the Poisson curve. Consider the elution curve as shown in Figure 9. The origin of the Poisson curve is the point of injection, whereas the origin of the Gaussian curve is at the peak maximum, which will be (n) plate volumes from the injection point.  Now, a point X, (v)  plate  volumes  from the point of injection will be (v-n) = w plate volumes from the peak maximum. Consequently, v = (n+w). This change of origin is depicted in Figure 9. Now, the Poisson form of the elution equation is as follows,                                      Figure 9.  The Different Axes of The Poisson Elution Curve and the Gaussian Elution Curve

onto a column. The first component to elute, (A), will be that solute held least strongly in the stationary phase. Then the second solute, (B), will elute but it will be mixed with the first solute. Finally, the third solute (C), will elute in conjunction with (A) and (B). It is clear that only solute (A) is eluted in a pure form and, thus, frontal analysis would be quite inappropriate for most practical analytical applications. This development technique has been completely superseded by elution development. Elution Development Elution development is best described as a series of absorption-extraction processes which are continuous from the time the sample is injected into the distribution system until the time the solutes exit from it. The elution process is depicted in Figure 1. The concentration profiles of the solute in both the mobile and stationary phases are depicted as Gaussian in form. Equilibrium occurs between the two phases when the

loop) and then to the column. The layout of a suitable apparatus for gradient elution that can be employed with low volume small bore columns, including the low dead volume T mixer,  is shown in figure 26. It should be emphasized that modern chromatographs may, or may not, be designed for use with very small capacity, fast, high resolution columns and therefore, the specifications of the instrument must be closely examined if it is intended for such use. Rapid Separations Employing Gradient Elution Rapid LC separations are relatively easy to accomplish with isocratic development assuming low dispersion instrumentation with a fast response is available. However, if a sample mixture contains components that extend over a wide polarity or molecular weight range then gradient elution development will be necessary and fast gradients are almost impossible to form with conventional LC solvent programmers. As a consequence, for high speed gradient separations, a unique procedure must be

Low Dispersion Gradient Elution Apparatus Gradient elution with high efficiency low dispersion columns, particularly small bore columns that operate at very low flow rates, present another instrumental problem for the designer of the modern chromatograph. Not only must the gradients be formed accurately and precisely, they must be also formed at very low flow rates and sometimes the total volume employed for the analysis will be less than 1 ml.  The apparatus can be basic and accommodate only to solvents systems. It

Column Efficiency The column efficiency is defined as the number of theoretical plates in the column. As discussed in the plate theory, the faster the equilibrium process, the smaller the plates and thus, the greater the number of plates in the column. It is therefore important to know how to determine the number of plates a column possesses and the relationship of the number of theoretical plates in the column to the properties of the chromatogram. Starting with the Poisson form of the elution equation, the peak width at the points of inflexion (which corresponds to twice the standard deviation of the normal elution curve) can be found by equating the second differential of the elution equation to zero and solving in the usual manner. Thus, at the points of inflexion,                                    

as small as 1.5, and the first solute is eluted at a (k') value of 2.0, a volume as large as 66 ml can be still placed on the column.      At the other extreme if the first solute is eluted at a (k') value of 10 and the separation ratio is 2.5 then the sample volume can be over 1 liter. Nevertheless, it should be emphasized that the mathematical argument tacitly assumes that sample is injected onto the column as a solution, in the mobile phase. (i.e. the sample solvent does not change theelution conditions in any way). In addition, it also assumes that the solute concentration in the sample solution is not strong enough to produce significant solute/solute interactionin the mobile phase and consequently, also effect the conditions of elution. The effect of volume overload on the elution profiles of solutes separated on an LC column was examined by Scott and Kucera (3) and the results they obtained are shown as elution curves in figure 3. After, J. Chromatogr., Ref