Separation of phenylureas by capillary electrochromatography on porous graphitic carbon

Summary In this work the potential of porous graphitic carbon (PGC) as a new stationary phase in, capillary electrochromatography has been explored. Its behavior under the action of an applied potential is described for the separation of phenylureas. First, it was shown that porous graphitic carbon enables high efficiency in capillary electrochromatography over a wide range of mobile phase velocities. It was then demonstrated that this material might be responsible for degradation of the solutes at frit-PGC interfaces. Although electrolytic degradation reactions are suspected to occur on this type of conductive material, voltamperometric measurements furnished no clear evidence. A specific injection procedure is proposed for avoiding degradation of the solutes at the inlet interface before their chromatographic separation. Comparison of the retention behavior of phenylureas on PGC in liquid chromatography and in capillary electrochromatography show that the retention propertiets of PGC are altered by application of an electrical field, because this modifies the donor-acceptor interactions between the solutes and the stationary phase.     

Calculation of retention factors in capillary electrochromatography from chromatographic and electrophoretic data

Retention factors in capillary electrochromatography (CEC) were calculated by means of theoretically derived equations and experimentally determined parameters in microcolumn liquid chromatography and capillary zone electrophoresis. It was found that the retention factor of uncharged components in CEC was about 20% higher than was calculated. The derived equations do not take into account alteration of the nature of the stationary phase or distribution constant by the applied electric field. However, the influence of the electric field on the retention in CEC can be estimated. Individual field contributions could not be determined.     

Separation of enantiomers by capillary electrochromatography

The current popularity of capillary electrochromatography (CEC) has led to an increasing number of studies on the development and evaluation of enantioselective CEC systems. These studies clearly demonstrate that the most prominent advantage of electrically driven separation methods, the vastly increased column efficiency as compared to pressure-driven chromatography, can also be experimentally achieved for the separations of enantiomers. In analogy to high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), several approaches have been used. The addition of a chiral selector to the mobile phase is the simplest method. Less erroneous and more elegant approaches are those that use open-tubular, conventional packed, and monolithic columns containing chiral stationary phases that stereoselectively interact with enantiomers. This review evaluates the new techniques and compares them to enantioselective HPLC and CE. Further, it describes the various concepts of enantioselective CEC and focuses on the current ‘state-of-the-art' column technology.     

Coupling of capillary electrochromatography to coordination ion spray mass spectrometry, a novel detection method.

Miniaturized separation techniques such as capillary electrochromatography (CEC), pressurized capillary electrochromatography (pCEC) and capillary high performance liquid chromatography (CHPLC) have been coupled to a new detection technique: coordination ion spray mass spectrometry (CIS-MS). Electrospray ionization (ESI) has found widespread applications in mass spectrometry (MS) for the analysis of polar compounds such as peptides or nucleotides. However, for weakly polar or nonpolar substances, ESI-MS yields poor sensitivity since, in the absence of basic or acidic groups, protonation or deprotonation is not possible. CIS is a universal ionization technique capable of detecting these compounds. Through the addition of a central complexing ion, charged coordination compounds are formed, enabling the detection with good sensitivity. Using the coaxial sheath flow interface commonly employed in CE-MS coupling, we were able to separate and detect various important natural compounds such as unsaturated fatty acid methyl esters, vitamins D2 and D3, and four different estrogens. A central ion solution of 100 microg/mL AgNO3 in water was used as sheath flow liquid, resulting in the formation of positively charged coordination compounds.     

Separation of sulfonamides by capillary electrochromatography

Summary The dual separation mechanism exhibited by capillary electrochromatography is demonstrated by the simultaneous separation of sulfonamides as charged and neutral species over a pH range of 2.5 to 6.9. The neutral sulfonamides were separated according to the difference in their hydrophobicities while charged components were separated by the differences in their electrophoretic mobilities. The limitation of capillary electrochromatography for acidic components was also examined.     

Separation of rhubarb anthraquinones by capillary electrochromatography

Summary A rapid, simple method for packing capillary electrochromatography (CEC) columns with HPLC stationary phases is described. The basis of the method is the use of a vacuum to suck a slurry of stationary phase into the fused-silica tubing, a procedure which takes approximately ten seconds only, then compression of the stationary phase by means of an HPLC pump. These packed CEC columns have been investigated for the separation of five anthraquinones from rhubarb. Separation of the anthraquinones inRheum palmatum L. under optimized conditions is presented.     

Graphene oxide coated capillary for the analysis of endocrine‐disrupting chemicals by open‐tubular capillary electrochromatography with amperometric detection

A graphene oxide‐coated capillary was fabricated by using 3‐aminopropyltriethoxysilane as the cross‐linking agent. It was used for the separation and detection of three endocrine‐disrupting chemicals, including bisphenol A, 4‐nonylphenol, and 4‐octylphenol by capillary electrochromatography. Due to the hydrophobicity, hydrogen bonding, and π–π interaction between graphene oxide and the analytes, the three analytes could be well separated in pH = 11.0, 20 mmol/L Na₂B₄O₇‐NaOH/methanol mobile phase (50:50, v/v) within 950 s. After preconcentration, the detection limits were 6.7 × 10^?10, 3.3 × 10^?9, and 6.7 × 10^?10 mol/L (S/N = 3) for bisphenol A, nonylphenol, and octylphenol, respectively. The developed method was successfully applied to the determination of the above analytes in water samples. The satisfactory result demonstrated that the graphene oxide coated capillary used in capillary electrochromatography with amperometric detection was convenient to prepare, highly stable, and had good reproducibility.     

Instrumentation for capillary electrochromatography

One of the reasons for the immense interest in capillary electrochromatography (CEC) is its feature to combine chromatographic selectivity with the high efficiency and the miniaturization potential of capillary electrophoresis (CE). The capability of commercial CE instruments to run CEC has enforced the readiness of users and researchers to work on this separation technique. Nevertheless, to fully exploit the potential of CEC, a routine CE device can certainly not fulfill all requirements. Two different approaches have been made to overcome this problem. The first was to modify commercial CE instruments for various demands. Pressurization of the packed capillary to prevent "air" bubble formation, gradient elution capabilities and thermostating devices allowing a greater flexibility in column designs have been implemented in CE instruments of several manufacturers. A completely different approach is the development of modular laboratory-made instrumentation dedicated to special CEC requirements. In order to increase mobile phase velocity and thus the speed of analysis the availability of voltages higher than 30 kV was accomplished in some of these devices. Gradient elution was achieved by either coupling of gradient LC systems or an electroosmotic generation of the changing eluent composition. When a pressure gradient is applied between both column ends in addition to the voltage gradient, a hybrid between capillary HPLC and CEC results. This chromatographic mode is named pressure-assisted electrochromatography (PEC). Either CE instruments equipped with additional HPLC pumps or modular laboratory-made devices are suitable for PEC. In CEC, sensitivity for UV detection is rather poor due to the short optical path length for on-column detection in capillary separation techniques. A special cell design with enhanced light path is presented and further principles like, e.g., fluorescence detection and coupling to mass spectrometry are discussed.     

磁场辅助毛细管电色谱

磁场辅助毛细管电色谱是液相色谱研究领域中出现的新技术．它利用外加磁场的引力将置于毛细管内的具有磁响应性的硅胶微球或四氧化三铁微球固定在管内任意位置．磁场固定微球聚集体既可用作填充柱,直接用于电色谱分离;也可用作柱筛,用于填装由商品色谱填料组成的色谱柱．这一技术的优势在于制备简便易行,柱管可以再生使用,适合于微流控芯片上柱筛或柱床的制作．本文简要评述磁场辅助毛细管电色谱的进展,包括磁性色谱填料的制备,磁场固定柱床电色谱,磁性柱筛电色谱及毛细管柱内柱结构参数的测定等方面．     

Open tubular capillary column for the separation of cytochrome C tryptic digest in capillary electrochromatography

A silica capillary of 50 μm internal diameter and 500 mm length (416 mm effective length) was chemically modified with 4‐(trifluoromethoxy) phenyl isocyanate in the presence of dibutyl tin dichloride as catalyst. Sodium diethyl dithiocarbamate was reacted with the terminal halogen of the bound ligand to incorporate the initiator moiety, and in situ polymerization was performed using a monomer mixture of styrene, N‐phenylacrylamide, and methacrylic acid. The resultant open tubular capillary column immobilized with the copolymer layer was used for the separation of tryptic digest of cytochrome C in capillary electrochromatography. The sample was well eluted and separated into many components. The elution patterns of tryptic digest of cytochrome C were studied with respect to pH and water content in the mobile phase. This preliminary study demonstrates that open tubular capillary electrochromatography columns with a modified copolymer layer composed of proper nonpolar and polar units fabricated by reversible addition‐fragmentation transfer polymerization can be useful as separation media for proteomic analysis.     

Perphenylcarbamoylated beta-cyclodextrin bonded-silica particles as chiral stationary phase for enantioseparation by pressure-assisted capillary electrochromatography

Perphenylcarbamoylated beta-cyclodextrin bonded-silica particles (5 microm) were packed into 75-mum fused-silica capillaries, and used for the enantiomer separation of neutral and basic solutes by pressure-assisted capillary electrochromatography. Triethylammonium acetate and phosphate buffer were employed as the BGEs. A cathodic EOF was observed with these two BGEs. Seven chiral analytes were successfully resolved into their enantiomers under optimized conditions, and five of them could be baseline-separated within 12 min due to their high electrophoretic mobility. Better results were achieved with phosphate buffer as the BGE. The effects of organic content and pH on the enantioseparation were also investigated.     

Sensitivity enhancement in capillary electrochromatography by on-column preconcentration

Summary The potential of on-column preconcentration in capillary electrochromatography (CEC) to improve the detection limit was investigated. Two test mixtures containing a pharmaceutically relevant steroid (Desogestrel or Tibolon) together with several structurally related compounds were used for evaluation. For both test mixtures, the mobile phase composition was optimised resulting in a baseline separation of all components and plate numbers of up to 1.2·10⁵ plates m⁻¹ within 15 min. An equation was derived which describes the obtainable gain in injection time as function of the analyte retention factor in the mobile phase and in the injection solvent. The proposed model accounts for the focussing of the analytes due to both the retention during injection and the step-gradient during elution. For the experimental study, the least hydrophobic component of the Desogestrel mixture was used. When the mobile phase was used as injection solvent, a considerable decrease in plate number was observed when the injection time exceeded 5 s. By dissolving the analyte in a less-eluting solvent, injection times could be increased up to 60 s without causing significant extra band broadening. Two mixtures containing a relatively high amount of Desogestrel or Tibolon, and the related components at the 0.1% level were analysed to study the potential of the system for impurity profiling. With the mobile phase as injection solvent, the low level components could hardly be detected. By applying large volume injection from a less-eluting injection solvent, a gain in sensitivity of a factor of 7–9 was achieved.     

Simultaneous determination of dihydroxybenzene positional isomers by capillary electrochromatography using gold nanoparticles as stationary phase

The paper describes the enhanced separation of o-, m-, p-dihydroxybenzene by capillary electrochromatography (CEC) using gold nanoparticles (AuNPs) as stationary phase. The effect of the AuNPs concentration upon separation was investigated. The experimental parameters, including separation voltage, pH, and concentration of running buffer, were optimized. Under the optimum conditions, a good resolution of three dihydroxybenzene isomers was obtained within 15 min in a 50 cm effective length capillary modified with 0.02 nmol/L AuNPs at a separation voltage of 16 kV in a 50 mmol/L acetate buffer (pH 5.0). The linear ranges were from 10(-6) to 10(-4) mol/L and the detection limits were as low as 10(-7) mol/L. This method was successfully used to analysis two kinds of hair coloring agent sample with recoveries in the range of 90-105% and relative standard deviations (RSD) less than 5.0%.     

On-line coupling of pressurized capillary electrochromatography with end-column amperometric detection for analysis of estrogens

An improved technique, pressurized capillary electrochromatography (pCEC) coupling with end-column amperometric detection (AD), was developed and used for the separation and determination of estrogens. The effects of pH value, composition of mobile phase, concentration of the surfactant sodium dodecyl sulfate (SDS) and applied voltage on separation were investigated. The electrochemical oxidation of diethylstilbestrol (DES), dienestrol (DE), and hexestrol (HEX) could be reliably monitored with a carbon electrode at 0.9 V (vs. Ag/AgCl). The pCEC analyses were performed on a capillary separation column packed with 3 microm C18 particles with an acetonitrile/water (31%: 69%) mobile phase containing Tris buffer (5 mmol/L, pH 4.5) and 4 mmol/L SDS. High voltage up to 12 kV reduced the retention time dramatically and still provided a baseline resolution. In addition, supplementary pressure prevented bubble formation and provided reliability and reproducibility of the pCEC performance. The detection limits for the three estrogens ranged from 1.2 to 2.2x10(-7) mol/L, about 10 20-fold lower than those obtained with pCEC-UV detection. To evaluate the feasibility and reliability of this system, the proposed pCEC-AD method was further demonstrated with fish muscle samples spiked with estrogens.     

Recent trends in open-tubular capillary electrochromatography

Capillary electrochromatography (CEC), which combines the advantages of the high efficiency of capillary electrophoresis (CE) and the high selectivity of liquid chromatography (LC), has recently received considerable attention. Most CEC experiments have been performed with capillary columns packed with small LC packing materials (1.5–5 μm particle diameter). However, problems such as difficulties in packing the small LC packing materials and fabricating the frits still exist in preparing the CEC column. The use of open-tubular columns in CEC is therefore an alternative approach that can eliminate the problems encountered in packed-column CEC. So far, several types of open-tubular columns have been proposed for CEC separations and in this article recent progress in this area is reviewed.     

Underivatized cyclic olefin copolymer as substrate material and stationary phase for capillary and microchip electrochromatography

We report, for the first time, the use of underivatized cyclic olefin copolymer (COC, more specifically: Topas) as the substrate material and the stationary phase for capillary and microchip electrochromatography (CEC), and demonstrate chromatographic separations without the need of coating procedures. Electroosmotic mobility measurements in a 25 microm id Topas capillary showed a significant cathodic EOF that is pH-dependent. The magnitude of the electroosmotic mobility is comparable to that found in glass substrates and other polymeric materials. Open-tubular CEC was employed to baseline-separate three neutral compounds in an underivatized Topas capillary with plate heights ranging from 5.3 to 12.7 microm. The analytes were detected using UV absorbance at 254 nm, thus taking advantage of the optical transparency of Topas at short wavelengths. The fabrication of a Topas-based electrochromatography microchip by nanoimprint lithography is also presented. The microchip has an array of pillars in the separation column to increase the surface area. The smallest features that were successfully imprinted were around 2 microm wide and 5 microm high. No plasma treatment was used during the bonding, thus keeping the surface properties of the native material. An RP microchip electrochromatography separation of three fluorescently labeled amines is demonstrated on the underivatized microchip with plate heights ranging from 3.4 to 22 microm.     

Monomer surface modifications for rapid peptide analysis by capillary electrophoresis and capillary electrochromatography coupled to electrospray ionization-mass spectrometry

In this study, positively charged alkylaminosilyl monomers were used to modify the inner surface of fused silica capillaries, which subsequently were employed in capillary electrophoresis (CE) and capillary electrochromatography (CEC). The obtained surfaces yield a reversed electroosmotic flow (EOF) and have varying carbon chain lengths, that interact with the analytes and give chromatographic retention. The coating procedure is very simple and fast. The performance of the modified capillaries was evaluated regarding pH influence on EOF and chromatographic interactions. The experiments were conducted with UV and mass spectrometry (MS) and applied to the separation of various neuropeptides. The derivatized surfaces showed a linear (R(2) approximately 0.99) pH dependence with isoelectric points (pI) at 8.6-8.8. Rapid separations of peptide standards and a protein digest with efficiencies as high as 5 x 10(5) plates/m were performed.     

Analysis of flavanone-7-O-glycosides in citrus juices by short-end capillary electrochromatography

The separation of the major flavanone-7-O-glycoside constituents of Citrus was carried out by isocratic reversed phase capillary electrochromatography using a 75 microm i.d. silica fused column packed with 5 microm ODS silica gel. In comparison to HPLC mode, capillary electrochromatography resolution of flavanone glycosides was obtained with a high selectivity factor. Optimum separation conditions were found using a mixture of ammonium formate (pH 2.5)--acetonitrile (8:2, v/v) as the mobile phase by the short-end injection mode. Under these conditions all the investigated flavanones were baseline-resolved within short analysis time (i.e. between 5 and 10 min). A study, evaluating the intra- and inter-day repeatability as well as limit of detection and method linearity, was developed in accordance with the analytical procedures for method validation. The developed method was applied for the quantitative analysis of flavanone glycosides in commercial fruit juices (sweet orange, lemon and grapefruit).     

Enantioseparations on amylose tris(5-chloro-2-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Amylose tris(5-chloro-2-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSP) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the nature of silica, the particle size and pore diameter, the chiral selector loading onto silica, the mobile phase composition and pH, as well as separation variables such as a linear flow rate of the mobile phase, applied voltage in CEC, etc. on the separation of enantiomers was studied. It was found that CSPs based on amylose tris(5-chloro-2-methylphenylcarbamate) can be used for preparation of stable capillary columns for enantioseparations by nano-LC and CEC in combination with polar organic and aqueous–organic mobile phases. Higher peak efficiency was observed in CEC than in nano-LC.     

Coupling capillary electrochromatography with mass spectrometry by using a liquid-junction nano-spray interface

Capillary electrochromatography (CEC) was coupled with mass spectrometry (MS) for the separation of some selected pesticides and drug enantiomers. CEC separations were carried out in fused silica capillaries packed with either 5 μm RP₁₈ silica or 5 μm silica modified vancomycin particles. The capillary column was connected with the MS utilizing a laboratory-made liquid-junction interface equipped with a 50 μm I.D. capillary-tip positioned at a few mm from the orifice of the MS. The CEC–MS set-up was operated without external pressure assistance during the electrochromatographic run commonly used to avoid bubble formation. However a hydrostatic pressure of a few kPa was applied only to the liquid-junction interface to optimize the ion-spray due to the low I.D. of the capillary-tip. In order to optimize the CEC–MS method, several experimental parameters were studied, namely the inlet pressure, the hydrostatic pressure into the liquid-junction interface, the type of sheath-liquid and the mobile phase. The application of an inlet pressure influenced only analyte retention times that were shortened by increasing the pressure. On the contrary the hydrostatic pressure applied to the interface increased the flow rate into the tip also increasing the ion-signal recorded in the mass spectrometry. The ion-signal raised almost linearly by increasing the outlet pressure till 3.5 kPa and then decreased. The separation of the selected pesticides was not influenced at all changing the hydrostatic pressure on the interface. Some basic enantiomeric compounds of pharmaceutical interest were successfully separated by CEC achieving good resolution. They were detected by MS with limit of detection in a range of 0.24–0.60 μg/mL.