Migration behavior and separation of active components in Glycyrrhiza uralensis Fisch and its commercial extract by micellar electrokinetic capillary chromatography

1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and 2-(p-toluidino)naphthalene-6-sulfonic acid (2,6-TNS) were evaluated as additives in different buffers for the detection of bovine whey proteins using laser-induced fluorescence (LIF) monitoring in capillary electrophoresis (CE). These N-arylaminonaphthalene sulfonates furnish a large fluorescence emission when associated to some proteins whereas their emission in aqueous buffers, such as those used in CE separations, is very small. To select the best detection conditions, the fluorescence of these probes was first compared using experiments carried out in a fluorescence spectrophotometer. Using bovine serum albumin (BSA) as a model protein, it was demonstrated that 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 8 and pH 10.2) and the fluorescent probe 2,6-TNS gave rise to the highest increase in fluorescence for BSA. When the composition of these separation buffers was optimized for the electrophoretic separations, CHES buffer, pH 10.2 was chosen as the most suitable buffer to detect bovine whey proteins. The limit of detection obtained for some whey proteins in CE separations was about 6.10(-8) M for BSA, 3.10(-7) M for beta-lactoglobulin A (beta-LGA), 3.10(-7) M for beta-lactoglobulin B (beta-LGB), and 3.10(-6) M for alpha-lactalbumin (alpha-LA). These detection limits were compared to those achieved using UV detection under the same separation conditions. The results showed that the detection limits of BSA, beta-LGA and beta-LGB were twice as good using LIF than with UV detection. However, the limit of detection for alpha-LA was better when UV was used. The applicability of LIF detection to CE separation of whey proteins in bovine milk samples was also demonstrated.     

Construction and analytical application of an on-column photo reactor for improved detection of iron-species as plant metabolites in capillary flow injection and capillary electrophoresis

An on-column photo reactor for CE, which is constructed from UV-transparent capillaries and a small Pen-Ray UV lamp, is applied to the analysis of small, non-covalent iron-species. These iron-species, e.g. phytosiderophores (PS) in grasses and the non-protein amino acid nicotianamine (NA), play an important role in plant metabolism. The photo reactor is placed directly in front of the on-column absorbance detector, illuminating only some centimeters of the capillary. The photo reactor is used for capillary electrophoresis (CE) and also for capillary flow injection analysis (CFIA). Photoinduced sensitivity changes for model iron-species and for plant extracts are investigated, using UV detection and capacitively coupled contactless conductivity detection (C(4)D). The detection sensitivity for iron-species is enhanced in CFIA; the enhancement factor depends on the type of iron-species. In CE, the sensitivity of iron-species is kinetically dependent on the type of iron-species, decreasing with both detectors, but a photo reaction product is detectable. The relationship between irradiation window length and sensitivity is investigated quantitatively using EDTA-Fe(III). Pure ligands without iron are little affected by the photo reaction in both CFIA and CE. In CE, the detector signals of iron-species in real plant samples are selectively decreased with the proposed photo reactor, thus enabling a simple screening method for such photoactive iron-species.     

Comparison of high-performance liquid chromatography and capillary electrophoresis for the determination of some bee venom components

HPLC and capillary electrophoretic (CE) methods were compared for the determination of phospholipase A₂ and melittin in bee venom. Size-exclusion chromatography on a Tessek Separon HEMA-BIO 40 column requires the use of a denaturing eluent (0.2% trifluoroacetic acid in 20% acetonitrile) to overcome non-specific interactions of some components, e.g., melittin. Reversed-phase HPLC on a HEMA-BIO 1000 C₁₈ column with gradient elution using water-acetonitrile mobile phases containing trifluoroacetic acid and UV spectrophotometric detection at 215 nm permits the identification and determination of the main bee venom components and their preparative chromatography. CE analysis for bee venom components is optimum with electrolyte system of 150 mM phosphoric acid (pH 1.8) with UV spectrophotometric detection at 190 nm. In comparison with HPLC, the CE method is cheaper and faster (6 min vs. 45 min) and the separation is more efficient.     

Capillary liquid chromatography-mass spectrometry and micellar electrokinetic chromatography as complementary techniques in environmental analysis

Applications of capillary electrophoresis (CE) and capillary liquid chromatography (LC) to environmental analysis have been limited. In this work we present applications of micellar electrokinetic chromatography (MEKC) to the analysis of environmental matrices for synthetic dyes. Separations obtained by capillary LC are compared with those obtained under MEKC for seven selected dyes. Both techniques are capable of resolving the subject compounds at high efficiency. Recovery data for spiked water and soil matrices were obtained for four dyes using solid-phase extraction cartridges and disks with determination by MEKC-UV detection. Both pH adjustment via acid and ion-pairing via a cationic surfactant were investigated for isolating dyes. Capillary LC detection was by continuous-flow liquid secondary ion mass spectrometry (CF-LSI-MS) whereas MEKC used UV detection (214 nm). Application of peak-profiling at high mass resolution is illustrated with the capillary LC-MS technique. Interfacing capillary LC under CF-LSI-MS using the coaxial arrangement is easier than interfacing CE with this arrangement. MEKC provides a powerful screening and determinative technique, while capillary LC-MS provides a confirmatory tool.     

Direct analysis of polyols using 3-nitrophenylboronic acid in capillary electrophoresis: thermodynamic and electrokinetic principles of molecular recognition

The design of boronic acid sensors for photometric detection of carbohydrates has relied on exploiting differences in the thermodynamic stability of complex formation for molecular recognition. Herein, we introduce a direct method for analysis of sugar alcohols using 3-nitrophenylboronic acid (NPBA) as an electrokinetic probe in capillary electrophoresis (CE). Dynamic complexation of neutral polyols by NPBA during electromigration allows for their simultaneous resolution and UV detection based on formation of an anionic ternary boronate ester complex in phosphate buffer. Unlike conventional boronic acid sensors, thermodynamic and electrokinetic processes in CE allow for improved selectivity for the resolution of sugar alcohol stereoisomers having different vicinal polyol chain lengths even in cases when binding affinity is similar due to differences in their complex mobility. Three complementary approaches were investigated to compare the thermodynamics of polyol chelation with NPBA, namely direct binding assays by CE, UV absorbance spectroscopy and an indirect pK _a depression method. Overall, CE offers a convenient platform for characterization of reversible arylboronic acid interactions in free solution while allowing for direct analysis of complex mixtures of neutral/UV-transparent polyols without complicated sample handling.     

UV- and visible-excited fluorescence of nucleic acids separated by capillary electrophoresis

UV- and visible-excited fluorescence detection strategies were compared for nucleic acids separated by capillary electrophoresis (CE). A dual-polymer sieving matrix consisting of hydroxypropylmethylcellulose and poly(vinylpyrrolidone) was used to separate DNA fragments from a 100-base pair ladder and RNA from individual cells. Two nucleic acid dyes, SYBR Gold and SYBR Green I, were evaluated for their performance at both UV (275 nm) and visible (488 nm) excitation wavelengths. While SYBR Gold-bound RNA from single cells yielded a substantially reduced UV-excited signal compared to that with visible excitation (as expected), the sensitivity of SYBR Gold-bound double-stranded DNA was comparable for UV and Vis excitation wavelengths. This study reveals the first demonstration of using SYBR Gold dyes for DNA detection following separation with CE and also the first example of SYBR-based detection of RNA sampled and separated from individual cells.     

Separation and determination of 10-hydroxy-2-decenoic acid in royal jelly by capillary electrophoresis

Summary The application of capillary electrophoresis (CE) to the separation and determination of the active ingredient, 10-hydroxy-2-decenoic acid, in royal jelly with direct on-column UV detection at 214 nm is described. Using a cathodic injection and anodic detection scheme, 10-hydroxy-2-decenoic acid (10-HDA) was separated and detected in less than 10 min in a fused silica capillary column with a phosphate buffer at pH 7.3 with an applied voltage of 20 KV followed by direct UV detection. The use of cetyltrimethylammonium bromide (CTAB) as electroosmotic flow modifier allows the rapid separation of 10-HDA from other constituents in royal jelly by reversing the direction of electroosmotic flow. The influence of organic solvents in the electrolyte on separation selectivity is also discussed.     

Separation and detection of angiotensin peptides by Cu(II) complexation and capillary electrophoresis with UV and electrochemical detection

The use of capillary electrophoresis (CE) with on-capillary Cu(II) complexation for the determination of angiotensin and its metabolites is described. The resulting copper-peptide complexes can be detected using either UV or electrochemical (EC) detection. Optimal reaction and separation conditions for the angiotensin peptides were first determined using CE with UV detection. With UV detection, the limit of detection (signal-to noise ratio S/N = 3) for native angiotensin II was 18 microM, while the limit of detection (LOD) obtained for the copper-angiotensin II complex is 2 microM. CE with EC detection was then evaluated, yielding significantly lower LODs--2 microM for native angiotensin II and 200 nM for the copper-angiotensin II complex. The addition of copper to the run buffer improved the separation and sensitivity for both CE-UV and CE-EC detection. The method was demonstrated by monitoring the conversion of angiotensin I to angiotensin II in plasma via angiotensin-converting enzyme (ACE) and subsequent inhibition of ACE by captopril.     

Online preconcentration of arsenic compounds by dynamic pH junction-capillary electrophoresis

An online preconcentration technique by dynamic pH junction was studied to improve the detection limit for anionic arsenic compounds by CE. The main target compound is roxarsone, or 3-nitro-4-hydroxyphenylarsonic acid, which is being used as an animal feed additive. The other inorganic and organoarsenic compounds studied are the possible biotransformation products of roxarsone. The arsenic species were separated by a dynamic pH junction in a fused-silica capillary using 15 mM phosphate buffer (pH 10.6) as the BGE and 15 mM acetic acid as the sample matrix. CE with UV detection was monitored at a wavelength of 192 nm. The influence of buffer pH and concentration on dynamic pH junction were investigated. The arsenic species focusing resulted in LOD improvement by a factor of 100-800. The combined use of C18 and anion exchange SPE and dynamic pH junction to CE analysis of chicken litter and soils helps to increase the detection sensitivity. Recoveries of spiked samples ranged between 70 and 72%.     

Improving detection in capillary electrophoresis with laser induced fluorescence via a bubble cell capillary and laser power adjustment

Bubble cells have been frequently employed in capillary electrophoresis (CE) to increase the light path length with UV detection to provide an increase in the observed sensitivity of CE; however this approach has not been commonly used for laser-induced fluorescence detection (LIF) with CE. In this paper we study the influence of laser power on the sensitivity of detection in using conventional and enlarged fused silica capillaries for CE with LIF. When using the bubble cell capillary, the laser power must be decreased relative to use of the conventional capillary to reduce the effects of photodegradation of the species being illuminated by the laser. Even though the light intensity was decreased, an increase in sensitivity of detection was observed for most compounds when a bubble cell was used. This increase ranged from a factor of 8 for riboflavin (410 nm excitation) to 3.2 for most aromatic compounds (266 nm excitation), when using a 3x bubble cell compared with a conventional capillary. The bubble cell capillary was used for native detection of IgG by LIF at 266 nm. A limit of detection of 60 ng mL(-1) was obtained from a 20 pg injection, which was 40 times more sensitive than silver staining in conventional SDS/PAGE.     

Speciation of organomercury compounds by capillary electrophoresis with pre-column derivatization and on-line stacking

A simple capillary electrophoresis method was developed to separate and quantify methylmercury, ethylmercury, and phenylmercury with the enhancement of pre-column derivatization and on-line stacking.     

Determination of low-molecular-mass quaternary ammonium compounds by capillary electrophoresis and hyphenation with mass spectrometry

The determination of quaternary ammonium ions by capillary electrophoresis (CE) is reviewed. The analytes include tetraalkylammonium and alkylbenzyldimethylammonium compounds frequently used as antiseptic and antibacterial agents as well as in various household products, several plant growth regulators and herbicides, by-products in bile acid sequestrants, and a range of anticholinergic drugs. Besides direct and indirect UV detection, hyphenation with electrospray mass spectrometry is particularly suited for quaternary ammonium ions and may lower the detection limits by two orders of magnitude. In comparison with established liquid chromatographic techniques, CE may exhibits superior separation efficiency. Applications in routine analysis have demonstrated that CE is reliable and robust enough to represent a real alternative to chromatography.     

Development and validation of a capillary electrophoresis-indirect photometric detection method for the determination of the non-UV-absorbing 1,4-dideoxy-1,4-imino-D-arabinitol in active pharmaceutical ingredients,solutions and tablets using an internal standard

A high speed, selective, and robust capillary electrophoresis (CE) method with high capacity was developed and validated for determination of assay of 1,4-dideoxy-1,4-imino-D-arabinitol in active pharmaceutical ingredients, solutions, and tablets during the development work at preclinical and Phase I and II clinical studies. 1,4-Dideoxy-1,4-imino-D-arabinitol, tartrate has (almost) no UV absorption. Therefore, the developed CE method for quantification was based on indirect UV detection. A cation CE principle was chosen using an electrolyte at pH 4.0 containing dimethyldiphenylphosphonium hydroxide, which has a strong UV absorbance. The quantification was performed using internal standard technique, by which piperidine was used as internal standard. The method was validated. The validation results showed that the CE method was suitable for the assay (and dissolution) analysis.     

Determination of Phenols,Inorganic Anions,and Carboxylic Acids in Kraft Black Liquors by Capillary Electrophoresis

Two methods are presented for the quantitative capillary electrophoretic (CE) determination of phenolic lignin degradation compounds as well as of inorganic anions and organic acids in Kraft black liquors. Important phenolic lignin degradation compounds can be rapidly separated by co-electroosmotic CE after acidification of the liquors and subsequent extraction of the compounds with chloroform. A capillary electrophoretic separation of phenolic compounds is performed by using a phosphate/borate electrolyte system and UV detection at 214 nm. In addition, a HPLC method using a gradient with water, methanol, and acetic acid is also developed. Inorganic ions which are of importance to the pulping process can be determined by simply diluting the black liquors after sampling and subsequent analysis with a chromate electrolyte system and indirect UV detection at 185 nm. In addition, the concentration of low molecular aliphatic carboxylic acids can be determined simultaneously within the same run. By method optimization it is possible to separate the anions within one minute and, at the same time, to increase the resolution of the solutes. The electrolyte systems for the CE separations were optimized by varying the pH value and by adding organic solvents. Short separation times are obtained by adding a polycationic EOF modifier (hexadimethrine bromide) to the electrolyte which reverses the electroosmotic flow. A migration of the anionic analytes in the same direction as the electroosmotic flow is thus established.     

Simultaneous Determination of Glyphosate,Glufosinate, and Aminomethylphosphonic Acid by Capillary Electrophoresis After 9‐Fluorenylmethyl Chloroformate Derivatization

A capillary electrophoresis (CE) with UV absorption detection method is described for the simultaneous determination of glufosinate, glyphosate, and aminomethylphosphoric acid. The 9‐fluorenylmethyl chloroformate (FMOC‐Cl) was used for precolumn derivatization of the non‐absorbing herbicides. The three analytes were separated by CE in 9 min with 25 mM borate buffer at pH 9, followed by detection with a UV detector at 260 nm. We demonstrate how the detection limit can be enhanced by using acetonitrile‐salt mixtures. With acetonitrile‐salt mixtures, the limit of detection (LOD) was in the 10^?7 M range. Linearity of more than two orders of magnitude was generally obtained. Precisions of migration times and peak areas were less than 0.9% and 7.5%, respectively. The applicabilities of the method for the analysis of ground water and lake water were examined.     

On-line sample preconcentration with chemical derivatization of bacterial biomarkers by capillary electrophoresis: a dual strategy for integrating sample pretreatment with chemical analysis

Simple, selective yet sensitive methods to quantify low-abundance bacterial biomarkers derived from complex samples are required in clinical, biological, and environmental applications. In this report, a new strategy to integrate sample pretreatment with chemical analysis is investigated using on-line preconcentration with chemical derivatization by CE and UV detection. Single-step enantioselective analysis of muramic acid (MA) and diaminopimelic acid (DAP) was achieved by CE via sample enrichment by dynamic pH junction with ortho-phthalaldehyde/N-acetyl-L-cysteine labeling directly in-capillary. The optimized method resulted in up to a 100-fold enhancement in concentration sensitivity compared to conventional off-line derivatization procedures. The method was also applied toward the detection of micromolar levels of MA and DAP excreted in the extracellular medium of Escherichia coli bacterial cell cultures. On-line preconcentration with chemical derivatization by CE represents a unique approach for conducting rapid, sensitive, and high-throughput analyses of other classes of amino acid and amino sugar metabolites with reduced sample handling, where the capillary functions simultaneously as a concentrator, microreactor, and chiral selector.     

Rapid determination of cyclamate in foods by solid-phase extraction and capillary electrophoresis

A method for the determination of cyclamate in food was developed using solid-phase extraction (SPE) and capillary electrophoresis (CE) with indirect ultraviolet (UV) detection. A 5-10 g sample in 0.1 mol/L hydrochloric acid was homogenized and made up to a volume of 50 mL with 0.1 mol/L hydrochloric acid. After the sample was centrifuged, 25 mL of supernatant was loaded into an Oasis HLB SPE cartridge. The cartridge was washed with 2 mL of demineralized water followed by 2 mL of 50% aqueous methanol, and cyclamate was eluted with 4.5 mL of 50% aqueous methanol. The eluate was added to a solution of sodium propionate (internal standard) for CE analysis. The cyclamate in the eluate was electrophoresed on a fused-silica capillary using 1 mmol/L hexadecyltrimethylammonium bromide and 10 mmol/L potassium sorbate as a running buffer. Detection and reference wavelengths of cyclamate determined with a UV detector were 300 and 254 nm, respectively. The calibration curves for cyclamate showed good linearity in the range of 2-1000 microg/mL and the limits of detection in beverage, fruit in syrup, jam, pickles and confectionary are sample dependent and ranged from 5-10 microg/g. The recovery of cyclamate added at a level of 200 microg/g to various kinds of foods was 93.3-108.3% and the relative standard deviation was less than 4.9% (n=3). A number of commercial samples were analyzed using the proposed method. Cyclamate was detected in one waume, two pickles, and two sunflower seeds. The quantitative values determined with CE correlated to those from high-performance liquid chromatography (HPLC) (the detected values of cyclamate in a sunflower seed measured by CE and HPLC were 3.40 g/kg and 3.51 g/kg, respectively). This analytical method for cyclamate using CE is especially suitable for use in the field.     

Large-volume sample stacking for analysis of ethylenediaminetetraacetic acid by capillary electrophoresis

A simple, quick, and sensitive capillary electrophoretic technique-large volume stacking using the electroosmotic flow (EOF) pump (LVSEP) - has been developed for determining ethylenediaminetetraacetic acid (EDTA) in drinking water for the first time. It is based on a precapillary complexation of EDTA with Fe(III) ions, followed by large-volume sample stacking and direct UV detection at 258 nm. The curve of peak response versus concentration was linear from 5.0 to 600.0 microg/L, and 0.7 to 30.0 mg/L. The regression coefficients were 0.9988 and 0.9990, respectively. The detection limit of the current technique for EDTA analysis was 0.2 microg/L with an additional 10-fold preconcentration procedure, based on the signal-to-noise ratio of 3. As opposed to the classical capillary zone electrophoresis (CE) method, the detection limit was improved about 1000-fold by using this LVSEP method. To the best of our knowledge, it represents the highest sensitivity for EDTA analysis via CE. Several drinking water samples were tested by this novel method with satisfactory results.     

A highly sensitive method for enantioseparation of fenoprofen and amino acid derivatives by capillary electrophoresis with on-line sample preconcentration

A highly sensitive method for enantioseparation of trace fenoprofen and amino acid derivatives by capillary electrophoresis (CE) with vancomycin as the chiral selector was developed. Several CE techniques, such as the partial filling, large-volume sample stacking with EOF as pump plus anion-selective exhaustive injection (LVSEP-ASEI) were involved in the present method to improve the detection sensitivity. With on-column concentration, enantioseparation of racemic fenoprofen and six 9-fluorenylmethyl chloroformate (FMOC)-amino acid derivatives (at the concentration level of ng/mL) with the background electrolyte composed of 100 mmol/L Tris-H(3)PO(4) (pH 6.0) and 2 mmol/L vancomycin was detected readily with the UV detection at 214 nm. Successfully performing LVSEP-ASEI needs a very low EOF that could be depressed by coating the capillary with poly(dimethylacrylamide) solution. The coating also played a role to minimize the adsorption of vancomycin onto the capillary wall. Effect of the injected sample volume and the electrokinetic injection time on the peak area of the enantiomers and their resolution factor were investigated and optimized. Under the optimized conditions, more than 1000-fold enhancement in detection sensitivity compared with the normal injection was achieved.     

Capillary electrophoresis using a surfactant-treated capillary coupled with offline matrix-assisted laser desorption ionization mass spectrometry for high efficiency and sensitivity detection of proteins.

A method of combining capillary electrophoresis (CE) using a surfactant-modified capillary with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is described for protein analysis. The CE-MALDI-MS coupling is based on CE fraction collection of nanoliter volume samples in less than 5 microl of dilute acid. This offline coupling does not require any special instrumentation and can be readily performed with commercial instruments. Protein adsorption during CE separation is prevented by coating the capillary with the surfactant didodecyldimethylammonium bromide. This surfactant binds strongly with the capillary wall, hence it does not desorb significantly to interfere with subsequent MALDI-MS analysis. It is shown that the use of a dilute acid for CE fraction collection is advantageous in lowering the detection limit of MALDI-MS compared to using an electrophoretic buffer. The detection limit for proteins such as cytochrome c is 23 fmol injected for CE, or 1.2 fmol spotted for MALDI-MS. This sensitivity is comparable to alternative CE-MALDI-MS coupling techniques using direct CE sample deposition on the MALDI target. In addition, the fraction collection approach has the advantage of allowing multiple reactions to be carried out on the fractioned sample. These reactions are very important in protein identification and structure analysis.