Phosphate Analysis Ascorbic Acid-Molybdate Method

Based on SMEWW Method

Adapted from Murphree 2002

Theory
In the ascorbic acid-molybdate method orthophosphate reacts with molybdate to form phosphomolybdic acid. Phosphomolybdic acid is reduced by ascorbic acid to form a blue complex:

Phosphate + Molybdate Phosphomolybdic Acid Phosphopmolybdic Acid + Ascorbic Acid Reduced Phosphomolybdate complex
Blue

We can determine the amount of phosphate in the solution by measuring how blue it turns in the presence of molybdate and ascorbic acid because the absorbance of the solution is directly related to the concentration (Beers Law). However, to do this we first need to determine the quantitative relationship between absorbance and concentration for this type of solution by using a standard curve (calibration curve). In this case our standards are known concentrations of phosphate.

Materials

10mg PO4/L stock solution to make standards, 0-1mg PO4/L 10ml graduated pipette

5ml graduated pipette 200 l micropipette and tips 5 50 or 100ml volumetric flasks 1 13x100mm test tube for each sample, standard, and blank 1 small square of parafilm for each test tube Ascorbic acid reagent Molybdate reagent Spectrophotometer Computer

Procedure
Standards

1. 2. 3. 4. 5. 6. 7.

Prepare standards between 0-1mg PO4/L from the 10mg PO4/L stock solution. Pipet 5ml of the solution to be tested into a small test tube. Carefully use the micropipet to add 100 l of ascorbic acid to the small test tube. Cover the test tube with parafilm and mix. Carefully use the micropipet to add 100 l of the molybdate reagent to the small test tube. Cover the test tube with parafilm and mix. Let the solution react for 20min then measure the absorption at 880nm using DI water as a blank.
Samples

8. Establish Phosphate Range The chemistry above illustrates that the amount of color the solution develops is proportional to the amount of phosphate present in the sample. Unfortunately, while the spectrophotometers we use can give us very good precision, they only work within fairly narrow absorption values (From close 0 to close to 1). The method above works just fine for samples between zero and 1 part per million (ppm) phosphate, but more concentrated samples must be diluted with DI water. To determine how much each sample must be diluted take one replicate from each of your samples (Not all three replicates). Shake the sample and add 5 mls of sample to the first test tube, and 0.5mls to a second. Add 4.5 mls of DI water to the second tube (you have diluted the contents of the second tube by a factor of 10. This procedure will tell you if you can analyze your samples full straight, or whether you need to dilute them before hand (see number 8). 9. Record the absorption from each tube BEFORE YOU DO THE FOLLOWING (see number 9). 10. Proceed through steps 3 through 7 above. 11. You have two tubes for one replicate of each sample. If the absorbtion of the undiluted tube is 1 or less, you do not
need to dilute your other two replicates and should complete steps 3 though 7 on the other two replicates of each sample. If the absorption of the diluted sample is between 0.1 and 1, youll need to dilute the other two replicates for that sample by adding 4.5 mls of DI water to 0.5 mls of sample and following steps 3 through 7. If the absorbtion of the diluted tube is greater than 1, see your instructor regarding how to dilute the sample further.

12. Analyze all of your standards (0-1mg PO4/L) and all of your samples after reacting with ascorbic acid and molybdate reagents. The 0mg/L standard will tell you if there is any absorbance at 880nm from the reagents alone. This absorbance will be accounted for in your standard curve. Also analyze all of your samples without adding reagents. This will tell you if there is any absorbance at 880nm from other non-phosphate parts of the sample. You must account for this absorbance when you calculate your concentrations. Keep track of your data in a table like the sample below. Each cell is required information.

13. Plot the standard curve and calculate the concentrations of the samples. 14. If you diluted any of your samples, be sure to take the dilution into account when calculating the concentration. For instance, if you diluted by a 10 to 1 ratio, you must multiply the concentration by 10. 15. Dispose of waste in the appropriate waste beakers. 16. Clean up all of your materials and turn the spec off.

Sample Number/

Dilution? Absorption Absorption Equation Concentration for Standard If yes, Without With Curve Replicate how Number much? Reagents Reagents