Sampling and extra-column effects in high-performance liquid chromatography; influence of peak skew on plate count calculations.

Chromatographic resolution and analysis accuracy can be seriously affected by broadening of very sharp bands associated with both high-performance size-exclusion chromatography and other liquid chromatography methods using less than 5 micron particles. The effect on column plate count by extra-column band broadening from injection systems, detectors, connectors and "guard" columns is discussed and exemplified with experimental data. The special cases of tailing and doublet peaks for a single species are investigated. These effects are eliminated by a special sample injection technique. Computer simulation studies demonstrated that serious errors can result from calculating column plate count with non-Gaussian peaks. The maximum allowable peak tailing is suggested and a new method for quantitating peak skew is proposed.