With an enormous growth in the application of hydrophilic interaction liquid chromatography (HILIC), there has also been significant progress in HILIC method development. HILIC is a chromatographic ...method that utilises hydro-organic mobile phases with a high organic content, and a hydrophilic stationary phase. It has been applied predominantly in the determination of small polar compounds. Theoretical studies in computer-aided modelling tools, most importantly the predictive, quantitative structure retention relationship (QSRR) modelling methods, have attracted the attention of researchers and these approaches greatly assist the method development process. This review focuses on the application of computer-aided modelling tools in understanding the retention mechanism, the classification of HILIC stationary phases, prediction of retention times in HILIC systems, optimisation of chromatographic conditions, and description of the interaction effects of the chromatographic factors in HILIC separations. Additionally, what has been achieved in the potential application of QSRR methodology in combination with experimental design philosophy in the optimisation of chromatographic separation conditions in the HILIC method development process is communicated. Developing robust predictive QSRR models will undoubtedly facilitate more application of this chromatographic mode in a broader variety of research areas, significantly minimising cost and time of the experimental work.
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•Review of method development in hydrophilic interaction chromatography.•Focus on chemometric methods, particularly quantitative structure-retention relationships.•Use of computational methods for characterisation of stationary phases in hydrophilic interaction chromatography.•Application of quality by design to the optimisation of chromatographic separation in hydrophilic interaction chromatography.
A general approach for the manipulation of anion-exchange selectivity through derivatization of hydroxyl groups adjacent to quaternary anion-exchange sites with glycidol is described. Repetitive ...reactions with glycidol result in dramatic shifts in the retention of divalent anions. Unique selectivities are observed for specific divalent species resulting in shifts in elution order. Modification of anion-exchange materials with glycidol has a small effect on the selectivity of monovalent anions, but in some cases, significant shifts in selectivity are observed. Use of the synthetic approach for modification of commercially available ion-exchange materials is demonstrated.
•An overview of glycidol reaction chemistry with β-hydroxyl substituted quaternary anion-exchange sites is presented.•The effect of reaction temperature and glycidol concentration are investigated and optimized.•Unique anion exchange selectivity is demonstrated using a new synthetic route.•Manipulation of anion exchange selectivity through the use of successive glycidol reactions is demonstrated.
•Hybrid grafted and hyperbranched anion-exchangers based on EVB-DVB substrate.•Using N-vinylformamide grafting and hydrolysis to form amino polymer on the resin surface.•Controlled uniform crosslink ...distribution in the hyperbranched anion-exchange layer.•Unique selectivity as compared to electrostatically bonded hyperbranched phases.•Column capacity and selectivity control independent of hyperbranching process.
Novel grafted anion exchangers with covalently bonded hyperbranched functional layers were prepared and evaluated for the separation of monovalent standard inorganic anions and oxyhalides. Preparation of base coating included grafting highly polar N-vinylformamide to the ethylvinylbenzene-divinylbenzene (EVB-DVB) substrate surface in highly polar solvent (methanol) with subsequent hydrolysis of grafted amide polymer in basic media, which resulted in preparation of polymer chains with multiple primary amino groups. Those amino groups were used as attachment points for forming hyperbranched anion-exchange layers using 1,4-butanediol diglycidyl ether and primary mono- or diamine (methylamine or 1,3-diaminopropane, respectively). The effects of hyperbranching reaction cycles number on selectivity were evaluated which revealed that selectivity and capacity can be controlled independently for the covalently bonded stationary phases in contrast to electrostatically bonded phases. It was demonstrated that unlike for electrostatically bonded phases, the intentional increase of crosslink by using primary diamine instead of primary monoamine doesn't cause the shift of selectivity coefficients. It was also shown that crosslink distribution throughout the hyperbranched layer is an important factor determining selectivity of hyperbranched anion exchangers.
A new synthesis approach for the preparation of highly branched anion exchange materials utilizing diamine and diepoxide reagents is described. Unlike previously reported condensation polymers ...prepared from primary amine and diepoxide reagents, anion exchange polymers prepared from diamines and diepoxide reagents exhibit exceptionally low affinity for polyvalent ions. Use of anion-exchange materials synthesized utilizing this new synthetic method for the analysis of common inorganic anions is demonstrated.
•An investigation into the utility of diamines as the reagent for hyperbranched anion-exchange phases is presented.•The effect of two different diamines on anion-exchange selectivity are investigated.•Unique anion-exchange selectivity is demonstrated for polyvalent anions using diamine reagents.•The influence of anion concentration and cation type on retention is investigated.
Charge variant analysis is a widely used analytical tool in characterization of monoclonal antibodies (mAbs). It depicts the heterogeneity of charge variant forms, some of which may differ by only ...minor modifications of a single amino acid. The analysis ensures product consistency with no unwanted changes to the protein. With increasing numbers of new mAb drug products emerging in the market, the need for a robust charge variant analysis has intensified. The charge variant profiles often display partially resolved peaks on shoulders of larger peaks. This puts considerably more pressure on the robustness of the method to maintain the suboptimum selectivity. New products and techniques have emerged to address these requirements, in addition to the pre-existing older methods that may not have been optimized correctly in the past. This has led to some confusion as to the best approach and strategies in optimization of charge variant analysis. We show studies from several different approaches using on-line pH monitoring to check the performance characteristics of the methods. This has led to new insights on the interactions between the protein, column, and buffer constituents. We dispel some inaccurate assumptions about the different ion-exchange elution mechanisms and suggest ways to develop high-throughput methods that remain robust and of high resolution. Streamlined automatable method development tools are presented that will result in more efficient method optimization. The mechanisms behind poor chromatography design have provided an alternative explanation behind some methods failing when in the QC laboratories.
This study involved three commercial reversed-phase (RP)/anion-exchange (AEX)/cation-exchange (CEX) trimodal columns, namely Acclaim Trinity P1 (Thermo Fisher Scientific), Obelisc R (SIELC ...Technologies) and Scherzo SM-C18 (Imtakt). Their chromatographic properties were compared in details with respect to hydrophobicity, anion-exchange capacity, cation-exchange capacity, and selectivity, by studying retention behavior dependency on organic solvent, buffer concentration and pH. It was found that their remarkably different column chemistries resulted in distinctive chromatography properties. Trinity P1 exhibited strong anion-exchange and cation-exchange interactions but low RP retention while Scherzo SM-C18 showed strong reversed-phase retention with little cation-exchange and anion-exchange capacities. For Obelisc R, its reversed-phase capacity was weaker than Scherzo SM-C18 but slightly higher than Trinity P1, and its ion-exchange retentions were between Trinity P1 and Scherzo SM-C18. In addition, their difference in selectivity was demonstrated by examples of determining the active pharmaceutical ingredient (API) and counterion of drug products.
Characterization and quality control of biotherapeutic proteins commonly require the application of several orthogonal separation techniques in order to establish product identity and purity. Many of ...the techniques used rely on a buffered aqueous mobile phase system to maintain the native conformation of the protein and its variants. Optimal pH, buffer substance(s), and chromatography methods vary with each protein of interest and result in tedious method development for each new drug product. Linear controlled pH gradient systems from pH 5.6 to pH 10.2 has been shown to provide a global method for the separation of charge variants of monoclonal antibodies. This can be realized using two balanced zwitterionic buffer blends. The pH linearity of the resulting system, with a cation ion exchange column in place, can generate any pH value in this accessible pH range. This study expands the scope of this buffer system and demonstrates its application in conjunction with a quaternary HPLC pump for several analytical techniques: the pH optimization of salt gradient-based anion and cation exchange during method development, as well as performing pH gradient elution. In addition, the same universal buffers are used for hydrophobic interaction and size exclusion chromatography. This eluent system omits the need to prepare different buffers for each method and flushing of the HPLC system between method changes. The implementation of this concept is further demonstrated to allow an automated method scouting approach and selection of different methods that requires minimal manual intervention.
Preparation of columns using electrostatic attachment of anion exchange latex particles with charge density gradients is demonstrated. When such columns are oriented with the highest charge density ...at the column outlet, the chromatographic performance at low linear velocity is enhanced. When multiple successive charge density gradients are prepared along the length of the column with the highest capacity oriented at the inlet end of the column, significant improvement in chromatographic performance is observed during gradient elution chromatography.
Anion-exchange, Latex, Electrostatic attachment, Ion chromatography.
•QSRR is performed to accelerate the screening of five different stationary phases in HILIC method development.•QSRR is established based on DFT computed molecular descriptors utilizing a GA-PLS for ...variable selection.•GA descriptor sets are optimized to improve prediction accuracy.•QSRR is used to obtain some insight into the HILIC mechanism.
Quantitative structure-retention relationship (QSRR) models are developed to predict the retention times of analytes on five hydrophilic interaction liquid chromatography (HILIC) stationary phases (bare silica, amine, amide, diol and zwitterionic), with a view to selecting the most suitable stationary phase(s) for the separation of these analytes. The study was conducted using six β-adrenergic agonists as target analytes. Molecular descriptors were calculated based only on chemical structures optimized using density functional theory. A genetic algorithm (GA) was then used to select the most relevant molecular descriptors and these were used to build a retention model for each stationary phase using partial least squares (PLS) regression. This model was then used to predict the retention of the test set of target analytes. This process created an optimized descriptor set which enhanced the reliability of the developed QSRR models. Finally, the QSRR models developed in the work were utilized to provide some insight into the separation mechanisms operating in the HILIC mode. Three performance criteria – mean absolute error (MAE), root mean square error of prediction scaled to retention time (RMSEP), and the number of selected descriptors, were used to evaluate the developed models when applied to an external test set of six β-adrenergic agonists and showed highly predictive abilities. MAE values ranged from 13 to 25s on four of the stationary phases, with a somewhat higher error (50s) being observed for the zwitterionic phase. RMSEP values of 4.88–11.12% were recorded. Validation was performed through Y-randomization and chemical domain applicability, from which it was evident that the developed optimized GA-PLS models were robust. The high levels of accuracy, reliability and applicability of the models were to a large extent due to the optimization of the GA descriptor set and the presence of relevant structural and geometric molecular descriptors, together with descriptors based on important physicochemical properties, which establish a strong connection between retention time and meaningful chemical properties. The present strategy, while it is a pilot study, holds great promise for broader screening of HILIC stationary phases for desired separation, as well as for acquisition of information about molecular mechanisms of separation under chromatographic conditions.
The hydrophilic interaction liquid chromatography behavior of a novel trimodal stationary phase with reversed-phase/cation-exchange/anion-exchange characteristics was studied. This material is based ...on nanopolymer silica hybrid technology and provides reversed-phase, strong cation-exchange and weak anion-exchange properties at the same time. It is constructed with high-purity porous spherical silica particles coated with charged organic polymer nano-beads (dp ~100 nm). The inner-pore area of the silica gel is covalently modified with an organic layer that provides both reversed-phase and weak anion-exchange properties. The outer surface is modified with strong cation-exchange functionality. This chemistry ensures distinctive spatial separation of the anion-exchange and cation-exchange regions, which allows both retention mechanisms to function simultaneously and provides more flexibility in selectivity control. The new phase exhibits hydrophilic interaction liquid chromatography characteristics: at high acetonitrile levels, the retention for charged analytes is governed by an ion-exchange process, accompanied by hydrophilic interaction (partitioning mechanism). This feature provides good flexibility in pharmaceutical method development such as simultaneous separation of hydrophilic drug molecule and its counterions (e.g. penicillin G potassium salt).
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