult.
Tissue analysis
Tissue analysis is more rapid as a large number of plants scattered over a wide area can be
covered with a relatively small volume of sample. Tissue analysis is based on the assumption that for
each plant species certain plant parts will have uniform composition for at least a given period of time.

Sample collection
What to Sample
• Proper sampling requires a specific plant part to be taken such as a particular leaf, group of leaves
or portion of the plant. Instructions also include number of individual parts, as well as the number
of plants to sample. This will ensure that a sufficient quantity of plant tissue is submitted for
analysis and that the collected sample is statistically representative of the area under study.
• When no specific sampling instructions are given for a particular crop, the general rule of thumb is
to sample the uppermost recently mature leaves.
• Young emerging leaves, older mature leaves and seed are not usually suitable plant tissues for
analysis since they do not ordinarily reflect the general nutrient status of the whole plant.
• The recommended time to sample usually occurs just prior to the beginning of the reproductive
stage for many plants. However, sampling earlier or even later than the specified time may be
recommended for specific plants or circumstances.
• Sample plants that are showing a suspected nutrient deficiency symptom at the time or shortly after
the visual symptoms appear.
What Not to Sample
Do not include diseased or dead plant material in a sample. Do not sample or include plants or
leaf tissue that have been damaged by insects or mechanically injured in a sample. When whole plants
are sampled, remove the roots and wash the upper portion to remove soil particles. Do not sample
plants, which have been stressed extensively by cold, heat, moisture deficiency, or by excess moisture.
Examine both the below ground as well as the above ground portion of the plant. The presence of
nematodes or roots damaged by other insects or diseases should preclude the need to sample.

Plant tissue sampling guidelines for different crops

Crop Index tissue (reflect) Growth stage
Rice 3rd leaf from apex Tillering
Wheat Flag leaf Before head emergence
Sorghum 3rd leaf from inflorescence Blooming stage
Pulse Recently matured leaf Bloom initiation stage
Potato Most recently fully developed leaf Half growth stage
Cassava 4th leaf from apex Juvenile phase
Ground Recently matured leaf Maximum tillering
nut
Sunflower Young mature leaf blade Flower initiation stage
Sugarcane 3rd leaf 3 – 5 month after planting
Avocado Fully expanded leaf from non-fruiting 5 – 7 month after planting
branches
Banana Petiole of the 3rd leaf Bud differentiation stage
Cocoa 3rd leaf Bloom initiation stage
Cashew 4th leaf from tip of matured branch Beginning of flowering
Custard 5th leaf 2 months after new growth
apple
Grapes 5th petiole from base Bud differentiation stage for yield &
bloom stage for quality
Mango Leaves & petiole 4 – 7 month old leaves from middle of
the tree
Papaya 6th petiole 6 month after planting
Passion Mature leaf opposite to last fully opened Blooming stage
fruit flower
Pine apple Middle 1/3rd white portion of 4th leaf 4 – 6 month after planting
Tomato Leaf adjacent to inflorescence Mid bloom
Coconut 2nd leaf for seedling Repeated every year
14th leaf for mature palms
 Coffee 3rd or 4th pair of leaves from apex of lateral Blooming stage
shoots
Oil palm Middle 1/3rd mid rib of 3 upper & 3 lower Repeated every year
leaflets from the 17th frond of mature palms
and 3rd frond of young palms
Tea 3rd leaf from young shoot 3 – 4 month after planting
Clove 10 – 12 leaves from non fruiting shoots End of blooming
Onion Centre mature leaf Middle of growth period
Water Young fully developed leaf Middle of growth period
melon
Cucumber Middle fully developed leaves Blossom development stage

Sample preparation
After sample collection the fresh tissue should be decontaminated from dust or other foreign
material by washing in sequence in the following solutions taken in plastic containers
1. 2% liquid detergent like teepol: removes any waxy coating on leaf surface and any soil
particle.
2. 0.1 N HCl: Acid washing will help to remove the metallic contaminants.
3. Deionised water (distilled water): will wash out the previous solutions.

The extra moisture is wiped out and the sample placed in paper bags and dried in a hot air oven at 60
+ 5OC for 48 hrs. In case the sampling site is far away from laboratory requiring longer travel period,
the sample may be placed in perforated polythene bags and transported in refrigerated boxes. The low
temperature transportation will reduce the physiological activity to the minimum and will check the
spoilage of sample due to high temperature and humidity.
After drying the samples are milled or ground using a grinder which will not give any metabolic
contamination. The regular metallic mill with iron cutting blades & brass sieves will contaminate with
Fe, Cu, Zn. So instead, a mill made of hard plastic is the best especially if micro nutrient estimations
are to be done. The samples are then dried at a temperature of 70 0C before weighing for individual
analysis.

Critical nutrient concentration

The critical nutrient concentration (CNC) is the level of the nutrient below which crop yield,
quality and performance is unsatisfactory. Since the level of other nutrients in the plant affect this
concentration, it is difficult to choose this level. A more realistic approach is to use the concept of
critical nutrient range (CNR) which is the range of nutrient concentration at a specified growth stage
above which the crop is amply supplied and below which the crop is deficient. CNR values have been
developed for most of the essential elements for may crops. A combination of soil test data and leaf
nutrient levels can be used to arrive at a more realistic fertilizer recommendation for crops. However,
a lot of research data on leaf critical levels of nutrients for various crops/soils/climatic situations are
required. Approximate nutrient concentrations in the index leaf tissues of crop plants are given in the
table.
Table 1. Approximate nutrient concentrations in the index leaf tissue of crops

Nutrient Deficient Sufficient Average Toxic

N (%) - 1–5 1.5 -
P (%) - 0.1 – 0.4 0.2 -
K (%) - 1–5 1.0 -
Ca (%) - 0.2 – 1 0.5 -
Mg (%) - 0.1 – 0.4 0.2 -
S (%) - 0.1 – 0.4 0.1 -
Fe (mg kg -1) < 50 100 – 500 100 >500
Mn (mg kg -1) 15- 25 20 - 300 20 300- 500
Zn (mg kg -1) 10- 20 20 – 150 20 100 – 400
Cu (mg kg -1) 2-5 5 – 30 6 100 – 200
B (mg kg -1) 5 – 30 10 – 20 20 50 – 200
Mo (mg kg -1) 0.03 – 0.15 0.1 – 2.0 0.1 >100
-1
Cl (mg kg ) < 100 100 – 500 100 500 – 1000
Ni (mg kg -1) < 0.1 - 0.1 -
Source: Jones (1991) Tisdale et al., (1997)
 Determination of organic carbon in soil
(Walkley and Black’s rapid titration method)
Principle
Organic carbon present in the organic matter is oxidized to CO2 in the presence of K2Cr2O7 and H2SO4.
K2Cr2O7 produces nascent oxygen which combines with organic carbon producing CO 2. Excess
K2Cr2O7 which is not reduced by the organic matter of the soil is determined by titration with standard
ferrous ammonium sulphate (FAS).
Apparatus: Balance, 0.2mm sieve, beaker (250ml), Measuring cylinder(100ml), Burette
Reagents
1) 1 N K2Cr2O7 (Potassium dichromate): 49.04 g AR grade K2Cr2O7 dissolved in distilled water
and made up to 1 L
2) 0.5 N FAS: 196.1 g FAS is dissolved in 800ml distilled water to which 20 ml Con. H 2SO4 is
added, cooled the solution and made up to 1 L.
3) Con. H2SO4 : concentration not less than 96% (Sp.Gr .1.84).
4) Ferroin indicator (1,10-Orthophenanthroline ferrous sulphate): Dissolve 14.85 g of O-
phenanthroline monohydrate and 6.95 g ferrous sulphate heptahydrate in distilled water. Make
up the solution to 1000 mL.
5) Diphenyl amine indicator: Dissolve 0.5 g diphenyl amine in a mixture of 100 ml con. H 2SO4
and 20 ml distilled water. Indicator is colourless, store in a coloured bottle.
6) Orthophosphoric acid (85%)-used if diphenylamine is the indicator
Procedure
1) Take 1g soil (sieved through 0.2mm sieve) into a 250 ml beaker.
2) Add 10 ml 1N K2Cr2O7 and 20 ml Con. H2SO4 and stir for 1 min.
3) Keep it for 30 minutes.
4) Add 200 ml distilled water to this
5) Add either 2 drops of ferroin indicator or, 10 ml orthophosphoric acid and 1ml diphenyl amine
indicator.
6) Titrate against 0.5 N FAS.
7) End point is the appearance of brickred colour for ferroin indicator and brilliant green colour
for diphenyl amine indicator.
8) A blank titration is carried out with all reagents and without soil.

Calculation
▪ Wt. of soil = ‘W’g
▪ Vol. of FAS used for the blank= ‘B’ ml
▪ Vol. of FAS used for the sample = ‘S’ ml
▪ 1 ml of 1 N K2Cr2O7 =0.003 g carbon
➢ % organic carbon present in the sample = (B-S) x 0.5 x 0.003 x 100
W

Rating of Soil Organic Carbon

Sl.No. Soil O.C % Rating
1. <0.75 Low
2. 0.75-1.5 Medium
3. >1.5 High

Note: Since organic matter (O.M) contains 58% of organic carbon (O.C), multiplying O.C % with
Bemlen factor = 1.724 (or 100/58) will give % of O.M in soil.
O.M (%) = O.C (% ) × 1.724
Determination of soil available nitrogen

Principle

• Organic matter present in the soil is oxidized by nascent O2 liberated by KMnO4 in the presence
of alkali.
• Organic carbon will be lost as CO2 and organic N is converted to NH3 which reacts with Boric
acid and form Ammonium borate which is neutralized by titration with a standard acid using
Methyl red indicator.

Reagents

1. 0.32% KMnO4 – 3.2g KMnO4 is dissolved and made up to 1 L using distilled water.
2. 2.5% NaOH – 25 g NaOH is dissolved and made up to 1L using distilled water.
3. 2.5% Boric acid – 25 g boric acid is dissolved and made up to 1L using distilled water.
4. Mixed indicator-Dissolve 0.066 g of Methyl red and 0.099 g of Bromocresol green in 100 mL
of Ethyl alcohol.
5. 0.02 N H2SO4 – Dissolve 2.77 ml Conc.H2SO4 in distilled water and make up the volume to 1
L. This gives 0.1 N H2SO4. Take 200 ml of the 0.1 N solution and make up to 1 L with distilled
water to give 0.02 N H2SO4.

Procedure

1) Make sure that bottle on the top of the distillation assembly unit is filled with enough distilled
water.
2) Dip the tubes of alkali and KMnO4 in 2 conical flasks separately containing distilled water.
3) Place a blank long tube in the space provided in the instrument.
4) Open the tap for water supply to the instrument.
5) Switch on the instrument, press POWER button and wait for red light to blink in the ready
button.
6) Press RUN button near alkali and then press 50 ml KMnO4 button one by one to rinse the
tubes with distilled water already taken in conical flasks.
7) Then replace the conical flasks with original alkali bottle and KMnO 4 bottle, put the tubes in
respective bottles.
8) Take 5 g sample in the long tube and load the tube on the left side of the instrument.
9) Take 25 ml of 2.5 % boric acid in 250 ml conical flask and add 2 drops mixed indicator (colour
of the solution is pink). Place the conical flask on the right side of the unit.
 10) Set the time in alkali for 28 sec and press RUN button or add 50 ml of 2.5 % NaOH to
the long tube manually.
11) Press 50 ml KMnO4 button or add 50 ml of 0.32 % KMnO4 manually to the long tube.
12) Set the processing time for 8-9 minutes and press RUN key. (After complete processing,
colour of the solution in the conical flask turn from pink to green).
13) Take out the conical flask and titrate against 0.02
N H2SO4 taken in the burette.
14) End point is the appearance of light pink colour
(After doing analysis of all samples, rinse the tubes with
distilled water as described above and switch off the
instrument. Then close the tap 15 min later).

Calculation

1 ml of 1 N H2SO4= 0.014 g N2

Available N in kg/ha in the soil sample=TV x N x 0.014 x 100

x10,000 x 2.24
W
N – Normality of acid (0.02 N)

W – Weight of soil (5g)

Note:

Rating of Soil Available Nitrogen

Sl. N (kg/ha) Rating

No.
1. <280 Low
2. 280-560 Medium
3. >560 High
Determination of available phosphorus in soil

Principle
In an acid molybdate solution, the orthophosphate ions get precipitated as phosphomolybdate
complex that can be reduced by ascorbic acid, stannous chloride and other reducing agents. This
reduced phosphomolybdate has blue colour. The intensity of the blue colour varies with the P
concentration, which is measured using spectrophotometer at 660 nm using red filter. The intensity of
colour is directly proportional to P concentration of the medium.
Available P in acidic soils: Available P is commonly extracted using Bray No.1 (Bray and Kurtz,
1945), which consists of 0.03 N NH4F and 0.025 N HCl. The combination of HCl and NH4F is
designed to remove easily acid soluble P forms, largely calcium phosphates, and a portion of the Al
and Fe phosphates. The NH4F dissolves Al and Fe phosphates by its complex ion formation with these
metal ions in acid solution.

Reagents
1) Bray No.1 (extracting solution): Dissolve 2.22 g of solid NH4F, add 4.16 ml of Con. HCl and
made up to 2 litre of distilled water. This can be stored only in polythene bottles not in glass
bottles
2) Ammonium molybdate: Weigh 12 g Ammonium molybdate and dissolve in 250 ml of distilled
water
3) Antimony Potassium Tartarate: Weigh 0.2908 g antimony potassium tartarate and dissolve in
100 ml of distilled water
4) 2.5 M H2SO4: 140 mL of Con. H2SO4 is to be taken and make up to 1 L using distilled water
5) Reagent A (colourless): Add solutions mentioned in 2. (Ammonium molybdate) and 3.
(Antimony Potassium Tartarate) to 1000 mL of 2.5 M H2SO4 and make up the whole solutions
to 2 L using distilled water. It is stable and can be stored in brown pyrex bottle.
6) Reagent B (yellow or straw colour): 1.056 g of ascorbic acid is to be dissolved in 200 mL of
Reagent A. This reagent is only stable for 24 hrs. Prepare it freshly.
7) Standard P stock solution: Dissolve 0.439 g of pure dry KH2PO4 (Potassium dihydrogen
orthophosphate) in 500 ml distilled water and stir well. Add 25 ml of 7 N H 2SO4 and make up
the solution to 1L with distilled water. This gives 100 ppm of P (KH2PO4). From this, working
standards of 0,2,4,6,8,10 ppm are to be made.

Procedure
a) Preparation of working standards
1) Pipette out 0.5, 1, 1.5, 2 & 2.5 ml of 100 ppm P solution to separate 25 ml volumetric
flask to produce working standards of 2,4,6,8,10 ppm.
2) To these add 4 ml of Reagent B and makeup the volume with distilled water.
3) Blank: Add 5mL Bray no.1 and 4 ml of Reagent B and make up with distilled water.
4) After 10 min, blue colour of the soln. is read in spectrophotometer at 660 nm.
b) Extraction of P from soil sample
1) Weight out 5 g sieved soil (2mm) in a 100 ml shaking bottle or 250ml conical flask.
2) Add 50 ml of Bray solution
3) Shake for 5 minutes in a mechanical shaker.
4) Filter the soln. using Whatman no. 42 filter paper.
 5) Collect the filtrate into a beaker.
(To avoid interference of fluoride, 7.5 ml of 0.8 M Boric acid (i.e, 50 g of H3BO3 per litre) can
be added to 5 ml of the extract if necessary.)
c) Estimation of P in sample
1) Pipette out 5 ml of filtered soil extract into a 25 ml volumetric flask.
2) Add 4 ml of Reagent B and makeup the volume with distilled water.
3) Mix well and keep for colour development for 10 minutes and colour intensity is read in
a spectrophotometer at 660 nm (Colour is stable only for 20 min.)

Calculation
Amount of available P in soil (kg/ha) =’X’ x 50 x 25 x 2.24 where X→Con. of available P
5x5

Rating of Soil Available Phosphorous

Sl. No Soil Available P Rating

1. <11 Kg/ha Low
2. 11-24 g/ha Medium
3. > 24 Kg/ha High
Determination of available Potassium in soil

Principle

Exchangeable plus water soluble K contributes to the plant available pool of K in the soil which is
extracted with neutral 1N ammonium acetate and is determined using flame photometer. Soil to
extractant in the ratio 1:5 is commonly followed.

Reagents

1) Neutral 1 N ammonium acetate solution: Dissolve 77.08 g solid ammonium acetate in 900
ml of distilled water and adjust the pH at 7.0 by adding NH4OH or acetic acid as required and
make the volume to 1 L.
2) Standard KCl solution: Stock solution (1000ppm K)- Dissolve 1.908 g KCl (potassium
chloride) in distilled water and make up to 1L.
3) Working standard
i. 100ppm-Take 10ml from 1000ppm and make up to100 ml with distilled water
ii. 50 ppm- Take 5ml from 1000ppm and make up to100 ml with distilled water
iii. 10 ppm- Dilute 1 ml of 1000 ppm to 100 ml with distilled water.
4) Blank solution-Take 25 ml neutral normal ammonium acetate in a beaker

Procedure

A. Standardization of flame photometer

• Feed 10, 50, 100 ppm std. solutions and blank solution to the flame photometer and
calibrate the instrument.
B. Preparation of soil extract
1) Take 5 g in a 100 ml polythene shaking bottle
2) Add 25 ml neutral normal ammonium acetate
3) Shake it in a shaker for 5 minutes.
4) Samples should be filtered through a Whatman No. 1 filter paper immediately.
5) Then samples are fed to flame photometer and readings (ppm) are noted. (Fluctuation in
gas and air pressure does not allow steady reading in the meter and must be taken care
of)

Calculation

• Wt. of soil = 5 g
• Vol. of extractant added = 25 ml
 ➢ Available K in soil (kg/ha) = ’X’ ppm x 25 x 2.2 ; where, X-ppm of K obtained from the
instrument
5

Note:

Rating of Soil Available Potassium

Sl. No. Soil available K Rating

1. <110 Kg/ha Low
2. 110-280 Kg/ha Medium
3. > 280 Kg/ha High

➢ Available K2O (kg/ha soil) = Available K x 1.2

➢ Available K (kg ha-1) = Available K2O x 0.83
 Dry ashing and Wet digestion of plant samples
Prior to the determination of inorganic plant nutrients, the organic matter has to be destroyed to release
the nutrient elements from their combinations and to solubilize them. This is accomplished through
i. Dry ashing or dry oxidation
ii. Wet digestion or wet oxidation
(a) Dry ashing or dry oxidation
This method can be used for sample preparation for the determination of K, Ca, Mg, Cu, Fe,
Mn, Zn, B and Mo in plant tissue. It is the preferred technique for B & Mo. It provides good precision
and is an easy, rapid method requiring minimum analytical attention. It is also free from reagent
contamination. The main disadvantage of this procedure is that it cannot be used for elements such as
N, P, S which are volatile at the ashing temperature. The vessels used for ashing ranges from silica to
platinum dishes/crucibles. The sample size can vary from 0.5 to 2 g depending upon the expected
concentration of the elements to be determined. The ashing temperature used ranges from 475 - 600
O
C and duration of 4 – 12 hrs depending on the weight and type of the sample.. The ash residue is
usually dissolved in HNO3 or HCL and diluted to volume with distilled water. While determining Ca
and Mg the final sample solution should contain 1% Lanthanum to overcome potential anionic
interference.
Procedure for dry ashing
1. Place 1g of dried ground plant sample in a silica crucible.
2. Place the crucible in a muffle furnace at a temp of 550OC for 5 hrs.
3. The ash is then cooled & dissolved in 5 ml of 20% HCl, warming the solution if necessary to
dissolve the residue. Care should be taken to see that crucibles are at least 2cm away from the
walls and bottom of the furnace to avoid localized heating.
4. The solution is filtered through an acid washed filter paper into a 50ml volumetric flask.
5. The filter paper is washed & the solution is diluted with deionised water and make up to definite
volume.
6. Aliquots from this are used for individual analysis.
(b) Wet Digestion or wet oxidation
Wet digestion is done by boiling the plant sample with an acid or a mixture of acids as per
requirement as shown below.
1. Single acid digestion using H2SO4 alone:- for N estimation
2. Diacid digestion (HNO3:HClO4 in 3:1 ratio):- This is preferred digestion agent at almost
all nutrient elements except N.
3. Triacid digestion (HNO3:HClO4:H2SO4 in 9:3:1 ratio):- Triacid mixture is recommended
when P & K alone are to be estimated. This acid mixture cannot be used for the estimation
of S. H2SO4 may contribute some micronutrient elements. The use of HClO4 in the
mixture result in the formation of sparingly soluble KClO3 resulting in lower estimation
of K.

Procedure for single acid digestion

1. Accurately weigh 0.5 g of dried and ground sample
2. Feed the sample into tubes of Kelplus digestion assembly
3. Put a pinch of Digestion mixture (K2SO4/ Na2SO4, CuSO4 and Selenium powder in 100:10:1
ratio)
4. Add 10 ml of Con.H2SO4
5. Open the tap for water supply to the instrument
 6. Switch on the instrument and wait for the temperature to attain 350 0C which has already set in
the instrument
7. When it reaches 3500C, or when the digestion is completed, the solution becomes clear
8. Switch off the instrument
9. Then close the tap only after 15 min for cooling the instrument
10. Transfer the contents into 100 ml volumetric flask and make up to 100 ml (after cooling of
sample)
Procedure for Di acid digestion
1. Prepare digestion mixture with HNO3 and HClO4 (perchloric acid) in the ratio 9:4
2. Take 1 g of ground plant sample in a 100 ml conical flask
3. Add 10 ml digestion mixture and shake gently to mix well
4. Place the conical flask on hot plate and allow it to boil till the solution becomes clear and
colourless
5. Allow the flask to cool and transfer the solution to 100 ml volumetric flask and make up to
volume
*If the sample is high in fats and oils, pre-digestion using 25 mL HNO3/g sample is
recommended
DETERMINATION OF POTASSIUM IN THE PLANT SAMPLE
Principle
Potassium is estimated by Flame photometry. It is based on the principle that atoms or some
specific elements (alkali metals), take energy from flame and gets excited to higher energy orbits
These excited atoms before falling back to its original low energy orbit will release energy in the form
of light having specific wavelength which is unique for that element in the sample. The light emitted
is directly proportional to concentration of atoms of these elements in the sample.

Procedure

1. Preparation of K standard solution and K standard curve

Dissolve 1.906g KCl in 1 litre of distilled water to get 1000 ppm standard K solution.
From this, pipette 10ml into a 100ml volumetric flask and make up to get 100 ppm K.
From this, pipette out 2, 4, 6, 8, 10 ml to different 100ml volumetric flasks and make up with
distilled water to give working standards of 2,4,6,8 and 10 ppm K respectively.
Set violet filter (766 nm) for K in flame photometer. Aspirate 0 ppm (distill water), turn ‘blank’
knob and set the flame photometer to ‘zero’. Aspirate 10 ppm K standard, turn the ‘sensitivity’
knob and set the flame photometer to ‘100’. Repeat aspirating 0 ppm and 10 ppm solution
alternately until 0 and 100 readings are obtained automatically.
Now aspirate 2, 4, 6 and 8 ppm K solutions into flame photometer and note down the flame
photometer readings. Plot the readings against respective concentrations on graph paper (X axis:
K concentration in ppm, Y axis: Flame Photometer reading) and connect points with a straight
line.
NOTE: Readings can be read directly in concentration mode also.
2. Determination of K in plant digest
Pipette out 5 ml of digested sample (diacid) into 50 ml volumetric flask and make up the volume.
Now aspirate the plant sample into flame photometer and note down the flame photometer
readings.

Calculation

K in the given plant sample = Reading from the standard graph X dilution factor

OR

% K in plant sample
X Con. of K in plant sample from the instrument
 ESTIMATION OF SULPHUR IN PLANT SAMPLES

Principle
Sulphur (S) is estimated by Barium sulphate turbidimetry method. During di-acid digestion of
the plant sample, all the plant S is converted to sulphate form, which when treated with BaCl 2 is
precipitated as BaSO4. This provides white turbidity to the solution which is proportional to the amount
of sulphate present. Gum acacia solution is added to stabilize the turbidity. The turbidity developed is
read either on a photoelectric colorimeter using blue filter or on a spectrophotometer using 420 nm
wavelength.

Reagents
1. Sodium acetate- Acetic acid buffer (pH 4.8)
-Dissolve 100 g of Sodium acetate in about 500 ml of distilled water
-Add 31 ml of 99.5% Acetic acid (AR)
-Mix the contents and add 450 ml of distilled water
-Adjust the pH of solution to 4.8 and make the volume to 1 litre with distilled water
2. 0.25 % Gum acacia solution
Transfer 0.25 g of finely ground gum acacia into a 100 ml volumetric flask
-Make up the volume using distilled water (shake to dissolve the contents) and leave it
overnight.
-Then filter the solution and collect the filtrate.
3. 100 ppm SO4 S solution
Dissolve 0.5370 g of CaSO4. 2 H2O in acetate -acetic acid buffer and dilute to 1000 ml
with the buffer solution.
4. 1.0 ppm SO4 S solution
Dilute 1ml of 100 ppm SO4 S solution to 100 ml with sodium acetate-acetic acid buffer.
Procedure
• Prepare plant sample test solution from 1.0 g finely ground sample following wet digestion
procedure
• Similarly prepare the blank test solution without taking the plant sample
• Transfer 5 ml of sample and blank test solution into separate 25 ml volumetric flasks.
• Add to each 10 ml of Sodium acetate-Acetic acid buffers and 1 g of BaCl2 crystals (0.25 to 0.5
mm size)
• Allow it to stand for a minute and then cool the contents until the crystals are dissolved
• Then add to each 1 ml of 0.25 % Gum acacia solution (if the concentration of SO4 S is < 20
ppm. If the concentration of SO4 S is between 20-40 ppm, add 2 ml of 0.25 % gum acacia
solution)
• Mix the contents and dilute it to 25 ml with distilled water
• Measure concentration of BaSO4 turbidity developed with aliquot of the sample and blank test
solution using spectrophotometer at 420nm after 5 to 30 minutes of developing turbidity
• Prepare working standards of following con. and feed to spectrophotometer.
 0 ppm- Take 0 ml of 1 ppm SO4 S solution and make up to 25 ml with distilled water.
0.08 ppm- Take 2ml of 1 ppm SO4 S solution and make up to 25 ml with distilled water.
0.16ppm- Take 4 ml of 1 ppm SO4 S solution and make up to 25 ml with distilled water.
0.24ppm- Take 6 ml of 1 ppm SO4 S solution and make up to 25 ml with distilled water.
0.32ppm- Take 8 ml of 1 ppm SO4 S solution and make up to 25 ml with distilled water.
0.4ppm- Take 10 ml of 1 ppm SO4 S solution and make up to 25 ml with distilled water.
Calculation
S in plant sample (ppm) = A x 100 x 25
5x1
% S in plant sample = S in plant sample (ppm)/10000
Where A is con. of BaSO4 turbidity developed with the sample test solution which is read from
spectrophotometer.
 ESTIMATION OF PHOSPHORUS IN PLANT SAMPLE
(Vanadomolybdate phosphoric acid method in HNO3 system)

Principle
Vanadate, molybdate and orthophosphate radicals react together to give a yellow colour
heteropoly compound in HNO3 acid medium. The exact chemical composition is not known. Colour
is due to the substitution of oxyvanadium / oxymolybdate radical by the orthophosphate radical to give
a heteropoly compound which is chromogenic (yellow colour). Intensity of colour is read using
photoelectric colorimeter at 470nm. The main advantage of this method are its extra simplicity,
moderate sensitivity, freedom from interference from a wide range of other ionic species and stability
of colour. Colour develops in about 10 min.

Reagent:
Barton’s reagent (Vanadomolybdate reagent)
It is prepared by mixing the following solutions.
Solution A: Prepared by dissolving 25g of ammonium molybdate in 400 ml distilled water.
Soultion B: Prepared by dissolving 1.25g of ammonium metavanadate in 300 ml boiling water.
Solution B is cooled and 250ml of concentrated HNO3 added. Solution is again cooled to room
temperature. Solution A is then poured into solution B and the mixture is diluted to 1litre.

Procedure

Preparation of P standard solution and P standard curve

Prepare a 50ppm standard P solution by dissolving 0.2195g of potassium dihydrogen
orthophosphate (KH2PO4) in 1L distilled water. From that prepare 1, 2, 3, 4 and 5ppm P standards by
pipetting out 1ml, 2ml, 3ml, 4ml, 5ml aliquot from 50ppm standard solution into 5 different volumetric
flask of 50 ml capacity, add 10ml of vanadomolybdate reagent and diluting each to 50 ml. Read the
colour developed after 10 min in a photoelectric colorimeter with blue filter (470nm). Plot readings
against respective concentrations on graph paper (X axis: P concentration in ppm, Y axis: photoelectric
colorimeter reading) and connect points with a straight line.

Determination of P in plant digest

Take 10ml of the digest (diacid) in a 50ml volumetric flask. Add 10ml of vanadomolybdate
reagent and dilute to 50ml with distilled water. Read the colour developed after 10 min in the same
photoelectric colorimeter. Concentration of P in the given plant sample is calculated from the standard
curve.
Calculation

P in the given plant sample = Reading from the standard graph X dilution factor
Dilution = 50 x 100
factor
0.5 10
i.e. 0.5g plant sample made upto 100 ml; from this 10 ml aliquot is taken and made up to 50 ml
Dividing the value with 10,000 gives P content in the given plant sample in percentage
OR
% total P in plant samples = A x 50x 100
10x0.5x10000

=A x 0.1

(A=Con. of P from Spectrophotometer)

 Estimation of nitrogen in plant samples
Principle
N is present in oven dried powdered sample in the organic form, which is converted into inorganic ammoniacal form
(NH4)2SO4 by digestion. The digest when distilled in presence of an alkali will release ammonia which can be estimated. So after
digestion, this is followed by macro distillation, liberating Ammonia (NH 3) from the compound. This liberated NH3 is absorbed
in 4 % solution of boric acid containing mixed indicator. Back titrate the mixture with a standard acid taken in burette.

Apparatus required – Kjelplus Distillation Assembly

Reagents
1) 40% NaOH – 40 g NaOH dissolved and made up to 100 ml distilled water.
2) 4 % Boric acid – 4 g Boric acid dissolved and made up to 100 ml distilled water.
3) Mixed indicator – Dissolve 0.07g methyl red and 0.1 g bromocresol green in 100 ml of 95 % ethanol. (95
% ethanol – 95 ml ethanol dissolved and made up to 100 ml distilled water). (it is red in pH under 4.4, yellow
in pH over 6.2).
4) 0.02 N H2 SO4 – For preparing 0.02 N H2SO4, follow the calculation given below.
Volume (ml) of acid to be taken from conc. H2SO4 to get required normality

Procedure
• Make sure that bottle on the top of the distillation assembly unit is filled with enough distilled water
• Dip the tube of alkali in conical flask containing distilled water
• Place a blank long tube in the space provided in the instrument
• Open the tap for water supply to the instrument
• Switch on the instrument, press POWER button and wait for red light to blink in the ready button
• Then press RUN button near alkali to rinse the tube with distilled water already taken in conical flask
• Next replace the conical flask with alkali bottle and dip the alkali tube in alkali bottle
• Take 10 ml of digested and made up plant sample in the long tube and load the tube on the left side of the
instrument
• Take 10 ml of boric acid in 250 ml conical flask and add 2 – 3 drops of mixed indicator (Colour of solution
is pink)
• Place the conical flask on the right side of the unit
• Set the time in alkali for 15 sec and press RUN button or add 10 ml of 40 % NaOH to the long tube manually
• Set the processing time for 8 min and press RUN key. (After complete processing, colour of the solution in
the conical flask will turn from pink to green)
• Take out the conical flask and titrate against 0.02 N H 2SO4 taken in the burette
• End point is the appearance of light pink colour
(After doing analysis of all samples, rinse the tubes with distilled water as above and switch off the
instrument. Also close the tap 15 min later)

Calculation
1 ml of 1 N H2SO4 = 0.014 g N2
% of total N in the plant sample

N – Normality of acid (0.02 N)

W – Weight of dried and ground plant sample taken (0.5 g)

________________________________________________________________________________________________________
Agron 502_A1

Commercial fertilizers-composition, relative fertilizer value and cost, fertilizer mixtures and grades

Fertilizers

Fertilizers are industrially manufactured inorganic chemicals containing plant nutrients in higher amount than the organic manures; also,
release the nutrients immediately.

Classification

Supply of Total Concentration of Physical form Nutrient present Leaving residue on soil
Primary nutrient Primary nutrient
Straight High analysis : > 25% Solid Nitrogenous Acid-forming
Complex Low analysis : <25% Liquid Phosphatic Alkaline-forming/Basic
Mixed Potassic
Etc

Composition of commercial fertilizers

Mixed fertilizers (Complex fertilizers- supply 2 or more elements)

Factory/home mixed ores.

Factory:- thought on mixing compatibility & may be stored for long term.

Home:- Compatibility nit considered & have to be used soon after preparation.

References

Singh, S. S., Dr, S.S. Singh, Soil fertility and Nutrient management pages 78-117
NOTES
08/02/2024 – Shalini Ma’am
Magnesium
The only mineral constituent of chlorophyll molecule located at the centre.
Accounts for 15-20% of chlorophyll formation.

Functions:

• Synthesis of chlorophylls, protein; energy.

• Structural components of chlorophylls and ribosomes
• Has an indirect role in photosynthesis.
• Stabilizes ribosomal particles in the configuration necessary for protein synthesis.
• Activates polypeptide chain formation from amino acids.
• Involved in various physiological and biochemical functions.
• Associated with transfer reactions involving phosphate reactive groups.
• Involved in regulation of cell pH, cation-anion balance and the cell turgor.
• Enzyme activator for carbohydrate and ATP metabolism.
• Plays vital role in the activation of RUBISCO in chloroplast; enhances the enzyme affinity to CO2.
• Essential for the formation of oils and fats.
• Essential for the stabilization of cell membranes.
Deficiency:
As it is mobile, deficiency symptoms appear first on older leaves (lower leaves).