Recent developments in supercritical fluid chromatography (SFC) have highlighted the applicability of this technique for the analysis of highly polar compounds. The combination of polar stationary ...phases and CO2-based mobile phases with an increasing presence of liquid co-solvent (up to 100%) has enabled to further expand the application field of SFC towards a variety of samples such as polar endogenous metabolites, plant extracts, water-soluble vitamins, pesticides, sugars, peptides and so on. In this evolution, a key role was played by the addition of up to 5–10% of water in the liquid co-solvent. Moreover, the presence of water enabled higher concentrations of additives, up to 75–100 mM in some cases. These improved conditions were fundamental in expanding the applicability range of SFC. Overall, SFC has demonstrated its evolution into a mature technique capable of offering a true alternative to liquid chromatography for the analysis of polar compounds.
•Systematic addition of water with increased concentration of additives have shown to greatly extend the polarity range of UHPSFC.•Higher percentages of co-solvent are more frequently employed in UHPSFC to analyze polar substances.•Risk of exceeding the system pressure limit when sub-2 μm stationary phases are used with a large presence of modifier.•Recent UHPSFC applications illustrate the potential of SFC as a complementary tool to UHPLC for highly polar analytes.
Supercritical fluid chromatography (SFC) has recently experienced renovated impulse from research groups. Its hyphenation to mass spectrometers (MS) proved to be of significant importance in ...catalysing interest from researchers. In contrast to liquid chromatography (LC), the coupling of SFC-MS requires the use of an interface in order to deal efficiently with the decompression of supercritical CO2 and possible precipitation issues of samples while entering the ionization chamber. The most common SFC-MS interfaces employ an additional sheath pump that reduces sample precipitation. However, there are still issues in dealing with the CO2 decompression phenomenon, with different solutions being given. Matrix effects (MEs) under SFC-MS have proved to be quite different from those generally observed in LC-MS, with ion suppression being the main form of ME. Nonetheless, SFC-MS is capable of reaching comparable sensitivity values to LC-MS, and in some cases performing even better. Several applications have been recently developed for SFC-MS, spacing from the analysis of plant extracts, biological matrices for anti-doping and forensic purposes, as well as highly polar compounds such as carbohydrates and endogenous metabolites.
•A detailed overview on SFC-MS interfaces is given, with emphasis on current issues and potential solutions.•Differences between LC-MS and SFC-MS in terms of matrix effects are highlighted.•Sensitivity in SFC-MS has been demonstrated to be comparable to LC-MS conditions.•Applications for SFC-MS are shifting towards the analysis of compounds with increasing polarity.
Ion mobility spectrometry is an analytical technique known for more than 100 years, which entails separating ions in the gas phase based on their size, shape, and charge. While ion mobility ...spectrometry alone can be useful for some applications (mostly security analysis for detecting certain classes of narcotics and explosives), it becomes even more powerful in combination with mass spectrometry and high‐performance liquid chromatography. Indeed, the limited resolving power of ion mobility spectrometry alone can be tackled when combining this analytical strategy with mass spectrometry or liquid chromatography with mass spectrometry. Over the last few years, the hyphenation of ion mobility spectrometry to mass spectrometry or liquid chromatography with mass spectrometry has attracted more and more interest, with significant progresses in both technical advances and pioneering applications. This review describes the theoretical background, available technologies, and future capabilities of these techniques. It also highlights a wide range of applications, from small molecules (natural products, metabolites, glycans, lipids) to large biomolecules (proteins, protein complexes, biopharmaceuticals, oligonucleotides).
In the past few years, there has been significant interest within the forensic community regarding the deployment of portable solutions that provide real‐time results. This article introduces an ...innovative technology or technology architecture that enables the integration of a handheld device, specifically, Viavi MicroNIR, with a cloud‐based system. This cloud system encompasses a server responsible for data processing and a mobile application acting as a user interface.
To demonstrate the transformative impact of this technology on field operators, the analysis of cannabis specimens has been utilized. System's capacity to distinguish between CBD‐type and THC‐type cannabis has been particularly highlighted, along with the remarkable congruence observed between the near‐infrared (NIR) spectra and the reference analytical method involving ultra‐high‐performance liquid chromatography (UHPLC)
The article will present the advantages of this application primarily focusing on its potential to alleviate the burden on laboratories by expediting routine illicit drug analysis. Viavi MicroNIR technology provides laboratory personnel with additional time to handle more complex cases, thereby enhancing overall efficiency.
•The theoretical aspects of IEX are overviewed.•The analytical conditions for performing modern IEX are reviewed.•The recent advances in IEX are discussed.•Several examples of IEX for ...biopharmaceutical applications are provided.
Ion-exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge heterogeneity. The goal of this review is to provide an overview of theoretical and practical aspects of modern IEX applied for the characterization of therapeutic proteins including monoclonal antibodies (Mabs) and antibody drug conjugates (ADCs). The section on method development describes how to select a suitable stationary phase chemistry and dimensions, the mobile phase conditions (pH, nature and concentration of salt), as well as the temperature and flow rate, considering proteins isoelectric point (pI). In addition, both salt-gradient and pH-gradient approaches were critically reviewed and benefits as well as limitations of these two strategies were provided. Finally, several applications, mostly from pharmaceutical industries, illustrate the potential of IEX for the characterization of charge variants of various types of biopharmaceutical products.
•The theoretical aspects of HIC are overviewed.•The analytical conditions for performing modern HIC are reviewed.•The recent advances in HIC are discussed.•Several examples of HIC for ...biopharmaceutical applications are provided.
Hydrophobic interaction chromatography (HIC) is a historical strategy used for the analytical purification and characterization of proteins. Similarly to what can be done in reversed-phase liquid chromatography (RPLC), HIC is able to separate protein species based on their hydrophobicity, but using different conditions. Compared to RPLC, the main benefit of HIC is its ability to perform separations under non denaturing conditions (i.e. physiological pH conditions, ambient mobile phase temperature and no need for organic solvents) and so an orthogonal method. The goal of this review is to provide a general overview of theoretical and practical aspects of modern HIC applied for the characterization of therapeutic protein biopharmaceuticals including monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) and bispecific antibodies (bsAbs). Therefore, method development approaches, state-of-the-art column technology, applications and future perspectives are described and critically discussed.
The transport of pyruvate, the end product of glycolysis, into mitochondria is an essential process that provides the organelle with a major oxidative fuel. Although the existence of a specific ...mitochondrial pyruvate carrier (MPC) has been anticipated, its molecular identity remained unknown. We report that MPC is a heterocomplex formed by two members of a family of previously uncharacterized membrane proteins that are conserved from yeast to mammals. Members of the MPC family were found in the inner mitochondrial membrane, and yeast mutants lacking MPC proteins showed severe defects in mitochondrial pyruvate uptake. Coexpression of mouse MPC1 and MPC2 in Lactococcus lactis promoted transport of pyruvate across the membrane. These observations firmly establish these proteins as essential components of the MPC.
•The analytical conditions for performing modern size exclusion chromatography (SEC) are described.•The detectors that can be employed in SEC are critically reviewed.•The recent advances in SEC are ...discussed.•Possible pitfalls of fast SEC.•SEC for biopharmaceutical applications.
Size exclusion chromatography (SEC) is a historical technique widely employed for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of aggregates. The main advantage of this approach is the mild mobile phase conditions that permit the characterization of proteins with minimal impact on the conformational structure and local environment. Despite the fact that the chromatographic behavior and peak shape are hardly predictable in SEC, some generic rules can be applied for SEC method development, which are described in this review.
During recent years, some improvements were introduced to conventional SEC that will also be discussed. Of these new SEC characteristics, we discuss (i) the commercialization of shorter and narrower columns packed with reduced particle sizes allowing an improvement in the resolution and throughput; (ii) the possibility of combining SEC with various detectors, including refractive index (RI), ultraviolet (UV), multi-angle laser light scattering (MALLS) and viscometer (IV), for extensive characterization of protein samples and (iii) the possibility of hyphenating SEC with mass spectrometry (MS) detectors using an adapted mobile phase containing a small proportion of organic modifiers and ion-pairing reagents.
Mass spectrometry (MS) is considered today as one of the most popular detection methods, due to its high selectivity and sensitivity. In particular, this detector has become the gold standard for the ...analysis of complex mixtures such as biological samples. The first successful SFC-MS hyphenation was reported in the 80’s, and since then, several ionization sources, mass analyzers and interfacing technologies have been combined. Due to the specific physicochemical properties and compressibility of the SFC mobile phase, directing the column effluent into the ionization source is more challenging than in LC. Therefore, some specific interfaces have to be employed in SFC-MS, to i) avoid (or at least limit) analytes precipitation due to CO2 decompression, when the SFC mobile phase is not anymore under backpressure control, ii) achieve adequate ionization yield, even with a low proportion of MeOH in the mobile phase and iii) preserve the chromatographic integrity (i.e. maintaining retention, selectivity, and efficiency). The goal of this review is to describe the various SFC-MS interfaces and highlight the most favorable ones in terms of reliability, flexibility, sensitivity and user-friendliness.
•The five different interfaces for coupling SFC and MS are presented.•These interfaces were compared in terms of reliability, flexibility, sensitivity and user-friendliness.•The amount of co-solvent entering MS was calculated for various conditions.•The expected changes in sensitivity using ESI and APCI sources were critically discussed.•The achievable sensitivities in SFC-MS and LC-MS were compared.
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