Liquid Chromatography

The intrinsic efficiency per unit length of packed columns in LC increases as the particle diameter is reduced. It can also be increased by using solvents of low viscosity, which result in smaller contributions to the column plate height from resistance to mass transfer and flow anisotropy. Operation at low mobilephase velocities compared to GC further minimizes the contributions from resistance to mass transfer in the mobile phase at the expense of longer separation times. The pressure drop required to maintain a constant mobile-phase velocity is proportional to the ratio of the column length to the particle diameter squared. Since the available operating pressure is finite, the column length must be reduced as the particle diameter is decreased. Consequently, most separations in LC are performed with a total of about 5000-20000 theoretical plates that is largely independent of the particle size. However, since the retention time at a constant (optimum) mobile-phase velocity is proportional to the column length, this arbitrary fixed number of plates is made available in a shorter time for shorter columns packed with smaller diameter particles. Thus the principal virtue of using particles of a small diameter is that they permit a reduction in the separation time for those separations that do not require a large number of theoretical plates.

Conventional column diameters in analytical LC at 3-5 mm are comparatively large so as to minimize zone broadening from extracolumn effects in earlier instrument designs and have become the de facto standard dimensions, even though instrument capabilities have improved over time. Smaller diameter columns have been explored to reduce mobile-phase consumption (which is proportional to the square of the column radius) and to enhance mass detection through reduction in peak volumes, but offer no improvement in the intrinsic column efficiency, except perhaps for columns with a low column diameter-to-particle size ratio. Capillary columns of 0.1 to 0.5 mm internal diameter packed with 3-10 |im particles can be used in relatively long lengths for the separation of complex mixtures, where a large number of theoretical plates is required. Such columns probably minimize the contribution form flow anisotropy while at the same time providing a better mechanism for the dissipation of heat caused by the viscous drag of the mobile phase moving through the packed bed. The operation of these columns is still pressure-limited and separation times an order of magnitude greater than for GC have to be accepted as the price for high efficiency.

The enhancement of intraparticular mass transport is particularly important for the rapid separation of biopolymers, whose diffusion coefficients are perhaps 100-fold smaller than those of low molecular weight compounds in typical mobile phases used in LC. Also, the high surface area porous packings used for small molecules may be too retentive for biopolymers with a significant capacity for multisite interactions. For these compounds short columns packed with 1.5 and 2 |im pellicular or porous particles are used for fast separations. Longer columns containing perfusive particles of a large size with large diameter through-pores to promote convective transport can also be used for fast separations. Per-fusive particles are also used for the preparative-scale separation of biopolymers.

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