DIGESTION FOR PARTICULATE PHOSPHORUS

 

Particulate matter collected on a glass fiber filter is ignited at low temperature to destroy organic matter.  The ignited filter is heated with dilute HCl which extracts the phosphorus and converts it to ortho-phosphate.  The phosphorus is then analyzed by a version of the reactive phosphorus method.

 

Apparatus and supplies

                              Ammonium Para - molybdate (A674-500) (NH₄)₆Mo₇O₂₄ *4H₂O

Antimony potassium tartrate

Sulfuric acid (A300-212)

L-Ascorbic Acid (A61-100)

Phosphate standard (LC 186-007)

HCL (A1444-212)

                              Spectrophotometer set to read @ 885 nm with a 1 cm flow cell

Muffle furnace set 450-550 C

Oven capable of being set at 70 C and 104 C

Pyrex screw cap test tubes (Fisher 149330 10E)

HDPE lined caps (Fisher 14930 15E)

      Glass scint vials will also work (OK for muffle furnace), caps will withstand drying oven temps (<104)

Metal rack capable of withstanding 550 C (14-790)

Re- pipettes/Pipettes

2 ml (1 N HCL)

10 ml (DI water)

2.5 ml (mixed molybdate reagent)

1 liter amber bottle to store mixed para molybdate solution

100 ml volumetric for ascorbic acid

500 ml graduated cylinder to mix ascorbic acid and mixed molybdate

1000 ml volumetric

100 ml graduated cylinder (sulfuric acid)

Phosphorus standard (1000uM)

       Anhydrous potassium phosphate KH₂PO₄ mw 136.09

NIST Apple leaf standard reference material

 

REAGENTS

 

1)     1 N HCl

            82.64ml of 12.1 N HCl to 1000 ml with DI (you will need some for flushing the spec also)

 

2)     Ammonium Molybdate

In 500ml volumetric add 15g of ammonium paramolybdate, (NH₄)₆Mo₇O₂₄ *4H₂O and approximately 350ml DI water, shake/stir till dissolved, make to volume (500ml) with DI and transfer to plastic amber bottle (transparent bottle can be used, just keep out of sunlight).

 

3)     4.8N Sulfuric Acid (H₂SO₄)

In ice bath Add 140ml of 36N H₂SO₄ to 900ml DI water.  Allow solution to cool and store in a glass bottle.

 

4)     Ascorbic  Acid Solution

In 500ml volumetric add 27g ascorbic acid and approx. 350ml DI water, shake/stir till dissolved, make to volume with DI (500ml).  Store in plastic bottle in freezer.  Thaw for use but refreeze.  Stable for many months but should not be left at room temperature.  Tip: separate solution into plastic disposable scint vials or similar containers, in this way you can thaw out only the amount needed.

 

5)     Potassium Antimonyl Tartrate (careful, toxic)

0.34g potassium antimonyl tartrate, fill to volume with 250ml DI water, warm if necessary.  Store in glass or plastic, stable for many months.

 

6)     Mixed Reagent 500ml (have samples ready, make daily, only keeps 6-8 hours)

Final amount can be modified depending on number of samples.  Reagent should be made from the following proportions in the order given.

100ml               Ammonium Molybdate

250ml               4.8N Sulfuric Acid

100ml               Ascorbic  Acid Solution                        Should turn pale yellow color

50ml                 Potassium Antimonyl Tartrate   

 

 

PROCEDURE

 

1)     Filter an adequate amount of water through an ashed 2.5 cm GF/F filter, recording amount of water filtered.  This amount will very with sediment load of the water being analyzed.  Filter should be “clogged” to ensure enough particulate phosphorus for an accurate measurement.  Place filters in labeled petri-dishes.

2)     Cover tubes with tin foil (or petri-dish lids loosely) and dry at 70º C for 1 hour, I typically dry the blank and std filters in bulk in tin foil

3)     Prepare standards and blanks by placing ashed GF/F filters in marked reaction tubes and adding appropriate amount of primary stock standard (see below).  Stock is added directly to blank/std tubes which contain filters.

4)     Place tubes in muffle furnace at 500º C for 1 hour (if samples have been in petri-dishes, transfer to tubes before placing in muffle furnace), cover with foil

5)     After sample has cooled, add 2 ml of 1 N HCl and 10 ml of DI, Vortex

6)     Cap samples tightly and place in oven at 104º C for 2 hours

7)     After samples have cooled, add 2.5 ml of mixed reagent (see above) to each tube, Vortex

8)     Re-cap and place tubes in the dark

9)     Allow 30 minutes for color reaction to occur, std should be noticeably blue, and read standards and samples at 885 nm on a spectrophotometer within 2 hours.

 

STANDARDS AND CALCULATIONS

 

Standards for PPO₄ are added to ashed GF/F filters prior to ashing (before step 4 above).  Prepare a standard curve by adding varying amounts of 1000 uM primary stock standard (see below) to filters that have been placed in reaction tubes.  The following is a table of the number of tubes and filters needed, amount of primary (1000uM)  stock to add, and mmols of PO₄ in each tube after standard is added:

Number            1º Std (ml)        mmols PO₄          1000um KH₂PO₄ Stock

            4                      0                      0.0                   Anhydrous potassium phosphate KH₂PO₄ 

            3                      25                    0.025               Dissolve 0.1361g KH₂PO₄ in DI water, bring to

            3                      50                    0.050               volume in 1L volumetric.

            3                      100                  0.10

            3                      150                  0.15

            3                      200                  0.20

Obtain a regression equation y=mx+b where y is the absorbance, m is the slope of the linear regression, x is the mmols of PO4, and b is the intercept.  Calculate the amount of PPO₄ in each sample tube (in mmols) using this standard curve:

mmols = (y - b) / x

 

The concentration of PPO₄ in the water sample (in mmols/L) is then determined by dividing the mmols by the amount of water filtered for each sample (in liters).