Digestion of Soil and Plant Materials for Maco- and Microntrients Analysis

A. Soil Digestion procedures

Total nitrogen Determination in soil

(Handouts already provided)

Total phosphorus determination in soil

Total P determination involves digestion of soil sample with a strong acid and the
dissolution of all insoluble inorganic minerals and organic P forms.

Reagents
A.. Perchloric acid (HClO4), 60%

B. Ammonium heptamolybdate-Ammonium metavanadate in nitric acid

- Dissolve 22.5 g ammonium heptamolybdate [(NH4)6Mo7O24.4H2O] in 400 ml
distilled water (a).
- Dissolve 1.25 g ammonium metavanadate (NH4VO3) in 300 ml hot distilled water
(b).
- Add (b) to (a) in a 1L volumetric flask and let the mixture cool to room temp.
- Slowly add 250 ml conc. nitric acid to the mixture, cool the solution to room
temp.and dilute to 1L volume with distilled water.

C. Standard stock solution

- Dry about 2.5 g potassium dihydrogen phosphate (KH2PO4) in an oven at 105 oC
for 1 h, cool in a desiccator and store in a tightly stoppered bottle.
- Dissolve 0.4393 g dried potassium dihydrogen phosphate in distilled water and
bring to 1 L volume with distilled water. This solution contains 100 ppm P (Stock
solution).
- Prepare a series of standard solutions as follows: Dilute 1, 2, 3, 4 and 5 ml Stock
solution to 50 ml final volume by adding distilled water. These solutions contain
2, 4, 6, 8 and 10 ppm P, respectively.
Procedure

Digestion
- Weigh 2 g air-dry soil (0.15 mm) into a 250 ml digestion tube.
- Add 30 mL 60% perchloric acid.
- Place the tubes rack in the block digester and gently heat at about 100 oC.
- Slowly increase the temp. to 180 oC and digest the samples until dense white
fumes of acid appear. Use a little extra perchloric acid to wash down the sides of
the digestion tubes as necessary.
- Continue heating at the boiling temp. for 15- 20 min. longer. At this stage the
insoluble material becomes like white sand. The total digestion with perchloric
acid usually requires about 40 min.

1
 - Cool the mixture, add distilled water to obtain a volume of 250 ml, mix the
contents and filter through Whatman No. 1 filter paper.

Note
If the soil samples are high in organic matter content, add 20 mL conc. HNO3 before
Step 2 and cautiously heat to oxidize organic matter.

Measurement
- Pipette 5 mL of the sample digest into a 50 mL volumetric flask.
- Add 10 mL ammonium-vanadomolybdate and dilute the volume with distilled
water.
- Prepare a standard curve as follows:
1. Pipette 5 ml of each standard (2-10 ppm) and proceed as for the samples.
2. Also make a blank with 10 mL ammonium-vanadomolybdate reagent and
proceed as for the samples. Read the absorbance of blank, standards and
samples after 10 min. at 410 nm wavelength.
3. Prepare a calibration curve for standards, plotting absorbance against the
respective P concentrations.
4. Read P concentration in the unknown samples from the calibration curve.

Calculations
A 50
Total P (ppm) = ppm P (from calibration curve) x -------- x ---------
Wt V

Where: A = Total volume of the digest (mL)

Wt = Weight of air-dry soil (g)
V = Volume of digest used for measurement (mL)

B. Plant Digestion Procedures

Digestion of plant for nitrogen determination

Procedure:
- Transfer weighed amount of finely ground plant material (0.2 g in case of straw
and 0.1 g in case of grain) in 100 mL digestion tubes.
- Add 4 mL of sulphuric acid and 1.1 g digestion mixture (K2SO4, CuSO4 and Se in
ratio of 100 : 10 : 1 ).
- Heat the mixtures cautiously in the Block Digester.
- Heat the tube contents on higher temperature (375 oC) until the digest clears. The
digestion is continued for 1 h after clearing.
- Cool the tubes and add 20 mL of water to each.
- For distillation of the digest with sodium hydroxide, proceed as per procedure
given for soil.

2
Note:
For total N determination in plant material, the modified Kjeldahl method to include
nitrates can be followed. [Consult Soil and Plant Analysis Manual by Ryan et. al. (2001)
p. 129].

Digestion of plant material for phosphorus determination

Phosphorus in plant material can be determined by wet digestion or dry ashing
procedure. Phosphorus in the digests or dissolved ash is measured colorimetrically.

Dry Ashing (High Temperature Oxidation) for Macro- and Micronutrients

Analysis
Plant analysis by dry ashing is simple, non-hazardous and less expensive, compared with
HNO3-HClO4 wet digestion. The method is appropriate for analyzing P, K, Ca, Mg and
Na and micronutrients i.e. Zn, Cu, Fe and Mn but only in plants containing low silica
contents like legumes. The HNO3-HClO4 wet digestion is required for full recovery of
micronutrients in high silica plant tissues like wheat, barley, rice and sugarcane etc. In
dry ashing, for B, use of glassware should be avoided.

Reagent
Hydrochloric acid (HCl), 2 N
Dilute 165.6 mL concentrated hydrochloric acid in DI water, mix well, let it cool and
bring to 1L volume with DI water.

Procedure
The procedure is that of Chapman and Pratt (1961) with slight modifications.
- Weigh 0.5 - 1.0 g portions of ground plant material in a 30 -50 mL porcelain
crucibles or Pyrex glass beakers.
- Place the crucibles in a cool muffle furnace and increase temperature gradually to
550 oC and continue heating for 5 h.
- Switch off the muffle furnace..
- When cool , take out the crucibles carefully.
- Dissolve the cooled ash in 5 mL portions of 2 N HCl and mix with a plastic rod.
- After 15-20 min., make up the volume (usually to 50 mL) using DI water.
- Mix thoroughly, allow to stand for about 30 min. and use the supernatant or filter
through Whatman No. 42 filter paper, discarding the first portions of the filterates.
- Analyse the filtrates for P by colorimetry (by Ammonium vanadate-Ammonium
molybdate yellow colour method) for K and Na by Flame photometery, and for
Ca, Mg, Zn, Cu, Fe and Mn by Atomic Absorption Spectroscopy.

Note:
For Ca and Mg measurement, the final dilution should contain 1 % w/v Lanthanum (La)
and the determinations should be against standards and blank containing similar La
concentration to overcome an ionic interference.
Reference
Chapman, H. D. and F. P. Pratt. 1961. Methods of analysis for soils, plants and water.
Univ. California, Riverside, CA., USA..

3
Wet Digestion of Plant Material for Macro- and Micronutrients Analysis

1. Nitric Acid - Perchloric Acid Digestion Procedure

Full recovery of micronutrients in high silica containing plant tissues like wheat, barley,
rice, sugarcane etc. is not possible by dry ashing procedure. Therefore, this kind of plant
materials should be wet digested using HNO3-HClO4 (diacid mixture) as described by
Rashid (1986). Many other elements (P, K, Ca, Mg and Na) can also be determined in the
same digest.

Reagents
Nitric acid - Perchloric acid (HNO3-HClO4), 2:1 ratio
Add 500 mL concentrated perchloric acid to 1 L concentrated nitric acid.

Procedure

Digestion
- Transfer 1 g dry plant material to a 100-mL Pyrex digestion tube.
- Add 10 mL nitric-perchloric acid mixture and allow to stand overnight for
preliminary digestion.
- Place small, short-stemmed funnels in the mouth of the tubes to reflux acid.
- Place tubes in the digester and raise temperature to 150 oC for 1 h.
- Place U-shaped glass rods under each funnel to permit exit of volatile vapors.
- Increase temperature slowly until all traces of nitric acid disappear, and then
remove U shaped glass rods.
- Raise temperature to 235 oC.
- Note time, when white fumes of perchloric acid appear in the tubes, and continue
digestion for 30 min. more.
- Take out tube rack out of block digester.
- After vapors condense, add DI water in small increments for washing down walls
of tubes and funnels.
- Bring to volume with DI water. Mix the solution of each tube and then leave
undisturbed for few hours.
- Each batch of samples should contain at least one reagent blank.

Measurement
Decant the supernatant liquid and analyse digests for Zn, Cu, Fe, Mn, Ca and Mg by
Atomic Absorption Spectrophotometery and K and Na by Flame Photometery.
Calculations
A
Elements in plant (ppm) = (ppm in extract – Blank) x --------------
Wt

4
Where: A = Total volume of the extract (mL)
Wt = Weight of dry plant material (g)
Reference
Rashid, A. 1986. Mapping zinc fertility of soils using indicator plants and soils analyses.
Ph. D. Dissertation. University of Hawaii, HI, USA.

2. Plant Digestion with Sulphuric Acid and 30 % Hydrogen Peroxide

Procedure [as described by Ryan et al. (2001)]

Reagents
A. Sulphuric Acid (H2SO4), conc.
B. Hydrogen peroxide (H2O2), 30%

Procedure
1.Weigh 0.5 g dry plant material (20 mesh) into a 100 ml digestion tube.
2.Add 5 ml conc. sulphuric acid and mix.
3.Keep overnight
4.Add 2 mL 30% hydrogen peroxide and heat slowly on block digester to
280 oC.
5. Heat the tubes for 30 - 60 min.
6. Cool the tubes, then add 2 mL hydrogen peroxide.
7. Raise the temp. of the block digester to 280 oC.
8. Heat the tubes for 10 min. at 280 oC.
9. Cool, then add 2 mL hydrogen peroxide and heat for 10 min.
10. Repeat Steps 8 and 9 until solution remains clear after 10 min. of heating.
11. Cool and make up to 100 mL volume with distilled water.
.
Suggested Reading

1. Page, A.L., D. E. Baker and D. R. Keeney (eds). 1982. Methods of soil Analysis.
Part 2, 2nd edn. Am. Soc. Agron., Inc. Madison, Wis.
2. Ryan, J., G. Estefan and A. Rashid (eds.). 2001. Soil and Plant Analysis Laboratory
Manual. 2001.2nd edn. Intenational Centre for Agricultural Research in Dry Areas,
Aleppo, Syria & National Agricultural Research Centre, Islamabad, Pakistan.
3. Jones, Jr., J. B. and V. W. Case. 1990. Sampling, handling and analyzing plant tissue
samples. In: R. L. Westerman Soil Testing and Plant Analysis. 3rd edn. pp. 389-427.
Soil Sci. Soc. of America, Madison, Wis.

Soil Extraction Procedures for Available Micronutrients

Determination of micronutrients in soils

For the determination of micronutrient status of soils, DTPA test of Lindsay and Norvell
(1978) is commonly used. A universal soil test i.e. AB-DTPA is also an effective method
for determining micronutrients in soils.

5
DTPA Method:

Reagents

DTPA Extraction Solution

- Weigh 1.97 g diethylene triamine pentaacetic acid (DTPA) and 1.1 g calcium
chloride or 1.47 g calcium chloride dihydrate (Ca Cl2.2H2O) in a beaker.
Dissolve with distilled water and transfer to a 1 L volumetric flask.
- Weigh 14.92 g or 13.38 ml Triethanolamine (TEA), transfer with distilled water
into the 1 L volume and make up to about 900 ml with distilled water.
- Adjust pH to exactly 7.3 with 6 N HCl and make to 1 L volume with distilled
water. The final extraction solution is 0.005 M DTPA, 0.1 M TEA, 0.1 M Ca Cl2.

Standard Stock Solutions

Prepare standard solutions for micronutrients in DTPA extraction solution:
Zn: 0, 0.2, 0.4, 0.6, 0.8, 1.0 ppm Cu: 0, 1, 2, 3, 4, 5 ppm
Fe: 0, 1, 2, 3, 4, 5 ppm Mn: 0, 1.0, 1.5, 2.0, 2.5 ppm

Procedure:
- Weigh 10 g air-dry soil in 125-mL Erlenmeyer flask.
- Add 20 mL extraction solution. Shake for 2 h on a reciprocal shaker.
- Filter the suspension through a Whatman No. 42 filter paper.
- Measure Zn, Cu, Fe and Mn in the filtrate by an Atomic Absorption Spectro-
photometer using appropriate lamp for each element.

Calculations:
A
Zn, Cu, Fe or Mn (ppm) = (ppm in extract – Blank) x ------------
Wt.

Where: A = Total volume of the extract (mL)

Wt. = Weight of soil (g)

Reference
Lindsay, W.L. and W.A. Norvell. 1978. Development of a DTPA soil test for zinc, iron,
manganese and copper. Soil Sci. Soc. Am. J. 42: 421- 428.

Ammonium Bicarbonate- DTPA Method

It is a multi-element soil test for alkaline soils developed by Sultanpour and
Schwab (1977) and later modified by Soltanpour and Workman (1979). NO3-N, P and K
can also be determined in the same extract.

6
Reagents

Extracting Solution
Prepare 0.005 M DTPA solution by dissolving 1.97 g DTPA in 800 mL DI water. Add
approx. 2 mL of 1:1 ammonium hydroxide (NH4OH) to facilitate dissolution and to
prevent effervescence when bicarbonate is added. When most of the DTPA is dissolved,
add 79.06 g ammonium bicarbonate (NH4HCO3) and stir gently until dissolved. Adjust
pH to 7.6 with ammonium hydroxide. Dilute the solution to 1 L with DI water. Either use
it immediately or store it under mineral oil.

Procedure

Extraction method
Weigh 10 g air-dry soil (2 mm) into 125 mL conical flask. Add 20-mL extracting solution
and shake on a reciprocal shaker for 15 min. at 180 cycles/min. with flasks kept open.
Filter extracts through Whatman No. 42 filter paper.

Micronutrients determination
Zinc, Cu, Fe and Mn in the extracts are determined by Atomic Absorption
Spectrophotometer. The standard solution of these metals are prepared in the extracting
solution.

Calculations
Zn, Cu, Fe and Mn (ppm) = ppm of metal in extract x Dilution factor

Note
The DTPA reagent should be of the acid form and not a disodium salt. Extracting
solution can be stored for 2 weeks under mineral oil, and then the pH adjusted to 7.6, if
necessary.

References
Soltanpour, P. N. and A. P. Schwab. 1977. A new soil test for simultaneous extraction of
macro- and micronutrients in alkaline soils. Commun. Soil Sci. Plant Anal. 8: 195 – 207.
Soltanpour, P. N. and S. Workman. 1979. Modification of the NH4HCO3-DTPA soil test
to omit carbon black. Commun. Soil Sci. Plant Anal. 10: 1411-1420.

Suggested Reading

1. Page, A.L., D. E. Baker and D. R. Keeney (eds). 1982. Methods of soil Analysis.
Part 2, 2nd edn. Am. Soc. Agron., Inc. Madison, Wis.
2. Ryan, J., G. Estefan and A. Rashid (eds.). 2001. Soil and Plant Analysis Laboratory
Manual. 2nd edn. Intenational Centre for Agricultural Research in Dry Areas, Aleppo,
Syria & National Agricultural Research Centre, Islamabad, Pakistan.