Research Project

Phosphate Determination
If we knew what it was we were doing, it would not be called
research, would it?
Albert Einstein
Physicist

Abstract

A spectrophotometric technique for determining [phosphate] in solution. Ammonium molybdate and

ammonium metavanadate are added to a sample. Any phosphate present reacts to produce a colored
product, vanadomolydbophosphoric acid. The reaction is performed individually on the samples and on a
series of standards. A calibration plot is generated from the absorbance readings for the standards. The
sample concentrations are read from this plot.

Application

Effective for determining [phosphate] in the range 2 x 10-4 M to 1 x 10-5 M. This technique is commonly
employed in agricultural applications to determine phosphate in soil and water samples. It can also be
applied to other samples such as food stuffs and medications.
.

Sample Issues

The reaction is a hardy one and not susceptible to very many interferences. Silica and arsenate interfere
in heated samples. A blue color is formed by ferrous iron, but does not interfere if the iron concentration
is < 2x 10-3 M. It is reliable over a wide range of pH values.
Because this is a spectrophotometric technique, anything in the sample that affects the light passing
through the sample cell will adversely affect its measurement. Such interferences include other colored
compounds and turbid (cloudy) solutions. Suitable samples include:
fruit juices
colorless commercial products
soil samples
water samples

Background
Phosphorous, the eleventh-most abundant element on the surface of the earth, is essential for all forms of
life. It is predominately found in nature as phosphate (PO3-3) which is also the form so critical to living
systems. Phosphate is a component of many biological systems. Its role is primarily as a energy conduit.
The interconversion of adenosine diphosphate (ADP) and adenosine triphosphate (ATP) is the primary
mechanism by which energy is conserved and used in living organisms. Thus phosphate is a critical
nutrient for plants and animals. The availability of phosphate readily affects the ability of plants to
perform photosynthesis and of both plants and animals to respire.

Phosphate is a tri-protic weak acid that is readily soluble in water. The predominate form is pH
dependent.

H3PO4 H2PO4- HPO42- PO43pH 2

pH 7

pH 12

Phosphate can form complexes with many di and tri-valent metals. It can also be tied up in organic
molecules such as ATP. Any evaluation of phosphate content must take these factors into account.
Phosphate is usually not readily available in soil for plant uptake. In the soil it usually reacts to form
insoluble calcium or iron phosphates. Most of the phosphorus in soils is present as insoluble soil particles
or incorporated into organic matter. As a result, an evaluation for total phosphate can provide results that
are very different from an evaluation for soluble phosphate.
Standard tests for phosphate evaluate only soluble phosphates. An analysis for total phosphate requires
two procedural steps: 1) conversion of all forms of phosphorus into dissolved phosphate by a digestion
method, and 2) evaluation of the dissolved phosphate concentration.
The technique available for your use is the vanadomolybdophosphoricacid spectrophotometric method
(VM method). In this procedure, ammomium molybdate is reacted with a phosphate sample under acid
conditions to form a heteropolyacid. In the presence of vanadium, yellow vanadomolybdophosphoric acid
is formed, the intensity of which is directly proportional to the amount of phosphate present. This
technique is based on the spectrophotometric phosphate determination method taken from Standard
Methods for the Examination of Water and Wastewater, a comprehensive reference covering all aspects
of water analysis techniques and published jointly by the American Public Health Association and the
Water Environment Federation.
Much information regarding testing for phosphate and the vanadomolybdophosphoric acid method is
available on the web.

Procedure
This test uses absorption spectrometry to determine concentrations. A thorough understanding of the
Beer-Lambert Law and its use is necessary to analyze the data collected. Refer to chapter 10-7 of the
technique book for details on this technique.
This test also require the use of a quantitative dilution to prepare standards. See chapter 11 for details.
Although this is primarily a measurement lab, there are many observations you can make to help you with
your analysis. In particular, you should be noting what changes occur in your samples every time you
perform an operation. Unusual observations often explain unusual numbers. Remember that to be able to
describe any changes that occur, you will need to have accurate descriptions of the starting conditions as
well as the ending conditions.
Reagents & Other Materials.
The main reagent, the vanadate-molybdate (VM) reagent, will have to be made. All other required
reagents are available as reagents.
In addition to the normal lab items found in your locker and in the room, the materials that will be
provided by the preproom are detailed in the Research Project Materials

You are responsible for providing your own samples to evaluate. Additional, non-sample materials may
be requested of the preproom. Such requests must be submitted by your TA.
Making the VM reagent. Prepare two solutions and then mixing them.
Solution A: Dissolve 2.5 g ammonium molybdate ((NH4)6Mo7)24 4 H20) in 30.0 mL distilled water.
Solution B: Add 0.125 g of ammonium metavanadate (NH4VO3) to 30.0 mL of distilled water. Heat just
to boiling and stir until dissolved. Cool to room temperature and add 33 mL concentrated HCl.
With both solutions at room temperature, mix them and dilute to 100 mL with distilled water. This is the
VM reagent.
Design Issues Determining concentrations via absorption spectroscopy requires a calibration plot.
Construct such a plot requires a series of phosphate solutions of known concentration to test. These can
be made from pure, solid samples of known molecular weight. The easiest way to make a series of
solutions of known concentrations is to prepare the most concentrated one and then perform a serial
dilution. When deciding what solutions to make, keep the following in mind:
The concentrations of your standard solutions should cover a range of values that includes the
expected concentrations of your samples.
Determining linearity of the calibration plot is crucial for proper evaluation of its reliability. A
minimum of four standard solutions should be employed. Note that the more samples you test,
the more detailed your analysis can be.
The following is a description of the steps required to obtain results for a generic sample containing
phosphate. This procedure will need to be modified, and more detail added, based on the nature and
number of samples to be evaluated.
Successful performance of the VM phosphate determination requires the following sequence of events.
Preparation of Standards. A sufficient number of standard phosphate solutions must be prepared.
These will be used to generate a calibration plot. Anhydrous KH2PO4 will be available for this
application.
Preparation of Samples. The following issues need to be addressed.
If the expected [PO4-3] is not in the effective range (see Application above) the sample will have
to be concentrated or diluted as appropriate. If the sample is a solid, it needs to be dissolved to an
appropriate approximate concentration.
The pH must be between 2 and 10. If not, it needs to be adjusted. Note: when an acid or base is
added to adjust pH, the increase in volume will dilute the sample!
If the sample is biological or soil and total phosphate is being evaluated, a digestion will have to
be performed. A procedure is given below.
Preparation of Reagents. The VM reagent needs to be made as described
above.
Mixing the VM reagent and samples. 4.00 mL of sample is thoroughly
mixed with 1.00 mL of the VM reagent. Each reaction must be performed
separately.

WARNING
It is recommended that
you keep all solutions
until you are sure you
have reliable
measurements.

Mixing the reaction solution. After mixing, allow all samples to stand for
10 minutes to ensure complete reaction. There should be little or no apparent color change in the test
tubes during this time.

Absorption Measurements. Prepare a suitable spectrophotometer. Zero the instrument using a blank
consisting of one mL VM reagent and four mLs distilled water. Measure the absorbance of all standards
and samples at 470 nM.

Sample Digestion
The following is a description of a digestion designed to free up bound phosphate.
Acidify the sample. Take 50 mL of sample. Check the pH. If greater than 7, add 6 M H2SO4
with mixing until the pH is less than 7.
Add one mL 6 M H2SO4 and 0.5 g solid K2S2O8. Mix to dissolve.
Using a hot plate, boil gently for 40 minutes or until the sample volume is 10 mL or less.
Cool and dilute to 30 mL. Add 1.0 M NaOH, with mixing, until the sample is distinctly basic.
Dilute to 100 mL with distilled water.
Reagents
ammonium molybdate ((NH4)6Mo7)24 4 H2O)
ammonium metavanadate (NH4VO3)
12 M (concentrated) HCl
Anhydrous KH2PO4
6 M H2SO4
K2S2O8.
1 M NaOH