In this study, polyacrylamide nanocomposite hydrogels were formulated in combination with sodium montmorillonite (MMT) in the presence of a 2-T magnetic field. This top-down nanomanufacturing approach led to interesting changes in the internal structure of the gel and ultimately to a dramatic improvement in the ability of the composite to separate the two protein probes, ovum serum albumin and carbonic anhydrase. The morphology of the nanocomposite hydrogel was analyzed with cryogenic scanning and transmission electron microscopy, wide-angle X-ray diffraction, and small-angle scattering to determine whether morphological changes would correlate with this improved separation. As the volume fractions of MMT were well under 1% in this study (because of aqueous swelling), scattering data were dominated by the polymer structure. Significant morphological changes were noted at two length scales: (1) the hydrogel cell structure, at hundreds of nanometers, appeared to exhibit changes in the anisotropic orientation with magnetization, and (2) the polyamide structure, at tens of nanometers, exhibited decreasing pore size (small-angle X-ray scattering). The separation data correlated most closely with a reduction in pore size; however, an additional contribution to separation from local electrostatic effects from the presence of charged MMT in the cell walls could not be discounted. The change in the pore size associated with processing may have been due to the MMT presence altering the diffusion rates of the reactants during polymer formation. The method demonstrated herein could be used ultimately to separate proteins in their native state, with the potential retention of function for downstream applications, such as novel detection techniques or purification. (c) 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012