Thermal Gradients for the Control of Elution in RP-LC: Application to the Separation of Model Drugs

The use of temperature as a variable in liquid chromatography enables the facile alteration of eluotropic strength without the need to change solvent composition. The ability to change eluotropic strength via temperature alone means that thermal gradients can be used to mimic the effects of solvent gradients but without many of the unwanted effects of changes in solvent composition. Here we illustrate the use of thermal gradients as a means of controlling chromatographic separations using either constant flow or, with the flow rate increased to maintain isobaric conditions, constant pressure, performed using columns packed with 1.7 μm particles. A model is described that can be used to used to predict flow, pressure and temperature under gradient conditions. Practical experimental factors such as the need for post column cooling and the use of frit restrictors in order to obtain optimum results are described.     

Thermal Gradients for the Control of Elution in RP-LC: Application to the Separation of Model Drugs

The use of temperature as a variable in liquid chromatography enables the facile alteration of eluotropic strength without the need to change solvent composition. The ability to change eluotropic strength via temperature alone means that thermal gradients can be used to mimic the effects of solvent gradients but without many of the unwanted effects of changes in solvent composition. Here we illustrate the use of thermal gradients as a means of controlling chromatographic separations using either constant flow or, with the flow rate increased to maintain isobaric conditions, constant pressure, performed using columns packed with 1.7 μm particles. A model is described that can be used to used to predict flow, pressure and temperature under gradient conditions. Practical experimental factors such as the need for post column cooling and the use of frit restrictors in order to obtain optimum results are described.

     

Quantification of the In Vitro and In Vivo Metabolic Fates of 2-, 3- and 4-Bromobenzoic Acids Using High Temperature LC Coupled to ICP-MS and Linear Ion Trap MS

High temperature liquid chromatography (HTLC), with water as the mobile phase, combined with ICP-MS tuned to the detection of Br, for quantification, and a linear ion trap MS, for structural identification, were applied to determine the disposition and metabolic fate of 2-, 3- and 4-bromobenzoic acids (BBAs) following in vitro incubation with rat hepatocytes at 4 mM. The separation of the metabolites was performed using a thermal gradient to increase the eluotropic strength of the aqueous mobile phase through the run to elute less polar components. The use of highly aqueous solvents for separations involving ICP-MS is advantageous because the water does not change the conductivity of the plasma thereby providing a more stable system. The improved system stability resulted in better sensitivity, as shown by the increased signal intensity for HTLC compared to conventional reversed-phase separations. Using HTLC to investigate the in vitro metabolic fate of the BBAs showed the major route of metabolism to be glycine conjugation, irrespective of the structure of the parent, but with different amounts produced depending on the positional isomer. The comparison of HTLC with the conventional methodology showed that chromatography at elevated temperatures had no effect on the observed metabolite profile. HTLC was also applied to urine obtained from an in vivo sample and showed an improved chromatographic peak shape compared to conventional liquid chromatography (LC) whilst providing the same analytical result.     

Application of High Temperature LC to the Separation of AZD5438 (4-(1-Isopropyl-2-methyl-1H-imidazol-5-yl)-N-[4-(methylsulfonyl)phenyl]pyrimidin-2-amine) and Its Metabolites: Comparison of LC, UPLC and HTLC

A comparison is made of the separation and analysis of a test probe, AZD5438 (4-(1-isopropyl-2-methyl-1H-imidazol-5-yl)-N-[4-(methylsulfonyl)phenyl]pyrimidin-2-amine), and its metabolites using conventional LC, UPLC and high temperature liquid chromatography (HTLC). LC and UPLC separations were performed using reversed-phase water:acetonitrile solvent systems whilst HTLC was performed with an entirely aqueous mobile phase, using both isothermal and temperature gradient chromatography. Substantial reductions in run times were observed with both UPLC and HTLC compared to the conventional LC approach without loss in chromatographic resolution for the major metabolites. At temperatures in excess of 100 °C, thermal degradation of some of the metabolites was observed in an isothermal separation, however, the application of a thermal gradient proved successful in maintaining the structural integrity of the analytes and simultaneously separating the parent compound and its major metabolites.     

Temperature as a variable in liquid chromatography: development and application of a model for the separation of model drugs using water as the eluent

The use of high temperatures in liquid chromatography allows for the use of a purely aqueous mobile phase. At elevated temperatures water possesses many of the characteristics of organic solvents in terms of eluotropic strength, as well as having a lower viscosity. A model is developed, based on data obtained using a range of model drugs, which demonstrates the relationship between temperature, flow and pressure. Experimental data from different column types, at temperatures from 40 degrees C to 180 degrees C, is presented which matches well with the predicted data from the model.