A high-performance liquid chromatographic technique for the separation of triglycerides in natural mixtures is described. The influence of column temperature on separation is investigated, and a linear relationship between log k' and 1/T is demonstrated. Operating this reversed-phase technique with a LiChrosorb RP-18 packing at 14.5 degree C, separation of triglycerides with the same equivalent carbon numbers (ECN), but different degrees of saturation, is obtained. The composition of an eluent which permits separation of saturated triglycerides with carbon numbers as high as 54 (e.g., tristearin) is given.