Plate Theory and Extensions - The Maximum Sample Volume that Can Be Placed on a Chromatographic Column > Page 69
The acceptable extent to which the performance of the column is denigrated is an arbitrary decision. In 1957 Klinkenberg (18) recommended that the maximum extra-column dispersion that should be tolerated without serious loss in resolution was a 5% increase of the standard deviation (ca. 10% increase in peak variance). This criterion, recommended by Klinkenberg as the limit for extra-column dispersion, is now generally accepted.
The variance of a rectangular distribution of sample volume (V1) is . If it is assumed that the peak width can be increased by 5% as a result of the dispersing effect of the sample volume, then applying the principle of the summation the variances,
where, from the plate theory, dispersion due to the column alone is
Recalling that ,
then, . (60)
The maximum sample volume that can be used without seriously denigrating the column performance can be calculated using equation (60) from the retention volume of the solute and the column efficiency. In any separation, there will be one pair of solutes that are eluted closest together and it is the retention volume of the first of these that is employed in equation (60) to calculate the maximum acceptable sample volume.