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Liquid chromatography (LC) is the single largest analytical field in terms of people involved and money spent. LC is crucial for almost all public and private sectors and the technique has seen tremendous technological advancements. Nevertheless, separations are often performed under suboptimal conditions and technological capabilities remain unused. Because expert knowledge and method development time are increasingly scarce, methods are often inefficient. Exploiting the full technological capabilities of liquid-phase separation technology requires deep knowledge and great time investments. Method optimization strategies that can simultaneously optimize the large number of parameters involved are therefore of great interest to chromatographers. This review examines different workflows that have been designed and used to facilitate and/or automate method development. In particular, focus is paid to the implementation of computer-aided workflows for the optimization of kinetic and thermodynamic parameters in LC, as well as on the possibilities to conduct this in a closed-loop fashion. Finally, the opportunities to use machine learning to achieve these goals is addressed. [ABSTRACT FROM AUTHOR]

Commercialization of columns that provide multiple modes of chromatographic separations have recently been on the rise. For example, combinations of retention modes such as ion-exchange and reversed-phase, often enable the separation of complex mixtures of analytes not possible using single-mode columns. In this work, recent trends in what is often referred to as "mixed-mode" phase are investigated. Particular attention is made to recent fundamental research, stationary phase development and design, and areas of application. [ABSTRACT FROM AUTHOR]

The article focuses on the International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-18), scheduled for May 28—31st, 2024, in Leuven, Belgium, serving as a significant meeting point for scientists and practitioners to exchange knowledge.

The challenges we face in troubleshooting problems with liquid chromatography (LC) separations are highly diverse. This month we take a closer look at topics that have garnered more attention recently. [ABSTRACT FROM AUTHOR]

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?. [ABSTRACT FROM AUTHOR]

The quality of a gas chromatogram depends heavily on the quality of the separation and detection gases, among many other factors. In this month's instalment, "GC Connections" discusses ways in which chromatographers can ensure a safe working environment while delivering gases that are up to the requirements of the separations at hand, in the context of moving a laboratory to a new location. [ABSTRACT FROM AUTHOR]

Column-based liquid phase separation techniques, such as liquid chromatography (LC) in reversed phase separation mode and capillary electromigration techniques, using continuous electrolyte systems, are widely used for the identifi cation and quantifi cation of phenolic compounds in plants and food matrices of plant origin. This paper is the fi rst of a two-part review article discussing fundamental and practical aspects of both LC and capillary electromigration techniques used for the analysis of phenolic compounds occurring in plant-derived food and in edible and medicinal plants. The chemical structure and distribution of the major phenolic compounds occurring in the plant kingdom, as well as the main methods used for their extraction and sample preparation, are also discussed. Part 1 will focus on liquid chromatography. [ABSTRACT FROM AUTHOR]

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? [ABSTRACT FROM AUTHOR]

Comprehensive two-dimensional gas chromatography (GC x GC) offers significant improvement for volatile chemical separation. Selecting suitable first (¹D) and second dimension (²D) columns normally requires consideration of the chemical composition of a sample. Replacing one of these dimensions with a two-column ensemble (for example, ¹D1 + ¹D2 for the ¹D column), provided with a pressure tuning makeup gas between them, varies the relative retentions of compounds before the modulation step according to the junction pressure. This makes it possible to alter the apparent polarity of the ¹D ensemble, and this alters peak positions in the 2D GC x GC space. This article presents an account of studies that suggest this offers potential for improved operation for a GC x GC laboratory. [ABSTRACT FROM AUTHOR]

The use of superfi cially porous particles (SPPs) for modern high performance liquid chromatography (HPLC) is now very common. Initially, SPPs rose as an alternative to sub-2-µm fully porous particles (FPPs). In recent years, many column manufacturers have developed 2-µm and smaller SPP-based products. This article investigates the practical utility of these smaller SPP designs. [ABSTRACT FROM AUTHOR]

Compared to the progress made in reversed phase separations in terms of speed and efficiency, chiral chromatographers have traditionally focused on improving the selectivity of enantiomeric separations by synthetic procedures. As a result, more than 50 different types of advanced chiral stationary phase chemistries are available today. Traditionally, speed and efficiency of chiral chromatography has not received attention until recently. With the availability of superfi cially porous particles and sub-2-μm particles, sub-minute enantiomeric separations have been achieved with the help of improved particle technology with narrow size distribution, and systematic studies on packing columns. This article covers advances made in the fi eld of ultrafast chiral chromatography in the last decade. The development of instrumentation technology has also contributed immensely to making sub-second chiral separations a reality. Enantiomeric separations can now compete with the speed of sensors. Future directions and unanswered questions in the fi eld of ultrafast enantiomeric separations are highlighted. [ABSTRACT FROM AUTHOR]

Comprehensive two-dimensional liquid chromatography (LC×LC) is a powerful technique for separating highly complex samples. However, the proliferation of this technique is hindered by a range of challenges, including the possible impact of the additional separation on the detection sensitivity, concerns that mobile phase incompatibility problems will limit the applicability, and the complexity of the system and associated method development costs. This article addresses these issues and describes how modern modulators and software tools are overcoming the barriers associated with this technique. [ABSTRACT FROM AUTHOR]

LC practitioners are faced with an overwhelming number of choices for fittings that can be used to connect the components of LC systems. Users must ultimately decide which fitting technology offers the best compromise of performance and cost for each particular situation. For dedicated LC systems where system reconfiguration is infrequent-and especially for connections between instrument components excluding the column- traditional metal, ferrule-based fittings usually offer the best compromise of cost, reliability, and quality of connections. For connections to columns, particularly when changing columns frequently, reusable fittings that do not involve swaging of a metal ferrule to the capillary are very convenient. Technologies introduced by several manufacturers over the past few years have improved the design of these reusable fittings, and now enable repeated connections of high quality and at very high pressures using a single fitting. These technologies come at increased costs, but the performance gains and ease of use they provide will outweigh the costs in many practical situations. [ABSTRACT FROM AUTHOR]

The article looks on the alternatives for liquid chromatography columns. It highlights the development of hydrophilic-interaction chromatography (HILIC) columns and reversed-phase analysis of large molecules. An overview on the procedures complementary to reversed-phase mode to ion-exchange chromatography, is also mentioned.

Chromatographic method development for pharmaceutical analysis can benefit from in silico steered serial coupling of column segments containing different stationary phases of varying length. Contrary to column coupling through trial and error, in stationary-phase optimized selectivity (SOS)-based chromatography the retention of all solutes is predicted for all possible column combinations allowing a rational selection of the optimal column combination. The possibilities of the strategy now surpass the initial usage in isocratic high performance liquid chromatography (HPLC) on dedicated commercial column segments, and allow applications in gradient-, green-, preparative-, and in supercritical fluid chromatography (SFC) on conventional column hardware. Current possibilities, pharmaceutical applications, a downloadable algorithm, and weaknesses of the approach are discussed to allow broader implementation of this methodology in separation science. [ABSTRACT FROM AUTHOR]

The article focuses on the development in the liquid chromatography instruments with the introduction of fully porous particles rather than the old columns. It discusses how the ultra high-performance liquid chromatography (UHPLC) should be improved with separation quality and operating pressure, and analyze the separation performance while applying kinetic plot methodology.

The article focuses on the development of fast gas chromatography (GC) and its significance in the separation technology. It discusses the difference between fast GC and high speed separations, the use of GC simulation software including calculations needed for electronic pressure control and compound retention behavior.

This instalment in our series on ultrahigh-pressure liquid chromatography (UHPLC) highlights its benefits in fast analysis, high-resolution separations, high performance liquid chromatography (HPLC) method development, reduced solvent and sample usage, and enhanced sensitivity and precision performance. [ABSTRACT FROM AUTHOR]

Dual flow chromatography (DFC) separations are performed with back and forth flow for rapid method development, design of experiments (DOE), quality-by-design (QbD), or high-throughput chromatographic purification. Although different than conventional unidirectional flow through chromatography, chromatographic principles still control the separations. Selectivity coefficients and Langmuir adsorption isotherms control the separation chemistry properties of the column and dictate the mobile phase conditions needed to achieve separation. However, the kinetic rates of diffusion and interaction of mobile phase molecules with the stationary phase, column channeling, and other column properties are not germane to the practice of DFC. Chromatographic conditions developed with DFC can be scaled to any size, including laboratory and industrial preparative columns. One practical use of DFC is in the pharmaceutical industry where multidimensional method development and high-throughput, parallel processing has been used to improve R&D productivity and decrease the time to manufacturing of biopharmaceutical drugs. [ABSTRACT FROM AUTHOR]

The article offers information on a study on the comparison of the separation speed of gas chromatography (GC), liquid chromatography (LC), and supercritical fluid chromatography (SFC). Topics discussed include using the kinetic plot method neglecting differences in viscous heating or extra-column band broadening; obtaining fixed length kinetic plot curves; and role of thermodynamic resistance factor in LC.

Comprehensive two-dimensional liquid chromatography (LC×LC) is evolving and becoming more commonly used in practice, but there are some specific problems still present that hamper the widespread use of this technology. One key aspect is the coupling of an on-line LC×LC system to a mass spectrometer. Generally, on-line LC×LC is based on a very fast second dimension separation to achieve low cycle times. This often results in flow rates that are far above the optimum for electrospray ionization mass spectrometry (ESI-MS). This month's "Multidimensional Matters" looks at the benefits of miniaturization in the first and second dimension for coupling with a high-resolution mass spectrometer (HRMS) and describes an environmental analysis application. [ABSTRACT FROM AUTHOR]

The article focuses on using the chromatography fundamentals on liquid chromatography (LC) separations in order to increase resolution (R s). Topics discussed include retention factor (k) influences on resolution, increase in K due to increase in retention time, and effect of percentage change in organic content of the mobile phase to find optimized separation.

Quality by design (QbD) has gained in importance in the pharmaceutical industry and is supported by several regulatory documents (ICH, FDA). The aim is to ensure product quality through a better understanding of products and processes during pharmaceutical development. As analytical procedures are critical processes of pharmaceutical product development and quality control (QC), QbD has become a key tool in the development of analytical methods. This article outlines the general trends observed when applying QbD to the development of separation methods in pharmaceutical analysis. The main pharmaceutical applications are reviewed along with a detailed description of tools involved in QbD methodology. A focus on QbD benefits for the pharmaceutical industry is provided. [ABSTRACT FROM AUTHOR]

Sample preparation techniques in bioanalysis are multistep, time-consuming, and labour-intensive procedures that can take up 60-80% of the total analysis time. Sample preparation is often the limiting step of fast bioanalysis and the most error-prone part of the analytical method. There is currently a focus on improving the sample preparation process by shortening sample preparation time, cutting the cost of analysis, decreasing sample volume and solvent consumption, reducing the number of sample preparation steps, and adapting the whole process for automation. This article explores microextraction techniques, selective approaches, on-line sample preparation, and dried matrix spots that aim to provide solutions to sample preparation problems in bioanalysis. [ABSTRACT FROM AUTHOR]

Cartenoid compounds can be used as probes for studying bonded stationary phases for reversed-phase liquid chromatography, such as C18, phenyl-hexyl, and cholesteryl. From one supercritical fluid chromatography (SFC) analysis that favours the chromatographic behaviours related to the stationary phase properties, bonding density, ligand type (monomeric or polymeric), and endcapping treatment, two separation factors are calculated allowing us to build a two-dimensional map. These two axes are related either to the shape selectivity or the polar surface activity (residual silanols). Each point on the map corresponds to a column. The retention factor of (3-carotene, which describes the phase hydrophobicity, is indicated by the size of the point. More than 200 stationary phases were studied, including small particle sizes and superficially porous ones. [ABSTRACT FROM AUTHOR]

Interfering peaks or high baseline background can compromise the results of gradient liquid chromatography (LC) separations. [ABSTRACT FROM AUTHOR]

A theme for The Chromatographic Society Diamond Jubilee Year is to increase public awareness of chromatography. Often the general public, and even chromatographers themselves, fail to appreciate the importance of chromatography. Here Tony Edge, The Chromatographic Society Vice-President, discusses just how much chromatography underpins everyday life. [ABSTRACT FROM AUTHOR]

A popular feature of The Chromatographic Society's Golden Jubilee publication was an article by Waters describing their heritage and setting out their contributions to the development of chromatography. In this special feature, some of the companies who are regular exhibitors at Society events answer questions on the current and future status of chromatography. [ABSTRACT FROM AUTHOR]

The 44th International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2016), chaired by Professor Robert Kennedy, was held 19-24 June in San Francisco, California, USA, at the Marriott San Francisco Marquis. This instalment of "Column Watch" covers some of the highlights observed at the symposium, including stationary-phase developments, particle technology, and areas of growing application of high performance liquid chromatography (HPLC). In addition, trends and perspectives on future developments in HPLC noted from the conference are presented. [ABSTRACT FROM AUTHOR]

This fourth and last instalment in the "Separation Science in Drug Development" series provides an overview of modern practices of quality control in small-molecule drug development, including activities such as setting specifications, method validation and transfer, release and stability testing, and authoring chemistry, manufacturing, and controls (CMC) sections of regulatory filings. [ABSTRACT FROM AUTHOR]

The article evaluates several chromatographic analysis products including YMC-Triart Bio C4 column from YMC Europe GmbH, Gerstel PyroVial pyrolysis product from Gerstel GmbH & Co. KG and C2MAP-2000 automated pretreatment module for cell culture media analysis from Shimadzu.

The third instalment in this series provides an overview of modern practices of separation science in small-molecule drug development. It highlights approaches in high performance liquid chromatography (HPLC) method development and physical or chemical characterization to support process chemistry and formulation development, and for assessment or control of clinical trial materials. The roles of separation scientists in analytical development and salient chromatographic methodology trends are also discussed. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) have been increasingly used as biotherapeutic agents, and a number of new mAbs are currently in the drug pipeline. Over the next five years the patent on at least nine major biotherapeutic mAbs will expire, opening the door for development and marketing of generic forms known as biosimilars. This article reviews the central role mass spectrometry (MS) coupled to liquid chromatography (LC) plays in characterizing these antibodies. Contemporary top-down and middle-up approaches using MS and various novel separation techniques to measure the intact masses of mAbs and their subunits or domains are highlighted. Example data of an innovator mAb, adalimumab, are presented that show the identities and relative abundances of the isoforms associated with this mAb. Similarly, the current state of classical peptide mapping using reversed-phase LC and tandem MS with scan-dependent acquisition is briefly reviewed. Novel approaches that speed up analysis and provide information on post-translational modifications, glycosylation, and disulfide mapping are discussed. Example data of stressed and unstressed samples of adalimumab are also presented to demonstrate peptide mapping and modifications to the antibody. Lastly, the current use of MS in glycoprofiling of mAbs is reviewed. Example glycan data for adalimumab generated by a novel labeling scheme and sensitive to detection by both fluorescence and MS are presented. [ABSTRACT FROM AUTHOR]

The article presents several terms related to gas chromatography. These include the Active flow technology, a concept that merge two types of column designs, the curtain flow technology and the parallel segmented flow. It notes the active site, a strong attracting site on the surface of chromatographic packing. It mentions the adsorption chromatography, a basic separation mode that relies on adsorption method to effect a separation.

Recent progress in supercritical fluid chromatography (SFC) instrumentation has led to renewed interest in the technique as a powerful tool for chiral and achiral separations of pharmaceutical molecules, for both analytical and preparative purposes. The "green" aspect of the technique and low running costs make SFC technology particularly attractive for preparative chromatography because it considerably reduces the consumption of organic solvents. These factors led to a revised strategy for purification and to a general interest in evaluating possible extensions for the application of packed SFC (pSFC). The results of this extensive evaluation have led to the establishment of SFC platforms for preparative achiral purifications as a standard practice, alongside its use for preparative chiral separations. [ABSTRACT FROM AUTHOR]

This instalment provides an overview of high-throughput characterization techniques of drug leads to support small-molecule drug discovery programmes in a pharmaceutical company. Myriad analytical chemistry techniques including separation science methodologies are used to confirm the structures and identities, quantitating the concentrations of stock solutions, and measuring key physicochemical properties of the new chemical entities (NCE). A case study is used here to illustrate the details of these applications in high-throughput characterization. [ABSTRACT FROM AUTHOR]

The article presents questions and answers related to liquid chromatography and separation science asked to chromatography vendors like Advanced Chromatography Technologies Ltd, Anthias Consulting Ltd and Biotech AB.

This instalment provides an overview of modern practices of high-throughput purification to support small-molecule drug discovery. It describes the use of reversed-phase liquid chromatography (LC) and supercritical fluid chromatography (SFC) for purifying diverse samples in a centralized laboratory setting. A case study is used to illustrate the principles and rationales for selecting operating parameters for these applications. This is the first instalment of four articles on "Separation Science in Drug Development" to describe the modern practice of separation science in supporting small-molecule drug discovery and development. The complete series will be published throughout the year in LCGC Europe. [ABSTRACT FROM AUTHOR]

Nonconventional Alternatives to LC-MS An important attribute of a novel ionization process for use in mass spectrometry (MS) is its simplicity and flexibility to be hyphenated to conventional liquid-based separation methods. The new solution-based ionization process described here provides exciting opportunities for nonconventional alternatives to interfacing liquid chromatography (LC) with MS. Combining LC separation with ion mobility spectrometry (IMS) provides additional separation of gas-phase ions. These technology developments have potential for extending MS to areas of high-performance characterization using advanced fragmentation technology (for example, electron-transfer dissociation) as well as to areas where cost, robustness, and simplicity are important assets such as clinical settings and field-portable instruments. [ABSTRACT FROM AUTHOR]

The development of sub-2-pm particles, core-shell particles, and monolithic columns has improved separation performance in liquid chromatography (LC) over the past 20 years. However, a key limitation that still prevents LC columns from reaching their full potential is the heterogeneity in the flow profile of solute bands as they travel through the chromatographic packing material within the column. Solutes travel through the column in bowl-like migration profiles, and, as a consequence, more separation power is needed to separate closely eluting solute bands. This limitation can be overcome by using a new type of column technology referred to as active flow technology (AFT). These columns use a new type of column end fitting that negates the problems associated with the bowl-like solute profiles caused by heterogeneity in the fluid flow through the column, which improves efficiency, sensitivity, and therefore increases sample throughput. This review will provide a brief introduction to AFT with a particular focus on improvements in separation performance that can be achieved when using these columns and coupling LC with mass spectrometry (MS). [ABSTRACT FROM AUTHOR]

Supercritical (subcritical) fluid chromatography (SFC) was evaluated as an alternative to high performance liquid chromatography (HPLC) for the enantiomeric separation of primary amines on a cyclofructan-based chiral stationary phase. The effect of various organic modifiers, acidic and basic additives, as well as instrumentation-specific parameters such as column temperature, flow rate, and back pressure were evaluated. The results were compared to normal-phase and polar organic modes. [ABSTRACT FROM AUTHOR]

Miniaturization is one of the emerging trends in modern analytical chemistry. Capillary electrophoresis (CE), capillary electrochromatography (CEC), and more recently nano-liquid chromatography (nano-LC) have been successfully miniaturized and applied to forensic drug analysis. This review will discuss the recent applications of miniaturized separation techniques for the analysis of illicit drugs and new psychoactive substances in seized materials and biological fluids. Particular attention is given to chiral discrimination of abused drugs, which is an important topic in forensic analysis. [ABSTRACT FROM AUTHOR]

In this article, we discuss the role of the gradient in protein separations by reversed-phase and ion-exchange high performance liquid chromatography (HPLC). To illustrate the key points, we show data from two products. Granulocyte colony stimulating factor (GCSF) is a microbial protein that is expressed in E. coli. For this molecule, reversed-phase HPLC is examined for separation of the product-related variants. The other molecule is a biosimilar monoclonal antibody product and, in this case, ion-exchange HPLC is explored as a tool for analysis of the acidic, main, and basic variant species. [ABSTRACT FROM AUTHOR]

The article presents suggestions to prevent the early demise of liquid chromatography (LC) columns. It is noted that somewhere around 500-600 dollars is the typical cost of reversed-phase column. It is mentioned that even though the price of an individual column still seems high, nothing much has been done to reduce the cost of analysis.

Synthetic oligonucleotides have become increasingly popular as a result of the recent discovery of ribonucleic acid interference (RNAi), a natural process for silencing gene expression. As biomedical researchers evaluate the use of antisense and small interfering RNAs (siRNAs) as potential therapies for the treatment of disease, the need for improved methods for the chromatographic separation and analysis of oligonucleotides has become apparent. This article presents a review of different liquid chromatography (LC) methods and strategies for the chromatographic separation of short RNA oligonucleotides. [ABSTRACT FROM AUTHOR]

In this instalment of "Sample Prep Perspectives", we cover some of the basic scientific principles behind solid-phase extraction (SPE) to allow the correct mode of extraction to be selected through an understanding of how analytes interact with and are separated by the sorbent. We also look at some of the most common causes of low recovery in SPE and how they can be investigated and remedied. Finally, we examine some of the emerging technologies within the area. [ABSTRACT FROM AUTHOR]

The article discusses the effect of using an injection solvent that is stronger or weaker than the mobile phase for liquid chromatography separation. Topics discussed include the allowed injection volume for a peak or a given retention and the concept of a sample plug injected in mobile phase as the injection solvent as it moves through the column. Also mentioned is the initial slowdown of molecules relative to their normal phase migration rates when the solvent is weaker than the mobile phase.