A new strategy for optimizing sensitivity, speed, and resolution in capillary electrophoretic separation of DNA

DNA separations were performed in poly(ethylene oxide) (PEO) solutions prepared in 100 mM Tris-boric acid (TB) buffers using a capillary filled with TB buffers with concentrations up to 2.5 M, pH 10.0. The electroosmotic flow (EOF) increased with increasing the concentration of TB buffers till 1.5 M as a result of decreasing PEO adsorption on the capillary wall. At high TB concentrations (> 1.5 M), the peaks corresponding to small DNA fragments (11 and 8 base pairs) became sharper and were detected. Relative standard deviations of the EOF coefficient and the migration times of the DNA fragments were all less than 1% using a capillary filled with TB buffers at concentrations higher than 1.5 M. When separations were performed at different pH values of PEO solutions and TB buffers, better results in terms of sensitivity, speed, and resolution were generally achieved. The fluorescence intensity of the 2176 bp fragment obtained at pH values of TB buffers/PEO solutions 10.0/8.2 was 27-fold of that at pH values 8.2/8.2. The enhancement was related to effects of pH and borate on fluorescence intensity, DNA conformation, stacking, and interactions with the capillary wall. Using a capillary filled with 400 mM TB buffers, pH 10.0, the separation of DNA (pBR 322/HaeIII digest, pBR 328/Bg/I digest and pBR 328/HinfI digest) in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 9.0, at 375 V/cm was accomplished in less than 18 min.     

Maximization of injection volumes for DNA analysis in capillary electrophoresis

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.     

Stacking and separation of fluorescent derivatives of amino acids by micellar electrokinetic chromatography in the presence of poly(ethylene oxide)

A new approach for the analysis of large-volume naphthalene-2,3-dicarboxaldehyde (NDA) derivatives of amino acids by micellar electrokinetic chromatography (MEKC) in conjunction with a purple light-emitting diode-induced fluorescence detection is described. In order to optimize resolution, speed, and stacking efficiency, a discontinuous condition is essential for the analysis of NDA-amino acid derivatives. The optimum conditions use 2.0M TB (pH 10.0) buffer containing 40mM sodium dodecyl sulfate (SDS) to fill the capillary, deionized water to dilute samples, and 200mM TB (pH 9.0) containing 10mM SDS to prepare 0.6% poly(ethylene oxide) (PEO). Once high voltage is applied, PEO solution enters the capillary via electroosmotic flow and SDS micelles interact and thus sweep the NDA-amino acid derivatives having smaller electrophoretic mobilities than that of SDS micelles in the sample zone. When the aggregates between SDS micelles and NDA amino acid derivatives enter PEO zone, they are stacked due to decrease in electric field and increases in viscosity. Under the optimum conditions, the concentration and separation of 0.53-microL 13 NDA-amino acid derivatives that are negatively charged has been demonstrated by using a 60-cm capillary, with the efficiencies 0.3-9.0x10(5) theoretical plates and the LODs at signal-to-noise ratio 3 ranging from 0.30 to 2.76nM. When compared to standard injection (30-cm height for 10s), the approach allows the sensitivity enhancements over the range of 50-800 folds for the derivatives. The new approach has been applied to the analysis of a red wine sample, with great linearity of fluorescent intensity against concentrations (R(2)>0.98) and the RSD (three repetitive runs in one day) values of the migration times for the ten identified amino acids less than 2.8%.     

Discontinuous electrolyte systems for improved detection of biologically active amines and acids by capillary electrophoresis with laser-induced native fluorescence detection

On-line concentration and separation of biologically active amines and acids by capillary electrophoresis (CE) in conjunction with laser-induced fluorescence using an Nd:YAG laser at 266 nm under discontinuous conditions is presented. The suitable conditions for simultaneous analysis of amines and acids were: samples were prepared in a solution (pH* 3.1) consisting of 10 mM citric acid, 89% acetonitrile (ACN), and water; a capillary was filled with 1.5 M Tris-borate (TB) buffer (pH 10.0); and the anodic vial contained PTG10 buffer (pH* 9.0) that consists of 50 mM propanoic acid, Tris, 10% glycerol, and water. After injecting a large-volume sample, amines and acids were separately stacked at the front (cathodic side) and back (anodic side) of the acidic sample zone, mainly because of changes in their electrophoretic mobilities as a result of changes in pH, viscosity, and electric field when high voltage was applied. When the sample was injected at 15 kV for 360 s, the concentration limits of detection (LODs) for 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) were 0.27 and 0.31 nM, respectively, which are about 400- and 800-fold sensitivity improvements when compared to those injected at 1 kV for 10 s. For the analysis of amines, samples were prepared in 100 mM citric acid (pH* 1.8) containing 89% ACN and both the capillary and anodic vial were filled with 400 mM PTG20 (propanoic acid, Tris, 20% glycerol, and water) at pH* 4.5. Using a large injection volume (15 kV for 360 s), we achieved concentration LODs of 17 pM and 0.3 nM for tryptamine and epinephrine, which are about 5200- and 14,000-fold sensitivity improvements, respectively, in comparison with those injected at 1 kV for 10 s. The features of simplicity (no sample pretreatment), rapidity (12 min), and sensitivity for identification of amines and acids of interest in urine samples show diagnostic potential of the two approaches developed in this study.     

Analysis of amino acids and biogenic amines in breast cancer cells by capillary electrophoresis using polymer solutions containing sodium dodecyl sulfate

We describe simultaneous analysis of naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and NDA-biogenic amine derivatives by CE in conjunction with light-emitting diode-induced fluorescence detection using poly(ethylene oxide) (PEO) solutions containing sodium dodecyl sulfate (SDS). After sample injection, via EOF 0.1% PEO prepared in 100 mM TB solution (pH 9.0) containing 30 mM SDS entered a capillary filled with 0.5 M TB solution (pH 10.2) containing 40 mM SDS. Under this condition, 14 NDA-amino acid and NDA-amine derivatives were separated within 16 min, with high efficiency ((1.0–3.2) × 10⁵ theoretical plates) and sensitivity (LODs at S/N = 3 ranging from 2.06 to 19.17 nM). In the presence of SDS and PEO, analytes adsorption on the capillary wall was suppressed, leading to high efficiency and reproducibility. The intraday analysis RSD values (n = 3) of the mobilities for the analytes are less than 0.52%. We have validated the practicality of this approach by quantitative determination of 9 amino acids in breast cancer cells (MCF-7) and 10 amino acids in normal epithelial cells (H184B5F5/M10). The concentrations of Tau and Gln in the MCF-7 cells were different than those in the H184B5F5/M10 cells, respectively. Our results show the potential of this approach for cancer study.     

Stacking and separation of protein derivatives of naphthalene-2,3-dicarboxaldehyde by CE with light-emitting diode induced fluorescence detection

We describe the stacking and separation of proteins by CE under discontinuous conditions in conjunction with light-emitting diode induced fluorescence (LEDIF) detection using a violet LED at 405 nm. The proteins were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to form NDA-protein derivatives prior to CE-LEDIF analysis. During the separation, poly(ethylene oxide) (PEO) solution containing CTAB enters from the cathodic inlet to the capillary via electroosomotic flow (EOF). The optimum conditions are: the capillary was filled with 50 mM glycine buffer (pH 9.0) containing 1.0 mM CTAB, NDA-protein derivatives were prepared in deionized water containing 1.0 mM CTAB, and 0.6% PEO was prepared in 50 mM glycine (pH 9.0) containing 2.0 mM CTAB. The analysis of four NDA-protein derivatives is fast (<3 min), with RSD <1.5% in terms of migration time. In order to improve the sensitivity of NDA-protein derivatives, a stacking approach based on increases in viscosity and electric field, as well as sieving was applied. The efficient stacking approach provides LODs (S/N = 3) of 2.41, 0.59, 0.61, and 4.22 nM for trypsin inhibitor, HSA, beta-lactoglobulin, and lysozyme, respectively. In addition, we also applied the stacking approach to determination of the concentration of HSA in one urine sample, which was determined to be 0.31 +/- 0.05 microM (n = 3).     

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.     

Comparison of the separation of large DNA fragments in the presence and absence of electroosmotic flow at high pH

This paper describes the analysis of large DNA fragments at pH > 10.0 by capillary electrophoresis (CE) in the presence of electroosmotic flow (EOF) using hydroxyethylcellulose (HEC) solution. HEC solution in the anodic reservoir enters the capillaries filled with high-pH buffer by EOF after sample injection. With respect to resolution, sensitivity, and speed, separation conducted under discontinuous conditions (different pH values of HEC solutions and buffer filling the capillary) is appropriate. Using HEC solution at concentrations higher than its entanglement threshold ensures a good separation of large DNA fragments in the presence of EOF at high pH. In addition to pH and HEC, the electrolyte species, dimethylamine, methylamine, and piperidine, play different roles in determining the resolution. The separation of DNA fragments ranging in size from 5 to 40 kilo base pairs was completed in 6 min using 1.5% HEC prepared in 20 mM methylamine-borate, pH 12.0, and the capillary filled with 40 mM dimethylamine-borate, pH 10.0. In comparison, this method allows faster separations of large DNA fragments compared with that conducted in the absence of EOF using dilute HEC solutions.     

Analysis of large-volume DNA markers and polymerase chain reaction products by capillary electrophoresis in the presence of electroosmotic flow

We have demonstrated on-line concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large-volumes DNA samples, PEO solutions entered a capillary filled with 400 mM Tris-borate (TB) buffers by EOF and acted as sieving matrices. DNA fragments stacked between the sample zone and PEO solutions. Because sample matrixes affected PEO adsorption on the capillary wall, leading to changes in EOF, migration time, concentration, and resolving power varied with the injection length. When injecting phiX174 RF DNA-HaeIII digest prepared in 5 mM Tris-HCl buffer, pH 7.0, at 250 V/cm, peak height increased linearly as a function of injection volume up to 0.9 microl (injection time 150 s). The sensitivity improvement was 100-fold compare to that injected at 25 V/cm for 10 s (0.006 microl). When injecting 1.54 microl of GeneScan 1000 ROX, the sensitivity improvement was 265-fold. The sensitivity improvement was 40-fold when injecting 0.17 microl DNA sample containing pBR 322/HaeIII, pBR 328/BglI, and pBR 328/HinfI digests prepared in phosphate-buffered saline. This method allows the analysis of polymerase chain reaction (PCR) products amplified after 17 cycles when injecting 0.32 microl (at 30 cm height for 300 s). The total analysis time was shorter (91.6 min) than that (119.6 min) obtained from injecting PCR products after 32 cycles for 10 s.     

A simple,rapid, and sensitive method for analysis of SYPRO Red labeled sodium dodecyl sulfate-protein complexes by capillary electrophoresis with laser-induced fluorescence

We describe a segmental filling method for the analysis of SYPRO Red labeled sodium dodecyl sulfate (SDS)-proteins (SRSPs) by capillary electrophoresis-laser induced fluorescence (CE-LIF) with electroosmotic counterflow of poly(ethylene oxide) (PEO). It is shown that SDS and salt play a crucial role in determining the fluorescence intensity of the SRSP. Although the fluorimetric measurements reveal that the SRSPs fluoresce strongly in Tris-borate (TB) buffer containing 0.1% SDS and high concentrations of NaCl (100 mM), these conditions are not appropriate to CE in view of Joule heating. To overcome that impediment, we applied a plug of 0.1% SDS (1/5 to 1/3 of the injection volume) prior to injection of samples (0.64 microL) prepared in TB buffer containing 50 mM NaCl and SYPRO Red. When using a background electrolyte of 0.6% PEO in TB buffer containing NaCl, electroosmotic counterflow of the analytes allows one to concentrate large sample volumes (up to 1/3 of effective capillary length) in 21 min, with detection of 0.35 and 0.10 nM for bovine serum albumin and casein, respectively. With a linear dynamic range from 10 nM to 5 microM, this method provides the capability of determining the concentration of casein in cow's milk as 0.45 +/- 0.03 mM (n = 5).     

Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions

Separations of phiX-174/HaeIII DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5-5 microg/mL), (ii) voltage programming (125-375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris-borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8,000,000) under isocratic conditions have been completely resolved by either stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 mM TB buffers, pH 10.0, high-speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/ Bg/l digest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.     

Continuous on-line concentration based on dynamic pH junction for trimethoprim and sulfamethoxazole by microfluidic capillary electrophoresis combined with flow injection analysis system

A novel, rapid and continuous on-line concentration approach based on dynamic pH junction for the analysis of trimethoprim (TMP) and sulfamethoxazole (SMZ) by microfluidic capillary electrophoresis (CE) combined with flow injection analysis is developed in this paper. Stacking is due to decreases in the velocity of analytes when migrating from the low-pH sample zone (sample was dissolved in 50 mM HCl) to a relatively high-pH buffer (30 mM phosphate buffer, pH 8.5) filled in the capillary. This results in 2.9-4.7-fold improvement in concentration sensitivity relative to conventional capillary electrophoresis methods. The separation could be achieved within 2 min and sample throughput rate can reach up to 38 h(-1).     

Fast and sensitive diagnosis of thalassemia by capillary electrophoresis

This paper demonstrates the diagnosis of -thalassemia by capillary electrophoresis in conjunction with laser-induced fluorescence using poly(ethylene oxide) (PEO) solutions in the presence of electroosmotic flow (EOF). During the electrophoretic separation, PEO solution entered a capillary from the anodic vial by EOF. The separation of a mixture of the polymerase chain reaction (PCR) products (330 and 334 base pairs) from a healthy person and a -thalassemia patient was accomplished within 15 min at 15 kV using 1.5% PEO containing 2 M urea at 30 °C. The electropherogram patterns instead of migration times were used to diagnose -thalassemia, with an accuracy of 100% for the analyses of 11 blood samples from suspected patients. After injecting a large volume of the mixture to the capillary filled with 800 mM Tris-borate buffer (pH 10.0), the DNA fragments stacked due to increases in viscosity and sieving when migrating into 1.5% PEO solution. As a result of improved sensitivity, only 15 PCR cycles were required when using 500 ng of DNA templates. The results shown in this study indicate the potential of this simple, rapid, and cost-effective method for the diagnosis of -thalassemia.Abbreviations CE Capillary electrophoresis - EOF Electroosmotic flow - EtBr Ethidium bromide - LIF Laser-induced fluorescence - PCR Polymerase chain reaction - PEO Poly(ethylene oxide) - TRIS Tris(hydroxymethyl)aminomethane - TB TRIS-borate     

Impact of halides on the simultaneous separation of aromatic amines and their acidic metabolites by capillary electrophoresis with laser-induced native fluorescence detection under acidic conditions

This paper describes a simple, sensitive, efficient, and rapid method for simultaneous analysis of biologically active amines and acids by capillary electrophoresis in conjunction with laser-induced native fluorescence detection (CE-LINF) using a diode pumped solid state nanolaser at 266 nm. In order to optimize resolution of the amines that were prepared in 10.0 mM formate-Tris (FT) solutions, 10.0 mM FT solutions with and without containing halides were used to fill the capillary and reservoirs, respectively. The electrophoretic mobilities of tryptamine (TA) and serotonin (5-HT) at pH 4.0 decrease with the increase in halide concentration (0-10.0 mM). Taken together with a great effect of iodide than other halides, we suggest that the formation of ion pairs is a main contributor for altering the migration of the amines. In order to simultaneously analyze the amines and their metabolites (acids) at low pH, a high bulk EOF is required. The analysis of 10 anlytes including amines and acids was completed within 12 min by CE-LINF using a capillary treated with 0.5M NaOH and then filled with 10.0 mM FT solutions (pH 4.0) containing 10.0 mM KCl prior to analysis. The limits of detection for TA and 5-hydroxyindole-3-acetic acid (5-HIAA) are 0.12 and 6.0 nM, respectively. The present method has been further validated by analyzing urine samples, with an RSD less than 3.1% (migration times) and 3.9% (concentration).     

Bis-indole derivatives for polysaccharide compositional analysis and chiral resolution of D-, L-monosaccharides by ligand exchange capillary electrophoresis using borate-cyclodextrin as a chiral selector

A series of aldo-bis-indole derivatives (aldo-BINs) was prepared by aromatic C-alkylation reactions of aldoses and indole in acetic acid solution. Common monosaccharides such as glucose, mannose, galactose, fucose, xylose, rhamnose, ribose, arabinose and N-acetylglucosamine were smoothly derivatized to form the UV absorbing aldo-BINs. The use of a capillary electrophoretic method to separate these novel aldo-BIN derivatives was established. The capillary electrophoresis conditions were set by using borate buffer (100 mM) at high pH (pH 9.0). The limit of determination was assessed to be 25 nM. The enantioseparation of D, L-pairs of aldo-BINs based on chiral ligand-exchange capillary electrophoresis technology was also achieved by using modified hydroxypropyl-β-cyclodextrin as the chiral selector in the presence of borate buffer. This aldose labeling method was applied successfully to the compositional and configurational analysis of saccharides, exemplified by a rapid and efficient method to simultaneously analyze the composition and configuration of saccharides from the medicinal herbs Cordyceps sinensis and Dendrobium huoshanense.     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.     

On-column preconcentration and separation of DNA fragments using polymer solutions in the presence of electroosmotic flow

We demonstrated DNA preconcentration and separation in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large volumes of DNA samples into a capillary filled with free tris(hydroxymethyl)aminomethane (Tris)-borate (TB) buffers, PEO solutions entered the capillary by EOF and acted as sieving matrices. In contrast to conventional methods (in the absence of EOF), controlling the EOF was also useful for resolution optimization. We have found that PEO adsorption on the capillary wall was more pronounced when low ionic strength buffers were used. Thus, the EOF decreased with increasing injection length, which led to longer migration times and changes in resolution and stacking efficiency. All resolution values were higher than 1.5 when 1.0 microg/mL DNA samples were injected at 240 V/cm for 60 s (0.67 microL). In addition, as low as 0.015 microg/mL DNA samples (an about 66-fold increase in sensitivity) were detected when the injection was performed at 250 V/cm for 60 s.     

Polymer solution-filled column for the analysis of antisense phosphorothioates by capillary electrophoresis

A capillary electrophoresis (CE) column filled with 13% poly(ethylene glycol) (PEG) solution is demonstrated to resolve different lengths of antisense phosphorothioates in 100 mM Tris-borate (pH 9.0) buffer containing 30% formamide at 50 degrees C. Two sets of mixtures composed of 15-20 mers of either antisense phosphorothioate or phosphodiester oligonucleotides were synthesized based on a sequence of the antisense orientation directed against DNA-methyltransferase (denoted as MT-AS) and were used as model compounds. It was found that column coating reduced electroosmotic flow, as well as wall adsorption, and led to the separation of both phosphorothioate and phosphodiester molecules. Substantial peak broadening, however, specifically occurred to the phosphorothioates and was reduced statisfactorily by the addition of formamide into the buffer solution, raising the temperature, and raising the pH value. Under experimental conditions, a linear relationship between the migration time and the base number was observed, indicating that no peak compression artifacts existed. Without tedious pretreatment, antisense phosphorothioates were spiked into human serum, followed by water dilution, and then directly injected into the column. Separation of different lengths of phosphorothioates was observed using pressure injection, which did not suffer from injection bias.     

Stacking and separation of aspartic acid enantiomers under discontinuous system by capillary electrophoresis with light-emitting diode-induced fluorescence detection

We describe the stacking and separation of d- and l-aspartic acid (Asp) by capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF). In the presence of cyanide, d- and l-Asp were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to form fluorescent derivatives prior to CE-LEDIF. The separation of NDA-derivatized d- and l-Asp was accomplished using a discontinuous system - buffer vials contained a solution of 0.6% poly(ethylene oxide) (PEO), 150 mM sodium dodecyl sulfate (SDS), and 60 mM hydroxypropyl-β-cyclodextrin (Hp-β-CD), while a capillary was filled with a solution of 150 mM SDS and 60 mM Hp-β-CD. The role of PEO, Hp-β-CD, and SDS is to act as a concentrating media, as a chiral selector, and as a pseudostationary phase, respectively. This discontinuous system could be employed for the stacking of 600 nL of NDA-derivatized d- and l-Asp without the loss of chiral resolution. The stacking mechanism is mainly based on the difference in viscosity between sample zone and PEO as well as SDS sweeping. The limits of detection at signal-to-noise of 3 for d- and l-Asp were down to 2.4 and 2.5 × 10⁻¹⁰ M, respectively. Compared to normal sample injection volume (25 nL), this stacking approach provided a 100- and 110-fold improvement in the sensitivity of d- and l-Asp, respectively. This method was further applied for determining d- and l-Asp in cerebrospinal fluid, soymilk, and beer.     

On‐line Concentration and Separation of Parabens by Micellar Electrokinetic Chromatography Using Polymer Solutions

Simultaneous separation and quantification of seven parabens commonly used as preservatives in cosmetic products, by micellar electrokinetic chromatography with a stacking technique has been demonstrated. An effective on‐line concentration strategy involving a combination of sweeping and the use of polymer solutions is a key feature of the proposed method, which successfully determined individual parabens. The analysis parameters such as injection time, pH and concentration of phosphate solution, and concentration of sodium dodecyl sulphate (SDS) and poly(ethylene oxide) (PEO) were examined. The optimum conditions were found to be as follows: a 15 mM phosphate solution (pH 9.5) containing 20 mM SDS for filling the capillary, and for the separation electrolytes, 0.100% PEO (8 MDa) added to the phosphate and SDS solution of the same composition as for the capillary. The entire analysis process was completed in 13 min and a 930‐2200‐fold enhancement factor was achieved. The LODs (S/N = 3) for this approach were in the range from 4.32 to 7.78 nM. The linear range for each paraben was between 50 nM and 5.0 μM (R² > 0.990). The optimized method was then successfully applied to the determination of parabens in commercial cosmetic products.