The column dead volume was assumed to be ca 2.5 ml. The chromatographic properties of the three solutes chromatographed on the column at a flow rate of 1 ml/min. together with the respective efficiencies are shown in table 1.
Table 1 Chromatographic Properties of the Three Solutes Separated on the Column Used for Overload Experiments
|Capacity Ratio k||1.18||2.33||4.31|
|Retention Ratio a||—||1.97||1.85|
|Sample Volume VL||3.1 ml||6.1 ml||—|
It is seen that the column was operated well above its optimum flow rate so the maximum efficiencies obtainable for each solute were not realized. It also appears that the dispersion that occurs in the sample loop from Newtonian Flow (see Dispersion in Chromatography Columns ) seriously reduces the efficiency of the two first peaks and, consequently, it is not until the capacity ratio of the solute reaches 4, or more, could reasonable efficiencies be obtained. Notwithstanding, the results unambiguously demonstrate the advantages of column overload.
In the chromatograms on the left hand side of figure 3, 10 ml, 1 ml, 2 ml, and 3 ml of sample was placed on the column each containing 176 mg of benzene, 9 mg of naphthalene and 0.3 mg of anthracene. So all the samples contained the same mass of each solute. It is seen that a sample volume of 3 ml (theoretical value calculated from equation (2), was 3.1 ml) just allows benzene and naphthalene to be separated.