High Sample Load Capacity Research Articles

Reversed-Phase Functionalised Multi-lumen Capillary as Combined Concentrator, Separation Column, and ESI Emitter in Capillary-LC–MS

For the first time, a multi-lumen capillary (MLC) (126 parallel channels of 4.2 µm i.d) has been modified to produce a C18-functionalised silica porous layer open tubular (PLOT) capillary column for both on-capillary preconcentration and separation. The modified multi-lumen capillary used in this dual mode provided significant advantages over typical nano/capillary-LC–MS systems, in that it facilitated both higher sample loading capacity, the use of elevated flow rates, and simplified equipment requirements. Following modification, 100% of the channels displayed a homogenous porous silica layer, 257 ± 36 nm thick. The PLOT-MLC was first evaluated for on-capillary solid-phase extraction. Extraction of caffeine, ofloxacin, atrazine, and diuron was carried out offline using an 8-cm-long PLOT-MLC, with quantification achieved using HPLC coupled to a quadrupole-time of flight (QTOF) mass spectrometer. The results confirmed reversed-phase selectivity and average recoveries obtained were around 70%. Subsequently, a 65-cm-long PLOT-MLC was evaluated as a separator column using a capillary liquid chromatography (Cap-LC) system equipped with a nano-injector and coupled to the mass spectrometer. The short PLOT-MLC provided a baseline separation in isocratic mode [water:acetonitrile (each with 0.1% formic acid) = 70:30, v/v] of ofloxacin, atrazine, and diuron. Finally, direct coupling of the PLOT-MLC with the QTOF via a capillary electrosprayer facilitated the simultaneous use of the modified capillary as a solid-phase concentrator, separator column (carrying out concentration-focusing-separation on the PLOT-MLC) and electrospray emitter. This configuration greatly simplifies the traditional capillary-LC–MS equipment requirements, via the removal of all connectors and additional capillary between injector and MS inlet, and is demonstrated herein with large volume sample loading and step-gradient elution/separation with sensitive MS detection.

Controlled Release of DEET Loaded on Fibrous Mats from Electrospun PMDA/Cyclodextrin Polymer.

Electrospun beta-cyclodextrin (βCD)-based polymers can combine a high surface-to-volume ratio and a high loading/controlled-release-system potential. In this work, pyromellitic dianhydride (PMDA)/βCD-based nanosponge microfibers were used to study the capability to host a common insect repellent (N,N-diethyl-3-toluamide (DEET)) and to monitor its release over time. Fibrous samples characterized by an average fibrous diameter of 2.8 ± 0.8 µm were obtained and subsequently loaded with DEET, starting from a 10 g/L diethyl ether (DEET) solution. The loading capacity of the system was assessed via HPLC/UV–Vis analysis and resulted in 130 mg/g. The releasing behavior was followed by leaving fibrous DEET-loaded nanosponge samples in air at room temperature for a period of between 24 h and 2 weeks. The releasing rate and the amount were calculated by thermogravimetric analysis (TGA), and the release of the repellent was found to last for over 2 weeks. Eventually, both the chemical composition and sample morphology were proven to play a key role for the high sample loading capacity, determining the microfibers’ capability to be applied as an effective controlled-release system.

Fabric Sol–gel Phase Sorptive Extraction Technique: A Review

Since the introduction in 2014 of fabric phase sorptive extraction (FPSE) as a sample preparation technique, it has attracted the attention of many scientists working in the field of separation science. This novel sorbent extraction technique has successfully utilized the benefits of sol–gel derived hybrid sorbents and a plethora of fabric substrates, resulting in a highly efficient, sensitive and green sample pretreatment methodology. The proposed procedure is an easy and efficient pathway to extract target analytes from different matrices providing inherent advantages such as high sample loading capacity and short pretreatment time. The present review mainly focuses on the background and sol–gel chemistry for the preparation of new fabric sorbents as well as on the applications of FPSE for extracting target analytes, from the time that it was first introduced. New modes of FPSE including stir FPSE, stir-bar FPSE, dynamic FPSE, and automated on-line FPSE are also highlighted and commented upon in detail. FPSE has been effectively applied for the determination of various organic and inorganic analytes in different types of environmental and biological samples in high throughput analytical, environmental, and toxicological laboratories.

Gram-Scale Purification of Dihydrorobinetin from Robinia pseudoacacia L. Wood by Centrifugal Partition Chromatography

Robinia pseudoacacia L. is a tree widely dispersed in France that is characterized by good growth rates and important biomass production, which produces wood with very high natural durability used for outdoor fence posts, timber, and barrels to age vinegars and wines. Its mature heartwood presents high resistance against wood fungi decay and contains two main flavonoid extractives, dihydrorobinetin—the most abundant—and robinetin that present interesting biological activities. The aim of the present study was to optimize a procedure allowing an important recovery of purified dihydrorobinetin from R. pseudoacacia wood, representing an interesting sustainable, local, highly available, and, consequently, economical source of bioactive components. The extraction of dihydrorobinetin was first optimized by evaluating the influence of various extraction parameters such as temperature, extraction time, solvent nature, and wood/solvent mass ratio to obtain an efficient, safe, and low cost extraction. Then, dihydrorobinetin was purified over 95% using centrifugal partition chromatography (CPC). CPC purification was first developed on a small volume column with low amounts of injected extract, then scaled-up on a 200 mL column with higher sample loading capacity in order to purify more than 1.3 g of dihydrorobinetin in one run.

Valveless gated injection for microfluidic chip-based liquid chromatography system with polymer monolithic column

In this work, we developed a microfluidic chip-based liquid chromatography (LC) system with valveless gated sample injection method and monolithic columns. The valveless LC system consisted of a microchip with a separation column and a balance column, two syringe pumps and a laser induced fluorescence detector. Nanoliter-scale sample injection was achieved by simply switching the ON and OFF states of the syringe pumps under gated injection mode, without the need of any mechanical valve. The balance column facilitated the gated injection process by balancing the back pressure in the sample channel with that in the separation channel. Under the gated injection mode, the sample injection volumes could be varied in the range of 0.4–2.4nL. We investigated the effects of the mobile-phase composition, flow rate, and the sample injection volume on the performance of the LC system. Under the optimized conditions, two fluorescently labeled amines were successfully separated in less than 220s with a column length of 1cm. Theoretical plates ranging from 35,700 to 29,400 per meter, corresponding to plate heights ranging from 28 to 34μm were obtained. The advantages of the present system include simple system structure, ease of operation, convenience for varying injection volume, and high sample loading capacity.

Direct determination of celiprolol in human urine using on‐line coupled ITP‐CZE method with fiber‐based DAD

The present work illustrated possibilities of column-coupling electrophoresis combined with DAD for the direct quantitative determination of trace drug (celiprolol, CEL) in clinical human urine samples. ITP, on-line coupled with CZE, served as an ideal injection technique (high sample load capacity, narrow and sharp drug zone). Moreover, the ITP provided an effective on-line sample pretreatment (preseparation, purification and preconcentration of the drug) producing analyte zone suitable for its direct detection and quantitation in CZE stage. Spectral DAD in comparison with single wavelength ultraviolet detection enhanced value of analytical information (i) verifying purity (i.e., spectral homogeneity) of drug zone (according to differences in spectrum profiles when compared tested and reference drug spectra) and (ii) indicating zones/peaks with spectra similar to the drug spectrum (potential structurally related metabolites). The characterization of trace analyte signals superposed on the baseline noise was more definite thanks to the application of background correction and smoothing procedure to the raw DAD spectra (producing relevant spectral response). The proposed ITP-CZE-DAD method was characterized by favorable performance parameters for CEL in urine matrices {e.g., the lower limit of quantification was 9.7 ng/mL, RSD and relative error of the determinations were lower than 3% (precision) and 1% (accuracy), respectively, analyte peak exhibited spectral homogeneity (reflecting separation selectivity), separation efficiency was 84,500 theoretical plates} and successfully applied in a trial pharmacokinetic study of CEL.

Possibilities of column coupling electrophoresis provided with a fiber-based diode array detection in enantioselective analysis of drugs in pharmaceutical and clinical samples

The present work illustrated possibilities of column coupling electrophoresis combined with ionizable chiral selector and diode array detection (DAD) for the enantioselective analysis of trace drugs (pheniramine and its analogs) in pharmaceutical and clinical samples. Isotachophoresis (ITP), on-line coupled with capillary zone electrophoresis (CZE), served as an ideal injection technique (high sample load capacity, narrow and sharp drugs zones) of on-line pretreated samples (preseparation, purification and preconcentration of drugs) for the CZE stage. Enhanced (enantio)separation selectivity of CZE with ionizable chiral selector (carboxyethyl-β-cyclodextrin recognized between drugs enantiomers on one hand as well as between drugs and sample matrix constituents on the other hand) enabled to obtain pure zones of the drugs enantiomers, suitable for their detection and quantitation. DAD in comparison with single wavelength UV detection enhanced value of analytical information verifying purity of drugs enantiomers zones (indicating interferents with different spectra to those of drugs). Obtained results indicated pure zones of interest confirming effective ITP–CZE (enantio)separation process. Distinguishing the trace analytes signals superposed on the baseline noise was provided with sufficient reliability (for this purpose the background correction and smoothing procedure had to be applied to the raw DAD spectra). The proposed ITP–CZE–DAD methods were characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, selectivity) and successfully applied for (i) enantiomeric purity testing of dexbrompheniramine in commercial pharmaceutical tablets and (ii) enantioselective metabolic study of pheniramine in human urine.

Potentialities of ITP-CZE method with diode array detection for enantiomeric purity control of dexbrompheniramine in pharmaceuticals

The present work illustrates potentialities of on-line combined isotachophoresis-capillary zone electrophoresis (ITP-CZE) separation techniques coupled with on-capillary diode array detector (DAD) for enantiomeric purity testing of drugs in pharmaceuticals. The general advantages of the proposed method are its (i) high selectivity, (ii) low concentration limit of detection (LOD) obtainable, (iii) enhanced sample loadability, and (iv) enhanced reliability. For separation of brompheniramine (BP) enantiomers, serving as model analytes, carboxyethyl-β-cyclodextrin (CE-β-CD) was appropriate chiral selector providing complete enantioresolution. Given by a high sample load capacity (30 μl sample injection volume) and preconcentration of the analytes in ITP stage, concentration LOD of levobrompheniramine (LBP), serving as model impurity, was 2.5 ng/ml (8 × 10 −9 mol/l). Such separation and detection conditions enabled to easily determine LBP in samples containing a 10 3 excess of dexbrompheniramine (DBP). DAD detection in comparison with single wavelength detection can enhance value of analytical information when analytes and interferents have different spectra (distinguishing impurities in analyte zone, confirmation of migration positions of migrants). In this context purity of BP zones was confirmed with higher reliability in pharmaceutical sample. Moreover, distinguishing the trace analyte signal superposed on the baseline noise was provided with sufficient reliability (for this purpose the background correction and smoothing procedure had to be applied to the raw DAD spectra). Successful validation and application of the proposed ITP-CZE-DAD method suggest its routine use for the enantiomeric purity testing of pharmaceuticals.

Miniaturised isotachophoresis analysis

The application of miniaturized total analysis systems (microTAS) has seen rapid development over the past few years. Isotachophoresis (ITP) has been transferred into microchip format for both electrophoretic separation and pretreatment purposes, due to its advantageous features including separation parameters controlled by electrolyte composition and high sample load capacity. The primary focus of this concise review is to summarize the basic features of microchip based ITP and its applications to the analysis and pretreatment of ionic compounds and biomolecules that have arisen since 1998.

Determination of oxalate in beer by zone electrophoresis on a chip with conductivity detection

AbstractThe use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel combined with a 500 nL sample injection channel) and a pair of on‐chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in beer was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (3.8), implemented by aspartic acid and bis‐tris propane, provided an adequate selectivity in the separation of oxalate from anionic beer constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 0.5 μmol/L concentration also in samples containing chloride (a major anionic constituent of beer) at a 1800 higher concentration. Such a favorable analyte/matrix concentration ratio made possible accurate and reproducible [typically, 2–5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample] determination of oxalate in 500 nL volumes of 20–50‐fold diluted beer samples. Short analysis times (about 200 s), minimum sample preparation, and reproducible migration times of this analyte (0.5–1.0% RSD values) were characteristic for ZE on the chip.

Determination of bromate in drinking water by zone electrophoresis-isotachophoresis on a column-coupling chip with conductivity detection.

The use of capillary zone electrophoresis (CZE) on-line coupled with isotachophoresis (ITP) sample pretreatment (ITP-CZE) on a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to the determination of bromate in drinking water was investigated. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the ITP-CZE separations. A high sample load capacity, linked with the use of ITP in this combination, made possible loading of the samples by a 9.2 microL sample injection channel of the chip. In addition, bromate was concentrated by a factor of 10(3) or more in the ITP stage of the separation and, therefore, its transfer to the CZE stage characterized negligible injection dispersion. This, along with a favorable electric conductivity of the carrier electrolyte solution, contributed to a 20 nmol/L (2.5 ppb) limit of detection for bromate in the CZE stage. Sample cleanup, integrated into the ITP stage, effectively complemented such a detection sensitivity and bromate could be quantified in drinking water matrices when its concentration was 80 nmol/L (10 ppb) or slightly less while the concentrations of anionic macroconstituent (chloride, sulfate, nitrate) in the loaded sample corresponding to a 2 mmol/L (70 ppm) concentration of chloride were still tolerable. The samples containing macroconstituents at higher concentrations required appropriate dilutions and, consequently, bromate in these samples could be directly determined only at proportionally higher concentrations.

Determination of oxalate in urine by zone electrophoresis on a chip with conductivity detection.

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel coupled to a 500 nL sample injection channel) and a pair of on-chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in urine was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (4.0) provided an adequate selectivity in the separation of oxalate from anionic urine constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 8 x 10(-8) mol/L concentration also in samples containing chloride (a major anionic constituent of urine) at 3.5 x 10(-3) mol/L concentrations. Such a favorable analyte/matrix concentration ratio (in part, attributable to a transient isotachophoresis stacking in the initial phase of the separation) made possible accurate and reproducible (typically, 2-5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample) determination of oxalate in 500 nL volumes of 20-100-fold diluted urine samples. Short analysis times (about 280 s), no sample pretreatment (not considering urine dilution) and reproducible migration times of this analyte (0.5-1.0% RSD values) were characteristic for ZE on the chip. This work indicates general potentialities of the present chip design in rapid ZE analysis of samples containing the analyte(s) at high ionic matrix/analyte concentration ratios.

Determination of oxalate in urine by zone electrophoresis on a chip with conductivity detection

The use of a poly(methylmethacrylate) capillary electrophoresis chip, provided with a high sample load capacity separation system (a 8500 nL separation channel coupled to a 500 nL sample injection channel) and a pair of on-chip conductivity detectors, for zone electrophoresis (ZE) determination of oxalate in urine was studied. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the separations performed on the chip. A low pH of the carrier electrolyte (4.0) provided an adequate selectivity in the separation of oxalate from anionic urine constituents and, at the same time, also a sufficient sensitivity in its conductivity detection. Under our working conditions, this anion could be detected at a 8×10–8 mol/L concentration also in samples containing chloride (a major anionic constituent of urine) at 3.5×10–3 mol/L concentrations. Such a favorable analyte/matrix concentration ratio (in part, attributable to a transient isotachophoresis stacking in the initial phase of the separation) made possible accurate and reproducible (typically, 2–5% relative standard deviation (RSD) values of the peak areas of the analyte in dependence on its concentration in the sample) determination of oxalate in 500 nL volumes of 20–100-fold diluted urine samples. Short analysis times (about 280 s), no sample pretreatment (not considering urine dilution) and reproducible migration times of this analyte (0.5–1.0% RSD values) were characteristic for ZE on the chip. This work indicates general potentialities of the present chip design in rapid ZE analysis of samples containing the analyte(s) at high ionic matrix/analyte concentration ratios.

Isolation of oligogalacturonic acids up to DP 20 by preparative high-performance anion-exchange chromatography and pulsed amperometric detection

Milligram quantities of oligogalacturonic acids up to a degree of polymerization (DP) of 20 were purified by high-performance anion-exchange chromatography utilizing a preparative-scale (21-mm i.d.) CarboPac PA1 column and a nonlinear potassium acetate (pH 7.5) gradient. Detection was accomplished by pulsed amperometry without post-column addition of hydroxide. Pulsed amperometry at near-neutral pH is an excellent detection method for preparative separations of carbohydrates because it avoids base-catalyzed degradation reactions that can occur at high pH. This method was simpler, faster, had higher sample loading capacity and allowed for the isolation of higher DP oligogalacturonic acids than other methods reported previously. With this improved method, multi-milligram quantities of valuable oligogalacturonic acids (up to DP 20) can be readily isolated.

Capillary zone electrophoresis separations of enantiomers present in complex ionic matrices with on-line isotachophoretic sample pretreatment

Analytical capabilities of capillary zone electrophoresis (CZE) with on-line coupled capillary isotachophoresis (ITP) sample pretreatment in the column-coupling capillary electrophoresis equipment to separate and determine enantiomers present in multicomponent ionic matrices were studied. Tryptophan was used as a model analyte in the ITP-capillary zone electrophoresis experiments performed in this context while a 90-component model mixture of UV-light absorbing organic anions and urine served as multicomponent sample matrices. Various working modes in which the on-line coupled capillary isotachophoresis–capillary zone electrophoresis combination in the column-coupling separation system can operate were employed in the anionic regime of the separation with direct injections of the samples. Advantages and limitations of these working modes in the separations of enantiomers present in model and urine matrices were assessed. Experiments with model mixtures of tryptophan enantiomers revealed that the two were resolved in the capillary zone electrophoresis stage with the aid of α-cyclodextrin also when their concentration ratio in the sample was 1:200 while the concentration of L(−)-tryptophan was 25 nmol/l. The limits of detection for the enantiomers were at ∼10 nmol/l (∼1.5 ng/ml) concentrations for a 220 nm detection wavelength of the UV detector employed in the capillary zone electrophoresis stage and for a 30 μl sample load. A high sample load capacity of the on-line coupled capillary isotachophoresis stage was effective in separating the samples corresponding to 3–6 μl volumes of undiluted urine. The results from the runs with urine samples showed that only the capillary isotachophoresis–capillary zone electrophoresis combination with a post-column on-line coupled capillary isotachophoresis sample clean-up (responsible for a removal of more than 99% of the sample anionic constituents migrating in the on-line coupled capillary isotachophoresis stack and detectable in the capillary zone electrophoresis stage) provided a universal alternative for the detection and quantitation of the model analyte ( L(−)-tryptophan).

High performance liquid chromatography characterization of macromolecules

AbstractA new method, temperature gradient interaction chromatography(TGIC) is employed for the characterization of macromolecules. Fine and reproducible control of interaction between polymer chains and the alkyl chain bonded silica packing material can be achieved by varying the temperature of the column. This method provides a far superior resolution to the conventional size exclusion chromatography. In addition, this method has a high sample loading capacity to be effective for preparative purpose. Furthermore, this method can be used to characterize binary polymer mixtures, where one component of a polymer mixture is separated by the size exclusion mechanism and the other is by the interaction mechanism simultaneously from single isocratic elution.

Moleclar imprinting by noncovalent interactions: Tailor‐made chiral stationary phases of high selectivity and sample load capacity

AbstractA moleclar imprinting technique based on electrostatic and hydrogen bonding interactions was used to prepare polymers of high selectvity for the original print molecule (D or L form of an amino acid derivative). In the chromatographic mode ig enantioselectivity was observed, in particular for amino acid amides and basic amino acid esters. As indicated y he broad peaks obtained, the mass transfer, including the kinetics of the binding and dissociation process, was slow and appeared to be slower in systems where a higher number of interactions between the solute and the stationary phase could be expected. In such systems enhanced selectivity was observed. For polymers prepared at a lower temperature the mass transfer was more rapid and a higher selectivity was observed, wich allowed the separations to be performed at room temperature. A more rapid mass transfer and a higher selectivity could also be achieved by increasing the column temperature. Furthermore the polymers showed a high sample load capacity and a high stability, and the can easily be prepared.