UNIVERSITY OF NAIROBI

COLLEGE OF AGRICULTURE AND VETERINARY SCIENCE

DEPARTMENT OF LAND RESEOURCE MANAGEMENT AND AGRICULTURAL TECHNOLOGY

CONTENTS PAGE

INTRODUCTION 1

Determination of total nitrogen in the soil 2

The determination of soil phosphorus 6

Total phosphorus and extractable phosphorus 6

Table showing extracting solutions, solution/soil ratios

and shaking in six methods of establishing phosphorus 8

The Olsen (sodium bicarbonate) method 9

The Bray 1 and Bray II methods 11

The Mehlich (north Carolina) method 3

INTRODUCTION

Laboratory procedures currently taught to second year students were described in Technical
communication No. 1 In this communication, technical Communication No.1 In this
communication, Technical Communication No. 5, information is given on some of the laboratory
determination taught in the third year of the undergraduate soil science course. The third year
syllabus include about 20 lectures on soil chemistry I(including a discussion of the forms in which
nutrients are stored in the soil and made available to the plant) and on manures, fertilizers and
their use. The laboratory practical are in general designed to reinforce and sometimes amplify
material taught in the lectures at about the same time.

As explained in the introduction to Technical Communication No. 1, students beginning their soil
science course in the second year are required to sample soil horizons in the fields, and to
describe them, prior to analyzing the soil they have sampled in the laboratory in various ways. In
year 3 students continue with this procedure, determining total nitrogen and extractable
phosphorus by at least two different methods in order to appreciate the empirical nature of the
results and the fact that different extractants may give quite different results for the same soil. The
Olsen method is included because, in the experience of many soil chemists working on tropical
soils, it is one of the best extractants when a wide range of soils is being analyzed. The Mehlich
method is included because if has been adopted as a routine analysis by the Ministry of
Agriculture National Agricultural Laboratories at Nairobi, and thus some information has already
been acquired on its use under local conditions. By extracting soils by these two methods students
learn both the molybdenum blue and the vanadium yellow methods for the colorimetric
determination of small concentrations of phosphorus in solution

DETERMINATION OF TOTAL NITROGEN IN THE SOIL

Most of the nitrogen in the soil occurs in the organic matter (humus) of the soil, and while in
organic combination is not available to plants.

Unavailable nitrogen in the soil is mineralized to available forms –NH4 and N03-by the action of
bacteria, but the available nitrogen in the soil at any one time forms a very small fraction of the total
and, moreover, can fluctuate quite quickly. For these reasons the total nitrogen in the soil is far
more commonly determined than the nitrate nitrogen.

The most commonly used method of determining total nitrogen is a wet digestion method in which
the sample is digested for several hours with concentrated sulphuric acid so that all the nitrogen is
converted to ammonium. This method was first described by J. Kjeldahl in 1883 and the simplicity
of the method and speed and completeness of the conversion of N to NH4 have resulted in the fact
that the basic method is still the most widely used today, though several modifications have been
introduced. Kjeldahl originally used sulphuric acid alone for the digestion, but nowadays the
efficiency of the original method has been improved by adding salts to raise the temperature of
digestion, and by adding a selenium catalysis to promote oxidation of the organic matter.

The Kjedahl method gives very satisfactory, reproducible results provided that the digestion
procedure is containing long enough. Almost all combined forms of N are converted to NH4,
though the nitrate and nitrate in the soil is not included unless the method is modified. In most
soils, the amounts of nitrite and nitrate present at any one time are generally too small to have any
appreciable effect on the result.

The standard Kjeldahl analysis involves two steps:-

a) digestion of the sample to convert N to ammonium
b) b)determination of the ammonium in the digest.

If it is desired to include in the analysis those forms of N (including nitrates and nitrites)not
determined by the standard method, an appropriate pre-treatment (prior to the digestion) can be
included.(1)

A. DIGESITON OF SAMPLE

The term digestion is used here to describe the process in which the sieves soil sample is heated
with concentrated H4S04 to covert organic N to ammonium

1) See Bremner, J.M. 1965. Total Nitrogen (chapter in Methods of Soil Analysis Part II, edited by
C.A. Black et alia, and published by the American Society of Agronomy, Madison, as number 9 in
the Agronomy series).

1.Weigh out exactly 0.500 or 1.00g of air dry soil ground to pass through a 1/2mm (0.5mm) sieve,
and transfer carefully to a 300 or 500ml Kjeldahl flask.

the amount to be weighed out depends on the N content. Where this is relatively high , as
in the case of many topsoil samples then 5.00g should be weighed out, but with samples
containing less than a about 1% organic carbon, a 10.00g samples is preferable.

2.Add 10.00g of mixed catalyst, and wash in with 5-7ml of distilled water.

the mixed catalyst contains potassium sulphate and copper sulphate, which raise the
boiling point of the acid and therefore the temperature of the digest, and a small quantity of
selenium, a catalyst. Temperature shortens the required digestion time.

3.Add 20.0ml of concentrated sulphuric acid (36N) from a graduates cylinder. Mix acid and soil by
gentle shaking; allow to stand for 20minutes.

4.Heat the flask gently and cautiously on a digestion stand or in fume cupboard (caution: sulphur
trioxide fumes are given off). At first there will be some frothing after this has ceased the
temperature can be increased until the digest “clears”, i.e. the digested material ceases to lose
colour and is relatively free of suspended material. After clearing, the mixture should boil gently for
at least another 2 ½ hours. The heat should be so regulated that the H 2S04 condenses about one
third of the way up the neck of the flask.
5.After completion of the digestion, allow the flask to cool,. Then slowly add about 100ml of water,
and shake to mix. The liquid is then transferred to 250ml volumetric flask and made up to the
mark. In order to do this, the contents of the digestion flask are first transferred to a large conical
flask, and the liquid digest is that decanted off, followed by about four further washing of the
residue and subsequent decantation and filtering, in order to get all the digest into volumetric flask
and to leave only the washed residue.

B. DETERMINATION OF NH4 IN THE DIGEST BY DISTILLATION

The second step involves the determination of the NH 4 in the digest by distillation with an alkali,
and subsequent titration of the ammonium given off.

The distillation can either be of the whole digest, in which cases all the ammonium is distilled over
and measured, or only a small aliquot of the digest can be distilled, in which case a known
proportion of the total ammonium is measured. The former procedures is known as macro-
distillation, the latter as macro-distillation. Distillation of a very small quantity of digest, sometimes
as little as 2ml, can be done in specially designed apparatus and has advantage of speed macro-
distillation methods are slower but generally more accurate.

If only part of the digest is distilled the remainder can, if desired, ,be used for determination of the
total quantity of other elements in the soil sample (e.g. total phosphorus).

1.Pipette 50.0ml of the digest into a 500ml Kjeldahl flask, add 200-250ml of distilled water,
and two glass beads.

2.Set up the distillation apparatus with the lower and of the Liebig condenser leading into a
500ml conical flask containing 50ml of 2% boric acid solution and a few drops of mixed
indicator.

3.Hold the Kjedahl flask at about 45 o and pour 25ml of ION NaOH gently down the neck of
the flask and immediately connect to the distillation apparatus. Swirl the flask to mix the
contents and commence distillation. The flame should be regulated so that the amount of
distillate collected is about 10ml per minute: the rate of low of cold water is also regulated
so that the distillate is not too hot (not above about 35%C). Continue with the distillation
until about half of the liquid in the Kjeldahl flask has been distilled over. Lower the
receiver flask so that the end of the condenser is out of the liquid before stopping the
distillation, in order to prevent sucking back

4.Determine the ammonium-N in the distillate by titrating with 0.01N HCl or H 2S04. The
colour change at the end point is from green to grey to pink.

5.Calculate the %N in the soil. 1ml of 0.01N acid in the titration is equivalent to 0.14mg
ammoniumN. If a 10.0g sample was used to give 250ml of digest from which a 50ml
aliquot was distilled (as above), then the percentage N in the sample is:

ml acid x 5 x 0.14 (=mg N) x 100

wt of soil (in mg)
assuming a reading of 30ml of 0.01 N acid and 10.0g of soil, then the calculation is

mg N = 30 x 5 x 0.14 = 21 = 0.21%
mg soil 10,000 10,000
As a very rough guide it should be remembered that the C/N ratio in m ost soil samples is
approximately 5-15, so that if a very approximate figure of 10 is assumed, then the % nitrogen
should not be far different from one-tenth of the organic matter percentage. For example, a soil
with 3% organic matter would be expected to have something like 0.2 –0.6% nitrogen.

REAGENTS

1. Concentrated (36N) H2 S04

-----------------------------------------------------

2. Mixed catalyst: Prepare by mixing 160.0g of K2S04, 10.0g of CuS0 4. 5H20 and 3.0 of
selenium powder.

3. Sodium hydroxide: approximately ION. Dissolve 420g of NaOH in 1 litre of C)2 – free
water.

4. Boric acid: 20.0g boric acid (H3P03) per litre.

5. Indicator solution: a mixed indicator is prepared by dissolving 0.12g methyl red and 0.8g
methylene blue in 100ml of 95% methonal, or alternatively, by dissolving the above
together with 0.1g bromcresol green.

THE DETERMINATION OF SOIL PHOSPHORUS

Most determination of soil phosphorus – either of total phosphorus, or of any particularly fraction of
the total (including “available” phosphorus) –consist of two distinct phases:

a) the preparation of a solution containing the phosphorus

b) the quantitative determination of the phosphorus in solution.

The second phase, the determination of the phosphorus in solution, is usually carried out by a
colorimetric method. The choice of method is determined by the contraction of phosphorus in the
solution, the amounts and nature of interfering substances present and the particular system
involved in the preparation of the phostphorus containing solution.

The molybdenum blue methods are the most sensitive and therefore the best for determining small
concentration of P In an acid molyodate solution containing orthophosphate ions, a
phosphomolybdate complex forms that can be reduced by Shannon chloride at a molybdenum blue
colour. It is the intensity of this blue colour which is measured to give the P concentration.
However, the colour developed may be also affected by other factors such as acidity and the
presence of arsenates and silicates and other substances influencing the oxidation-reduction
conditions in the system.
The vanadium yellow method (the vanadomolybdophosphoric yellow colour method) has a much
lower sensitivity than the blue colour method. This permits determination of larger concentrations
of phosphorus. The method has the advantages of simplicity, stability of colour, freedom from
interferences from a range of ionic species, and adaptability to various extracting systems.

TOTAL PHOSPHORUS AND EXTRACTABLE PHOSPHORUS

The total phosphorus present in the soil can be determined in the laboratory by a choice of
methods which include digesting the soil with perchloric acid HCl0 4) cr fusing the soil with sodium
carbonate (Na2C03). Digestion with perchloric acid is the simpler of these two procedures.

Such a procedure would give the total elemental phosphorus present in the soil in all forms, both
organic and inorganic. The organic phosphorus would include the phosphorus present in insoluble
forms and the so-called occluded (hidden) phosphorus present, for example, in ironstone
concretions. The total phosphorus bears little regular relationship to the amount of phosphorus
actually available or likely to become available to plants. The “available phosphorus is that
phosphorus capable of entering the soil solution and being absorbed by plants (as HP0 4 or H2P04
orthophosphate anions).

Since the “available” phosphorus in practice depends on environmental factors and the
nature and rooting system of the plant or crop involved, it is not possible to measure
“available” phosphorus in the soil. What is commonly done instead is to measure the
phosphorus which can be extracted by a particular method. The amount of phosphorus
extracted by a suitable method gives an empirical “availability index” which is
interpreted by correlating amounts extracted from a range of soils with actual crop
responses in the field.

A number of different extractants have been used to give an indication of phosphorus

availability to the plant. These extractants generally remove a very small proportion of
the total. Phosphorus in the soil, i.e. a small part of the more exposed reactive
phosphorus, in order to give an empirical indication of amounts likely to be available to
plants under field conditions. The extractants commonly used include very weak solution
of strong acids (e.g. 0.002N H2 S04 in the Truog method), or stronger solution of weak
acids such as citric or acetic acid, or weak solutions of substances such as ammonium
fluoride (NH4F)
Or sodium bicarbonate (NaHC03) which bring part of the more reactive soil phosphorus
into solution. Information on a selection of commonly used methods is given in table 1.

It is important to remember that these methods are empirical. They do not extract any
particular, definable fraction of the soil phosphorus. All we can say of the result
obtained is that it represents the amount of phosphorus extracted by the particular method
used. Different extractants vary in the types and amounts of soil phosphorus they extract
from different soils. Extractants. A for example might extract more P from an acid soil
than extractant B, but with a different soil then extractants B may remove more than
extractant A.
In order to interpret the “availability index” given by using a particular extractant, it is
necessary to have some information on the correlation between the amounts of P
extracted from a range of soils by the method used, and the results of applying
phosphatic fertilizers to the soils tested. One might as a result be able to any that certain
ranges of results would be regarded as very low, moderate or high. Nevertheless the
result is still little more than a general guide, and close and reliable correlations between
extractable phosphorus and crop responses are not to be expected unless considerable
experience of a particular soil has already been acquired.

METHOD EXTRACTION WEIGHT (SOLUTION/ SOIL SHAKER

SOIL RATIO TIME
SOIL

VOLUME
SOLUTION
MEHLICH 0.1N HCl 5.0g 5 10 minutes
+ 25.0ml (1) (3)
0.025N H2S04
Truog 0.002 N H2S04 1.0g 200 30 minutes
at pH 3.0 200ml (2)
Olsen 0.5N NaHC03 5.0g 20 30 minutes
at pH 8.5 100ml (2)

Bray I 0.03N NH4 F 2.85g 7 1 minute

in 0.025N HCl 20.0ml (3)
Bray II 0.03N NH4F 2.85g 7 40 minutes
in 0.1 N HCl 20.0ml (3)
Water H20 5.0g 10 5 minutes
50ml

NOTES:
1) after adding solution to soil, allow to stand 1 hour before shaking
2) shake in a 250ml conical flask
3) shake in a 50ml extraction bottle or tube

TABLE 1 Extracting solutions, solution/soil ratios and shaking times used in six
methods of establishing phosphorus availability indexes
THE OLSEN (SODIUM BICARBONATE) METHOD
The Olsen method of phosphorus extraction employs an 0.5M sodium bicarbonate
solution adjusted with sodium hydroxide to a pH of exactly 8.5. A 5.0gram sample of
soil is shaken with 100ml of extractant (so giving an extractant/soil ratio of 20) for 30
minutes.

This method was primarily designed for use in calcareous soils (in which the usual
weak acid extraction methods often gave unreliable results). In fact, it has now proved
on of the best all round extractants for both acid and alkaline soils and when a wide range
of soils has no to be tested is probably the best single extractant available.

In calcareous , alkaline or neutral soils containing calcium phosphates, the bicarbonate

solution increases the P in solution by decreasing the Ca concentration. This it does by
precipitating some Ca as CaC03. In acid soils containing iron and aluminum phosphates,
P is released as the rises.

Method

A. Extraction

1. Add 5.0g of soil, 1 teaspoon of carbon black (or leached, P-free charcoal) and
100ml of the extracting solution to a 250ml conical flask.

2. Shake the flask for 30 minutes.

3. Filter through a whatman no. 40 paper.

B. Development & Measurement of Colour

4. Pipette a 10ml aliquot into a 50ml volumetric flask.

5. Add10ml of ammonium molybdte –HCL solution. After evolution of C02 has

ceased, shake the flask gently to mix the contents, washing the ammonium
molybdate down the neck of the flask with distilled water, and diluting the
contents to about 40ml.

6. Add 1ml of dilute .025M) SnCl2 solution, shake immediately, make up to

volume, and mix contents.

7. Measure the blue colour (660mu wavelength) about 10minutes after adding the
stannous chloride.
C. Standard curve and calculation of results
Prepare a standard curve by pipetting 10ml aliquots of standard phosphorus solution
containing 0.1-5ppm p into 50ml volumetric flasks and develop the colour as above. Plot
the colorimeter readings against the P concentrations in the standards on place graph
paper.

In order to calculate the ppm P in the soil, use the following formula:

ppm P in coil = ppm P in solution x 100

5

The factor 100 represents the ratio of extracting solution to soil, so that 1 ppm in the
5
100ml of solution is equivalent to 20 ppm in 5.0g of soil. The standard curve has a range
of 1.0-5ppz P in the solution, corresponding to 2-100 ppm P in the soil.

REAGENTS

1. Extracting solution (0.5M NaHCO3

-------------------------------------------------------
Dissolve 2.0gg NaHC03 in 1 lire of distilled water, adjusting the pH of the solution to
exactly pH 8.5 with NaOH.

2. Phosphorus free lamp black or activated charcoal

3. Ammonium molybdate –HCl solution

Dissolve 15.0g ammonium molybdate, (H4) M07024, in about

300ml distilled water warmed to about 50 0C, when cool, add 410ml of 10.0N HCl,
stirring rapidly. When cool, make up to 1 litre with distilled water and store in an amber
bottle.

NB: This solution has a little more HCl than the ammonium molybdate solution used
with some other extractants. This is in order to neutrals the Na HC03 in the 10ml eliquot.
4. Stannous chloride solution (0.25m)

Dissolve 6.25g reagent grade SnCl2 2H20 in 12.5ml concentrated HCl and then make up
to 500ml with distilled water. This stannous solution is then further diluted with an equal
quantity of 1.2N HCl to make it up to 1 litre. The final solution is 0.025 molar Sn ++ but
should be checked byy titration with 0.1N standard iodine solution. In the Olsen method
as described by Jackson (M.L Jackson, 1958; Soil Chemical Analysis) 5 drops of 0.1
molar stannous chloride solution are used, but it has been found in student practicals that
if students put in a few extra drops of solution the colour developed is affected. For this
reason it was considered advisable to use 1.0ml of 0.025 molar solution rather than 5
drops of 1.0 molar. Erratic results are caused if the procedure outlined above in steps 5
&6 is not followed exactly.

BRAY I AND II METHODS

These methods employ ammonium fluoride (NH4F) in weak (0.025N or 0.1N)

hydrochloric acid.

The F (fluoride) ion has the property of complexing Fe 3+ and AI 3+ ions. It thus can
release some of the phosphate held by these trivalent ions which is present in the soil as
iron and aluminum phosphates.

Inclusion of the HCL in the extractant results in the solution of the more active calcium
phosphates. It also prevents the calcium released by the fluoride being precipated as
calcium phosphate.

In effect the method removes “the more readily soluble portion of each form of available
soil phosphorus”.

In effect the method removes “the more readily soluble portion of each form of available
soil phosphorus”.

The Bray I method employee 0.025N HCl. In the Bray II method the strength of the acid
is increased to 0.IN. This is to include more of the soil anatine: this would be present in
near neutral or calcareous soils, or having received rock phosphate.
As a general guide to crop responses, results for the Bray I methods are commonly
interpreted in the U.S. follows:

Below 3pppm - very low

3-7ppm - low
-20ppm - medium
above 20 ppm - adequate to high

BRAY I METHOD

A Extraction
1. Weigh out 2.85 g of the sieved air dry soil into a 50ml extraction bottle or tube.

2. Pipette 20.0ml of extracting solution (0.03N NH4F in 0.025 HCI) into the 50ml
extraction bottle or tube shake for one minutes.

3. Immediately after shaking, filter through a moist Whatman no. 42 filter paper.
The filtrate should be clear (if not refiler

B. Development and -----of colour

4. Transfer a 2ml aliquot of clear filtrate (after rinsing pipette with 1ml of filtrate) to
A colorimeter test tube. Add 5ml distilled water.
5. Add 2ml of ammonium molybdate reagent and mix.
6. Add 1m of fresh diluted stannous chloride reagent and mix immediately
7. read colour in a colorimeter after five minutes and before 20 minutes.
NB -A reagent blank is made up with each batch of determinations and used for the 100%
transmission setting.

C. Preparation of standard curve and calculation of results

Phosphorus standards are prepared at 0.5ppm intervals in the range 0.5 –5.0 ppm by
diluting a higher concentration of p with the NH4F –HCl extracting solution. (The F
content of the extracting solution has some influence on the colour developed, so that it
must be included in the preparation of standards.)

The concentration of Bray I extractable phosphorus in the soil is calculated as follows:

Ppm P in soil = ppm P in solution x 20

2.85

= ppm P in solution x 7
REAGENTS

Extracting solution
(Bray I) 1.11g solid NH4F and 4.16ml 6N HCl in one
litre. (0.03N NH4F in 0.025 N HCl).

Ammonium molybdate 15.0g (NH4)6 Mo7024 is dissolved in 300ml warm

distilled water; when cool slowly add 250ml 10.0 N HCl, stirring rapidly.
When cool, make up to 1 litre. Store in an amber bottle. When needed,
make 3ml stock solution up to 1 litre with freshly boiled, cooled distilled
water.
BRAY II METHOD

As above except that the extracting solution is 0.03N NH 4F in 0.IN HCl and the shaking
time is 40 seconds.

THE MENLICH (NORTH CAROLINA) METHOD

The Mehlich method is a modification of the developed in North Carolina by Nelson et

alia to extract phosphorus from some North Carolina soils which fix phosphate strongly.
The method is not widely used, but was introduced by North Carolina soil scientists into
parts of /South America, and was introduced into Kenya by Mehlich when working at
the National Agricultural Laboratories, Nairobi. The extractant is a general purpose
extractant designed to extract a number of nutrient elements in a single shaking. At the
National Agricultural Laboratories, Nairobi, the extractant is used to determine both
phosphorus and extractable Ca,Mg,K, Na and Mn.

The extracting solution recommended by Mehlich for use in Kenya is a mixture of 0.1N
HCl and 0.025N H2S04. The mixed acids are more effective than HCl alone as a P
extractant from soils fixing phosphorus as iron and aluminum phosphates. In North
Carolina the extracting solution used has a lower concentration of HCl.

At the National Agricultural Laboratories, Nairobi, P levels obtained by this method are
classified as follows:

0-20ppm - defiance
20-80ppm - sufficient
0ver 80 ppm - high

However, the test is not considered sensitive enough to indicate, in the case of soil
thought to be deficient, how phosphatic fertilizer might be needed.

METHOD

A Extraction

1. Weigh 5.0g of soil into a 50ml bottle. (NB for routine use at the National ?
Agricultural Laboratories, time is saved by using a 5ml scoop rather than
weighing the soil. The result obtained is therefore for a volume of soil rather
than for a weight of soil).
 2. Add one small scoop of P-free charcoal (about 200mg) and 25ml of extracting
solution

3. Mix and allow to soak for one hour

4. ?shake in a mechanical shaker for 10 minutes

5. filter through an 11 cm whatman No. 42 filter paper.

B. Development of the colour by the vanadium yellow method

6. Pipette 5.0ml of the filters extractant into a test tube or colorimeter tube.
7. Add 1ml of ammonium vanadate-ammonium molybdate solution.
8. read density of colour developed, after 20 minutes and before six hours, at 430mu
(11 ford 6621 filter).

C. Standard curve and calculation of results

Prepare a standard curve by pipetting 5.0ml of standard solutions containing 1-20 ppm P
in the extracting solution into a tube and then carrying out steps 7 and 8 above.

The concentration of P in the extractant, as read from the standard curve, is converted to
ppm P in the soil by multiplying by 25, i.e. by 5. This represents the ratio of
5
extracting solution to soil. Standards on the curve in the range 1-20 ppm P therefore
correspond to 5-100ppm P in the soil.

REAGENTS

1. Extracting solution: Prepare a stock solution by adding 330 ml concentration HCl

and 28ml concentration H2S04 to 500ml of water in a 1 litre volumetric flask;
shake, cool and make up to volume. To prepare the extracting solution (0.1N HCl
and 0.025N H2S04) dilute 25ml of stock solution to 1 litre with distilled water.

2. Ammonium vanadate –ammonium molybdate solution : Dissolve 1.25g of

ammonium vanadate (NH4V03) in 500ml of IN HN03. dissolve 25g of ammonium
molybdate (NH4)6 M07024. 4H20) in 500ml of distilled water. Add the two
solutions together to make 1 litre of vanadete-molybdaate solution.