Effect of Subambient Column Temperature on Resolution of Flouorouracil Metabolites in Reversed-Phase High Performance Liquid Chromatography

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Abstract
Reversed-phase high performance liquid chromatography (HPLC) was used to separate in the same sample the pyrimidine base, 5-fluorouracil, the nucleosides, 5-fluorouridine and 5-fluoro-2'-deoxyuridine, as well as the nucleotides, 5-fluorouridine monophosphate and 5-fluoro-2'-deoxyuridine monophosphate. After the resolution of these compounds had been optimized using conventional HPLC techniques at ambient temperature (∼30°C), chromatography was performed at 60°C, 40°C, 30°C, 20°C, and 5°C, and the resultant effect on resolution was analyzed. The capacity factor (k') of each compound was observed to increase with decreasing temperature. The extent of change in k' with temperature varied with each of the five compounds with resultant changes in resolution between adjacent peaks being associated with marked differences in the separation factor (α). The optimal temperature for resolving all five compounds (particularly the two nucleotides) was subambient (below 30°C). This study demonstrates that column temperature should be reconsidered as a chromatographic parameter that may be varied to improve resolution in HPLC when conventional methods are unsuccessful and furthermore suggests that subambient temperature may be useful with selected resolution problems in HPLC.

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