14N to 15N Isotopic Exchange of Nitrogen Heteroaromatics through Skeletal Editing.

The selective modification of nitrogen heteroaromatics enables the development of new chemical tools and accelerates drug discovery. While methods that focus on expanding or contracting the skeletal structures of heteroaromatics are emerging, methods for the direct exchange of single core atoms remain limited. Here, we present a method for 14N → 15N isotopic exchange for several aromatic nitrogen heterocycles. This nitrogen isotope transmutation occurs through activation of the heteroaromatic substrate by triflylation of a nitrogen atom, followed by a ring-opening/ring-closure sequence mediated by 15N-aspartate to effect the isotopic exchange of the nitrogen atom. Key to the success of this transformation is the formation of an isolable 15N-succinyl intermediate, which undergoes elimination to give the isotopically labeled heterocycle. These transformations occur under mild conditions in high chemical and isotopic yields.

Selective Plate-Based Assay for Trace EDTA Analysis via Boron Trifluoride-methanol Derivatization UHPLC-QqQ-MS/MS Enabling Biologic and Vaccine Processes.

The employment of ethylenediaminetetraacetic acid (EDTA) across several fields in chemistry and biology has required the creation of a high number of quantitative assays. Nonetheless, the determination of trace EDTA, especially in biologics and vaccines, remains challenging. Herein, we introduce an automated high-throughput approach based on EDTA esterification in 96-well plates using boron trifluoride-methanol combined with rapid analysis by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Derivatization of EDTA to its methyl ester (Me-EDTA) serves to significantly improve chromatographic performance (retention, peak shape, and selectivity), while also delivering a tremendous enhancement of sensitivity in the positive ion mode electrospray ionization (ESI+). This procedure, in contrast to previous EDTA methods based on complexation with metal ions, is not affected by high concentration of other metals, buffers, and related salts abundantly present in biopharmaceutical processes (e.g., iron, copper, citrate, etc.). Validation of this assay for the determination of ng·mL-1 level EDTA in monoclonal antibody and vaccine products demonstrated excellent performance (repeatability, precision, and linear range) with high recovery from small sample volumes while also providing an advantageous automation-friendly workflow for high-throughput analysis.

Dual-Gradient Unified Chromatography: A New Paradigm for Versatility in Simultaneous Multicomponent Analysis.

Generality in analytical chemistry can be manifested in impactful platforms that can streamline modern organic synthesis and biopharmaceutical processes. We herein introduce 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. This concept enables simultaneous multicomponent analysis of both small and large molecules across a wide polarity range in single experimental runs. After a careful investigation of its fundamental aspects, a DGUC-DAD-MS screening workflow that combines multiple orthogonal column and mobile phase choices across a far-reaching universal elution profile is also reported. The power of this framework is demonstrated with new analytical applications guiding academic and industrial laboratories in the development of new (bio)pharmaceutical targets (e.g. synthetic intermediates, nucleosides, cyclic and linear peptides, proteins, antibody drug conjugates).

The Emergence of Universal Chromatographic Methods in the Research and Development of New Drug Substances.

Manufacturing process development of new drug substances in the pharmaceutical industry combines numerous chemical challenges beyond the efficient synthesis of complex molecules. Optimization of a synthetic route involves the screening of multiple reaction variables with a desired outcome that not only depends on an increased product yield but is also highly influenced by the removal efficacy of residual chemicals and reaction byproducts during the subsequent synthetic route. Consequently, organic chemists must survey a wide array of synthetic variables to develop a highly productive, green, and cost-effective manufacturing process. The time constraints of developing robust quantitative methods prior to each processing step can easily lead to sample analysis becoming a bottleneck in synthetic route development. In this regard, conventional "on demand" analytical method development and optimization approaches, traditionally used for guiding synthetic chemistry efforts, become unsustainable. This Account introduces recent efforts to address the aforementioned challenges through the development and implementation of generic or more universal chromatographic methods that can cover a broad spectrum of targeted compound classes. Such generic methods require significant resolving power to enable baseline resolution of multicomponent mixtures in a single experimental run without additional method customization but must be simple enough to allow for routine use by chemists, chemical engineers and other researchers with little experience in chromatographic method development. These powerful analytical methodologies are often employed to minimize the time spent developing new analytical assays, while also facilitating method transfer to manufacturing facilities and application in regulatory settings. Diverse examples of universal and fit-for-purpose analytical procedures are presented herein, illustrating the power of modern readily available analytical technology for streamlining the development of new drug substances in organic chemistry laboratories across both academic and industrial sectors. With recent advances in analytical instrumentation and column technologies, universal chromatographic methods are quickly becoming a proactive and effective strategy to accelerate the discovery and implementation of new synthetic methodologies, especially but not limited to laboratories where the synthetic process route is undergoing rapid change and optimization. Targets of these generic methods include analysis of organic solvents, acid and basic additives, nucleotide species, palladium scavengers, impurity mapping, enantiopurity, synthetic intermediates, active pharmaceutical ingredients and their counterions, dehalogenation byproducts, and mixtures of organohalogenated pharmaceuticals, among other chemicals used or formed in process chemistry reactions.

Comprehensive online multicolumn two-dimensional liquid chromatography-diode array detection-mass spectrometry workflow as a framework for chromatographic screening and analysis of new drug substances.

The analysis of complex mixtures of closely related species is quickly becoming a bottleneck in the development of new drug substances, reflecting the ever-increasing complexity of both fundamental biology and the therapeutics used to treat disease. Two-dimensional liquid chromatography (2D-LC) is emerging as a powerful tool to achieve substantial improvements in peak capacity and selectivity. However, 2D-LC suffers from several limitations, including the lack of automated multicolumn setups capable of combining multiple columns in both dimensions. Herein, we report an investigation into the development and implementation of a customized online comprehensive multicolumn 2D-LC-DAD-MS setup for screening and method development purposes, as well as analysis of multicomponent biopharmaceutical mixtures. In this study, excellent chromatographic performance in terms of selectivity, peak shape, and reproducibility were achieved by combining reversed-phase (RP), strong cation exchange (SCX), strong anion exchange (SAX), and size exclusion chromatography (SEC) using sub-2-µm columns in the first dimension in conjunction with several 3.0 mm × 50 mm RP columns packed with sub-3-µm fully porous particles in the second dimension. Multiple combinations of separation modes coupled to UV and MS detection are applied to the LC × LC analysis of a protein standard mixture, intended to be representative of protein drug substances. The results reported in this study demonstrate that our automated online multicolumn 2D-LC-DAD-MS workflow can be a powerful tool for comprehensive chromatographic column screening that enables the semi-automated development of 2D-LC methods, offering the ability to streamline full visualization of sample composition for an unknown complex mixture while maximizing chromatographic orthogonality. Graphical Abstract.

Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale.

Chromatographic separation, analysis and characterization of complex highly polar analyte mixtures can often be very challenging using conventional separation approaches. Analysis and purification of hydrophilic compounds have been dominated by liquid chromatography (LC) and ion-exchange chromatography (IC), with sub/supercritical fluid chromatography (SFC) moving toward these new applications beyond traditional chiral separations. However, the low polarity of supercritical carbon dioxide (CO2) has limited the use of SFC for separation and purification in the bioanalytical space, especially at the preparative scale. Reaction mixtures of highly polar species are strongly retained even using polar additives in alcohol modifier/CO2 based eluents. Herein, we overcome these problems by introducing chaotropic effects in SFC separations using a nontraditional mobile phase mixture consisting of ammonium hydroxide combined with high water concentration in the alcohol modifier and carbon dioxide. The separation mechanism was here elucidated based on extensive IC-CD (IC couple to conductivity detection) analysis of cyclic peptides subjected to the SFC conditions, indicating the in situ formation of a bicarbonate counterion (HCO3-). In contrast to other salts, HCO3- was found to play a crucial role acting as a chaotropic agent that disrupts undesired H-bonding interactions, which was demonstrated by size-exclusion chromatography coupled with differential hydrogen-deuterium exchange-mass spectrometry experiments (SEC-HDX-MS). In addition, the use of NH4OH in water-rich MeOH modifiers was compared to other commonly used basic additives (diethylamine, triethylamine, and isobutylamine) showing unmatched chromatographic and MS detection performance in terms of peak shape, retention, selectivity, and ionization as well as a completely different selectivity and retention behavior. Moreover, relative to ammonium formate and ammonium acetate in water-rich methanol modifier, the ammonium hydroxide in water additive showed better chromatographic performance with enhanced sensitivity. Further optimization of NH4OH and H2O levels in conjunction with MeOH/CO2 served to furnish a generic modifier (0.2% NH4OH, 5% H2O in MeOH) that enables the widespread transition of SFC to domains that were previously considered out of its scope. This approach is extensively applied to the separation, analysis, and purification of multicomponent reaction mixtures of closely related polar pharmaceuticals using readily available SFC instrumentation. The examples described here cover a broad spectrum of bioanalytical and pharmaceutical applications including analytical and preparative chromatography of organohalogenated species, nucleobases, nucleosides, nucleotides, sulfonamides, and cyclic peptides among other highly polar species.

Multi-column ultra-high performance liquid chromatography screening with chaotropic agents and computer-assisted separation modeling enables process development of new drug substances.

Modern process research and development can often be hampered by the tedious method development required to chromatographically resolve mixtures of chemical species with very similar physical properties. Herein, we describe a simple approach for the development and implementation of an efficient ultra-high performance liquid chromatography (UHPLC) assay that is extensively applied to the separation and analysis of multicomponent reaction mixtures of closely related pharmaceutical intermediates and impurities. Methods are optimized using multi-column and multi-solvent UHPLC screening in conjunction with chromatography simulation software (ACD Labs/LC Simulator). This approach is implemented to enable the separation, identification, mapping and control of impurities formed within the process chemistry optimization of the dimeric catalyst used in the synthesis of new drug substances. The final method utilized a sub-2 µm C18 stationary phase (2.1 mm I.D. × 50 mm length, 1.7 µm particle size ACQUITY UPLC BEH C18) with a non-conventional chaotropic mobile phase buffer (35 mM potassium hexafluorophosphate in 0.1% phosphoric acid/acetonitrile) in order to achieve baseline separation of all reaction components. The chromatographic simulation and modeling strategy served to generate 3D resolution maps with robust separation conditions that match the outcome of subsequent experimental data (overall ΔtR < 0.35%). Our multi-column UHPLC screening with computer-assisted chromatographic modeling is a great addition to the toolbox of synthetic chemists and can be a powerful tool for streamlining process chemistry optimization in organic chemistry laboratories across both academic and industrial sectors.

High-throughput infrared spectroscopy for quantification of peptides in drug discovery.

Peptides have gained an increasing importance in drug discovery as potential therapeutics. Discovery efforts toward finding new, efficacious peptide-based therapeutics have increased the throughput of peptide development, allowing the rapid generation of unique and pure peptide samples. However, high-throughput analysis of peptides may be still challenging and can encumber a high-throughput drug discovery campaign. We report herein a fit-for-purpose method to quantify peptide concentrations using high-throughput infrared spectroscopy (HT-IR). Through the development of this method, multiple critical method parameters were optimized including solvent composition, droplet deposition size, plate drying procedures, sample concentration, and internal standard. The relative absorbance of the amide region (1600-1750 cm-1) to the internal standard, K3Fe(CN)6 (2140 cm-1), was determined to be most effective at providing lowest interference for measuring peptide concentration. The best sample deposition was achieved by dissolving samples in a 50:50 v/v allyl alcohol/water mixture. The developed method was used on 96-well plates and analyzed at a rate of 22 min per plate. Calibration curves to measure sample concentration versus response relationship displayed sufficient linearity (R2 > 0.95). The repeatability and scope of detection was demonstrated with eighteen peptide samples that were measured with most values below 20% relative standard deviation. The linear dynamic range of the method was determined to be between 1 and 5 mg/mL. This developed HT-IR methodology could be a useful tool in peptide drug candidate lead identification and optimization processes.

Surprising formation of p-cymene in the oxidation of α-pinene in air by the atmospheric oxidants OH, O3, and NO3.

Anthropogenic sources release into the troposphere a wide range of volatile organic compounds (VOCs) including aromatic hydrocarbons, whose major sources are believed to be combustion and the evaporation of fossil fuels. An important question is whether there are other sources of aromatics in air. We report here the formation of p-cymene [1-methyl-4-(1-methylethyl) benzene, C6H4(CH3)(C3H7)] from the oxidation of α-pinene by OH, O3, and NO3 at 1 atm in air and 298 K at low (<5%) and high (70%) relative humidities (RH). Loss of α-pinene and the generation of p-cymene were measured using GC-MS. The fractional yields of p-cymene relative to the loss of α-pinene, Δ [p-cymeme]/Δ [α-pinene], were measured to range from (1.6±0.2)×10(-5) for the O3 reaction to (3.0±0.3)×10(-4) for the NO3 reaction in the absence of added water vapor. The yields for the OH and O3 reactions increased by a factor of 4-8 at 70% RH (uncertainties are ±2s). The highest yields at 70% RH for the OH and O3 reactions, ∼15 times higher than for dry conditions, were observed if the walls of the Teflon reaction chamber had been previously exposed to H2SO4 formed from the OH oxidation of SO2. Possible mechanisms of the conversion of α-pinene to p-cymene and the potential importance in the atmosphere are discussed.

Ultra-high-throughput SPE-MALDI workflow: Blueprint for efficient purification and screening of peptide libraries.

At the forefront of synthetic endeavors in the pharmaceutical industry, including drug discovery and high-throughput screening, timelines are tight and large quantities of pure chemical targets are rarely available. In this regard, the development of novel and increasingly challenging chemistries requires a commensurate level of innovation to develop reliable analytical assays and purification workflows with rapid turnaround that enables accelerated pharmacological evaluation. A small-scale automation platform enabling high-throughput analysis and purification to streamline the selection of candidate leads would be a transformative advance. Herein, we introduce an automation-friendly solid-phase extraction-matrix-assisted laser desorption/ionization (SPE-MALDI) platform applied to the high-throughput purification and analysis of peptide libraries. This advance enabled us to purify peptides from microgram levels in less than a day with results comparable to traditional high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS).

In silico method development for the reversed-phase liquid chromatography separation of proteins using chaotropic mobile phase modifiers.

Recent advances in biomedical and pharmaceutical processes has enabled a notable increase of protein- and peptide-based drug therapies and vaccines that often contain a higher-order structure critical to their efficacy. Hyphenation of chromatographic and spectrometric techniques is at the center of all facets of biopharmaceutical analysis, purification and chemical characterization. Although computer-assisted chromatographic modeling of small molecules has reached a mature stage across the pharmaceutical industry, software-based method optimization approaches for large molecules has yet to see the same revitalization. Conformational changes of biomolecules under chromatographic conditions have been identified as the major culprit in terms of sub-optimal modeling outcomes. In order to circumvent these challenges, we herein investigate the outcomes generated via computer-assisted modeling from using different chaotropic and denaturing mobile phases (trifluoroacetic acid, sodium perchlorate and guanidine hydrochloride in acetonitrile/water-based eluents). Linear and polynomial regression retention models using ACD/Labs software were built as a function of gradient slope, column temperature and mobile phase buffer for eight different model proteins ranging from 12 to 670 kDa (holo-transferrin, cytochrome C, apomyoglobin, ribonuclease A, ribonuclease A type I-A, albumin, y-globulin and thyroglobulin bovine). Correlation between experimental and modeled outputs was substantially improved by using strong chaotropic and denaturing modifiers in the mobile phase, even when using linear regression modeling as typically observed for small molecules. On the contrary, the use of conventional TFA buffer concentrations at low column temperatures required the used of polynomial regression modeling indicating potential conformational structure changes of proteins upon chromatographic conditions. In addition, we illustrate the power of modern computer-assisted chromatography modeling combined with chaotropic agents in the developing of new RPLC assays for protein-based therapeutics and vaccines.

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility.

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

Introducing online multicolumn two-dimensional liquid chromatography screening for facile selection of stationary and mobile phase conditions in both dimensions.

Baseline separation and analysis of multicomponent mixtures of closely related pharmaceuticals using single column selectivity can often be challenging, requiring the combination of orthogonal stationary and mobile phase methods to monitor all the species and optimize reaction outcomes. In recent years, two-dimensional liquid chromatography (2D-LC) has become a valuable tool for improving peak capacity and selectivity. Though powerful, standard 2D-LC instrumentation and software can often lead to tedious method development and has a requirement for very specific expertise that is poorly suited for a fast-paced industrial environment. In this regard, the introduction of an automated online 2D-LC setup that could screen multiple columns in both dimensions without manual intervention will undeniably serve to streamline column/mobile phase selection and secure the viability of 2D-LC as a mainstay instrument for industrial applications. Herein, we introduce and investigate a multicolumn online 2D-LC approach that simplifies column screening and method development dramatically. This setup incorporates 6-position column selection valve technology whose functionality enables us to combine multiple columns in the first and second dimensions. This strategy in conjunction with diode array detection (DAD) in both dimensions and mass spectrometry (MS) acquisition in the second dimension serves to explore different columns and mobile phases as a framework for screening targeted compounds in multicomponent mixtures without having to perform chromatographic purification. Multiple online heart cutting achiral RPLC - achiral RPLC and achiral RPLC - chiral RPLC coupled to DAD and ESI-MS methods combining several stationary phase selectivity in an automated fashion are successfully applied to the separation and analysis of complex mixtures of drug substances, where in many instances, traditional 1D-ultra-high performance liquid chromatography (UHPLC) fails or delivers sub-optimal results. This automated online multicolumn 2D-LC workflow enables rapid and efficient identification of column/eluent combinations, as well as sample analysis across multiple columns in both dimensions overnight with a single click.

Doing more with less: Evaluation of the use of high linear velocities in preparative supercritical fluid chromatography.

The evaluation of higher than typical linear velocities is discussed for supercritical fluid chromatographic purifications on the preparative scale. SFC separation efficiency suffers far less at high linear velocities than HPLC by the rapid mass transfer of analytes carried by compressed CO2 through the stationary phase. The technique is discussed using chiral test compounds and columns. In many cases, running at high linear velocities can yield significant time savings and decreased consumption of mobile phase solvent, while also lowering energy consumption. Within the practical limitations of commercial instrumentation, using 20 µm particles can aid in achieving higher linear velocities not attainable with smaller 5 µm particles, particularly when running with high percentages of organic co-solvent. Use of larger particles for the stationary phase also lowers the associated column cost. These benefits can yield an overall purification process that is more productive and environmentally friendly.

Enhanced fluidity liquid chromatography: A guide to scaling up from analytical to preparative separations.

The evolution of supercritical fluid chromatography (SFC) instrumentation, improved detection capability, and expanded modifier range has led to extending the reach of SFC to the analysis of a broader spectrum of analytes beyond enantioselective separations. However, preparative SFC has yet to see the same technological revitalization, especially in regards to the purification of highly polar analytes. Enhanced fluidity liquid chromatography (EFLC) has been demonstrated as one of the ways to extend the applicable range of SFC instrumentation to highly polar analytes such as proteins, carbohydrates, and nucleotides. Despite recent applications of EFLC for challenging mixtures of hydrophilic metabolites and analogs, its viability in preparative purification, which is of great importance to the pharmaceutical industry, remains unknown. Herein, multiple chromatographic parameters that are critical to achieve feasible EFLC purification methods were investigated, including system pressure as a function of modifier composition (for several MeOH:H2O ratios), effect of diluent injection conditions on peak shape, and optimization of mass load with diluent composition. The usage of 50% acetonitrile or methanol diluents provided the most volumetric loading capacity. In the case of sucrose, leveraging higher analyte solubility in water proved to be more favorable than the volumetric loading capacity of diluents with higher organic content. In fact, an 80 mg injection of sucrose was possible on a 2 cm preparative HILIC column with minimal peak shape degradation. The combined information led to the successful demonstration of EFLC for the preparative separation of sugars using readily available MS-directed SFC instrumentation.

Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography.

Supercritical fluid chromatography (SFC) has become the fastest growing analytical tool for chiral and achiral small-molecule pharmaceutical separations. The benefits from savings in cost (as a result of lower solvent and energy consumption), and time have made SFC a proven effective tool for solving many analytical problems for small-molecules over the years. There is, however, a gap in the application of SFC for larger biomolecules, proteins and peptides. There has been a notable increase of protein- and peptide-based drug therapies that contain a higher-order structure important to their efficacy. These studies leverage the use of size exclusion chromatography coupled with hydrogen-deuterium exchange (SEC-HDX) methodology and circular dichroism (CD) spectroscopy to probe global conformational structures of model peptides and proteins following purification by preparative SFC. It was demonstrated that bradykinin and insulin can be used in SFC purification, and moreover, insulin was able to recover its original higher-order structure when compared to pre-purification insulin by three orthogonal techniques: 1) calculated percent alpha-helicity based on CD spectra, 2) alpha-helix - temperature hysteresis analysis by CD and 3) SEC-HDX-MS at different temperatures. However, it was shown that the higher order structures of the other three model proteins used in the study (ubiquitin, cytochrome C, and apomyoglobin) were significantly modified during SFC purification and were unable to re-fold to their original conformations. The present workflow was applied successfully to several peptide therapeutic programs at our comp any and in addition can be applied for small proteins.

Enhanced fluidity liquid chromatography of inulin fructans using ternary solvent strength and selectivity gradients.

The value of exploring selectivity and solvent strength ternary gradients in enhanced fluidity liquid chromatography (EFLC) is demonstrated for the separation of inulin-type fructans from chicory. Commercial binary pump systems for supercritical fluid chromatography only allow for the implementation of ternary solvent strength gradients which can be restrictive for the separation of polar polymeric analytes. In this work, a custom system was designed to extend the capability of EFLC to allow tuning of selectivity or solvent strength in ternary gradients. Gradient profiles were evaluated using the Berridge function (RF1), normalized resolution product (NRP), and gradient peak capacity (Pc). Selectivity gradients provided the separation of more analytes over time. The RF1 function showed favor to selectivity gradients with comparable Pc to that of solvent strength gradients. NRP did not strongly correlate with Pc or RF1 score. EFLC with the hydrophilic interaction chromatography, HILIC, separation mode was successfully employed to separate up to 47 fructan analytes in less than 25 min using a selectivity gradient.

Protein separations using enhanced-fluidity liquid chromatography.

Enhanced-fluidity liquid chromatography (EFLC) methods using methanol/H2O/CO2 and hydrophilic interaction liquid chromatography (HILIC) were explored for the separation of proteins and peptides. EFLC is a separation mode that uses a mobile phase made of conventional solvents combined with liquid carbon dioxide (CO2) in subcritical conditions. The addition of liquid CO2 enhances diffusivity and decreases viscosity while maintaining mixture polarity, which typically results in reduced time of analysis. TFA additive and elevated temperature were leveraged as key factors in the separation of a 13-analyte intact protein mixture in under 5min. Under these conditions EFLC showed modest improvement in terms of peak asymmetry and analysis time over the competing ACN/H2O separation. Protein analytes detected by electrospray ionization - quadrupole time of flight, were shown to be unaffected by the addition of CO2 in the mobile phase. Herein, the feasibility of separating hydrophilic proteins up to 80kDa (with transferrin) is demonstrated for CO2-containing mobile phases.

Gradient separation of oligosaccharides and suppressing anomeric mutarotation with enhanced-fluidity liquid hydrophilic interaction chromatography.

Enhanced fluidity liquid chromatography using the hydrophilic interaction retention mechanism (EFLC-HILIC) is studied as an alternative separation mode for analyzing oligosaccharides and other sugars. These carbohydrates, which are important for the study of foods and biological systems, are difficult to comprehensively profile and either require a non-green, expensive solvent (i.e. acetonitrile) or derivatization of the analytes at the expense of time, sample loss, and loss of quantitative information. These difficulties arise from the diverse isomerism, mutarotation, and lack of a useable chromophore/fluorophore for spectroscopic detection. Enhanced fluidity liquid chromatography is an alternative separation method that involves the use of conventional polar solvents, such as methanol/water mixtures, as the primary mobile phase component and liquid carbon dioxide (CO2) as the modifier in subcritical conditions. The addition of liquid CO2 enhances diffusivity and decreases viscosity while maintaining mixture polarity, which typically results in reduced time of analysis and higher efficiency. This work illustrates an optimized EFLC-HILIC separation of a test mixture of oligosaccharides and simple sugars with a resolution greater than 1.3 and an analysis time decrease of over 35% compared to a conventional HPLC HILIC-mode analysis using acetonitrile/water mobile phases.