Performance of Markers and the Homologous Series Method for Dead Time Estimation in Reversed-Phase Liquid Chromatography

Two methods for dead time estimation (the use of markers and the homologous series mathematical method) are revised. Out of twelve assayed common markers, only KBr, KI, tartrazine, thiourea, uracil, and urea yielded retention times independent of the mobile phase composition in the range 10-90% acetonitrile, using a Zorbax Eclipse XDB-C18 column. On the other hand, the quality of the estimations provided by the homologous series method was limited by the mathematical approach and the data quality. With this method, the estimated dead time is an extrapolated value, which is severely affected by the data of the most retained compounds that act as leverage points, biasing the result. The sequential elimination of the most retained compounds attenuates this problem. This means that the homologous series should contain at least four compounds with low retention. Otherwise, overestimations of dead time are yielded.