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column volumes were considered to be the interstitial volume (the volume between the particles), the column pore volume (the volume within the particle) and the volume of stationary phase. However, in using these basic column volumes, certain invalid assumptions were made. Firstly. it was assumed that the mobile phase in the interstitial volume was moving phase and none was static, which, although possibly true in GC (particularly in capillary columns), is certainly not true for packed column in LC. Secondly, the pore contents had the same composition as the bulk mobile phase which, for solvent mixtures has been proved not to be so (10). Thirdly, it was assumed that all the stationary phase was available to the solute and, thus, exclusion effects were ignored. The different column volumes that are chromatographically pertinent can be logically broken down into the individual volumes involved. The column contains three materials, the mobile phase, the stationary phase and

mobile phase, followed by (v) plate volumes of equilibrated mobile phase carrying a solute concentration (Xo) of solute, will be given by                .                (63) The sum expressed by equation (63) also lends itself to a digital solution and with an appropriate computer program the actual peak profiles can be calculated for different volumes of pure mobile phase injected onto the equilibrated column. Values of (XE) were calculated for a column having 500 theoretical plates and for sample volumes of 20, 50, 100 and 200 plate volumes, respectively. The curves relating solute concentration (XE) to plate volumes of mobile phase passed through  the column are shown in Figure 19

nbsp;                                                         Xo(e-p–1) After the addition of each plate volume of charge, a new concentration of solute exists in plate (1), and its contents will be eluted through the column in the normal manner. Consider a total of (p) plate volumes of pure mobile phase are injected onto the column followed by a further (v) plate volumes of equilibrated mobile phase. After the injection of (r) plate volumes of pure mobile phase, the new concentration of solute in plate 1 will be eluted by a further (p-r) plate volumes of sample followed by (v) plate volumes of equilibrated mobile phase. Therefore, the concentration of solute leaving the (n)th plate due to the (r)th

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

The column was operated close to its optimum velocity and, even with a 2 m column the analysis time extended over 40 hr. The number of peaks disclosed by the chromatogram is about 150. The separation was developed isocratically by a 75% v/v acetonitrile/water mixture. It is seen that there is now a clear resemblance to a GC separation carried out on a capillary column. Unfortunately the analysis times are far from comparable. It is seen that extra-column dispersion can arise in the sample valve, unions, frits, connecting tubing, and the sensor cell of the detector. The maximum sample volume, i.e., that volume that contributes less than 10% to the column variance, is determined by the type of column, dimensions of the column and the chromatographic characteristics of the solute. In practice, the majority of the permitted extra-column dispersion should be allotted to the sample volume, as a large sample volume may be necessary to handle a

and the same solvent was used at a flow rate of 40 ml/min Benzene was also used a the solute. It is seen that the reduction in cell volume has a dramatic effect on both peak width and peak shape. The large 25 ml cell causes significant peak asymmetry as well as excessive peak dispersion A result which is predicted by the work of Atwood and Golay (11) which is discussed below. It is seen that the large sensor cell has a disastrous effect on the band width of the solute eluted from the microbore column. Clearly, even cell volumes of 3 ml are too large for use with 1 mm I.D. columns and relatively few contemporary detectors have cell volumes less than 3 ml.    J. Chromatogr. 169(1979)51 Figure 17. Peak Profiles from Detector Having Different Cell Volumes