Practical implications of the "Tanaka" stationary phase characterization methodology using ultra high performance liquid chromatographic conditions.

The practical implications of performing column characterization protocols (i.e. Tanaka) and their resultant chromatographic selectivity parameters using small dimension columns (i.e. 50 × 2.1 mm I.D.) at high pressures have been critically compared to those obtained using conventional LC methodology. Retention factors should be corrected for the system extra column volume even when determined on ultra high performance liquid chromatographic (UHPLC) systems with low system volumes. An increase in pressure resulted in a general increase in the retention factor for most analytes, the degree being dependent on the physico/chemical properties of each analyte and the chromatographic conditions employed. However, analytes chromatographed at pH values close to their pK(a) values exhibited a substantial decrease in retention factor. Performing the Tanaka and extended column characterization procedures at pressures that would be encountered during the characterization of small particle sizes packed into 50 × 2.1 mm I.D. column formats at a constant linear velocity according to standard protocols, resulted in comparable chromatographic selectivity parameters to those determined using standard HPLC systems and column formats. However, due to the wide structural diversity of analytes employed in other popular column characterization protocols, it is imperative to demonstrate comparable results when small columns packed with small particle sizes are chromatographed at increased pressure and compared to standard column formats--otherwise erroneous comparisons and conclusions may be made.