3.3: : Determination of Phosphate by a Colorimetric Method

A3: Determination of Phosphate by a Colorimetric Method

Normal laboratory safety rules should be observed throughout this experiment. Lab coats and safety spectacles must be worn at all times. Mouth pipetting is forbidden. Benches must be kept clean and tidy. Eating, drinking and smoking are not allowed in the laboratory. The vanadate-molybdate reagent used in this practical is in strong acid solution. Handle with care, avoid skin contact and clear up any spillages immediately.

Introduction

In a dilute orthophosphate solution, ammonium molybdate reacts under acid conditions to form the heteropoly acid, molybdophosphoric acid. In the presence of vanadium, yellow vanadomolybdo-phosphoric acid is formed. The intensity of the yellow color is proportional to phosphate concentration. Absorbance may be measured at 400, 420 or 470 nm depending on the sensitivity required, the absorbance at 400 nm being ten times that at 490 nm for a given P concentration. However, due to potential interference from ferric ions at low wavelengths, 470 nm is normally chosen. In this experiment, you will determine the phosphorus content of various samples of waters. No sample pre-treatment is required, except for dilution and addition of the colorimetric reagent. To avoid any possible phosphate contamination from detergents, it is very important that you use only the acid-washed glassware supplied in the cupboard for this experiment.

Apparatus

UV/visible spectrophotometer & glass cells

Volumetric flasks: 13 x 50 ml

Beakers: 6 x 100 ml

Pipettes: 10 & 25 ml bulb pipettes

Reagents

Sample of water from the lungs of the victim.

Samples of water from the bath, swimming pool, tap, river and sea.

Vanadate-molybdate reagent: a mixture of ammonium molybdate [(NH4)6Mo7O24] and ammonium metavanadate [NH4VO3] in HCl.

Standard phosphate solution: KH2PO4 containing 50.0 !g P per ml.

Preparation of Calibration Standards

Prepare a series of calibration standards by transferring 0, 5, 10, 15 and 20 ml aliquots of the standard phosphate solution to 50 ml volumetric flasks, adding 10 ml of the vanadate-molybdate reagent to each and diluting to the mark with deionized water. The standards thus diluted contain 0, 5, 10, 15 and 20 !g P per ml (the one containing no phosphate acts as a reagent blank).

Procedure

1. Shake the ‘bath water sample’ to ensure thorough mixing, then transfer a 25-ml aliquot to each of two 50-ml volumetric flasks.
2. Add 10 ml of the vanadate-molybdate reagent to one of these flasks - the second acts as a sample blank - and dilute each to the mark with de-ionised water.
3. Shake to mix.
4. Repeat 1-3 for the other water samples.
5. Leave all prepared solutions (including calibration standards) to stand for at least 10 minutes for the color to develop fully before taking absorbance readings.
6. Measure the absorbance of each standard, sample and blank at 470 nm.
7. Plot a calibration graph of absorbance vs. phosphorus concentration (in !g/ml) for the standard solutions.
8. Use this graph to determine the phosphate (as P) concentration in each water sample (remember to allow for the dilution of the sample in your calculations) and subtract the sample blank reading from the corresponding sample.

Thinking About Making Valid Measurements

To give evidence in court you have to show that the analytical method is producing valid results.

1. Are you confident that the data you have obtained can discriminate between the samples?
2. Are you certain that any sample pre-treatment has not invalidated the analysis?
3. How might you determine the concentration of organically bound phosphorus in a water sample?

Improving Your Practical Technique

• The instrument should be allowed to warm up before use.
• A wavelength check should be carried out to check the instrument is correctly set up.
• Instrument needs to be calibrated using a set of standards of differing concentration for quantitative analysis.
• Cuvettes must be scrupulously clean especially in the region where the light beam passes through.
• Cuvettes should always be placed the same way round to so subtle differences in the cuvette shape are avoided.
• Matched cuvettes or the same cuvette should be used throughout the series of analysis.
• Sample may require degassing before analysis to remove air bubbles.
• Solid material must be removed from the sample before analysis.
• Beer Lambert law no longer applies above an absorbance of 1.
• A double beam spectrophotometer permits the reference beam to have a cuvette containing solvent.
• Measurements can easily be repeated and there should not be any significant difference between the readings (<±0.003 absorbance).
• Consider doing duplicate samples.
• Analyze blank sample (i.e. solvent blank and sample blank).
• In many cases samples may need to be diluted so that the measured absorbance is between the lowest and highest standard on the calibration curve.
• Wavelength scan should normally be performed to determine the absorbance maximum ("max) however, the determination was not carried out at the "max due to the potential interference from iron (Fe).
• Qualitative identification of UV/Vis is very crude but it is ideal for quantification using a colored complex in this experiment.
• Where practicable use a more specific technique such as ion chromatography.