Imprinting effects were assessed by competitive binding assays by incubating the MIPs in equimolar peptide mixtures in different acetonitrile/water mixtures or aqueous buffers. First, we probed the polymer's ability to recognize their templates in an acetonitrile rich solvent system. This compensates for network solvation effects on recognition and is the standard gauge of the polymer's memory for their templates. shows that the polymers also crossreact with the complementary decapeptides but that this effect is strongly solvent dependent. In the buffered acetonitrile/water mixtures only in 90% acetonitrile buffered with TFA is this effect strongly manifested (Fig. , Fig. ). Under these conditions more than 90% of the complementary peptides were bound with selectivity factors all exceeding 2. The discrimination was lost when replacing the strong acid TFA with the weaker acid (FA) or basic (TEA) modifiers. Meanwhile, increasing the aqueous content completely suppressed binding of the peptides. Binding tests using aqueous buffers showed that both uptake and selectivity depended strongly on the buffer pH. Hence, whereas binding of the peptides was elevated in low pH 1 and pH 3 buffers (Fig. ) it dropped overall with increasing pH (Fig. ). At low pH, binding increased inversely with the number of phosphate groups of the peptide with YY displaying the strongest binding. This trend was no longer present at pH 5, although still with no imprint related discrimination to be seen. On the contrary, at neutral and basic pH, the MIPs specifically recognized their peptide complements, an effect seen most clearly in Fig. and Fig. . The above observations can be understood when taking a closer look at the peptide ionization states in the different buffers (Table ) and ionic strength effects. In the acetonitrile/water media (Fig. , Fig. ) the acidic modifiers TFA and FA reduces the charge of the phosphopeptides, while leaving the phosphate groups partly ionized for interacting with the urea N-H hydrogen bond donors. This facilitates their interactions with the templated sites explaining the pronounced phosphosite selectivities observed. This contrasts with results obtained with the basic modifier TEA, which renders all peptides multiply negatively, charged and strongly solvated. In addition, nonspecific effects due to the three carboxylic acid groups contribute to overall enhanced binding. In the aqueous buffers the binding dependence on the ionization state show clear trends. At pH 1, only the terminal amino acids are charged whereas the acidic side chains are protonated (pYpY being a possible exception) and charge neutral rendering all peptides an equal +2 charge. At pH 3, pTyr sites of the phosphopetides are ionized reducing the charge of the latter in comparison with the nonphosphorylated peptide. The latter, carrying a positive net charge, is still tightly bound. At pH 5, which is situated between the pKa of the Asp side chains and the pKa2 of the phosphotyrosines, the phosphate groups are only partially charged with only one H-bond acceptor free to interact with the MIP sites. Increasing pH from 5 to 7.4