In this practical you will analyse different water samples for the presence of phosphate and ammonia and measure their concentration.

Determination of ammonia and phosphate in water samples

Introduction

In this practical you will analyse different water samples for the presence of phosphate and ammonia and measure their concentration.

Surface water quality in England and Wales is monitored by the Environment Agency (EA) under the European Water Framework Directive (WFD).  Water quality is described for ecological, chemical, biological and overall quality with reference levels for each of ca. 30 parameters that are analysed.

(http://ec.europa.eu/environment/water/water-framework/index_en.html) (https://www.gov.uk/government/publications/list-of-chemicals-for-water-framework-directive-assessments/environmental-quality-standards-directive-eqsd-list-for-wfd-assessments)

Ammonia (NH₃) is one of the key physico-chemical water quality elements.  The EA classifies the water quality for ammonia on a scale from high to poor.  Ammonia occurs in natural waters as a result of the microbially mediated degradation of organic matter.  A key process in the nitrogen cycle, the ammonification reaction occurs anoxically and, as such, significant quantities of ammonia present in water is a strong indicator of poor water quality.  The EA therefore test water courses regularly for the amount of ammonia present as a method of determining water quality.  Ammonia is also of concern as an air pollutant.

http://webarchive.nationalarchives.gov.uk/20130402151656/http://archive.defra.gov.uk/environment/quality/air/airquality/p

ublications/ammonia/documents/ammonia-in-uk.pdf

In this experiment you will use a standard procedure for measuring ammonia using Nessler’s Reagent (an alkaline solution of potassium tetraiodomercurate).  The Nessleriser system is based on a colorimetric reaction and colour comparison using a reference is used.  The equipment consists of a light box to view the samples and to compare the colour change in a sample with and without the reagent added.  Reference discs with coloured glass are used to convert the observed colour into a quantitative measure of ammonia concentration.  This method can also be used by EHPs in field tests to see if standing water, which is often located in both residential and commercial settings is in fact sewage or contains sewage.  Armed with this information the EHP can then investigate the likely sources and pathways from the defective pipe work.  Since the level of ammonia will be much higher than in environmental samples it is only necessary to get a qualitative view of the presence of ammonia. As you will be operating in a fully set-up laboratory, you can use spectrophotometry to quantify the product of the Nessler reactions. This will involve measuring the absorbance at a specific wavelength and converting this result into an ammonia concentration.

Phosphate (PO₄^3-), also referred to as orthophosphate, is measured under the physico-chemical water quality elements.  Phosphate can be introduced into water bodies from a number of sources.  The main problem associated with elevated phosphate levels is that of over fertilisation or eutrophication of water bodies.  This can lead on to decay of vegetative matter leading to deterioration in dissolved oxygen levels in the water.  Phosphate levels are classified according to the revised standards in the Water Framework Directive.

(http://www.wfduk.org/resources/new-and-revised-phosphorus-and-biological-standards)

(http://www.wfduk.org/resources/rivers-phosphorus-standards)

In this experiment you will use a quantitative colorimetric method to measure the phosphate concentration in water samples. When phosphate containing water sample is reacted with sodium molybdate in the presence of ascorbic acid and sodium pyrosulfate a chemical complex is formed that has an intensive blue colour. Ready mixed reagent powders are available for phosphate testing that can directly be added to the water sample to be tested.  The intensity of the colour is proportional to the amount of phosphate present in the water sample. With a spectrophotometer the absorbance (“intensity” of the blue colour) can be measured at a specific wavelength and phosphate concentration calculated from the result.

Notes on the Experiments

You will test a range of water samples and quantify ammonia and phosphate contents in each of those samples. Each sample will be tested multiple times in independent tests (done by different students) and statistical analysis used to calculate the concentrations of ammonia and phosphate.

You will also analyse solutions of know ammonia/phosphate content as quality control. These will be treated in the same way as the water samples for experimental analysis. The obtained result for ammonia and phosphate concentrations can then be compared with the actual values (provided) to assess the quality of the analysis.

Each student will be allocate a selection of samples and standard solutions to test for both ammonia and phosphate content in each.

We will collate all results and share these via MyLearning so that all students have a full set of results available for their report.

Experiment 1: Determination of ammonia

Equipment and Materials

Water samples from different sources; make sure to note the exact information on the origins of these.

Standard concentration solutions as quality controls; make sure you note the concentrations and understand the calculations needed for quality control of your method.  

Nessler’s reagent (ready-made, containing 10-20 % potassium hydroxide, KOH, and 10 - 20 % dipotassium tetraiodomercurate (II), K₂HgI₄)

Pasteur pipettes, micropipettes and tips

Glass tubes

Cuvettes

Spectrophotometer

Nessler’s reagent contains mercury, which is toxic in small concentrations.  All solutions containing Nessler’s reagent must be collected separately to recycle or dispose of safely.  Do not pour anything down the sink that might contain mercury!  The solution also contains potassium hydroxide, which is a strong base, PPE including gloves and goggles must be worn throughout.

Experimental procedure

1. Fill 5 mL of each of the water samples and standard solutions into a glass test tube (label tubes).
2. Add 200 mL of Nessler’s reagent to each of the tubes and mix.
3. Transfer contents from each tube into a corresponding labelled cuvette filling the cuvette to about ¾. If you notice any turbidity or precipitate forming after adding Nesslers Reagent, you need to let this settle and only transfer clear liquid into the cuvette (use a pipette and take carefully from top).
4. Fill a second set of cuvettes with each of the water samples and standard solutions as blank.

For each water sample and standard concentration you now have a pair of cuvettes, one containing only the sample and one containing the sample reacted with Nessler’s reagent.

5. Take your cuvettes to the spectrophotometer and make sure it is set to the correct wavelength for the ammonia test, 425 nm.
6. For each sample zero the instrument using the cuvette containing the water sample  or standard solution only (blank), then measure the absorbance in the cuvette containing the sample or standard with added Nesslers reagent.
7. Record the results for all samples and standards in a table.

Experiment 2: Determination of phosphate

Equipment and Materials

Water samples from different sources; make sure to note the exact information on the origins of these.

Standard concentration solutions as quality controls; make sure you note the concentrations  and understand the calculations needed for quality control of your method. 

Pasteur pipettes, micropipettes and tips

Glass tubes

Phosphate test powder pouches for 10 mL sample volume

Cuvettes

Spectrophotometer

Experimental procedure

1. Fill 10 mL of each of the water samples and standard solutions into separate glass tubes (label tubes).
2. To each tube add the entire contents of one phosphate reagent powder pouch, make sure not to spill any.
3. Invert each tube carefully to mix.
4. After 2 min reaction time invert the tubes once more and then transfer contents from each tube into a corresponding labelled cuvette filling the cuvette to about ¾.
5. Prepare a second set of cuvettes containing the water samples and standard solutions only as blanks.  

For each water sample and standard concentration you now have a pair of cuvettes, one containing only the sample and one containing the sample reacted with phosphate reagent.

8. Take your cuvettes to the spectrophotometer and make sure it is set to the correct wavelength for the phosphate test, 880 nm.
9. For each sample zero the instrument using the cuvette containing the water sample  or standard solution only (blank), then measure the absorbance in the cuvette containing the sample or standard with added phosphate reagent.
10. Record the results for all samples and standards in a table.

Supplementary information

Table 2: Molecular / atomic weights (g mol^-1).

NH4+	18.039
NH3	17.031
N	14.007
HPO42–	95.978
PO43–	94.970
P	30.974

Example for a calculation of analyte concentration in a solution.

You can calculate the nitrogen or phosphorous equivalents from measured ammonia or phosphate concentrations in a solution. This might be required to evaluate your results with environmental standards. Please note that the high pH of the Nesslers reagent results in any ammonium ions (NH₄⁺) to become deprotonated to form ammonia (NH₃), so this is what was measured. To measure total nitrogen a pre-reaction step is required to reduce any oxidised nitrogen species (nitrates, nitrites) into ammonia. The example below shows the calculation of nitrogen equivalents for a result of 0.4 mg L^-1 ammonia: