Methanol, ethanol, 1-propanol, 2-propanol, dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4) and potassium phosphate (K3PO4) were purchased at Vetec (Rio de Janeiro, Brazil). The alcohols have purities higher than 99 wt.%. MEK inhibitor The phosphate salts present had purity levels

higher than 98 wt.%. The l-ascorbic acid (>98 wt.%) was acquired at Labsynth (São Paulo, Brazil) and vanillin (>99 wt.%) was purchased at Sigma–Aldrich. Ultrapure water, double distilled, passed by a reverse osmosis system and further treated with a Milli-Q plus 185 water purification apparatus, was used. The vanilla diet pudding Dr. Oetker was purchased at a regular supermarket in Aracaju, Brazil (http://www.oetker.com.br/?actA = 2111&produtoID = 138). PCI-32765 research buy The ATPS were formed using aqueous solutions of alcohols (methanol, ethanol, 1-propanol and 2-propanol) at 80 wt.% and distinct aqueous solutions of inorganic potassium phosphate salts (K3PO4, K2HPO4 and the phosphate buffer solution KH2PO4/K2HPO4 – Henderson–Hasselbalch equation equivalents = 1.087) at ca. 40 wt.%.

The phase diagrams were determined through two different experimental methodologies well described in literature, the cloud point titration method ( Merchuck et al., 1998, Neves et al., 2009, Ventura et al., 2009, Ventura et al., 2011 and Ventura et al., in press) and the turbidometric titration method ( Aqueous Two-Phase Systems, 2000 and Freire et al., 2012) at (298 ± 1) K

and atmospheric pressure. The tie-lines (TLs) were obtained using a gravimetric method originally applied by Merchuck and co-workers (Merchuck et al., 1998) and already validated in previous studies (Neves NADPH-cytochrome-c2 reductase et al., 2009 and Ventura et al., 2009). Each tie-line (TL) was determined by the application of the lever-arm rule. For that purpose, the experimental solubility curves were correlated using the following Eq. (1), equation(1) Y=Aexp[(BX0.5)-(CX3)]where Y and X, are the alcohol and salt mass fraction percentages, respectively, and A, B and C are the regression constants. The partitioning systems for l-ascorbic acid were prepared using graduated centrifuge tubes by weighing the appropriate amounts of alcohol (at ca. 50 wt.%), inorganic salt (at ca. 15 wt.%) and l-ascorbic acid (2.8 mg). To prepare the vanillin partitioning systems, vials with the same weight fractions of alcohol and inorganic salt were prepared. An aqueous solution of vanillin (concentration of ca. 1.0 g.dm−3) was used as the aqueous phase. Afterwards, the mixtures were gently stirred and centrifuged at 3,000 × g for 10 min. The extraction systems were placed at (298 ± 1) K, for at least 18 h, to reach the equilibrium and the consequent and complete partitioning of the antioxidants. The vials were closed during this period to avoid the alcohol vaporisation.