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The Phosphate Assay Virtual Lab

Malachite Green Reaction Protocol for measuring phosphate concentration

Nitrate and phosphate-containing compounds create a serious problem when released in water bodies without treatment. Phosphate and nitrate are major nutrients needed by living microorganisms for their physiological processes. However, they are considered pollutants if their concentration is more than the recommended limit. 

Heavy nutrient load (nitrate and phosphate) containing water bodies favor the growth of aquatic plants and create negative effects on water quality by accelerating the growth of algal clump, bad odor, and decoloration.


In the lab one day...


Can someone please explain this...



 Make the following 4 reagents labeled as A-D 

  • Reagent A: 4.2% Ammonium molybdate in 4.5M HCL 
    • Made by adding 2.1g of ammonia molybdate to 50mL of 4.5M HCl
  • Reagent B: malachite green reagent solution 
    • 1%, Aqueous, 100 mL
    • Company: Flinn scientific Item #: M0100 
  • Reagent C: 1:3 reaction mixture of A and B
    • To make this mix 25mL of A and 75mL of B 
  • Reagent D: 3% volume by volume triton x-100
    • To make this mix 3 uL of triton x-100 in 100uL of di water 


  • Reagents B and C are extremely light-sensitive so keep them covered with aluminum foil.
  • Reagent C and D are temperature sensitive so keep them in cold temperature or on ice when using them 
  • Reagent C is only stable for up to 5 weeks so make sure to only make the amount you plan to use in this allowed time frame.

How do we create the solutions for that standard curve...



Making the reagents


Performing the Experiment and Interpreting the Plot 


This lab was created through funding from NSF Award #1708823.

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