Peak dispersion

The dead point is the position of the peak maximum of an unretained solute. It is not the initial part of the dead volume peak as this represents a retarded portion of the peak that is caused by dispersion processes. The importance of employing the peak maximum for such measurements as dead volume and retention volume will be discussed in later chapters of the book that deal with peak dispersion. 

Thus, the variance of the peak is inversely proportional to the number of theoretical plates in the column. Consequently, the greater the value of (n), the more narrow the peak, and the more efficiently has the column constrained peak dispersion. As a result, the number of theoretical plates in a column has been given the term Column Efficiency. From the above equations, a fairly simple procedure for measuring the efficiency of any column can be derived. 

Peak dispersion can happen in any part of the chromatographic system, from the... 

Having established that a finite volume of sample causes peak dispersion and that it is highly desirable to limit that dispersion to a level that does not impair the performance of the column, the maximum sample volume that can be tolerated can be evaluated by employing the principle of the summation of variances. Let a volume (Vi) be injected onto a column. This sample volume (Vi) will be dispersed on the front of the column in the form of a rectangular distribution. The eluted peak will have an overall variance that consists of that produced by the column and other parts of the mobile phase conduit system plus that due to the dispersion from the finite sample volume. For convenience, the dispersion contributed by parts of the mobile phase system, other than the column (except for that from the finite sample volume), will be considered negligible. In most well-designed chromatographic systems, this will be true, particularly for well-packed GC and LC columns. However, for open tubular columns in GC, and possibly microbore columns in LC, where peak volumes can be extremely small, this may not necessarily be true, and other extra-column dispersion sources may need to be taken into account. It is now possible to apply the principle of the summation of variances to the effect of sample volume. 

Diffusion plays an important part in peak dispersion. It not only contributes to dispersion directly (i.e., longitudinal diffusion), but also plays a part in the dispersion that results from solute transfer between the two phases. Consider the situation depicted in Figure 4, where a sample of solute is introduced in plane (A), plane (A) having unit cros-sectional area. Solute will diffuse according to Fick s law in both directions ( x) and, at a point (x) from the sample point, according to Ficks law, the mass of solute transported across unit area in unit time (mx) will be given by... 

The dispersion of a solute band in a packed column was originally treated comprehensively by Van Deemter et al. [4] who postulated that there were four first-order effect, spreading processes that were responsible for peak dispersion. These the authors designated as multi-path dispersion, longitudinal diffusion, resistance to mass transfer in the mobile phase and resistance to mass transfer in the stationary phase. Van Deemter derived an expression for the variance contribution of each dispersion process to the overall variance per unit length of the column. Consequently, as the individual dispersion processes can be assumed to be random and non-interacting, the total variance per unit length of the column was obtained from a sum of the individual variance contributions. 

Equations that quantitatively describe peak dispersion are derived from the rate theory. The equations relate the variance per unit length of the solute concentration... 

To reiterate the definition of chromatographic resolution a separation is achieved in a chromatographic system by moving the peaks apart and by constraining the peak dispersion so that the individual peaks can be eluted discretely. Thus, even if the column succeeds in meeting this criterion, the separation can still be destroyed if the peaks are dispersed in parts of the apparatus other than the column. It follows that extra-column dispersion must be controlled and minimized to ensure that the full performance of the column is realized. 

Unfortunately, any equation that does provide a good fit to a series of experimentally determined data sets, and meets the requirement that all constants were positive and real, would still not uniquely identify the correct expression for peak dispersion. After a satisfactory fit of the experimental data to a particular equation is obtained, the constants, (A), (B), (C) etc. must then be replaced by the explicit expressions derived from the respective theory. These expressions will contain constants that define certain physical properties of the solute, solvent and stationary phase. Consequently, if the pertinent physical properties of solute, solvent and stationary phase are varied in a systematic manner to change the magnitude of the constants (A), (B), (C) etc., the changes as predicted by the equation under examination must then be compared with those obtained experimentally. The equation that satisfies both requirements can then be considered to be the true equation that describes band dispersion in a packed column. 

Figure 6. Graph of Corrected Peak Dispersion Reciprocal of the Diffusivity...

Figure 7. Graph of Number of Compounds against Error % obtained from the Linear Regression of the Graph of the Corrected Peak Dispersion against the Reciprocal of the Diffusivity...

Thus, a practical procedure would be as follows. Initially the HETP of a series of peptides of known molecular weight must be measured at a high mobile phase velocity to ensure a strong dependence of peak dispersion on solute diffusivity. 

The problem is made more difficult because these different dispersion processes are interactive and the extent to which one process affects the peak shape is modified by the presence of another. It follows if the processes that causes dispersion in mass overload are not random, but interactive, the normal procedures for mathematically analyzing peak dispersion can not be applied. These complex interacting effects can, however, be demonstrated experimentally, if not by rigorous theoretical treatment, and examples of mass overload were included in the work of Scott and Kucera [1]. The authors employed the same chromatographic system that they used to examine volume overload, but they employed two mobile phases of different polarity. In the first experiments, the mobile phase n-heptane was used and the sample volume was kept constant at 200 pi. The masses of naphthalene and anthracene were kept... 

As the individual components of a mixture are moved apart on the basis of their differing retention, then the separation can be partly controlled by the choice of the phase system. In contrast, the peak dispersion that takes place in a column results from kinetic effects and thus is largely determined by the physical properties of the column and its contents. 

At this point, it is important to stress the difference between separation and resolution. Although a pair of solutes may be separated they will only be resolved if the peaks are kept sufficiently narrow so that, having been moved apart (that is, separated), they are eluted discretely. Practically, this means that firstly there must be sufficient stationary phase in the column to move the peaks apart, and secondly, the column must be constructed so that the individual bands do not spread (disperse) to a greater extent than the phase system has separated them. It follows that the factors that determine peak dispersion must be identified and this requires an introduction to the Rate Theory. The Rate Theory will not be considered in detail as this subject has been treated extensively elsewhere (1), but the basic processes of band dispersion will be examined in order to understand... 

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