CE Separation Research Articles

Nanometer‐sized alumina packed microcolumn solid‐phase extraction combined with field‐amplified sample stacking‐capillary electrophoresis for the speciation analysis of inorganic selenium in environmental water samples

In this paper, a new method of nanometer-sized alumina packed microcolumn SPE combined with field-amplified sample stacking (FASS)-CE-UV detection was developed for the speciation analysis of inorganic selenium in environmental water samples. Self-synthesized nanometer-sized alumina was packed in a microcolumn as the SPE adsorbent to retain Se(IV) and Se(VI) simultaneously at pH 6 and the retained inorganic selenium was eluted by concentrated ammonia. The eluent was used for FASS-CE-UV analysis after NH₃ evaporation. The factors affecting the preconcentration of both Se(IV) and Se(VI) by SPE and FASS were studied and the optimal CE separation conditions for Se(IV) and Se(VI) were obtained. Under the optimal conditions, the LODs of 57 ng L⁻¹ (Se(IV)) and 71 ng L⁻¹ (Se(VI)) were obtained, respectively. The developed method was validated by the analysis of a certified reference material of GBW(E)080395 environmental water and the determined value was in a good agreement with the certified value. It was also successfully applied to the speciation analysis of inorganic selenium in environmental water samples, including Yangtze River water, spring water, and tap water.

Novel covalently coated diazoresin/polyvinyl alcohol capillary column for the analysis of proteins by capillary electrophoresis

A novel method for the preparation of covalently linked capillary coatings of PVA was demonstrated using photosensitive diazoresin (DR) as coupling agents. Layer-by-layer self-assembly film of DR and PVA based on hydrogen bonding was first fabricated on the inner wall of capillary, then the hydrogen bonding was converted into covalent bonding after treatment with UV light through the unique photochemistry reaction of DR. The covalently bonded coatings suppressed basic protein adsorption on the inner surface of capillary, and thus a baseline separation of lysozyme, cytochrome c and BSA was achieved using CE. Compared with bare capillary or noncovalently bonded DR/PVA coatings, the covalently linked DR/PVA capillary coatings not only improved the CE separation performance for proteins, but also exhibited good stability and repeatability. Due to the replacement of highly toxic and moisture-sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide a green and easy way to make the covalently coated capillaries for CE.

On‐line CE/ESI/MS interfacing: Recent developments and applications in proteomics

After shining as the ultimate separation - sequencing technique used for the successful completion of the Human Genome Project, in the early 2000s CE experienced lowered popularity among separation scientists. The renewed interest in recent years relates to the separation needs, especially in proteomics, metabolomics, and glycomics, where CE complements liquid chromatography techniques. This interest is further boosted by the regulators requiring additional separation techniques for characterization of newly developed pharmaceuticals. This paper gives a short overview of recent developments in the on-line interfacing of CE separation techniques with electrospray ionization/mass spectrometric analysis. Both the instrumentation and selected CE/ESI/MS applications including analyses of peptides, proteins, and glycans are discussed with the stress on research published in the past 3 years. Techniques related to the proteomic and glycomic analyses such as sample preconcentration, on-line protein digestion, and analyte derivatization prior CE/ESI/MS analysis are also included.

Development and characterization of a novel semiautomated arrangement for electrochemically assisted injection in combination with capillary electrophoresis time‐of‐flight mass spectrometry

Electrochemically assisted injection (EAI) is an attractive injection concept for CE that enables the separation of neutral analytes via electrochemical generation of charged species during the injection process. A new semiautomated EAI configuration was developed and applied in conjunction with CE-MS (EAI-CE-MS). The EAI cell arrangement consists of an integrated buffer reservoir for CE separations and a compartment holding screen-printed electrodes. A drop of sample solution (50 μL) was sufficient to cover the three-electrode structures. A piezo motor provided a fast and precise capillary positioning over the screen-printed electrode assembly. Using ferrocene methanol as a model system, the EAI arrangement was characterized regarding coulometric efficiency, precision, and sensitivity of electrospray ionization-time-of-flight-MS. The formation of the cationic oxidation product of ferrocene methanol enhanced the sensitivity of CE-MS determination by two orders of magnitude and the electrochemically formed product showed a migration time corresponding to its individual electrophoretic mobility. Preliminary studies of EAI-CE-MS in the field of the analysis of nitroaromatic compounds were carried out. The formation of corresponding hydroxylamines and amines paved the way for selective and sensitive CE-MS determinations without the need of adding surfactants to the electrophoresis buffer.

Characterization of Yew Tree (Taxus) Varieties by Fingerprint and Principal Component Analyses

The composition of varieties of Taxus growing in Estonia was analyzed by capillary electrophoresis with diode array detection (CE-DAD) for the separation of phenolic compounds, and by high performance liquid chromatography-mass spectrometry (HPLC-MS) for the determination of taxoids. The main purpose of this study was the chemotaxonomic differentiation of varieties of Taxus by using data from these analyses. Fingerprints scanned at 214 nm on the basis of CE separation at pH 9.3 were used to characterize seven varieties of yew. The contents of four key taxoids (10-deacetylbaccatin, baccatin III, cephalomannine and paclitaxel) in six Taxus varieties were comparatively determined by HPLC-MS. The set of electropherograms/chromatograms of the various Taxus extracts were subjected to principal component analysis (PCA), using the peak areas of 16 phenolic compounds and 14 taxoids as characteristics. The formation of distinct clusters in accordance with botanical classification proves the suitability of PCA for differentiating varieties of Taxus.

Pressure-Assisted Capillary Electrophoresis Coupling with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging for Quantitative Analysis of Complex Peptide Mixtures

Herein, we report a pressure-assisted capillary electrophoresis-mass spectrometric imaging (PACE-MSI) platform for peptide analysis. This new platform has addressed the sample diffusion and peak splitting problems that appeared in our previous groove design, and it enables homogeneous deposition of the CE trace for high-throughput MALDI imaging. In the coupling of CE to MSI, individual peaks (m/z) can be visualized as discrete colored image regions and extracted from the MS imaging data, thus eliminating issues with peak overlapping and reducing reliance on an ultrahigh mass resolution mass spectrometer. Through a PACE separation, 46 tryptic peptides from bovine serum albumin and 150 putative neuropeptides from the pericardial organs of a model organism blue crab Callinectes sapidus were detected from the MALDI MS imaging traces, enabling a 4- to 6-fold increase of peptide coverage as compared with direct MALDI MS analysis. For the first time, quantitation with high accuracy was obtained using PACE-MSI for both digested tryptic peptides and endogenous neuropeptides from complex biological samples in combination with isotopic formaldehyde labeling. Although MSI is typically employed in tissue imaging, we show in this report that it offers a unique tool for quantitative analysis of complex trace-level analytes with CE separation. These results demonstrate a great potential of the PACE-MSI platform for enhanced quantitative proteomics and neuropeptidomics.

Simultaneous determination of lappaconitine hydrobromide and isopropiram fumarate in rabbit plasma by capillary electrophoresis with electrochemiluminescence detection

A CE electrochemiluminescence (CE-ECL) method for simultaneous determination of lappaconitine hydrobromide (LH) and isopropiram fumarate (IF) has been first established, with a chemically modified platinum electrode by europium (III)-doped Prussian blue analogue film as a working electrode. The conditions for CE separation and ECL detection are discussed and optimized in detail. It has been proved that 20 mmol/L phosphate buffer (pH 8.5) containing 5% (v/v) ACN and 0.17 mol/L SDS could achieve the most favorable resolution, and the high sensitivity of detection was obtained by maintaining the detection potential at 1.23 V. Under optimized conditions, a baseline separation for the two analytes was achieved within 6 min, and the standard curves were linear in the range of 1.0×10(-7) ~ 5.0 × 10(-5) g/mL for LH and 4.0 × 10(-8) ~ 1.0 × 10(-5) g/mL for IF with the detection limits (3σ) of 6.6 × 10(-8) g/mL for LH and 3.7 × 10(-8) g/mL for IF, respectively. The precisions of intra- and interday measurements for LH and IF were less than 4.21 and 2.61%, respectively. The applicability of the proposed method was illustrated in the determination of LH and IF in rabbit plasma with recoveries between 95.6 and 103.0%.

Monitoring potential prostate cancer biomarkers in urine by capillary electrophoresis–tandem mass spectrometry

Current prostate cancer (PCa) diagnosis based on prostate-specific antigen (PSA) has been gradually losing its credibility over the last decade due to contradictory results in published literature and clinical practice. Recently, a group of potential PCa biomarkers in urine, particularly sarcosine, was found to increase significantly as the cancer progressed to metastasis. We report a simple, robust, and reproducible CE–ESI-MS/MS method for the determination of sarcosine and other representative potential biomarkers in pooled urine. The pooled urine was obtained from 20 healthy adult volunteers between the ages of 23–30 years old. A solid phase extraction (SPE) technique was optimized for maximum recovery of sarcosine. With no derivatization step, excellent resolution between sarcosine and its isomers (α-alanine and β-alanine) was achieved. A separate non-SPE method was also developed for quantitative determination of highly concentrated urinary metabolites. CE separation was performed on a positively-charged, polyethyleneimine (PEI)-coated capillary using 0.4–2% formic acid in 50% methanol. Precision for intra- and inter-day standard addition calibration of sarcosine were found to be within 15%, whereas intra-day precisions for the rest of the metabolites varied from 0.03 to 13.4%. Acceptable intra-day and inter-day accuracies, ranging from 80 to 124%, were obtained for sarcosine and the other metabolites.

Enantioseparation of chiral acids and bases on a clindamycin phosphate-modified zirconia monolith by capillary electrochromatography

Porous zirconia monolith modified with clindamycin phosphate (CLIP-ZM) was used as chiral stationary phase (CSP) to separate a set of six acidic and basic chiral compounds in capillary electrochromatography (CEC). Resolutions and chiral selectivity factors of the chiral compounds were measured in ACN/MeOH mobile phases of varying compositions of MeOH and ammonium acetate as the electrolyte. In contrast to the CE separations where only chiral separations of acidic compounds were achieved enantiomers of both acidic and basic compounds were separated with acidic compounds better resolved than basic ones by CEC on the CLIP-ZM CSP. Best chiral separations of the chiral compounds were obtained with an eluent containing 10mM ammonium acetate and 35vol.% methanol.

Determination of gabapentin in human plasma by capillary electrophoresis-laser induced fluorescence detection with and without solid-phase extraction

We have developed two methods for the quantitation of gabapentin in human plasma. They are based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) with and without solid-phase extraction (SPE) and the derivatizing reagent 5-(4,6-dichlorotriazinyl)amino fluoresencin. The conditions for derivatization, separation and extraction were investigated in detail, and the optimal labeling conditions include a temperature of 40 °C, a reaction time of 30 min, and the use of a borate buffer of pH 9.0 as the reaction medium. A borate buffer of pH 9.2 served as a background electrolyte for CE separations. The CE-LIF and SPE-CE-LIF methods have linear ranges of 5–200 nmol L−1 and 0.2–10 nmol L−1, respectively, and the limits of detection are 0.5 and 0.02 nmol L−1, respectively. The SPE-CE-LIF method was successfully applied to the determination of gabapentin in blood plasma samples.

Dispersive liquid–liquid microextraction using a low density extraction solvent for the determination of 17 N-methylcarbamates by micellar electrokinetic chromatography–electrospray–mass spectrometry employing a volatile surfactant

A new analytical method based on micellar electrokinetic chromatography tandem mass spectrometry (MEKC–ESI–MS/MS) employing a MS friendly surfactant (ammonium perfluorooctanoate) is proposed and validated for the identification and simultaneous quantification of 17 N-methylcarbamate pesticides in environmental and drinking water samples. MS/MS detection using an ion trap as analyzer operating in the multiple reaction monitoring mode was used. Different parameters were optimized in order to obtain an adequate CE separation combined with the highest sensitivity in MS/MS. Dispersive liquid–liquid microextraction (DLLME) using a low-density extraction solvent has been proposed for extraction, obtaining a preconcentration factor of 10. Under optimum conditions, recoveries for fortified samples ranged from 83% to 101%, with relative standard deviations lower than 8%. The limits of detection ranged from 1 to 144ngl−1, demonstrating the sensitivity and applicability of this fast, simple, and environmentally friendly method.

Recentchiral selectors for separation in HPLC and CE

Abstract Enantiomers (stereoisomers) can exhibit substantially different properties if present in chiral environments. Since chirality is a basic property of nature, the different behaviors of the individual enantiomers must be carefully studied and properly treated. Therefore, enantioselective separations are a very important part of separation science. To achieve the separation of enantiomers, an enantioselective environment must be created by the addition of a chiral selector to the separation system. Many chiral selectors have been designed and used in various fields, such as the analyses of drugs, food constituents and agrochemicals. The most popular have become the chiral selectors and/or chiral stationary phases that are of general use, i.e., are applicable in various separation systems and allow for chiral separation of structurally different compounds. This review covers the most important chiral selectors / chiral stationary phases described and applied in high performance liquid chromatography and capillary electrophoresis during the period of the last three years (2008–2011).

On-Column Labeling of Gram-Positive Bacteria with a Boronic Acid Functionalized Squarylium Cyanine Dye for Analysis by Polymer-Enhanced Capillary Transient Isotachophoresis

A new asymmetric, squarylium cyanine dye functionalized by boronic acid ("SQ-BA") was designed and synthesized for on-capillary labeling of gram-positive bacteria to provide for high sensitivity detection by way of a modified form of capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-based separation employed a polymer-enhanced buffer with capillary transient isotachophoresis in a new hybrid method dubbed "PectI." It was found that the addition of various monosaccharides to SQ-BA in a batch aqueous solution greatly enhanced the emission of the boronic acid functionalized dye by a factor of up to 18.3 at a long wavelength (λ(ex) = 630 nm, λ(em) = 660 nm) with a high affinity constant (K = ~10(2.80) M(-1)) superior to other sugar probes. Semiempirical quantum mechanics calculations suggest that the mechanism for this high enhancement may involve the dissociation of initially nonemissive dye associates (stabilized by an intramolecular hydrogen bond) upon complex formation with sugars. The fluorescence emission of SQ-BA was also significantly enhanced in the presence of a gram-positive bacterial spore, Bacillus globigii (Bg), which serves as a simulant of B. anthracis (or anthrax) and which possesses a peptidoglycan (sugar)-rich spore coat to provide ample sites for interaction with the dye. Several peaks were observed for a pure Bg sample even with polyethyleneoxide (PEO) present in the CE separation buffer, despite the polymer's previously demonstrated ability to focus microoorganisms to a single peak during migration. Likewise, several peaks were observed for a Bg sample when capillary transient isotachophoresis (ctITP) alone was employed. However, the new combination of these techniques as "PectI" dramatically and reproducibly focused the bacteria to a single peak with no staining procedure. Using PectI, the trace detection of Bg spores (corresponding to approximately three cells per injection) along with separation efficiency enough to separate Bg from another gram-positive bacteria, Saccharomyces cerevisiae (resolution, R(s) = 6.09, and apparent plate number, N = 2.7-3.3 × 10(5)), were successfully achieved.

CE‐ESI‐MS separation of divalent organic and inorganic anions using a tricationic complexing reagent

A tricationic ion-pairing reagent, 1,3,5-1-butyl-3-methyl-1H-imidazol-3-ium-2,4,6-trimethylbenzene, was used to form complexes with doubly charged anions for their subsequent analysis by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in the positive-ion mode. This methodology offers the advantages of greater versatility and sensitivity relative to direct detection of the anions in negative-ion mode, and it can be realized by a number of possible complexation strategies, including pre-column, on-column, and post-column modes. Three model anions, sulfate [SO₄²⁻], thiosulfate [TSFA, S₂O₃²⁻], and benzenedisulfonate [BZDS, C₆H₄(SO₃)₂²⁻], were amenable to complexation with the tricationic reagent, yielding singly charged cations with greater mass-to-charge (m/z) ratios than the native analytes. By utilizing optimized parameters obtained through previous work with dicationic reagents and singly charged anions, including the CE separation buffer composition and pH, the concentration of the dicationic reagent, the mode of complexation, the nebulizing gas pressure, and the sheath liquid composition, it was possible to develop a robust CE-ESI-MS method appropriate for the analysis of divalent anions in a mixture.

Analysis of alendronate in human urine and plasma by magnetic solid‐phase extraction and capillary electrophoresis with fluorescence detection

A simple, rapid and sensitive CE-fluorescence (FL) detection method for the analysis of alendronate (ALEN), a bisphosphonate drug, has been developed. Using a buffer solution of 20 mM sodium phosphate (pH 10.0) and a voltage of 24 kV, separation of ALEN in a 55-cm length (35-cm effective length) capillary was achieved in 5 min. FL detection of ALEN was performed via pre-column derivatization with 2,3-naphthalene dicarbox-yaldehyde (NDA). Linear correlation (r=0.9981, n=6) between FL intensity and analyte concentration was obtained in the range of 7-200 ng/mL ALEN. The developed CE-FL method was applied to the analysis of ALEN in human urine and plasma samples. In order to eliminate the interfering matrix components, SPE using magnetic Fe(3) O(4) @Al(2) O(3) nanoparticles as solid sorbents was employed to clean the biological fluids before CE-FL analysis. The linear ranges of ALEN in urine and plasma were 5-100 ng/mL (r = 0.9982, n = 7) and 5-70 ng/mL (r = 0.9954, n = 7), respectively. The LOD and LOQ in both urine and plasma samples were 1.5 and 5 ng/mL ALEN, respectively. Total analysis time including sample pre-treatment and CE separation was less than 1.5 h.

Repeated capillary electrophoresis separations conducted on a commercial DNA chip

Multi-sample microchip capillary electrophoresis separations of a DNA sample (i.e. pUC18 DNA Hae III restriction digest) have been conducted on a commercial bioanalyzer. According to the vendor's specifications, a new chip must be used and the gel–dye mix must be used within 2 weeks for the best possible results. However, such a requirement of new chips and fresh reagents is quite costly, especially during preliminary research work, or instructional experiments in teaching labs. Therefore, we have developed a method to clean the microchip, so that the same DNA chip can be inspected and re-used. Our experimental data show that the CE separations conducted on a new chip and on a cleaned chip are comparable without loss in sizing accuracy and reproducibility. The gel–dye mix can also be used many times, longer than 2 weeks as specified by the vendor until the gel–dye changes color. This method will provide cost-saving advantage to laboratories during training and preliminary research work.

PH-mediated stacking in capillary electrophoresis for analysis of opiates in saliva

Both acid-mediated and base-mediated stacking in capillary electrophoresis were developed and validated in the present paper for opiates analysis in saliva. Optimum conditions for the determination of morphine, codeine and 6-monoacetylmorphine in both acid-mediated and base-mediated stacking CE were investigated. 100 mmol L−1 phosphate buffer (pH 3) containing 20% methanol and 5% isopropanol (v/v) was used as the background electrolyte for CE separation. The samples were introduced electrokinetically for 99 s at 10 kV in both stacking modes. An acid plug (100 mmol L−1 HCl) was introduced for 2 s at 2 kV after sampling in acid-mediated stacking. 12.5% aqueous ammonia and methanol (50 : 50, v/v) was introduced for 3 s under a pressure of 0.2 psi before sampling in base-mediated stacking. The methods showed a significant decrease in LODs (≤7 ng mL−1), while providing good separation efficiency, and reproducibility (RSD ≤ 19%, n = 5). The methods provided 1000-fold sensitivity enhancement compared with the normal hydrodynamic injection method. Beyond this, sample pretreatment was simple, which only involved diluting with methanol. Even in acid back liquid extraction in base-mediated stacking, the procedure for sample preparation was effort- and time-saving. These methods can be used to analyze trace amount of opiates in human saliva.

Elimination of suction effect in interfacing microchip electrophoresis with inductively coupled plasma mass spectrometry using porous monolithic plugs

A suction-free interfacing method was developed for microchip electrophoresis hyphenated with inductively coupled plasma mass spectrometry (MCE-ICP-MS). The hyphenated system was composed of a microchip, a demountable capillary microflow nebulizer (d-CMN) combined with a heated single pass spray chamber, a negative pressure sampling device, a high voltage power supply, a syringe pump and an ICP-MS. To eliminate the nebulizer suction generated by the pneumatic nebulizer and to ensure that the makeup solution flowed into the nebulizer, two porous polymer plugs were fabricated in the microchip. As a result, reasonably true electropherograms were obtained when compared to the CE separation performed in the traditional MCE-ICP-MS mode without porous polymer plugs. Electrophoretic separation of I(-) and IO(3)(-) was achieved within 25 s in a microchip with an effective separation length of only 15 mm at an electric field of 857 V cm(-1) using 10 mmol L(-1) borate (pH 9.2) as the running buffer. A resolution of 1.3 was obtained and the absolute detection limits for I(-) and IO(3)(-) were 0.12 and 0.13 fg, respectively. The precisions (RSD, n = 10) of the migration time and peak height for I(-) and IO(3)(-) were in the range of 1.1-1.6% and 2.5-2.8%, respectively. Two table salt samples were analyzed by an external calibration method. The iodate contents were in accordance with their labeled values. The recoveries of I(-) and IO(3)(-) in the table salt samples were in the range of 92-105%.

Capillary electrophoresis with electrospray ionisation-mass spectrometry for the characterisation of degradation products in aged papers

A methodology for capillary electrophoresis/electrospray ionisation mass spectrometry (CE/ESI-MS) was developed for the simultaneous analysis of degradation products from paper among two families of compounds: low molar mass aliphatic organic acids, and aromatic (phenolic and furanic) compounds. The work comprises the optimisation of the CE separation and the ESI-MS parameters for improved sensitivity with model compounds using two successive designs of experiments. The method was applied to the analysis of lignocellulosic paper at different stages of accelerated hygrothermal ageing. The compounds of interest were identified. Most of them could be quantified and several additional analytes were separated.

Highly sensitive capillary electrophoresis analysis of N‐linked oligosaccharides in glycoproteins following fluorescence derivatization with rhodamine 110 and laser‐induced fluorescence detection

We describe a highly sensitive CE with laser-induced fluorescence (LIF) detection for the analysis of N-linked oligosaccharides in glycoproteins using rhodamine 110 as a fluorescence derivatization reagent. One CE separation is performed using a fused-silica capillary and neutral pH buffer conditions and allows for the separation of sialo-oligosaccharides according to the number of sialic acids. An alternate separation is performed using the same capillary and acidic pH buffer conditions, enabling the separation of asialo-oligosaccharides according to their sizes. The derivatization and separation conditions for the analysis of sialo- and asialo-oligosaccharides were optimized. Furthermore, we applied the proposed method for the analyses of N-linked sialo- and asialo-oligosaccharides in glycoproteins (ribonuclease B, fetuin, and recombinant human erythropoietin).