Determination of Phosphate by Absorption Spectroscopy

Determination of Phosphate by Absorption Spectroscopy


0.05 mg/mL (or 0.05g/L) phosphate [PO43-] standard from using NaPO4 or KPO4 reagents, Molybdate-Antimony Reagent, 1 g/L ascorbic acid, reagent water, concentrated sulfuric acid, 1:10 dilute nitric acid (or hydrochloric acid)

Four 10mL volumetric flasks, three 100mL vol flask, one 500mL vol flask, pipette bulb, two 100mL beaker, one 500mL beaker, one 1000mL beaker, three Erlenmeyer flask, 2 droppers, hot plate, glass rod, three volumetric pipettes (1mL, 2mL, 5mL)


  1. Check if we have everything on hand and ready to use 
  2. Clean all glassware with dilute acid (HCl or HNO3
  3. Prepare phosphate standards: (use 10mL Erlenmeyer flasks) add XmL (X=0mL, 1mL, 2mL) of 0.005 mg/mL phosphate standard, 2mL Molybdate-Antimony reagent, 1mL ascorbic acid, then dilute to volume with reagent water 
  4. Blank the instrument (see baseline correction instructions) with solvent only
      a. In this case, 0mL PO4 Standard put at back of sample holder
      b. Set range from start:900nm to end:200nm 
  5. Sample preparation (use Alcanox, saltwater from aquarium, Tap water)
      a.Weigh approximately Y grams in Erlenmeyer flask (Y=10g Alcanox
      b. Dilute to approximately 100mL
      c. Add 1 drop of 0.2% phenolphthalein indicator and 0. 1mL (2 drops) concentrated sulfuric acid        and boil for an hour
      d. Put a glass rod against the beaker (one end at the bottom, inside beaker) to prevent bumping 
      e. Cool to room temperature, neutralize with strong base (NaOH) 
  6. Quantitatively transfer and dilute to 100mL volumetric flask (saltwater aquarium and tap water) 
  7. Quantitatively transfer and dilute to 500mL vol flask (Alcanox solution) 
  8. Pipette 5mL aliquot (for each dilution, separately) add 2mL Molybdate-Antimony Reagent, 1mL ascorbic acid, mix, dilute to volume and mix again 
  9. Wait 15 minutes for color to develop completely 
  10. Measure absorbance of each 3 samples 
  11. Repeat steps 8-10 three times

                                                                   The Many Attempts:

My original plan was to adapt this lab by miniaturizing a pre-made procedure from another online source. This meant I would perform a 10-fold dilution on all required reagents, which would significantly cut down my waste and create a “green chemistry” technique.

The first few attempts failed because the wrong resources and calculations were used to create the concentration of reagents, these attempts are:

1. First try to make the phosphate standard was based on the weight-volume composition or (mg/mL) of the monopotassium phosphate to acquire target goal of 0.05mg/mL phosphate. This is wrong. Correct way is to prepare the standard based on phosphate [PO4], otherwise phosphate concentration is too low to be detectable.
    a. Reagents prepared: 0.005175mg/mL KH2PO4 (from 0.5175g then series of 100-fold                        dilutions), 0.1g/100mL ascorbic acid, molybdate-antimony reagent (prepared from                          outside vendor)
The resulting 10mL volumetric flasks showed no color change due to low concentrations of phosphate.

  1. Second attempt to make the phosphate was to use phosphate [PO4] to acquire the target concentration of 0.05mg/mL phosphate. This is correct.
        a. However, no color change occurred because ascorbic acid concentrations were more than              10 order of magnitude lower than prescribed. 
  2. Consequent trials were done to fix this issue of no color change. These changes include increasing the concentrations of phosphate by increasing KH2PO4 (1.4329g/L, 0.14329 g/L)
        a. Increasing phosphate still did not work, ascorbic acid concentrations were not corrected                and still far too low. 
  3.  Subsequent trials further were made based on target concentration of 0.5g/L of Phosphorous, which resulted in 2.197g/L, 0.02197g/L, 0.002197g/L of KH2PO4.
        a. Ascorbic acid was also corrected upon stumbling on the Molybdate-antimony (MA) vendor
            supplied procedural instructions for a similar method. Two different concentrations of                    ascorbic acid were used to get the final results: 10.67g/100mL and 1.67g/100mL
        b. Lowest concentration (0.002197g/L) of phosphorous was used to make results of this                  experiment, which surprisingly worked. Surprising because phosphate concentration is                  significantly lower than the detection limit called for by the MA vendor’s instructions.


Recommendation for Future Experiments:

  1. Use the manufacturer’s procedure guideline, and then modify it to fit my goal 
  2. Experiment reaction time with 10.67 g/100mL ascorbic acid took 5-10 minutes compared to 1.67 g/100mL ascorbic acid required 1 hour for completion
        b. No difference in absorbance between the two ascorbic acid (high concentrations of acid do           result in yellow form of blank standard)
        a. Therefore, use 10.67 g/100mL ascorbic acid

General Recommendation:

  1. If a pre-made reagent (made by an outside vendor) is going to be used for the experiment, look for, and use their procedure while making changes, if necessary. 
  2. Lab notebook needs organization:
      a. Make tables before the experiment
      b. Leave extra room for observations (maybe, on left or right hand-side) 
  3. If an experiment does not work, there should be a scientific method to deducing fixes, such as:
      a. Checking references and other outside resources
      b. Changing one variable while keeping everything else constant

Weighing reagents

Diluting a standard to volume

    Standards in their respective cuvettes, ready to be analyzed for absorbance measurements

More standards with accidental spill that needs to be cleaned 

Graph of the calibration standards density (ug/L) against absorbance measurements at the 880nm. Note that only 3 different standards are graphed. Also, the phosphate concentrations are significantly lower than the manufacturer of Molybdate-antimony recommends. The experiment will need to be redone to fit a 19 to 3000ug/L range of phosphate.