Fast Separations Research Articles

Development of a superheated water chromatography method for the sustainable determination of preservatives in foods and cosmetics

The present research focused on the use of high temperatures to achieve fast separations in Liquid Chromatography (LC) and minimize the consumption of organic solvents. Particularly, superheated water was employed, by exploiting the decrease in dielectric constant of water when increasing the temperature. To realize this kind of application, the availability of LC columns resistant at high temperature is mandatory. Recently, porous graphitic carbon (PGC) columns have been proposed as stationary phases resistant up to 250 °C. Hardware modifications were also necessary to guarantee the efficient heating of the mobile phase prior of entering the column, as well as its rapid cooling before detection by photodiode array (PDA). Specifically, an LC system was interfaced to a GC oven, which hosted the LC column; to achieve a fast and efficient heating of the eluent coming from the autosampler prior to enter into the column, a pre-heating tube was interposed between the autosampler outlet and the column inlet. Instead, a cooling loop was connected between the column outlet and the PDA cell. A mixture of 1-nitroalkanes was used to evaluate the performance of the system in terms of dead volumes, band broadening and peak shape, as well as chromatographic efficiency and resolution. The use of higher temperatures allowed to perform analyses at higher flow rates in reduced analysis time and was also in terms of peak bandwidth resulting to increased efficiency for the most retained compound. Moreover, van't Hoff plots were obtained at different temperature, confirming that the most retained analyte greatly benefited of the temperature increase.The system was applied to the analysis of parabens in cosmetic and food products. The method combined eco-sustainable sample preparation with high-temperature LC separation on a PGC column and was fully validated in terms of linearity, precision, accuracy and detection and quantification limits. The developed method was critically evaluated by comparing the output of different metric tools implemented in the last decade for the quantitative assessment of the greenness.

Sensitive and rapid analysis of plasmid DNA topology isoforms by capillary gel electrophoresis with laser induced fluorescence in uncoated capillary.

The work describes the use of SYBR Gold to improve the detection sensitivity of plasmid DNA topoisomers by capillary gel electrophoresis with laser induced fluorescence in an uncoated capillary. The impact of different dyes, including ethidium bromide, SYBR Green and SYBR Gold, was compared based on detection and separation of DNA plasmid topoisomers. Use of SYBR Gold enabled improvement of detection sensitivity by 15-fold while maintaining good separation resolution of the different topoisomers. The baseline dropped with the use SYBR Gold but was overcome by the employment of a capillary with longer ineffective length (40 vs. 20cm). Separation resolution and reproducibility were impacted by the concentration of SYBR Gold and hydroxypropyl methylcellulose. With the use of a short capillary (10cm effective length and 50cm total length), fast separations of supercoiled, linear, open circular, and other isoforms were accomplished within 8min. Appropriate capillary cleaning with 0.1M sodium hydroxide/0.1M hydrochloric acid and capillary storage with 0.1M hydrochloric acid ensured good separation reproducibility of 217 runs during an extended period of use.

Photo-Induced Geometry and Polarity Gradients in Covalent Organic Frameworks Enabling Fast and Durable Molecular Separations.

Azobenzene, which activates its geometric and chemical structure under light stimulation enables noninvasive control of mass transport in many processes including membrane separations. However, producing azobenzene-decorated channels that have precise size tunability and favorable pore wall chemistry allowing fast and durable permeation to solvent molecules, remains a great challenge. Herein, an advanced membrane that comprises geometry and polarity gradients within covalent organic framework (COF) nanochannels utilizing photoisomerization of azobenzene groups is reported. Such functional variations afford reduced interfacial transfer resistance and enhanced solvent-philic pore channels, thus creating a fast solvent transport pathway without compromising selectivity. Moreover, the membrane sets up a densely covered defense layer to prevent foulant adhesion and the accumulation of cake layer, contributing to enhanced antifouling resistance to organic foulants, and a high recovery rate of solvent permeance. More importantly, the solvent permeance displays a negligible decline throughout the long-term filtration for over 40 days. This work reports the geometry and polarity gradients in COF channels induced by the conformation change of branched azobenzene groups and demonstrates the strong capability of this conformation change in realizing fast and durable molecular separations.

Multicapillary columns with ionic liquids as stationary liquid phase

The study demonstrates the possibility of using ionic liquids (IL) as a stationary liquid phase (SLP) for gas chromatographic (GC) multicapillary columns (MCC). Three types of IL of three classes were employed as SLP: Imidazolium, Pyridinium and Quinolinium. Dependences of the MCCs efficiency on the carrier gas flow rate were obtained. Highest efficiency was achieved on the column with 1,2-Dimethyl-3-propylimidazolium bis(trifluoromethylsulfonyl)imide (DiMPrIm). For this column, dependence of the efficiency on the sample volume has been investigated. Also the loading capacity of the MCC with DiMPrIm was determined. Separation of fatty acid esters and phenols served as an example to demonstrate that using ionic liquids as SLP for МСС make it possible to combine fast separations with high selectivity.

Employment of chiral columns with superficially porous particles in chiral separations of cobalt bis (dicarbollide) and nido-7,8-C2 B9 H12 (1-) derivatives.

Derivatives of the nido-7,8-C2 B9 H12 (1-) (dicarbollide ion) and [3,3'-Co-(1,2-C2 B9 H11 )2 ](1-) cobalt sandwich (COSAN) ion represent groups of extremely chemically and thermally stable abiotic compounds. They are being investigated in many research areas, that is, medicinal chemistry, material sciences, analytical chemistry, and electrochemistry. The chirality of these compounds remains still grossly overlooked, what is also reflected in limited number of reports on their chiral separations. Continued progress depends on reliable, fast, and cost-effective methods for such separations. Recently, chiral separations of COSAN derivatives were achieved in liquid chromatography and supercritical fluid chromatography. Only five anionic derivatives of nido-7,8-C2 B9 H12 (1-) were successfully enantioseparated in liquid chromatography. Efforts to separate anionic nido-7,8-C2 B9 H12 (1-) in supercritical chromatography have failed, and only a few dicarbollide ions were separated using liquid chromatography. Generally, all chiral separations in liquid chromatography took about 30 min. Herein, we identify a versatile column capable of separating both COSAN and nido-7,8-C2 B9 H12 (1-) derivatives and achieve faster analyses times employing commercially available superficially porous chiral stationary phases. The semisynthetic hydroxypropyl β-cyclodextrin-based column (CDShell-RSP) is identified as the column of choice from the tested columns by separating 19 of 27 compounds from each structural motifs tested mainly in less than 10min. The dihydroxyalkyl, oxygen-bridged hydroxyalkyl, and bisphenylene-bridged COSAN derivatives were baseline separated in less than 5min exceeding the results of supercritical fluid chromatography. Methods developed herein will aid synthetic chemists without the possession of a supercritical fluid chromatograph to achieve fast chiral separations of COSAN and derivatives of nido-7,8-C2 B9 H12 (1-) on a common liquid chromatograph without the need of dedicated instrumentation.

Automated Regularized Deconvolution for Eliminating Extra-Column Effects in Fast High-Efficiency Separations.

With the introduction of ultrahigh efficiency columns and fast separations, the need to eliminate peak deformation contributed by the instrument must be effectively solved. Herein, we develop a robust framework to automate deconvolution and minimize its artifacts, such as negative dips, wild noise oscillations, and ringing, by combining regularized deconvolution and Perona-Malik (PM) anisotropic diffusion methods. A asymmetric generalized normal (AGN) function is proposed to model the instrumental response for the first time. With no-column data at various flow rates, the interior point optimization algorithm extracts the parameters describing instrumental distortion. The column-only chromatogram was reconstructed using the Tikhonov regularization technique with minimal instrumental distortion. For illustration, four different chromatography systems are used in fast chiral and achiral separations with 2.1 and 4.6 mm i.d. columns. Ordinary HPLC data can approach highly optimized UHPLC data. Similarly, in fast HPLC-circular dichroism (CD) detection, 8000 plates were gained for a fast chiral separation. Moment analysis of deconvolved peaks confirms correction of the center of mass, variance, skew, and kurtosis. This approach can be easily integrated and used with virtually any separation and detection system to provide enhanced analytical data.

Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

Currently, fast liquid chromatographic separations at low temperatures are exclusively used for the separation of peptides generated in hydrogen deuterium exchange (HDX) workflows. However, it has been suggested that capillary electrophoresis may be a better option for use with HDX. We performed in solution HDX on peptides and bovine hemoglobin (Hb) followed by quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for benchmarking a laboratory-built HDX-MS platform. We found that capillaries with a neutral coating to eliminate electroosmotic flow and adsorptive processes provided fast separations with upper limit peak capacities surpassing 170. In contrast, uncoated capillaries achieved 30% higher deuterium retention for an angiotensin II peptide standard owing to faster separations but with only half the peak capacity of coated capillaries. Data obtained using two different separation conditions on peptic digests of Hb showed strong agreement of the relative deuterium uptake between methods. Processed data for denatured versus native Hb after deuterium labeling for the longest timepoint in this study (50,000 s) also showed agreement with subunit interaction sites determined by crystallographic methods. All proteomic data are available under DOI: 10.6019/PXD034245.

Ultra-Fast Middle-Up Reversed Phase Liquid Chromatography Analysis of Complex Bispecific Antibodies Obtained in Less Than One Minute.

This work illustrates the benefits and limitations of using ultra-short reversed phase liquid chromatography (RPLC) columns for the characterization of various complex bispecific antibodies after prolonged thermal stress at the middle-up level of analysis. First, we have demonstrated that alternative organic modifiers, such as isopropanol, can be used in RPLC mode without generating excessive pressure, thanks to the prototype 10 × 2.1 mm, 2.7 µm particle column. However, compared to acetonitrile, the selectivity was not improved, at least for the selected biopharmaceutical products. Importantly, very fast separations (sub-1 min) of high quality were systematically obtained for the different samples when using a spectroscopic detector, but a severe loss of performance was observed with mass spectrometry (MS) detection due to dispersion effects. Based on these results, there is a clear need to improve the interfacing between LC and MS (shorter/thinner tubing) to mitigate band broadening.

Rapid and simultaneous determination of dichloroacetic acid, trichloroacetic acid, ClO3−, ClO4−, and BrO3− using short-column ion chromatography-tandem electrospray mass spectrometry

A simple, accurate and reliable analytical method has been developed for the simultaneous determination of dichloroacetic acid, trichloroacetic acid, chlorate, perchlorate and bromate by using ion chromatography coupled with electrospray ionization tandem mass spectrometry (IC-ESI-MS/MS). To reduce the sample analysis time, a short column AG18 was evaluated to perform fast separations of the target analytes. Using the combination of 0.1 M aqueous methylamine solution and acetonitrile as the mobile phase under the optimized gradient elution mode, all 5 analytes could be resolved satisfactorily from the column within 5 min. The limits of detection and limits of quantification for 5 analytes were in the range of 0.09–0.28 μg/L and 0.28–0.84 μg/L, respectively. The method recoveries for the analytes spiked into tap water at a concentration of 5 μg/L were in the range of 69.6–118.3%; the precision values (as intra- and inter-day relative standard deviations) were in the range of 5.1–9.9%. The proposed method was subject to very limited interference from the common substances in the water matrix, such as Cl− (25–200 mg/L), SO4− (25–200 mg/L), HCO3−/CO32− (1–6 mM) and natural organic matter (1–8 mg/L). For environmental applications, the method successfully monitored haloacetic acids and oxyhalides yields in laboratory chlorination experiments.

Stability-Indicating Analytical Method Development and Validation of Thiocolchicoside and Ibuprofen in Tablet Dosage Form by RP-HPLC Method

Objective: First time, a simple, specific, accurate and economic stability-indicating reverse phase high performance liquid chromatographic method was reported for the simultaneous estimation of THIO and IBU in tablet dosage form. Method: The method has shown adequate separation of THIO and IBU from their degradation products. Separation was achieved on an Inertsil, 3V ODS C18, 4.6 mm x 250 mm, 5μ column at wavelength of 248 nm, using a mobile phase Methanol: Dist. water (50:50, v/v) in a mode of isocratic elution at a flow rate of 1.0 ml/min. Results: This method results has minimum retention time for THIO and IBU i.e. 2.317 and 1.075 min. correspondingly, which gives fast separations of drugs furthermore both drug combinations are subjected to acidic, base, oxidation, thermal and photolytic stress environment. Thus stressed samples of these drugs are analyzed by the proposed analytical method. Quantitation was achieve with UV detection at 248 nm based on peak area with linear calibration curve at concentration range 100-600 ppm for THIO and 400-2400 ppm for IBU. The LOD's found 2.00 and 0.54 for THIO and IBU in addition to that LOQ's were found to be 6.08 and 1.63 resp. Conclusion: The statistical analysis proved that this novel proposed method was established to be specific, accurate, precise and stability-indicating study do not shows interfering peaks of degrades and excipient. The proposed method is therefore suitable for purpose in quality-control laboratories for quantitative analysis of THIO and IBU drugs individually and in combined dosage form, as it is performed and validated as per ICH Q2 (R1) and Q1A (R2) guideline and it meets to specific acceptance criteria.
 Keywords: Thiocolchicoside (THIO), Ibuprofen (IBU), RP-HPLC, Stability-indicating, ICH.

Considerations for Generating Frequency Modulation Waveforms for Fourier Transform-Ion Mobility Experiments.

By casting the information regarding an ion population's mobility in the frequency domain, the coupling of time-dispersive ion mobility techniques is now imminently compatible with slower mass analyzers such as ion traps. Recent reports have detailed the continued progress toward maximizing the efficiency of the Fourier transform ion mobility-mass spectrometry (FT-IM-MS) experiments, but few reports have outlined the intersection between the practical considerations of implementation against the theoretical limits imposed by traditional signal processing techniques. One of the important concerns for Fourier-based multiplexing experiments is avoiding signal aliasing as a product of undersampled signals that may occur during data acquisition. In addition to traditional considerations such as detector sampling frequency, the limitations (i.e., maximum measurable drift time) imposed by experimental mass scan duration and the frequency sweep used for ion gate modulation must also be assessed. This work aims to connect the fundamental underpinnings of FT-IM-MS experiments and the associated experimental parameters that are encountered when coupling the comparatively fast separations in the mobility domain with the slower m/z scanning common for ion-trap mass analyzers. In addition to stating the relevant theory that applies to the FT-IM-MS experiment, this report highlights how aliased signals will manifest post Fourier transform in reconstructed arrival time distributions and calculated mobilities.

An Overview of Monolithic Column: Types, Parameters and Applications

The column is the main component for chromatographic separation. Nowadays, monolithic columns are graining more popularity in the field of separation media for liquid chromatography. The monolith columns possess great potential as compared to the conventional packed column in terms of preparing complex mixtures. These columns provide various properties like higher permeability, high-efficiency fast separations, high flow rate with lower backpressure, fast mass transfer kinetics with a high binding capacity. It is categories into three columns and they are organic monolithic column, inorganic monolithic column and hybrid monolithic column and all three types of monolithic column differ through their porous properties. In this review, the various advantage of the high-efficiency monolithic column with recent advances, the origin of the concept, the various parameter of the monolithic stationary phase and the application of monolithic columns are illustrated. It is better column in comparison of selectivity, reproducibility and performance.
 Keywords: Monolithic column, Packed columns, Inorganic and organic monolithic column, Column parameters, Pharmaceutical Applications

Underwater Unidirectional Cellular Fluidics.

The unidirectional fluidics underwater promises the manipulation of gas/liquid for various significant applications. Inspired by the unique stomata on the surface of hornwort stems and leaves that enable the transport and storage of oxygen underwater, we propose a bionic cell with porous membranes fabricated by the projection microstereolithography based 3D printing technique. Different Laplace forces coming from different contact angles for the respectively superhydrophilic outside and hydrophobic inside promise unidirectional fluidic performance, which stop water flowing inside of the bionic cell while exhausting gas and liquid outside of it. In addition, geometric parameters of the bionic cell make a big difference in its unique unidirectional fluidic performance. Simultaneously, the underlying mechanisms of the unidirectional penetration of liquid in our 3D printed bionic cell are theoretically revealed. Moreover, we demonstrate potential applications of our bionic cell with underwater anaerobic chemical reactions to fully apply its outstanding unidirectional fluidics underwater. Our bionic cell opens a gate for potential applications in chemical and microfluidic engineering underwater, such as the storage of flammable materials, fast solid-liquid separations, and anaerobic chemical reactions.

Investigation of the effects of solvent-mismatch and immiscibility in normal-phase × aqueous reversed-phase liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive separation technique that allows achieving high peak capacities and information on chemical correlations. Unfortunately, its application in industrial practice is still not widespread due to limiting factors such as complex method development, tedious method optimization and solvent-incompatibility (such as solvent-strength mismatch or immiscibility experienced during fraction transfer).A severe case of solvent-incompatibility is encountered in the comprehensive coupling of normal-phase LC and reversed-phase LC (NPLC × RPLC). NPLC × RPLC is considered a desirable LC × LC system, especially for the characterization of synthetic polymers, due to the high orthogonality of the two retention mechanisms. However, its experimental realization often suffers from solvent-injection effects in the RPLC dimension, such as peak-deformation, peak-splitting, or even unretained elution (“breakthrough”) of sample components. Such a decrease in performance or loss of retention is highly dependent on the types of solvents used.To explore the boundaries of solvent compatibility, we applied large-volume injections (LVI) of reference analytes (e.g. alkyl benzenes; ethoxylate and propoxylate polymers) dissolved in water-immiscible sample solvents, such as dichloromethane, n-hexane, and isooctane in fast water-based gradient RPLC separations (using methanol or acetonitrile as eluent). It was found that, when using highly aqueous initial gradient conditions, hydrophobic sample diluents were retained and eluted during the applied gradient. Depending on the relative retention of the retained diluent and the sample analytes, good chromatograms for LVI of immiscible solvents were obtained, comparable with injections under ideal conditions. The conclusions from injection experiments in aqueous RPLC were verified by coupling an NPLC system with a gradient from isooctane to tetrahydrofuran and an RPLC system with a gradient from water to acetonitrile in an online comprehensive NPLC × RPLC separation of a mixture of propoxylate polymers. The separation provided separation of the polymers based on their number of hydroxyl end-groups (NPLC) and oligomer chain-length (RPLC), without suffering from significant band-broadening effects due to solvent-mismatch upon injection in the second-dimension RPLC system.

Realizing “net-zero-carbon” sustainable aviation fuel

Realizing “net-zero-carbon” sustainable aviation fuel

The Utilization of Triacontyl-Bonded Silica Coated with Imidazolium Ions for Capillary Ion Chromatographic Determination of Inorganic Anions

This paper describes the separation and detection of five inorganic anions (I - , BrO3 - , NO2 - , Br - , and NO3 -) by capillary ion chromatography using 1-alkyl-3-methylimidazolium-coated columns and UV detection at 210 nm. The salts of imidazolium (IM) ions (C2-, C4-, C6-, C8-, C10-, and C18-IMs) are a family of ionic liquids and were newly examined as permanent coating reagents on a triacontyl (C30) -bonded silica reversed phase column. When 40 mM NaCl aqueous solution was used as an eluent, the elution time of these anions was increased with the length of alkyl chain between C2- and C10-IMs, but turned to decrease with C18-IM. Good and faster separations were attained using a C18-IM-coated column with 150 mM NaCl in 25%(v/v) acetonitrile-75%(v/v) water. The method was successfully applied to the determination of nitrite, bromide, and nitrate in seawater and river water.

Ultra-short ion-exchange columns for fast charge variants analysis of therapeutic proteins

The purpose of this work was to study the potential of recently developed ultra-short column hardware for ion exchange chromatography (IEX). Various prototype and commercial columns having lengths of 5, 10, 15, 20 and 50 mm and packed with non-porous 3 µm particles were systematically compared. Both pH and salt gradient modes of elution were evaluated. Similarly, what has been previously reported for reversed phase liquid chromatography (RPLC) mode, an “on-off” retention mechanism was observed in IEX for therapeutic proteins and their fragments (25–150 kDa range).Because of the non-porous nature of the IEX packing material, the column porosity was relatively low (ε = 0.42) and therefore the volumes of ultra-short columns were very small. Based on this observation, it was important to reduce as much as possible all the sources of extra-column volumes (i.e. injection volume, extra-bed volume, detector cell volume and connector tubing volume), to limit peak broadening. With a fully optimized UHPLC system, very fast separations of intact and IdeS digested mAb products were successfully performed in about 1 min using an IEX column with dimensions of 15 × 2.1 mm. This column was selected for high-throughput separations, since it probably offers the best compromise between efficiency and analysis time. For such ultra-fast separations, PEEK tubing was applied to bypass the column oven (column directly connected) to the optical detector via a zero dead volume connection.

Large-pore covalent organic frameworks for ultra-fast tight ultrafiltration (TUF)

Tight ultrafiltration (TUF) membranes featuring specific pore sizes are increasingly developed to bridge the gap between nanofiltration and ultrafiltration. So far, a wealth of efforts has been devoted to tackle the limitation found in TUF, but there still lacks a facile accessibility to upgrade TUF membranes with simultaneously improved permeance and selectivity. Herein, we report a large-pore covalent organic framework (LP-COF) as the building material to prepare ultra-permeable TUF membranes for fast separations. The LP-COF layers with an exceptionally large aperture size of up to ~3.6 nm are synthesized on macroporous substrates through a unidirectional diffusion method. The resultant LP-COF layers show a moderate crystallinity and low thickness down to ~60 nm, and allow ultrafast water permeation. Surprisingly, the optimal LP-COF membrane exhibits an unprecedented water permeance of ~3147 L m−2 h−1 MPa−1 with a high Congo red rejection (~92.6%), which is basically unchanged after several cycles of filtration. Moreover, the large-pore channels enable an unimpeded pass of ions, thus affording the membrane an excellent dye/salt separation competence, and largely exceeding state-of-the-art membranes. Therefore, this work opens up a new opportunity in producing high-performance TUF membranes by large-pore COFs for rapid and precise separations.

Donaldson Co Inc, USA

We examine the use of a z2 flow distribution and collection feature for enhancing the separation efficiency of a laterally-fed membrane chromatography (or LFMC) device. The new device is designated z2LFMC. Two devices were fabricated using two different ion-exchange membranes: strong anion exchange (Q) with 0.8-micron pore size, and strong cation exchange (S) with pore size in the 3–5 µm range. The number of theoretical plates per unit membrane bed height in these z2LFMC devices were higher than those reported for other membrane chromatography devices housing similar membranes, including the older versions of LFMC devices. The enhancement in separation efficiency is explained based on computational fluid dynamic (CFD) simulations. Model protein separation experiments showed that the z2LFMC device gave similar resolution as an equivalent resin-packed column, at 40-time greater flow rate than that used with the column. At a comparable flow rate, the resolution obtained with the z2LFMC device was significantly higher than that obtained with the equivalent resin-packed column. Therefore, the z2LFMC device combines high-speed with high-resolution. We demonstrate the suitability of the z2LFMC devices for carrying out fast and efficient biopharmaceutical separations through two case studies, i.e. the fractionation of monoclonal antibody charge variants, and monoclonal antibody aggregates separation.

Fundamental to achieving fast separations with high efficiency: A review of chromatography with superficially porous particles.

Types of particles have been fundamental to LC separation technology for many years. Originally, LC columns were packed with large-diameter (>100 μm) calcium carbonate, silica gel, or alumina particles that prohibited fast mobile-phase speeds because of the slow diffusion of sample molecules inside deep pores. During the birth of HPLC in the 1960s, superficially porous particles (SPP, ≥30 μm) were developed as the first high-speed stationary-phase support structures commercialized, which permitted faster mobile-phase flowrates due to the fast movement of sample molecules in/out of the thin shells. These initial SPPs were displaced by smaller totally porous particles (TPP) in the mid-1970s. But SPP history repeated when UHPLC emerged in the 2000s. Stationary-phase support structures made from sub-3-μm SPPs were introduced to chromatographers in 2006. The initial purpose of this modern SPP was to enable chromatographers to achieve fast separations with high efficiency using conventional HPLCs. Later, the introduction of sub-2-μm SPPs with UHPLC instruments pushed the separation speed and efficiency to a very fast zone. This review aims at providing readers a comprehensive and up-to-date view on the advantages of SPP materials over TPPs historically and theoretically from the material science angle.