Generality in analytical chemistry can be manifested in impactful platforms that can streamline modern organic synthesis and biopharmaceutical processes. We herein center on a hybrid separation technique named Dual-Gradient Unified Chromatography (DGUC), which is built upon an automated dynamic modulation of CO2, organic modifier, and water blends with various buffers.
Current optimization of high performance liquid chromatography separations relies on inefficient trial-and-error approaches that group many molecular phenomena into three constants: A, B, C within the van Deemter equation. While iterative experiments can determine the balance between diffusional and mass transfer components, there is a lack of insight into the molecular dynamics that lead to the success or failure of a separation and, hence, ways to improve the process.
Significant efforts have been made to automate and rationalize the process of method development in liquid chromatography. Most method development strategies start by executing a number of screening runs, wherein the type of stationary and mobile phase are varied. The combination of stationary and mobile phase type resulting in the best separation is then selected and further fine-tuned in an optimization step, wherein continuous chromatographic parameters, such as the gradient conditions, are optimized. To speed up the optimization, retention models can be used to predict the outcome of a separation. Retention models can be empirical or mechanistic in nature, and require executing a number of scouting runs to determine the parameters of the model. A careful definition of these scouting runs is crucial to obtain accurate retention models. When suitable retention models are available, the retention times of all analytes in the sample can be predicted to determine conditions that will lead to a satisfactory separation. Since the accuracy of the predictions depends on the quality of the retention models, manual optimization is often still required to obtain an adequate separation.
The push for “greener” analytical methods often includes a reduction in solvent usage to minimize waste generation. In liquid chromatography, one way this can be achieved is by translating methods to capillary-scale columns (typically 0.05 – 0.3 mm inner diameter) which operate at flow rates that are 100-1,000 times lower than standard analytical-scale columns (typically 1.0 – 4.6 mm inner diameter). However, smaller column volumes mean that extra-column broadening effects can play a more significant role in the observed chromatographic efficiency of a separation. In this presentation, efforts to develop methods using a compact capillary LC instrument with integrated components that reduce extra-column volumes in order to better accommodate these columns will be described. Applications for both small molecule and large molecule separations have been developed, primarily focused on therapeutic and illicit drug compounds. Recent progress on the design and use of a new capillary-scale column format that can further be integrated into LC instrumentation while still achieving high separation pressures will also be discussed.
Enzyme inhibitors are a major class of pharmaceutical compounds, making inhibitor characterization a significant area of therapeutic development. Indeed, enzyme inhibition measurements are foundational to drug discovery, requiring rapid assays with low sample and reagent consumption. Capillary electrophoresis is a miniaturized analytical method used to quantify the amount of product generated off-line in an enzyme reaction or in-line in the separation capillary using immobilized enzymes or electrophoretically mediated microanalysis.
Hydrophilic Interaction Chromatography (HILIC), demonstrates an interesting selectivity and resolving power for compounds found in many biological systems. Typically, acetonitrile/water solvent mixtures are used in the low-water domain with a polar stationary phase.
When oil or gas is extracted from the ground, it is typically accompanied by a greater volume of water than hydrocarbon resource. This so-called produced water (PW) is one of the most complex mixtures on the planet; it must be extensively treated for any reuse application. Disposing of water by pumping it back underground is not only wasteful, it is known to induce seismic activity.
We are developing integrated micro- and nanofluidic devices to probe individual nanoscale particles, e.g., virus capsids, extracellular vesicles, and inorganic particles, with improved spatial and temporal resolution. To develop these measurement systems, we design in-plane architectures into which a range of functions can be integrated, e.g., reactions, filters, separations, sensors, and sorters.
The cycle time of a liquid chromatography (LC) system is the sum of the time for the chromatographic run and the autosampler injection sequence. Although LC separation times in the 1-10 s range have been demonstrated, injection sequences are commonly >15 s, limiting throughput possible with LC separations. Further, such separations are performed on relatively large bore columns requiring flow rates of >5 mL/min, thus generating large volumes of mobile phase waste when used for large scale screening and increasing the difficulty in interfacing to mass spectrometry. In this work, a droplet system that replaces the autosampler with a two-position, 20 nL internal loop valve coordinating with a three-axis positioner to sample droplets from a well plate was established. In the system, sample and immiscible fluid are pulled alternately from a well-plate into a capillary interfaced through the injection valve. The valve is actuated when sample fills the loop to allow sequential injection of samples at high throughput. Capillary LC columns with 300 µm inner diameter were used to reduce the consumption of mobile phase and sample. The system achieved 96 separations of 20 nL droplet samples containing 3 components in as little as 7.8 min with 4-s cycle time. This system was coupled to a mass spectrometer through an electrospray source for high-throughput chemical reaction screening.
Protein conformation is dynamic as it is influenced by post-translational modifications (PTMs) and interactions with other proteins, small molecules or RNA, for example. However, in vivo characterization of protein structures and protein structural changes after perturbation is a major challenge. Therefore, experiments to characterize protein structures are typically performed in vitro and with highly purified proteins or protein complexes, revealing a static picture of the protein.
In free flow electrophoresis (FFE) a thin stream of sample is continuously introduced into a planar flow chamber. An electric field is applied perpendicularly to the flow through the separation chamber. Analyte streams are defected laterally according to their electrophoretic mobility giving rise to individual paths through the separation chamber. FFE has recently been miniaturized into a microfluidic format (FFE), requiring less sample and reagents, a simplified flow profile, and better heat dissipation.
Liquid chromatography forms a key component in modern chemical research and analysis. However, most this work is based on general separation modes that employ adsorption, reversed-phase, normal-phase, ion-exchange, or size-exclusion media. A more specific approach is utilized in affinity chromatography, in which a biologically-related binding agent is used as a stationary phase for the isolation, analysis, or study of chemicals and biochemicals in complex samples. The binding agent, or affinity ligand, may be of biological or non-biological origin and may be either highly-specific or more general in its selectivity. There is a vast range of affinity ligands that may be employed in this technique. Examples of binding agents that are utilized in this method span from antibodies, immunoglobulin-binding proteins, lectins, and aptamers to dye-ligands, boronates, metal-ion chelates, and molecularly-imprinted polymers. This large set of selective stationary phases makes affinity chromatography the most diverse form of modern chromatography for analytical-scale and preparative-scale separations.
Lipidomics and metabolomics are analytical approaches widely adopted in biomedical research with the aim to obtain mechanistic insights in biochemical processes, to find diagnostic and prognostic biomarkers, and generate new hypothesis about biological processes or validate concepts from other experimentation. Although common untargeted methodologies allow simultaneous analysis of hundreds of metabolites, they often fall short in full coverage of pathways in which isomeric species play a major role, e.g. the phosphoinositide network, glycolysis and pentosephosphate pathways. This presentation will focus on challenging lipid and metabolite classes in targeted and untargeted lipidomics and metabolomics, respectively, for which biological interpretations rely on a separation of their structural isomers.
It is about 20 years since the introduction of sub-2 um particle columns and UHPLC instrumentation. The initial race towards higher and higher system pressures appears to have paused as far as commercial instrumentation is concerned.
Separations play a key role in biochemical analysis, particularly proteomic analyses in which tens of thousands of species must be rapidly quantified. Single-cell proteomics (SCP) aims to quantify the heterogeneity within cellular populations that is obscured in bulk-scale measurements, thus enabling protein expression within biological systems to be studied with a new level of granularity. We aim to optimize separations for SCP in several ways. First, we have developed a multicolumn nanoLC system that enables one column to actively elute peptides, while the inactive column(s) undergo sample loading, column regeneration, etc.
In this study, we focus on the theoretical understanding for π interactions due to the separation of aromatic halogens, H/D isotopes, sugar chains and glycoproteins. Additionally, we focus on the molecularly imprinted polymers (MIPs) using the weak intermolecular interactions.
Human health is influenced by the daily exposure to a variety of chemicals through nutrition, treatments, and lifestyle that may have either positive or harmful effects. Untargeted metabolomic investigation based on liquid chromatography- tandem mass spectrometry (LC-MS/MS) followed by multivariate analysis is an approach that can produce evidence of different health status mainly by features comparison (e.g. retention time, m/z and intensity). These features can be common to all study samples, mainly endogenous metabolites, or present in a single or a few samples, mainly pharmaceuticals or pesticides,.
In this presentation I will discuss the current state of mass spectrometry-based proteomics and the crucial role separations play in obtaining the highest performance possible. As mass spectrometer scan rates have continued to increase, so too have the demands on the separation.
Asymmetrical flow field-flow fractionation (AF4) is a versatile open-channel separation technique. It can resolve analytes with sizes ranging from tens of nanometers to a few microns while preserving fragile structures and reversible molecular interactions. The performance of AF4 could be further enhanced by coupling it with other orthogonal separation systems as well as detectors that can provide further size differentiation.
Supercritical fluid chromatography (SFC) has evolved significantly over the recent years, expanding its applicability across various fields. Different types of stationary phases, including those designed specifically for SFC and those for high-performance liquid chromatography, have been effectively employed in SFC allowing this wide range of applications with the same type of CO2 based mobile phase. The interactions in SFC using different types of stationary phases have been extensively studied, mainly by means of linear solvation energy relationship (LSER) model. However, other tools, such as artificial neural networks (ANN) may currently be used to define molecule properties responsible for a particular interaction and to describe the interactions in SFC more precisely.
Modern vaccine development often employs large biopolymers or/and nanoparticles as antigens or adjuvants. These large molecules often present a wide range of analytical challenges. However, extensive characterization of multi critical quality attributes (CQA) for these large molecules are required for vaccine quality, purity and efficacy. Furthermore, multi-valent vaccines are required to effectively combat complex diseases caused by viral/bacterial infections, such as infection by bacteria Streptococcus pneumoniae or human papillomavirus (HPV).
In recent years, there has been a significant investment and success in nucleic acid based medicines (NABM), encompassing ASO, siRNA, mRNA, as well as gene-editing technologies like guide RNA and plasmid DNA. Alongside with advancements of the different NABM modalities is the advanced drug delivery technologies such as nanoparticles. The rapid clinical success and increasing expectations from Health Authorities have highlighted gaps in analytical technologies that require attention.
Biologically produced messenger RNA and plasmid DNA are large nucleic acids utilized as gene-editing materials for cell and gene therapy. The fast development of vaccines and CRISPR gene editing technology has somewhat outpaced the analytical development and characterization technologies. To our knowledge, it was only in 2023 that mRNA aggregate characterization was evaluated via the use of a novel size exclusion chromatography (SEC) column.
Proteoforms - encompassing the diverse protein products arising from alternative splice isoforms, genetic variations, and posttranslational modifications (PTMs) originating from a single gene - are fundamental drivers in biology. Top-down mass spectrometry (MS)-based proteomics (TDP), analyzing whole proteins without digestion, offers a comprehensive perspective of proteoforms, which is invaluable in deciphering proteoform function, uncovering disease mechanisms, and advancing precision medicine.
The advent of activity-based probes (ABPs) combined with mass spectrometry-based proteomics enables selectivity profiling of small molecules that bind mechanistically related enzyme families. This approach for chemoproteomic activity-based protein profiling (ABPP) has been used extensively to accelerate development of inhibitors against clinically validated targets such as kinases as well as other enzyme families in the functional proteome.
The use of analytical methods and techniques ensures the high quality, safety, and compliance of materials and products cycled back in circular economy systems. However, despite this connection, the analytical chemistry sector still largely adheres to the linear "take-makeconsume and dispose" model, requiring a continuous supply of resources and generating hazardous waste.
Flow based sample treatment derives from concepts incepted by flow injection analysis, aiming at the handling of solutions or suspensions in a confined environment with high controlled conditions in time and space. This precise control of operations allows for an increase in reproducibility of events, allowing kinetic control of processes.
High-resolution LC and two-dimensional liquid chromatography (2D-LC) are strongly advancing the resolving power of separation technology. It is our vision that advanced separation technology must become accessible throughout society and our mission to develop tools that make this possible. The chemometrics and chromatography communities have developed a plethora of useful techniques that – when combined – can be used to enable automation of various components of method development. However, this innovation demands several scientific challenges to be overcome.
Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty®, an mRNA vaccine immunizing against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping provides direct primary structure characterization of mRNA, through rapid, one-pot, one-enzyme digestion, followed by optimized LC-MS/MS. Data analysis utilizing semi-automated software generates a highly reproducible and completely annotated UV chromatogram with 100% sequence coverage, and a microheterogeneity assessment of 5´ terminus capping and 3´ terminus poly(A) tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of Comirnaty® by providing confirmation of construct identity, characterization of primary structure and assessment of product comparability following manufacturing process changes.
Therapeutic peptides are considered one of the most promising class of biopharmaceuticals. Their industrial production (upstream processing) has exceptionally advanced in the last ten years, especially for what regards solid-phase synthesis. However, these advancements have not been matched by equivalent improvements in purification procedures (downstream processing) which still represents the bottleneck, in terms of both cost, time and sustainability in the entire production process.
There has been increasing concern over the human cost and the analytical challenges resulting from the increasing use and abuse of novel psychoactive substances. In 2023 alone over 100,000 Americans lost their lives to drug overdoses with up to 65,000 of these coming from synthetic opioids such as fentanyl.
The extraction of small molecules from complex samples presents a significant challenge in analytical method development, whether for targeted or non- targeted analysis. Recent trends in microextraction techniques development have shifted towards greener and faster approaches, ensuring sustainability and high throughput during the extraction process. Solid Phase Microextraction (SPME) is an ideal method that aligns with these features, offering simultaneous extraction and enrichment of targeted analytes.
While biomolecular damage plays a critical role in human chronic diseases, there is currently no drug available to address this issue. Protein undergoes progressive damage through spontaneous chemical reactions like oxidation, deamidation, carbamylation, and glycation, collectively referred to as 'degenerative protein modification' (DPM). These modifications can lead to protein malfunction, but in some cases, acquire harmful new functions to cause age-linked chronic diseases in humans. In our research, we employ a mixed-mode liquid chromatograph coupled with tandem mass spectrometry (ERLIC-MS/MS) to separate and identify DPM targets in samples from patients and animal models of cardiovascular disease and vascular dementia.
Two-dimensional liquid chromatography (2D-LC) separations are increasing in popularity, both for the analysis of relatively simple, but hard-to-separate mixtures (e.g., mixtures of achiral and chiral compounds), and the analysis of highly complex mixtures such as those encountered in the biological samples and natural products.
Nucleic acid extraction and purification represents a significant bottleneck in DNA analysis. Magnetic bead-based extraction methods can rapidly extract DNA from sample solutions; however, these methods suffer from particle aggregation and sedimentation resulting in diminished extraction efficiencies. Circulating tumor DNA (ctDNA) is a promising biomarker for the diagnosis of cancer and monitoring of treatment.
Sample preparation is a critical step in complex sample analysis which effect to sensitivity, selectivity, speed, and accuracy of analytical results. The goal of sample preparation is separation and enrichment. Separation and enrichment are entropy reduction procedures which cannot happen spontaneously. On account of consuming over two thirds of analysis time, sample preparation becomes the bottleneck issue in analytical chemistry. Therefore, fast sample preparation has been received much attention. In this work, a comprehensive survey on progress in fast sample preparation techniques for complex sample has been composed. Four approaches for entropy reduction of sample preparation system were generalized, they are energy exchange acceleration, materials-based acceleration, size reduction acceleration, and integration acceleration. Moreover, we highlight the most interesting acceleration techniques, including field-assisted, materials-based rapid mass transfer, microfluidic, and multistep integration techniques. Furthermore, analytical applications of these acceleration approaches were discussed. Meanwhile, further utilization potentiality of these approaches was offered.
3D printing provides a powerful process for making novel, miniaturized systems to facilitate separation science. Our research is directed at developing and utilizing 3D printing to create integrated microfluidic devices for separation-based bioanalysis. We use a custom-built 3D printer with digital light processing–stereolithography to form microfluidic channels, pumps, and valves, which together can carry out various chromatographic operations.
Glycosylation is the most abundant post-translational modification (PTM) in mammals, mediating essential biological functions. Aberrant protein glycosylation and isomeric distribution are associated with many diseases and impact the efficacy of biologics and biosimilars. The presence of distinct isomeric glycans on a glycosylation site can fine-tune the biological functions of a protein. However, studying glycomics remains challenging due to the microheterogeneity of glycan structures and the limited identification of glycan isomers. Notably, isomeric glycans are linked to various diseases, including cancers, neurodegenerative diseases, and inflammatory conditions, highlighting the need for both qualitative and quantitative analyses of glycans.
The different behavior of the enantiomers of chiral compounds in a non-isotropic environments (among them in living organism) is well known. On the other hand, the importance of a kinetic isotope effect in the biomedical field has become evident during the past decades.
Monoclonal antibodies (mAbs) have revolutionized the pharmaceutical landscape offering safe and effective therapeutic options for life-threatening diseases including cancer, autoimmune and infectious diseases. Well beyond 100 antibodies, spanning multiple formats, such as canonical mAbs, antibody-drug conjugates (ADCs), bispecifics and fragments, have been approved by regulatory agencies for therapeutic use in the United States and Europe.
