Regeneration of Tetrabutylammonium Ion-Pairing Reagent Distribution in a Gradient Elution of Reversed Phase Ion-Pair Chromatography

Abstract

The regeneration of ion-pairing reagent distribution on liquid chromatography columns after gradient elution has been well recognized as the cause for long column equilibration time, a major drawback associated with gradient elution reverse phase ion-pair chromatography. To date, the majority of studies have focused on optimizing the separation conditions to shorten the equilibration time. There is limited understanding of the ion-pairing reagent distribution process between the mobile phase and stationary phase in the course of gradient elution, and subsequent column re-equilibration. The focus of this work is to gain a better understanding of this process. An ion-pair chromatographic system, equipped with a YMC ODS C(18) column and a mobile phase containing tetrabutylammonium (TBA) hydroxide as the ion-pairing reagent, was used in the study. The TBA distribution profile was established by measuring its concentration in the eluent fractions collected during the gradient cycle using different column equilibration times with an ion chromatographic method. Furthermore, the analyte retention time was evaluated as the function of the column equilibration time and TBA concentration in the mobile phase. The column equilibration and its impact on the method robustness will also be discussed.

Comprehensive quantitative analysis of purines and pyrimidines in the human malaria parasite using ion-pairing ultra-performance liquid chromatography–mass spectrometry

“…Reproducibility in retention times among different days was evaluated since the ion pairing approach could be problematic especially due to changes in the concentration of the ion-pairing reagent on the surface of the column material [24,25]. We found that nucleobases and nucleosides presented, on average, less than 0.15 minutes of variability in retention time compared to their phosphorylated counterparts, which varied from 0.5 to 0.9 minutes over the period of 24 hours when only 1.25 mM DBAA was present in eluent B.…”

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC‐MS/MS

“…For method robustness, we utilized mixed mode chromatography coupled with multiple‐reaction‐monitoring (MRM) instead of ion‐pairing chromatography (e.g. ion pairing reagent can have deteriorating effects on mass spectrometer) . The applicability of the developed method was demonstrated on a study of feed medium stability over a typical life span in fed‐batch cell culture processing.…”

“…ion pairing reagent can have deteriorating effects on mass spectrometer). 34 The applicability of the developed method was demonstrated on a study of feed medium stability over a typical life span in fed-batch cell culture processing. In addition, flexibility of our method was demonstrated by inclusion of additional compounds of interest to identify CDM-related reaction products described in the literature including methionine sulfone, cysteic acid, lumichrome, N-formylkynurenine, 3-hydroxy-DL-kynurenine, L-cysteinsulfinic acid, 3-aminopropionamide, hydroxocobalamin, 5-hydrody-L-tryptophan, DL-o-tyrosine, DL-methionine sulfoxide and L-citrulline.…”

Boosting basic-peptide separation through dynamic electrostatic-repulsion reversed-phase (d-ERRP) liquid chromatography

“…inuences the chromatographic performance of the system can be attributed to their ranking in the Hofmeister series. 37 The rationalization of the different equilibria that can be in such system is not trivial; indeed, while the adsorption of IP agent on the hydrophobic stationary phase has been and profoundly evaluated, 38,39 and a rationale of the behavior of inorganic additive in RPLC has been assessed, [40][41][42][43][44] the presence of both additives has not yet been taken into consideration, as well as a comprehensive theoretical model. However, it is not a technique that can be interfaced with mass spectrometry due to the low volatility of IP agents.…”