Jeroen Billen

Influence of pressure and temperature on the physico-chemical properties of mobile phase mixtures commonly used in high-performance liquid chromatography

To fulfil the increasing demand for faster and more complex separations, modern HPLC separations are performed at ever higher pressures and temperatures. Under these operating conditions, it is no longer possible to safely assume the mobile phase fluid properties to be invariable of the governing pressures and temperatures, without this resulting in significantly deficient results. A detailed insight in the influence of pressure and temperature on the physico-chemical properties of the most commonly used liquid mobile phases: water-methanol and water-acetonitrile mixtures, therefore becomes very timely. Viscosity, isothermal compressibility and density were measured for pressures up to 1000 bar and temperatures up to 100 degrees C for the entire range of water-methanol and water-acetonitrile mixtures. The paper reports on two different viscosity values: apparent and real viscosities. The apparent viscosities represent the apparent flow resistance under high pressure referred to by the flow rates measured at atmospheric pressure. They are of great practical use, because the flow rates at atmospheric pressure are commonly stable and more easily measurable in a chromatographic setup. The real viscosities are those complying with the physical definition of viscosity and they are important from a fundamental point of view. By measuring the isothermal compressibility, the actual volumetric flow rates at elevated pressures and temperatures can be calculated. The viscosities corresponding to these flow rates are the real viscosities of the solvent under the given elevated pressure and temperature. The measurements agree very well with existing literature data, which mainly focus on pure water, methanol and acetonitrile and are only available for a limited range of temperatures and pressures. As a consequence, the physico-chemical properties reported on in this paper provide a significant extension to the range of data available, hereby providing useful data to practical as well as theoretical chromatographers investigating the limits of modern day HPLC.

Towards a solution for viscous heating in ultra-high pressure liquid chromatography using intermediate cooling

A generic solution is proposed for the deleterious viscous heating effects in adiabatic or near-adiabatic systems that can be expected when trying to push the column operating pressures above the currently available range of ultra-high pressures (i.e., 1200 bar). A set of proof-of-principle experiments, mainly using existing commercial equipment, is presented. The solution is based on splitting up a column with given length L into n segments with length L/n, and providing an active cooling to the capillaries connecting the segments. In this way, the viscous heat is removed at a location where the radial heat removal does not lead to an efficiency loss (i.e., in the thin connection capillaries), while the column segments can be operated under near-adiabatic conditions without suffering from an unacceptable rise of the mobile phase temperature. Experimental results indicate that the column segmentation does not lead to a significant efficiency loss (comparing the performance of a 10 cm column with a 2 cm x 5 cm column system), whereas, as expected, the system displays a much improved temperature stability, both in time (because of the shortened temperature transient times) and in space (reduction of the average axial temperature rise by a factor n). The method also prevents a large backflow of heat along the column wall that would lead to large efficiency losses if one would attempt to operate columns at pressures of 1500 bar or more. A real-world pharmaceutical example is given where this improved temperature robustness could help in moderating the changes in selectivity during method transfer from a low to a high pressure operation, although the complex non-linear behavior of the viscous heating and high pressure effects result in lower than expected improvement.

High performance liquid chromatography column packings with deliberately broadened particle size distribution: Relation between column performance and packing structure

The effect of the addition of 25%, 50% and 75% (weight percent, wt%) of larger particles (resp. 3 and 5 μm) to a commercial batch of 1.9 μm particles has been investigated as an academic exercise to study the effects of particle size distribution on the kinetic performance of packed bed columns in a magnified way. Comparing the performance of the different mixtures in a kinetic plot, it could be irrefutably shown that the addition of larger particles to a commercial batch of small particles cannot be expected to lead to an improved kinetic performance. Whereas the addition of 25 wt% of larger particles still only has a minor negative effect, a significantly deteriorated performance is obtained when 50 or 75 wt% of larger particles are added. In this case, separation impedance number increases up to 200% were observed. Studying the packing structure through computational packing simulations, together with the experimental determination of the external porosity, helped in understanding the obtained results. This showed that small particles tend to settle in the flow-through pores surrounding the larger particles, leading to very high packing densities (external porosities as low as 32% were observed) and also negatively influencing the column permeability as well as the band broadening (because of the broadened flow-through pore size range).