Stirring was continued for another hour after complete addition. The resulting white suspension was washed three times with water by centrifugation and twice with acetone. Finally, the precipitate was dried in an oven at 37 °C for 2 days. For the final dispersion, 0.56 g of the intermediate was dissolved in 15 ml 1 M HCl and filtered over a Minisart disposable cellulose acetate filter (0.2 μm pore size, 16534-K). The solution was injected into 35 ml 0.39 M NaOH solution while stirring vigorously with a magnetic stirrer. The turbid white dispersion was stirred for another 10 min after injection, the pH of the final dispersion was 7. The sample was

GW786034 nmr washed twice by centrifugation and redispersed in a final volume of 50 ml water. It has been shown previously that the stability of metal-pyrophosphate dispersions is strongly dependent on the ionic strength

of the solution (van Leeuwen et al., 2012a). Therefore, mixed systems at a fixed concentration of pyrophosphate were prepared as this set the concentration of the counterions. Mixed systems were prepared by substituting part of the iron in the precursor solution with calcium or magnesium (together referred to as M2+), the LY2109761 amounts of Fe3+ and M2+ in the mixture are then determined in stoichiometry with the concentration of PPi. This resulted in the following Fe:M2+ ratios: Fe10M2+PPi8 (10:1 ratio), Fe16 M2+2PPi13 (8:1), Fe8 M2+2PPi7 (4:1), Fe4M2+4PPi5 (1:1), Fe2M2+11PPi7 (1:5) or Fe2M2+19PPi11 (1:10). Here complete precipitation without inclusion of the Na+ and Cl− from the reactants was assumed. Iron pyrophosphate prepared without any substitution was referred to

as ‘pure FePPi’. Full substitution of iron results in the pure M2+ pyrophosphate, M2+PPi. For Fe:Na, the following ratios were prepared: Fe22Na2PPi17 (10:1), Fe32Na4PPi25 (8:1), Fe16Na4PPi13 (4:1). Samples containing a lower iron content remained clear and no particles were pheromone formed. All samples were stored in plastic (Teflon™) bottles. Mixed systems prepared using the pH dependent precipitation method only resulted in stable dispersions when prepared using Magnesium. Colloidal (mixed) iron pyrophosphates were coated with zein protein through an antisolvent precipitation method (Velikov & Pelan, 2008). As colloidal iron pyrophosphate aggregates over time in water (van Leeuwen et al., 2012a), the nanoparticles were prepared either immediately before (in case of the NP-Z system) or simultaneously with the zein precipitation. The concentrations were also lowered: the final dispersion contained 2 mM iron and 1.5 mM pyrophosphate, in order to prevent aggregation during the addition of zein. After complete precipitation of the iron pyrophosphate, the 30 ml dispersion was removed and 40 ml zein solution (1 g zein in 80 vol.% ethanol) was slowly poured into the dispersion, which turned more turbid and slightly yellow. Some aggregates were formed, which were filtered out of the dispersion before further analysis.