Pressure-assisted Electrokinetic Injection Research Articles

Nanoemulsion supported microemulsion electrokinetic chromatography coupled with selected preconcentration techniques as an approach for analysis of highly hydrophobic compounds

In this paper, an oil-in-water (O/W) nanoemulsion (NE) prepared by water cold dilution of an O/W microemulsion (ME) was introduced as a sample matrix in microemulsion electrokinetic capillary chromatography (MEEKC) for the highly hydrophobic compounds analysis. Several model compounds with log PO/W values in the 4.1–10.9 range, from different chemical groups, including retinol, α-tocopherol, cholecalciferol, phylloquinone, menaquinone-7, dichlorodiphenyltrichloroethane, ivermectin have been tested. As a proof of the concept of NE formation, a dynamic light scattering technique was employed to determine the size distribution profile of NE particles. Moreover, due to relatively low conductivity of the NE matrix (50–100 times lower in comparison to the separation buffer) and a negative electric charge provided to hydrophobic compounds through NE dispersed phase, NE matrices have been combined with preconcentration techniques based on electrokinetic dosing, namely field amplified sample injection (FASI) and pressure assisted electrokinetic injection (PAEKI). The detection limits for vitamin K1 and K2-MK7 in the NE matrix in combination with FASI (NE–MEEKC–FASI) as well as PAEKI (NE–MEEKC–PAEKI) were up to 42.9 and 12.1 ng mL−1, respectively. In comparison to standard hydrodynamic injection for microemulsion sample matrix NE–MEEKC–PAEKI grant 45-fold improvement in signal sensitivity. The study presents an innovative approach, as it enables the use of preconcentration techniques for highly hydrophobic compounds (log PO/W > 4), which was not previously possible for implementation in the electromigration techniques. Likewise, the use of organic solvents has been reduced by using ME as a solvent for stock solutions and diluting with water prior to the analysis. The application to real samples was investigated using a dietary supplement containing vitamin K2-MK7 obtained from the fermentation product of soybeans.

Pressure-assisted electrokinetic injection for the stacking of biogenic amines gives enhancement factor up to 1000 in CE with UV detection.

Pressure-assisted electrokinetic injection (PAEKI) was applied for stacking of positively charged biogenic amines (BAs) to improve the sensitivity of capillary electrophoresis (CE). It is well known that the essential step for PAEKI is finding a stationary state of the running buffer such that the movement of the running buffer due to electroosmotic flow (EOF) is counterbalanced by external pressure in the opposite direction of the EOF under a given electric field. In order to find the balance point systematically and integrally, we studied the velocity of the whole BGE in the capillary by the impetus of opposite direction pressure (-0.1 to -0.6 psi), and the velocity of EOF with different voltages. According to the two sets of linear data, the EOF of CE coupled with PAEKI could be counterbalanced at the opposite direction pressure (-0.1 psi) and voltage (7.8 kV). In this study, the injection time was extended up to 0.35 min for all BAs and 0.70 min for the direct ultraviolet (UV) detection of BAs. Compared with hydrodynamic injection (HDI), the enrichment factors for sample injection times of 0.35 min and 0.70 min were 480-fold and 970-fold, respectively. The limits of detection (LODs) (S/N = 3) of indirect and direct UV detection were respectively 8.7-24.3 nmol L-1 and 0.4-4.5 nmol L-1, which reaches the sensitivity of high-performance liquid chromatography-mass spectrophotometry (HPLC-MS). With appropriate sample dilution, PAEKI can be used in the analysis of BAs in chicken.

Simultaneous enrichment/determination of six sulfonamides in animal husbandry products and environmental waters by pressure-assisted electrokinetic injection coupled with capillary zone electrophoresis

Pressure-assisted electrokinetic injection (PAEKI) as the online method was developed for the simultaneous enrichment of six sulfonamides (SAs), including sulfamethazine (SMZ), sulfamerazine (SMR), sulfamonomethoxine (SMM), sulfadizine (SDZ), sulfamethoxazole (SMX) and sulfacetamide (SFA) in different animal husbandry products and environmental waters samples, followed by capillary zone electrophoresis (CZE) determination. Various parameters affecting the separation performance of CZE and the enrichment efficiency of PAEKI were optimized in detail. Under optimal conditions, the six SAs were completely separated within 8.5 min, and the enrichment factors for SMZ, SMR, SMM, SDZ, SMX and SFA were 39, 47, 53, 50, 54 and 62 folds, respectively, compared with the direct CZE. The limits of detection (LOD) ranged from 0.0018–0.0163 μg/mL, 0.0083–0.0638 μg/mL and 0.0052–0.0478 μg/mL in milk, pork and egg samples, respectively, as well as limits of quantification (LOQ) within 0.0061–0.0503 μg/mL, 0.0253–0.1826 μg/mL and 0.0164–0.1475 μg/mL, respectively. Tap water, lake water and seawater were also examined for investigation of applicability of the proposed method. Satisfactory recoveries in the range of 89–113 % were obtained with the three spiked food samples, and the relative standard deviations were from 1.6 to 7.3%. Simple and effective online enrichment method provides a promising perspective for CZE measurements of SAs in complex matrices with high accuracy, sensitivity and rapidity.

Dispersive liquid-liquid microextraction coupled with pressure-assisted electrokinetic injection for simultaneous enrichment of seven phenolic compounds in water samples followed by determination using capillary electrophoresis.

Offline dispersive liquid-liquid microextraction combined with online pressure-assisted electrokinetic injection was developed to simultaneously enrich seven phenolic compounds in water samples, followed by determination using capillary electrophoresis, namely phenol, 4-chlorophenol, pentachlorophenol, 2,4,6-trichlorophenol, 2,4-dichlorophenol, 2-chlorophenol, and 2,6-dichlorophenol. Several parameters affecting separation performance of capillary electrophoresis and the enrichment efficiency of pressure-assisted electrokinetic injection and dispersive liquid-liquid microextraction were systematically investigated. Under the optimal conditions, seven phenolic compounds were completely separated within 14min and good enrichment factors were obtained of 61, 236, 3705, 3288, 920, 86, and 1807 for phenol, 4-chlorophenol, pentachlorophenol, 2,4,6-trichlorophenol, 2,4-dichlorophenol, 2-chlorophenol, and 2,6-dichlorophenol, respectively. Good linearity was attained in the range of 0.1-200μg/L for 2,4-dichlorophenol, 0.5-200μg/L for 4-chlorophenol, pentachlorophenol, 2,4,6-trichlorophenol, 2-chlorophenol, and 2,6-dichlorophenol, as well as 1-200μg/L for phenol, with correlation coefficients (r) over 0.9905. The limits of detection and quantification ranging from 0.03-0.28 and 0.07-0.94μg/L were attained. This two step enrichment method was potentially applicable for the rapid and simultaneous determination of phenolic compounds in water samples.

Pressure-assisted electrokinetic injection stacking for seven typical antibiotics in waters to achieve μg/L level analysis by capillary electrophoresis with UV detection

An effect sensitive CE method for the analysis of four fluoroquinolones (FQs) antibiotics (ofloxacin (OFL), enrofloxacin (ENR), ciprofloxacin (CIP) and norfloxacin (NOR)) and three sulfonamides (SAs) antibiotics (sulfamethazine (SMZ), sulfamethoxazole (SMX) and sulfadiazine (SDI)) in waters was developed by using pressure-assisted electrokinetic injection (PAEKI) stacking technique. The optimal background electrolyte (BGE) was 25.0 mmol/L Na2B4O7, 15.0 mmol/L methyl-β-cyclodextrin (M-β-CD) at pH 10.0 (adjusted by 1.0 mol/L NaOH). The addition of M-β-CD made significant contributions to the separation of OFL, CIP, and NOR, otherwise they co-migrated due to the similar electrophoretic mobilities in the borate buffer. To avoid a cumbersome optimization process of PAEKI, we built a simple method to determine the balance conditions. Herein, the applied voltage and external pressure values to immobilize the bulk flow of BGE could be clearly read from two curves of the BGE velocity respectively induced by pressure and electroosmotic flow (EOF). Under the selected injection condition during PAEKI (0.2 psi vs. −5.2 kV), the sample injection time could sustain 3.0 min without compromising separation efficiency. The sensitivity was improved to 75–110 times in comparison with normal hydrodynamic injection (HDI) mode. The obtained LODs (S/N = 3) of seven antibiotics were in the range of 1.96–4.06 μg/L at UV detection (272 nm), which approached the level of the antibiotics analyzed by CE with fluorescence detection. The precision was characterized by the RSDs of migration times and peak areas, which were averaged at 0.84% and 5.8% for the proposed PAEKI method. Finally, the recoveries of PAEKI were investigated at 83.3–98.7% by spiking FQs and SAs in the actual lake water of Donghua campus to illuminate its feasibility.

Determination of four phenolic estrogens in water samples by pressure-assisted electrokinetic injection coupled with capillary electrophoresis

A capillary electrophoresis (CE) method was developed for the simultaneous separation and determination of four phenolic estrogens (PEs), in which pressure-assisted electrokinetic injection (PAEKI) was adopted as the on-line concentration method to enrich the PEs. Several parameters affecting PAEKI conditions such as injection voltage and injection time, were systematically investigated and compared with the usual parameters. Under the optimized PAEKI conditions (-9 kV, 0.3 psi (ca. 2.1 kPa), 0.4 min), the four PEs separated completely within 7 min. Good linearities were attained in the range of 0.05-5 mg/L for hexestrol and dienestrol, and 0.1-10 mg/L for bisphenol A and diethylstilbestrol, with correlation coefficients (r) over 0.9936. The limits of detection (S/N=3) were 0.0071-0.017 mg/L, and enrichment factors ranged from 11 to 15 compared to normal hydrodynamic injection. The combined micellar electrokinetic chromatographic-PAEKI method developed was used to determine the PEs in tap water and lake water samples; limits of quantification (S/N=10) of 0.029-0.064 mg/L and 0.033-0.079 mg/L were attained, respectively, by the two sample types. Furthermore, recoveries ranged from 75.6% to 110.1% with relative standard deviations (n=5) within 4.6%-11.8%. To use this PAEKI method, researchers would only need to adjust the parameters of the CE apparatus to perform on-line enrichment of analytes, without using other reagents; this demonstrates the simplicity, rapidity, and highly automated nature of this method.

Expanding the scope of pressure-assisted electrokinetic injection for online concentration of positively charged analytes in capillary electrophoresis-mass spectrometry.

Sample injection is a crucial step in CE. In past, many efforts have been focused on concentrating the analytes into a sharp sample plug. In 2006, pressure-assisted electrokinetic injection (PAEKI) was proposed as a new preconcentration technique for anions. In this study, we expanded the applicability of PAEKI to online preconcentrate positively charged analytes. L-Arginine, L-lysine, and imidazole were chosen as target analytes for method development. The enhancement factor of PAEKI was over 3000-fold. When CZE was coupled with a Q-TOF system, PAEKI delivers a detection limit of 18-28 pg/mL and a dynamic calibration range over four orders of magnitude. The RSD was less than 6.4% (n = 4) on both peak area and migration time, indicating a good repeatability.

Pressure-assisted electrokinetic injection stacking for citalopram drug to achieve highly sensitive detection and enantioseparation by capillary electrophoresis.

Pressure-assisted electrokinetic injection (PAEKI) was applied to the highly sensitive enantioseparation of the positively charged drug citalopram (CIT) by capillary electrophoresis (CE). It was found that the injection discrimination occurred in electrokinetic injection (EKI) process due to the different dynamic equilibrium constant between chiral selector (sulfated-β-cyclodextrin, S-β-CD) and two isomers of CIT. Herein, it was proposed to use the background electrolyte (BGE) without chiral selector to fill the capillary, and then start the EKI step to eliminate the injection discrimination of free analytes. The critical parameters in PAEKI could be optimized in two steps to seek the balance between the electroosmotic flow (EOF) and the counterbalance pressure. Under the optimized PAEKI conditions (+10 kV, 0.2 psi (ca. 1.4 kPa)), the obtained LODs (S/N = 3) of the two isomers were 1.1 and 2.2 ng/mL under UV detection (205 nm), which was averaged 62-fold improved in comparison with normal hydrodynamic injection (HDI). The proposal offered ng/mL (ppb) level sensitivity of CIT determination and could be an effective method in the applications in human body biofluids.

Pressure-assisted electrokinetic injection stacking for verteporfin drug to achieve highly sensitive enantioseparation and detection in artificial urine by capillary electrophoresis

Pressure-assisted electrokinetic injection (PAEKI) was applied for negatively charged verteporfin (VER) overloading and inline stacking, which targeted highly sensitive enantioseparation by CE. The essential step of PAEKI is a constant pressure used to counterbalance the electroosmotic flow (EOF), consequently, the large amount of analyte could be permitted into capillary and concentrated at the motionless boundary of the sample zone and background electrolyte (BGE). Aiming to know the balance, the velocity of the whole BGE in capillary by the impetus of pressure (0.2–2.0psi), and the velocity of EOF depending on the length of sample plug and voltage (5.0–20kV) was investigated, respectively. The velocity of bulk flow in capillary has good linearity with the pressure or applied voltage. Through the pattern of EOF marked peak and analyte peaks (dissolved in pure water), the constant pressure (0.8psi) vs. the added voltage (−10.3kV) during PAEKI was confirmed to immobilize the bulk flow of BGE, thus the sample injection time could sustain 2.0min without compromising separation efficiency. The obtained LOD (S/N=3) of each isomer at UV detection (428nm) was around 10.3μg/L, which was improved to 116 and 39-fold in comparison with normal hydrodynamic injection (HDI) and electrokinetic injection (EKI). The LOD is far below the reported value with LIF detection of VER. The RSD (n=5) of migration time and peak area was, respectively, around 3.5% and 5.7% for the proposed PAEKI method. Finally, PAEKI was used for the detection of VER in artificial urine to investigate the matrix interference.

Application of Pressure-assisted Electrokinetic Injection-Tandem Mass Spectrometry in the Determination of Perchlorate in Water and Soil Samples

A new method for the determination of perchlorate in water and soil samples using on-line enrichment in a capillary zone electrophoresis-mass spectrometry is presented. The target analytes in the sample solutions were introduced into the capillary column by pressure-assisted electrokinetic injection (PAEKI) with the simultaneous application of -18 kV and +50 mbar external pressure to the sample vial at the capillary inlet for 4 min. The injected sample zone was flushed out with a running buffer and analyzed by a tandem mass spectrometer in a negative selected reaction monitoring mode. The influence of the matrix in both water and soil samples was eliminated by a clean-up step by passing the sample through Ba/Ag/H cartridges. The method showed good linearity in the dynamic range of 20 to 1000 ng/L, and achieved a detection limit of 18.7 ng/L for water samples and 3.3 ng/g for soil samples, respectively. The recovery of perchlorate in spiked samples, at three different levels, ranged over 79-127%. The method reproducibility was found to be 11% RSD for water samples and 7% RSD for soil samples. Perchlorate was found in 25 out of 28 water samples analyzed, with the levels ranging from 19.8 to 192 ng/L, and was not detected in the 10 soil samples analyzed.

Pressure-assisted electrokinetic injection for on-line enrichment in capillary electrophoresis–mass spectrometry: A sensitive method for measurement of ten haloacetic acids in drinking water

Haloacetic acids (HAAs) are by-products of the chlorination of drinking water containing natural organic matter and bromide. A simple and sensitive method has been developed for determination of ten HAAs in drinking water. The pressure-assisted electrokinetic injection (PAEKI), an on-line enrichment technique, was employed to introduce the sample into a capillary electrophoresis (CE)–electrospray ionization–tandem mass spectrometry system (ESI-MS/MS). HAAs were monitored in selected reaction monitoring mode. With 3 min of PAEKI time, the ten major HAAs (HAA10) in drinking water were enriched up to 20,000-fold into the capillary without compromising resolution. A simple solid phase clean-up method has been developed to eliminate the influence of ionic matrices from drinking water on PAEKI. Under conditions optimized for mass spectrometry, PAEKI and capillary electrophoresis, detection limits defined as three times ratio of signal to noise have been achieved in a range of 0.013–0.12 μg L −1 for ten HAAs in water sample. The overall recoveries for all ten HAAs in drinking water samples were between 76 and 125%. Six HAAs including monochloro- (MCAA), dichloro- (DCAA), trichloro- (TCAA), monobromo- (MBAA), bromochloro- (BCAA), and bromodichloroacetic acids (BDCAA) were found in tap water samples collected.

Understanding mechanisms of pressure-assisted electrokinetic injection: Application to analysis of bromate, arsenic and selenium species in drinking water by capillary electrophoresis-mass spectrometry

The mechanism underlying the enrichment power by pressure-assisted electrokinetic injection (PAEKI) in capillary electrophoresis (CE) was investigated for on-line pre-concentration of arsenic [As(III) and As(V)], selenium [Se(IV) and Se(VI)] and bromate (BrO 3 −). Analyte diffusion behaviour from PAEKI sample plugs were evaluated by monitoring peak broadening as a function of stagnant time and position in the capillary. During PAEKI, anionic analytes accumulate at the sample-separation buffer boundary. We proposed that a counter-ion layer formed in PAEKI, where a cation layer was formed at the separation buffer side of boundary. The cation layer served as a soft boundary which impeded zone broadening via electrostatic attraction between layers. This effect likely played an important role in maintaining focused analyte bands by suppressing diffusion. Comparison of analyte behaviour in PAEKI injected sample plugs to behaviour in hydrodynamically injected ones proved the existence of a counter-ion layer. The dependence of analyte diffusion in PAEKI plugs on electrochemical properties (viscosity, conductivity, electrophoretic mobility) further supported the hypothesis. Additionally, it was noted that analytes with low electrophoretic mobility were more efficiently pre-concentrated by PAEKI and were less subject to forces of dispersion than analytes with greater electrophoretic mobility. PAEKI-CE coupled to electrospray tandem mass spectroscopy (ESI-MS/MS) was then optimized and validated for detection of arsenic, selenium and bromate in water samples. On-line enrichment of the target analytes was achieved with 1–3 ng mL −1 detection limits, which was below the maximum contaminant levels in drinking water for all five anions studied. Noteworthy, the potential of the method for unbiased detection of molecular species in untreated water was demonstrated. No contamination was detected in the water samples tested; however, recovery was 90–118% for spiked samples. The method was demonstrated be comparable to current methods for detection of inorganic contaminants in drinking water and is a good alternative method to ion chromatography/liquid chromatography–MS.

On-line Enrichment and Measurement of Four Halogenated Phenols in Water Samples Using Pressure-Assisted Electrokinetic Injection–Tandem Mass Spectrometry

A rapid and sensitive method using pressure-assisted electrokinetic injection (PAEKI) as the on-line sample enrichment technique and electrospray-tandem mass spectrometry as the monitor has been developed for the determination of four halogenated phenols, 2,4,6-trichlorophenol, 2,4-dichlorophenol, 2,3,4,6-tetrachlorophenol and tetrabromobisphenol A, in water samples. In 8-min of injection, the four halogenated phenols in water samples were enriched up to several thousand times. With the aid of a solid-phase extraction clean-up procedure, the method achieved a detection limit in the range of 7.4 to 37.1 ng L(-1) for the four phenols by multiple reaction monitoring in a negative mode. The overall recoveries for all four halogenated phenols in water samples were in the range of 68 to 114%. The good method repeatability has been demonstrated by the relative standard deviations (RSDs) of less than 10% for water samples spiked with standard mixtures. The four target phenols in all the water samples were below the detection limits.

Constant pressure‐assisted electrokinetic injection for on‐line enhanced detection of monophthalates in capillary electrophoresis‐mass spectrometry with application to human urine

Electrophoresis characteristics of several monophthalates in the sample zone and EOF variation in a fused-silica capillary column during a constant pressure-assisted electrokinetic injection (PAEKI) in an on-line CE-MS were studied in an effort to reconcile the mobility theory and field amplification with the enhancement achieved in present work. Influences of capillary length on the amount injected using PAEKI were investigated in detail and except for the injection time, the amount injected was found to increase linearly with capillary column length. A longer capillary provides a longer linear increase time range with PAEKI injection. The results show that smaller m/z analytes generate a large enhancement power using PAEKI, which is in agreement with the mobility theory. ACN was used as an example to investigate influences of organic additives on the amount injected and was found to decrease the amount injected with PAEKI injection, which is in agreement with an increase of resistivity in running buffer by organic additives. The peak width obtained with PAEKI injection proved to be independent of the amount injected. The band size of the sample zone was estimated by comparison with conventional hydrodynamic injection. A 240 s PAEKI injection achieved the same size of sample zone as a 2 s of hydrodynamic injection. Existance of two ion layers around the boundary of the buffer and sample solutions in sample zone was hypothesized to contribute the narrow sample zone with a long time of PAEKI injection. With a 240 s on-line PAEKI injection in CZE-MS, five monophthalates were enriched several hundred times without compromise in their separation efficiency and peak shape. With appropriate sample cleanup, PAEKI was applied to the analysis of monophthalates in urine samples, achieving detection limits ranging between 0.53 and 1.3 ng/mL.

Application of pressure assisted electrokinetic injection technique in the measurements of DNA oligonucleotides and their adducts using capillary electrophoresis–mass spectrometry

The identification and measurement of negatively charged DNA oligonucleotides and their benzo[a]pyrene-7,8,9,10-tetrahydro-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts by capillary zone electrophoresis (CZE) hyphenated mass spectrometry (MS) system using an on-line enrichment technique, the constant pressure assisted electrokinetic injection (PAEKI), is described here. With optimized PAEKI conditions, an on-line sample concentration power of 300–800 times could be reached for both single-stranded (ss) and double-stranded (ds) oligonucleotides during a 90-s PAEKI injection. The detection limits using single quadrupole MS in the scan mode were 0.01–0.03 μM for ss and 0.04–0.08 μM for ds oligonucleotides, respectively. The relative standard deviations at 1 μM of oligonucleotides were from 7.6 to 15.8%. A dynamic linear calibration range of about two orders of magnitude were observed. Good mass spectra of oligonucleotides and BPDE–oligonucleotide adducts at low micromolar levels could be obtained using single quadrupole MS which could be a helpful tool in DNA adducts studies.

On-Line Enhancement Technique for the Analysis of Nucleotides Using Capillary Zone Electrophoresis/Mass Spectrometry

A new on-line capillary zone electrophoresis/mass spectrometry (CZE/MS), constant pressure-assisted electrokinetic injection (PAEKI), for the analysis of negatively charged nucleotides is reported. PAEKI uses an applied pressure to counterbalance the reverse electroosmotic flow in the capillary column during sample injection, while taking advantage of the field amplification in the sample medium. At balance, the running buffer in the column is stationary, permitting potentially unlimited injection time, and hence unlimited sample enrichment power. The ability of PAEKI to maintain a narrow sample zone over a long injection time seems to be a result of the formation of a high ion concentration band at the boundary of the two media due to rapid deceleration of the migrating ions at the boundary. The injected amount of analytes proved to be linearly proportional to both the field amplification factor, which is expressed as the ratio of resistivities of sample medium to running buffer, and the injection time, which extended up to 1200 s in CZE/MS and 3600 s in CZE/UV. For a 300-s on-line PAEKI injection in CZE/MS, 3 orders of magnitude sample enhancement (5000-fold enrichment) could be observed for the four single nucleotides without compromising separation efficiency and peak shape, and an achievement of detection limits between 0.04 and 0.07 ng/mL. With appropriate sample cleanup, PAEKI can be used in the analysis of single nucleotides in enzyme-digested DNA.