Hydrophobic interaction chromatography (HIC) is considered a native LC mode complementary to reversed-phase liquid chromatography, where biomolecules largely maintain their 3D protein conformation. In HIC, protein adsorption on a mildly hydrophobic stationary phase is modulated via an inverse salt gradient. The impact of various salt types on HIC retention is discussed, and the potential of ternary mobile phases for improved selectivity is examined. The most commonly-used HIC stationary phases are highlighted and the effect of particle diameter on resolving power is critically discussed. Emerging approaches for the direct coupling of HIC to electrospray ionization (ESI)-MS detection are demonstrated and discussed as well as the integration of HIC into a multi-dimensional LC set-up.

Ion Chromatography (IC), the gold standard for the separation and analysis of ionic species, has been extensively used for detection of ions in industrial applications. Pharmaceutical samples with limited water solubility often require the use of organic solvents as diluents, which can pose significant challenges on IC analysis. Herein, the effects of various organic solvents as sample diluents in IC for anion analysis are systematically investigated. Electrochemically stable solvents such as 2-propanol (IPA), acetone, and dimethyl sulfoxide (DMSO) were found to have the least impact on the IC baseline. For solvents with a greater impact, a solvent dilution device (SDD) has been designed to minimize their effects. The device effectively diluted the organic solvents before they reached the suppressor electrode, reducing the generation of oxidized products. The results showed that the baseline stability and separation efficiency of IC can be maintained when using a wide range of organic solvents as diluents. Notably, the use of a solvent dilution device enabled good recovery of ions in various active pharmaceutical ingredients and significantly enhanced the signal to noise ratio of early-eluting ions compared to analyses conducted without the device. Guidelines are proposed to minimize the impact of organic solvents in IC sample preparation and anion analysis. The study also investigates the origin of impurity peaks introduced by the organic solvents and demonstrated the effectiveness of an ion-exchange resin treatment for their removal. Overall, this study provides insights for the use of organic solvents as IC diluents and offers practical solutions to overcome their challenges in pharmaceutical applications.

Ion Chromatography (IC) is one of the most widely used methods for analyzing ionic species in pharmaceutical samples. A universal IC method that can separate a wide range of different analytes is highly desired as it can save a lot of time for method development and validation processes. Herein we report the development of a universal method for anions in active pharmaceutical ingredients (APIs) using computer-assisted chromatography modeling tools. We have screened three different IC columns (Dionex IonPac AS28-Fast 4 µm, AS19 4 µm and AS11-HC 4 µm) to determine the best suitable column for universal IC method development. A universal IC method was then developed using an AS11-HC 4 µm column to separate 31 most common anionic substances in 36 mins. This method was optimized using LC Simulator and a model which precisely predicts the retention behavior of 31 anions was established. This model demonstrated an excellent match between predicted and experimental analyte retention time (R2 =0.999). To validate this universal IC method, we have studied the stability of sulfite and sulfide analytes in ambient conditions. The method was then validated for a subset of 29 anions using water and organic solvent/water binary solvents as diluents for commercial APIs. This universal IC method provides an efficient and simple way to separate and analyze common anions in APIs. In addition, the method development process combined with LC simulator modeling can be effectively used as a starting point during method development for other ions beyond those investigated in this study.

This study presents a comprehensive investigation of the mechanistic understanding of retention and selectivity in hydrophobic interaction chromatography. It provides valuable insights into crucial method-development parameters involved in achieving chromatographic resolution for profiling molecular variants of trastuzumab. Retention characteristics have been assessed for three column chemistries, i.e., butyl, alkylamide, and long-stranded multialkylamide ligands, while distinguishing column hydrophobicity and surface area. Salt type and specifically chloride ions proved to be the key driver for improving chromatographic selectivity, and this was attributed to the spatial distribution of ions at the protein surface, which is ion-specific. The effect was notably more pronounced on the multialkylamide column, as proteins intercalated between the multiamide polymer strands, enabling steric effects. Column coupling proved to be an effective approach for maximizing resolution between molecular variants present in the trastuzumab reference sample and trastuzumab variants induced by forced oxidation. Liquid chromatography-mass spectrometry (LC-MS)/MS peptide mapping experiments after fraction collection indicate that the presence of chloride in the mobile phase enables the selectivity of site-specific deamidation (N30) situated at the heavy chain. Moreover, site-specific oxidation of peptides (M255, W420, and M431) was observed for peptides situated at the Fc region close to the CH2-CH3 interface, previously reported to activate unfolding of trastuzumab, increasing the accessible surface area and hence resulting in an increase in chromatographic retention.

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.

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.

This chapter provides a comprehensive overview of ion chromatography (IC) columns developed first at Dionex and then later at Thermo Fisher Scientific. The chapter includes a discussion of virtually all IC columns introduced at Dionex, including columns for the separation of inorganic anions, organic acids, inorganic cations, amines, carbohydrates, and amino acids. While most of the columns described are either anion-exchange columns or cation-exchange columns, the chapter also covers mixed-mode, ion exclusion, and ion pair stationary phases. Older columns are described to provide context for the development of newer phases even though most of the oldest columns are no longer in wide use. The chapter also discusses the mechanism and practice of both ion-exclusion chromatography and ion pair chromatography. The chapter ends with a discussion of methods for cleaning columns that have become contaminated.

To date, few tools are available for the analysis of the glycome without derivatization, a process which is known to introduce issues such as differential loss of sialic acid and incomplete labeling. We have previously reported the use of ion chromatography-mass spectrometry (IC-MS) to analyze native sialylated and sulfated glycans. Here, we introduce improvements to IC column technology, enabling the separation of neutral glycans while maintaining charge separation capabilities. When implemented in an IC-MS workflow, this enables the structural characterization of a broad array of chemically distinct glycans. With the newly developed IC column and modified IC-MS instrumentation configuration, we qualitatively investigated O-glycome profiles in bovine fetuin and porcine gastric mucins. The improved chromatographic resolution in combination with high-resolution MS data present a powerful tool for glycan structural identification.

This chapter provides a historical overview of the first 25 years of commercial development of ion chromatography (IC) columns and suppressors at Dionex Corporation. This includes a discussion of the major technology trends in stationary phases, suppressor technology, and the evolution of mobile phases in IC. Many of the most important trends in IC, such as the development of hydroxide selective columns and hydronium selective columns, began during the first 25 years of ion chromatography. Likewise, the transition from packed-bed suppressors to first fiber suppressors and then to membrane suppressors began during the early years of ion chromatography. The chapter also provides a backdrop for understanding these trends and how they have influenced the development of modern IC instruments and consumables to this day.

A new analytical method was developed to extract and determine 25 perfluorinated and polyfluorinated alkyl substances from aqueous samples using an automated solid phase extraction system and liquid chromatography-tandem mass spectrometry. Full method validation including assessment of background signal, lowest concentration minimum reporting level, calibration, limit of quantitation, precision, and accuracy was performed. The analysis of drinking water samples was performed for all target analytes. This method also incorporates the use of isotope dilution in which isotopically labeled analogs of the target analytes are added to the sample prior to extraction in a known amount to correct for sample matrix interference and improve data quality. We demonstrated that the automated solid-phase extraction system provided reliable extraction of the target perfluorinated and polyfluorinated alkyl substances analytes from large- of use, reduced human errors, avoidance of massive lab contaminations and saving up to 50% operational time to manual operation while ensuring high reproducibility and productivity for analytical testing laboratories.

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.

Ion chromatography-electrospray tandem mass spectrometry (IC-ESI-MS/MS) is used to determine nine haloacetic acids (HAAs), bromate, and dalapon in drinking water samples in U.S. EPA Method 557. In this method, all target analytes are separated and measured with good sensitivity without the need for sample preconcentration or derivatization. However, the separation time is relatively long. In order to reduce the sample analysis time in EPA Method 557, a new anion exchange column has been developed to perform fast separation of the target analytes. Using this new anion exchange column, nine HAAs, bromate, and dalapon can be resolved and separated from interfering matrix ions within 40 minutes, about 33% faster than the analysis time obtained using an earlier anion exchange column reported in EPA Method 557. The new anion exchange column has unique selectivity and high exchange capacity. Method optimization, simplification and improvements in robustness are demonstrated while validating the new column suitability for the determine of HAAs, bromate and dalapon according to EPA Method 557.

The hydrophobic subtraction model (HSM) combined with quantitative structure-retention relationships (QSRR) methodology was utilized to predict retention times in reversed-phase liquid chromatography (RPLC). A selection of new analytes and new RPLC columns that had never been used in the QSRR modeling process were used to verify the proposed approach. This work is designed to facilitate early prediction of co-elution of analytes in pharmaceutical drug discovery applications where it is advantageous to predict whether impurities might be co-eluted with the active drug component. The QSRR models were constructed through partial least squares regression combined with a genetic algorithm (GA-PLS) which was employed as a feature selection method to choose the most informative molecular descriptors calculated using VolSurf+ software. The analyte hydrophobicity coefficient of the HSM was predicted for subsequent calculation of retention. Clustering approaches based on the local compound type and the local second dominant interaction were investigated to select the most appropriate training set of analytes from a larger database. Predicted retention times of five new compounds on five new RPLC C18 columns were compared with their measured retention times with percentage root-mean-square errors of 15.4 and 24.7 for the local compound type and local second dominant interaction clustering methods, respectively.

The use of ultrahigh pressures in combination with columns packed with 2.5 μm microporous and supermacroporous (perfusive) stationary phase particles coated with nanobeads has been successfully explored in ion chromatography with online eluent generation and suppressed conductivity detection. Isocratic separations of inorganic anions and organic acids yielding reduced plate heights as low as 2.1 were achieved, corresponding to efficiencies up to 190000 plates/m, using an optimized system configuration with respect to injection parameters, considering volume and mass loadability, and extra-column dispersion. Viscous-heating effects have been assessed for PEEK-lined stainless steel columns operated at 70 MPa, and effects of thermal gradients on separation efficiency and retention are demonstrated. Whereas the PEEK-lined column hardware acts to some extent as an insulator, a 10% increase in plate number could be obtained when applying a still-air column oven configuration. In the forced-air mode, an increase in retention was observed for polyvalent ions. Finally, the kinetic performance limits of ultrahigh-pressure ion chromatography applying 2.5 μm particle-packed columns operated at 70 MPa were compared to conventional ion-chromatography technology using columns packed with 4 μm particles operated at a maximum pressure of 35 MPa. Downscaling the particle size and increasing the operating pressure led to a maximum time gain with a factor of 3.4, without compromising separation efficiency (N = 10000).

Quantitative structure-retention relationships (QSRR) predicting the values of solute "hydrophobicity" coefficient η' in the approximate hydrophobic subtraction model (HSM) can be used to predict retention times of compounds on numerous reversed-phase (RP) columns, provided that column parameters on the corresponding stationary phases are available. In the present study, we propose a new dual clustering-based localised QSRR approach, combining P-ratio clustering (where P is the octanol-water partition coefficient) with second dominant interaction (SDI)-based clustering, to produce predictive models with an acceptable level of prediction accuracy for in silico column scoping in RP method development. QSRR models for η' values were derived for 49 compounds out of 63 in a dataset extracted from the literature, where retention data were measured under one isocratic mobile phase condition (i.e., acetonitrile-water, 50:50 [v/v]). These models gave a predictive squared correlation coefficient Qext(F2)2 of 0.83 and a root mean square error of prediction (RMSEP) of 0.14. For the modelling, a genetic algorithm-partial least square regression (GA-PLS) approach was performed using the η' values and their relevant molecular descriptors. The corresponding retention times were predicted by applying the predicted η' values of the models and the stationary phase "hydrophobicity" parameter H values for the corresponding columns to the approximate HSM, resulting in excellent accuracy and predictability (Qext(F2)2 of 0.90 and RMSEP of 0.72 min). The established QSRR approach was experimentally verified for six Thermo Scientific columns (Acclaim™ 120 C18, Acclaim Polar Advantage, Acclaim Polar Advantage II, Accucore™ aQ, Accucore Phenyl-X, and Hypersil Gold C18 columns) using two types of datasets. The first dataset consisted of eight model compounds extracted from the original dataset and retention time predictions for those compounds were then evaluated on the above columns. The result showed good agreement between predicted and observed retention times with an acceptable error in retention time predictions (slope of 0.97, Qext(F2)2 of 0.95, a mean absolute error (MAE) of 0.43 min and RMSEP of 0.61 min). The second dataset included eight test compounds not included in the original dataset, which were all classified into the η' cluster by applying a Tanimoto similarity (TS) threshold of 0.7. Similarly, predicted retention times of the test compounds were compared with their corresponding observed retention times, resulting in acceptable retention time predictions with the slope of 0.99, Qext(F2)2 of 0.93 and RMSEP of 0.52 min. Comparisons of resolution values between columns were utilised to select the most suitable columns for separations of the compounds in the respective test sets. Actual chromatograms obtained on the chosen columns showed the feasibility for effective column scoping without experimentation on numerous RP stationary phases available in the USP website, based on the predicted resolution values.

The research on oligosaccharides is growing and gaining in importance at a rapid pace. The efforts to understand their bioactivity and to develop new products based on oligosaccharides in biotherapeutics and food industry require effective and reliable tools for analysis of oligosaccharides. Here we present a dual electrolytic eluent generation platform for the analysis of oligosaccharides by high-performance anion-exchange liquid chromatography (HPAE) in both analytical and capillary column formats. The system consists of one eluent generator producing methanesulfonic acid (MSA) connected in series with a second eluent generator producing potassium hydroxide (KOH). Through manipulating the concentration output of both eluent generators, chromatographic performance comparable to that obtained using the conventional sodium acetate/sodium hydroxide (NaOAc/NaOH) eluents is achieved using the electrolytically generated potassium methanesulfonate/potassium hydroxide (KMSA/KOH) eluent. This platform utilizes deionized water as the only carrier stream through a single isocratic pump, overcomes the various drawbacks associated with manually prepared NaOAc/NaOH eluents, and offers an easy to use, simplified operation solution for oligosaccharides profiling with increased precision and accuracy.

Quantitative Structure-Retention Relationship (QSRR) methodology is a useful tool in chromatography of all kinds, allowing the prediction of analyte retention time and providing insight into the mechanisms of separation. The prediction of retention is useful in reducing method development time and identifying analytes in Non-Targeted Analysis. The varying methods used for geometry optimization, descriptor calculation, feature selection, and model generation in many different QSRR settings are investigated and compared. It is found that the method of geometry optimization and descriptor selection is of less importance than the chromatographic similarity of compounds in the training sets used for model building in order to reduce the error of the model.

Carbonate and bicarbonate based eluents have been applied for ion analysis from the inception of ion chromatography. The product of suppression with carbonate and/or bicarbonate eluent is carbonic acid which is weakly dissociated and tends to outgas. While the act of suppression enhanced the signal for fully dissociated ions and lowered the background to a weakly dissociated level, the overall noise performance, however, varied depending on the suppression mechanism. Chemical suppression with a membrane suppressor yielded low noise performance with carbonate and/or bicarbonate eluents. Electrolytic suppression, on the other hand, resulted in a relatively higher noise with carbonate based eluents when compared to chemical suppression. In this work, we investigated the root cause of noise with electrolytic suppressors and carbonate based eluents. Further, a new electrolytic suppressor design based on a three-electrode design is discussed in this paper and provided low noise performance with carbonate and/or bicarbonate eluents.