Migration Times Of Analytes Research Articles

Varying nanoparticle pseudostationary phase plug length during capillary electrophoresis

Capillary electrophoresis based separations of the hypothesized Parkinson's disease biomarkers dopamine, epinephrine, pyrocatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), glutathione, and uric acid are performed in the presence of a 1 nM 11-mercaptoundecanoic acid functionalized gold (Au@MUA) nanoparticle pseudostationary phase plug. Au@MUA nanoparticles are monitored in the capillary and remain stable in the presence of electrically-driven flow. Migration times, peak areas, and relative velocity changes (vs. no pseudostationary) are monitored upon varying (1) the Au@MUA nanoparticle pseudostationary phase plug length at a fixed separation voltage and (2) the separation voltage for a fixed Au@MUA nanoparticle pseudostationary phase plug length. For instance, the migration times of positively charged dopamine and epinephrine increase slightly as the nanoparticle pseudostationary phase plug length increases with concomitant decreases in peak areas and relative velocities as a result of attractive forces between the positively charged analytes and the negatively charged nanoparticles. Migration times for neutral pyrocatechol and slightly negative L-DOPA did not exhibit significant changes with increasing nanoparticle pseudostationary plug length; however, reduction in peak areas for these two molecules were evident and attributed to non-specific interactions (i.e. hydrogen bonding and van der Waals interactions) between the biomarkers and nanoparticles. Moreover, negatively charged uric acid and glutathione displayed progressively decreasing migration times and peak areas and as a result, increased relative velocities with increasing nanoparticle pseudostationary phase plug length. These trends are attributed to partitioning and exchanging with 11-mercaptoundecanoic acid on nanoparticle surfaces for uric acid and glutathione, respectively. Similar trends are observed when the separation voltage decreased thereby suggesting that nanoparticle-biomarker interaction time dictates these trends. Understanding these analyte migration time, peak area, and velocity trends will expand our insight for incorporating nanoparticles in separations.

Use of disposable open tubular ion exchange pre-columns for in-line clean-up of serum and plasma samples prior to capillary electrophoretic analysis of inorganic cations

A simple analytical system using disposable, open-tubular ion exchange clean-up precolumns coupled in-line to capillary electrophoresis for direct injection of biological samples is presented. The clean-up precolumns were prepared from fused silica capillaries by thermally initiated layer-by-layer polymerization of poly(butadiene-maleic acid) (PBMA) directly on the capillary wall. Typically, 6 cm long precolumns with 4-layers of PBMA were used for sample pretreatment. A robust and reproducible coupling between the precolumn (75 μm ID) and the analytical capillary (50 μm ID) was achieved using an inexpensive, commercially available low dead volume union. No extra dispersion of the analyte zones was observed. Proteins and other high molecular weight compounds from biological sample matrices were retained on the cation-exchanger sites of the precolumn, which eliminated their adsorption on analytical capillary walls and ensured stable electroosmotic flow and migration times of target analytes. Unretained small inorganic cations migrated freely into the analytical capillary for separation and detection. Applicability of the sample clean-up procedure was proved by determination of major inorganic cations in blood serum and plasma samples using capillary electrophoresis with contactless conductivity detection. Separations were performed in background electrolyte solution consisting of 15 mM l-arginine, 12.5 mM maleic acid, 3 mM 18-crown-6 at pH 5.5 and repeatabilities of migration times and peak areas were below 1.5% and 7.3%, respectively. Less than 1 μL of biological sample was required for injection.

Synthesis and application of a novel single‐isomer mono‐6‐deoxy‐6‐((2S,3S)‐(+)‐2,3‐O‐isopropylidene‐1,4‐tetramethylenediamine)‐β‐cyclodextrin as chiral selector in capillary electrophoresis

A novel single isomer of positively charged β-cyclodextrin, mono-6-deoxy-6-((2S,3S)-(+)-2,3-O-isopropylidene-1,4-tetramethylenediamine)-β-CD (MIPTACD) was designed and synthesized in seven steps starting from commercially available (2R,3R)-tartaric acid. The chiral resolution abilities of the new cationic chiral selector were studied by capillary electrophoresis using 10 different dansyl (Dns)-amino acids and N-acetylphenylalanine (N-Ac-Phe) as model analytes. The effects of running buffer pH and chiral selector concentration on the separation selectivity, resolutions, and migration times of analytes were studied in detail. MIPTACD shows a very good chiral recognition ability even at very low concentrations at the investigated pH values, as shown by the very large values of selectivity and resolution towards amino acids enantiomers to be assessed.

Polymeric ionic liquid as additive for the high speed and efficient separation of aromatic acids by co-electroosmotic capillary electrophoresis

A simple and reliable co-electroosmotic capillary electrophoresis system for the fast determination of aromatic acids has been developed by employing poly (1-vinyl-3-butylimidazolium bromide) as the background electrolyte modifier. The polymeric ionic liquid was synthesized by the conventional radical polymerization. The reversed electroosmotic flow was obtained by adding a small amount of the polymeric ionic liquid (0.0006%, w/v) to the electrolyte. To further improve the resolution of aromatic acids, conditions including the concentration of polymeric ionic liquid and pH of background electrolytes were optimized. All eight aromatic acids were baseline resolved in one measurement in a short time (less than 3.5 min) under optimized conditions, 100 mM NaH 2PO 4 buffer containing 0.006% (w/v) polymeric ionic liquid, pH 6.0. Separation efficiencies were in the range from 355,000 to 943,000 (plates/m). Satisfactory reproducibility on the basis of the migration time of analytes was achieved. RSDs ( n = 3) were less than 0.33% except the p-aminobenzoic acid (0.9%). The applicability of the present method has been demonstrated for the determination of water-soluble aromatic acids in a common drug for external use.

Analysis of Phosphorus-containing Amino Acid-type Herbicides by Capillary Electrophoresis/Mass Spectrometry Using a Chemically Modified Capillary Having Amino Groups

We describe a simple and practical method for the analysis of phosphorus-containing amino acid-type herbicides and their decomposition products without derivatization by capillary electrophoresis/mass spectrometry using a chemically modified capillary having amino groups. The compounds were glyphosate (GLYP), glufosinate (GLUF), bialaphos (BIAL), aminomethylphosphonic acid (AMPA) and 3-methylphosphinicopropionic acid (MPPA). AMPA and MPPA are the decomposition products of GLYP and GLUF, respectively. The optimum running conditions were found to be 100 mM formic acid adjusted to pH 3.4 with 100 mM ammonia with an applied voltage of −30 kV using an amino capillary (FunCap-CE/Type A) and mass spectrometry. The five compounds were separately determined within 10 min. Relative standard deviations of the migration times of analytes were less than 0.52%. Total analysis time of the proposed method was 1/6 to 1/3 of that of gas chromatography/mass spectrometry methods. The method was applicable for the analysis of these compounds in soil and tea beverage samples. The decomposition rates of GLYP, GLUF and BIAL in soil are discussed.

Reproducible chiral capillary electrophoresis of methamphetamine and its related compounds using a chemically modified capillary having diol groups

Excellent reproducibility of chiral capillary electrophoresis for methamphetamine (MA) and its related compounds was obtained using a chemically modified capillary having diol groups. The data were compared with those using an untreated fused-silica capillary and a poly(vinyl alcohol) (PVA)-coated capillary. For five replicate analyses of a standard mixture, relative standard deviations (RSDs) of the migration times of analytes with diol or PVA-coated capillaries were less than 0.1%, which were lower than those obtained with an untreated fused-silica capillary (ca. 0.4%). The diol capillary gave reproducible migration times especially in analyses of real samples, such as crude human urine. In the analyses of five spiked urine samples, the RSD values of migration times of analytes with the diol capillary were not greater than 0.14%, which were much lower than those obtained with the PVA-coated capillary (ca. 1.5%). The proposed method was successfully used for chiral analyses of actual urine samples of MA addicts.

A determination of submicromolar concentrations of glycine in periaqueductal gray matter microdialyzates using capillary zone electrophoresis with contactless conductivity detection

CE with contactless conductivity detection has been used to determine the glycine neurotransmitter in periaqueductal gray matter (PAG) of rats. The LOD for glycine has been decreased to a value of 0.2 microM by adding 75% v/v of ACN to the samples and increasing the sample zone introduced to a value of 20% of the overall capillary length. The repeatabilities of the analyte migration times and the zone areas amount to 2.1 and 2.7%, respectively. The optimized CE/contactless conductivity detection method makes it possible to determine the micromolar concentrations of glycine in PAG microdialyzates without the necessity of sample derivatization. It follows from a pharmacological study that a local inflammation initiated by an application of carrageenan increased the glycine concentration in the rat PAG seven times, compared with a control. The glycine level in PAG can be decreased and the pain suppressed by administering paracetamol.

Combining capillary electrophoresis with laser‐induced fluorescence detection for the analysis of Escherichia coli lysates

With the goal of improving water quality control, in this study we combined CE-LIF for the analysis of Escherichia coli lysates under discontinuous conditions. Prior to injecting a large-volume protein sample, a plug of 0.2% w/v SDS was injected into the capillary filled with 2.0 M Tris-borate (TB) solution (pH 10.3). During the courses of the process stacking, proteins migrated against the electroosmotic flow (EOF) and entered a 1.7% w/v poly(ethylene oxide) solution that has been prepared in 200 mM TB (pH 9.0). The stacked proteins were then separated through sieving and then detected at the cathodic end. The use of TB solutions containing 0.01% w/v poly(ethylene oxide) was essential for the preparation of the protein samples and E. coli lysates to minimize the extent of protein adsorption on the capillary wall. Under the optimized CE-LIF conditions, we detected beta-Galactosidase with and without concentration at levels as low as 0.23 and 7.52 nM, respectively. Our approach allowed the detection of 3 x 10(2) E. coli cells based on the characteristic peaks of its lysates; in addition, we could detect the presence of 20 E. coli cells in a 50 mL sample of pond water within 24 h. In terms of the analyte migration time, the relative standard deviation of this CE-LIF method was less than 1.3%. We also extended the practicality of this technique by applying it to the separation of mixtures of E. coli, Enterobacter aerogenes, and Salmonella enterica.

Enantiomeric separation of baclofen by capillary electrophoresis tandem mass spectrometry with sulfobutylether-β-cyclodextrin as chiral selector in partial filling mode

Capillary electrophoresis (CE) coupled to tandem mass spectrometry was applied to the chiral separation of baclofen using sulfobutylether-β-cyclodextrin chiral selector in partial filling counter current mode. On-line UV detection was simultaneously used. Method optimization was performed by studying the effect of cyclodextrin and BGE concentration as well as sheath liquid composition on analyte migration time and enantiomeric resolution. The cyclodextrin showed stereoselective complexation towards baclofen enantiomers, allowing chiral resolution at low concentration. The CE capillary protrusion from the ESI needle relevantly affected the chiral resolution and the analyte migration time. Complete enantiomeric separation was obtained by using 0.25 M formic acid BGE containing 1.75 mM of chiral selector and water/methanol (30:70, v/v) 3% formic acid as sheath liquid. The method exhibited a LOD of 0.1 μg/mL (racemic concentration) in MS 3 product ion scan mode of detection and was applied to the analysis of racemic baclofen in pharmaceutical formulations.

Intracellular adenosine 5′‐triphosphate, adenosine 5′‐diphosphate, and adenosine 5′‐monophosphate detection by short‐end injection capillary electrophoresis using methylcellulose as the effective electroosmostic flow suppressor

We present a new rapid CE method to measure adenine nucleotides adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in cells. The short-end injection mode allows a decrease in the analysis time by injecting samples at the outlet end of a silica capillary closest to the detection window, reducing the migration distance. Moreover, the use of methylcellulose (MC) as run buffer additive to suppress EOF permits to further reduce the migration times of analytes. Thus, when a capillary with an effective length of 10.2 cm was used with a 60 mmol/L sodium acetate buffer pH 3.80 in the presence of 0.01% of MC, the migration time of analytes were 1.35 min for ATP, 1.85 min for ADP, and 4.64 min for AMP. These conditions gave a good reproducibility for intra- and interassay (CV <4 and 8%, respectively) and all the procedure demonstrated an excellent analytical recovery (from 98.3 to 99 %). The method suitability was proved both on red blood cells and in spermatozoa. We compared our proposed method to a spectrophotometric assay, by measuring ATP levels in 40 spermatozoa samples. The obtained data were analyzed by the Passing and Bablok regression and Bland-Altman test.

The effect of conditioning of fused‐silica capillaries on their electrophoretic performance

In CE practice, conditioning of the capillary tube with strong base, acid, or both in sequence, has been recognized as essential to obtain reasonable precision in electroosmotic mobility (EOM), and consequently, in the migration times of analytes. This report focuses on the comparative study of three different approaches for capillary conditioning: etching with HF, etching with NaOH, and leaching with HCl after NaOH treatment. EOM-based measurements, including the hysteresis effect that characterizes the dependence of EOM with pH, were used to evaluate the conditioned silica surface. Additionally, indirect inspection of the conditioned surface was carried out by examining the electrophoretic profile of the cationic probe Ru(bpy)(3) (2+), known to strongly interact with the ionized silica surface while displaying no significant acid-base activity. It was shown that, once conditioned and prior to any CE measurement, extensive rinse of the capillary with the running electrolyte at high flow rate was essential to attain relatively rapid re-equilibration of the tube with the fluid. Insufficient rinse with the running electrolyte would result in negatively biased EOM measurements and significant drift in migration times. It was also established that relatively high flow rates of 1 M NaOH conditioning solution (4-5 column volumes per minute) was required to attain capillaries with reproducible electrophoretic performance within a reasonable conditioning time (typically, 1 h). In addition to relatively more extensive rehydroxylation of the silica surface, evidenced by the highest EOM values obtained, the sequential use of NaOH-etching and HCl-leaching provided better precision than HF-etching or NaOH-etching alone.

Evaluation of poly{‐N‐isopropylacrylamide‐co‐[3‐(methacryloylamino)propyl]trimethylammonium} as a stationary phase for capillary electrochromatography

A cationic polyacrylamide-based stationary phase was synthesized and characterized for CEC. The stationary phase was prepared by radical copolymerization of N-isopropylacrylamide (NIPAAm) and (3-(methacryloylamino)propyl)trimethylammonium chloride (MAPTA), producing a copolymer attached to 5 microm porous silica particles. Fourier transform infrared spectroscopy and thermogravimetric analysis were used to characterize the copolymer. Under capillary electrochromatographic conditions, the poly-NIPAAm-co-MAPTA stationary phase showed to be stable in a wide pH range. The amino groups in the MAPTA provided an anodic EOF for CEC separation. The electroosmotic mobility changed less than 10% when the pH of the mobile phase was changed from 2 to 12. The run-to-run RSD of analyte migration time was less than 1.5% (n = 3), and the RSD of peak area was less than 3% (n = 3). The day-to-day RSD for migration time was less than 2% (n = 3). The polar groups present in the stationary phase contributed to the selectivity of the phase providing for hydrophilic interactions. In the separation of a series of neutral and acidic compounds, the stationary phase shows a mixed-mode separation mechanism with both hydrophobicity and hydrophilicity contributing to the separation.

Fabrication of microfluidic devices using dry film photoresist for microchip capillary electrophoresis

An inexpensive, disposable microfluidic device was fabricated from a dry film photoresist using a combination of photolithographic and hot roll lamination techniques. A microfluidic flow pattern was prefabricated in a dry film photoresist tape using traditional photolithographic methods. This tape became bonded to a poly(methyl methacrylate) (PMMA) sheet with prepouched holes when passed through a hot roll laminator. A copper working electrode and platinum decoupler was readily incorporated within this microchip. The integrated microchip device was then fixed in a laboratory-built Plexiglas holder prior to its use in microchip capillary electrophoresis. The performance of this device with amperometric detection for the separation of dopamine and catechol was examined. The separation was complete within 50 s at an applied potential of 200 V/cm. The relative standard deviations (RSD) of analyte migration times were less than 0.71%, and the theoretical plate numbers for dopamine and catechol were 3.2 × 10 4 and 4.1 × 10 4, respectively, based on a 65 mm separation channel.

Separation of nicotinic acid and its structural isomers using 1-ethyl-3-methylimidazolium ionic liquid as a buffer additive by capillary electrophoresis

The growing interest in application of ionic liquids (ILs) in analytical chemistry has been observed. The aim of presented investigation was to verify whether ILs would be a suitable modifier of the background electrolyte (BGE) for pharmaceutical analysis of the closely related drug analogues. The study demonstrates the use of 1-ethyl-3-methylimidazolium tetrafluoroborate (1E-3MI-TFB) ionic liquid as modifiers in the separation of nicotinic acid and its structural isomers by capillary electrophoresis. Dependences of the ionic liquid concentration in a BGE on the separation parameters like migration time, resolution factor and width at peak's baseline have been compared. The separation mechanism involves the free imidazolium ions, which can interact with inner surface of the capillary wall. Increased 1E-3MI-TFB concentration to 150 mmol/L caused decrease of migration times of analytes, improve peaks shape and increase of separation performances. At this ionic liquid concentration in a BGE resolution factor between nicotinic and isonicotinic acids increased to 1.86. The proposed CE separation procedure is highly reproducible and can be applied in qualitative and quantitative analysis of carboxylic acids.

A sulfonated capillary that gives reproducible migration times for capillary zone electrophoresis and micellar electrokinetic chromatography

To obtain reproducible migration times and rapid analyses of analytes, sulfonate groups were chemically introduced to the inner wall of untreated fused-silica capillary with 2-(4-chlorosulfonylphenyl)ethyltrichlorosilane. The sulfonated capillary showed relatively constant electroosmotic mobility which was greater than that obtained by an untreated fused-silica capillary over the pH range studied (pH 2-9). In both CZE and MEKC, the RSDs of the migration times of analytes with the sulfonated capillary were less than 0.2% which were significantly lower than those obtained with an untreated fused-silica capillary (0.5-3.5%). When BGE were set at pH 7.0 for CZE and MEKC, the analysis times with the sulfonated capillary were about half those obtained with an untreated fused-silica capillary. These results indicate that the sulfonated capillary can provide highly reproducible and rapid analyses in CE.

Enantiomeric separation of 8 hydroxy, 10 carboxylic and 6 dansyl amino acids by mono(6-amino-6-deoxy)-β-cyclodextrin in capillary electrophoresis

The enantioseparation of a wide spectrum of anionic analytes was successfully performed by using mono(6-amino-6-deoxy)-β-cyclodextrin (β-CD-NH 2). The effects of buffer pH and selector concentration on migration time and resolution of analytes were studied in detail. Good results were obtained for the chiral separations of hydroxy and carboxylic acids. A clear maximum in selectivity was found for most analytes. The introduced positive charge can greatly improve the chiral recognition ability of β-CD-NH 2 towards anionic analytes. The binding constants of eight analytes with β-CD-NH 2 were determined and suited well with the mobility difference model (by Wren). Moreover, a standard mixture of five acids was baseline separated within 23 min.

Enumeration Algorithm for Determination of Binding Constants in Capillary Electrophoresis

With more accurate simulation models and more efficient algorithms becoming available, the binding constants of an affinity interaction can be obtained from much simpler experiments using capillary electrophoresis. With the enumeration algorithm, all possible combinations of the binding constant and the complex mobility in certain ranges that could result in the experimental migration time of an injected analyte are extracted from a 3-D surface, which depicts the migration times resulting from different values of the binding constant and the mobility of the complex formed between the interacting pair, to form a 2-D curve. When the experimental conditions are changed, the analyte migration time will also change. A new 2-D curve can be constructed from another 3-D surface on the basis of the pairs of binding constants and complex mobility values that could result in the new migration time. Because the true binding constant and complex mobility values have to be the same for both experimental conditions under the same temperature, there has to be a point where both 2-D curves will converge. The coordinates of the converging point give the values for a binding constant and a complex mobility that will fit all 2-D curves generated under certain experimental conditions. p-Nitrophenol is used as the analyte, beta-cyclodextrin is used as the additive, and a one-cell model is used to simulate affinity CE. The experimental conditions that can improve the accuracy of the binding constants are discussed.

Sol−Gel Modified Poly(dimethylsiloxane) Microfluidic Devices with High Electroosmotic Mobilities and Hydrophilic Channel Wall Characteristics

Using a sol-gel method, we have fabricated poly(dimethylsiloxane) (PDMS) microchips with SiO2 particles homogeneously distributed within the PDMS polymer matrix. These particles are approximately 10 nm in diameter. To fabricate such devices, PDMS (Sylgard 184) was cast against SU-8 molds. After curing, the chips were carefully removed from the mold and sealed against flat, cured pieces of PDMS to form enclosed channel manifolds. These chips were then solvated in tetraethyl orthosilicate (TEOS), causing them to expand. Subsequently, the chips were placed in an aqueous solution containing 2.8% ethylamine and heated to form nanometer-sized SiO2 particles within the cross-linked PDMS polymer. The water contact angle for the PDMS-SiO2 chips was approximately 90.2 degrees compared to a water contact angle for Sylgard 184 of approximately 108.5 degrees . More importantly, the SiO2 modified PDMS chips showed no rhodamine B absorption after 4 h, indicating a substantially more hydrophilic and nonabsorptive surface than native PDMS. Initial electroosmotic mobilities (EOM) of (8.3+/-0.2)x10(-4) cm2/(V.s) (RSD=2.6% (RSD is relative standard deviation); n=10) were measured. This value was approximately twice that of native Sylgard 184 PDMS chips (4.21+/-0.09)x10(-4) cm2/(V.s) (RSD=2.2%; n=10) and 55% greater than glass chips (5.3+/-0.4)x10(-4) cm2/(V.s) (RSD=7.7%; n=5). After 60 days of dry storage, the EOM was (7.6+/-0.3)x10(-4) cm2/(V.s) (RSD=3.9%; n=3), a decrease of only 8% below that of the initially measured value. Separations performed on these devices generated 80,000-100,000 theoretical plates in 6-14 s for both tetramethylrhodamine succidimidyl ester and fluorescein-5-isothiocyanate derivatized amino acids. The separation distance was 3.5 cm. Plots of peak variance vs analyte migration times gave diffusion coefficients which indicate that the separation efficiencies are within 15% of the diffusion limit.

A new two-chip concept for continuous measurements on PMMA-microchips

A new concept for continuous measurements on microchips is presented. A PMMA (polymethylmethacrylate) based capillary electrophoresis chip with integrated conductivity detection is combined with a second chip, which undertakes the task of fluid handling and electrical connections. The combination of electrokinetic and hydrodynamic flows allows long-term continuous stable analyses with good reproducibilities of migration time and peak heights of analytes. The two-chip system is characterized in terms of stability and reproducibility of separation and detection of small ions. Relative standard deviations of <1% and 3% respectively for retention times and peak heights during long-term measurements can be achieved. The new system combines simple handling and automated analysis without the need for refilling, cleaning or removal of the separation chip after one or several measurements.

Microsecond electrophoresis.

Although analysis strategies exist for probing a diverse array of molecular properties, most of these approaches are not amenable to the study of reaction intermediates and other transient species. Separations in particular can provide detailed information on attributes not readily measured by spectroscopy but typically are performed over time scales much longer than the life span of highly unstable compounds. Here we report the development of an electrophoretic strategy that dramatically extends the practical speed limit for fractionations and demonstrate its utility in examining transient hydroxyindole photoproducts. Fluorescent reaction intermediates are optically generated in femtoliter volumes within a flowing reagent stream and are differentially transported at velocities as large as 1.3 m.s(-1), thereby minimizing band variance and allowing multicomponent reaction mixtures to be resolved over separation paths as short as 9 microm. Analyte migration times and band variances do not deviate significantly from basic theory for separations performed with fields that exceed 0.1 MV.cm(-1), indicating that effects from Joule heating are minor. We demonstrate the feasibility of achieving baseline resolution of a binary mixture in <10 micros, nearly 100-fold faster than previously possible. Application of this approach to the study of a range of short-lived molecules should be feasible.