Analysis of Phosphate in a Water Sample

Experiment #5

Conducted 09/26/2024

Belen Kohl, Austin Acharya

Department of Chemistry, Binghamton University, Binghamton, NY 13902

Phosphorus is a vital nutrient for aquatic plants and algae, but too much will become a pollutant

to the water. This leads to eutrophication, which causes overgrowth of plants and algae and

lowers the oxygen levels in the water, making it uninhabitable for other organisms. Phosphorus is

present in surface water as orthophosphates, organically bound phosphates, and condensed

phosphates. Orthophosphates can be measured to give a rough estimation of phosphate levels in

the water. The specific orthophosphate measured in this lab is PO₄, which is colorless in an

aqueous solution on its own. This is done by inducing a chemical reaction, which will result in a

color change based on the concentration of phosphates in the water. The chemical reaction which

results in the formation of phosphomolybdic acid is shown below:

72H⁺ + 7PO₄³⁻ + 12Mo₇O₂₄⁶⁻ → 7PMo₁₂O₄₀³⁻ + 36H₂O

The subsequent reaction causes the formation of molybdenum blue from phosphomolybdic acid:

PMo₁₂O₄₀³⁻ + 2C₆H₈O₆ → PMo₁₂O₄₀⁷⁻ + 2C₆H₈O₆ + 4H⁺

Then, the colored solution is placed into a spectrophotometer, which is a device that emits

radiation reflected on a monochromator. The monochromator disperses the radiation into a

spectrum and a section of that spectrum illuminates the sample solution, through the solution and

to a detector to measure the intensity of the beams. The specific model of spectrophotometer

used in this experiment is the SpectroVis Plus, which covers the visible light range of

wavelengths. Through use of the SpectroVis Plus, the objective of the experiment is to log the

concentrations and absorptions of sample solutions with different concentrations. With this data,

the concentration of an unknown sample of the same solution can be obtained using a linear

regression model.
Experimental Section

To execute this experiment, the students cleaned and prepared five Erlenmeyer flasks to put the

different sample solutions into. To measure out the right concentration of each Phosphate

Standard solution, they first measured 25.00 mL of the 20.0 mg/L Phosphate Standard with a

pipet, relocated it into a volumetric flask, and added 75.00 mL of distilled water to create a 5.00

mg/L Phosphate Standard solution. The 5.00 mg/L solution was then poured into one Buret, with

distilled water in another. The students used the Burets to measure the different concentrations

for each of the four sample solutions so the final volume of all of them was 25.00 mL and

numbered each Erlenmeyer flask accordingly, then filled the final flask with the unknown

solution and labeled it with an S. After thoroughly mixing each solution, the students calculated

the final concentrations in each flask. Then, a PhosVer 3 Phosphate Powder Pillow was added to

each solution including the unknown and the mixtures were mixed until all powder dissolved and

left to rest for ten minutes so the reaction could occur. The fourth flask –with a concentration of

2.50 mg/L PO₄– did not react initially, despite waiting the allotted ten minutes, so the students

cleaned the flask and remeasured the concentration of PO₄. The second attempt was successful

and the solution turned a deep blue. It was observed that as the concentration of PO₄ in the

solution increased, the blue color deepens and grows in intensity. The unknown sample was

noted to be a similar shade of blue to the 2.50 mg/L PO₄ sample. The students calibrated the

SpectroVis Plus by first running a sample of distilled water through Logger Pro. Then, the

spectrophotometric analysis of the absorbance vs. wavelength spectrum for the first four

solutions were collected, and the students selected an operating wavelength of 711.9 nm. Finally,

by plotting the absorbance and concentration values of the known solutions and creating a linear
regression model with a zeroed line of best fit, the students determined the concentration of the

unknown sample solution.

Figure 1: Calculations of the Final Concentrations of Each Known Sample

Calculations conducted during the experiment.

C₁ × V₁ = C₂ × V₂

5. 00 𝑚𝑔/𝐿 ✕ 2. 50 𝑚𝐿 = 0. 50 𝑚𝑔/𝐿 ✕ 25. 00 𝑚𝐿

5. 00 𝑚𝑔/𝐿 ✕ 5. 00 𝑚𝐿 = 1. 00 𝑚𝑔/𝐿 ✕ 25. 00 𝑚𝐿

5. 00 𝑚𝑔/𝐿 ✕ 10. 00 𝑚𝐿 = 2. 00 𝑚𝑔/𝐿 ✕ 25. 00 𝑚𝐿

5. 00 𝑚𝑔/𝐿 ✕ 12. 50 𝑚𝐿 = 2. 50 𝑚𝑔/𝐿 ✕ 25. 00 𝑚𝐿

Concentration and volume are inversely proportional, meaning as volume increases,

concentration decreases proportionally, and vice versa. This relationship can be used to

determine the final concentration of a solution, given the initial concentration, initial volume, and

final volume is known. The accuracy of this method is not guaranteed due to human

measurement errors, but it is a simple calculation and is commonly used in chemistry.

Table 1: Concentrations of Phosphate Standard in Water

Data values recorded before and during the experiment.

Flask # 5.0 mg/L PO₄ (mL) Final Volume (mL) Concentration Absorption
(mg/L PO₄)

1 2.50 25.00 0.50 0.063

2 5.00 25.00 1.00 0.184

3 10.00 25.00 2.00 0.358

4 12.50 25.00 2.50 0.430

The absorption of each sample was found using the SpectroVis Plus’s spectrophotometric

analysis in Logger Pro. Each value matches a data point of that sample’s absorbance vs.
wavelength spectrum, at the operating wavelength of 711.9 nm. When run through Logger Pro,

the unknown sample showed an absorption peak at 0.424.

Figure 2: Calculating Concentration of the Unknown Sample

Calculations conducted during the experiment.

𝑦 = 0. 1745𝑥

0. 424 = 0. 1745𝑥

𝑥 = 2. 43

Using the equation obtained by the Absorbance vs. Concentration linear regression model on

Excel –with absorbance on the y-axis and concentration on the x-axis– the value of the unknown

sample’s concentration was found to be 2.43 mg/L PO₄. While the linear regression model is a

less accurate form of deriving concentration, the unknown sample’s similarity in color to the 2.50

mg/L PO₄ concentration sample supports the data, as 2.43 and 2.50 are very close in value, and

therefore should be almost identical shades of blue.

Overall, the objective of this lab experiment to determine the concentration of the unknown

sample was successfully completed. The concentration of the unknown solution was found to be

2.43 mg/L PO₄ at an operating wavelength of 711.9. With this experiment, the students learned

the importance of natural water sources keeping a low concentration of phosphates to protect

water dwelling organisms. The concepts of concentration, absorbance, and spectrophotometric

analysis learned in this lab will be vital information to know in future experiments.
References

CHEM 111 Laboratory Manual, Department of Chemistry, Binghamton University, Binghamton,

New York, 2019, pp. xx-xx.