Resolution

We now turn to comparison of resolution (RS) in the different fractionation processes: we shall examine RS value and its variation with elution volume.

Reprinted with permission from Revillon A (1994) Journal of Liquid Chromatography 17: 2991-3023. CHDC, Capillary HDC; DCP, centrifugation under disc; TFFF, Thermal FFF; SFFF, steric FFF; SFC, supercritical fluid chromatography.

Firstly, we define a specific resolution (Rsp) = Rs/ratio of the diameter of the species under consideration. Secondly, we define resolution per unit time (Rt) = Rsp/time of measurement, in order to compare different samples and different elution conditions, respectively. To obtain polymer dimensions in solution, molecular mass, M, is converted to diameter, applying the equation: diameter is proportional to M0 6.

RS depends on chemical or physical factors, packing size being one of them. As a general rule in chromatography, resolution increases when the particle size of the packing decreases. In HDC with packed columns, the RF ratio is increased by using fine packings and resolution is increased. Pore size and pore volume, being the origin of molecular separation in SEC, strongly affect elution, whereas the effect of porosity is controversial in HDC, where the main phenomenon takes place essentially between - and not in the porous part of - the packing particles. Combined HDC and SEC action increases separation, in terms of sample mass and elution volume (see above).

For SEC, HDC, supercritical fluid chromatography and TFFF, theoretical plate height generally increases with flow rate according to the van Demter equation, so that resolution decreases. Sometimes Rt may increase, particularly if a gradient is used properly or when mass transfer does not play a role.

For all separations, it is possible to obtain an accurate molecular weight by using deconvolution to remove system dispersion.

Table 4 indicates meaningful values of R, RS, Rt and analysis time with the various separation systems.

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