Determination of Phosphorus In Milk – Principle, Procedure, Result

Determination of phosphorus in milk is a biochemical test in which the total phosphorus present in milk is converted into inorganic orthophosphate and then it is measured by colour reaction. It is the process in which different form of phosphorus present in milk matrix are first released from organic matter and then it is estimated by molybdate method.

In this test the milk sample is first mineralized by wet digestion (acid oxidation) using strong acids like nitric acid (HNO3) sulfuric acid (H2SO4) or perchloric acid (HClO4). The organic matter is destroyed and bound phosphorus ions are released and it is converted into inorganic phosphate form. After digestion clear solution is obtained and phosphate is present in measurable form.

The inorganic phosphate is reacted with ammonium molybdate reagent in highly acidic condition and phosphomolybdic acid complex is formed. This is referred to as molybdate complex formation. The complex is treated with mild reducing agent like ascorbic acid or 1,2,4-aminonaphtholsulfonic acid and blue colour is produced (molybdenum blue). In some official method vanadium–ammonium molybdate reagent is used and yellow complex compound is formed instead of blue colour. The intensity of blue or yellow colour is directly proportional to the concentration of phosphorus present in milk sample.

The coloured solution is then measured by spectrophotometer and absorbance is taken at specific wavelength. Usually 820 nm is used for blue complex and 440 nm is used for yellow complex. Standard phosphorus solutions are prepared and calibration curve is made and sample absorbance is compared with standard curve and final phosphorus content is calculated. In some alternative biochemical protocols the phosphorus–molybdate precipitate is filtered and washed and then it is subjected to titration using standard acid and alkali solutions and phosphorus percentage is calculated.

Principle

Principle of biochemical test for determination of phosphorus in milk is based on complete destruction of milk organic and colloidal structure and releasing the bound phosphorus into inorganic orthophosphate form. It is the process called mineralization in which heat and strong oxidizing acids are used and all phosphorus present in milk is converted into measurable phosphate.

After mineralization the orthophosphate is reacted with ammonium molybdate in strongly acidic condition and phosphomolybdic acid complex is formed. In molybdenum blue method a mild reducing agent like ascorbic acid or aminonaphtholsulfonic acid is added and this complex is converted into intense blue coloured compound. In some standard method vanadium–ammonium molybdate reagent is used in nitric acid solution and yellow complex compound is formed and reduction step is not needed.

The coloured solution is measured in spectrophotometer at specific wavelength and absorbance is taken. The colour intensity is directly proportional to concentration of phosphorus present in milk sample and by comparing with standard value the quantitative calculation of phosphorus is done.

Requirements

Requirements are as follows–

Reagents and chemicals–

• Nitric acid (HNO3) sulfuric acid (H2SO4) and perchloric acid (HClO4) for mineralization of milk sample.
• Trichloroacetic acid (TCA) for precipitation of milk proteins and to get clear filtrate.
• Ammonium molybdate reagent for formation of phosphomolybdic acid complex.
• Vanadium–ammonium molybdate reagent (in nitric acid) for yellow colour method.
• Reducing agent– ascorbic acid or 1,2,4-aminonaphtholsulfonic acid (ANSA) for molybdenum blue colour development.
• Sodium bisulfite and anhydrous sodium sulfite for stabilizing ANSA reagent.
• Potassium dihydrogen phosphate (KH2PO4) for preparation of standard phosphorus solution.
• Alkaline solution– sodium hydroxide (NaOH) or ammonium hydroxide for pH adjustment and neutralization.
• pH indicators– phenolphthalein or dinitrophenol (2,6-dinitrophenol or 2,4-dinitrophenol).
• Standard acid solution (HCl) and 2% sodium nitrate wash solution if titration method is followed.

Apparatus and equipment–

• Spectrophotometer or colorimeter for measuring absorbance (440 nm or 680 nm or 820 nm).
• Analytical balance (sensitive up to 0.1 mg) for accurate weighing.
• Heating equipment– electric hot plate autoclave muffle furnace or microwave digestion system for digestion/ashing.
• Acid washed glassware– beakers Erlenmeyer flask volumetric flask test tubes and pipettes.
• Phosphorus free filter paper (Whatman No.1 or No.42) or centrifuge for getting clear solution.

Procedure

Procedure are as follows–

Titrimetric method (precipitation and titration)–

1. 10 ml milk is taken in wide mouthed test tube and about 5 ml conc. sulfuric acid is added carefully along the side so that acid layer is formed below milk.
2. 20 ml nitric acid is added for breaking the organic matrix and phosphorus is released in solution.
3. Ammonium hydroxide is added slowly with vigorous stirring and the precipitate formed is dissolved.
4. The solution is diluted up to 75–100 ml and temperature is adjusted to 25–30°C. If solution is not neutral it is made slightly alkaline by ammonium hydroxide and then slightly acidic by diluted nitric acid.
5. 20–25 ml freshly prepared molybdate solution is added and precipitate is formed. It is agitated on stirring apparatus for 30 minutes at room temperature.
6. The mixture is decanted through filter paper (Whatman No.42) and precipitate is washed twice by decantation with 25–30 ml of 2% sodium nitrate solution.
7. The precipitate is transferred to filter paper and it is washed with cold 2% sodium nitrate until filtrate becomes colourless when phenolphthalein and one drop standard alkali is added.
8. The precipitate is dissolved by slight excess of standard alkali (NaOH or KOH). Few drops phenolphthalein is added and pink colour is produced.
9. The solution is titrated with standard acid till pink colour disappears completely and end point is noted.

Colorimetric method (Fiske–Subbarow)–

1. 2 ml milk sample is mixed with 8 ml of 10% or 20% trichloroacetic acid (TCA).
2. The mixture is shaken and it is kept for 5 minutes and then it is filtered or centrifuged to obtain clear protein free filtrate (PFF).
3. 1 ml of clear PFF is taken in test tube and it is diluted with 4 ml distilled water.
4. 1 ml ammonium molybdate reagent is added and immediately 0.4 ml ANSA reducing agent is added.
5. The tube is mixed and final volume is made with distilled water and blue colour is allowed to develop for 10 minutes at room temperature.
6. Absorbance is measured at 680 nm (or red filter) and phosphorus concentration is calculated by standard.

Results

Titration method–

The result is calculated to determine the percentage of phosphorus present in given milk sample. The calculation is done by using volume of standard alkali and standard acid used in titration.

Formula–
Phosphorus (%) = 0.4364 × (V1 − V2) / 2W

Where–
V1 = Volume (ml) of standard alkali used for dissolving the phosphorus containing precipitate.
V2 = Volume (ml) of standard acid used for neutralizing the excess alkali.
W = Weight (g) of milk sample ash taken for test.
0.4364 = It is the amount of phosphorus (mg) corresponding to 1 ml standard alkali in titration.

Colorimetric / spectrophotometric method–

In this method the phosphorus concentration is determined by comparing absorbance of test with absorbance of standard and it is based on Beer–Lambert relationship. The result is obtained from standard calibration curve and final value is calculated by dilution factor.

General formula–
Inorganic phosphorus (mg/100 ml) = (Absorbance of test / Absorbance of standard) × Concentration of standard × (Final volume / Sample volume) × 100

AOAC type formula–
Phosphorus (mg/100 g) = Y × 125 / W
Where Y is phosphorus concentration obtained from standard curve and W is weight of test portion.

Reporting–
Final result is reported as arithmetic mean of two independent test results. The difference between two results should not exceed 5% of their mean. The final value is reported in 3 significant figures or nearest milligram as per lab standard.

Uses

Uses are as follows–

• It is used for determination of nutritional phosphorus content in milk dairy products and infant formula and it is checked for legal standard compliance.
• It helps in monitoring cheese and yogurt production and it is useful for predicting technological property during fermentation (gelation curd tension and final texture).
• It is used for controlling food additives and monitoring addition of polyphosphates in processed cheese and it is also checked for unauthorized mineral addition.
• It is used for verification of pasteurization efficacy by measuring alkaline phosphatase activity and confirming proper heating and absence of raw milk contamination.
• It is used in veterinary diagnosis and phosphorus level in milk and blood is determined for metabolic disorders like milk fever (hypocalcemia) which is associated with low phosphorus.
• It helps in optimizing cattle diet and monitoring partitioning of phosphorus into milk and it prevents negative phosphorus balance in herd.
• It is used for managing environmental impact and phosphorus secreted in milk is assessed and it reduces excess phosphorus excretion in manure and pollution is minimized.
• It is used for research purpose to assess breed variation and seasonal variation and effect of pasture diet on mineral composition and processing quality of milk.

Limitations

Limitations are as follows–

• Strong acids are required for wet digestion like conc. sulfuric acid nitric acid and perchloric acid and these chemicals are toxic corrosive and hazardous for handling.
• The method is time consuming and labour intensive because complete mineralization of organic matter take long time and large sample is required in some standard procedure.
• In dry ashing method (500°C–550°C) loss of phosphorus can occur due to volatilization of phosphate compounds if proper fixative like zinc oxide is not used.
• Reducing agents used for colour development specially 1,2,4-aminonaphtholsulfonic acid (ANSA) are unstable and it is easily oxidized by air and potency is lost fast so fresh reagent and proper dark refrigerated storage are required.
• In molybdenum blue method timing is very strict because blue colour can fade or it can increase if it is kept for long time and absorbance reading can be affected.
• Spectrophotometer reading can be interfered by turbidity and sample colour and vanadium–molybdate yellow method is less sensitive and it is affected by natural yellow colour of digest.
• Milk matrix interference occur due to proteins and lipids and turbidity and non specific colour development is produced if deproteinization is not done properly (TCA is used). High iron can precipitate phosphorus and loss is occurred and high arsenate can mimic phosphate and it interfere in reading.
• Complete digestion is necessary because colour reaction is specific for inorganic orthophosphate only and if digestion is incomplete then total phosphorus value is underestimated.

References

1. BenchChem Technical Support Team. (2025). The discovery of phosphocreatine: A technical retrospective on the foundational work of Fiske and SubbaRow. BenchChem.
2. Cornell CALS. (n.d.). Alkaline phosphatase (ALP) testing for milk pasteurization (Dairy Foods Science Notes). Cornell University.
3. Cupisti, A., Benini, O., Ferretti, V., Gianfaldoni, D., & Kalantar-Zadeh, K. (2012). Novel differential measurement of natural and added phosphorus in preserved versus non-enhanced ham. Journal of Renal Nutrition, 22(6), 533-540.
4. Dabkowski, B., & White, M. (2015). Understanding the different phosphorus tests. Hach Company.
5. Dairy Knowledge Portal. (n.d.). Alkaline phosphatase test for pasteurized milk. National Dairy Development Board.
6. Editorial Team. (2024, May 13). Determination of phosphorus in milk. Biology Notes Online.
7. Fatum, T., & Higgins, D. (2008). Process for producing cheese (U.S. Patent Application Publication No. US 2008/0299252 A1). U.S. Patent and Trademark Office.
8. Fieser, L. F. (1931). 1-Amino-2-naphthol-4-sulfonic acid. Organic Syntheses, 11, 12.
9. Hwang, J., Jang, H. W., Namgung, B., Oh, M., Kim, S., Seo, D., Kim, S.-N., Choi, Y., Nam, J.-S., & Yang, M.-O. (2015). Determination of phosphorus in foods by inductively coupled plasma optical emission spectrometry (ICP-OES). Food Engineering Progress, 19(2), 161-166.
10. International Organization for Standardization. (2006). Milk — Determination of total phosphorus content — Method using molecular absorption spectrometry (ISO Standard No. 9874:2006 / IDF 42:2006).
11. Kay, H. D., & Graham, W. R. Jr. (1933). 73. Phosphorus compounds of milk. VI. The effect of heat on milk phosphatase. A simple method for distinguishing raw from pasteurised milk, raw from pasteurised cream, and butter made from raw cream from that made from pasteurised cream. Journal of Dairy Research, 5(1), 63-74.
12. Keršienė, M., Jasutienė, I., Kubiliūtė, M., Ruzgas, T., & Leskauskaitė, D. (2019). Composition and processing characteristics of milk from Lithuanian black and white cows. Journal of Food and Nutrition Research, 7(2), 114-121.
13. Khan, M. K. U., & Appanna, T. C. (1966). Compounds of phosphorus in camel milk. Indian Journal of Physiology and Pharmacology, 10(3), 61-65.
14. Klop, G., Ellis, J. L., Blok, M. C., & Brandsma, G. G. (2014). Variation in phosphorus content of milk from dairy cattle as affected by differences in milk composition. The Journal of Agricultural Science, 152(5), 860-869.
15. Latimer, G. W. Jr. (Ed.). (2023). Subchapter 7 Cheese. In Official Methods of Analysis of AOAC INTERNATIONAL (22nd ed.). AOAC International.
16. Lemon, K. (n.d.). What’s the difference between phosphate tests for orthophosphate, total phosphorous, acid hydrolyzable phosphates and organic phosphates? Camlab.
17. Lima, M. J. R., Fernandes, S. M. V., & Rangel, A. O. S. S. (2002). Colorimetric determination of phosphorus in milk by flow injection analysis using a thermal/UV induced digestion. Italian Journal of Food Science, 14(1), 9-16.
18. Merck KGaA. (2024). Total phosphorus in milk and dairy products (Application Note). Sigma-Aldrich.
19. Ministry of Health of the People’s Republic of China. (2010). National food safety standard: Determination of phosphorus in foods for infants and young children, milk and milk products (GB 5413.22-2010).
20. Mirajkar, A. S. (n.d.). Estimation of inorganic phosphorus by Fiske Subbarow method [PowerPoint slides]. Slideshare.
21. Moreno-Torres, R., Navarro-Alarcon, M., Ruiz-López, M. D., & Artacho, R. (2000). Comparison of wet and dry mineralisation procedures for determining calcium and phosphorus in cow’s milk. Australian Journal of Dairy Technology, 55(1), 23-27.
22. O’Dell, J. W. (Ed.). (1993). Method 365.1: Determination of phosphorus by semi-automated colorimetry (Revision 2.0). U.S. Environmental Protection Agency.
23. OI Analytical. (2020). Determination of nitrate/nitrite in milk products (FIA) according to ISO 14673-3 (Method Abstract 4389-01). Xylem Inc.
24. Setiawati, T. A., Guyomarc’h, F., Beaucher, E., & Gaucheron, F. (2015). Ion exchange chromatography with conductivity detection compared to spectrophotometry to assay phosphate in milk and milk products. Jurnal Teknologi dan Industri Pangan, 26(1), 115-123.
25. Shreyas9224. (n.d.). Serum inorganic phosphorus estimation. Scribd.
26. Simoni, R. D., et al. (2002). The determination of phosphorus and the discovery of phosphocreatine and ATP: The work of Fiske and SubbaRow. Journal of Biological Chemistry.
27. U.S. Environmental Protection Agency. (2012, March 6). 5.6 Phosphorus | Monitoring & assessment.
28. Unknown Author. (n.d.). Analytical biochemistry of phosphorus in dairy matrices: Principles, methodologies, and systematic quantification.