Capillary Column Liquid Research Articles

High-Throughput Capillary Liquid Chromatography Using a Droplet Injection and Application to Reaction Screening.

The cycle time of a standard 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. Here, a droplet injector system was established that replaces the autosampler with a four-port, two-position valve equipped with a 20 nL internal loop interfaced to a syringe pump and a three-axis positioner to withdraw sample droplets from a well plate. In the system, sample and immiscible fluid are pulled alternately from a well plate into a capillary and then 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 8.1 min with 5-s cycle time. This system was coupled to a mass spectrometer through an electrospray ionization source for high-throughput chemical reaction screening.

Organic-silica hybrid monolithic sorbents for sample preparation techniques: A review on advances in synthesis, characterization, and applications

Organic-silica hybrid monolithic materials have attracted considerable attention as potential stationary phases in separation science. These materials combine the advantages of organic polymer and silica-based monoliths, including easy preparation, lower back pressure, high permeability, excellent mechanical strength, thermal stability, and tunable surface chemistry with high surface area and selectivity. The outstanding chromatographic efficiency as stationary phase of hybrid monolithic capillary columns for capillary liquid chromatography and capillary electrochromatography has been reported in many papers. Organic-silica hybrid monolithic materials have also been extensively used in the field of sample preparation. Owing to their surface functionalities, these porous sorbents offer unique selectivity for pre-concentration of different analytes in the most complex matrixes by fast dynamic transport. These sorbents not only improve the analytical method sensitivity, but also introduce novelties in terms of extraction devices and instrument coupling strategies. The current review covers the period spanning from 2017 to 2023 and describes the properties of organic-inorganic hybrid monolithic materials, the present status of this technology and summarizes recent developments in their use as innovative sorbents for microextraction sample preparation techniques (solid phase microextraction with pipette tip, offline in-tube SPME, in-tube SPME online with LC, and in-tube SPME directly coupled with mass spectrometry). Aspects such as the synthesis methods (sol–gel process, one-pot approach, and polyhedral oligomeric silsesquioxanes-based procedure), characterization techniques, and strategies to improve extraction efficiency in various applications in different areas (environmental, food, bioanalysis, and proteomics) are also discussed.

Practical aspects of preparation, optimization, and evaluation of poly(styrene-divinylbenzene)porous-layer open tubular (PLOT) columns for capillary liquid chromatography

Using open tubular (OT) capillary columns in the nano and micro liquid chromatography scales has several advantages over conventional large-bore packed columns. Among the open tubular columns, those of the PLOT (porous layer open tubular) type are more suitable for liquid chromatography due to the porous character of the stationary phase that provides more significant interaction with the analytes. In addition, recent instrumental advances in capillaryLC coupled with mass spectrometry (MS) have opened up new avenues for developing and applying OT-LC-MS. This work discusses the possibility of applying PLOT columns in the capillary LC flow range (0.5–2 μL min−1) and describes the preparation, optimization, and evaluation of those columns. Poly(styrene-divinylbenzene) (PS-DVB) was used as the stationary phase, and the internal diameter of the columns discussed in this work was 10 and 25 μm. A comparison between these diameters is presented regarding fabrication and practical aspects of the operation. Additionally, for the 25 μm PLOT column, the gradient window and the flow rate effects on the system's performance were investigated. Flow rates of 1 μL min−1 and a gradient window of 60% (from 20 to 80% of acetonitrile) were the best conditions to work on this column in capillary LC conditions.

Investigation of the applicability of silica-graphene hybrid materials as stationary phases for capillary liquid chromatography

Graphene and graphene-derived substances are cutting-edge materials receiving increasing attention in the analytical chemistry field. Graphene oxide sheets bonded to amino silica particles functionalized with octadecyl (C18) groups and endcapped, also known as SiGO-C18ec, have been successfully employed as extraction phases and in analytical columns associated with conventional liquid chromatography (LC). In this work, SiGO-C18ec particles of 3, 5, and 10 µm nominal id were employed to pack capillary LC columns (100 mm long x 0.3 mm id), and their performance in the gradient mode was evaluated and compared. A 3 µm C18 capillary LC column (50 x 0.3 mm) was used as a reference column. Eight analytes having different polarities and topological surface areas were selected as a probe in this study: carbofuran clomazone, hexazinone, carbamazepine, citalopram, clomipramine, desipramine, and ochratoxin A. Studies about orthogonality were performed to investigate the orthogonality between the SiGO-C18ec and C18 phases. Among the SiGO-C18ec phases investigated, the column packed with 5 µm SiGO-C18ec particles presented the best peak capacity (29) in 15 min.Additionally, the performance of the columns packed with 5 µm SiGO-C18ec particles overcame the performance of the C18 columns used. Significant orthogonality was found between C18 and SiGO-C18ec packed columns; however, no significant differences were found between columns packed with SiGO-C18ec particles of different diameters.

Phenyl-bonded monolithic silica capillary column liquid chromatographic separation and detection of fluorogenic derivatized intact proteins.

Prior to the identification of proteins for proteomics analysis in human cells, separation of fluorogenic derivatized proteins with a fluorogenic reagent, 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide, has typically been performed by using a conventional reversed-phase HPLC column. However, the number of proteins in human cells (HepaRG) that are separated by this conventional approach is limited to approximately 500. In this study, a nanoflow liquid chromatography system with an evaluated phenyl-bonded monolithic silica capillary column (0.1 mm i.d., 700 mm length) was used to increase the number of separated fluorogenic derivatized proteins. This system was used to separate derivatized human cell proteins (K562) and yeast (Saccharomyces cerevisiae) proteins as model cell proteomes. More than 1,300 protein peaks were separated/detected from both cell proteomes. We present a straightforward comparison of multiple separation profiles using a novel chromatogram display approach, termed the "spiderweb" chromatogram. In addition, to validate that the detected peaks are derived from proteins, a mass spectrometer was connected to the capillary column and deconvolution of the obtained mass spectra was performed. Furthermore, different molecular weight distribution profiles of the expressed proteins were observed between the two cell proteomes.

A Sol–gel Method for Preparing Poly (N-Isopropyl Acrylamide) Hybrid Column and Its Application in Small Molecular Analysis Using Capillary Liquid Chromatography

A poly (N-isopropyl acrylamide) (polyNIPA)-based hybrid column for capillary liquid chromatography (cLC) was prepared with N-isopropyl acrylamide (NIPA) and alkoxysilanes through a sol–gel “one-pot” method. In this process, the hydrolyzed tetramethoxysilane (TMOS) and vinyltrimethoxysilane (VTMS) were the precursors for the synthesis of a silica-based monolith and subsequently reacted with organic monomer (NIPA) via free radical polymerization within the confines of a capillary. The effects of polymerization temperature, the volume of TMOS, VTMS, and acetic acid (HAc) as well as the amount of progens [polyethylene glycol 10,000 (PEG10000) and urea] on the morphology and permeability were investigated in detail. Uniform pore structure and good mechanical stability were obtained for columns prepared under optimized conditions. Separation of various neutral, acidic and basic analytes was achieved by cLC in reversed-phase liquid chromatography (RP-LC) mode. Moreover, the resulting hybrid column was also applied for the analysis of small organic pollutants, including polycyclic aromatic hydrocarbons (PAHs), perfluorinated compounds (PFCs) and chlorophenols (CPs). The successful application demonstrated its potential in the separation of small molecules.

Portable capillary liquid chromatography for pharmaceutical and illicit drug analysis.

A newly developed portable capillary liquid chromatograph was investigated for the separation of various pharmaceutical and illicit drug compounds. The system consists of two high-pressure syringe pumps capable of delivering capillary-scale flow rates at pressures up to 10000psi. Capillary liquid chromatography columns packed with sub-2μm particles are housed in cartridges that can be inserted into the system and easily connected through high-pressure fluidic contact points by simply applying a specific, predetermined torque rather than using standard fittings and less precise sealing protocols. Several over-the-counter analgesic drug separations are demonstrated, along with a simple online measurement of tablet dissolution. Twenty illicit drug compounds were also separated across six targeted drug panels. The results described in this study demonstrate the capability of this compact liquid chromatography instrument to address several important drug-related applications while simplifying system operation, and greatly reducing solvent usage and waste generation essential for onsite analysis.

Multiplicative On-Column Solute Focusing Using Spatially Dependent Temperature Programming for Capillary HPLC.

The benefits of capillary liquid chromatography columns are truly realized when small, limited sample volumes require signal enhancement, but the available sample volume does not permit on-column focusing during injection onto a larger column. This dilemma is common when samples are naturally small or precious (such as in biological, forensic, art, and archeological investigations) and analyte concentrations are low. Signal enhancement by solvent-based focusing is effective with capillary columns, but it is limited to a single band-compression step and can only be achieved at the inlet. Here we evaluate multiplicative temperature-assisted solute focusing using a linear array of ten independently controlled 1.0 × 1.0 cm thermoelectric cooling elements (TECs) to generate dynamic temperature changes along the length of the column. The evaluation has two prongs: simulation and experimental. Simulation is required to understand the effect of a particular temperature change at a particular place and time on the column to determine optimal timing of temperature changes. Because the accuracy of the simulations is good, as long as the effect of temperature on retention factor is known, experimental conditions required to achieve a particular focusing objective can be estimated. We evaluated the capability of the technique to selectively focus only one of two solutes. This was achieved using three adjacent zones with temperature controlled by (upstream first) four, two, and one TECs. The three focusing steps occurring on column gave a 20-fold increase in peak height without solvent-based focusing for a solute with modest retention enthalpy.

Controlled crosslinking of trimethylolpropane trimethacrylate for preparation of organic monolithic columns for capillary liquid chromatography.

Organic monolithic columns based on single crosslinking of trimethylolpropane trimethacrylate (TRIM) monomer were prepared in a single step by living/controlled free-radical polymerization. Full optimization of the preparation, such as using different percentages of TRIM and different amounts of radical promoter as well as various porogen solvents were explored. The resulting monolithic columns were characterized by scanning electronic microscopy and nitrogen sorption for structure morphology studies and surface area measurements, respectively. Using capillary liquid chromatography, 150μm i.d. columns were applied to separate a mixture of small hydrophobic molecules. The results indicated that column performance is highly sensitive to the type and the amount of porogen solvents used in the polymerization mixture composition. Good resolution factors and methylene selectivity were obtained, indicating the promising potential of this material for capillary liquid chromatography separations.

Highly sensitive method for the quantification of trans-linolenic acid isomers in trilinolenin of edible oils using an ionic liquid capillary column.

The polarities of linolenic acid isomers are very similar, and only a few studies to date have attempted to separate α-linolenic acid (ALA) isomers completely. The aim of this study was to fill this gap by developing and validating an accurate method for the analysis of ALA isomers in trilinolenin at 200, 220 and 240 °C using a gas chromatograph-flame ionization detector equipped with an SLB-IL111 capillary column. Results showed that eight ALA isomer standards were separated effectively using these optimized gas chromatographic conditions. The coefficient of determination was r2 > 0.9994 in the linear range of each ALA isomer. The obtained limits of detection and limits of quantification of the ALA isomers were 0.02-0.08 ppm and 0.05-0.22 ppm, respectively. A high degree of reproducibility and percent recoveries between 96.2% and 106.5%, with coefficients of variation ranging from 0.82% to 0.97%, were achieved. The developed method has been successfully applied to the analysis of ALA isomers in heated pure trilinolenin as well as to trilinolenin in various edible oils, and the TALA isomerization pathways in heated trilinolenin were verified. © 2017 Society of Chemical Industry.

Effect of sporopollenin microparticle incorporation into the hexyl methacrylate-based monolithic columns for capillary liquid chromatography

ABSTRACTSporopollenin microparticles have been prepared form Lycopodium clavatum spores, defatted and incorporated into a porous methacrylate polymer monolith to enhance liquid chromatographic performance of different sets of small neutral molecules. A stable suspension between sporopollenin microparticles and porogenic solvents composed of 1-propanol and 1,4-butandiol has proved before preparation, and seven compositions with increasing sporopollenin microparticles were prepared inside fused silica tubing. After optimizing of the preparation conditions, the structure of the stationary phase was characterized by scanning electron microscopy, surface area analysis, thermodynamic study, short- and long-term precision, and hydrodynamic properties including mechanical stability, porosity, and permeability. The columns were successfully applied to improve the separation efficiency of different mixtures using capillary liquid chromatography. Addition of very small amount of sporopollenin microparticles to the methacrylate mixture enhanced the column efficiency from 3 to 5 times for ketonic and phenolic compounds and reduced the retention with the corresponding better resolution and peak shapes for all studied compounds.

New evidences on efficacy of boronic acid-based derivatization method to identify sugars in plant material by gas chromatography–mass spectrometry

This work presents an analytical procedure based on gas chromatography–mass spectrometry which allows the determination of aldoses (glucose, mannose, galactose, arabinose, xylose, fucose, rhamnose) and chetoses (fructose) in plant material.One peak for each target carbohydrate was obtained by using an efficient derivatization employing methylboronic acid and acetic anhydride sequentially, whereas the baseline separation of the analytes was accomplished using an ionic liquid capillary column. First, the proposed method was optimized and validated. Successively, it was applied to identify the carbohydrates present in plant material. Finally, the procedure was successfully applied to samples from a XVII century painting, thus highlighting the occurrence of starch glue and fruit tree gum as polysaccharide materials.

Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry

Methodology for sequence analysis of ∼150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8murea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3-9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous post-translational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms ofN-linked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion-ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides.

Gas chromatographic separation of fatty acid esters of cholesterol and phytosterols on an ionic liquid capillary column.

Steryl esters are high molecular weight compounds (600-700g/mol) regularly present as a minor lipid class in animal and plant lipids. Different sterol backbones (e.g., cholesterol, β-sitosterol and brassicasterol) which can be esterified with various fatty acids can result in highly complex steryl ester patterns in food samples. The gas chromatographic (GC) analysis of intact steryl esters is challenging, since high elution temperatures are required for their elution. On nonpolar GC phases, steryl esters with fatty acids with differing degree of unsaturation (e.g., oleate and linoleate) cannot be separated and there are only few polar columns available with sufficient temperature stability. In this study, we used gas chromatography with mass spectrometry (GC/MS) and analyzed intact steryl esters on a commercial room temperature ionic liquid (RTIL) column which was shortened to a length of 12m. The column separated the steryl esters both by total carbon number and by degree of unsaturation of the fatty acid. For instance, cholesteryl esters with stearic acid (18:0), oleic acid (18:1n-9), linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) could be resolved (R≥1.3) from each other. By analysis of synthesized standard substances, the elution orders for different steryl backbones and different fatty acids on a given sterol backbone could be determined. Analysis of spreads and plant oils allowed to determine retention times for 37 steryl esters, although a few co-elutions were observed. The ionic liquid column proved to be well-suited for the analysis of intact steryl esters.

Capillary Action Liquid Chromatography: New Chromatographic Technique for the Separation and Determination of Colour Substances

The aim of this study is to compare the chromatographic separation of dyes, used in food industry, by thin-layer chromatography and capillary column liquid chromatography techniques. The same separation systems (stationary phase containing adsorbent type RP-8; and mobile phase containing methanol–water, with the modifier) were used for both techniques.

Cholesterol-based polymeric monolithic columns for capillary liquid chromatography. Part II

This paper presents a second part of our research devoted to cholesterol-based polymeric monolithic stationary phase. The obtained capillary columns were successfully used for separations of alkylbenzenes, steroid hormones and polycyclic aromatic hydrocarbons during isocratic or gradient elutions. The columns showed excellent thermal stability. Increasing the temperature resulted in decrease of the retention factors and peak widths, but selectivity and efficient separations have been retained in the studied temperature range of 20 to100°C. Additionally, the van’t Hoff model presented a non-linear relationship of lnk versus 1/T plots, which is likely the result of liquid crystal properties of cholesterol. The studied capillary monolithic columns showed extremely fast re-equilibration after gradient elution and found to be stable under such conditions as: fast flow rate, high acetonitrile content in the mobile phase (no swelling) and high temperature.

Regiospecific Distribution of trans-Octadecenoic Acid Positional Isomers in Triacylglycerols of Partially Hydrogenated Vegetable Oil and Ruminant Fat.

It is revealed that binding position of fatty acid in triacylglycerol (TAG) deeply relates to the expression of its function. Therefore, we investigated the binding positions of individual trans-octadecenoic acid (trans-C18:1) positional isomers, known as unhealthy fatty acids, on TAG in partially hydrogenated canola oil (PHCO), milk fat (MF), and beef tallow (BT). The analysis was carried out by the sn-1(3)-selective transesterification of Candida antarctica Lipase B and by using a highly polar ionic liquid capillary column for gas chromatography-flame ionization detection. Trans-9-C18:1, the major trans-C18:1 positional isomer, was selectively located at the sn-2 position of TAG in PHCO, although considerable amounts of trans-9-C18:1 were also esterified at the sn-1(3) position. Meanwhile, trans-11-C18:1, the major isomer in MF and BT, was preferentially located at the sn-1(3) position. These results revealed that the binding position of trans-C18:1 positional isomer varies between various fats and oils.

A Novel Fluorinated Boron Nitride Nanotube Organic Monolithic Column for Capillary Liquid Chromatography

A novel HPLC stationary phase based on fluorinated boron nitride nanotubes (F-BNNTs) incorporated into a monolithic polymeric material was developed. This F-BNNT stationary phase was synthesized to combine the analytical performance of boron nitride nanotubes and the fluorine-based unique selectivity for polar compounds. This F-BNNT column appeared to work well when fluorinated or halogenated compounds were encountered.

Nanostructured porous polymer monolithic columns for capillary liquid chromatography of peptides

The macroporous structure of poly(styrene-co-divinylbenzene) monolithic capillary columns has been optimized for the gradient separation of peptides. To exploit monolithic supports with porosity exceeding 70%, the thermodynamic properties of the polymerization mixture were carefully tailored to yield homogeneous monolithic materials featuring macropore and polymer microglobule sizes in the range of 50–200nm. The effects of (i) initiator content, (ii) composition of porogenic mixture, comprising tetrahydrofuran and 1-decanol, (iii) percentage of divinylbenzene crosslinker, and (iv) monomers to porogen ratio on the morphology was investigated. The resulting column structures were investigated using scanning electron microscopy and the prepared monolithic columns were tested for the separation of a tryptic digest of cytochrome c while applying a fixed flow rate and gradient time. To obtain a better understanding of the effects of macropore and microglobule size, and structure homogeneity on the separation performance in gradient elution, both in terms of peak capacity and gradient plate height, separations were also carried out at different flow rates while maintaining a constant gradient steepness. Furthermore, performance limits were determined applying ultra-high pressure conditions up to the maximum system pressure of 80MPa. The potential of monolithic nanostructured columns is demonstrated for the separation of tryptic digests of cytochrome c and bovine serum albumin.

Cholesterol-based polymeric monolithic columns for capillary liquid chromatography

A novel, cholesterol-based polymeric monolithic stationary phase for capillary liquid chromatography, was prepared by thermally initiated in-situ polymerization. Cholesteryl methacrylate (CholMA) was used as a functional monomer and trimethylolpropane trimethacrylate (TRIM) was a cross-linker, while azobisisobutyronitrile (AIBN) was an initiator. Isooctane and toluene were chosen as “poor” and “good” solvent, respectively, as constituents of the porogen solvent. Isocratic elutions of alkylbenzenes and separation of the testing mixture of o-terphenyl and triphenylene were conducted for all of the monoliths to assess their hydrophobicity and planar selectivity characteristic for cholesterol-based stationary phases. The synthesized columns demonstrated efficiency exceeding N=10,000 plates and a plate height of ca. H=30μm. Column preparation was found to be highly reproducible; the relative standard deviation (RSD) values (n=3) for day-to-day and column-to-column were less than 4.08 and 2.02%, respectively, based on retention factor of alkylbenzenes.