The group-resolving power of cascade-mode multiaffinity column chromatography (CASMAC), was demonstrated with human serum as a model mixture. More than 99% of the serum proteins were adsorbed in the same high salt-containing buffer on a tandem column consisting of (1) immobilized Zn2+ on triscarboxymethyl diamine gel followed by (2) thiophilic (T) gel, (3) Zn2+ bound to the new tridentate chelating adsorbent dipicolylamine (DPA) agarose, (4) hexyl-thioether C6-S agarose and (5) Ni(2+)-DPA agarose. After the adsorption step the immobilized metal ion affinity gels were attached to the top of tandem columns of other adsorbents (T gel, Sephadex G-25 for desalting and Mono-Q) and the elution conditions were selected such that further group separation was achieved. High resolution, high recovery, easy manipulation and high capacity are characteristic features of the cascade process with these adsorbents. The advantage of CASMAC is particularly striking when, with a given number of adsorbents, the overall number of operations involving adsorption, desorption, washing, buffer change and substance concentration can be effectively minimized.