Manipulation of the Electroosmotic Flow in Glass und PMMA Microchips with Respect to Specific Enzymatic Glucose Determinations

The determination of glucose in microfluidic chips made of glass or PMMA was used as a model for the combination of an enzymatic reaction with the separation of compounds. It was based on the enzymatic oxidation of glucose and the amperometric detection of hydrogen peroxide. Real samples frequently contain compounds, such as ascorbic acid, which may interfere with quantitative glucose determinations. Thus, electrophoretic separation of specific from unspecific signals was envisaged by applying electric fields which are also used to control the flow of liquid via electroosmotic effects. Surface charge densities of the capillaries influence the electroosmotic flow (EOF). They are dependent on the chip material and on the adsorption of components from the background electrolyte. Reversal of the EOF after addition of cetyltrimethylammonium bromide (CTAB) and an increase in EOF after addition of sodium dodecylsulfate (SDS) were observed at lower surfactant concentrations with the PMMA chips rather than with the glass chips. For both chip materials these concentrations were below the critical micelle concentration. Effective separation of H₂O₂ and ascorbic acid was achieved with low CTAB concentrations, which lead to a reduction, but not to a reversal of the EOF. Reversal of the EOF by higher CTAB concentrations or the increase in cathodic EOF by SDS accelerated ascorbic acid transportation and reduced the differences in migration times. Thus, for the specific determination of glucose, glucose oxidase was added together with low CTAB concentrations to the background electrolyte. This avoided interference from ascorbic acid, and data obtained from the analysis of fruit juices showed a good correlation to data obtained from a reference method.     

Indirect amperometric measurement of electroosmotic flow rates and effective mobilities in microchip capillary electrophoresis

End-channel indirect amperometry is based on the principle of Kohlrausch regulating function (KRF). A dilute electroactive ionic species is added to the background electrolyte as a continuously eluting electrophore, which is used as probe. The probe concentration variation with the omega value of KRF in the sampling zone was described schematically in this report. Either cathodic or anodic electroosmotic flow (EOF) rates were monitored in microchip. There was no significant difference between the values of EOF rates measured by present method and current-monitoring method. Detection of electroactive and nonelectroactive analytes can also be accomplished by indirect amperometric method. Hence, the effective mobility of analytes can be accurately calculated. And the response mechanism of nonelectroactive analytes K(+), Na(+) and Li(+) in the indirect method was speculated.     

Polymeric ionic liquid-coated capillary for capillary electrophoresis

A new application of the polymeric ionic liquid (PIL) in capillary electrophoresis is reported. Poly(1-vinyl-3-butylimidazolium bromide) was physically adsorbed on silica capillary as the simple and effective coating for capillary electrophoresis (CE) analysis, in which the PIL is not present in the background electrolyte. The electroosmotic flow (EOF) of the PIL-coated capillary as compared with that of the bare fused-silica capillary shows a different dependence on electrolyte pH values. The EOF is reversed over a wide pH range from 3.0 to 9.0 and shows good repeatability. It is also found that the coated capillary has a good tolerance to some organic solvents, 0.1 M NaOH and 0.1 M HCl. The PIL-coated capillary has been employed in different areas. Both the basic proteins and anionic analytes can be well separated by PIL-coated capillaries in a fast and easy way. The PIL-coated capillary is also able to separate organic acid additives in a grape juice. The results showed that this type of coating provides an alternative to the CE separation of anions and basic proteins.     

Enantiomer separation by nonaqueous and aqueous capillary electrochromatography on cyclodextrin stationary phases

Native beta- and gamma-cyclodextrin bound to silica (ChiraDex-beta and ChiraDex-gamma) were packed into capillaries and used for enantiomer separation by capillary electrochromatography (CEC) under aqueous and nonaqueous conditions. Negatively charged analytes (dansyl-amino acids) were resolved into their enantiomers by nonaqueous CEC (NA-CEC). The addition of a small amount of water to the nonaqueous mobile phase enhanced the enantioselectivity but increased the elution time. The choice of the background electrolyte (BGE) determined the direction of the electroosmotic flow (EOF). With 2-(N-morpholino) ethanesulfonic acid (MES) or triethylammonium acetate (TEAA) as BGE an inverse EOF (anodic EOF) was observed while with phosphate a cathodic EOF was found. The apparent pH (pH*), the concentration of the BGE, and the nature of the mobile phase strongly influenced the elution time, the theoretical plate number and the chiral separation factor of racemic analytes.     

Success and failure with phthalate buffers in capillary zone electrophoresis

Phthalate buffers are currently used in capillary electrophoresis as robust electrolyte systems for indirect detection. This contribution demonstrates that these buffers show regularly not only successful regions of mobilities of analytes (sample window) but also regions of failure where the migration of analytes is strongly deteriorated due to the presence of a system zone. System zones in phthalate buffers may be easily detected by UV detection and manifest themselves as peaks or dips. Peak shape diagrams are advantageously used for the prediction of the migration behavior of system zones in phthalate background electrolyte (BGE) systems at various pH. It is shown that the mobility of the system zone varies strongly with pH, is practically zero at pH values below 4 and above 7, and shows a maximum at pH 5. Thus, the system peak may coincide either with the peaks of various analytes or with the electroosmotic flow (EOF) peak. Experiments are given showing the effects of such coincidences as, e.g., zigzag detection patterns, double EOF peaks, and/or unusually broad peaks/dips. The message of this contribution is to show how to understand the electrophoretic properties of phthalate BGEs that, regardless of possible failure regions, may be successfully used in the analytical practice of capillary zone electrophoresis (CZE).     

Comparison of surfactants for dynamic surface modification of poly(dimethylsiloxane) microchips

In the present report, the use of negatively charged surfactants as modifiers of the background electrolyte is reported using poly(dimethylsiloxane) (PDMS) microchips. In particular, the use of anionic surfactants, such as sodium dodecyl sulfate, phosphatidic acid, and deoxycholate, was studied. When surfactants were present in the run buffer, an increase in the electroosmotic flow (EOF) was observed. Two additional effects were also observed: (i) stabilization of the run-to-run EOF, (ii) an improvement in the electrochemical response for several biomolecules. In order to characterize the analysis conditions, the effects of different surfactant, electrolyte, and pH were studied. EOF measurements were performed using either the current monitoring method or by detection of a neutral molecule. The first adsorption/desorption kinetics studies are also reported for different surfactants onto PDMS. The separation of biologically important analytes (glucose, penicillin, phenol, and homovanillic acid) was improved decreasing the analysis time from 200 to 125 s. However, no significant changes in the number of theoretical plates were observed.     

Characteristics of the electroosmotic flow of electrolyte systems for nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) is a powerful tool for the analysis of surface-active substances, which represent a broad class of analytes containing cationic and anionic species, such as surfactants, phosphoric acid esters, and amines. In order to conduct an efficient method development in NACE, the influence of the electrolyte composition on the electroosmotic flow (EOF) of organic separation systems was systematically investigated. Background electrolytes and background chromophores appropriate for direct and indirect UV-detection were considered, as the majority of surface-active substances do not absorb UV-light. It was found that theoretical models developed to describe the EOF in aqueous electrolyte systems are insufficient for organic electrolyte systems. Experimental data on electroosmosis in a variety of organic solvents and mixtures of methanol and acetonitrile applying different background chromophores and basic or acidic additives are given. Differences between them are discussed with relation to the physicochemical properties of the organic solvents.     

Chiral anion exchangers applied to capillary electrochromatography enantioseparation of oppositely charged chiral analytes: investigation of stationary and mobile phase parameters

Weak anion-exchange (WAX) type chiral stationary phases (CSPs) based on tert.-butyl carbamoyl quinine as chiral selector (SO) and different types of silica particles (porous and non-porous) as chromatographic support are evaluated in packed capillary electrochromatography (CEC). Their ability to resolve the enantiomers of negatively charged chiral analytes, e.g., N-derivatized amino acids, in the anion-exchange mode and their electrochromatographic characteristics are described in dependence of several mobile phase parameters (pH, buffer type and concentration, organic modifier type and concentration) and other experimental variables (electric field strength, capillary temperature). The inherent "zwitterionic" surface character of such silica-based WAX type CSPs (positively charged SO and negatively charged residual silanols) allows the reversal of the electroosmotic flow (EOF) towards the anode at pH values below the isoelectric point (pI) of the modified surface, whereas a cathodic EOF results at pH values above the pI. Since for negatively charged analytes also an electrophoretic transport increment has to be considered, which can be either in or against the EOF direction, several distinct modes of elution have been observed under different stationary phase and mobile phase conditions: (i) co-electrophoretic elution of the negatively charged solutes with the anodic EOF in the negative polarity mode, (ii) counter-electrophoretic elution with the cathodic EOF in the positive polarity mode, and (iii) electrophoretically dominated elution in the negative polarity mode with a cathodic EOF directed to the injection end of the capillary. Useful enantioseparations of chiral acids have been obtained with all three modes. Enantioselectivity values as high as under pressure-driven conditions and theoretical plate numbers up to 120000 per meter could be achieved under electrically driven conditions. A repeatability study yielded RSD values below 2% for retention times and RSD values in the range of 5-10% for theoretical plate numbers and resolution, thus clearly establishing the reliability of the investigated anion-exchange type CEC enantioseparation methods.     

Electrophoretic separations in poly(dimethylsiloxane) microchips using a mixture of ionic and zwitterionic surfactants

The use of mixtures of ionic and zwitterionic surfactants in poly(dimethylsiloxane) (PDMS) microchips is reported. The effect of surfactant concentration on electroosmotic flow (EOF) was studied for a single anionic surfactant (sodium dodecyl sulfate, SDS), a single zwitterionic surfactant (N-tetradecylammonium-N,N-dimethyl-3-ammonio-1-propanesulfonate, TDAPS), and a mixed SDS/TDAPS surfactant system. SDS increased the EOF as reported previously while TDAPS showed an initial increase in EOF followed by a reduction at higher concentrations. When TDAPS was added to a solution containing SDS, the EOF decreased in a concentration-dependent manner. The EOF for all three surfactant systems followed expected pH trends, with increasing EOF at higher pH. The mixed surfactant system allowed tuning of the EOF across a range of pH and concentration conditions. After establishing the EOF behavior, the adsorption/desorption kinetics were measured and showed a slower adsorption/desorption rate for TDAPS than SDS. Finally, the separation and electrochemical detection of model catecholamines in buffer and reduced glutathione in red blood cell lysate using the mixed surfactant system were explored. The mixed surfactant system provided shorter analysis times and/or improved resolution when compared to the single surfactant systems.     

Optimization of separation and migration behavior of chloropyridines in micellar electrokinetic chromatography

The separation and migration behavior of pyridine and eight chloropyridines, including three monochloropyridines, four dichloropyridines, and 2,3,5-trichloropyridine were investigated by micellar electrokinetic chromatography using either sodium dodecyl sulfate (SDS) as an anionic surfactant or SDS-Brij 35 mixed micelles. Various parameters such as buffer pH, SDS concentration, Brij 35 concentration and methanol content that affect the separation were optimized. Complete separation of these chloropyridines was optimally achieved with a phosphate buffer containing SDS (30 mM) and methanol (10%, v/v) at pH 7.0. The resolution and selectivity of analytes could be considerably affected by the addition of methanol and/or Brij 35 to the background electrolyte. The migration order of these chloropyridines depends primarily on their hydrophobicity. However, electrostatic interactions may also play a significant role in the determination of the migration order of the positional isomers of chloropyridines.     

Migration behaviour and separation of tramadol metabolites and diastereomeric separation of tramadol glucuronides by capillary electrophoresis

Capillary electrophoresis with UV detection was used to separate tramadol (TR), a centrally acting analgesic, and its five phase I (M1, M2, M3, M4, M5) and three phase II metabolites (glucuronides of M1, M4 and M5). Several factors were evaluated in optimisation of the separation: pH and composition of the background electrolyte and the influence of a micellar modifier, sodium dodecyl sulfate. Baseline separation of TR and all the analytes was obtained with use of 65 mM tetraborate electrolyte solution at pH 10.65. The lowest concentrations of the analytes that could be detected were below 1 microM for the O-methylated, below 2 microM for the phenolic and ca. 7 microM for the glucuronide metabolites. The suitability of the method for screening of real samples was tested with an authentic urine sample collected after a single oral dose (50 mg) of TR. After purification and five-fold concentration of the sample (solid-phase extraction with Oasis MCX cartridges), the parent drug TR and its metabolites M1, M1G, M5 and M5G were easily detected, in comparison with standards, in an interference-free area of the electropherogram. Diastereomeric separation of TR glucuronides in in vitro samples was achieved with 10 mM ammonium acetate-100 mM formic acid electrolyte solution at pH 2.75 and with basic micellar 25 mM tetraborate-70 mM SDS electrolyte solution at pH 10.45. Both separations showed that glucuronidation in vitro produces glucuronide diastereomers in different amounts. The authentic TR urine sample was also analysed by micellar method, but unambiguous identification of the glucuronide diastereomers was not achieved owing to many interferences.     

Capillary electrochromatography with zwitterionic stationary phase on the lysine-bonded poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column

A polymer-based neutral monolithic capillary column was prepared by radical polymerization of glycidyl methacrylate and ethylene dimethacrylate in a 100 mum id fused-silica capillary, and the prepared monolithic column was subsequently modified based on a ring opening reaction of epoxide groups with 1 M lysine in solution (pH 8.0) at 75 degrees C for 10 h to produce a lysine chemically bonded stationary phases in capillary column. The ring opening reaction conditions were optimized so that the column could generate substantial EOF. Due to the zwitterionic functional groups of the lysine covalently bonded on the polymer monolithic rod, the prepared column can generate cathodic and anodic EOF by varying the pH values of running buffer during CEC separation. EOF reached the maximum of -2.0 x 10(-8) m2v(-1)s(-1) and 2.6 x 10(-8) m2v(-1)s(-1) with pH of the running buffer of 2.25 and 10, respectively. As a consequence, neutral compounds, ionic solutes such as phenols, aromatic acids, anilines, and basic pharmaceuticals were all successfully separated on the column by CEC. Hydrophobic interaction is responsible for separation of neutral analytes. In addition, the electrostatic and hydrophobic interaction and the electrophoretic migration play a significant role in separation of the ionic or ionizable analytes.     

Microemulsion electrokinetic chromatography applied for separation of levetiracetam from other antiepileptic drugs in polypharmacy

Microemulsion electrokinetic chromatography was applied for the separation of levetiracetam from other antiepileptic drugs (primidone, phenobarbital, phenytoin, lamotrigine and carbamazepine) that are potentially coadministered in therapy of patients. The influence of the composition of the microemulsion system (with sodium dodecyl sulfate as charged surfactant) was investigated, modifying the kind of cosurfactant (lower alcohols from C3 to C5), the pH (and salinity) of the aqueous background electrolyte, and the ratio of aqueous phase to organic constituents forming the microdroplets of the oil-in-water emulsion. Separation selectivity was depending on all these parameters, resulting even in changes of the migration sequence of the analytes. Only moderate correlation was observed for the microemulsion system compared with a micellar system, both consisting of the aqueous borate buffer (pH 9.2) and SDS as micelle former (linear correlation coefficient for analyte mobilities is 0.974). The sample solvent plays an important role on the shape of the resulting chromatograms: methanol at concentrations higher than 35% impairs peak shape and separation efficiency. The microemulsion method (with 93.76% aqueous borate buffer (pH 9.2, 10 mM), 0.48% n-octane, 1.80% SDS, 3.96% 1-butanol, all w/w) is suitable for the determination of levetiracetam in human plasma (combined with a sample pretreatment based on solid-phase extraction).     

Modification of the electroosmotic flow and separation selectivity of anions in electrochromatography with pseudo-stationary phases of C14-alkyldimethylammoniopropane sulfonate zwitterionic surfactants by addition of salts to the background electrolyte

The effects of salts (NaCl, NaClO4, MgCl2, CeCl3) added to background electrolyte (BGE) solutions (10 mmol L(-1) sodium phosphate, pH 7.2) on electroosmotic flow (EOF) and the separation selectivity of anions (chloride, bromide, iodide, nitrite, nitrate, chlorate, thiocyanate, iodate, chromate, and molybdate ion) by capillary electrochromatography using the zwitterionic surfactant 3-(N,N-dimethylmyristylammonio)propane sulfonate (C14N3S) as a pseudo-stationary phase were investigated. There are two mechanisms affecting the separations: 1. the cations and anions of the added salts interact with the zwitterionic surfactant to varying degrees, thus changing the overall retention of the analytes; and 2. they change the EOF and the resulting apparent mobilities. It was shown that a BGE containing perchlorate and a low concentration of zwitterionic surfactant (2 mmol L(-1)) gave a stable and reproducible EOF and the concentration of perchlorate could be used to manipulate the separation selectivity for polarizable anions, such as iodide and thiocyanate. These effects are discussed in terms of measured association constants describing the interaction of anions and cations with the zwitterion.     

Preparation and characterization of mixed-mode monolithic silica column for capillary electrochromatography

A silica-based monolithic stationary phase with mixed-mode of reversed phase (RP) and weak anion-exchange (WAX) for capillary electrochromatography (CEC) has been prepared. The mixed-mode monolithic silica column was prepared using the sol–gel technique and followed by a post-modification with hexadecyltrimethoxysilane (HDTMS) and aminopropyltrimethoxysilane (APTMS). The amino groups on the surface of the stationary phase were used to generate a substantial anodic EOF as well as to provide electrostatic interaction sites for charged compounds at low pH. A cathodic EOF was observed at pH above 7.3 due to the full ionization of residual silanol groups and the suppression in the ionization of amino groups. A variety of analytes were used to evaluate the electrochromatographic characterization and column performance. The monolithic stationary phase exhibited RP chromatographic behavior toward neutral solutes. The model anionic solutes were separated by the mixed-mode mechanism, which comprised RP interaction, WAX, and electrophoresis. Symmetrical peaks can be obtained for basic solutes because positively charged amino groups can effectively minimize the adsorption of positively charged analytes to the stationary phase.     

Method development approaches for capillary ion electrophoresis

Capillary ion electrophoresis (CIE) is a capillary electrophoretic technique optimized for rapid determination of low-molecular-mass inorganic and organic ions. CIE predominantly employs indirect UV detection since the majority of the analytes lack specific chromophores. Described are three methods for detection and electrolyte optimization. The first method discussed approaches for optimizing sensitivity, selectivity and peak confirmation using a chromate electrolyte and selected detection wavelengths. Peak confirmation is aided by using both direct detection of analytes. The second and third methods involve an unattended electrolyte development approach for instruments that only provide fresh electrolyte on the injection side of the capillary. The electrolyte composition is changed in both the injection side vial and in capillary before each sample injection while leaving the receiving side electrolyte vial constant at the initial electrolyte composition. In one mode, the concentration of the electroosmotic flow (EOF) modifier used to induce anodic flow is varied while keeping the background electrolyte composition constant. In a second experiment, the background electrolyte co-ion is sequentially changed from high mobility to low mobility while keeping the EOF modifier concentration constant. The end effect is to achieve a broad range of controlled peak symmetry for analytes in a simple matrix. The results are compared to separations obtained when the injection side and receiving side electrolytes are manually matched.     

Different sample stacking strategies to analyse some nonsteroidal anti-inflammatory drugs by micellar electrokinetic capillary chromatography in mineral waters

Three on-column preconcentration techniques were compared to analyse a group of nonsteroidal anti-inflammatory drugs (NSAIDs) using micellar electrokinetic capillary chromatography (MEKC) under pH-suppressed electroosmotic flow (EOF) in water samples. The analysed drugs were ibuprofen, fenoprofen, naproxen, ketoprofen, and diclofenac sodium. The micellar background electrolyte (BGE) solution was formed by 75 mM sodium dodecyl sulfate (SDS), 40% (v/v) acetonitrile, and 25 mM sodium phosphate at pH 2.5. When this BGE solution was used the applied voltage was reversed, -10 kV, and the drugs were separated within 20 min. The on-column preconcentration modes, characterised all of them for the sample matrix removal out of the capillary by itself under a reverse potential at the same time as the EOF was reduced, were stacking with reverse migrating micelles (SRMM), stacking with reverse migrating micelles-anion selective exhaustive injection (SRMM-ASEI), and field-enhanced sample injection with reverse migrating micelles (FESI-RMM). The sensitivity was improved up to 154-, 263-, and 63-fold, respectively when it was calculated through the peaks height. The optimised methods were validated with spiked mineral water by combining off-line solid-phase extraction (SPE) and the proposed on-line sample stacking strategies. The detection limits (LODs) of NSAIDs in mineral water were at ng/L levels.     

Microchip electrophoretic protein separation using electroosmotic flow induced by dynamic sodium dodecyl sulfate-coating of uncoated plastic chips

Separation of sodium dodecyl sulfate (SDS)-protein complexes is difficult on plastic microchips due to protein adsorption onto the wall. In this paper, we elucidated the reasons for the difficulties in separating SDS-protein complexes on plastic microchips, and we then demonstrated an effective method for separating proteins using polymethyl methacrylate (PMMA) microchips. Separation difficulties were found to be dependent on adsorption of SDS onto the hydrophobic surface of the channel, by which cathodic electroosmotic flow (EOF; reversed flow) was generated. Our developed method effectively utilized the reversed flow from this cathodic EOF as a driving force for sample proteins using permanently uncoated but dynamic SDS-coated PMMA microchips. High-speed (6 s) separation of proteins and peptides up to 116 kDa was successfully achieved using this system.     

Electroosmotic flow changes due to interactions of background electrolyte counter-ions with polyethyleneimine coating in capillary zone electrophoresis of proteins

The properties and behavior of polyethyleneimine (PEI) covalently coated capillaries with respect to different background electrolytes used in capillary zone electrophoresis (CZE) are described. The coating stability and changes of inner surface charge in the capillary were followed by measurement of electroosmotic flow (EOF). Interest was focused mainly on conjugate bases of carboxylic acids as anionic background electrolyte components (acetate, citrate, malate, malonate, tartrate, and succinate). An interesting phenomenon was observed in PEI-coated capillaries: The direction (and the magnitude) of EOF depends on the composition of the background electrolyte and at a certain pH it can undergo reversible change. Ionic complex formation was suggested as a hypothesis to explain this behavior. With this knowledge, the PEI-coated capillary was used for the separation of basic proteins in the above-mentioned background electrolytes. A standard protein mixture of cytochrome c, ribonuclease A, and lysozyme at a concentration of 0.25 mg/mL each was chosen as model sample.