Thin layer chromatography (TLC) of cobalt and nickel has been performed on silica gel layers induced with alkali mediated cellulose extract. A novel combination of 10% aqueous solutions of Tween-20 and potassium thiocyanate in 1:1 (v/v) was identified as the best mobile phase for the selective separation of Co2+ from Ni2+ on the impregnated Silica Gel G layers. The chromatographic characteristics of the cations were studied and the limits of detection as well as the limits of quantification for Co2+ and Ni2+ were determined. The quantitative estimation of the cations was achieved from the digital image analysis of respective chromatograms. The proposed quantitative method was successfully applied with 0-0.50% error for the determination of Co2+ from Ni2+ in spiked samples of bauxite, soil and rock containing common cations such as Al3+, Fe2+, Ti4+, Zn2+, Mn2+, Cu2+, Cr6+, Mg2+ etc. under the optimized chromatographic conditions.