2.2.12. Determination of Calcium Concentration Using the EDTA Complexometric Titration Method

The soluble calcium concentration in the skim milk and HA added skim milk samples was determined [29,30,31]. 10 mL aliquots of the samples, both skim milk and HA-added hyaluronic acid, were transferred into 10.4 mL, 16 × 76 mm polycarbonate capped centrifuged bottles (Beckman Coulter, Inc., Brea, CA, USA, cat. 355603). Subsequently, the samples were centrifugated at 21,500× g at 20 °C in an ultracentrifuge (Optima MAX-E, Beckman Coulter, Inc., Brea, CA, USA) equipped with a fixed angle rotor (MLA-55, Beckman Coulter, Inc., Brea, CA, USA) for a duration of 90 min. This resulted in the separation of milk samples into supernatant (serum) and precipitation layers. The serum samples were then carefully transferred and stored in plastic tubes before proceeding with titration. For titration, one milliliter of serum samples was diluted by adding 50 mL of glass-distilled water. Subsequently, 3 mL of 8 M NaOH was added into the diluted sample to elevate the pH to ≥12.5. The samples were kept aside for 5 min with periodic swirling to facilitate the precipitation of any magnesium present in the solution. After that, 2–3 drops of a 0.5% w/v Patton-Reeder indicator (calconcarboxylic acid) (Sigma Aldrich, St. Louis, MO, USA) solution, prepared in 1 M NaOH, were added into the sample prior to titration. The samples were titrated using a 0.01 M EDTA until color change occurred, transitioning from pink to blue. The amount of EDTA required for titration was recorded. Here, moles of EDTA utilized during the titration is equivalent to the moles of calcium originally present in the serum sample. Hence, the mass of soluble calcium (mg) present in serum samples was determined using the following equation:

The quantification of total calcium concentration in skim milk was performed following the identical procedure as previously described. The mass of insoluble calcium (present in the precipitate) was calculated by subtracting the concentration of serum calcium from the total calcium concentration.