Affinity macroligands (AML) comprising a reversibly water soluble poly-N-alkylacrylamide linked to a ligand, and their use in the separation of a target substance from a mixture comprising the target substance and other materials are disclosed. Preferred AMLs have number average molar masses of less than 7,000 g/mol (chain length n typically about 25), mass distributions of less than 1.2 and narrow precipitation-solubility temperature (NPST) interval, typically in the range of 1-2° C.