Determination of Phosphorus In Milk


Table of Contents

Phosphorus is an essential mineral found in many foods, including milk. It plays a critical role in various bodily functions such as the formation of bones and teeth, energy production, and the synthesis of RNA and DNA, which are crucial for the growth and repair of cells.

In milk, phosphorus is naturally present and contributes to the nutritional value of milk and dairy products. Dairy products are among the best sources of readily absorbable phosphorus in the human diet. The phosphorus in milk is bound in a complex form known as phosphoproteins, which include casein, the main protein in milk. This form of phosphorus is efficiently absorbed by the human body.


The amount of phosphorus in milk can vary depending on the type of milk (such as whole, skim, or non-fat), but on average, a cup of cow’s milk contains about 233 milligrams of phosphorus, which is roughly 23% of the recommended daily intake for an adult. It’s important to note that while phosphorus is necessary for health, excessive intake, especially from phosphorus additives in processed foods, can be harmful, particularly for people with kidney disease or other conditions that affect phosphorus metabolism.

Including milk and dairy products as part of a balanced diet can help meet the daily requirements for phosphorus, along with other essential nutrients like calcium, vitamin D, and protein.



Determining the phosphorus content in milk is an essential process in nutritional analysis and quality control in the dairy industry. This process involves precise chemical procedures to accurately measure the amount of phosphorus present. The following components and steps are crucial for the successful determination of phosphorus in milk:

  1. Nitric Acid: A strong oxidizing agent, with a specific gravity of 1.42, is used in the process. It aids in breaking down organic compounds in the milk, facilitating the release of phosphorus ions.
  2. Ammonium Hydroxide: With a specific gravity of 0.88, ammonium hydroxide is used to adjust the pH of the solution during the analysis. Proper pH adjustment is crucial for the accurate determination of phosphorus.
  3. Diluted Nitric Acid: A diluted version of nitric acid, prepared at a volume-to-volume ratio of 1:3, is utilized for specific steps in the process where a less concentrated acid is necessary.
  4. Phenolphthalein Indicator Solution: This 1% solution, prepared in rectified spirit, serves as a pH indicator. When the solution reaches a neutral pH, phenolphthalein changes color, indicating the endpoint of the titration process.
  5. Standard Sodium or Potassium Hydroxide Solution: This solution is prepared by diluting 324.03 ml of carbonate-free alkali to 1 litre. It is used in the titration process, where 100 ml of this solution should neutralize 32.40 ml of a standard acid.
  6. Standard Acid Solution: A hydrochloric acid (HCl) solution is prepared to match the strength of the alkali. It is standardized by titrating against the alkali solution using phenolphthalein as the indicator.


  1. Sample Preparation:
    • Begin by measuring 10 ml of milk into a wide-mouthed test tube. Carefully add approximately 5 ml of concentrated sulfuric acid (H₂SO₄) along the inner side of the tube so that it forms a distinct layer beneath the milk. A violet or blue coloration at the interface of the two liquids may indicate the presence of formaldehyde, which is important to note as it could affect the results.
  2. Acidification:
    • To the milk sample, add 20 ml of nitric acid (specific gravity 1.42). This step helps in breaking down the organic matrix of the milk, releasing phosphorus ions into the solution.
  3. Neutralization and Precipitation:
    • Gradually introduce ammonium hydroxide (specific gravity 0.88) to the acidic mixture until any precipitate that forms dissolves, albeit slowly, even with vigorous stirring. This step is crucial for ensuring that the phosphorus compounds are fully available for quantification.
  4. Dilution and Temperature Adjustment:
    • Dilute the solution to a volume between 75 to 100 ml and adjust its temperature to fall within the range of 25 to 30°C. If the solution does not form a precipitate with ammonium hydroxide, indicating it is not yet neutral, adjust the pH first to slightly alkaline with more ammonium hydroxide, and then back to slightly acidic with a diluted nitric acid solution (1:3 ratio).
  5. Molybdate Reaction:
    • Add 20 to 25 ml of freshly prepared molybdate solution to the mixture. This reacts with the phosphorus in the sample to form a precipitate. Place the mixture on a stirring apparatus and agitate for 30 minutes at room temperature to ensure complete reaction.
  6. Filtration and Washing:
    • Immediately decant the mixture through a Whatman No. 42 filter paper. Wash the precipitate twice by decantation with 25-30 ml of a 2% sodium nitrate solution, ensuring thorough agitation and settling before each decantation.
  7. Final Washing:
    • Transfer the precipitate onto the filter and continue washing with cold water containing 2% sodium nitrate. Continue this process until the filtrate remains colorless upon the addition of phenolphthalein indicator and a single drop of standard alkali, indicating the removal of excess reagents.
  8. Titration:
    • Dissolve the precipitate with a slight excess of standard alkali (either sodium or potassium hydroxide solution). Add a few drops of phenolphthalein indicator to the solution, which will turn pink in alkaline conditions. Titrate this solution with a standard acid solution until the pink color disappears, indicating the solution has reached neutrality. The amount of standard acid used in this titration is directly related to the phosphorus content in the original milk sample, with 1 ml of the standard alkali solution equating to 0.4364 mg of phosphorus.


The process of determining the phosphorus content in milk culminates with the calculation of the percentage of phosphorus present in the sample. This is achieved through a specific formula that takes into account the volumes of the standard alkali and acid solutions used in the titration process, as well as the weight of the milk sample ash prepared for analysis. The formula is as follows:


Phosphorus (%)=0.4364×(V1−V2)​/2W


  • V1 is the volume (in milliliters) of the standard alkali solution used to dissolve the phosphorus-containing precipitate.
  • V2 is the volume (in milliliters) of the standard acid solution that was required to neutralize the excess alkali.
  • W is the weight (in grams) of the milk sample ash that was prepared for the determination of phosphorus content. This weight is typically based on a specific quantity of milk sample, such as 10 grams, that was ashed in a previous step of the analysis.

This calculation provides the percentage of phosphorus in the milk sample, reflecting the amount of this essential mineral present. The coefficient 0.4364 in the formula represents the milligram amount of phosphorus that corresponds to one milliliter of the standard alkali solution used in the titration. The division by 2W accounts for the dilution and sample size, ensuring the result reflects the percentage concentration of phosphorus in the original milk sample.

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