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Environmental Measurements Lab—Colorimetric Phosphate Determinations


Chapter 12 of your lab manual gives background information on spectroscopic measurements. Read this chapter paying special attention to Beer’s Law (aka the Beer-Lambert Law). Section 25.2 of your lab manual describes procedures for measuring the phosphorus content of water samples using a spectroscopic method. You can find more information on spectroscopic analysis using visible light at

Phosphorus can be present in a water sample in at least three forms: orthophosphate, acid-hydrolyzable phosphate and total or organic phosphate. Orthophosphate can be measured directly. The measurement is carried out by adding reagents which form a deep blue complex with any orthophosphate ion in the sample. The blue color is read on a spectrophotometer. The other two forms must be converted to ortho before measuring. Total phosphorus can be digested to ortho using ammonium persulfate, sulfuric acid and heat. A method in which you measure color proportional to an analyte is called a colorimetric method.

I think that we will work in two groups for this lab. One set of reagents should be enough for the whole class. We should divide up the work among everyone. You have two weeks to complete this lab assignment.

Week One:

  1. Read this entire handout and ask me any questions you have about what you are supposed to do for this assignment.
  2. Acid-wash all the glassware that you will need for reagents and samples.
    1. Assemble the glassware you need. It should be soap and water clean. Rinse each piece with distilled water.
    2. Fill with ~6 M hydrochloric acid (1:1 concentrated acid and distilled water) and let soak for a minute or so. Note: You can make the acid right in the item being rinsed.
    3. Pour the acid into the next item to be cleaned and rinse the empty item three times with distilled water.
    4. Clean about eight to sixteen 125 mL Erlenmeyer flasks, five 500 mL Erlenmeyers, a small volumetric flask (100 to 250 mL), 500 mL volumetric flask, 1 L volumetric flask, at least one pipette, 2 reagent bottles, and small graduated cylinders (25 and 100 mL), a large graduated cylinder, a 1 L beaker.
    5. Divide this work up among everyone in class.
  3. Make the reagents listed on page 289 and page 291, (don't make the 0.01 M Ascorbic Acid or the Combined Reagent until you are about to use them).
    1. We have crystalline potassium dihydrogen phosphate (KH2PO4) which can be used instead of potassium dihydrogen phosphate.
    2. The potassium antimonyl tartrate solution is commercially available (premade). We may be using that.
  4. If you have time, you can run an orthophosphate standard curve (see number 6 below).

Week Two:

  1. Use only acid-washed glassware.
  2. Before you can measure the Total Phosphorus, you need to digest the samples with ammonium persulfate and sulfuric acid to convert all the phosphorus to orthophosphate. Set up digestions on four or five solutions: a blank (distilled water), a QC standard (supplied by the instructor), untreated sewage, treated sewage and another sample (optional). Check with the instructor for appropriate volumes to run for each. Use the hot plate procedure on page 289. We may need to use bunsen burners in place of some of the hot plates. This digestion may have already been done for you by your humble professor. While the total phosphorus samples are digesting, continue by setting up and running an orthophosphate standard curve.
  3. Run an orthophosphate standard curve using the procedure on page 292.
    1. Make up the 0.01 M Ascorbic Acid solution and the Combined Reagent.
    2. You will run a series of dilutions of the 2.5 mg/L Standard Phosphate Solution and an orthophosphate reference standard (0.5 mg/L) made from the 25 mg/L Reference Solution.
    3. Instead of the standard values listed on page 292, use the following:
    4. mgP/L

      mL standard solution
      (2.5 mgP/L)

      0 (Blank)

      0 mL


      1 mL


      5 mL


      10 mL


      15 mL


      20 mL

      Dilute all to 50 mL with distilled water.

    5. Add 8 mL of combined reagent to each flask and read the blue color at 850 nm at least 10 minutes but no more than 30 minutes after adding the reagent.
  4. The absorbance values (read on the Spec 20) for these standards should form a line (plot mgP/L vs absorbance).
    1. You can use your TI 82 to calculate a slope and intercept for the absorbance and concentration values. Alternatively, you can sketch a line by eye on a piece of graph paper.
    2. Calculate a phosphorus concentration for the reference standard (or read it by eye off your sketch). This value should fall within ten percent of the known value (ie, 0.5± 0.05 mgP/L).
  5. Go back to your persulfate digestions. When a sample has decreased in volume to less than 10 mL, or has been boiling at least 40 minutes and has a volume less than 50 mL (whichever comes first), it is done. Remove from the heat and let it cool.
  6. Dilute and neutralize as described on pages 289-290 except make up to a final volume of 50 mL (NOT 100 mL).
  7. Measure the orthophosphate concentration as before. In addition to your four or five digested solutions, run a non-digested blank and a non-digested standard (0.5 mgP/L).
  8. Calculate the total phosphorus concentration using the "Calculating Concentrations" example attached as a guide.
  9. I will give you data for calculating an MDL as described on page 32. If you have time (ha-ha) you can generate your own data by running seven low standards—0.10 mgP/L would be good. Also run a blank and a mid-range standard (0.5 mgP/L).
  10. You have two weeks to complete your lab report as described below. Ask any questions you have about what you did or about the report.

Required in Lab Report #4

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Anthony Benoit