Indirect fluorescence detection of phenolic compounds by capillary electrophoresis on a glass device

A micromachined capillary electrophoresis system has been fabricated on a glass device for the separation and indirect fluorescence detection of phenols. Using this device two phenols viz., 2,4-dichlorophenol and pentachlorophenol, were separated within 12 s compared to under 19 min on a conventional capillary electrophoresis system using direct ultraviolet detection. The precision of the glass device ranged from 12.7%-16.7% compared to 0.42%-4.9% for the conventional system. Both systems showed good linearity in the concentration range of 0.8-6.38 mM for the glass device and 5-130 microM for the conventional system. The relationship between temperature and high voltage with baseline drift was also investigated. These results provide a foundation for the development of a miniaturised chemical analysis system for the on-line analysis of phenols in water.     

Gas-chromatographic determination of chlorostyrene and dichlorostyrene isomers in workplace air

Conditions for a gas-chromatographic determination of vapors and aerosols of p-chlorostyrene and 2,6-dichlorostyrene contained in workplace air samples were determined. The method is based on the adsorption of p-chlorostyrene and 2,6-dichlorostyrene on activated charcoal and fiberglass, desorption with toluene and analysis of the obtained solution by capillary gas chromatography with flame ionization detection (FID). The determination limit of the method is 5 mg m^–3 for each substance. Received: 13 December 1999 / Revised: 6 March 2000 / Accepted: 7 March 2000     

Determination of Phenolic Acids in Herba Epilobi by ITP–CE in the Column-Coupling Configuration

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column-coupling configuration has been optimized in a mode in which the background electrolyte employed in the CZE step was different from the leading and terminating electrolytes of the ITP step. The optimum composition of the electrolyte system was 0.01 M HCl, 0.02 M IMI, 0.2% HEC, pH 7.2 (leading electrolyte), 0.01 M HEPES, pH 8.2 (terminating electrolyte), and 25 mM MES, 50 mM TRIS, 30 mM boric acid, 0.2% HEC, pH 8.3 (background electrolyte). All solutions contained 20% methanol. The timing of the transfer of isotachophoretically stacked analyte zones into the CZE column was also optimized. An ITP–CZE method with UV detection at 270 nm was developed for separation of nine phenolic acids (protocatechuic, syringic, vanillic, cinnamic, ferulic, caffeic, ρ-coumaric, chlorogenic, and gentisic acids) in a model mixture and used for assay of some of these acids in a methanolic extract of herba epilobi. Application of ITP–CZE resulted in 100-fold better sensitivity than conventional CZE; limits of detection ranged between 10 and 60 ng mL⁻¹. When MES–TRIS–borate-based buffer, pH 8.3, was used in the CZE separation step the linearity of the ITP–CZE response was satisfactory (correlation coefficients were from 0.9937 to 0.9777). Repeatability was also satisfactory (RSD values ranged between 0.77% and 1.28% for migration times and between 1.65% and 13.69% for peak area). Revised: 23 March and 27 April 2006     

Integrated microfluidic device for the separation and electrochemical detection of catechol estrogen-derived DNA adducts

Catechol estrogen-derived DNA adducts are formed as a result of the reaction of catechol estrogen metabolites (e.g., catechol estrogen quinones) with DNA to form depurinating adducts. Developing a new methodology for the detection of various DNA adducts is essential for medical diagnostics, and to this end, we demonstrate the applicability of on-chip capillary electrophoresis with an integrated electrochemical system for the separation and amperometric detection of various catechol estrogen-derived DNA adducts. A hybrid PDMS/glass microchip with in-channel amperometric detection interfaced with in situ palladium decoupler is utilized and presented. The influence of buffer additives along with the effect of the separation voltage on the resolving power of the microchip is discussed. Calibration plots were constructed in the range 0.4–10 μM with r ² ≥ 0.999, and detection limits in the attomole range are reported. These results suggest that on-chip analysis is applicable for analyzing various DNA adducts as potential biomarkers for future medical diagnostics.     

Capillary waveguide fluoroimmunosensor with improved repeatability and detection sensitivity

An optical capillary waveguide fluoroimmunosensor based on glass capillaries internally coated with an ultrathin poly(dimethylsiloxane) (PDMS) film is presented. The evaluation of the capillaries developed was done in comparison with aminosilanized [3-(aminopropyl)triethoxysilane, APTES] glass and poly(methylpentene) (PMP) capillaries by immobilizing rabbit γ-globulins on the internal capillary wall. Following reaction with (R)-phycoerythrin-labelled antibody, the capillary was scanned with a laser beam and the fluorescence waveguided through the capillary wall was detected by a photomultiplier placed at one of its ends. The capillaries developed provided considerably improved protein coating homogeneity (intracapillary coefficients of variation 2.9–6.6%) and repeatability (intercapillary coefficients of variation 2.1–5.0%) compared with APTES-treated ones (7.9–13.4 and 8.5–15.2%, respectively). With use of these capillaries in a sandwich-type immunosensor for the determination of rabbit γ-globulins, the assay detection limit was improved eightfold (4.4 ng/mL) compared with that obtained using PMP capillaries (35.3 ng/mL), whereas the assay repeatability was improved threefold (intra-assay coefficients of variation 5.9–13.1%) compared with APTES-treated capillaries (15.6–36%). Optoelectronic set-up used to scan the capillaries (left) and representative fluorescence scannings of dual-band poly(methylpentene) (PMP), PDMS-modified glass and APTES treated glass capillaries     

Potencies of ligand-exchange capillary electrophoresis in the determination of biologically active compounds

The analytical potencies of ligand-exchange capillary electrophoresis (LECE) in regard to the determination of bioactive substances are discussed. As it is shown, complexation with copper(II) in the electrophoretic determination with UV detection makes possible the determination of amino acids, amines, and sugars, which do not absorb in the UV region, with the limits of detection 5–10 mg/L, and reduce the detection limits of the absorbing analytes, such as tryptophane, tyrosine, and histamine by 2–3 times. Prospects for using online preconcentration for the reduction of c _min are shown on an example of aliphatic amino acids, i.e., thirtyfold preconcentration is attained. LECE is shown to be applicable to the simultaneous determination of glucose, Na(⁺), and K(⁺) in human blood serum. Also versions of ligand-exchange capillary electrophoresis with direct UV detection and capillary zone electrophoresis with indirect one are compared in terms of efficiency, time of analysis, and limit of detection. The potencies of the LECE method are compared with those of ligand-exchange chromatography.     

Routine serum protein analysis by capillary zone electrophoresis: Clinical evaluation

Summary To measure the five classical protein fractions in human serum several electrophoretic techniques are available. Besides separation on cellulose acetate membrane or agarose gel, capillary zone electrophoresis (CZE) may be a useful analytical alternative in clinical routine. We have compared the Dionex CES I capillary electrophoresis system with that of the Olympus Fractoscan using specimens submitted for routine analysis. For clinical evaluation 102 samples from patients with various diseases have been analysed. Serum protein fractions were judged on separation performance, precision and the regression method ofBablok-Passing. Regression analysis revealed variable agreement between both methods with a slope ± intercept of 2.10–0.52 (α₁-fraction) and 1.0–0.20 (α₂-fraction) as worse and best, resectively; and the coefficient of variation of migration time: 5.9 %–6.8 % (between-run imprecision). Differences in the comparison of fractions are mainly caused by the improved resolution of CZE; e.g. one β-globulin peak on cellulose acetate is separated into two distinct protein fractions in CZE, including more detailed diagnostic information—as is also the case with γ-fraction. In some cases monoclonal gammopathy with low concentrations of immunglobulin clone can only be detected in CZE, whereas the cellulose acetate membrane (CAME) electropherogram is inconspicuous. The within-run precision (N=18) gave coefficients of variation of peak areas 1.3–5.9 % (CZE) and 1.0–3.8 % (cellulose acetate membrane). This is the first time that a complete clinical evaluation of CZE serum protein fraction analysis has been performed. CZE with its higher resolution and hence more detailed diagnostic information in some cases, showed good separation patterns, precision and correlation. Interchangeability of results showed that this CZE method is well suited for analysis of serum protein fractions in clinical routine. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Determination of inorganic oxyhalides in drinking water by on-line coupled capillary isotachophoresis— capillary zone electrophoresis

Some oxyhalides can be found in drinking waters as inorganic disinfection byproducts. An on-line coupled capillary isotachophoresis—capillary zone electrophoresis (CITP-CZE) method was developed for the analysis of chlorate, chlorite and bromate in water. The optimized CITP-CZE electrolyte system consisted of the following: 10 mM—HCl+20 mM—β-Alanine (leading electrolyte), 5 mM—succinic acid (terminating electrolyte), and 10 mM—succinic acid +5 mM—β-Alanine +0.1% HPMC (carrier electrolyte). A clear separation of oxyhalides from other components of drinking water was achieved within 25 min. Method characteristics, i.e., linearity (0–200 ng/mL), accuracy (88–110%), intra-assay (3–5%), quantification limit (5–15 ng/mL), and detection limit (2–5 ng/mL), were determined. Minimum labor requirements, sufficient sensitivity and low running cost are important attributes of this method. It was found that the developed method is useful for the routine analysis of oxyhalides in water.     

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometry for determination of drugs of abuse in human urine

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometric detection (SPE–CE–MS) has been used for determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine (COD), hydrocodeine (HCOD), and 6-acetylmorphine (6AM) in urine. The preconcentration system consists of a small capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoresis capillary. The SPE–CE–MS experimental conditions were optimized as follows: the sample (adjusted to pH 6.0) was loaded at 930 mbar for 60 min, elution was performed with methanol at 50 mbar for 35 s, 60 mmol L⁻¹ ammonium acetate at pH 3.8 was used as running buffer, the separation voltage was 30 kV, and the sheath liquid at a flow rate of 5.0 μL min⁻¹ was isopropanol–water 50:50 (v/v) containing 0.5% acetic acid. Analysis of urine samples spiked with the four drugs and diluted 1:1 (v/v) was studied in the linear range 0.08–10 ng mL⁻¹. Detection limits (LODs) (S/N = 3) were between 0.013 and 0.210 ng mL⁻¹. Repeatability (expressed as relative standard deviation) was below 7.2%. The method developed enables simple and effective determination of these drugs of abuse in urine samples at the levels encountered in toxicology and doping.     

CE Determination of 2-(9-Carbazole)ethyl Chloroformate-Labeled Oligopeptides

A pre-column derivatization method for sensitive determination of oligopeptides, using the tagging reagent 2-(9-carbazole)ethyl chloroformate (CEOC-Cl) followed by capillary electrophoresis (CE) with diode-array detection, has been developed. Maximum yield close to 100% were observed when a three to fourfold molar excess of reagent was used at pH 9.0–10.0. Excess reagent was extracted with n-hexane–ethyl acetate 9:1–10:1 (v/v); this enabled direct analysis using CE with no significant disturbance from the major fluorescent reagent degradation by-products. The effects on the results of buffer pH and of SDS and organic modifier concentrations were examined. Good baseline resolution in the separation of five CEOC-peptides was achieved with a 48.5-cm total length (effective length 40 cm) 50-μm inner diameter capillary column.     

Development of a non-aqueous capillary electrophoresis method with UV-visible and fluorescence detection for phenolics compounds in olive oil

Response surface methodology has been applied to the optimization of a simple and rapid non-aqueous capillary electrophoresis method for the separation and determination of several phenolic compounds belonging to the different families present in olive oil. A Box–Behnken design was employed and a total of 27 experiments were performed using olive oil samples spiked with the phenols and injected directly in the capillary after dilution 1:1 with 1-propanol. Finally, the background electrolyte (BGE) was constituted of 25 mM boric acid and 18 mM KOH in a mixture of 74:26 (v/v) 1-propanol/methanol. The hydrophobicity of the BGE allows its miscibility with the olive oil and, as a consequence, the possibility of characterizing and determining these kinds of compounds in this sample without any pretreatment. A hydrodynamic injection (6 s, −30 mbar) was applied and the separation was carried out using 35 °C and +20 kV of separation temperature and voltage, respectively. A capillary with two detection windows for serial online UV and fluorescence detection was satisfactorily employed. The validation of the method was carried out by setting the calibration curves, and the figures of merit were finally obtained. A lineal relationship between the corrected peak area and concentration and limits of detection in the order of micrograms per milliliter were found.     

On-line determination of aliphatic amines in water using in-tube solid-phase microextraction-assisted derivatisation in in-valve mode for processing large sample volumes in LC

This paper describes a cost-effective procedure for the analysis of short-chain aliphatic amines in water samples using a solid-phase microextraction device. Analyte preconcentration and derivatisation were effected into a capillary column coated with 95% polydimethylsiloxane–5% polydiphenylsiloxane, which was used as the injection loop of a Rheodyne injection valve. The coating was previously loaded with the derivatisation reagent, 9-fluorenylmethyl chloroformate. A volume of 1 mL of samples was then drawn into the capillary column, and the extracted analytes were left to react on the capillary coating for 5 min. Next, the capillary column was cleaned by passing water. Finally, the injection valve was rotated, and the derivatives formed were dynamically desorbed and transferred to the analytical column into the mobile phase. Methylamine, ethylamine, propylamine, n-butylamine and n-pentylamine were selected as model compounds. Excellent sensitivity was achieved, being the limits of detection of 15–200 μg/L when using UV detection and of 0.1–0.4 μg/L by fluorescence.     

Detection of transferrin isoforms in human serum: comparison of UV and ICP–MS detection after CZE and HPLC separations

Two methods for separation of transferrin (Tf) sialoforms, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) with conventional UV absorbance detection, have been investigated and compared. First, conditions affecting the separation of the Tf isoforms by capillary zone electrophoresis and HPLC were carefully optimized. The use of 15 mmol L⁻¹ borate buffer (pH 8.4) containing 3 mmol L⁻¹ diaminobutane (DAB) as additive enabled good separation of the Tf isoforms by CE (75 cm×50 μm i.d. fused silica capillary) at 25 kV. In HPLC, a gradient of ammonium acetate (from 0 to 250 mmol L⁻¹ in 45 min) buffered at pH 6 (Tris-HCl) proved suitable for separation of Tf isoforms on a Mono-Q HR 5/5 anion-exchange column. On-line specific detection of the iron associated with the different Tf isoforms, after Fe saturation, by inductively coupled plasma mass spectrometry (ICP–MS) was studied in detail to compare its analytical performance with UV detection. For both CE and HPLC an octapole reaction system (ORS) ICP–MS instrument was used to minimize polyatomic interferences on the ⁵⁶Fe major isotope. Limits of detection of the different isoforms were in the range of 0.02–0.04 μmol L⁻¹ Tf for HPLC–ICP (ORS)–MS. This hybrid technique proved more selective and reliable detection of transferrin isoforms with 2, 3, 4, 5, and 6 sialic acid residues (S₂, S₃, S₄, S₅, and S₆) in real serum samples. Interesting results from iron speciation of Tf in serum from healthy individuals and from pregnant women are given.     

Use of Multiplexed CE for Pharmaceutical Analysis

This report describes the evaluation of a multiplexed, 96-capillary array instrument for analytical performance with a view to implementation for routine, high-throughput use. The need for higher throughput analytical capability for today’s busy pharmaceutical laboratory continues to rise. The use of multiplexed capillary electrophoresis is discussed, and the 96 multiplexed capillary array instrument with UV absorbance detection was used to perform high throughput pharmaceutical analysis. Pharmaceutical product assay, pK _a and log P determinations were achieved. Reproducibility of relative migration time and relative peak area between capillaries was found to be acceptable. The multiplexed instrument offered equivalent performance for tablet assay to a conventional CE instrument. Using the multiplexed CE, pK _a values approaching those of the literature values were obtained for a range of drugs. A microemulsion electrokinetic chromatography (MEEKC) method was successfully used without resulting in excessive operating current. The use of multiplexed CE for high throughput analysis has been shown to be a highly viable alternative to HPLC and other conventional analytical techniques.Presented at: CE in the Biotechnology & Pharmaceutical Industries: 7th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, Montreal, Canada, August 12–16, 2005.     

Integrated microfluidic device with an electroplated palladium decoupler for more sensitive amperometric detection of the 8-hydroxy-deoxyguanosine (8-OH-dG) DNA adduct

8-Hydroxy-deoxyguanosine (8-OH-dG) DNA adduct is one of the most frequently used biomarkers reporting on the oxidative stress that leads to DNA damage. More sensitive and reliable microfluidic devices are needed for the detection of these biomarkers of interest. We have developed a capillary electrophoresis (CE)-based microfluidic device with an electroplated palladium decoupler that provides significantly improved detection limit, separation efficiency, and resolving power. The poly(dimethylsiloxane) (PDMS)/glass hybrid device has fully integrated gold microelectrodes covered in situ with palladium nanoparticles using an electroplating technique. The performance and coverage of the electrodes electroplated with palladium particles were evaluated electrochemically and via scanning electron microscope (SEM) imaging, respectively. The performance of the device was tested and evaluated with different buffer systems, pH values, and electric field strengths. The results showed that this device has significantly improved resolving power, even at separation electric field strengths as high as 600 V cm⁻¹. The detection limit for the 8-OH-dG adduct is about 20 attomoles; the concentration limit is on the order of 100 nM (S/N = 3). A linear response is reported for both 8-OH-dG and dG in the range from 100 nM to 150 μM (≈100 pA μM⁻¹) with separation efficiencies of approximately 120,000–170,000 plates m⁻¹.     

Use of lignins as components of background electrolyte in capillary electrophoresis

The electrophoretic behavior of two lignins of different compositions, i.e., spruce dioxane lignin and lignosulfonate, is studied. The lignins are shown to affect the electrophoretic behavior of negatively charged analytes, such as carboxylic acids and phenols; their migration time increases. The addition of lignins improves the analytical parameters of phenol quantification by capillary electrophoresis. By means of a simple non-modified capillary, a mixture of six phenols was separated (simple phenol, 2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, and perchlorophenol) with the high resolution (up to 20) and efficiency [(1–5) × 10⁵ TPM]. The separation of six phenols takes 10 min, the lower limit of the analytical range makes 1 μg/mL, the relative standard deviation does not exceed 3%. The potency for the determination of simple phenol and m-cresol is shown on an example of the Verrukatsid medication within 7 min.     

Comparison of enantioseparation of amino acid derivatives in aqueous and mixed aqueous-organic media by capillary zone electrophoresis

The chiral separation of dansyl-amino acids has been performed by capillary zone electrophoresis using ?β-cyclodextrin as a chiral selector, urea as an additive and 2-propanol and methanol as organic modifiers. The enantiomeric separations of dansyl-amino acids were investigated in aqueous medium and compared with the separation in mixed aqueous-organic medium as background electrolytes. The separation conditions, (concentration of buffer, β-cyclodextrin, methanol, urea and the pH value of buffer) were optimized. In the absence of organic modifier, only five pairs of 8 separated dansyl-amino acids were resolved when run separately. A mixture of up to eight chiral amino acids can be baseline resolved in less than 19 min by β-cyclodextrin-modified capillary zone electrophoresis with a buffer of 60 mmol L^–1 H₃BO₃-KCl/40 mmol L^–1 NaOH (pH 9.0), 4 mol L^–1 urea, 100 mmol L^–1β-cyclodextrin and 10% (v/v) methanol. Received: 15 March 1999 / Revised: 10 May 1999 / Accepted: 12 May 1999     

Determination of vitamin B12 in pharmaceutical preparations by a highly sensitive fluorimetric method

A fluorimetric method for the determination of vitamin B₁₂ has been developed. The fluorescence emission was measured at λ_ex/λ_em275/305 nm in phosphate buffer solution (pH 7.0), and the experimental variables and possible interference were studied. The linear calibration range was 1.000 ng/mL to 100.0 ng/mL with a correlation coefficient of 0.9994 and a detection limit of 0.1 ng/mL. The method is rapid, simple and highly sensitive. It was used to determine vitamin B₁₂ in pharmaceutical preparations. The recovery was 96%–98% and the relative standard deviation was in the range of 1.8%–2.7%. The results agreed with those obtained by spectrophotometry. Received: 17 July 2000 / Revised: 4 September 2000 / Accepted: 7 September 2000     

Optimization of a laser ablation-inductively coupled plasma “time of flight” mass spectrometry system for short transient signal acquisition

Simultaneous ion sampling and sequential detection offered by inductively coupled plasma ‘time of flight’ mass spectrometry (ICP-TOFMS) provides advantages for the analysis of short transient concentration-variable signals as produced in laser ablation. In order to investigate the capabilities of ICP-TOFMS in combination with an excimer laser ablation system, ablation studies on reference materials and geological samples were carried out. Various ICP-TOFMS parameters were optimized for laser-induced aerosols. Transverse rejection ion pulse was used to extend the dynamic range in concentration. A reduced volume ablation cell was designed and used in order to increase the sample density in the ICP. Results for 63 simultaneously measured isotopes (SRM 610 from NIST) lead to limits of detection in the 1–100 μg/g range for a 80 μm crater diameter (10 Hz, 1.2 mJ pulse energy). The reproducibility of signal ratios was determined to be better than 2% RSD for transient signals using 102 ms integration time. These optimized parameters were then used for the analysis of tin-rich fluid inclusions. Preliminary results of multielement analysis and isotopic ratio determinations on individual fluid inclusions (63 isotopes, 102 ms integration time) demonstrate the capabilities of ICP-TOFMS in combination with laser ablation. Received: 6 March 2000 / Revised: 11 May 2000 / Accepted: 14 May 2000     

A multichannel native fluorescence detection system for capillary electrophoretic analysis of neurotransmitters in single neurons

A laser-induced native fluorescence detection system optimized for analysis of indolamines and catecholamines by capillary electrophoresis is described. A hollow-cathode metal vapor laser emitting at 224 nm is used for fluorescence excitation, and the emitted fluorescence is spectrally distributed by a series of dichroic beam-splitters into three wavelength channels: 250–310 nm, 310–400 nm, and >400 nm. A separate photomultiplier tube is used for detection of the fluorescence in each of the three wavelength ranges. The instrument provides more information than a single-channel system, without the complexity associfated with a spectrograph/charge-coupled device-based detector. With this instrument, analytes can be separated and identified not only on the basis of their electrophoretic migration time but also on the basis of their multichannel signature, which consists of the ratios of relative fluorescence intensities detected in each wavelength channel. The 224-nm excitation channel resulted in a detection limit of 40 nmol L⁻¹ for dopamine. The utility of this instrument for single-cell analysis was demonstrated by the detection and identification of the neurotransmitters in serotonergic LPeD1 and dopaminergic RPeD1 neurons, isolated from the central nervous system of the well-established neurobiological model Lymnaea stagnalis. Not only can this system detect neurotransmitters in these individual neurons with S/N>50, but analyte identity is confirmed on the basis of spectral characteristics. Lapainis and Scanlan contributed equally to this work.