Use of individual retention modeling for gradient optimization in hydrophilic interaction chromatography: Separation of nucleobases and nucleosides

Abstract

a b s t r a c tIn this study, the separation of twelve nucleobases and nucleosides was optimized via chromatogram simulation (i.e., prediction of individual retention times and estimation of the peak widths) with the use of an empirical (reversed-phase) non-linear model proposed by Neue and Kuss. Retention time prediction errors of less than 2% were observed for all compounds on different stationary phases. As a single HILIC column could not resolve all peaks, the modeling was extended to coupled-column systems (with different stationary phase chemistries) to increase the separation efficiency and selectivity. The analytical expressions for the gradient retention factor on a coupled column system were derived and accurate retention time predictions were obtained (<2% prediction errors in general). The optimized gradient (predicted by the optimization software) included coupling of an amide and an pentahydroxy functionalized silica stationary phases with a gradient profile from 95 to 85%ACN in 6 min and resulted in almost baseline separation of the twelve nucleobases and nucleosides in less than 7 min. The final separation was obtained in less than 4 h of instrument time (including equilibration times) and was fully obtained via computer-based optimization. As such, this study provides an example of a case where individual retention modeling can be used as a way to optimize the gradient conditions in the HILIC mode using a non-linear model such as the Neue and Kuss model.

Accurate prediction of retention in hydrophilic interaction chromatography by back calculation of high pressure liquid chromatography gradient profiles

“…Prediction errors were ~1% (from 0.62 s to 2.5 s), which was 6–43 times more accurate than when gradients were assumed to be ideal and 3–5 times more accurate than when only gradient delay was taken into account. To the best of our knowledge, this is the most accurate gradient retention prediction in HILIC and approximately 5-fold more accurate than previous reports [14], [15], [18]. …”

“…This prediction accuracy is similar, if not more accurate than a report that, until now, showed the most accurate retention projections in HILIC. They also used an offset gradient profile [15]. In Table 5, which shows retention projection errors using the back-calculated gradient profiles, the prediction error was the smallest of all, ranging from ±0.62 s to ±2.5 s (0.53% to 1.83%), which was 6–43 times more accurate than when using the ideal gradient profiles and roughly threefold more accurate than when using the offset gradient profiles.…”

“…In previous studies using gradient retention projection from isocratic runs in HILIC where the back-calculation methodology was not used, the prediction accuracy was much worse than in RPLC [9], [14], [15], [21]. For example, Tyteca et al predicted gradient retention times in HILIC and found the prediction error was 3.7% to 9.5%, which is roughly 30-fold less accurate than retention projections in RPLC using back-calculation [14].…”

“…For example, Tyteca et al predicted gradient retention times in HILIC and found the prediction error was 3.7% to 9.5%, which is roughly 30-fold less accurate than retention projections in RPLC using back-calculation [14]. A later publication by Tyteca et al reported an improvement in the average prediction error to ~ 3% [15]. They attributed the improved accuracy to their use of a better equation to describe k vs. φ relationships, but it was probably also caused by their use of shallower gradients than in their previous work.…”

Computer-assisted multi-segment gradient optimization in ion chromatography

“…In RPLC using water-organic solvent systems, the retention is the result of solvophobic partitioning and the retention relationship can be described approximately by a semi-log linear relationship, referred to as the linear solvent strength (LSS) model: where is the fraction of organic solvent, k W is the extrapolated value of k for = 0 (i.e., pure water) and S is the solvent strength parameter which is a constant for a given compound, stationary phase and type of organic solvent [18,19]. For RPLC water-acetonitrile systems, and for other types of chromatography, such as hydrophilic interaction (HILIC) or supercritical fluid chromatography (SFC) a curvature is observed at higher percentages of eluting solvent when plotting ln(k) as a function of , and higher order retention models are therefore required to accurately describe the isocratic retention data [20][21][22][23].…”