It is seen that the maximum sample volume can vary over a very wide range of values. Employing the defined column packed with 20 mm particles, and operated at its optimum velocity to give 12,500 theoretical plates, if the separation ratio is as small as 1.5, and the first solute is eluted at a (k') value of 2.0, a volume as large as 66 ml can be still placed on the column.

At the other extreme if the first solute is eluted at a (k') value of 10 and the separation ratio is 2.5 then the sample volume can be over 1 liter. Nevertheless, it should be emphasized that the mathematical argument tacitly assumes that sample is injected onto the column as a solution, in the mobile phase. (i.e. the sample solvent does not change the elution conditions in any way).

In addition, it also assumes that the solute concentration in the sample solution is not strong enough to produce significant solute/solute interactionin the mobile phase and consequently, also effect the conditions of elution.

The effect of volume overload on the elution profiles of solutes separated on an LC column was examined by Scott and Kucera (3) and the results they obtained are shown as elution curves in figure 3.

After, J. Chromatogr., Ref. [3]

Figure 3 An Experimental Example of Volume Overload