Two cross linked polymers were synthesized, the first polymer was synthesized by condensation reaction between glycerol triethanol amine with maleic anhydride. The second polymer was synthesized by addition polymerization of free radical for acrylamide, bisacrylamide with ammonium persulphate and N,N,N,N-tetramethyldiamine (TEMED). The products were characterized by FTIR & X-ray. The resulted polymer have high rigidity and easily grinded, with high stability and used as stationary phase for HPLC. This stationary phase has been packed into stainless steel column. The chromatographic performances of the newly packed columns are characterized. The number of theoretical plates (N), high equivalent to theoretical plate (H), capacity factor (?) and selectivity factor (?) were measured by analyzing different analytes on the new packed columns using different mobile phase and flow rates. Some of drugs, such as amiloride, furosmide, and atenolol were separated with these columns. Gradient eluention of (0-20) % phosphate buffer adjusted at pH 8 with methanol, as a mobile phase, with flow rate of 1ml/min and UV detection at 233nm was used with first column. Gradient eluention of (0-10) % phosphate buffer adjusted pH at 6 in ethanol, as mobile phase with flow rate 1.2ml/min and UV detection at 233nm, was used with acrylamide-bisacrylamide column. Furthermore some amino acids (tyrosine, Tryptophane and Phenylalanine) were also examined with triethanolamine-glycerol-malate column with isocratic eluention 15% phosphate buffer adjusted pH at 6 in 85% ethanol as a mobile phase with flow rate of 1ml/min and UV detection of 245nm. Gradient eluention (0-15) % phosphate buffer adjusted pH at 6 in ethanol, as mobile phase with flow rate 1.2ml/min with UV detection at 245 nm, was used with acrylamide-bisacrylamide column. Vitamin E was also analyzed using triethanolamine-glycerol-malate column with 100% acetonitrile as a mobile phase at flow rate of 1ml/min and UV detection of 229nm. The separation of these compounds on these two columns was compared with their separation on C-18 commercial column. Calibration curves for all the analyzed compounds were linear from their detection limits to at least 10 ppm with correlation coefficient range from 0.9993 to 0.9999. The detection limits were ranged from 0.05 to 0.1 ppm at signal to noise ratio of three or more for most analyzed compounds.