2. Determination of available micronutrient cations in soil
There are two methods employed for the determination of
available micronutrient status of soil.
1. Colorimetric method
2. Atomic absorption spectroscopy method (AAS)
3. • The colorimetric method is quite tedious method and for each
element, separate method of extraction and estimation has to
be followed.
• Use of AAS is quite convenient for rapid method of
estimation. Irrespective of the method followed for
estimation, extraction of only the available quantity of
nutrients is important.
• The extractant used should extract all of the available forms
of a nutrient from soils with variable properties.
• The amount extracted should correlate with the crops
response to that nutrient under various conditions.
4. Principle
• Diethylene triamine penta acetic acid (DTPA) is a chelating
agent which extracts the available forms of iron, manganese,
zinc and copper when buffered at pH 7.3. The simultaneous
extraction of these elements is done by shaking the soil with
DTPA for two hours. The concentration of these elements in
solution can be determined by using Atomic Absorption
Spectrophotometer (AAS).
5. Principle of AAS
• When a solution having a mixture of metallic species is
introduced into a flame the solvent evaporates and vapors of
metallic ions are obtained. Some of the metal atoms can be
raised to an energy level sufficiently high (excited state) to
emit characteristic radiation -a phenomenon used in flame
photometry. These atoms return to the ground state at which
they absorb light energy characteristic of their own resonance
wave length (the radiation which they would emit when they
are excited).
6. • The light radiation emitting from a hallow cathode lamp
(light source) absorbed by the ground state atoms will be
proportional to the number of atoms in the flame. So in
AAS, the amount of light absorbed is determined as the
amount absorbed is directly proportional to the
concentration of the element.
7. Reagents
• DTPA extractant (0.005 M DTPA, 0.01 M CaCl2 and 0.1 M
Tri ethanol amine [TEA] with pH 7.3):
• Dissolve 149.2 g of reagent grade TEA, 19.67 g DTPA and in
approx. 200 mL glass distilled water. Dilute to about 9 L and
adjust the pH to 7.3 using dilute HCl or NaOH and make up
to 10 L. This solution is stable for several months.
8. Standard solutions of micronutrient cations
• The standard solutions are prepared using the extractant used
for extraction of soil.
1. Iron standard (100 ppm Fe): Dissolve 0.7022 g of fresh FAS
(ferrous ammonium sulphate) in glass distilled water and dilute
to 1 L. Prepare working standards ranging from 0 to10 ppm.
2. Manganese standard (100 ppm Mn): Dissolve 0.406 g of
MnSO4.4H2O in glass distilled water containing 1 mL of conc.
H2SO4 and dilute to 1 L. Prepare the standards ranging from 0
to 10 ppm.
9. 3. Zinc standard (1000 ppm Zn): Dissolve 0.4398 g of
ZnSO4.7H2O in glass distilled water and dilute to 1 L.
Dilute this to get 100 ppm and prepare working
standards ranging from 0 to 5 ppm.
4. Copper standard (100 ppm): Dissolve 0.393 g of
CuSO4.5H2O in glass distilled water and make up to 1 L.
Prepare working standards ranging from 0 to 5 ppm.
10. Procedure
Soil extraction
1. Weigh 10g of soil (2mm sieved) and transfer to a 100 ml
conical flask
2. Add 20 ml DTPA extractant (1:2 ratios) and shake on a
mechanical shaker for 2 hrs.
3. Filter the content using whatman No 42 filter paper
4. Prepare a blank in which all reagents are added similarly
except the soil
11. Estimation and standard curve preparation.
1. Feed this filtrate solution to the calibrated AAS using
standards and record the readings as absorbance value
2. Adjust the different wave length of light and change the
specified hallow cathode lamp for each element before taking
sample the readings.
3. Meanwhile prepare the standard curve for four elements using
standard solutions of respective compounds. Use highest
concentration of standards of each metal and calibrate it.
4. Compare the OD or Absorbance of unknown sample with
concentration of standards to get graph ppm value.
12. Element Salt/compound
Wt.of salt for
100ppm(g/L)
Range of
Std.conc. (ppm)
Wavelength
(nm)
Zn ZnSO4.7H2O 0.4398 0 - 5 213.9
Cu CuSO4.5H2O 0.3929 0 - 5 324.7
Mn MnCl2.4H2O 279.50 0 - 10 279.5
Fe FeSO4.7H2O 248.30 0 - 10 248.3
13. Calculation
Micro nutrient
conc. (ppm)
=
Graph value × volume of the extractant
Weight of the soil
Observation
Weight of soil = A gms
Volume of extractant = X ml
Volume made = Y ml
Aliquot taken = Z ml
14. Critical limits for micronutrients
(DTPA extractable at pH 7.3)
1. Zinc - 0.6 ppm
2. Copper - 2.0 ppm
3. Iron - 2.5-4.5 ppm
4. Manganese - 2.0 ppm
Below the critical concentrations indicated
for the above elements, deficiency symptoms in
plants can be occur.
