Milk contains high concentrations of calcium. It occurs in two forms, a free ionic form, and calcium associated with milk proteins (caseins). The latter association is called colloidal calcium phosphate. New Zealand Dairy Foods of Takanini is marketing a range of commercial milks in supermarkets. The company uses ultra filtration to concentrate milk proteins and calcium in different milk products. During ultra filtration, the fraction that is retained by the membrane is rich in calcium bound to proteins and the portion that passes through the membrane is richer in the free ionic form. The company wanted to develop a quick and an economical method that can be applied in industrial settings to determine calcium in both these fractions and in other milk products. This research aimed to develop a quick, wet chemistry method to measure calcium in milk fractions and to trial it in an industrial environment. Two methods, the so-called EDTA method and the atomic absorption spectrophotometric method (AA) were trialled as potential reference methods against which to compare results obtained by the method to be developed. The AA method was chosen due to its ease, accuracy and precision. (This could not be selected as the industrial method for a number of reasons.) A colorimetric method was favoured over other contenders. Two colorimetric dyes, Arsenazo I and o-cresolphthalein-complexone (CPC) were chosen to work with. Arsenazo I forms a purple complex with calcium in a suitable buffer at a range of pHs. o-Cresolphthalein-complexone also forms purple-coloured complexes at alkaline pHs. During method development with Arsenazo I, different buffers were trialled and a NaOH/ KCl buffer was selected for further development at pH 12. The method worked well during the development phase but with some inconsistent results at times. o-Cresolphthalein-complexone formed clear purple complexes with Clark and Lubs and 2-amino-2-methylpropanol (AMP) buffers. The key advantage of the CPC dye with AMP buffer was that when 8-hydroxyquinoline was included in the reaction mixture, it successfully masked coloured complex formation due to CPC with magnesium, which is present in milk at about 1/3 the calcium concentration. This effect did not work with Arsenazo I. However, the results obtained with the CPC method were lower than claimed values of most milks trialled during development. Both methods were compared for their precision and it was found that CPC method has better precision and was chosen for further development. To improve the accuracy and precision, various denaturing reagents were used to (hypothetically) release calcium from the caseins. Trichloroacetic acid at 25 % was more effective than the several other denaturing treatments tested. The finalised CPC method, using trichloroacetic acid, AMP and 8-hydroxyquinoline, was then used to monitor calcium concentration over four months in three milk products, skim, Xtra (retentate) and permeate. For all milks, the CPC values were lower than the AA reference values, and the values reported by a commercial analytical laboratory. The reasons for this are discussed, as are other changes in calcium concentration in the three milks throughout the trial. The correlation between the CPC and AA values was poor for Xtra, better for skim, and best for permeate. A chemical model to explain this is discussed. The method developed is cheap and quick, and sample and reagent preparation is simple. The method could be applied in an industrial environment, but a proportionality factor would have to be applied to account for the difference in mean values between the CPC and AA methods.

Milk contains high concentrations of calcium. It occurs in two forms, a free ionic form, and calcium associated with milk proteins (caseins). The latter association is called colloidal calcium phosphate. New Zealand Dairy Foods of Takanini is marketing a range of commercial milks in supermarkets. The company uses ultra filtration to concentrate milk proteins and calcium in different milk products. During ultra filtration, the fraction that is retained by the membrane is rich in calcium bound to proteins and the portion that passes through the membrane is richer in the free ionic form. The company wanted to develop a quick and an economical method that can be applied in industrial settings to determine calcium in both these fractions and in other milk products. This research aimed to develop a quick, wet chemistry method to measure calcium in milk fractions and to trial it in an industrial environment. Two methods, the so-called EDTA method and the atomic absorption spectrophotometric method (AA) were trialled as potential reference methods against which to compare results obtained by the method to be developed. The AA method was chosen due to its ease, accuracy and precision. (This could not be selected as the industrial method for a number of reasons.) A colorimetric method was favoured over other contenders. Two colorimetric dyes, Arsenazo I and o-cresolphthalein-complexone (CPC) were chosen to work with. Arsenazo I forms a purple complex with calcium in a suitable buffer at a range of pHs. o-Cresolphthalein-complexone also forms purple-coloured complexes at alkaline pHs. During method development with Arsenazo I, different buffers were trialled and a NaOH/ KCl buffer was selected for further development at pH 12. The method worked well during the development phase but with some inconsistent results at times. o-Cresolphthalein-complexone formed clear purple complexes with Clark and Lubs and 2-amino-2-methylpropanol (AMP) buffers. The key advantage of the CPC dye with AMP buffer was that when 8-hydroxyquinoline was included in the reaction mixture, it successfully masked coloured complex formation due to CPC with magnesium, which is present in milk at about 1/3 the calcium concentration. This effect did not work with Arsenazo I. However, the results obtained with the CPC method were lower than claimed values of most milks trialled during development. Both methods were compared for their precision and it was found that CPC method has better precision and was chosen for further development. To improve the accuracy and precision, various denaturing reagents were used to (hypothetically) release calcium from the caseins. Trichloroacetic acid at 25 % was more effective than the several other denaturing treatments tested. The finalised CPC method, using trichloroacetic acid, AMP and 8-hydroxyquinoline, was then used to monitor calcium concentration over four months in three milk products, skim, Xtra (retentate) and permeate. For all milks, the CPC values were lower than the AA reference values, and the values reported by a commercial analytical laboratory. The reasons for this are discussed, as are other changes in calcium concentration in the three milks throughout the trial. The correlation between the CPC and AA values was poor for Xtra, better for skim, and best for permeate. A chemical model to explain this is discussed. The method developed is cheap and quick, and sample and reagent preparation is simple. The method could be applied in an industrial environment, but a proportionality factor would have to be applied to account for the difference in mean values between the CPC and AA methods.