Preparative Chromatography - The Maximum Sample Volume > Page 5

The Maximum Sample Volume

Any finite volume of sample placed on to a chromatography column will have an intrinsic band variance, and this variance will be added to the variance from the usual dispersion processes that take place in the column to provide a value for the ultimate peak variance. Consequently, if the column efficiency is not to be seriously reduced the maximum volume of sample that can be placed on the column must be limited.

Consider a volume (Vi) of sample, injected onto a column. This sample volume will constitute a rectangular distribution on the front of the column. Now, (as discussed in Dispersion in Chromatography Columns of this series) the variance of the peak eluted from the column will be the sum of the variances of the injected sample plus the normal variance of the eluted peak.

Thus:

where s2 is the variance of the eluted peak,
si2 is the variance of the eluted sample,
and sc2 is the variance due to column dispersion.

The maximum increase in band width that can be accepted due to any (and all) extraneous dispersion process is clearly optional, and will be determined by the character of the separation, and the purity of the required product. Klinkenberg (2) suggested a 5% increase in standard deviation (or, ca. a 10% increase in peak variance) was the maximumextra-column dispersion that could be tolerated without serious loss in resolution. This criteria is the generally accepted standard in analytical LC.