Systematic investigations of different capillary electrophoretic techniques for separation of methylquinolines

The migration behaviour of isoquinoline, quinoline, and methyl derivatives of quinoline in different capillary electrophoretic modes has been systematically investigated. Optimised separation conditions were established by varying the key parameters (solvent, pH, temperature, surfactant concentration, core phase) for aqueous and non‐aqueous capillary zone electrophoresis (NACE), micellar electrokinetic chromatography (MEKC) with anionic or non‐ionic micelles (SDS, Brij 35), and microemulsion electrokinetic chromatography (MEEKC) with charged or uncharged microemulsion droplets. A separation of all quinolines could be achieved by MEEKC with charged droplets, by MEKC or by formamide‐based NACE. Comparing the separations with respect to separation selectivity, substantial changes in migration order could be observed between the different techniques. Regarding separation efficiency, the number of theoretical plates and limits of detection (LOD) have been compared. The best LODs were achieved using SDS as surfactant in MEKC, followed by MEEKC.     

Nanotube-grafted polyacrylamide hydrogels for electrophoretic protein separation

Multiwalled carbon nanotube-modified polyacrylamide gels have been employed for the electrophoretic separation of proteins. Two approaches are compared in this investigation, one using nanotubes only as fillers inside the gel matrix and the other using nanotubes as catalyst for polymerization of acrylamide. In both the cases, polymerization of acryl-amide/bisacrylamide has been carried out in situ in the presence of nanotubes dispersed in the gel buffer containing monomer and cross-linker. In the former case, initiator and catalyst have been added after ultrasonication of nanotubes in the gel buffer mixture where the nanotubes play the role of filler. On the other hand, the second approach precludes use of catalyst and involves addition of initiator alone during ultrasonication of nanotubes in the gel buffer containing monomer and cross-linker, which leads to the formation of nanotube-grafted gel after 25 min. When nanotubes are used as a catalyst instead of N,N,N',N'-tetramethylethylenediamine, pore size distribution of the gel matrix and linearity of molecular weight calibration plots are found to be improved. In addition, other issues associated with the use of an external catalyst like handling the moisture-sensitive and corrosive reagent and associated irreproducibility are addressed in this approach.     

Capillary electrophoretic separation of nanoparticles

In the present work, CdSe nanocrystals (NCs) synthesized with a trioctylphosphine surface passivation layer were modified using amphiphilic molecules to form a surface bilayer capable of providing stable NCs aqueous solutions. Such modified nanocrystals were used as a test solute in order to analyze new electrophoretic phenomena, by applying a micellar plug as a separation tool for discriminating nanocrystals between micellar and micelle-free zones during electrophoresis. The distribution of NCs between both zones depended on the affinity of nanocrystals towards the micellar zone, and this relies on the kind of surface ligands attached to the NCs, as well as electrophoretic conditions applied. In this case, the NCs that migrated within a micellar zone can be focused using a preconcentration mechanism. By modifying electrophoretic conditions, NCs were forced to migrate outside the micellar zone in the form of a typical CZE peak. In this situation, a two-order difference in separation efficiencies, in terms of theoretical plates, was observed between focused NCs (N ~ 10⁷) and a typical CZE peak for NCs (N ~ 10⁵). By applying the amino-functionalized NCs the preconcentration of NCs, using a micellar plug, was examined, with the conclusion that preconcentration efficiency, in terms of the enhancement factor for peak height (SEF_height) can be, at least 20. The distribution effect was applied to separate CdSe/ZnS NCs encapsulated in silica, as well as surface-modified with DNA, which allows the estimation of the yield of conjugation of biologically active molecules to a particle surface.     

Capillary electrophoretic separation of glycoproteins

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Paper electrophoretic separation of some diastereomers

Summary Gas chromatography (GC) and thin-layer chromatography (TLC) have received most attention as techniques for separation of stereoisomers. Indirect separations by GC of diastereomers have been extensively reviewed by Gil-Av and Nurok [1] while Lochmüller and Souter recently reviewed direct GC resolution methods [2]. TLC work involving diastereomeric compounds of various classes: -methyltryptophans [3], di- and tripeptides [4], some aliphatic compounds [5], and thymidine hydrates [6] have been reported. Although much attention is given to diastereomer separations by GC and TLC, no reports exist of such applications by electrophoresis. This report describes some high voltage electrophoretic separations of diastereomers of several pharmaceutically and biochemically useful compounds.     

Cyclic electrophoretic and chromatographic separation methods

A review is given of the application of cyclic analytical methods in capillary electroseparation (CE) and liquid chromatography (LC) systems. Cyclic methods have been used since the early sixties in chromatographic systems to overcome pressure limitations to resolution. From the early nineties on they have also been applied in capillary electroseparation systems to overcome voltage limitations. Some basic theory is given, outlining the temporal development of resolution in cyclic CE and LC systems and calculating the maximal resolution that can be obtained as a function of the operational parameters of pressure and electrical field. Simple equations are given for the temporal change in the peak capacity and the loss of peaks from the systems as it occurs in some cyclic systems. Finally, a circular open tubular chromatographic system is proposed using integrated pumping and continuous detection. The performance of such a system is discussed using magnetohydrodynamic and alternating current electroosmotic pumping as examples of integrated pumps and Shah Convolution Fourier transform detection as an example of a continuous detection method.     

Etched chemically modified capillaries: novel separation media for electrophoretic analyses

The fabrication, properties, and applications of etched chemically modified capillaries for electrophoretic analysis are reviewed. It is shown that the etching process creates a surface that is fundamentally different than a bare fused silica capillary. The new surface matrix produces unique electroosmotic flow properties and is more compatible with basic and biological compounds. After chemical modification of the surface, the bonded organic moiety (stationary phase) contributes to the control of migration of solutes in the capillary. Both electrophoretic and chromatographic processes take place in the etched chemically modified capillaries leading to a variety of experimental variables (pH, buffer type, presence and amount of organic modifier, and temperature) that can be used to optimize separations. A number of examples of separations on these capillaries are presented as well as data on column ruggedness and reproducibility.     

Theory for the capillary electrophoretic separation of DNA in polymer solutions

We present a mathematical model based on the models of Hubert et al. [Macromolecules 29 (1996) 1006] and Sunada and Blanch [Electrophoresis, 19 (1998) 3128] to describe the electrophoretic mobility of DNA by a transient entanglement coupling mechanism. The proposed model takes into account the interactions between molecules in the capillary and the cross-section of collision between DNA and polymer molecules. The results show that the calculated values agree remarkably well with our electrophoretic mobility data.     

Enhanced electrophoretic DNA separation in photonic crystal fiber

Joule heating generated by the electrical current in capillary electrophoresis leads to a temperature gradient along the separation channel and consequently affects the separation quality. We describe a method of reducing the Joule heating effect by incorporating photonic crystal fiber into a micro capillary electrophoresis chip. The photonic crystal fiber consists of a bundle of extremely narrow hollow channels, which ideally work as separation columns. Electrophoretic separation of DNA fragments was simultaneously but independently carried out in 54 narrow capillaries with a diameter of 3.7 μm each. The capillary bundle offers more efficient heat dissipation owing to the high surface-to-volume ratio. Under the same electrical field strength, notable improvement in resolution was obtained in the capillary bundle chip.     

Capillary electrophoretic separation of recombinant granulocyte-colony-stimulating factor glycoforms

Free zone capillary electrophoresis separated recombinant human granulocyte-colony-stimulating factor, expressed in Chinese hamster ovary cells, into two well-resolved species. Following incubation with neuraminidase, these species comigrated, eluting earlier than either of the original two species. This indicated that the observed heterogeneity was caused by different amounts of sialic acid present on the carbohydrate portion of the protein. It was determined that optimum separation occurred in the buffer pH range 7–9. Evidence is also presented to show that these glycoforms migrate in order of increasing numbers of sialic acids present.     

Capillary electrophoretic separation of polycyclic aromatic sulfur heterocycles

Capillary electrophoresis (CE) was used to separate polycyclic aromatic sulfur heterocycles (PASHs), a class of compounds that occurs in fossil fuels and refined products of petroleum. An electric charge was introduced into the compounds through methylation or phenylation of the sulfur atom. Separations of standard PASHs that are expected to be present in industrially desulfurized fuels showed that CE possessed a higher resolution than reversed phase liquid chromatography. The CE method can separate all the monomethylbenzothiophenes; this is not achieved in capillary gas chromatography. A linear relationship was found between migration time and the calculated volume of the compounds. The PASHs in deeply desulfurized diesel were separated after preconcentration, and the electropherogram was compared with the chromatograms from GC and HPLC. Finally, derivatized PASHs are often enantiomeric and the enantiomers can be separated if a suitable cyclodextrin is added to the running buffer.     

Capillary electrophoretic separation of phenolic diterpenes from rosemary

The major phenolic diterpenes responsible for the antioxidant properties of rosemary extracts, namely carnosol and carnosic acid, were separated by capillary zone electrophoresis (CZE) using a 56 cm long uncoated fused-silica capillary and a 50 mM disodium tetraborate buffer of pH 10.1. The effect of the buffer type, pH and concentration, and the capillary length on the separation, was studied. Carnosol and carnosic acid were identified in the electrophoregrams of rosemary extracts through their migration times and UV spectra obtained by CZE analysis of pure compounds isolated from a rosemary extract by HPLC fractionation. The CZE method had good reproducibility (relative standard deviation less than 5%) and was applied to compare the contents of carnosol and carnosic acid in solid and oil-dispersed commercial extracts of rosemary and in rosemary leaves. The separation of carnosol and carnosic acid was accomplished in less than 11 min.     

Preparatory investigations of fast separation for heavy ion reaction products

A method for separation and chemical identification of products formed in nuclear reactions or nuclear decay is to slow down the recoil products in a gas and to transport them to a trap, where a detecting system is arranged. The sources for the recoil products are²⁵²Cf and²²⁴Ra. As transport phase we used nitrogen or argon and added chemical reagents such as methyl and ethyl radicals, chlorine, oxygen, carbon monoxide or methane. The chemical additives lead to selective and fast separation of certain elements. As examples the results of two experiments are presented:²²⁴Ra with ethyl radicals, and²⁵²Cf with chlorine-oxygen-nitrogen mixtures. The registration of the recoil products at the trap was measured as a function of temperature, pressure and composition of the reactive gas.     

An integrated plastic microchip for enhancing electrophoretic separation using tunable pressure-driven backflows

Microfluidic CE (MCE) is an effective solution for rapid and sensitive determination of multiple analytes. Herein, a dynamic coated cyclic olefin copolymer microchip was developed having an on-chip micropump for fluid velocity adjusting in electrophoretic separations. This micropump was fabricated by constructing a polyacrylamide gel membrane at one channel terminal. Once applying electric field across the membrane, a pressure-driven flow generated automatically to balance the electroosmotic flow (EOF) mismatch at the channel-membrane interface. The influence of gel precursor concentration and operating voltages on the fluid velocity was carefully evaluated. Moreover, the highly integration of injection, separation, and pumping units of the MCE system minimized the dead volume and provides satisfied column efficiency. Experiments showed that by adjusting of pumping voltage reduced the fluid velocity by a factor of 6, resulting six- and threefold resolving power enhancements of rhodamine dye mixture and amino acid mixture, respectively. Furthermore, the developed MCE method was applied for rhodamines and amino acids quantitation in food and cosmetics, with standard addition recoveries of 87.3–106.9% and 89.9–117.4%, respectively. These results were also confirmed by standard HPLC method, revealing the application potential in fast and onsite analysis of complex samples.     

Open source simulation tool for electrophoretic stacking,focusing, and separation

We present the development, formulation, and performance of a new simulation tool for electrophoretic preconcentration and separation processes such as capillary electrophoresis, isotachophoresis, and field amplified sample stacking. The code solves the one-dimensional transient advection-diffusion equations for multiple multivalent weak electrolytes (including ampholytes) and includes a model for pressure-driven flow and Taylor–Aris dispersion. The code uses a new approach for the discretization of the equations, consisting of a high resolution compact scheme which is combined with an adaptive grid algorithm. We show that this combination allows for accurate resolution of sharp concentration gradients at high electric fields, while at the same time significantly reducing the computational time. We demonstrate smooth, stable, and accurate solutions at current densities as high as 5000 A/m² using only 300 grid points, and a 75-fold reduction in computational time compared with equivalent uniform grid techniques. The code is available as an open source for free at http://microfluidics.stanford.edu.     

On-chip pressure injection for integration of infrared-mediated DNA amplification with electrophoretic separation

Poly(dimethylsiloxane) (PDMS) membrane valves were utilized for diaphragm pumping on a PDMS-glass hybrid microdevice in order to couple infrared-mediated DNA amplification with electrophoretic separation of the products in a single device. Specific amplification products created during non-contact, infrared (IR) mediated polymerase chain reaction (PCR) were injected via chip-based diaphragm pumping into an electrophoretic separation channel. Channel dimensions were designed for injection plug shaping via preferential flow paths, which aided in minimizing the plug widths. Unbiased injection of sample could be achieved in as little as 190 ms, decreasing the time required with electrokinetic injection by two orders of magnitude. Additionally, sample stacking was promoted using laminar or biased-laminar loading to co-inject either water or low ionic strength DNA marker solution along with the PCR-amplified sample. Complete baseline resolution (Res = 2.11) of the 80- and 102-bp fragments of pUC-18 DNA marker solution was achieved, with partially resolved 257- and 267-bp fragments (Res = 0.56), in a separation channel having an effective length of only 3.0 cm. This resolution was deemed adequate for many PCR amplicon separations, with the added advantage of short separation time-typically complete in <120 s. Decreasing the amount of glass surrounding the PCR chamber reduced the DNA amplification time, yielding a further enhancement in analysis speed, with heating and cooling rates as high as 13.4 and -6.4 degrees C s(-1), respectively. With the time requirements greatly reduced for each step, it was possible to seamlessly couple IR-mediated amplification, sample injection, and separation/detection of a 278-bp fragment from the invA gene of <1000 starting copies of Salmonella typhimurium DNA in approximately 12 min on a single device, representing the fastest PCR-ME integration achieved to date.     

Capillary electrophoretic separation of inorganic and organic arsenic compounds

Capillary zone electrophoresis was used to separate arsenite, arsenate, dimethylarsinic and diphenylarsinic acid, methanearsonic acid, phenyl- and p-aminophenyl arsonic acid, phenylarsineoxide and phenarsazinic acid. Anionic and uncharged species were separated in a fused silica capillary with on-column UV detection at 200 nm. A 15 mM phosphate solution adjusted to pH 6.5 containing 10 mM sodium dodecylsulfonate served as background electrolyte. The influence of pH and applied voltage on separation efficiency, as well as the feasibility of identification of arsenic compounds in spiked urine, were investigated. Received: 18 March 1998 / Revised: 25 May 1998 / Accepted: 30 May 1998     

Capillary electrophoretic separation of glutamate enantiomers in neural samples

A micellar electrokinetic chromatographic (MEKC) separation of glutamate (Glu) enantiomers fluorescently tagged with naphthalene-2,3-dicarboxaldehyde (NDA) is described. Chiral selectors tested included alpha-, beta-, and gamma-cyclodextrins, modified cyclodextrins, D-glucosamine, sucrose, taurocholate, and their binary mixtures. NDA-labeled Glu enantiomers were best resolved with beta-cyclodextrin in the presence of methanol as an organic modifier. Under the separation conditions, no other amino acids coelute with Glu enantiomers. Using NDA as the reagent, the labeling reaction proceeded readily in aqueous solution, and the spectroscopic properties of NDA fluorophore were optimum for the sensitive laser-induced fluorescence (LIF) detection. Glu enantiomers present in mass limited samples such as single neurons could be adequately quantified by coupling this separation with LIF detection. A detection limit of 0.57 microM Glu was obtained. Using the present method, D-Glu was detected in rat brain, and, for the first time, in ganglia and single neurons of Aplysia californica, an extensively studied neuronal model. Interestingly, the level of D-Glu was found to be higher than that of L-Glu in many Aplysia neurons.     

Capillary electrophoretic separation of amino acids: Fraction collection

A simple method is described which enables solutes to be collected at an electrically isolated exit after they have been separated by a free solution capillary electrophoretic system. The method is illustrated by the separation of dansyl amino acids using multiple separation capillaries.