Phospholipids are emerging as a food ingredient due to their functional and nutritional properties. Our goal was to develop a new (non-solvent) separation process to produce an enriched dairy phospholipid (lecithin) fraction from whey protein phospholipid concentrate (WPPC). Two chemical treatments: (1) calcium-induced protein aggregation (pH 4.8) and (2) thermocalcic aggregation (pH 7.3) at 2 temperatures (50 and 80°C) were applied to rehydrated WPPC solutions. These experiments were done on a bench top scale to aid in precipitation of proteins and phospholipids. Chemically treated solutions were centrifuged and fractionated into 3 layers: (1) top fat layer (2) supernatant in middle zone and (3) sediment at bottom of centrifuge tubes. Compositional analysis was done on each layer to understand how the components (protein, phospholipid) fractionated. Treatment 1 did not aid in precipitation of proteins and about 70% of the proteins fractionated into the supernatant. Treatment 2 precipitated about 90% of the phospholipids at 80°C but the proteins precipitated along with the phospholipids. With both the treatments, proteins, fat and phospholipids always fractionated together in similar ratios. The proteins in each layer were characterized using SDS-PAGE and a range of proteins including whey proteins, caseins and milk fat globule membrane proteins were identified. The microstructure of protein and fat in WPPC was investigated using confocal laser scanning microscopy (CLSM). Dual staining of rehydrated WPPC solution with Fast Green FCF (proteins) and Nile Red (lipids) showed the presence of very large protein aggregates that varied in size from 20 to 150 μm with most fat trapped or associated with these aggregates, which explained why fat and protein co-separated with our chemical treatments. We are exploring other treatments to separate the components in these aggregates to identify a suitable separation process.