High-efficiency hydrophilic interaction chromatography by coupling 25cm×4.6mm ID×5μm silica columns and operation at 80°C

Abstract

Recently, hydrophilic interaction chromatography (HILIC) has emerged as a valuable orthogonal tool to reversed-phase liquid chromatography (RP-LC) as it allows for resolution of highly polar ionisable compounds. The relationships between separation efficiency, column length and speed of analysis for 4.6 mm ID x 5 µm silica particle columns in HILIC are demonstrated using kinetic plots. The kinetic plots constructed for conventional pressure systems operating at 350 bar and at 30 o C and 80°C are confirmed using experimental data for different column lengths. Efficiencies of more than 130,000 theoretical plates could be achieved by connecting up to six columns of 25 cm. As expected, a significant gain in analysis speed without loss of efficiency could be obtained by operating at 80°C compared to 30°C. The advantages of using long columns in HILIC in combination with elevated column temperature for the pharmaceutical industry are illustrated using test mixtures comprised of commercially available ionisable compounds (including some containing functional groups with potential genotoxic typical structural alerts) as well as real polar ionisable pharmaceuticals. Keywords:Hydrophilic interaction liquid chromatography, high efficiency, kinetic plots, temperature, speed 3 Correspondence:Professor Pat Sandra, Ghent University, Krijgslaan 281 S4-bis, B-9000, Ghent, Belgium E-mail: pat.sandra@richrom.comTel: +32 56 204031; Fax: +32 56 204858 IntroductionReversed-phase liquid chromatography (RP-LC) is by far the most widely employed analytical tool in drug discovery and development. In recent years, the efficiency of RP-LC has been drastically increased through various approaches including using sub-2µm particles in combination with ultra high pressure LC (UHPLC) [1][2][3], increasing the column length and operation at elevated temperature (ETLC) [4][5][6] or by combining both UHPLC and ETLC [7,8].These developments have certainly facilitated the development of generic methodologies for RP-LC for separations of active pharmaceutical ingredients (API's) and impurities [5].Furthermore, these advances have helped separation scientists to exploit early fundamental theories around the principles of separation optimisation with respect to efficiency and speed by utilisation of kinetic plots. For the pharmaceutical industry, which will certainly still have a plethora of conventional HPLC instrumentation in its good manufacturing practice (GMP) laboratories, kinetic plots can certainly serve a purpose in directing how conventional instrumentation and columns can be used more appropriately in combination with ETLC and long columns. This can help to optimize the use of conventional particle diameters (5 µm) to yield either maximum efficiency and to compared to sub-2µm particles, or to increased the speed of analysis.Since these developments, a chromatographic technique that has been enjoying recent attention in the pharmaceutical industry is hydrophilic interaction chromatography (HILIC).HILIC has become a valuable addition t...

Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter

“…Others have also reported effects on HILIC selectivity as function of organic solvent content, e.g. Jandera and coworkers who showed that the elution order under isocratic conditions may change as a function of the ACN mobile phase content [17], and Sandra and coworkers who observed that a gradient could increase the resolution of some compounds but decrease the resolution of others [18].…”

“…4). An increase in temperature usually leads to a shift in the equilibrium towards the mobile phase, shortening the retention time [18,20]. Inverse relationships between temperature and retention has been found and studied mostly regarding RP phases, and has been related to e.g.…”

Hydrophilic Interaction Liquid Chromatography Coupled to Mass Spectrometry and Multivariate Analysis of the De Novo Pyrimidine Pathway Metabolites

“…Johnsen and colleagues [34] also indicate higher efficiencies on the ZIC-p-HILIC column are obtained with high temperature and emphasize that the retention time also increases with the increase of temperature. Contrary to what occurs in RPLC, where the increase in temperature generally leads to a change in the equilibrium towards the mobile phase, which shortens the retention time [36,37]. It could be speculated that the temperature affects the hydration of the analytes to explain this behavior in polymeric zwitterionic columns, therefore affecting retention [38].…”

Graphical Data Representation Methods To Assess the Quality of LC Columns

“…Figure shows some examples of variable-length kinetic plots. Many other examples can be found in the literature. ,− Figure a,c displays the same data set, relating to two different sizes of a core-shell particle. Both plots clearly emphasize the complementary of both particle sizes, with the smaller particles performing best for short separations (small t 0 ), while the opposite holds in the range of separations requiring large times.…”