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Column extraction technique was applied to acid-base fractionation of urinary lipophilic components. The acidified urine was retained on an extraction column packed with diatomaceous earth granules. Extraction solvent passed through the column was introduced directly into alkaline columns to fractionate strong and weak acidic components, the former being trapped on sodium hydrogen carbonate and the latter on sodium hydroxide column. Neutral components were eluted out without any chemical interaction with the support reagents. Acidic components retained in the column were recovered after acidified with acetic acid. Each fraction thus obtained was analyzed by silica gel high performance liquid chromatography equipped with a multi-channel ultraviolet detector using a solvent system consisted of 0.2% distilled water, 0.2% acetic acid, 15% ethanol and appropriate volume of n-hexane. Thus the comparative chromatographic studies of these three fractions became very easy. In our experiment, urinary components were extracted and chromatographed in the order of their partition coefficients. The method was applied to xanthine derivatives after the administration of caffeine tablets to a healthy volunteer.