Electroendoosmotic Flow Research Articles

Fabrication of Graphene Oxide Nanosheets Incorporated Monolithic Column via One-Step Room Temperature Polymerization for Capillary Electrochromatography

Graphene oxide (GO) has received great interest for its unique properties and potential diverse applications. Here, we show the fabrication of GO nanosheets incorporated monolithic column via one-step room temperature polymerization for capillary electrochromatography (CEC). GO is attractive as the stationary phase for CEC because it provides not only ionized oxygen-containing functional groups to modify electroendoosmotic flow (EOF) but also aromatic macromolecule to give hydrophobicity and π-π electrostatic stacking property. Incorporation of GO into monolithic column greatly increased the interactions between the tested neutral analytes (alkyl benzenes and polycyclic aromatics) and the stationary phase and significantly improved their CEC separation. Baseline separation of the tested neutral analytes on the GO incorporated monolithic column was achieved on the basis of typical reversed-phase separation mechanism. The precision (relative standard deviation (RSD), n = 3) of EOF was 0.3%, while the precision of retention time, peak area, and peak height for the tested neutral analytes were in the range of 0.4-3.0%, 0.8-4.0%, and 0.8-4.9%, respectively. In addition, a set of anilines were well separated on the GO incorporated monolith. The GO incorporated monolithic columns are promising for CEC separation.

Analysis of trace degradation products (decarboxylated diastereoisomers) of S-adenosylmethionine by electrophoresis in capillaries with cationic coatings (N-methylpolyvinylpyridinium or divalent barium)

Commercial preparations of S-adenosylmethionine (SAM) when analyzed in uncoated capillaries show a minute impurity believed to be decarboxylated (dc) SAM. By using two types of cationic coatings, thus reducing the electro-endo-osmotic flow (EOF), it was possible to separate this impurity into two diastereoisomers of dcSAM. The coatings evaluated for this purpose were: (i) N-methylpolyvinylpyridinium, used under reversed EOF at acidic conditions (pH 4.0) and (ii) deposition of divalent barium at alkaline pH values (pH 9.4), providing reduced EOF. Under these conditions, it was possible to separate this impurity into two diastereoisomers, which by chemical synthesis were indeed proven to be dcSAM. It was further demonstrated that, in the alkylation of 5'-methylthioadenosine by 3-bromopropylamine in bromidric acid to dcSAM, another minute impurity was present, proven, via mass spectrometry, to consist of S-(5'-adenosyl)-3-thiopropylamine (decarboxylated and demethylated (dc-SAH)). The LOD for the two dcSAM diastereoisomers was assessed as 17.5 μg/mL and their LOQ as 25.5 μg/mL. By the barium-based protocol it was possible to quantify the dcSAM, present in a commercial sample of SAM, as a 0.1% impurity.

Separation of the Phenoxy Acid Herbicides and Their Enantiomers by Capillary Zone Electrophoresis in Presence of Highly Sulphated Cyclodextrins

AbstractThe study of the chiral compounds and their fate in the environment is receiving an increasing attention — enantiomeric ratios are being measured and enantioselective degradation processes are being reported. It is particularly important with the toxic compounds like the pesticides, which are being freely used in the environment to control the harmful pests. Capillary zone electrophoresis was used for the chiral and mutual separation of four phenoxy acid herbicides using highly sulphated cyclodextrins (HSCD) in the buffer. The CE runs were performed with reverse polarity (anode in the outlet vial) using the acidic ammonium formate buffer (20 mmol, pH 3). Under these conditions of suppressed the electroendoosmotic flow (EOF), the analytes are mobilized to the anode by entering into host guest relation with the migrating negatively charged sulphated cyclodextrin. The phenoxy acid herbicides selected for the purpose were fenoprop, dicloprop, mecoprop and 2,4‐DB. The α‐HSCD and β‐HSCD have been tested as resolving agents in the CE for the separation of the enantiomers of the herbicides. Though the chiral separation of the dicloprop and mecoprop were achieved with α‐HSCD but it was not able to resolve fenoprop. With β‐HSCD the required base line separation was achieved. Potential difference selected was 10 kV. The limit of detection (S/N= 3) achieved in present case is 0.15 ppm for fenoprop, 0.14 ppm for dicloprop and mecoprop and 0.11 ppm for 2,4‐DB.

Effect of barium tetraborate on the separation of tryptic digests of proteins by zone electrophoresis in uncoated capillaries

A simple, fast, efficient and reproducible method for peptide separations in CZE is reported. It consists in running tryptic digests of peptides in an uncoated capillary, in a BGE composed of tetraborate as a buffering ion, in which the typical sodium counterion is substituted with barium. Efficient absorption of this divalent cation to ionized silanols and barium silicate precipitation seem to be able to shield effectively the silica surface from separands. This is demonstrated by the fact that, when tBa(2+) ions are present in solution (from pH 8.5 up to pH 11.0), the electroendoosmotic flow is reversed; such reversal being progressively higher at higher pH values, by up to a four-fold. Separations become progressively better at higher pH values, whereas at pH 11 in sodium tetraborate they are dramatically worsened. It is further hypothesized that the barium silicate layer further protects the silica surface against dissolution and corrosion which is quite substantial at pH 11.

Capillary electrochromatography of biologically relevant flavonoids

Flavonoids were separated utilizing CEC technique. Baseline separation of biologically relevant flavonoids was obtained using a 100 microm ID fused-silica capillary filled with 3 microm Silica-C18 material and an optimized mobile phase comprising of 20 mM Tris-HCl (pH 6.5), ACN and water at a ratio of 10/40/50 v/v/v. Separations were carried out at 25 kV and a column temperature of 25 degrees C. The influence of relevant parameters for the CEC separation, such as buffer concentration, pH, separation voltage, and ACN concentration, was investigated and optimized. Dependencies of the electroendoosmotic flow (EOF) on these parameters and effects on the resolution of the analytes were studied. During analyses the solvents used for dissolving the samples turned out to have significant effects on the separation of flavonoids. The optimized system was then successfully used for the separation of the flavonoids epicatechin, myricetin, quercetin, naringenin, and hesperetin. CEC turned out to be a useful complementary tool for the economic analysis of flavonoids in addition to common HPLC, muHPLC, and CE methodologies. This method can be used for real applications in phytomics.

Piperazine quaternary diammonium salts as additives to background electrolytes in capillary zone electrophoresis.

The differential behavior of five different quaternary mono- and diammonium salts, among the 18 investigated, in modulating the electroendoosmotic flow (EOF) and analyte separations in capillary zone electrophoresis is evaluated. It is found that quaternary diammonium salts with positive charges separated by more than four carbon atoms, while exhibiting a very strong affinity for chromatographic silica beads, to the point of exhibiting Rf values close to zero, display, on the contrary, a very poor affinity for the silica wall of capillaries. Compounds separated only by a C2 unit (i.e., 1,4-dialkyl-1,4-diazoniabicyclo[2,2,2,]octane, salts 17 and 18) show high Rf values due to strong ion pair association. The unique behavior of quaternary monoammonium salts possessing an iodinated alkyl (butyl or octyl) tail (i.e., 1, 6, and 7) is attributed to their ability to be covalently affixed to the silica wall via alkylation of ionized silanols at alkaline pH values. They thus strongly modulate and typically invert the EOF, even when not present in the background electrolyte. On the contrary, all diammonium salts, devoid of such alkyl tails, are unable to modulate the EOF and to prevent analyte binding to the silica wall, since they are rapidly removed from the wall by the voltage gradient. However, if added in small amount to the background electrolyte, they offer excellent separations of mixtures of very similar organic acids and prevent any interaction with the capillary wall.

Protein analysis by capillary zone electrophoresis utilizing a trifunctional diamine for silica coating.

A novel method is here reported for the analysis of mixture of proteins with pI ranging from pH 3-9.5 in an ample pH interval (pH 2.5-9.0) without adsorption onto the naked silica wall. It consists of treating the capillary surface at alkaline pH, typically 9.0, with small amounts (2-4 mM) of a quaternarized piperazine derivative: (N-methyl-N-omega-iodobutyl)-N'-methylpiperazine (Q-PzI). It appears that this compound is able to dock onto the wall via trifunctional links: a salt bridge via the quaternary nitrogen, a hydrogen bond via the tertiary nitrogen, and finally, a covalent link via the terminal iodine in the butyl chain and a neighboring ionized silanol. This last reaction seems to be completed in a few minutes of incubation of the capillary at room temperature. Because the compound is permanently affixed to the wall, its presence is not needed during protein/peptide separations. By properly dosing the level of Q-PzI in the preconditioning step, it is possible to strongly reduce the electroendoosmotic flow (EOF), zero it, or reverse it. Unlike dynamic coatings with oligoamines, which are most effective only at acidic pH values and are required as additives during separations, Q-PzI is effective in an ample pH interval (pH 2.5-9.0) and is not needed during the CZE analysis. A broad pI (pH 3-10) protein mix can be separated according to protein mobility in free phase, suggesting a strong modulating capacity of the functionalized wall. The same separation is not obtained in capillaries permanently coated with neutral, hydrophilic polymers (such as polyacrylamide), even if the quality of a single protein/peptide profile in Q-PzI-conditioned capillaries is equivalent to those obtained in capillaries permanently coated. Although there is strong indirect evidence of the ability of Q-PzI to alkylate the silica wall, to which it is then irreversibly bound, such an alkylation event does not occur with proteins on potentially reacting sites, such as the free -SH of Cys or the -OH group of Tyr, as demonstrated by incubating them overnight in a large molar excess at strongly alkaline pH values and analyzing such proteins by MALDI-TOF mass spectrometry.

ω-Iodoalkylammonium salts as permanent capillary silica wall modifiers: Comparative analysis of their structural parameters and substituent effects

Following previous work on the modification and inversion of electroendoosmotic flow (EOF) of naked silica by a cyclic diamine [1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide] [J. Chromatogr. A 894 (2000) 53], the present report considerably expands previous data by describing additional compounds of the same series of ω-iodoalkylammonium salts. Four of them are able to instantaneously reverse the EOF, thus producing a cationic surface with a highly stable reverse EOF. All these compounds are believed to become covalently attached to the silica surface via alkylation occurring by nucleophilic substitution of ionized silanols on the silica wall by the ω-iodo functionality in the modifier. The unique advantage of such compounds, as compared to adsorbed polymers or oligoamine EOF quenchers, is that they are not needed any longer in the background electrolyte, after the initial conditioning step inducing the covalent bond. It is additionally demonstrated, by running a mixture of cinnamic acid compounds, that some of the ω-iodoalkylammonium salts can act as modulators of analyte migration, thus inducing separations of otherwise identical compounds, such as isomeric species. Such interactions can only occur when the analytes drift close to the silica wall, and must be rapidly reversible, since no peak tailing or broadening is experienced.

Quantitative studies on the adsorption of proteins to the bare silica wall in capillary electrophoresis: II. Effects of adsorbed, neutral polymers on quenching the interaction

A novel method is reported for quantifying protein adsorption to naked silica tubings and for assessing the efficacy of polymers added to the background electrolyte as dynamic wall modifiers. It consisted of flushing a fluorescently-labelled protein (myoglobin) into a capillary equilibrated in Tris–acetate buffer, pH 5.0, until full saturation of the potential adsorbing sites. Desorption was then affected by electrophoretically driving sodium dodecyl sulphate micelles into the capillary from the cathodic reservoir: the peak of eluted material is quantified by using a dual laser beam instrument able to read the fluorescein isothiocyanate-derivatized myoglobin at 520 nm and the internal standard (sulphorodamine) at 630 nm. Four polymers have been assessed as potential quenchers of interaction of proteins with the silica wall: hydroxypropylmethylcellulose (HPMC, M r=1 000 000), hydroxyethylcellulose (HEC, M r=27 000), poly(vinyl alcohol) (PVA, M r=49 000) and short-chain poly(dimethylacrylamide) [poly(DMA)] (average M r ca. 150 000). HPMC, poly(DMA) and PVA were effective in the 0.005 to 0.02% (w/v) range, whereas HEC was active in the 0.1 to 0.8% concentration range. All polymers, however, except for poly(DMA), exhibited a rather poor performance in suppressing protein interactions with the siliceous surface, and could inhibit adsorption only by, at most, 50% (contrary to oligoamines which can quench such interactions by >90%). It is hypothesized that dynamically adsorbed polymers leave ample regions of the capillary inner surface unmasked, thus allowing strong interactions of proteins with the silica wall. This is also confirmed by the modest reduction of electroendoosmotic flow upon polymer adsorption, as compared with an untreated silica surface. Although poly(DMA) can inhibit protein adsorption by as much as 85%, its hydrophobic nature could in turn provide more adsorption sites for less hydrophilic proteins than myoglobin.

Separation of selected peptides by capillary electroendoosmotic chromatography using 3 μm reversed-phase bonded silica and mixed-mode phases

The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm×100 μm, packed with 3 μm particles, and eluted with mobile phases composed of acetonitrile–triethylamine–phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentration of acetonitrile used and the buffer ionic strength. The retention and selectivity of these peptides under CEC conditions were compared to their behaviour in free zone capillary electrophoresis (CZE), where the separation mode was based on the electrophoretic migration of the analytes due to their charge and Stokes radius properties. In addition, their retention behaviour in RP-HPLC was also examined. As a result, it can be concluded that the elution process of this group of synthetic peptides in CEC with a TEAP buffer at pH 3.0 is mediated by a combination of both electrophoretic migration processes and retention mechanisms involving hydrophobic as well as silanophilic interactions. This CEC method when operated with these 3 μm reversed-phase and mixed-mode sorbents with peptides is thus a hybrid of two well-known analytical methods, namely CZE and RP-HPLC. However, the retention behaviour and selectivity of the selected peptides differs significantly in the CEC mode compared to the RP-HPLC or CZE modes. Therefore this CEC method with these peptides represents an orthogonal analytical separation procedure that is complimentary to both of these alternative techniques.

Chemiluminescence detection of heme proteins separated by capillary isoelectric focusing

Chemiluminescence detection was combined with capillary isoelectric focusing to perform protein analysis with high sensitivity. Luminol–H 2O 2 chemiluminescence was utilized, and heme proteins such as cytochrome c, myoglobin and peroxidase were analyzed. The proteins were focused by use of Pharmalyte 3-10 as ampholytes. Hydroxypropylmethylcellulose was added to the sample solution in order to easily reduce protein interactions with the capillary wall as well as the electroendoosmotic flow. The focused proteins were transported by salt mobilization to chemiluminescence detection cell equipped with an optical fiber. The present method showed significantly high sensitivity and wide dynamic range; the detection limit for cytochrome c was 6·10 −9 M and the linear dynamic range was greater than two-orders of magnitude (up to 2·10 −6 M).

Variety identification in maize lines via capillary electrophoresis of zeins in isoelectric acidic buffers.

Zeins (the prolamins or seed storage proteins in maize) have been used to characterize and identify different genotypes. Zeins were fractionated by capillary zone electrophoresis in acidic, amphoteric buffers, which represent a medium of moderate conductivity and are thus compatible with higher voltage gradients. The running buffer consisted of 40 mM isoelectric aspartic acid, in presence of 6 M urea and 0.5% hydroxyethyl cellulose (apparent pH: 3.8; pI in the absence of urea: 2.77). Thirty-one different zein peaks were mapped out of a total of 21 different maize genotypes. Each of them typically exhibited seven to twelve peaks, with some genotypes showing up to 20 zein bands. Due to slightly changing elution times, caused by a lack of reproducibility of the electroendoosmotic flow in uncoated silica surfaces, correct peak assignment and alignment among different runs was obtained by multivariate statistical analysis. The present method compares well, both in resolution and total number of peaks, with current protocols adopted for screening of maize inbreds, which consist of isoelectric focusing in agarose gels.

Determination of cis and trans isomers of tramadol hydrochloride by capillary zone electrophoresis.

Cis and trans isomers of tramadol hydrochloride were separated by capillary zone electrophoresis (CZE) in a fused-silica capillary with 40 mmol/L of borate as a carrier. The sample concentration-peak area plots were straight lines in the range 10-250 micrograms/mL for both isomers. Based on the electropherogram, the relationship between the structure of the two isomers and the elution order has been discussed. It is considered that the trans isomer has larger hydrodynamic radius than the cis isomer and lower electric charge density. The elution order can be predicted only according to the direction of electrophoresis and electroendo-osmotic flow. The technique was successfully applied to the industrial production and the research for utilization of industrial waste material.

Determination of cis and trans isomers of tramadol hydrochloride by capillary zone electrophoresis

Cis and trans isomers of tramadol hydrochloride were separated by capillary zone electrophoresis (CZE) in a fused-silica capillary with 40 mmol/L of borate as a carrier. The sample concentration-peak area plots were straight lines in the range 10–250 μg/mL for both isomers. Based on the electropherogram, the relationship between the structure of the two isomers and the elution order has been discussed. It is considered that the trans isomer has larger hydrodynamic radius than the cis isomer and lower electric charge density. The elution order can be predicted only according to the direction of electrophoresis and electroendo-osmotic flow. The technique was successfully applied to the industrial production and the research for utilization of industrial waste material. © 1998 John Wiley & Sons, Ltd.

Separation parameters via virtual migration distances in high-performance liquid chromatography, capillary zone electrophoresis and electrokinetic chromatography

Among the various differential migration processes of separation, high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) have emerged as the two major high-performance analytical techniques for separation of complex biological substances. In both HPLC and CZE with electroendoosmotic flow, the differential migration process can be divided into a separative component, which involves selective interactions with the stationary phase or differences in the electrophoretic migration velocities, and a non-separative component representing migration by convection that does not contribute directly to separation. The introduction of virtual migration distances leads to an additivity relationship for the two components that is applicable to both of the above techniques and facilitates the recognition of the underlying similarities as well as the expression and comparison of the various separation parameters. Examination of the key migration parameters led to the characterization and the classification of the various modes of CZE with electroendoosmotic flow. The treatment was extended to the analysis of capillary electrochromatography and micellar electrokinetic chromatography; two hybrid processes which exhibit features borrowed from HPLC and CZE. The use of virtual migration distances also led to a consistent and unified description of the characteristic parameters of these separation systems.

Determination of cations and anions by capillary electrophoresis combined with suppressed conductivity detection

Capillary electrophoresis combined with suppressed conductivity detection is a highly sensitive and selective method for the determination of UV-transparent ionic species. Using direct injection techniques (no electrostacking), minimum detection limits for common organic and inorganic ions are in the range of 1-10 ppb. Suppressors are made from short ion-exchange membrane capillaries and are placed between the separation capillary and the detector cell. The suppressor is placed in the destination vial, which contains regenerant and the ground electrode. The method is dependent on the presence of electroendoosmotic flow in the capillary to transport analyte and buffer ions through the suppressor

Capillary Electrophoretic Separation of Human Recombinant Erythropoietin (r-HuEPO) Glycoforms

Free zone capillary electrophoresis was investigated for the separation of glycoforms in recombinant erythropoietin (r-HuEPO). Reduction of electroendoosmotic flow through the addition of 1,4-diaminobutane and in the presence of urea resulted in optimum separation of all of the major glycoforms. Incubation of r-HuEPO with neuraminidase caused the slowest migrating species to diminish first in response, indicating that these contained the highest numbers of sialic acids. Additional evidence was obtained from spiking r-HuEPO with individual glycoforms isolated by preparative isoelectric focusing. These results indicate that the separation occurs in a predictable manner in order of increasing number of sialic acids contained in the glycoforms. As a consequence, the electropherograms provide the same information as that obtained from conventional gel isoelectric focusing, although separating by a different mechanism.

New polyacrylamide matrices for drift-free isoelectric focusing

The major cause for pH gradient decay and cathodic drift during isoelectric focusing in polyacrylamide gels has been found to be electroendo-osmotic flow generated by fixed charges in the gel matrix. These charges have the following causes: (a) trace impurities of acrylic acid in the co-monomers; (b) covalent incorporation of catalysts (persulphate and riboflavin 5'-phosphate) as terminal groups in polyacrylamide chains; (c) hydrolysis of amide groups to acrylic acid in the gel layer underneath the cathodic filter paper strip. The result of these fixed negative charges in the matrix is a movement of counter-ions with hydration water towards the cathode (i.e. electroendo-osmosis) with concomitant drift of pH gradient and focused protein zones in the same direction. It is impossible to cure the cathodic drift by increasing the pH of the anolyte, or decreasing the pH of the catholyte, or both, as previously suggested. One way to reduce the cathodic drift efficiently is to incorporate covalently in the matrix tertiary or quaternary groups (3-dimethylaminopropylmethacrylamide) in stoichiometric amounts as compared with the negative charges. This ‘balanced’ polyacrylamide displays zero drift for at least 5000V·h, which is considered to be an ample time for equilibrium separation of protein species in isoelectric focusing.