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Research Project

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


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.


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

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.

The technique available for your use is the vanadomolybdophosphoricacid spectrophotometric method (VM method). the intensity of which is directly proportional to the amount of phosphate present.⇌ PO43pH 2 pH 7 pH 12 Phosphate can form complexes with many di and tri-valent metals. Reagents & Other Materials. H3PO4 ⇌ H2PO4. In particular. there are many observations you can make to help you with your analysis. you should be noting what changes occur in your samples every time you perform an operation. See chapter 11 for details. Much information regarding testing for phosphate and the vanadomolybdophosphoric acid method is available on the web. This test also require the use of a quantitative dilution to prepare standards. ammomium molybdate is reacted with a phosphate sample under acid conditions to form a heteropolyacid.⇌ HPO42. An analysis for total phosphate requires two procedural steps: 1) conversion of all forms of phosphorus into dissolved phosphate by a digestion method. In addition to the normal lab items found in your locker and in the room. In the presence of vanadium. All other required reagents are available as reagents. will have to be made. The predominate form is pH dependent. Most of the phosphorus in soils is present as insoluble soil particles or incorporated into organic matter. In this procedure. Procedure This test uses absorption spectrometry to determine concentrations. the materials that will be provided by the preproom are detailed in the Research Project Materials . Any evaluation of phosphate content must take these factors into account. yellow vanadomolybdophosphoric acid is formed. and 2) evaluation of the dissolved phosphate concentration. Unusual observations often explain unusual numbers. This technique is based on the spectrophotometric phosphate determination method taken from Standard Methods for the Examination of Water and Wastewater. In the soil it usually reacts to form insoluble calcium or iron phosphates. an evaluation for total phosphate can provide results that are very different from an evaluation for soluble phosphate. The main reagent. Phosphate is usually not readily available in soil for plant uptake. the vanadate-molybdate (VM) reagent. Although this is primarily a measurement lab.Phosphate is a tri-protic weak acid that is readily soluble in water. 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. A thorough understanding of the Beer-Lambert Law and its use is necessary to analyze the data collected. As a result. Standard tests for phosphate evaluate only soluble phosphates. It can also be tied up in organic molecules such as ATP. a comprehensive reference covering all aspects of water analysis techniques and published jointly by the American Public Health Association and the Water Environment Federation. Refer to chapter 10-7 of the technique book for details on this technique.

allow all samples to stand for 10 minutes to ensure complete reaction. Such requests must be submitted by your TA. The following is a description of the steps required to obtain results for a generic sample containing phosphate. Design Issues Determining concentrations via absorption spectroscopy requires a calibration plot. based on the nature and number of samples to be evaluated. Solution A: Dissolve 2. Each reaction must be performed separately. After mixing. 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. Construct such a plot requires a series of phosphate solutions of known concentration to test. Successful performance of the VM phosphate determination requires the following sequence of events. Mixing the reaction solution. 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. Note: when an acid or base is added to adjust pH. it needs to be adjusted.125 g of ammonium metavanadate (NH4VO3) to 30. Anhydrous KH2PO4 will be available for this application. This procedure will need to be modified. These can be made from pure. 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. Heat just to boiling and stir until dissolved. Cool to room temperature and add 33 mL concentrated HCl.5 g ammonium molybdate ((NH4)6Mo7)24• 4 H20) in 30. If not. Mixing the VM reagent and samples. a digestion will have to be performed. When deciding what solutions to make. Making the VM reagent. solid samples of known molecular weight. Solution B: Add 0.0 mL distilled water. The VM reagent needs to be made as described above. mix them and dilute to 100 mL with distilled water. A minimum of four standard solutions should be employed. WARNING It is recommended that you keep all solutions until you are sure you have reliable measurements. If the sample is a solid.0 mL of distilled water. Preparation of Standards. Additional. The following issues need to be addressed. Preparation of Samples. There should be little or no apparent color change in the test tubes during this time. the increase in volume will dilute the sample! If the sample is biological or soil and total phosphate is being evaluated. This is the VM reagent. These will be used to generate a calibration plot. With both solutions at room temperature. the more detailed your analysis can be. . non-sample materials may be requested of the preproom.00 mL of the VM reagent. 4. The pH must be between 2 and 10.You are responsible for providing your own samples to evaluate. A procedure is given below. A sufficient number of standard phosphate solutions must be prepared. Prepare two solutions and then mixing them. and more detail added. Preparation of Reagents.00 mL of sample is thoroughly mixed with 1. Note that the more samples you test. it needs to be dissolved to an appropriate approximate concentration.

until the sample is distinctly basic. add 6 M H2SO4 with mixing until the pH is less than 7. Add 1. Prepare a suitable spectrophotometer. boil gently for 40 minutes or until the sample volume is 10 mL or less. 1 M NaOH . Add one mL 6 M H2SO4 and 0.Absorption Measurements. Take 50 mL of sample.5 g solid K2S2O8. Reagents ammonium molybdate ((NH4)6Mo7)24• 4 H2O) ammonium metavanadate (NH4VO3) 12 M (concentrated) HCl Anhydrous KH2PO4 6 M H2SO4 K2S2O8. Mix to dissolve.0 M NaOH. Dilute to 100 mL with distilled water. 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. Cool and dilute to 30 mL. Check the pH. Acidify the sample. If greater than 7. Using a hot plate. with mixing. Sample Digestion The following is a description of a digestion designed to free up bound phosphate.