. It should be pointed out that this injection procedure does result in some loss of sample, due to that retained at the walls of the ample tube, but this can be easily recovered and recycled if necessary. Thisn technique is strongly recommended for preparative sampling and should be employed wherever possible.      Sample Mass Overload The effect of excess mass of sample (mass overload) on the chromatographic process can be far more complex than volume overload. The theory of mass overload is, as one might expect, also complicated (5–7) and requires a considerable amount of basic physical chemical data, such as the adsorption isotherms of each solute measured over a wide range of concentration, before it can be applied to a practical problem. Only if the separation problem demands an extremely high through-put, and the process must be as economic as possible, will it be worthwhile to gather the necessary basic data. The problem of mass overload is more conveniently and

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

nbsp; These complex effects are best illustrated by experiment. The effect of mass overload was also investigated by Scott and Kucera (3) and the same column was used for mass overload experiments as that employed in the volume overload experiment. In the investigation of mass over load, the sample volume was kept constant at 200 ml, and a mixture of benzene, toluene and anthracene was placed on the column, the mass of benzene being increased progressively from 180 mg to 16.9  mg. An example of three of the chromatograms obtained are shown in figure 7. A chromatogram of the reference sample is shown on the left of figure 7 and contained 180 mg of benzene, 9 mg of

retention distance up to a sample volume of 0.5 ml for the three component mixtures, and up to 1 ml for the two component mixture. Subsequent to these limiting sample volume values, the retention of the back of the peak appears to increase linearly with charge volume. It is also interesting to note that peak dispersion is the same for each solute and is independent of the nature of the solute or its capacity ratio (k'). The peak dispersion towards greater retention is characteristic of volume overload which, as will be seen below, will not be true for mass overload. Sample volume overload distorts the normal elution profile derived from normal elution development to that of frontal analysis development. If elution development is carried out with sample of increasing volume, the distorted elution development concentration profile culminates in frontal analysis. After, J. Chromatogr., Ref. [3]   Figure 5. The Transition from Sample Volume Overload to Frontal Analysis

nbsp; The different effects of mass overload are now clearly revealed. Firstly, it must be emphasized that the curves in figure 8 represent the movement of the peak extremes and are in no way related to the peak shape. It is seen that the retention of the rear of the major peak, benzene, hardly changes with sample mass as this represents low concentrations of benzene and thus is eluted in the normal manner. However, the retention of the peak front is reduced progressively as the sample mass is increased. This results from both the

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 Benzene Naphthalene Anthracene Capacity Ratio k 1.18 2.33 4.31 Efficiency n 1850 4480 5470 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