Apparatus and initial results for pressurized planar electrochromatography

Pressurized planar electrochromatography (PPEC) is a new planar chromatographic technique in which the mobile phase is driven by electroosmotic flow, while the sorbent layer is pressurized in a manner that allows heat to flow from the layer through an electrically insulating, thermally conducting, sheet of aluminum nitride ceramic. A prototype apparatus for performing PPEC is described. Separation by PPEC is faster than by conventional TLC, and an example is presented of a 24-fold enhancement in the speed of separation. PPEC was performed on both regular and high-performance C18 layers, and the latter yield substantially faster separation. The sorbent layer requires conditioning at elevated temperature before use, and solute migration velocity increases with this temperature. The flow rate increases in a linear manner with increasing voltage and diminishes in a nonlinear manner with increasing pressure. Both electrical current and Joule heating diminish with increasing pressure, and the diminution of flow at high pressure can be compensated by an increase in voltage. PPEC is more efficient than classical TLC. Theoretical plate heights diminish with increasing Rf and are in the range 29-21 and 55-27 microm for the high-performance and regular plates, respectively. PPEC retains the advantages of classical TLC but has the ability to separate a substantially higher number of samples simultaneously. An example is presented on the separation of nine samples in 1 min on a 2.5 cm x 10 cm sorbent layer.     

Results with an apparatus for pressurized planar electrochromatography

Pressurized planar electrochromatography (PPEC) is a fast and efficient planar chromatographic technique. The mobile phase is driven by electroosmotic flow, while the system is pressurized in a manner that allows heat to flow between the sorbent layer and the pressurizing medium. The reproducibility of solute retention was not satisfactory in the initial report describing PPEC. In the current report, this reproducibility is improved by better control of several experimental variables. The pressure at which PPEC is performed is now free of drift, and the temperature at which the layer is preconditioned is maintained to within +/-1 degrees C. The best reproducibility of retention is obtained when the plate is soaked in the mobile phase for a defined time before each run. In the original prototype, the temperature of the sorbent layer was not controlled. In the present apparatus, water, at a constant temperature between 3 and 60 degrees C, is circulated through channels in the two die blocks that pressurize the layer. The highest efficiency is obtained at an intermediate temperature. This behavior is ascribed to high resistance to mass transfer at the lower temperatures and increased diffusion at higher temperatures. Efficiency, as measured by the number of theoretical plates, increases with increasing migration distance. The height equivalent of a theoretical plate diminishes with increasing migration distance, and values as low as 0.0106 mm are obtained under appropriate conditions. This extrapolates to 94 000 plates/m. Manual spotting was used in this report. Evidence is presented that substantially better efficiency would be obtained if the initial spot size were smaller. The efficiency of PPEC in its current form is illustrated by a chromatogram showing the separation of nine solutes in 2 min. PPEC was also performed with TLC plates in a back-to-back configuration, and this doubles the number of samples that can be simultaneously separated.     

Trapping of bead-based reagents within microfluidic systems: on-chip solid-phase extraction and electrochromatography

A 330-pL chromatographic bed was fabricated on a glass substrate as part of an electroosmotically pumped microfluidic system. Two weirs within a sample channel formed a cavity in which octadecylsilane (ODS) coated silica beads (1.5-4 microns diameter) were trapped. ODS beads were mobilized into and out of the cavity using electroosmotic flow through a bead-introduction channel which accessed the cavity. This procedure allowed the beads in the cavity to be repeatedly exchanged. A 1 nM solution of a nonpolar analyte (BODIPY 493/503) in buffer was loaded onto the beads for different lengths of time using an electroosmotic flow of 1.2 nL/s. The material retained on the ODS phase was then eluted by electroosmotic flow of acetonitrile with a concentration enhancement of up to 500 times. Capillary electrochromatography was conducted using a similar device. BODIPY and fluorescein were loaded onto a 200-micron-long chromatographic bed and then separated in an isocratic CEC run with an acetonitrile/buffer mobile phase. Complete separation was achieved in less than 20 s with a 2-micron plate height.     

Chemically L-phenylalaninamide-modified monolithic silica column prepared by a sol-gel process for enantioseparation of dansyl amino acids by ligand exchange-capillary electrochromatography

A new type of chiral monolithic column was successfully developed for the enantioseparation of dansyl amino acids by ligand exchange-capillary electrochromatography (LE-CEC) in this work. The monolithic column matrix was prepared by a sol-gel process and then chemically modified with the spacer (3-glycidoxypropyl)trimethoxysilane and the chiral selector L-phenylalaninamide. After being conditioned with Cu(II) aqueous solution, the ligand exchange-chiral stationary phase (LE-CSP) possesses positive charges. When the external electric field was applied in CEC, electroosmotic flow (EOF) was generated on the surface of LE-CSP in the direction from the cathode to the anode. The EOF was found to be dependent on the applied electric field strength and the composition of the mobile phase. With the increase of pH of the mobile phase, the EOF showed a tendency to decrease. Scanning electron microscopy showed that the chiral monolithic column has a continuous skeleton and large through-pore structure. The separation efficiency (theoretic plate numbers) for the separation of Dns-DL-Leu reached up to 9.0 x 10(4) plates m(-1) for the D-enantiomer and 6.6 x 10(4) plates m(-1) for the L-enantiomer, by using pH 5.5, acetonitrile/0.50 mM Cu(Ac)2-50 mM NH4Ac (7:3) as mobile phase. The reproducibility and lifetime were satisfactory. CEC was carried out with conventional capillary electrophoresis equipment without pressurizing the ends of the capillary. No bubble was formed during the operation, after degassing the mobile phase and conditioning the column.     

Reversed-phase electrochromatography with a monolithic microcolumn prepared in a 2.2-mm-inner diameter fused-silica tube

Capillary electrochromatography possesses advantages of high separation efficiency and velocity, but it also has its disadvantages due to its small inner diameter, such as poor detection sensitivity, low sample capacity, and some trouble in its column preparation. To overcome these shortcomings, a monolithic microcolumn with a surface area larger than 200 m2/g was prepared by sol-gel polycondensation of tetraethoxysilane-hydrochloric acid-poly(ethylene glycol) and filling with fine quartz sand in a 2.2-mm-i.d. fused-silica tube. The prepared microcolumn was used in the separation of aromatic compounds by reversed-phase electrochromatography. Some factors that affected electroosmotic flow were explored, such as electric field strength, buffer concentration, and buffer pH. Acetonitrile concentration in the mobile phase was investigated for phenol, benzene, and naphthalene separation. The separation results were satisfying with the electrochromatographic microcolumns. The detection limits of phenol, benzene, and naphthalene were 0.07, 0.26, and 0.04 mg/L, respectively.     

Performance of a monolithic silica column in a capillary under pressure-driven and electrodriven conditions

A continuous macroporous silica gel network was prepared in a fused-silica capillary and evaluated in reversed-phase liquid chromatography. Under pressure-driven conditions, the monolithic silica column derivatized to C18 phase (100 microns in diameter, 25 cm in length, silica skeleton size of approximately 2.2 microns) produced plate heights of about 23 and 81 microns at 0.5 mm/s with a pressure drop of 0.4 kg/cm2, and at 4.0 mm/s with 3.6 kg/cm2, respectively, in 90% acetonitrile for hexylbenzene with a k value of 0.7. The separation impedance, E, calculated for the present monolithic silica column was much smaller at a low flow rate than those for particle-packed columns, although higher E values were obtained at a higher flow rate. Considerable dependence of column efficiency on the linear velocity of the mobile phase was observed despite the small size of the silica skeletons. A major source of band broadening in the HPLC mode was found in the A term of the van Deemter equation. The performance of the continuous silica capillary column in the electrodriven mode was much better than that in the pressure-driven mode. Plate heights of 7-8 microns were obtained for alkylbenzenes at 0.7-1.3 mm/s, although the electroosmotic flow was slow. In HPLC and CEC mode, the dependency of plate height on k values of the solutes was observed as seen in open tube chromatography presumably due to the contribution of the large through-pores. Since monolithic silica capillary columns can provide high permeability, the pressure-driven operation at a very low pressure can afford a separation speed similar to CEC at a high electric field.     

High-performance capillary gel electrochromatography with replaceable media

Capillary gel electrochromatography is evaluated with an entangled polymer solution which is pumped into the capillary and run under fritless conditions. The polymer used has an acid backbone with grafted hydrophobic segments, the polyacid giving the electroosmotic flow and the hydrophobe segments providing the retentive component. Experimental evaluation of this type of system reveals performance similar to capillary electrophoresis and other forms of electrochromatography. The analysis of plate height data demonstrates that zone broadening is primarily due to diffusion with little contribution from nonequilibrium zone broadening. Hence, operation at high velocities (high voltages) is most desirable as opposed to most chromatographic methods. Some of the advantages of this type of experiment include being able to replace the retentive media in a few minutes, fast and reproducible high-performance separation, and having a retention mechanism similar to reversed-phase liquid chromatography. Disadvantages include a low retentive phase concentration and hence low sample loadability and limited solvent compatibility of the polymer. A number of different separations are demonstrated including separation of alkyl benzoates, alkylphenones, alkylbenzenes, oxidation inhibitors, and PAHs.     

High-speed electrochemically modulated liquid chromatography

The performance advantages of carrying out electrochemically modulated liquid chromatography (EMLC) at elevated temperatures and mobile-phase flow rates are investigated. EMLC has the unique ability to manipulate analyte retention and enhance separation efficiencies through changes in the potential applied to a conductive stationary phase. Operation of high-performance liquid chromatography systems at elevated column temperatures also provides pathways to improve chromatographic performance by enhancing analyte diffusivity and facilitating the use of higher mobile-phase flow rates than conventionally attainable. The results show that performing EMLC separations at elevated temperatures (e.g., 100 degrees C) reduces the analysis time of a mixture of aromatic sulfonates in a mixed mobile phase by more than a factor of 20. Moreover, use of higher operating temperatures enables the separation of this mixture with an entirely aqueous mobile phase in less than 2 min.     

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column.

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150,000 plates/meter) and good reproducibility (migration time with RSD <0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.     

Simultaneous separation of acidic, basic, and neutral organic compounds, including strong and moderate acids and bases, by capillary electrochromatography

The separation of strongly basic, moderately basic, weakly basic, strongly acidic, moderately acidic, weakly acidic, and neutral compounds in a single run using capillary electrochromatography (CEC) is presented. This is accomplished using a 3-μm CEC Hypersil C8 capillary with high organic content acetonitrile/phosphate (pH 2.5) mobile phases containing hexylamine. Fifteen basic, acidic, and neutral drugs of forensic interest are resolved using a step gradient. Strong and moderately basic drugs separate before t(o), apparently by a combination of free zone electrophoresis (CZE) and chromatographic phenomena. Weak bases separate after t(o), also by a combination of CZE and chromatographic processes. Due to large selectivity differences between CEC and CZE for bases, there is evidence that the stationary phase is playing a significant role in the separation of these solutes. The CEC approach presented offers unique selectivity, expanded peak capacity, and the ability to solubilize both hydrophilic and hydrophobic solutes in an injection solvent that is compatible with the chromatographic system.     

Capillary electrochromatography using a strong cation-exchange column with a dynamically modified cationic surfactant

A novel mode of capillary electrochromatography (CEC), called dynamically modified strong cation-exchange CEC (DMSCX-CEC), is described in this paper. A column packed with a strong cation-exchange (SCX) packing material was dynamically modified with a long-chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added to the mobile phase. CTAB ions were adsorbed onto the surface of the SCX packing material, and the resulting hydrophobic layer on this packing was used as the stationary phase. Using the dynamically modified SCX column, neutral solutes were separated with the CEC mode. The highest number of theoretical plates obtained was about 190,000/m, and the relative standard deviations (RSD's) for migration times and capacity factors of alkylbenzenes were less than 1.0% and 2.0% for five consecutive runs, respectively. The effects of CTAB and methanol concentrations and the pH value of the mobile phase on the electroosmotic flow and the separation mechanism were investigated. Excellent simultaneous separation of the basic and neutral solutes in DMSCX-CEC with a high-pH mobile phase was obtained. A mixture containing the acidic, basic, and neutral compounds was well separated in this mode with a low-pH mobile phase; however, peak tailing for basic compounds was observed in this mobile phase.     

Capillary electrochromatography of cholesterol and its ester derivatives

Separation of cholesterol and its ester derivatives using micellar electrokinetic chromatography is a challenge due to the extreme hydrophobicity of these compounds. In this work, an isocratic capillary electrochromatography (CEC) method has been developed to separate a complex mixture of cholesterol and its 12-ester derivatives. The proportions of mobile phase (tetrahydrofuran, acetonitrile, water), as well as the effects of acid modifiers, buffer concentrations, voltage, and temperature on the separation of cholesterol derivatives were investigated. Addition of a polymeric surfactant, poly(sodium N-undecanoyl-L-glycinate), to the mobile phase reduced migration time and improved resolution of the analytes. The CEC method developed allows baseline separation of a complex mixture of cholesterol and 12 ester derivatives in less than 40 min. Finally, the method is applied to the characterization of cholesterol, cholesterol linoleate, and cholesterol oleate extracted from atherosclerotic plaque deposits in the arterial walls of a human aorta.     

Chiral monolithic columns for enantioselective capillary electrochromatography prepared by copolymerization of a monomer with quinidine functionality. 2. Effect of chromatographic conditions on the chiral separations

The effect of chromatographic conditions on the performance of chiral monolithic poly(O-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroqui nidine-co-ethylene dimethacrylate-co-2-hydroxyethyl methacrylate) columns in the capillary electrochromatography of enantiomers has been studied. The flow velocity was found to be proportional to the pore size of the monolith and both the pH and the composition of the mobile phase. The length of both open and monolithic segments of the capillary column was found to exert a substantial effect on the run times. The use of monoliths as short as 8.5 cm and the "short-end" injection technique enabled the separations to be achieved in approximately 5 min despite the high retentitivity of the quinidine selector. Very high column efficiencies of close to 250000 plates/m and good selectivities were achieved for the separations of numerous enantiomers using the chiral monolithic capillaries with the optimized chromatographic conditions.     

Incorporation of single-wall carbon nanotubes into an organic polymer monolithic stationary phase for mu-HPLC and capillary electrochromatography

Single-wall carbon nanotubes (SWNT) were incorporated into an organic polymer monolith containing vinylbenzyl chloride (VBC) and ethylene dimethacrylate (EDMA) to form a novel monolithic stationary phase for high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The retention behavior of neutral compounds on this poly(VBC-EDMA-SWNT) monolith was examined by separating a mixture of small organic molecules using micro-HPLC. The result indicated that incorporation of SWNT enhanced chromatographic retention of small neutral molecules in reversed-phase HPLC presumably because of their strongly hydrophobic characteristics. The stationary phase was formed inside a fused-silica capillary whose lumen was coated with covalently bound polyethyleneimine (PEI). The annular electroosmotic flow (EOF) generated by the PEI coating allowed peptide separation by CEC in the counterdirectional mode. Comparison of peptide separations on poly(VBC-EDMA-SWNT) and on poly(VBC-EDMA) with annular EOF generation revealed that the incorporation of SWNT into the monolithic stationary phase improved peak efficiency and influenced chromatographic retention. The structures of pretreated SWNT and poly(VBC-EDMA-SWNT) monolith were examined by high-resolution transmission electron microscopy, Raman spectroscopy, scanning electron microscopy, and multipoint BET nitrogen adsorption/desorption.     

Sol-gel monolithic columns with reversed electroosmotic flow for capillary electrochromatography

Sol-gel chemistry was used to prepare porous monolithic columns for capillary electrochromatography. The developed sol-gel approach proved invaluable and generates monolithic columns in a simple and rapid manner. Practically any desired column length ranging from a few tens of centimeters to a few meters may be readily obtained. The incorporation of the sol-gel precursor, N-octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride, into the sol solution proved to be critical as this reagent possesses an octadecyl moiety that allows for chromatographic interactions of analytes with the monolithic stationary phase. Additionally, this reagent served to yield a positively charged surface, thereby providing the relatively strong reversed electroosmotic flow (EOF) in capillary electrochromatography. The enhanced permeability of the monolithic capillaries allowed for the use of such columns without the need for modifications to the commercial CE instrument. There was no need to pressurize both capillary ends during operation or to use high pressures for column rinsing. With the developed procedure, no bubble formation was detected during analysis with the monolithic capillaries when using electric field strengths of up to 300 V cm(-1). The EOF in the monolith columns was found to be dependent on the percentage of organic modifier present in the mobile phase. Separation efficiencies of up to 1.75 x 10(5) plates/m (87,300 plates/column) were achieved on a 50 cm x 50 microm i.d. column using polycyclic aromatic hydrocarbons and aromatic aldehydes and ketones as test solutes.     

Electroosmotic and pressure-driven flow in open and packed capillaries: velocity distributions and fluid dispersion

The flow field dynamics in open and packed segments of capillary columns has been studied by a direct motion encoding of the fluid molecules using pulsed magnetic field gradient nuclear magnetic resonance. This noninvasive method operates within a time window that allows a quantitative discrimination of electroosmotic against pressure-driven flow behavior. The inherent axial fluid flow field dispersion and characteristic length scales of either transport mode are addressed, and the results demonstrate a significant performance advantage of an electrokinetically driven mobile phase in both open-tubular and packed-bed geometries. In contrast to the parabolic velocity profile and its impact on axial dispersion characterizing laminar flow through an open cylindrical capillary, a pluglike velocity distribution of the electroosmotic flow field is revealed in capillary electrophoresis. Here, the variance of the radially averaged, axial displacement probability distributions is quantitatively explained by longitudinal molecular diffusion at the actual buffer temperature, while for Poiseuille flow, the preasymptotic regime to Taylor-Aris dispersion can be shown. Compared to creeping laminar flow through a packed bed, the increased efficiency observed in capillary electrochromatography is related to the superior characteristics of the electroosmotic flow profile over any length scale in the interstitial pore space and to the origin, spatial dimension, and hydrodynamics of the stagnant fluid on the support particles' external surface. Using the Knox equation to analyze the axial plate height data, an eddy dispersion term smaller by a factor of almost 2.5 than in capillary high-performance liquid chromatography is revealed for the electroosmotic flow field in the same column.     

Gold nanoparticle-modified etched capillaries for open-tubular capillary electrochromatography

The use of gold nanoparticles in conjunction with etched capillary-based open-tubular capillary electrochromatography (OTCEC) to improve the efficiency of separation and the selectivity between selected solutes is described. The fused-silica capillaries (50-microm i.d.) were etched with ammonium hydrogen difluoride, followed by prederivatization of the new surface with (3-mercaptopropyl)trimethoxysilane (MPTMS) for the immobilization of dodecanethiol gold nanoparticles, for OTCEC. The electrochromatography of a "reversed-phase" test mixture and of selected polycylic aromatic hydrocarbons was investigated, and efficient separations and high theoretical plate numbers per meter were obtained. The electroosmotic flow characteristics of the etched gold nanoparticle capillary, unetched gold nanoparticle capillary, bare capillary, and etched bare capillary were studied by varying the percentage of organic modifier in buffer, buffer pH, and separation voltage. Optical microscopy and scanning electron microscopy were used to examine the process of etching and modification and the surface features of the etched gold nanoparticle capillary. The results confirm that dodecanethiol gold nanoparticles bonded on the etched inner wall of the fused-silica capillary can provide sufficient solute-bonded phase interactions to obtain OTCEC separations with reproducible retention, as well as characteristic reversed-phase behavior, even with the inner diameter of the capillary of 50 microm.     

Imaging solute distribution in capillary electrochromatography with laser scanning confocal microscopy

A method for the direct observation of solute molecules interacting with a C18 stationary phase under real separation conditions in capillary electrochromatography (CEC) is investigated. The experiments were performed in a capillary electrochromatographic mode; however, the method and findings are useful both in CEC and revered-phase liquid chromatography. The distribution of solute molecules in the packed capillary is directly imaged with laser scanning confocal fluorescence microscopy. Conventional imaging techniques produce images where the C18 silica beads cannot be distinctively identified as a result of the deep depth of field. The optical sectioning capability of confocal imaging overcomes this problem to afford clearly defined images of the stationary-phase packing and the surrounding mobile phase. Fluorescein molecules are preferentially distributed in the mobile phase under reversed-phase chromatographic conditions. Nile Red and rhodamine 6G molecules prefer the environments of the porous C18 beads. Intensity distributions over time for areas within the stationary-phase beads differ from distributions of areas outside the beads in the mobile phase. Images taken at different depths into the capillary probe the internal structure of the C18 beads. While the internal structures of most beads are porous, confocal images show a small fraction (2%) of the silica beads have porous shells and nonporous cores. The capability of imaging the stationary phase distinctively from the mobile phase opens the possibilities of studying the quality of stationary phase, the structure of the column packing, and the mechanisms of separation.     

Fritless capillary columns for HPLC and CEC prepared by immobilizing the stationary phase in an organic polymer matrix

A new design of immobilized particle separation media for capillary liquid chromatography and electrochromatography has been developed. A mixture of porogenic solvents and methacrylate-based monomers is pumped through a packed column to provide, following a polymerization step, an organic matrix capable of holding the sorbent particles in place, thus rendering the end frits unnecessary. The new columns demonstrate excellent chromatographic performance in both CEC (reduced plate height [h]=1.1-1.5) and micro LC modes (h = 2.2-2.5), while minimizing secondary interactions encountered when silica-based entrapment matrixes are employed. In addition to delivering mechanically robust columns, the methacrylate matrix provides a mechanism for fine tuning of the electroosmotic flow velocity when 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) is incorporated into the polymerization mixture.     

A protein-encapsulation technique by the sol-gel method for the preparation of monolithic columns for capillary electrochromatography

A novel protein-encapsulation technique using sol-gels was developed for the preparation of monolithic capillary columns for capillary electrochromatography. Two chiral compounds, bovine serum albumin (BSA) and ovomucoid (OVM) from chicken egg white, were encapsulated in tetramethoxysilane-based hydrogel and their chiral selectivity was evaluated for the separation of some selected enantiomers (tryptophan, benzoin, eperisone, chlorpheniramine). The protein encapsulation was carried out within a capillary in a single step under mild conditions. The resultant monolithic columns showed adequate chromatographic performance, including mechanical strength, penetration of pressurized flow, and chiral separation. Two different proteins, BSA and OVM, were successfully encapsulated into the gel matrixes by changing the alkoxysilane compositions of the gel. Run-to-run repeatability was quite satisfactory. The consecutive analysis of the neutral compound, benzoin, by the OVM-encapsulated column showed good repeatability in the retention time (RSD = 1.23% for the first peak, N = 10). Under optimized conditions, the theoretical plate number for the first peak of benzoin reached 72,000 plates/m.