New stationary phase for high performance liquid chromatography had been prepared by the reaction of congo red solution with amberlite anion exchange. The F.T.I.R elemental analyses have configured the attachment of congo red on the resin as has indicated by the physical appearance of the new resin. The resin capacities of newly prepared resin has calculated the resin capacity for the amberlite-congo red resin was found to average 7.36 meq/ gm and for the amberlite resin 3.0 meq./gm. The new stationary phase has been packed into stainless steel column. This has been done by the special technique. The chromatographic performance of the packed column was characterized the number of plate numbers, height equivalent of a theoretical plates(HETP) capacity factors, selectivity factor and peak asymmetry were measured by analyzing different analysis on the new column using different mobile phases composition and flow rates. The parameters were measured from analysis of some amino acids such as phenyl alanine, tyrosine and tryptophan. The analysis of amino acids using amberlite-congo red column has performed with an aqueous mobile phase adjusted at pH 10. The average number of plates, N was(111.09-284.3) . The plate height H was (0.01343) cm, capacity factor, ḱ were ranged from(3.5-3.76). and peak asymmetry value was1.06. The analysis of some amino acid of the some column but with a mobile phase adjusted at pH 10 with 20% aceto nitrile, the chromatographic parameters were plate’s number, N was(176.3307). plate height H was(0.128) cm, capacity factor Ќ were ranged from(2.98-4.65).