Szabolcs Fekete

High performance liquid chromatography (HPLC) is a widely used and well-established technique, routinely employed by thousands of analytical scientists worldwide. Nonetheless, certain challenges—arising from the complex interplay of multiple factors affecting peak retention and separation—persist. In this context, multidimensional modeling approaches can provide valuable support.

Hydrophilic interaction liquid chromatography (HILIC) has emerged as a promising alternative to traditional ion-pair reversed phase liquid chromatography (IP-RPLC) methods for separating oligonucleotides (ON). This work investigates the application of HILIC to the separation of ON sequence and length variants, duplexes, and single-stranded components.

This article explores three case studies where autosampler injection programs help to (i) reduce the carry-over observed for large nucleic acids during anion-exchange chromatography (AEX), (ii) reduce peak distortion and breakthrough for monoclonal antibodies (mAbs) during hydrophilic-interaction chromatography (HILIC), and (iii) facilitate dissolution of lipid nanoparticles (LNPs) so that a size-exclusion chromatography (SEC) method can be applied for ribonucleic acid (RNA) payload analysis.

The purpose of this short tutorial article is to review the terms and official nomenclatures for size-exclusion separations and to provide some guidance and recommendations for practicing chromatographers. The interconversion between the different metrics is explained and some examples are presented.

Serial coupling of different column types can provide several important benefits to the chromatographer, including fine-tuning selectivity to separate complex mixtures.

Pressure-enhanced liquid chromatography (PE-LC) offers a new approach for improving selectivity for large molecule separations. Examples shown here include short oligonucleotides in ion-pairing reversed-phase (IP-RP) liquid chromatography and larger nucleic acids in ion-exchange (IEX) chromatography.

Multi-isocratic elution mode is a powerful chromatographic approach for characterizing minor isoforms of monoclonal antibodies.

There is a need to use more descriptive and precise terms to describe some of the properties of the products used for biopharmaceutical analysis. It is probably more correct to say “low adsorption” and “corrosion-resistant” systems.

In the second part of this review of the current state of HIC, some practical considerations are explained, including method development, selection of the phase system, combined salt systems, and possibilities to combine HIC with other chromatographic modes.

A review of the current state of HIC, focusing on retention and separation mechanisms with the aim of developing more robust methods. Can HIC be considered as a non-denaturing and non-destructive technique with advantages for protein analysis?

The impact of ionic strength, buffer capacity, and pH-response on the retention behavior and peak shape of mAb species characterization is evaluated for IEX-MS. The aim of the present study was to understand the impact of ionic strength, buffer capacity, and pH-response on the retention behavior and peak shape of mAb species.

Characterization of mAbs and related products requires the identification of chromatographic peaks with MS. However, the conventional salt- and pH-gradient elution techniques used in IEX are inherently incompatible with MS. Ammonium acetate- and ammonium carbonate-based mobile phase systems have been recently applied in IEX-MS, but the influence of the eluent composition on peak shape and retention has not been discussed nor studied systematically until now. The aim of the present study was to understand the impact of ionic strength, buffer capacity, and pH-response on the retention behaviour and peak shape of mAb species.

Several new materials and columns have been introduced in recent years for size-exclusion separations of proteins. How do I know which one to choose, and which separation conditions will be the best for my protein separation?

Much of the conventional wisdom regarding size-phase separations of proteins has been negated thanks to development of superior chemistries and advances in research. In this article, details that the authors have found to be especially beneficial in achieving effective SEC separations are examined.

Several new materials and columns have been introduced in recent years for size-exclusion separations of proteins. How do I know which one to choose, and which separation conditions will be the best for my protein separation?

Several new materials and columns have been introduced in recent years for reversed-phase separations of proteins. How do I know which one to choose, and which separation conditions will be best for my protein separation?

Several new materials and columns have been introduced in recent years for reversed-phase separations of proteins. How do I know which one to choose, and which separation conditions will be best for my protein separation?

For reversed-phase separations of proteins, you must consider pore size,column temperature, and stationary-phase chemistry. Here are some guidelines.

The aim of this article is to illustrate the current status of computer-assisted method development and retention modelling. This study focuses on the successful method development of typical small pharmaceutical compounds (impurity profiling) and large therapeutic proteins. By choosing appropriate initial conditions, the method development can be performed in less than one day. However, for small molecules possessing different physicochemical properties, the conditions can be multifarious, while for biopharmaceuticals (for example, monoclonal antibodies [mAbs], antibody–drug conjugates [ADCs]), a generic method can easily be developed. In addition to retention modelling and optimization, the potential of simulated robustness testing is also demonstrated. Depending on the applied retention model, the impact of any change among six experimental parameters (tG, T, pH, ternary composition, flow rate, and initial- and final mobile phase compositions) on the separation can be assessed using a 26 or 36 type virtual

The recent trends in column technology for reversed-phase LC, SEC, ion-exchange chromatography, and HIC for analysis of biopharmaceuticals are critically discussed.

The recent trends in column technology for reversed-phase LC, SEC, IEX, and HIC for analysis of biopharmaceuticals at the protein level is critically discussed.

Modern Column Technologies for the Analytical Characterization of Biopharmaceuticals in Various Liquid Chromatographic Modes

Experimental results and method validation data from two analytical case studies of complex small?molecule pharmaceuticals illustrates the utility and advantages of UHPLC in high-resolution separations.

Experimental results and method validation data from two case studies of complex small-molecule pharmaceuticals illustrate the utility and advantages of UHPLC for quality control.

This review provides an updated overview of the theory behind the success of SPP technology, trends, benefits, and limitations. It also summarizes the latest developments of sub-2-?m SPPs and instrumental constraints associated with their use.

An updated overview of the development of SPP technology, including benefits, limitations, and the theory behind its success

Two promising wide-pore stationary phases were recently introduced for the fast and high resolution separations of large biomoleculaes: the recently launched widepore 3.6?m superficially porous particle and the hybrid-type sub 2-?m fully porous particle. This article focuses on the achievable throughout and resolution for the characterization of biomolecules.

Two promising wide-pore stationary phases were recently introduced for the fast and high-resolution separations of large biomolecules: the recently launched wide pore 3.6 ?m superficially porous particle and the hybrid-type sub-2-?m fully porous particle. This article focuses on the achievable throughput and resolution for the characterization of biomolecules.