Casein or denatured whey is precipitated using acetic acid and sodium acetate solutions. A dye-binding difference technique is utilized to eliminate the effect of any non-protein nitrogen variation among milk sources.


Applicable to raw, homogenized, pasteurized, or diluted condensed milk as well as reconstituted milk powders. Also, the undenatured whey protein in non-fat dry milk can be readily measured.

Apparatus and Reagents

UDY Model MKP Protein Analyzer including An UDY Colorimeter, a suitable Balance, a Syringe-Pipet, and a Pressure Filter Syringe Pipet with 25 mm fiberglass filters. Acetic acid and sodium acetate along with standard concentrations of Reagent and Reference Dye Solutions. Also, a stirring Hot Plate with stir bars plus other common lab items are needed.


1. Place about 65 ml of milk into a clean dry 125 ml Erlenmeyer flask. Warm to 40C (104F) on stirrer-hot plate.

2. Add 2.00 ml of 33.3% (w/v) acetic acid while stirring.

3. After 10 minutes, add 2.00 ml of 27.35% (w/v) anhydrous sodium acetate while stirring.

4. After about 3 minutes, use a Pressure Filter Syringe-Pipet with 25 mm Whatman GF/B fiberglass filter disk to filter about 10 ml into a 20 ml vial. Cool to 20C.

5. Reading I: Shake 2.00 ml of above untreated milk plus 2.00 ml of distilled water with 40.00 ml Reagent Dye Solution in a 60 ml Sample Bottle and measure percent total protein using A = 6.39 and CI 1.20; PO = 3.45. %Protein= 6.39%(1.2 - C).

6. Reading II: Shake 2.00 ml of the same untreated milk plus 2.00 ml of the above non-casein filtrate with 40.00 ml of Reagent Dye Solution in a 60 ml Sample Bottle. Then, measure the percent total protein plus non-casein protein. Use the same settings as in Step 5.

7. Calculation of Percent Casein: Multiply Reading I by 2, then subtract Reading II to obtain Percent Casein.

8. To measure undenatured whey protein, reconstitute skim milk powder by mixing 10 to 12 g of powder with nine times as much distilled water. This is a 10-fold dilution. Stir for 30 minutes at 40C.

9. Proceed with this reconstituted milk by testing as above (Steps 1 thru 6).

10. Determine the UDY Protein Milk Difference (UPDM) by subtracting Reading I from Reading II. Classification equivalents to A.D.M.I. Specifications are:

a) Low Heat Powder - UPMD > 0.56

b) Medium Heat Powder - UPMD 0.24 to 0.56

c) High Heat Powder - UPMD < 0.24