Environmental Science 430

Fall 1999

Lab #4: Determination of orthophosphate by ascorbic acid method

Purpose: Prepare standard phosphorus solutions, develop a calibration curve for orthophosphate, and determine the orthophosphate concentration of an unknown water sample by using the ascorbic acid procedure and a Spec 20.

Materials:

• Spec 20
• Acid washed glassware
• 5.0 N H₂SO₄ - dilute 70 mL conc H₂SO₄ to 500 mL with distilled H₂O
• Potassium antimonyl tartrate - dissolve 1.3715g K(SbO)C₄H₄O^.½H₂O in 400 mL distilled water in a 500 mL volumetric flask and dilute to volume.
• Ammonium Molybdate - dissolve 20 g (NH₄)₆Mo₇O₂₄^.4H₂O in 500 mL distilled water.
• 0.01 M Ascorbic acid
• 50 ppm stock Phosphate solution - dissolve 219.5 mg KH₂PO₄ (FW=136.1) in distilled water and make up to 1.0 L
• Sample of unknown concentration
• Sample of French Creek from above sewage treatment plant
• Sample of French Creek from below sewage treatment plant

Procedure:

1. Place red filter and lamp in Spec 20, turn on and warm up the machine for 30 minutes.
2. Using acid washed glassware prepare the combined reagent by adding the following solutions to a 400 mL beaker.

   *** Use constant stirring; make sure to add the reagents in the order given.***

a. 50 mL 5.0 N H₂SO₄ using a volumetric flask.

 

b. First transfer 10-20 mL of Potassium antimonyl tartrate to a clean beaker. Using a five mL graduated pipette add 5.0 mL of this solution.

*** Be careful with this solution. It is TOXIC.***

c. After transferring 20-25 mL to a clean beaker, add 15 mL ammonium molybdate solution using a graduated pipette.

*** Be careful with this solution. It is TOXIC.***

d. 30 mL ascorbic acid, using a 10 mL graduated pipette.

 

3. This reagent is stable for about 4 hours.
4. Prepare a phosphorus standard from the stock solution by dissolving 50 mL of stock solution to 1000 mL of distilled water.
5. Prepare five phosphorus standards of known phosphorus concentrations, using volumetric glassware.Dilute in separate beakers to 100 mL with distilled water the following volumes of standard P solution: 2.0, 6.0, 10.0, 14.0, and 20.0 mL. This will give you 0.05, 0.15. 0.25, 0.35, and 0.5 mg/L P concentration.
6. Formulate a phosphorus mixture from the standard of unknown concentration, being sure, however, the concentration is within the range of your standards.
7. Transfer the 100 mL of each phosphorus standard and the unknown to an acid washed 250 mL beaker and add 8.0 mL of the combined reagent prepared in step 2.
8. Wait about 10 minutes, no longer than 30. A blue color should have formed. Set the wavelength scale to 880 nm and take absorbance readings of the six solutions.
9. Use 100 mL of distilled water with eight mL of the combined reagent to set zero absorbance before the readings are taken.

1. (10 points) One mL of the 50 ppm stock solution contains how many mg of PO₄^-3-P? Show calculations. Explain how 2 mL of the standard phosphate solution diluted in 100 mL of distilled water results in a concentration of 0.05 mg/L. Show calculations.
2. (10 points) Different sources recommend using different wave-lengths for molybdenum blue. These range from 830 to 880 nm. Why is this possible?
3. (10 points) What is the purpose of using a blank which contains eight mL of the reagent in distilled water, rather than distilled water alone? Be specific in your answer.
4. (10 points) Prepare a calibration curve and determine the concentration of your unknown. Compare the calculated concentration of your "unknown" to the value you get from your graph. Explain discrepencies.

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