Effects of high pressure in liquid chromatography

All the experimental parameters that the chromatographers are used to consider as constant (the column length and its diameter, the particle size, the column porosities, the phase ratio, the column hold-up volume, the pressure gradient along the column, the mobile phase density and its viscosity, the diffusion coefficients, the equilibrium constants, the retention factors, the efficiency parameters) depend on pressure to some extent. While this dependence is negligible as long as experiments, measurements, and separations are carried out under conventional pressures not exceeding a few tens of megapascal, it is no longer so when the inlet pressure becomes much larger and exceeds 100 MPa. Equations are developed to determine the extent of the influence of pressure on all these parameters and to account for it. The results obtained are illustrated with graphics. The essential results are that (1) many parameters depend on the inlet pressure, hence on the flow rate; (2) the apparent reproducibility of parameters as simple as the retention factor will be poor if measurements are carried out at different flow rates, unless due corrections are applied to the results; (3) the influence of the temperature on the equilibrium constants should be studied under constant inlet pressure rather than at a constant flow rate, to minimize the coupling effect of pressure and temperature through the temperature dependence of the viscosity; and (4) while reproducibility of results obtained at constant pressure and flow rate will not be affected, method development becomes far more complex because of the pressure dependence of everything.