REVISITING ELECTROOSMOTIC FLOW: AN IMPORTANT PARAMETER AFFECTING SEPARATION IN CAPILLARY AND MICROCHIP ELECTROPHORESIS

Electroosmotic flow mobility (EOF) is the movement of bulk liquid that provides an opportunity to separate charged molecules, either positive or negative, and transport all neutral molecules to the detector as a single peak. EOF originates on the silanol groups of the fused-silica capillary wall and is usually responsible for ions moving in the opposite direction of the electrostatic attraction. The interaction of the silanol groups with the electrolyte buffer leads to the formation of an electric double layer. Understanding double-layer theory and EOF is the first necessary step towards understanding many of the experimental observations in capillary and microchip electrophoresis. In this work, we introduce and validate a method to measure the EOF on both coated and uncoated capillaries by measuring the current time history, which has led to enhanced precision of the EOF measurement. We have also used the introduced method to study the fundamental parameters, such as the effect of electric field, temperature, buffer ionic strength, and pH on electroosmotic flow.     

REVISITING ELECTROOSMOTIC FLOW: AN IMPORTANT PARAMETER AFFECTING SEPARATION IN CAPILLARY AND MICROCHIP ELECTROPHORESIS

Electroosmotic flow mobility (EOF) is the movement of bulk liquid that provides an opportunity to separate charged molecules, either positive or negative, and transport all neutral molecules to the detector as a single peak. EOF originates on the silanol groups of the fused-silica capillary wall and is usually responsible for ions moving in the opposite direction of the electrostatic attraction. The interaction of the silanol groups with the electrolyte buffer leads to the formation of an electric double layer. Understanding double-layer theory and EOF is the first necessary step towards understanding many of the experimental observations in capillary and microchip electrophoresis. In this work, we introduce and validate a method to measure the EOF on both coated and uncoated capillaries by measuring the current time history, which has led to enhanced precision of the EOF measurement. We have also used the introduced method to study the fundamental parameters, such as the effect of electric field, temperature, buffer ionic strength, and pH on electroosmotic flow.     

Application of the Bifunctional Switch-Type Cryptand Ion Chromatographic Stationary Phase for Ion Separation

ABSTRACT

Poly(styrene/divinylbenzene) resin with cryptand 22 as an anchoring group was synthesized and applied in ion chromatography as a packing material for separations of organic anions as well as cations. The cryptand can not only form strong complexes with metal cations, but it has also shown a remarkable complexing ability with organic anions after protonation at pH < 7. The protonated cryptand resin has been applied as an anion exchanger to successfully separate some organic carboxylate anions including HCOO^?, CH₃COO^?, CH₃CH₂COO^?, and some geometric isomers, e.g., fumarate and maleate ions, with water as the eluent. Alternatively, after deprotonated at pH > 7, the cryptand resin can be switched as a cation exchanger to separate inorganic cations. The effects of solvents, temperature, pH values, and flow rates of eluents on the separation of organic anions were also investigated.     

Reactive extraction of 6-aminopenicillanic acid with aliquat-336: Equilibrium and kinetics

The reactive extraction of 6-aminopenicillanic acid (6-APA) from aqueous buffer solution has been studied using a liquid anion exchanger, tricaprylylmethyl ammonium chloride (Aliquat-336) in n-butyl acetate as the solvent. The extraction equilibrium constant and partition coefficient increase with increase in pH up to a limiting value of pH, beyond which extraction decreases due to coextraction of OH^? and buffer anions and hydrolytic decomposition of 6-APA. The experimental data could be predicted from an equilibrium model which takes into account the ideal behaviour of the two liquid phases. The coextraction of the buffer anion under low pH conditions was found to be negligible. The extraction rate measured in a stirred cell of constant interfacial area appears to be dependent on the interfacial mass transfer of the reactive species as well as on the interfacial reaction through its dependence on the concentration of 6-APA in the aqueous and organic phases, respectively.     

Quantitative Aspects of Rare Earth Metal Determinations Using Capillary Electrophoresis with Indirect Absorbance Detection

Abstract

The practical utility of capillary zone electrophoresis with indirect absorbance detection is examined for the separation and quantitation of rare earth metals. Various imidazole derivatives are investigated as to their suitability as running buffer (displaceable) detection ions with α-hydroxyisobutyric acid functioning as a chelating agent to enhance separations. Parameters important for quantitative analysis, such as limits of detection, relative standard deviation of peak areas, efficiency, resolution, peak shape and linear dynamic range are presented. The influences of sample matrix, method of injection, and background ion identity on these parameters are investigated and discussed.     

Optimization of conditions for single-step purification of recombinant hepatitis B surface antigen produced in Pichia pastoris using ion exchange chromatography

ABSTRACT

The adsorption and release of rHBsAg extracted from the final dosage form on various ion exchange resins and under different pH conditions were investigated after its peptide map and isoelectric point (PI) determination. Efficient antigen adsorption to the anion exchange resins occurred when the pH value of the protein buffer was adjusted to 5.0. In purification of rHBsAg derived from the yeast crude extract using Q Sepharose FF column, with adjusting the pH value of the crude extract to 5.0 (i.e., near to the target protein PI) and using 2M NaCl, rHBsAg with high purity (up to >95%) was obtained.

Abbreviations: rHBsAg, recombinant hepatitis B surface antigen; Alhydrogel, aluminum hydroxide; IEF, isoelectric focusing; PI, isoelectric point; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RP-HPLC, reversed-phase high-performance liquid chromatography; SE-HPLC, size-exclusion high-performance liquid chromatography; PBS, phosphate-buffered saline     

Quaternized biomass as an anion exchanger for the removal of nitrate and other anions from water

Dried Chinese Reed (Miscanthus sinensis), a fast growing plant, was used as a model biomass for the development of anion exchangers using a quaternization agent, N‐(3‐chloro‐2‐hydroxypropyl)trimethylammonium chloride (CHMAC), and a cross‐linking agent, epichlorohydrin. Anions studied include nitrate, phosphate, perchlorate and sulfate. Batch mode adsorption studies were conducted using aqueous solutions of anions. Detailed kinetics were studied for nitrate removal. Parameters studied included anion concentration, agitation time, adsorbent dose, and pH. Adsorption equilibrium occurred in 10 min for all the anions studied. Equilibrium adsorption data followed both Langmuir and Freundlich isotherms. The presence of phosphate, perchlorate, fluoride and sulfate lowered the adsorption significantly. The effects of pH on adsorption and desorption show that ion exchange is involved in the adsorption process. The adsorption capacity of the cross‐linked and quaternized Chinese Reed for nitrate, sulfate, phosphate and perchlorate was found to be 7.55, 13.25, 16.61 and 10.07 mg of anion per g of the anion exchanger, respectively. Copyright © 2004 Society of Chemical Industry     

Transport Phenomena in Zonal Centrifuge Rotors. XIII. Gradient Properties of Metrizamide

Abstract

The gradient properties of Metrizamide in three buffer solutions (0.005 M phosphate buffer at pH 6.2, 0.5 M acetate buffer at pH 4.6, and 1.0 M borate buffer at pH 8.2) were measured. Concentration-dependent diffusivities were measured by a microinterferometric method over a solute concentration range from 0.1054 to 0.4862 g/mL. Empirical formulas for the diffusivity and activity coefficient of Metrizamide in three buffer solutions are presented as a function of solute concentration. A generalization factor was obtained for the use of Metrizamide in different buffers at different pH values.     

Separation of Lens culinaris Hemagglutinin by Forced Flow Electrophoresis

Abstract

The usefulness and versatility of forced flow electrophoresis (FFE) for the separation of proteins were examined with Lens culinaris hemagglutinin (lentil lectins). FFE was operated both in the usual single stage and in a more effective, new double-stage scheme. The dependence of the resolution on the flow velocity through the permeable channel and on the electrical field were analyzed. Increased effectiveness in separation was obtained with various pretreatments such as electrofiltration, dialysis, and centrifugation.     

Synthesis of hyperbranched poly(3‐methyl‐3‐hydroxymethyloxetane) and their application to separate basic proteins by adsorption coated column

A series of hyperbranched poly(3‐methyl‐3‐hydroxymethyloxetane)s with different degree of branching were synthesized using BF₃·OEt₂ as initiator and coated on the inner surface of the fused‐silica capillaries. In the pH range of 3–9, the coated capillaries reduced electro‐osmotic flow by about four times lower than the bare fused‐silica capillary. The coated capillaries also displayed good resistance to adsorption of cationic proteins, providing clean separations of a mixture of Lysozyme, Cytochrome c, and Ribonuclease A around pH 3–6 in phosphate buffer. The separation efficiency in terms of peak shape was excellent compared with bare fuse‐silica capillary. The separation efficiency of hyperbranched poly(3‐methyl‐3‐hydroxymethyloxetane) with degree of branching of 0.43‐coated capillary column for Lysozyme reached 10⁶ plates/m with a resolution of 7.1, and the coated capillary column had good migration time reproducibility. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Comparison of analyses of surfactants in cosmetics using high-performance liquid chromatography and high-performance capillary electrophoresis

Separation of anionic, cationic, and amphoteric surfactants containing n-dodecyl groups and hydrophilic moieties was done by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE), and an ultraviolet-visible detector. Quantitation of surfactants in commercial cosmetic and toiletry products was also done using similar methods. Conditions used to separate mixtures of surfactants by HPLC are as follows: stationary phase: ODS 2; mobile phase: MeOH/H₂O (80∶20, vol/vol) containing 1.0M NH₄Cl, 0.003 M tetrabutylammonium hydrogen sulfate, and 0.005 M diammonium phosphate buffer solution at pH=6.0. Conditions for HPCE are as follows: an uncoated capillary (100 μm i.d., 110 cm in length, effective length of 75 cm) with 25 kV of applied voltage, and an aqueous buffer containing 80 mM sodium borate/20 mM NaOH at pH=9.2. Surfactants were eluted within 15 min. The accuracy of both techniques was evaluated by analyzing the recovery ratio of surfactants in the mixture.     

Adsorption and desorption properties of cationic polyethylene film gels to organic anions and their regeneration

An investigation was undertaken on the adsorption and desorption properties of 2‐(dimethylamino)ethyl methacrylate grafted polyethylene (PE‐g‐PDMAEMA) films to anionic dye anions with one to three sulfonic groups in response to pH and temperature changes. The amounts of dye anions adsorbed on the PE‐g‐PDMAEMA films passed through the maximum values at about pH 3 because of an increase in the protonation of dimethylamino groups caused by a decrease in the pH value. The amounts of adsorbed dye anions decreased below pH 3 because the ionic strength increased with the addition of HCl to adjust the initial pH values of the aqueous dye solutions. The amounts of adsorbed dye anions decreased with an increase in the number of sulfonic groups in the dye molecules at the same pH value because electrostatic repulsion was generated between free sulfonic groups of the dye anions adsorbed onto the PE‐g‐PDMAEMA films and free dye anions in the medium. A large number of dye anions adsorbed were desorbed from the PE‐g‐PDMAEMA film with initial pH values above 11.0. The cyclic processes of adsorption at pH 3.0 and desorption at pH 11.0 were repeated without considerable fatigue. The PE‐g‐PDMAEMA films showed practically regenerative adsorption and desorption behavior in response to the pH changes. In addition, when the dye‐anion‐adsorbed PE‐g‐PDMAEMA films were alternately immersed in water at two different temperatures, dye anions were desorbed in water at higher temperatures without any chemical agents because of the deprotonation of dimethylamino groups and thermosensitive contraction of grafted PDMAEMA chains. These results indicate that PE‐g‐PDMAEMA films can be applied as regenerative ion‐exchange membranes for adsorption and desorption processes of anionic compounds in response to the pH and temperature. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 381–391, 2006     

Experimental Validation of the Predicted Properties of a Multiphasic Buffer System Applied to Polyacrylamide Gel Electrophoresis

Abstract

The properties of a buffer system predicted by T. M. Jovin's theory of multiphasic (discontinuous) buffer systems have been tested experimentally both in free solution and in polyacrylamide gels. The most widely applied multiphasic buffer system, viz., the Tris-glycine system of Ornstein and Davis was used for this purpose. The properties of this system were computed on the basis of the Jovin theory. The pH, specific conductance, and boundary displacement were measured in the buffers corresponding to the original and the operative buffers of both the stacking (upper) gel and the separation (lower) gel in polyacrylamide gel electrophoresis. Values of pH and specific conductance in the absence of gels were found to be in reasonable agreement with theory. In polyacrylamide gels, after removal of residual reactants of the polymerization reaction by equilibration with the appropriate buffer, there was good agreement between observed and predicted values of pH and boundary displacement. However, the observed values for specific conductance were lower than predicted. The effects of pre-electrophoresis and of gel concentration on pH, conductance, and boundary displacement were also studied.

A new method has been developed for formation of stable pH gradients in polyacrylamide gel electrophoresis using multiphasic buffer systems. A concentration and pH gradient in the upper (stacking) gel as formed results in a pH gradient in the operative stacking gel (after passage of the stack). This pH gradient is stable for at least 8 h in the system investigated, and may be applicable to protein fractionation.     

An effective approach for nitrate removal from water using antimicrobial modified fish bone by silane groups containing quaternary ammonium salt

ABSTRACT

In this work, a new anion exchanger adsorbent was synthesized based on modified natural polymeric fish bone powder. The obtained adsorbent was investigated by Field Emission Scanning Electron Micrographs (FESEM), Thermo Gravimetric Analysis (TGA), and Fourier transform infrared (FT-IR) spectra. The effects of solution pH, contact time, initial concentration of nitrate, adsorbent dosage, temperature, and competing anions (ammonium chloride, phosphate, bicarbonate sodium, magnesium sulfate) on nitrate adsorption were investigated. The maximum nitrate removal efficiency was achieved 98.83%. Antimicrobial activity of adsorbent was carried out by disc diffusion method against two Gram-negative bacteria namely E. coli and P. aeruginosa, and also two fungi species of Aspergillus niger and Saccharomyces cerevisiae in vitro.     

Analysis of Low Molecular Weight Lignin-Derived Sulphonates by Capillary Zone Electrophoresis

A fast and simple analytical procedure, based on capillary zone electrophoresis (CZE), for separation of underivatized low molecular weight lignin-derived sulphonates has been developed. Optimal CZE-operatipn conditions for the separation and detection of several lignin-derived sulphonic acids was found when using a low pH (pH 1.7) phosphate buffer system, an applied voltage of 20 kV and UV-detection at the anode-side. At these conditions the electroosmotic flow is negligible and the analytes are transported through the capillary column by electromigration. The sulphonation and subsequent degradation of the lignin-model compound 1-(4-hydroxy-3-methoxyphenyl)-2-O-(2-methoxyphenyl)-glycerol has been studied by CZE-analysis of aliquots taken from a sulphite reaction mixture No sample pretreatment was needed before analyzing the sulphite reaction mixture with CZE. The method has also been found to be useful for detection of sulphonic acids in samples from sulphite cooking. Crude extracts of a sulphite cooking liquor could be directly analysed by using the technique developed. Based on migration time data and comparison with an authentic sample, 1-(4-hydroxy-3-meth6xyphenyl)-prop-2-ene-1-sulphonate was identified as one of the sulphonic acids present in the sulphite cooking liquor.     

Continuous Free Flow Electrophoresis in an Alternating Electric Field with a Variable Buffer Flow

Abstract

A novel method of continuous free flow electrophoresis in an alternating electric field with a variable buffer flow is proposed as a basis for continuous separation of biomolecules. A model is developed to evaluate the migration of charged particles as they move with the buffer flow. Numerical calculations on the trajectory and dispersion are presented for particles with different electrophoretic mobilities.     

柱内径对堆积边界法富集重现性的影响

Breadmore提出了一种新型毛细管电泳富集方法（电渗流控制下的逆流等速电泳堆积边界法富集）,可对阴离子实现十万倍在线富集效果。本文以NO3-为模型分子考察了不同内径毛细管柱对该富集重现性的影响。实验发现,使用内径为75μm的毛细管柱时富集重现性极不稳定,富集阶段电流对富集时间的积分面积和NO3-电泳峰面积的RSD（n=10）分别高达6.98%和105.43%,而使用内径为25μm的毛细管柱时富集重现性稳定,富集阶段电流对富集时间的积分面积和NO3-电泳峰面积的RSD（n=7）分别为0.96%和4.43%。实验结果表明这种富集方法的稳定性依赖于富集阶段电流对富集时间的积分面积的重现性,进而依赖于实验中采用的毛细管柱内径的大小尺寸。     

Electrophoretic Determination of Symmetric and Asymmetric Dimethylarginine in Human Blood Plasma with Whole Capillary Sample Injection

Asymmetric and symmetric dimethylarginines are toxic non-coded amino acids. They are formed by post-translational modifications and play multifunctional roles in some human diseases. Their determination in human blood plasma is performed using capillary electrophoresis with contactless conductivity detection. The separations are performed in a capillary covered with covalently bonded PAMAPTAC polymer, which generates anionic electroosmotic flow and the separation takes place in the counter-current regime. The background electrolyte is a 750 mM aqueous solution of acetic acid with pH 2.45. The plasma samples for analysis are treated by the addition of acetonitrile and injected into the capillary in a large volume, reaching 94.5% of the total volume of the capillary, and subsequently subjected to electrophoretic stacking. The attained LODs are 16 nm for ADMA and 22 nM for SDMA. The electrophoretic resolution of both isomers has a value of 5.3. The developed method is sufficiently sensitive for the determination of plasmatic levels of ADMA and SDMA. The determination does not require derivatization and the individual steps in the electrophoretic stacking are fully automated. The determined plasmatic levels for healthy individuals vary in the range 0.36–0.62 µM for ADMA and 0.32–0.70 µM for SDMA.     

Resolution and Peak Capacity in Equilibrium-Gradient Methods of Separation

Abstract

A theoretical analysis is made of the relative resolving power of equilibrium-gradient separation methods, such as isoelectric focusing and density-gradient sedimentation, and the corresponding kinetic methods, such as electrophoresis and kinetic centrifugatipn. Both general and specific equations are derived for resolution and peak capacity. It is concluded that peak capacity, the most general index of over-all resolving power, is of comparable magnitude for these two different approaches.

Two new equilibrium-gradient methods of separation are proposed, these employing dielectrical and thermal diffusion forces, respectively.     

A microelectrochemical scanning flow cell with downstream analytics

The combination of a capillary based microelectrochemical flow cell system and downstream UV–vis analytics allows obtaining synchronized electrochemical and spectroscopic data in a fully automated mode. This method combination can be generally applied to microelectrochemical studies in which an electrochemical species is released or consumed during the electrochemical reaction. For the example of pure zinc surfaces, the characterization of the integrated spectroscopic system is presented with a Zn²⁺ detection limit below 0.1 μmol l⁻¹ using Zincon as complexing agent. A parameter screening of the effect of pH in the range of 6.6–9.0 in borate buffer reveals a linear increase in zinc dissolution with proton concentration but a distinct step in the open circuit potential from the active state (around −700 mV SHE, pH 6.6–7.1) to the passive state (around −300 mV SHE, pH 7.4–9.0) indicating the formation of a closed passive layer. This mechanism is strongly influenced by sulfate anions which increase the dissolution rate of the passive film and promote the active state as monitored by the dissolution profile and OCP (open circuit potential) values. Within the scope of this parameter variation, the congruency between OCP transients, potentiodynamic sweeps and time resolved dissolution profiles is discussed.