Anion exchange silica monolith for capillary liquid chromatography

An anion exchange monolithic silica capillary column was prepared by surface modification of a hybrid monolithic silica capillary column prepared from a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS). The surface modification was carried out by on-column copolymerization of N-[3-(dimethylamino)propyl]acrylamide methyl chloride-quaternary salt (DMAPAA-Q) with 3-methacryloxypropyl moieties bonded as an anchor to the silica surface to form a strong anion exchange stationary phase. The columns were examined for their performance in liquid chromatography (LC) and capillary electrochromatography (CEC) separations of common anions. The ions were separated using 50 mM phosphate buffer at pH 6.6. Evaluation by LC produced an average of 30,000 theoretical plates (33 cm column length) for the inorganic anions and nucleotides. Evaluation by CEC, using the same buffer, produced enhanced chromatographic performance of up to ca. 90,000 theoretical plates and a theoretical plate height of ca. 4 μm. Although reduced efficiency was observed for inorganic anions that were retained a long time, the results of this study highlight the potential utility of the DMAPAA-Q stationary phase for anion separations. Figure Micro-LC performance evaluation of a strong anion exchange silica monolith column, 100H-MOP-DMAPAA-Q, 33 cm in length, with a mobile phase of 50 mM phosphate buffer, pH 2.8; linear velocity: u = 1.8 mm/s; UV-Vis detection at 254 nm. Sample solution (5 mg/mL of each component, 4 mL) was injected in split flow injection mode at a split ratio of ca. 1:1900 with a pump flow rate of 1.5 mL/min     

Miniaturized capillary ion chromatograph with UV light‐emitting diode based indirect absorbance detection for anion analysis in potable and environmental waters

A miniaturized, flexible, and low‐cost capillary ion chromatography system has been developed for anion analysis in water. The ion chromatography has an open platform, modular design, and allows for ease of modification. The assembled platform weighs ca. 0.6 kg and is 25 × 25 cm in size. Isocratic separation of common anions (F^–, Cl^–, NO₂^–, Br^–, and NO₃^–) could be achieved in under 15 min using sodium benzoate eluent at a flow rate of 3 μL/min, a packed capillary column (0.150 × 150 mm) containing Waters IC‐Pak 10 μm anion exchange resin, and light‐emitting diode based indirect UV detection. Several low UV light‐emitting diodes were assessed in terms of sensitivity, including a new 235 nm light‐emitting diode, however, the highest sensitivity was demonstrated using a 255 nm light‐emitting diode. Linear calibration ranges applicable to typical natural water analysis were obtained. For retention time and peak area repeatability, relative standard deviation values ranged from 0.60–0.95 and 1.95–3.53%, respectively. Several water samples were analysed and accuracy (recovery) was demonstrated through analysis of a prepared mixed anion standard. Relative errors of –0.36, –1.25, –0.80, and –0.76% were obtained for fluoride, chloride, nitrite, and nitrate, respectively.     

Automated high‐speed CE system for multiple samples

A high‐speed CE system for multiple samples was developed based on a short capillary and an automated sample introduction device consisting of a commercial multi‐well plate and an x‐y‐z translation stage. The spontaneous injection method was used to achieve picoliter‐scale sample injection from different sample wells. Under the optimized conditions, a 40 μm‐long sample plug (corresponding to 78‐pL plug volume) was obtained in a 50 μm id capillary, which ensured both the high separation speed and high separation efficiency. The performance of the system was demonstrated in the separation of FITC‐labeled amino acids with LIF detection. Five FITC‐labeled amino acids including arginine, phenylalanine, glycine, glutamic acid, and asparagine were separated within 15 s with an effective separation length of 1.5 cm. The separation efficiency ranged from 7.96 × 10⁵/m to 1.12 × 10⁶ /m (corresponding to 1.26–0.89 μm plate heights). The repeatability of the peak heights calibrated with an inner standard for different sample wells was 2.4 and 2.7% (n = 20) for arginine and phenylalanine, respectively. The present system was also applied in consecutive separations of 20 different samples of FITC‐labeled amino acids with a whole separation time of less than 6 min.     

Determination of betaine,l‐carnitine,and choline in human urine using a self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A simple method for the determination of betaine, l ‐carnitine, and choline in human urine was developed based on column‐switching ion chromatography coupled with nonsuppressed conductivity detection by using a self‐packed column. A pretreatment column (50 mm × 4.6 mm, id) packed with poly(glycidyl methacrylate‐divinylbenzene) microspheres was used for the extraction and cleanup of analytes. Chromatographic separation was achieved within 10 min on a cationic exchange column (150 mm × 4.6 mm, id) using maleic anhydride modified poly(glycidyl methacrylate‐divinylbenzene) as the particles for packing. The detection was performed by ion chromatography with nonsuppressed conductivity detection. Parameters including column‐switching time, eluent type, flow rates of eluent, and interfering effects were optimized. Linearity (r ² ≥ 0.99) was obtained for the concentration range of 0.50–100, 0.75–100, and 0.25–100 μg/mL for betaine, l ‐carnitine, and choline, respectively. Detection limits were 0.12, 0.20, and 0.05 μg/mL for betaine, l ‐carnitine, and choline, respectively. The intra‐ and interday accuracy and precision for all quality controls were within ±10.11%. Satisfactory recovery was observed between 92.5 and 105.0%. The validated method was successfully applied for the determination of betaine, l ‐carnitine, and choline in urine samples from healthy people.     

Use of a recently developed thermal modulator within the context of comprehensive two‐dimensional gas chromatography combined with time‐of‐flight mass spectrometry: Gas flow optimization aspects

The present research is based on the use of a recently developed comprehensive two‐dimensional gas chromatography thermal modulator, which is defined as solid‐state modulator. The transfer device was installed on top of a single gas chromatography oven, while benchtop low‐resolution time‐of‐flight mass spectrometry was used to monitor the compounds exiting the second analytical column. The solid‐state modulator was first described by Luong et al. in 2016, and it is a moving modulator that does not require heating and cooling gases to generate comprehensive two‐dimensional gas chromatography data. The accumulation and remobilization steps occur on a trapping capillary, this being subjected to thermoelectric cooling and micathermic heating. In this study, the effects of the gas linear velocity on the modulation performance were evaluated by using two different uncoated trapping capillaries, viz., 0.8 m × 0.25 mm id and 0.8 m × 0.20 mm id. Solid‐state modulator applications were carried out on a standard solution containing n‐alkanes (C_9, C_10, C₁₂), and on a sample of diesel fuel. The results indicated that the type of trapping capillary and gas velocity have a profound effect on modulation efficiency.     

Graphene/poly(ethylene‐co‐vinyl acetate) composite electrode fabricated by melt compounding for capillary electrophoretic determination of flavones in Cacumen platycladi

In this report, a graphene/poly(ethylene‐co‐vinyl acetate) composite electrode was fabricated by melt compounding for the amperometric detection of capillary electrophoresis. The composite electrode was fabricated by packing a mixture of graphene and melted poly(ethylene‐co‐vinyl acetate) in a piece of fused silica capillary under heat. The structure of the composite was investigated by scanning electron microscopy and Fourier transform infrared spectroscopy. The results indicated that graphene sheets were well dispersed in the composite to form an interconnected conducting network. The performance of this unique graphene‐based detector has been demonstrated by separating and detecting rutin, quercitrin, kaempferol, and quercetin in Cacumen platycladi in combination with capillary electrophoresis. The four flavones have been well separated within 9 min in a 50‐cm‐long capillary at a separation voltage of 12 kV using a 50 mM sodium borate buffer (pH 9.2). The graphene‐based detector offered significantly lower operating potentials, substantially enhanced signal‐to‐noise characteristics, lower expense of operation, high resistance to surface fouling, and enhanced stability. It showed long‐term stability and repeatability with relative standard deviations of <5% for the peak current (n = 15).     

Fast separation of enantiomers by capillary electrophoresis using a combination of two capillaries with different internal diameters

The combination of capillaries with different internal diameters was used to accelerate the separation of enantiomers in capillary electrophoresis. Separation of R ,S‐1,1′‐binaphthalene‐2,2′‐diyl hydrogen phosphate using isopropyl derivative of cyclofructan 6 was studied as a model system. The best separation conditions included 500 mM sodium borate pH 9.5 with 60 mM concentration of the chiral selector. Separation lasted approx. 1.5 min using the combination of 50 and 100 μm id capillaries of 9.7 cm and 22.9 cm, respectively. It allowed approx. 12‐fold acceleration in comparison to the traditional long‐end separation mainly due to the higher electroosmotic flow generated in the connected capillaries.     

Simultaneous determination of 13 carbohydrates using high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry

A simple, accurate, and highly sensitive method was developed for the determination of 13 carbohydrates in polysaccharide of Spirulina platensis based on high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry. Samples were extracted with deionized water using ultrasonic‐assisted extraction, and the ultrasound‐assisted extraction conditions were optimized by Box–Behnken design. Then the extracted polysaccharide was hydrolyzed by adding 1 mol/L trifluoroacetic acid before determination by high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and confirmed by high‐performance anion‐exchange chromatography coupled with mass spectrometry. The high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection method was performed on a CarboPac PA20 column by gradient elution using deionized water, 0.1 mol/L sodium hydroxide solution, and 0.4 mol/L sodium acetate solution. Excellent linearity was observed in the range of 0.05–10 mg/L. The average recoveries ranged from 80.7 to 121.7%. The limits of detection and limits of quantification for 13 carbohydrates were 0.02–0.10 and 0.2–1.2  μg/kg, respectively. The developed method has been successfully applied to ambient samples, and the results indicated that high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry could provide a rapid and accurate method for the simultaneous determination of carbohydrates.     

Affinity probe capillary electrophoresis of insulin using a fluorescence‐labeled recombinant Fab as an affinity probe

Affinity probe CE (APCE) separates and detects a target molecule as a complex using a fluorescence‐labeled affinity probe (AP) by CE. The electrophoretic separation of the complex ensures accurate identification of a specific signal among nonspecific ones, which often compromises the credibility of immunoassays. APCE of insulin using a recombinant Fab (rFab) as an AP was demonstrated as a model system in this report. Anti‐insulin rFab was expressed in Escherichia coli and labeled at a cysteine residue in the hinge region with a thiol‐reactive rhodamine dye. Electrophoretically pure labeled rFab was recovered from a focused band in slab‐gel IEF and used as an AP. A mixture of standard insulin and the AP with carrier ampholyte was introduced into a neutral‐polymer coated fused silica capillary (50 μm id, 120 mm long). IEF was carried out at 500 V/cm, and the capillary was scanned for laser‐induced fluorescence under focusing conditions. The insulin‐AP complex focused at pH 6.6 within 6 min along with the free AP at pH 7.6. The complex peak decayed according to the first‐order reaction kinetics with a half life of 3.8 min. A linear calibration line was obtained for standard insulin at a concentration range of 20 pM to 5 nM using the AP at 50 nM. These results demonstrate that rFab is useful for the preparation of an AP for APCE.     

Narrow Bore Ion Chromatography with Commercially Available Ion Chromatographic Equipment

Abstract

The use of narrow bore separator columns (inner diameter less than 1 μM) in high performance liquid chromatography has become a well established technique which has obvious advantages in specific situations over the use of traditional size separator columns. The work reported here investigates the use of traditional ion chromatographic equipment (essentially unmodified) for use in narrow bore ion chromatography. The only adjustments made to the equipment were some arrangements in the placement of valves and tubing in order to minimize extra-column dead volume. The columns used in this work were glass capillary columns of 1.0 mm inner diameter. A selected group of ion exchange resins were slurry packed into the column and the retention and separation characteristics of the narrow bore column were determined. The results of this investigation indicate that in certain cases the commercially available equipment is adequate for performing narrow bore ion chromatography. The distinct advantage of the use of narrow bore ion chromatography is the lower flow rates used in the chromatographic procedure with a corresponding decrease in eluant consumption, eluant cost, and eluant disposal.     

Large Volume Injection in Fast Gas Chromatography with On‐Column Injector

In this work a fast gas chromatography set‐up with on‐column injection was optimized and evaluated with a model mixture of C₈–C₂₈ n‐alkanes. Usual injection volumes when using narrow‐bore (e. g., 0.1 mm i.d.) analytical columns are ca. 0.1 μL. The presented configuration allows introduction of 10–30‐fold larger sample volumes without any distortion of peak shapes. In the set‐up a normal‐bore retention gap (1 m×0.32 mm i. d.) was coupled to a narrow‐bore (4.8 m×0.1 mm i. d.×0.4 μm film thickness) analytical column using a low dead volume column connector. The effects of the experimental conditions such as inlet pressure, sample volume, initial injection temperature, and oven temperature on a peak focusing are discussed. H‐u curves for helium and hydrogen are used to compare their suitability for high speed gas chromatography and to show the dependence of separation efficiency on the carrier gas velocity at high inlet pressures. In the fast gas chromatography system a baseline separation of C₁₀–C₂₈ n‐alkanes was achieved in less than 3 minutes.     

Open microfluidic gel electrophoresis: Rapid and low cost separation and analysis of DNA at the nanoliter scale

Gel electrophoresis is one of the most applied and standardized tools for separation and analysis of macromolecules and their fragments in academic research and in industry. In this work we present a novel approach for conducting on‐demand electrophoretic separations of DNA molecules in open microfluidic (OM) systems on planar polymer substrates. The approach combines advantages of slab gel, capillary‐ and chip‐based methods offering low consumable costs (<0.1$) circumventing cost‐intensive microfluidic chip fabrication, short process times (5 min per analysis) and high sensitivity (4 ng/μL dsDNA) combined with reasonable resolution (17 bases). The open microfluidic separation system comprises two opposing reservoirs of 2–4 μL in volume, a semi‐contact written gel line acting as separation channel interconnecting the reservoirs and sample injected into the line via non‐contact droplet dispensing and thus enabling the precise control of the injection plug and sample concentration. Evaporation is prevented by covering aqueous structures with PCR‐grade mineral oil while maintaining surface temperature at 15°C. The liquid gel line exhibits a semi‐circular cross section of adaptable width (∼200–600 μm) and height (∼30–80 μm) as well as a typical length of 15–55 mm. Layout of such liquid structures is adaptable on‐demand not requiring time consuming and repetitive fabrication steps. The approach was successfully demonstrated by the separation of a standard label‐free DNA ladder (100–1000 bp) at 100 V/cm via in‐line staining and laser induced fluorescent end‐point detection using an automated prototype.     

Preparation of a hybrid monolithic stationary phase with allylsulfonate for the rapid and simultaneous separation of cations in capillary ion chromatography

A hybrid monolithic column with sulfonate functionality was successfully prepared for the simultaneous separation of common inorganic cations in ion‐exchange chromatographic mode through a simple and easy single‐step preparation method. The strong cation‐exchange moieties were provided directly from allylsulfonate, which worked as an organic monomer in the single‐step reaction. Inorganic cations (Li⁺, Na⁺, K⁺, NH₄⁺, Cs⁺, Rb⁺, Mg²⁺, Ca²⁺, and Sr²⁺) were separated satisfactorily by using CuSO₄ as the eluent with indirect UV detection. The allysulfonate hybrid monolith showed a better performance in terms of speed and pressure drop than the capillary packed column. The number of theoretical plates achieved was 19 017 plates/m (in the case of NH₄⁺ as the analyte). The relative standard deviations (n = 6) of both retention time and peak height were less than 1.96% for all the analyte cations. The allysulfonate hybrid monolithic column was successfully applied for the rapid and simultaneous separation of inorganic cations in groundwater and the effluent of onsite domestic wastewater treatment system.     

In‐tube solid‐phase microextraction capillary column packed with mesoporous TiO2 nanoparticles for phosphopeptide analysis

In this study, an in‐tube solid‐phase microextraction column packed with mesoporous TiO₂ nanoparticles, coupled with MALDI–TOF–MS, was applied to the selective enrichment and detection of phosphopeptides in complex biological samples. The mesoporous TiO₂ nanoparticles with high specific surface areas, prepared by a sol–gel and solvothermal method, were injected into the capillary using a slurry packing method with in situ polymerized monolithic segments as frits. Compared with the traditional solid‐phase extraction method, the TiO₂‐packed column with an effective length of 1 cm exhibited excellent selectivity (α‐casein/β‐casein/BSA molar ratio of 1:1:100) and sensitivity (10 fmol of a β‐casein enzymatic hydrolysis sample) for the enrichment of phosphopeptides. These performance characteristics make this system suitable for the detection of phosphorylated peptides in practical biosamples, such as nonfat milk.     

Detection of S‐adenosylhomocysteine and methylation index in blood by capillary electrophoresis

This work proposes an approach to the direct analysis of S‐adenosylhomocysteine (SAH) and the methylation index in blood using CE with UV detection (CE‐UV). After application of meglumine postinjection, we achieved SAH in‐capillary preconcentration in the HClO₄ extracts of erythrocytes, which improved the detection limit (S/N = 3) of SAH up to 3 fmol or 180 nmol/L at the injection volume of 50 nL, taking into account the sample dilution rate. CE‐UV was carried out in 30 mM glycine and 45 mmol/L HCl (pH ～1.8) at 17 kV in a capillary 48 cm in length and 50 μm id. Accuracy of the technique was 101% and reproducibility was about 12%.     

Simultaneous determination of caffeine,paracetamol, and ibuprofen in pharmaceutical formulations by high‐performance liquid chromatography with UV detection and by capillary electrophoresis with conductivity detection

Paracetamol, caffeine and ibuprofen are found in over‐the‐counter pharmaceutical formulations. In this work, we propose two new methods for simultaneous determination of paracetamol, caffeine and ibuprofen in pharmaceutical formulations. One method is based on high‐performance liquid chromatography with diode‐array detection and the other on capillary electrophoresis with capacitively coupled contactless conductivity detection. The separation by high‐performance liquid chromatography with diode‐array detection was achieved on a C₁₈ column (250×4.6 mm², 5 μm) with a gradient mobile phase comprising 20–100% acetonitrile in 40 mmol L^?1 phosphate buffer pH 7.0. The separation by capillary electrophoresis with capacitively coupled contactless conductivity detection was achieved on a fused‐silica capillary (40 cm length, 50 μm i.d.) using 10 mmol L^?1 3,4‐dimethoxycinnamate and 10 mmol L^?1 β‐alanine with pH adjustment to 10.4 with lithium hydroxide as background electrolyte. The determination of all three pharmaceuticals was carried out in 9.6 min by liquid chromatography and in 2.2 min by capillary electrophoresis. Detection limits for caffeine, paracetamol and ibuprofen were 4.4, 0.7, and 3.4 μmol L^?1 by liquid chromatography and 39, 32, and 49 μmol L^?1 by capillary electrophoresis, respectively. Recovery values for spiked samples were between 92–107% for both proposed methods.     

Separation of cannabinoids on three different mixed‐mode columns containing carbon/nanodiamond/amine‐polymer superficially porous particles

Three mixed‐mode high‐performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine‐polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C₁₈), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed‐mode column (C₁₈) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed‐mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C₁₈) mixed‐mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution.     

Factors influencing chromatographic data in fast gas chromatography with on‐column injection

The use of larger volume injection with on‐column injection and fast GC commercial instrumentation was evaluated with the model mixture of n‐alkanes of a broad range of volatility (C₁₀–C₂₈). The presented configuration allows introduction of 40–80‐fold larger sample volumes without any distortion of peak shapes compared to “usual” fast GC set‐ups using narrow‐bore columns. A normal‐bore retention gap (1–5 m×0.32 mm ID) was coupled to a narrow‐bore (5 m×0.1 mm ID×0.4 μm film thickness) analytical column using a standard press‐fit connector. The connection was tight and reliable, and hence suitable for hydrogen as carrier gas. The effect of pre‐column and analytical column connector, injection volume, pre‐column length, column inlet pressure, and analyte volatility on peak shape, peak broadening, and focusing are discussed. The precision of chromatographic data measurements and peak capacity under optimised temperature programmed conditions for fast separations with large volume injection were found to be very good. The presented fast GC set‐up with on‐column injection extends the applicability of the technique to trace analysis.     

Characterization and kinetic performance of 2.1 × 100 mm production columns packed with new 1.6 μm superficially porous particles

The overall kinetic performance of three production columns (2.1 mm × 100 mm format) packed with 1.6 μm superficially porous CORTECS‐C₁₈+ particles was assessed on a low‐dispersive I‐class ACQUITY instrument. The values of their minimum intrinsic reduced plate heights (h_min = 1.42, 1.57, and 1.75) were measured at room temperature (295 K) for a small molecule (naphthalene) with an acetonitrile/water eluent mixture (75:25, v/v). These narrow‐bore columns provide an average intrinsic efficiency of 395 000 plates per meter. The gradient separation of 14 small molecules shows that these columns have a peak capacity about 25% larger than similar ones packed with fully porous BEH‐C₁₈ particles (1.7 μm) or shorter (50 mm) columns packed with smaller core–shell particles (1.3 μm) operated under very high pressure (>1000 bar) for steep gradient elution (analysis time 80 s). In contrast, because their permeabilities are lower than those of columns packed with larger core–shell particles, their peak capacities are 25% smaller than those of narrow‐bore columns packed with standard 2.7 μm core–shell particles.     

Temperature‐Programmed Packed Capillary Liquid Chromatography Coupled to Fourier‐Transform Infrared Spectroscopy

Temperature‐programmed packed capillary liquid chromatography has been coupled off‐line to Fourier‐transform infrared spectroscopy, utilizing a commercially available interface with a pneumatic nebulizer rebuilt to handle low flow rates at elevated temperatures. The modified interface showed excellent performance with regard to non‐aqueous reversed phase separations of polymer additives, resulting in constructed Gram‐Schmidt chromatograms comparable to chromatograms obtained using UV detection. The spray of the in‐house constructed nebulizer was not influenced by temperature changes of the column effluent, and hence temperature‐programmed gradient separations could be used successfully. The relative standard deviation of peak height was 4.4% (n = 5) and the mass limit of detection was determined to be about 40 ng, using a polymer antioxidant as model compound. The present instrumental coupling has been used for characterization of the antioxidant Irgafos P‐EPQ.