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PHOSPHATE DETERMINATION

Range = 0.03 to 5.00µ g-at/L

I. REAGENTS AND STOCK SOLUTIONS REQUIRED:

1. Ammonium Molybdate sol’n:


Dissolve 7.5g of ammonium paramolybdate (NH4)Mo7O24-4H2O in 250mL of DI
water. Store in plastic bottle out of sunlight.

2. Sulfuric Acid Sol’n:


Add 70mL of concentrated sulfuric acid to 450mL of DI water. Allow the solution
to cool and store it in a glass bottle.

3. Ascorbic Acid Solution:


Dissolve 13.5g of ascorbic acid in 250mL of DI water. Store the solution in a
plastic bottle in the freezer. This solution should not be kept at room temperature
for more than one week, but is stable in the freezer for many months.

4. Potassium antimonyl-tartrate solution:


Dissolve 0.34g of potassium antimonyl-tartrate in 250mL of DI water (if
necessary, warm and use a stirring rod to help dissolve). Store in a plastic bottle.

5. Mixed Reagent:
Mix together 100mL ammonium molybdate, 250mL sulfuric acid, 100mL
ascorbic acid, and 50mL of potassium antimonyl-tartrate solutions. Prepare this
solution fresh as needed. Quantity is suitable for about 50 samples. Discard
excess.

6. Phosphate Primary Stock:


Prepare a 10mM solution. Weigh out 0.6805g of KH2PO4 (fw = 136.09). Dissolve
in distilled, deionized water, making up to 100mL using a volumetric flask.

7. Phosphate Secondary Stock:


Prepare a 50µ M solution. Bring 100 uL of the primary stock up to 100mL with
distilled, deionized water using a volumetric flask.

8. Synthetic Seawater:
Mix together 31g NaCl, 10g MgSO4, and 0.5g Sodium Bicarbonate in 1000mL
distilled water.
II. BLANK DETERMINATION

Use synthetic seawater in place of a sample and carry out the steps outlined in section IV
to obtain the turbidity and extinction of the reagent blank. Reagent blanks should be less
than 0.02 in a 5cm cell.

III. STANDARDS
Phosphate Conc. (µ M) Phosphate 2º Standard (mL) Synthetic Seawater
0.05 0.10 ADD
0.10 0.20 TO
0.50 1.00 MAKE
0.75 1.50 100mL
1.00 2.00 FINAL
1.50 3.00 VOLUME
2.00 4.00
For example, to make a 0.1mM phosphate solution, add 0.20mL of the phosphate
secondary standard to a 100mL volumetric flask, then raise the volume to 100mL with
synthetic seawater.

Carry out the steps outlined in section IV (2-5) to obtain the extinction of the standards.

Plot the concentration of the standards vs. the measured absorbance and perform a linear
regression to obtain a standard curve.

IV. PROCEDURE
1. Warm the samples to room temperature. Using a spectrophotomer, measure the
turbidity of a sample at 885nm (using a a 5cm cell against DI water). If this value
is greater than 0.01, a correction should be applied to the final extinction value.

2. Measure out 100mL of sample using a graduated cylinder

3. add 10mL of the mixed reagent, using a volumetric pipette, and mix.

4. Wait at least 5 minutes (and not more than 2-3 hours)

5. Measure the extinction in a 5cm cell against DI water at 885nm. Correct the
absorbance measurement by subtracting the value obtained for the blank.

6. Use the standard curve to determine the concentration of your unknown.