Practical mobile phase velocity

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 multipath dispersion on a thin layer plate is the process most likely to be described by a function similar to that in the van Deemter equation. However, the actual mobile phase velocity is likely to enter that range where the Giddings function (3) applies. In addition, as the solvent composition is continually changing (at least in the vast majority of practical applications) the solute diffusivity is also altered and thus, the mobile phase velocity at which the Giddings function applies will vary. 

The curve exhibits a minimum, which means that there is an optimum mobile phase velocity at which the column will give the minimum HETP and consequently a maximum efficiency. In practice this usually means that reducing the flow rate of a column will increase the efficiency and thus the resolution. In doing so, however, the analysis time will also be increased. As seen in figure 5, however, there is a limit to this procedure, as reducing the column flow rate so that the mobile phase velocity falls below the optimum will result in an increase in the HETP and thus a decrease in column efficiency. 

It is seen that, providing the isomers are eluted at a (kf) value greater than about 2.0, the column will separate those solute pairs having separation ratios as low as about 1.03. This, however, assumes that the column is very well packed and is operated at about the optimum mobile phase velocity. In practice, a more realistic minimum separation ratio would be between 1.035 and 1.04. However, as it will be seen, the cyclodextrin bonded phases can easily provide separation ratios significantly greater than these values. This is achieved by... 

In practice, because of the slow diffusion rates in liquids, dispersion due to longitudinal diffusion becomes important only at very low velocities. Because the dispersion increases only slowly with increasing mobile phase velocity, flow rates used in hplc are considerably higher than the value corresponding to minimum dispersion. This gives us fast separations without too much loss of efficiency. 

Equation (5) is a hyperbolic function which indicates that there will be a minimum value of (H) for a particular value of (u). That is a maximum efficiency will obtained at a particular linear mobile phase velocity. An example of an HETP curve obtained in practice is shown in figure 1. 

In ideal chromatography, we assume that the column efficiency is infinite, or in other words, that the axial dispersion is negligibly small and the rate of the mass transfer kinetics is infinite. In ideal chromatography, the surface inside the particles is constantly at equilibrium with the solution that percolates through the particle bed. Under such conditions, the band profiles are controlled only by the thermodynamics of phase equilibria. In linear, ideal chromatography, all the elution band profiles are identical to the injection profiles, with a time or volume delay that depends only on the retention factor, or slope of the linear isotherm, and on the mobile phase velocity. This situation is unrealistic, and is usually of little importance or practical interest (except in SMB, see Chapter 17). By contrast, nonlinear, ideal chromatography is an important model, because the profiles of high-concentration bands is essentially controlled by equilibrium thermodynamics and this model permits the detailed study of the influence of thermodynamics on these profiles, independently of the influence of the kinetics of mass transfer... 

The reduced plate height, h, is independent of the particle diameter. The reduced fluid velocity (u), a concept conceived by Giddings, is a measure of the rate of flow over a particle relative to the rate of diffusion of solute within the particle. Since both reduced parameters, h and V, are normalized for the particle diameter, when h is plotted against v, the different size fractions of the same packing materials should give similar curves. This has been confirmed in practice, so that use of h vs v curves is preferred over that of plate height curves (H versus mobile phase velocity) when comparison of the efficiency of different columns is to be carried out. 

Series coupling of normal-length columns (30 cm) to increase efficiency or to optimize the pore-size range for the separation is common practice. Series coupling of columns is facilitated by the low optimum mobile phase velocity and thus lower column pressure drop per unit column length, typical for SEC separations. 

Resolution in forced flow thin-layer chromatography is not restricted by the same factors that apply to capillary flow. Resolution increases almost linearly with the solvent-front migration distance and is highest for separations at the optimum mobile phase velocity. Resolution has no theoretical limit for forced flow the upper bounds are established by practical constraints (plate length, separation time and inlet pressure). 

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