Sample Volume Overload

Consider the separation depicted in figure 1 (the retention parameters are labeled according to the plate theory as discussed in Plate Theory and Extensions ). Examination of the figure shows that the column could be heavily overloaded, to allow the peaks to spread until they touched at the base, before resolution would be lost. Under these conditions the principle of the summation of variances cannot be used, as when the sample volume becomes excessive, the dispersion of the peak becomes, to the first approximation, equal the volume of the sample itself.

Presented in a different way, the sample volume acts as a part of the mobile phase and contributes to the elution process in the same manner. Consequently, referring to figure 1, the peak separation in milliliters of mobile phase will be equivalent to the sample volume plus the sum of half the base widths of the respective peaks. Bearing in mind that half the peak width is equivalent to two standard deviations, then:

(KA) and (KB) are the distribution coefficients of solutes (A) and (B)
and (a) is the separation ratio of solutes B and A respectively.

Figure 1 Theory of Volume Overload