Electrophoretic mobility for peptides with post-translational modifications in capillary electrophoresis

Several semiempirical models for peptide electrophoretic mobility have been tested for capillary electrophoresis (CE) separation with a positively charged capillary using on-line CE combined with electrospray ionization-mass spectrometry (ESI-MS). In the current system with pH 2.7, the expression q/M(0.56) provided the best correlation with the electrophoretic mobility in the analysis of a set of 18 standard samples, where q is the calculated net charge and M is the molecular weight. The peptides resulting from various digests of horse heart myoglobin or bovine hemoglobin were used to demonstrate the validity of this correlation. Post-translationally modified peptides from tryptic digest of human myelin basic protein were also investigated and were found to provide excellent correlation with the linear plot when the total charge of the peptide was correctly calculated. If the total charge was not properly calculated then the post-translationally modified peptides fell off the linear plot. Using this method five arginine residues (residues 5, 49, 54, 97 and 130) were found to be partially citrullinated, four glutamine residues (residues 8, 103, 121 and 147) were found to be partially deamidated and both methionines at residues 21 and 167 were found to be partially oxidized. Three peptides were found with phosphorylation; TPPPSQGK (residue 98 to 105), FSWGAEGQR (residue 114 to 122), and SGSPMAR (residue 163 to 169) and Arginine at 107 was found to be partially monomethylated or dimethylated. The method may provide an excellent means of identifying the presence of peptides with post-translational modifications in conjunction with MS.     

Design and optimization of on-chip capillary electrophoresis

We present a systematic, experimentally validated method of designing electrokinetic injections for on-chip capillary electrophoresis applications. This method can be used to predict point-wise and charge-coupled device (CCD)-imaged electropherograms using estimates of species mobilities, diffusivities and initial sample plug parameters. A simple Taylor dispersion model is used to characterize electrophoretic separations in terms of resolution and signal-to-noise ratio (SNR). Detection convolutions using Gaussian and Boxcar detector response functions are used to relate optimal conditions for resolution and signal as a function of relevant system parameters including electroosmotic mobility, sample injection length, detector length scale, and the length-to-detector. Analytical solutions show a tradeoff between signal-to-noise ratio and resolution with respect to dimensionless injection width and length to the detector. In contrast, there is no tradeoff with respect to the Peclet number as increases in Peclet number favor both SNR and separation solution (R). We validate our model with quantitative epifluorescence visualizations of electrophoretic separation experiments in a simple cross channel microchip. For the pure advection regime of dispersion, we use numerical simulations of the transient convective diffusion processes associated with electrokinetics together with an optimization algorithm to design a voltage control scheme which produces an injection plug that has minimal advective dispersion. We also validate this optimal injection scheme using fluorescence visualizations. These validations show that optimized voltage scheme produces injections with a standard deviation less than one-fifth of the width of the microchannel.     

Separation of fluorescein dyes by capillary electrophoresis using β-cyclodextrin

Summary A capillary electrophoresis method for the separation and determination of five synthetic dyes used in pharmaceutical preparations, cosmetics and as food additives is described. The dyes, fluorescein, dichlorofluorescein, Rose Bengal erythrosine and eosine are well separated in less than 12 min using an electrolyte of 50 mM phosphate buffer (pH 7.5), 10 mM β-cyclodextrin and 5% (v/v) methanol. A linear relationship between concentration and peak area for each dye was obtained in the concentration range 0.3–500 μg mL⁻¹, with a correlation coefficient greater than 0.999. Intra- and inter-day precision of about 0.2–2.6% RSD (n=11) and 4.9–9.7% RSD (n=30), respectively, were obtained. The method has been used for determining the purity of fluorescein and erythrosine in practical samples.     

Monitoring of enzymatic reactions using conventional and on-chip capillary electrophoresis with contactless conductivity detection

The use of CE with contactless conductivity detection was evaluated for monitoring enzymatic reactions. The nonionic species ethanol, glucose, ethyl acetate, and ethyl butyrate were made accessible for analysis by CE via charged products or by-products obtained in enzymatic conversions using hexokinase, glucose oxidase, alcohol dehydrogenase, and esterase. Two of the reactions, namely the conversion of glucose with glucose oxidase and that of ethylacetate with esterase, were also successfully demonstrated on a microchip device. Quantification for ethyl acetate, taken as an example, was found possible with a detection limit of approximately 7 microM.     

Quantification and evaluation of Joule heating in on-chip capillary electrophoresis

We present the use of a novel, picoliter volume interferometer to measure, for the first time, the extent of Joule heating in chip-scale capillary electrophoresis (CE). The simple optical configuration for the on-chip interferometric backscatter detector (OCIBD) consists of an unfocused laser, an unaltered silica chip with a half-cylinder channel and a photodetector. Using OCIBD for millidegree-level noninvasive thermometry, temperature changes associated with Joule heating (2.81 degrees C above ambient) in on-chip CE have been observed in 90 microm wide and 40 microm deep separation channels. The temporal response of Joule heating in isotropically etched channels was exponential, with it taking an excess of 2.7 s to reach equilibrium. Buffer viscosity changes have also been derived from empirical on-chip thermometry data, allowing for the determination of diffusion coefficients for solutes when separated in heated buffers. In addition, OCIBD has allowed the reduction in separation efficiency to be estimated in the absence of laminar flow and due to increased molecular diffusion and lower buffer viscosity. A 7% reduction in separation efficiency was determined for a high current drawing buffer such as Tris-boric acid under an applied field of just 400 V/cm. Results indicate that heating effects in on-chip CE have been underestimated and there is a need to readdress the theoretical model.     

Determination of synthetic food dyes by capillary electrophoresis

A method for the determination of synthetic tar dyes used as food additives using capillary electrophoresis with photodiode-array detection was investigated. The dyes Erythrosine (R-3), Phloxine (R-104), Rose Bengal (R-105), Acid Red (R-106), Amaranth (R-2), New Coccine (R-102) and Allura Red AC (R-40) were separated on a capillary column (50 cm × 75 μm I.D.) and identified from the absorbance spectra of each peak. The electrophoresis buffer used was a mixture of 25 mM sodium phosphate buffer and 25 mM sodium borate buffer (1:1) (pH 8.0) containing 10 mM sodium dodecyl sulfate (SDS). Substitution of β-cyclodextrin for SDS in the electrolyte buffer was effective for the separation of R-2 and R-102. This modified method could be employed as an additional assay method for these two dyes.     

Analysis of sulphonated phthalocyanine dyes by capillary electrophoresis

Sulphonated phthalocyanine dyes may be prepared either by direct sulphonation or by mixed condensation methods. Both syntheses will produce mixtures of products. Such mixtures may be analysed by capillary electrophoresis to identify the various degrees of substitution and to indicate the approximate composition of the mixtures.     

Determination of sphingosine kinase 2 activity using fluorescent sphingosine by capillary electrophoresis

The study of sphingosine and sphingosine-1-phosphate is now widespread due to their immense role as intra- and extracellular messenger molecules. The balance and interplay of these ceramide metabolites is dependent on the activities of kinase and phosphatase enzymes. Sphingosine and sphingosine-1-phosphate are found in very minute quantities in cells; thus, they require highly sensitive techniques for quantitative analysis. In this study, we developed a quantitative assay for the determination of sphingosine kinase 2 (SphK2) activity both in vitro and with cell lysates, using CE-LIF. Sphingosine fluorescein was used as the substrate. The K(M) of SphK2 for sphingosine fluorescein was 2.8 ± 0.8 μM with a V(max) of 2490 ± 520?μM/min and a k(cat) of 1920 ± 402/s. The inhibition of SphK2 was also investigated using four different inhibitors for which 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole inhibitor was the most potent for the in vitro inhibition of SphK2 while N,N-dimethylsphingosine (DMS) did not inhibit but rather increased SphK2 activity. The fluorescence-based approach for the determination of the enzymatic activity of SphK2 proves to be useful for the quantitative determination of SphK2 activity in vitro and in cell lysates, and could be extended to single-cell analysis or applied in drug screening.     

Fabrication of calcium fluoride capillary electrophoresis microdevices for on-chip infrared detection

In this paper, we demonstrate microfluidic capillary electrophoresis (CE) devices made in CaF2 , for optical detection in a broad spectral range. We have designed methods for micromachining and enclosing capillaries in CaF2. The utility of these microdevices has been shown through CE analysis of fluorescently labeled amino acids. We have also performed infrared spectroscopy for analyte identification in microfluidic CaF2 channels. These CaF2 microdevices open the door to microchip separations with optical detection in the ultraviolet, visible, and infrared spectral regions.     

Infrared-based temperature measurements in capillary electrophoresis

In capillary electrophoresis (CE), the temperature inside the capillary is one of the most important parameters. In a concept for Analytical Instrument Qualification (AIQ) of CE systems, the temperature accuracy and stability have to be included. This fact requires an accurate look at the measurement of temperature which is generated by the applied electrical power. The generation of Joule heating is measured on the outside of the capillary using an infrared (IR) thermometer. The thermometer linearity is demonstrated over a wide range of various electrical field strength, buffer systems, and different capillary inner diameters. A slope of 6.3 °C m/W was found for the optimal thermometer capillary distance of 8 mm. Furthermore, the temperature measurements are highly precise, depending almost solely on the current variability. The proposed method is compared with three methods calculating the temperature from the conductance, the electroosmotic velocity, or the current. These indirect methods estimate slopes ranging from 7 to 10 °C m/W. In addition, the maximal suitable electrical power per unit length is estimated. Joule heating can often be tolerated up to 4 W/m. However, sensitive analytes can already be affected by using more than 1 W/m. In conclusion, the consideration of the temperature is essential for not only Analytical Instrument Qualification, but also certainly useful for method optimisation and method transfer.     

On-capillary fluorescent labeling of saccharides for capillary electrophoresis

The feasibility of on-capillary derivatization of saccharides by aromatic amine-based fluorescent labeling agents was tested. To avoid the problematic evolution of gaseous hydrogen cyanide, the Schiff base reduction by sodium cyanoborohydride, as the second step of the standard reductive amination protocol, was omitted. Glucose was used as a model analyte and 7-amino-1,3-naphthalenedisulfonic acid as the labeling agent. Our experiments showed that the direct reaction of the saccharide with the labeling agent in 2.5-M acetic acid yields a labeled product that is sufficiently stable to be separated from the labeling agent in 20-mM phosphate buffer, pH 3.5, and detected using UV detection. The glucose and label zones were introduced separately into the capillary and mixed using a negative voltage. Mixing voltage, its duration, the concentration of acetic acid in the reaction zone, and the waiting time between mixing and separation were optimized. To show the applicability of the procedure to a broader range of analytes, a mixture of different types of saccharides, that is, xylose (pentose), fucose (hexose), glucose (hexose), N-acetylglucosamine (N-acetylaminosaccharide), and lactose (disaccharide), was subjected to derivatization and analysis under the optimal conditions. The linearity and repeatability of the process were evaluated as critical parameters for its analytical applications. Six-point calibration dependences in the 1–50 mM range showed excellent determination coefficients of 0.9992 or higher for all five saccharides tested. The repeatability of the labeled saccharide peak areas was between 2.2% and 4.3%.     

Optimizing the separation of food dyes by capillary electrophoresis

In this work, the separation of eleven food dyes was evaluated by MEKC in electrolytes composed of tetraborate (TBS), Brij 35, and acetonitrile (ACN) using a factorial design at the following levels: TBS concentration (5 and 10 mmol L(-1)), pH (9.5 and 10.1), Brij concentration (5 and 20 mmol L(-1)), and ACN (5 and 15%). Several response functions were evaluated and indicated 10 mmol L(-1) TBS (pH 10.1), 15% ACN, and 20 mmol L(-1) Brij 35 as best values. However, baseline resolution was not achieved (R(cp) = 0.76) and the method lacked robustness. New conditions were sought by studying the dye mobility versus Brij concentration (5-20 mmol L(-1)). A set of well resolved and more uniformly spaced peaks was obtained with an electrolyte consisting of 7.5 mmol L(-1) TBS (pH 10.1), 10 mmol L(-1) Brij, and 15% ACN. Under these new conditions, complete resolution of the 11 dyes was achieved in less than 9 min. Migration time and peak area repeatabilities were better than 1.6% and 5% CV and the LODs were 0.47 to 2.3 microg mL(-1). The methodology was applied to fruit juice powders, lollipops, and other hard and soft chewable treats.     

Optimal MEMS device for mobility and zeta potential measurements using DC electrophoresis

We have developed a novel microchannel geometry that allows us to perform simple DC electrophoresis to measure the electrophoretic mobility and zeta potential of analytes and particles. In standard capillary geometries, mobility measurements using DC fields are difficult to perform. Specifically, measurements in open capillaries require knowledge of the hard to measure and often dynamic wall surface potential. Although measurements in closed capillaries eliminate this requirement, the measurements must be performed at infinitesimally small regions of zero flow where the pressure driven‐flow completely cancels the electroosmotic flow (Komagata Planes). Furthermore, applied DC fields lead to electrode polarization, further questioning the reliability and accuracy of the measurement. In contrast, our geometry expands and moves the Komagata planes to where velocity gradients are at a minimum, and thus knowledge of the precise location of a Komagata plane is not necessary. Additionally, our microfluidic device prevents electrode polarization because of fluid recirculation around the electrodes. We fabricated our device using standard MEMS fabrication techniques and performed electrophoretic mobility measurements on 500 nm fluorescently tagged polystyrene particles at various buffer concentrations. Results are comparable to two different commercial dynamic light scattering based particle sizing instruments. We conclude with guidelines to further develop this robust electrophoretic tool that allows for facile and efficient particle characterization.     

Photochemical properties of selected flavonol dyes: Effects on their separation using capillary electrophoresis

Flavonols are naturally occurring dyes that can be extracted from plants. Because of their antioxidant properties, they are thought to have health benefits. In this study, the photochemical degradation properties of selected flavonols were investigated. Dilute solutions of dyes were exposed to light from a broadband visible light source, and the rate of photodegradation was determined by measuring the decrease in fluorescence of the dyes with respect to time. At pH 9.24, the first-order rate constants for 10?µg?mL^?1 solutions of myricetin, quercetin, kaempferol, and morin were 0.468, 0.162, 0.108, and 0.126?s^?1, respectively. Interestingly, the stability of these historical dyes was also found to be greatly affected by pH. Awareness of the photochemical properties and stability of flavonol dyes is very important for capillary electrophoresis (CE) separations. Photodegradation of the flavonol dyes under the alkaline conditions (pH 9.2) used in CE can have a profound effect on the reproducibility of repeated separations. Even a modest decrease in pH (pH 8.5) greatly improved the stability of these dyes and enabled the successful separation of these flavonol dyes with minimal degradation over time.     

Determination of dyes in foodstuffs by capillary zone electrophoresis

A rapid method based on capillary zone electrophoresis coupled with photodiode-array detection has been developed to determine the dyes Tartrazine E-102, Sunset Yellow FCF E110, Amaranth E-123, New Coccine E-124, Patent Blue V calcium salt E-131 and Allura Red AC E-129 in foodstuffs. Separation was done by using a Bare CElect-FS75 CE column, using a 10 mM phosphate buffer at pH 11.0. Hydrodynamic injections at 0.5 p.s.i. for 4 s (21 nl of sample) and 20 kV separation voltage were used. The quantitation limits for the six dyes ranged from 3 to 6 microg/ml. A linear relationship between 3 to 95 microg/ml, with correlation coefficient better than 0.995 was obtained. This method has been applied to the determination of the studied dyes in beverages, jellies and syrups.     

基于毛细管电泳和离子迁移率经验公式测定洛伐他汀的绝对淌度和解离常数

作为一种可以预防动脉粥样硬化和冠心病的潜在药物洛伐他汀,其绝对淌度m₀和解离常数pK_a值的测定有助于其性质与应用的研究。在前期相关研究基础上,该文提出了一种基于毛细管区带电泳(CZE)和离子淌度经验公式测定洛伐他汀m₀和pK_a的新方法。首先,根据经验公式由实际淌度(m_act)、有效淌度(m_eff)和m₀之间的关系推导出m₀的计算公式。对于一元酸HA,根据之前m₀的计算公式,以氢离子的浓度为自变量,m_eff的倒数为因变量可得到一条直线。根据这条直线的斜率计算得到pK_a。为了验证该方法的可行性和可靠性,应用该方法测定了巴比妥酸、苯甲酸、苄胺、苯酚、间甲酚等有机酸碱的m₀和pK_a值。同时,对于pH值低于6的缓冲体系,采用反向毛细管电泳技术,测定其pK_a,并将测得的实验结果与理论参考值进行对比,发现两者具有较高的一致性,m₀的标准偏差小于6.0%, pK_a的标准偏差小于6.2%,且由线性回归方程的相关系数(R)可以看出测定pK_a时的线性回归直线拟合度较好,说明该文建立的新方法具有较高的可靠性。最后基于这种CZE与经验公式结合的新方法,采用二甲基亚砜(DMSO)作为电渗流标记物测定了洛伐他汀的m₀和pK_a,得到的值分别为-1.70×10^-8 m²/(V·s)和9.00。该方法适用于酸性和碱性分析物m₀和pK_a等理化参数的测定,在药物分析尤其是新药理化特性研究中具有重要意义。     

Determination of biogenic amines by capillary electrophoresis using a chameleon type of fluorescent stain

A method was developed for the determination of biogenic amines (BAs) via micellar electrokinetic chromatography along with laser induced fluorescence detection using the amino-reactive chameleon stain Py-1. A labeling protocol was established for seven primary BAs by optimizing the reaction conditions in terms of the amount of reagents, reaction temperature, reaction time and solvent. Derivatization was accomplished within 30 min and is visible by the naked eye because it is accompanied by a color change from blue to red. Separation of the labeled BAs was achieved within 15 min with a background buffer of pH 2.5 containing phosphate, Tween®80, and methanol. The LODs range from 0.1 to 0.9 µmol·L^?1, with RSDs ranging from 1.1 to 4.2% at 10 µmol·L^?1. The method was applied to the determination of histamine in various fish samples.     

Nonaqueous capillary electrophoresis using a titania-coated capillary

In this work, an ordered mesoporous titania film was introduced to coat a capillary by means of the sol-gel technique. Its electroosmotic flow (EOF) property was investigated in a variety of nonaqueous media (methanol, formamide and N,N'-dimethylformamide and mixtures of methanol and acetonitrile). The titania-coated capillary exhibited a distinctive EOF behavior, the direction and magnitude of which were strongly dependent on various parameters such as the solvent composition, apparent pH (pH*) and the electrolytes. The nonaqueous capillary electrophoresis separation of several alkaloids was investigated in the positively charged titania-coated capillary. Comparison of separation between coated and uncoated capillaries under optimal nonaqueous conditions was also carried out.     

Optimisation of probe concentration in indirect photometric detection in capillary electrophoresis using highly absorbing dyes

In indirect photometric detection in capillary electrophoresis, the concentration of the absorbing probe ion in the background electrolyte should be as high as possible in order to increase the dynamic range of the detection method. For relatively low absorptivity probes (epsilon < 2000 L mol(-1)cm(-1)) used under typical conditions (75 microm ID capillary) the maximum probe concentration is normally limited by the separation current. However, for medium (epsilon approximately/= 2000-15000 L mol(-1)cm(-1)) and especially for high (epsilon > 15000 L mol(-1)cm(-1)) absorptivity probes such as dyes, the maximum concentration may be limited by the background absorbance of the electrolyte which must fall within the linearity range of the detector. In this work, it is shown that another practical factor limiting the probe concentration is the adsorption of probe onto the capillary wall at higher concentrations, resulting in unstable baseline and increased noise. Use of a zwitterionic surfactant to suppress adsorption enabled the concentration of a model probe anion (tartrazine) to be increased by a factor of six times (to 3 mM). This resulted in significant improvements in peaks shapes, resolution between peaks, detection sensitivity and linear calibration range for the analyte anions. Baseline separation of a test mixture was maintained up to 7.5 mM total concentration of sample coions injected (13.7 nL) for the 3 mM electrolyte, with detection limits ranging from 0.63 to 0.94 microM. Peak height reproducibility (over 20 consecutive injections) was improved (values ranging from 1.1 to 1.9%) compared with electrolytes containing lower concentrations of the probe. Overall, the optimised, higher concentration probe electrolyte provided the sensitivity benefits of highly absorbing probes with the additional benefits of ruggedness and improved stacking, peak shapes and resolution.     

New approaches for fabrication of microfluidic capillary electrophoresis devices with on-chip conductivity detection

In practice, microfluidic systems are based on the principles of capillary electrophoresis (CE), for a large part due to the simplicity of electroosmotic pumping. In this contribution, a universal conductivity detector is presented that allows detection of charged species down to the microM level. Additionally, powderblasting is presented as a novel technique for direct etching of microfluidic networks. This method allows creation of features down to 50 microm with a total processing time (design to device) of less than one day. The performance of powderblasted devices with integrated conductivity detection is illustrated by the separation of lithium, sodium, and potassium ions and that of fumaric, malic, and citric acid.