One of the most cited limitations of capillary (and microchip) electrophoresis is the poor sensitivity. This review continues to update this series of biennial reviews, first published in Electrophoresis in 2007, on developments in the field of on-line/in-line concentration methods in capillaries and microchips, covering the period July 2014-June 2016. It includes developments in the field of stacking, covering all methods from field amplified sample stacking and large volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to on-line or in-line extraction methods that have been used for electrophoresis.
One of the most cited limitations of capillary and microchip electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of online/in-line concentration methods in capillaries and microchips, covering the period July 2016-June 2018. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to online or in-line extraction methods that have been used for electrophoresis.
A portable microchip electrophoresis (MCE) coupled with on-chip contactless conductivity detection (C(4)D) system was evaluated for the determination of vancomycin in human plasma. In order to enhance the detection sensitivity, a new online multi-stacking preconcentration technique based on field-enhanced sample injection (FESI) and micelle-to-solvent stacking (MSS) was developed and implemented in MCE-C(4)D system equipped with a commercially available double T-junction glass chip. The cationic analytes from the two sample reservoirs were injected under FESI conditions and subsequently focused by MSS within the sample-loading channel. The proposed multi-stacking strategy was verified under a fluorescence microscope using Rhodamine 6G as the model analyte and a sensitivity enhancement factor (SEF) of up to 217 was achieved. The developed approach was subsequently implemented in the aqueous-based MCE, coupled to C(4)D in order to monitor the targeted antibiotic (in this case, vancomycin) present in human plasma samples. The multi-stacking and analysis time for vancomycin were 50s and 250s respectively, with SEF of approximately 83 when compared to typical gated injection. The detection limit of the method for vancomycin was 1.2μg/mL, with intraday and interday repeatability RSDs of 2.6% and 4.3%, respectively. Recoveries in spiked human plasma were 99.0%-99.2%.
A new approach based on the integration of the free liquid membrane (FLM) into electrokinetic supercharging (EKS) was demonstrated to be a new powerful tool used in order to enhance online preconcentration efficiency in capillary electrophoresis (CE). A small plug of water immiscible organic solvent was used as a membrane interface during the electrokinetic sample injection step in EKS in order to significantly enhance the analyte stacking efficiency. The new online preconcentration strategy was evaluated for the determination of paraquat and diquat present in the environmental water samples. The optimised FLM-EKS conditions employed were as follows: hydrodynamic injection (HI) of 20mM potassium chloride as leading electrolyte at 50mbar for 75s (3% of the total capillary volume) followed by the HI of tris(2-ethylhexyl) phosphate (TEHP) as FLM at a 1mm length (0.1% of the capillary volume). The sample was injected at 10kV for 360s, followed by the HI of 20mM cetyl trimethylammonium bromide (CTAB) as terminating electrolyte at 50mbar for 50s (2% of the total capillary volume). The separation was performed in 12mM ammonium acetate and 30mM NaCl containing 20% MeOH at +25kV with UV detection at 205nm. Under optimised conditions, the sensitivity was enhanced between 1500- and 1866-fold when compared with the typical HI at 50mbar for 50s. The detection limit of the method for paraquat and diquat was 0.15-0.20ng/mL, with RSDs below 5.5%. Relative recoveries in spiked river water were in the range of 95.4-97.5%. A comparison was also made between the proposed approach with sole preconcentration of the field-enhanced sample injection (FASI) and EKS in the absence of the FLM.
Drug-drug cocrystalllization is a novel mechanism for effective pharmacological combination therapy. In this work, we have demonstrated the preparation of a drug-drug cocrystal of a hypertension drug (Telmisartan; TEL) with a hyperuricemia drug (Febuxostat; FEB) in 1:1 molar ratio using a solvent evaporation method for the first time. Generally, a multi-component system may yield either a eutectic, salt, and/or a cocrystal. This study adopted a methodical orthogonal framework to analyze the final solid form. A single crystal X-ray structural investigation revealed the formation of a heterosynthon with carboxylic and benzimidazole groups of FEB and TEL, respectively, in the triclinic P-1 space group. ΔpKa of the heterosynthon is ∼1.5, hence, based on the empirical rules, a salt-cocrystal continuum is hypothesized. Further, attenuated total reflectance Fourier transform infrared (ATR-FTIR), and Raman spectroscopy were employed to corroborate the hydrogen bond formation in the heterosynthon (-N---H-O-), which confirmed the propensity for cocrystal formation. An accelerated stability study and an in vitro biorelevant dissolution study of the cocrystal were performed, which demonstrated that it is physiochemically stable, but it resulted in a slower dissolution rate when compared with plain drugs.
One of the major drawbacks of electrophoresis in both capillary and microchip is the unsatisfactory sensitivity. Online sample preconcentration techniques can be regarded as the most common and powerful approaches commonly applied to enhance overall detection sensitivity. While the advances of various online preconcentration strategies in capillary and microchip employing aqueous background electrolytes are well-reviewed, there has been limited discussion of the feasible preconcentration techniques specifically developed for capillary and microchip using nonaqueous background electrolytes. This review provides the first consolidated overview of various online preconcentration techniques in nonaqueous capillary and microchip electrophoresis, covering the period of the last two decades. It covers developments in the field of sample stacking, isotachophoresis, and micellar-based stacking. Attention is also given to multi-stacking strategies that have been used for nonaqueous electrophoresis.
A simple adsorption/desorption procedure using a mixed matrix membrane (MMM) as extraction medium is demonstrated as a new miniaturized sample pretreatment and preconcentration technique. Reversed-phase particles namely polymeric bonded octadecyl (C18) was incorporated through dispersion in a cellulose triacetate (CTA) polymer matrix to form a C18-MMM. Non-steroidal anti-inflammatory drugs (NSAIDs) namely diclofenac, mefenamic acid and ibuprofen present in the environmental water samples were selected as targeted model analytes. The extraction setup is simple by dipping a small piece of C18-MMM (7 mm × 7 mm) in a stirred 10 mL sample solution for analyte adsorption process. The entrapped analyte within the membrane was then desorbed into 100 μL of methanol by ultrasonication prior to high performance liquid chromatography (HPLC) analysis. Each membrane was discarded after single use to avoid any analyte carry-over effect. Several important parameters, such as effect of sample pH, salting-out effect, sample volume, extraction time, desorption solvent and desorption time were comprehensively optimized. The C18-MMM demonstrated high affinity for NSAIDs spiked in tap and river water with relative recoveries ranging from 92 to 100% and good reproducibility with relative standard deviations between 1.1 and 5.5% (n=9). The overall results obtained were found comparable against conventional solid phase extraction (SPE) using cartridge packed with identical C18 adsorbent.
In this work, a new variation of the electromembrane extraction (EME) approach employing a hollow polymer inclusion membrane (HPIM) was developed. In this method, a HPIM was prepared by casting a solution of the desired proportions of cellulose acetate (CTA), tris(2-ethylhexyl)phosphate (TEHP) and di-(2-ethylhexyl)phosphoric acid (D2EHPA) in dichloromethane on glass capillary tubing. Three basic drugs namely amphetamine, methamphetamine, and 3,4-methylenedioxy-N-methylamphetamine (MDMA) were selected as model analytes to evaluate the extraction performance of this new approach. The drugs were extracted from human plasma samples, through a 20μm thickness HPIM, to an aqueous acceptor solution inside the lumen of the hollow membrane. Parameters affecting the extraction efficiency were investigated in detail. Under the optimized conditions, enrichment factors in the range of 97-103-fold were obtained from 3mL of sample solution with a 10min extraction time and an applied voltage of 300V across the HPIM. The detection limits of the method for the three drugs were in the range of 1.0-2.5ng/mL (at a signal/noise ratio of three), with relative standard deviations of between 6.4% and 7.9%. When the method was applied to spiked plasma samples, the relative recoveries ranged from 99.2% to 100.8%. Enrichment factors of 103, 99 and 97 were obtained for amphetamine, methamphetamine, and MDMA, respectively. A comparison was also made between the newly developed approach and EME using supported liquid membranes (SLM) as well as standard sample preparation methods (liquid-liquid extraction) used by the Toxicology Unit, Department of Chemistry, Malaysia.
A new electric-field driven extraction approach based on the integration of a bubbleless electrode into the electromembrane extraction (EME) across hollow polymer inclusion membranes (HPIMs) was demonstrated for the first time. The bubbleless electrode was prepared based on an in-situ synthesised polyacrylamide within a fused silica capillary. The electrode functions as a salt bridge, which conducts the electrical current between the acceptor phase in the lumen of the HPIM and the acceptor solution in the reservoir connected to a high voltage supply through a platinum electrode. Two types of HPIMs were employed, which consisted of desired proportions of cellulose acetate as base polymer, tris(2-ethylhexyl)phosphate as plasticizer, and di-(2-ethylhexyl)phosphoric acid as anionic carrier or Aliquat 336 as cationic carrier, respectively. The EME strategy was evaluated for the simultaneous determination of cationic quaternary ammonium and anionic chlorophenoxy acetic acid herbicides present in the river water, respectively. The analysis was carried out using capillary electrophoresis coupled with UV and contactless conductivity detection. Under the optimised conditions, enrichment factors in the range of 152-185-fold were obtained from 4mL of river water sample with a 20min extraction time and an applied voltage of 3000V. The proposed method provided good linearity with correlation coefficients ranging from 0.9982 to 0.9997 over a concentration range of 1-1000μg/L. The detection limits of the method for the herbicides were in the range of 0.3-0.4μg/L, with relative standard deviations of between 4.8% and 8.5%. The relative recoveries obtained when analysing the spiked river water ranged from 99.1% to 100%. A comparison was also made between the newly developed approach with the conventional EME setup by placing the platinum electrode directly in the lumen of the HPIMs.
In this study, the potential for carbonaceous nanomaterials to be used as adsorbents for the mixed matrix membrane (MMM) microextraction and preconcentration of organic pollutants was demonstrated. For this method, multiwall carbon nanotubes (MWCNT) and single layer graphene (SLG) nanoparticles were individually incorporated through dispersion in a cellulose triacetate (CTA) polymer matrix to form a MWCNT-MMM and SLG-MMM, respectively. The prepared membranes were evaluated for the extraction of selected polycyclic aromatic hydrocarbons (PAHs) present in sewage pond water samples. The extraction was performed by dipping a small piece of membrane (7 mm × 7 mm) in a stirred 7.5 mL sample solution to initiate the analyte adsorption. This step was followed by an analyte desorption into 60 μL of methanol prior to high performance liquid chromatography (HPLC) analysis. When the optimum SLG-MMM microextraction technique was applied to spiked sewage pond water samples, the detection limit of the method for the PAHs were in the range of 0.02-0.09 ng/mL, with relative standard deviations of between 1.4% and 7.8%. Enrichment factors of 54-100 were achieved with relative recoveries of 99%-101%. A comparison was also made between the proposed approach and standard solid phase extraction using polymeric bonded octadecyl (C18) cartridges.
A sample pre-treatment method based on a dynamic mixed matrix membrane tip extraction followed by capillary electrophoresis with contactless conductivity detection (CE-C4D) was evaluated for the determination of tobramycin in human plasma. The extraction tip device consisted of a cellulose triacetate membrane tip wall immobilised with 15% (w/w) of hydrophilic lipophilic balance (HLB) nanoparticles as adsorbent. The extraction was performed dynamically by withdrawing/dispensing the plasma sample through the tip device followed by desorption into 20 μL of acidified aqueous solution at pH 3 prior to the CE-C4D analysis. Under the optimum conditions, the detection limit of the method for tobramycin was 10 ng/mL, with intraday and interday repeatability RSDs of 3.5% and 4.5%, respectively. Relative recoveries in spiked human plasma were 99.6%-99.9%. The developed approach was successfully demonstrated for the quantification of tobramycin in human plasma samples.
An electrokinetic platform was developed for extracting small-molecule pharmaceuticals from a dried blood spot. Through the exclusion of liquid reagents and use of low field strength (6 V cm-1 ), the electroextraction of a drug from a dried blood spot, deposited on a polymer inclusion membrane (PIM), could be realised while in transit in the mail. In transit sample preparation provides a potential solution to in situ sample degradation and may accelerate the workflow upon arrival of a patient sample at the analytical facility. The electroextraction method was enabled through our discovery of the use of 15-20 μm thin PIMs as electrophoretic separation medium in absence of liquid reagents. Here, a PIM consisting of cellulose triacetate as polymer base, 2-nitrophenyl octyl ether as plasticizer and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide as carrier was used. The PIM, was packaged with two 12 V batteries to supply the separation voltage. A blood spot containing berberine chloride was deposited and dried before the applying the separation potential, allowing for the electroextraction while the packaged device was shipped in internal mail. Upon arrival in the analytical laboratory, the PIM was analysed using a fluorescence microscope with photon multiplier tube, quantifying the berberine extracted away from the sample matrix. This platform represents a new opportunity for processing clinical samples during transport to the laboratory, saving time and manual handling to accelerate the time to result.
Point-of-collection (POC) devices aim for a fast, on-site detection for medical and environmental purposes. In this area, microfluidic Paper-based Analytical Devices (μPADs) have recently gained popularity because these are potentially cheap and environmentally friendly to produce, and easy to use. From an analytical perspective, paper is well known for its use as a substrate for chromatography, but less known for its use in electrophoretic separations. With the recent interest in μPADs, most applications are based on rather simple assays with relatively few applications incorporating an analytical separation. The focus of this review is on paper-based electrophoresis, originating with the key developments in the 1940s and 1950s as well as the recent developments of electrophoretic μPADs, and concluding with a critical discussion of the opportunities and challenges for electrophoretic μPADS in the future.
A new sample pre-treatment technique termed cone-shaped membrane liquid phase microextraction (CSM-LPME) was developed and combined with micro-liquid chromatography (micro-LC) for the determination of selected pesticides in water samples. Four pesticides (hexaconazole, procymidone, quinalphos and vinclozolin) were considered as target analytes. Several important extraction parameters such as types of extraction solvent, agitation rate, pH value, total exposure time and effect of salt and humic acids were optimized. Enrichment factors of > 50 folds were easily achieved within 20 min of extraction. The analytical data demonstrated relative standard deviations for the reproducibility of the optimized CSM-LPME method ranging from 6.3 to 7.5%. The correlation coefficients of the calibration curves were at least 0.9995 across a concentration range of 2-100 microg/L. The detection limits for all the analytes were found to be in the range of 1.1-1.9 microg/L.
High temperature liquid chromatography using water-rich and superheated water eluent is evaluated as a new approach for the separation of selected triazole fungicides, hexaconazole, tebuconazole, propiconazole, and difenoconazole. Using a polybutadiene-coated zirconia column at temperatures of 100-150 degrees C, clear separations were achieved when 100% purified water was utilized as organic-free eluent. Excellent limits of detection down to pg level were obtained for the separation of the triazole fungicides under optimum conditions. Van't Hoff plots for the separations were linear suggesting that no changes occurred in the retention mechanism over the temperature range studied.
A dynamic supported liquid membrane tip extraction (SLMTE) procedure for the effective extraction and preconcentration of glyphosate (GLYP) and its metabolite aminomethylphosphonic acid (AMPA) in water has been investigated. The SLMTE procedure was performed in a semi-automated dynamic mode and demonstrated a greater performance against a static extraction. Several important extraction parameters such as donor phase pH, cationic carrier concentration, type of membrane solvent, type of acceptor stripping phase, agitation and extraction time were comprehensively optimized. A solution of Aliquat-336, a cationic carrier, in dihexyl ether was selected as the supported liquid incorporated into the membrane phase. Quantification of GLYP and AMPA was carried out using capillary electrophoresis with contactless conductivity detection. An electrolyte solution consisting of 12 mM histidine (His), 8 mM 2-(N-morpholino)ethanesulfonic acid (MES), 75 microM cetyltrimethylammonium bromide (CTAB), 3% methanol, pH 6.3, was used as running buffer. Under the optimum extraction conditions, the method showed good linearity in the range of 0.01-200 microg/L (GLYP) and 0.1-400 microg/L (AMPA), acceptable reproducibility (RSD 5-7%, n=5), low limits of detection of 0.005 microg/L for GLYP and 0.06 microg/L for AMPA, and satisfactory relative recoveries (90-94%). Due to the low cost, the SLMTE device was disposed after each run which additionally eliminated the possibility of carry-over between runs. The validated method was tested for the analysis of both analytes in spiked tap water and river water with good success.
Rapid and direct online preconcentration followed by CE with capacitively coupled contactless conductivity detection (CE-C(4)D) is evaluated as a new approach for the determination of glyphosate, glufosinate (GLUF), and aminophosphonic acid (AMPA) in drinking water. Two online preconcentration techniques, namely large volume sample stacking without polarity switching and field-enhanced sample injection, coupled with CE-C(4)D were successfully developed and optimized. Under optimized conditions, LODs in the range of 0.01-0.1 microM (1.7-11.1 microg/L) and sensitivity enhancements of 48- to 53-fold were achieved with the large volume sample stacking-CE-C(4)D method. By performing the field-enhanced sample injection-CE-C(4)D procedure, excellent LODs down to 0.0005-0.02 microM (0.1-2.2 microg/L) as well as sensitivity enhancements of up to 245- to 1002-fold were obtained. Both techniques showed satisfactory reproducibility with RSDs of peak height of better than 10%. The newly established approaches were successfully applied to the analysis of glyphosate, glufosinate, and aminophosphonic acid in spiked tap drinking water.
A novel microextraction technique termed solid phase membrane tip extraction (SPMTE) was developed. Selected triazine herbicides were employed as model compounds to evaluate the extraction performance and multiwall carbon nanotubes (MWCNTs) were used as the adsorbent enclosed in SPMTE device. The SPMTE procedure was performed in semi-automated dynamic mode and several important extraction parameters were comprehensively optimized. Under the optimum extraction conditions, the method showed good linearity in the range of 1-100 microg/L, acceptable reproducibility (RSD 6-8%, n=5), low limits of detection (0.2-0.5 microg/L), and satisfactory relative recoveries (95-101%). The SPMTE device could be regenerated and reused up to 15 analyses with no analyte carry-over effects observed. Comparison was made with commercially available solid phase extraction-molecular imprinted polymer cartridge (SPE-MIP) for triazine herbicides as the reference method. The new developed method showed comparable or even better results against reference method and is a simple, feasible, and cost effective microextraction technique.
A method for on-line matrix elimination to enable selective quantification of ultraviolet absorbing analytes by a flow-injection analysis procedure is described. Selectivity is achieved by electric field driven extraction across a polymer inclusion membrane. The method was demonstrated on the example of the determination of naproxen from spiked human urine. Membranes of 10 μm thickness were employed which consisted of 7.5 mg cellulose triacetate as base polymer, 5 mg of o-nitrophenyl octyl ether as plasticizer and 7.5 mg of Aliquat 336 as cationic carrier. Ten μL of sample was introduced into a continuous stream of background solution consisting of 100 µM aqueous NaClO₄ with a flow rate of 2 μL/min while applying a voltage of 150 V to the extraction cell. The target ion was electrokinetically transported across the membrane and enriched in 1.5 μL of a stagnant acceptor solution. This was subsequently pumped past a flow-through UV detector for quantification. The method showed a linear range from 5 to 200 µM with a correlation coefficient of 0.9978 and a reproducibility of typically 7% (n = 8). The detection limit of the method for naproxen was 2 µM.
A new multi-stacking pre-concentration procedure based on field-enhanced sample injection (FESI), field-amplified sample stacking, and transient isotachophoresis was developed and implemented in a compact microchip electrophoresis (MCE) with a double T-junction glass chip, coupled with an on-chip capacitively coupled contactless conductivity detection (C4 D) system. A mixture of the cationic target analyte and the terminating electrolyte (TE) from the two sample reservoirs was injected under FESI conditions within the two sample-loading channels. At the double T-junction, the stacked analyte zones were further concentrated under field-amplified stacking conditions and then subsequently focused by transient-isotachophoresis and separated along the separation channels. The proposed multi-stacking strategy was verified under a Universal Serial Bus (USB) fluorescence microscope employing Rhodamine 6G as the model analyte. This developed approach was subsequently used to monitor the target quinine present in human plasma samples. The total analysis time for quinine was approximately 200 s with a sensitivity enhancement factor of approximately 61 when compared to the typical gated injection. The detection and quantification limits of the developed approach for quinine were 3.0 μg/mL and 10 μg/mL, respectively, with intraday and interday repeatability (%RSDs, n = 5) of 3.6 and 4.4%. Recoveries in spiked human plasma were 98.1-99.8%.