Thin layer chromatography in combination with spectrophotometry has been used for the separation and determination of cobalt after its separation from nickel in aqueous samples. A chromatographic system consisting of a mixture containing stannic arsenate gel and silica gel G in 10:1, w/w ratio and impregnated with 0.2M tri-n-butyl phosphate(TBP) as stationary phase and IM aqueous potassium thiocyanate. as mobile phase has been used to separate cobalt from nickel. Effects of acidity and alkalinity of sample solution on the separation of Co2+ from Ni2+ have been examined. Microgram separation of cobalt from variable amounts of nickel has been achieved after pre-concentration of metal ions from aqueous solutions by, sulphide precipitation. Determination of cobalt after TLC separation provides a recovery of 92%.