Question

An HPLC column received from a manufacturer was specified to have 4000 plates measured under standard conditions with a test compound. When the customer repeated the test she found only 1500 plates, despite a symmetrical Gaussian peak shape. What might account for this?

Answer 1

Columns do not have fixed plate counts. For a given column, plate counts depend on the flow rate, the viscosity of the solvent and the molecular weight of the analyte. The instrument may affect the measurement as well, but we will ignore this for the time being.
The column manufacturer has set up standardized conditions by which the quality of the column is measured. In reversed-phase chromatography, the measurement is usually done with a simple hydrophobic analyte, like toluene, naphthalene or acenaphthene. The mobile phases used with
these compounds contain a high amount of organic solvent. Therefore they have a low viscosity. On the other hand, most HPLC users are dealing with more polar molecules, which
require mobile phases with a higher water content. These mobile phases have a higher viscosity. Since under normal HPLC conditions and normal HPLC flow rates, the plate count decreases when the viscosity increases,the plate count is lower under practical use conditions than under column testing conditions.

Gaussian curve - a standard error curve, based on a mathematical function, that is a symmetrical, bell-shaped band or peak. Most chromatographic theory assumes a Gaussian peak.