Ultra-short columns and ballistic gradients: considerations for ultra-fast chromatographic liquid...

Ultra-fast chromatographic separations has enabled fast chromatographic method development and rapid analysis for sample quantification. Decreasing over-all analytical time has become a factor of major importance for all aspects of drug discovery. However, merely decreasing chromatographic analysis time by decreasing k' can lead to inconsistent quantitative or qualitative results due to ineffective separations in complex matrices. We have found that by changing column length and gradient slope we can maintain chromatographic integrity of chemically diverse analytes and achieve the analytical speed required for bioanalytical drug discovery quantitative analysis. We have optimized method development strategy by performing separations on 2x20 mm HPLC columns at flow-rates of 1.5 ml/min to 2 ml/min with full linear gradients achieved in 1 min for the quantification of pharmaceuticals and their metabolites from biological matrices. This method development strategy can be readily adapted to other matrices. This paper will discuss the effects of column length and gradient time in ultra-fast chromatographic resolution.