Pressure-assisted Capillary Electrochromatography Research Articles

An underestimated technique. Does pressurized and pressure-assisted capillary electrochromatography have potential in drug and pharmacological-active compounds analysis?

Abstract Pharmaceutical analysis guarantees patient safety all over the world. Thus, continuous development of existing analytical techniques is still very important. Pressurized capillary electrochromatography and pressure-assisted capillary electrochromatography are hybrid separation techniques that combine the selectivity of liquid chromatography and the high separation efficiency of capillary electrophoresis. They use a smaller amount of reagents and samples, hence, reducing the total cost of analysis. Therefore they have found application in a number of pharmaceutical and biomedical analysis. This review article focuses on the use and importance of pressurized and pressure-assisted capillary electrochromatography in pharmaceutical and biomedical analysis, taking into account types of detectors and capillaries used. Despite the fact that pressurized capillary electrochromatography and pressure assisted capillary electrochromatography offer many possibilities and have been available for over a dozen years, they are still underdevelopment and not fully explored.

Enantiomeric separation of some chiral analytes using amylose 3,5-dimethylphenylcarbamate covalently immobilized on silica by nano-liquid chromatography and capillary electrochromatography

A new experimental chiral stationary phase containing amylose 3,5-dimethylphenylcarbamate immobilized onto silica gel (i-ADMPC) was packed in 100μm I.D. fused silica capillary and used for the chiral separation of eight selected flavanone derivatives in polar organic mobile phase by using different miniaturized techniques, such as nano-liquid chromatography (nano-LC), capillary electrochromatography (CEC) and pressure-assisted CEC (pCEC). A comparative study of different elution modes in terms of chromatographic efficiency, analysis time and enantiomeric resolution was carried out. In pCEC mode, the highest chromatography performance was obtained applying +2.5kV voltage and inlet pressure 10bar. Under these conditions, the analysis times were shorter than 20min, and chromatographic efficiencies were in the range 33,000–49,000 plates/m (first eluted peak).The solvent versatility of packed i- ADMPC capillary column was also investigated. In nano-LC, the CSP was stressed under the use of strong organic solvents such as ethyl acetate, acetone and methyl t-butyl ether (MTBE). After at least 40 working hours and over 50 sample injections, the CSP resulted to be very stable allowing to achieve good repeatability employing again the mobile phase containing polar organic solvent. Enantioresolutions and chromatographic efficiencies decreased by 1.60 and 10%, respectively as the result of using above mentioned strong solvents.

A new method for the analysis of β2-agonists in human urine by pressure-assisted capillary electrochromatography coupled with electrospray ionization-mass spectrometry using a silica-based monolithic column

A new pressure-assisted capillary electrochromatography coupled with electrospray ionization-mass spectrometry method using a silica-based monolithic column as separation media was developed for the analysis of beta(2)-agonists in human urine. Experimental conditions including the mobile phase, separation voltage, assisted pressure, and sheath liquid were optimized for the analysis: mobile phase composed of 82% (v/v) ACN and 18% (v/v) 20mmol/L ammonium acetate (pH 6.0); separation voltage 25kV; assisted pressure 2 bar; and the sheath liquid consisting of 7.5mmol/L acetic acid in isopropanol/water 50/50% (v/v) that was delivered at a flow rate of 3.0microL/min. Six beta(2)-agonists were separated within 12.5min with LODs (defined as S/N=3) in the range of 0.25-2.0ng/mL. The absolute LODs of the developed method for analyzing six beta(2)-agonists ranged from 5.75 to 46.0fg. Method repeatability of run-to-run and column-to-column was satisfactory. The recovery obtained from the analysis of spiked urine samples was between 88.2% and 106% with RSDs lower than 6.68%. The method was successfully applied to the analysis of real urine sample from volunteers.

Novel fabrication of on-column capillary inlet frits through flame induced sintering of stainless steel particles

A novel fritting technology was introduced for the fused-silica capillary. The technique involved sintering of stainless steel (SS) particles at the tip of capillary through flame heating. A simple butane gas based welding torch was used for sintering the SS particles. The new fritting technique, flame induced sintering of SS particles (FIS/SSP), was applied for making frits with different inlet diameters (75 μm, 100 μm, 250 μm and 530 μm). The changes in morphologies of SS particles during sintering process were identified by scanning electron microscopy (SEM). Frits with the length of 0.5–1 mm and capillaries with inner diameter about 50–100 μm were fabricated through suitable selection of experimental conditions (size of SS particles and heating mode). The frits prepared by FIS/SSP technique exhibited adequate separation properties and mechanical strength. Columns packed with C 18 particles were stable with these frits in a few important chromatographic operations. Frits prepared by FIS/SSP technique was used in three typical separation modes namely, capillary electrochromatography (CEC), p-assisted CEC (p-CEC) and low pressure liquid chromatography (LPLC). Importantly, no bubble formation was noticed with the frit over a period of one week. A good peak symmetry and high efficiency for separation were obtained using pressure-assisted CEC, p-CEC and low pressure-driven separation modes.

Chromatographic evaluation and comparison of three β‐cyclodextrin‐based stationary phases by capillary liquid chromatography and pressure‐assisted capillary electrochromatography

Enantiomer separations were performed on three beta-cyclodextrin-based chiral stationary phases (CSP) containing the pernaphthylcarbamoylated beta-cyclodextrin (CSP 1), peracetylated beta-cyclodextrin (CSP 2) and permethylated beta-cyclodextrin (CSP 3) as chiral selectors by capillary liquid chromatography and pressure-assisted capillary electrochromatography in this study. Triethylammonium acetate/MeOH or phosphate buffer/MeOH was used as the mobile phase. The experimental factors affecting chiral separations have been examined for each CSP, including pH of the buffers, methanol content and applied voltage. Under optimal separation conditions, a number of racemic compounds were resolved into their enantiomers on three cyclodextrin-based CSP. A comparative study on the performance of three CSP revealed the presence of carbonyl functional groups as well as aromatic rings in the cyclodextrin derivatives, enhanced the interaction between the analytes and CSP, and thus improved enantioselectivity of the CSP.

Rapid separation and sensitive detection method for β-blockers by pressure-assisted capillary electrochromatography–electrospray ionization mass spectrometry

A new method for rapid separation and sensitive detection of β-blockers by pressure-assisted capillary electrochromatography (pCEC) with electrospray ionization mass spectrometry (ESI-MS) using silica-based monolithic column was studied in this paper. The proposed method has been confirmed to be very powerful since the fast mass transfer property and good permeability of silica monolithic column was used in this pCEC–ESI-MS system. In this work, a silica monolithic column was prepared with sol–gel method for simultaneous fast separation of β-blockers. Furthermore, in order to obtain the highly selective and sensitive result of pCEC–ESI-MS, both the CEC separation and MS detection parameters were optimized in detail. Under the optimized conditions, namely 80% acetonitrile and 20% 20 mmol/L ammonium acetate (pH 6.0) as the mobile phase, 20 kV and 8 bar as the separation voltage and the assisted pressure, isopropanol/water (1:1, v/v) containing 7.5 mmol/L acetic acid as the sheath liquid, and 3 μL/min as the flow rate of sheath liquid, seven β-blockers were well separated within 11 min with detection limits in the range of 0.15–0.80 ng/mL (defined as S/N = 3). The recoveries of spiked urine samples of these β-blockers were between 86.3 and 103% with the RSDs lower than 8.0%. The real samples from some male volunteers were successfully analyzed and confirmed with the proposed method. Comparing with GC–MS or LC–MS, the new method has some superiority (such as fast analysis capacity and simple pretreatment) in clinical practice and doping control.

Novel method to prepare polystyrene-based monolithic columns for chromatographic and electrophoretic separations by microwave irradiation

Microwave irradiation can provide a viable alternative to the traditional means such as ultraviolet light and thermal initiation for the preparation of monolithic capillary columns. Polystyrene-based monolithic stationary phases were prepared in situ in fused-silica capillaries and simultaneously in vials. The column permeability, electrophoretic and chromatographic behavior were evaluated using pressure-assisted capillary electrochromatography (pCEC), capillary electrochromatography (CEC) and low pressure liquid chromatography (LPLC). With an optimal monolithic material, the largest theoretical plates for preparing the column could be close to 18,000 plates/m for thiourea in the mode of pCEC. Furthermore, the influence of the composition of the porogenic solvents (toluene/isooctane) on the morphology of organic-based monoliths [poly(styrene-divinylbenzene-methacrylic acid)] was systematically studied with mercury intrusion porosimetry and scanning electron microscopy. The monoliths which were prepared with a high content of isooctane had a bigger pore size and better permeability, and hence resulted in a faster separation.

Perphenylcarbamoylated β‐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.

Capillary electrochromatography of peptides and proteins.

This paper reviews recent progress in bioanalysis using capillary electrochromatography (CEC), especially in the field of separation of proteins and peptides. Fundamentals of CEC are briefly discussed. Since most of the recent developments on CEC have focused on column technology, i.e., design of new stationary phases and development of new column configurations, we describe here a variety of column architectures along with their advantages and disadvantages. Newly emerged column technologies in CEC for high speed and high efficiency separation are also discussed. Different analytical platforms of CEC such as pressure-assisted CEC or voltage-assisted micro- high-performance liquid chromatography (HPLC), CEC with different detection techniques, CEC on microchip platforms and multidimensional electrochromatography with their applications in peptide and protein analysis are presented.

Recent applications of capillary electrochromatography.

A review is presented of the most important recent applications of capillary electrochromatography (CEC) for the analysis of acidic, basic, and neutral compounds, of biomolecules, environmental substances, natural products, pharmaceuticals, and chiral compounds. Packed-column CEC (packed-CEC), open-tubular (OT-CEC), as well as pressure-assisted CEC (pseudo-CEC) are hereby considered. Papers published between July 1999 and April 2001 were taken into account. Applications before July 1999 have been reviewed in Electrophoresis 1999, 20, 3027-3065.

Separation and identification of etodolac and its urinary phase I metabolites using capillary electrochromatography and on-line capillary electrochromatography–electrospray ionisation mass spectrometry coupling

Capillary high-performance liquid chromatography (capillary HPLC), pressure-assisted capillary electrochromatography (pCEC) and capillary electrochromatography (CEC) were performed in the same capillary packed with 5 μm octadecylsilica (C 18) as stationary phase. These three separation modes were compared from the viewpoint of peak efficiency and separation selectivity in order to critically evaluate the advantages which CEC may offer compared to capillary HPLC for the solution of practical biomedical problems. The separation of the non-steroidal anti-inflammatory drug etodolac (ET, 1) and its phase I metabolites, 6-hydroxy etodolac (6-OH-ET, 2), 7-hydroxy etodolac (7-OH-ET, 3) and 8-(1′-hydroxyethyl) etodolac (8-OH-ET, 4) was selected as an example. Baseline separation of all compounds was achieved in different modes and conditions. The effect of pure electrophoretic separation mechanism on the overall separation selectivity observed in CEC has been shown. A high electroosmotic flow (EOF) was observed in C 18 packed capillary even at pH 2.5 in various buffers. Furthermore, these separations were coupled on-line with electrospray ionisation mass spectrometry (ESI-MS) and the parent drug and its metabolites were identified in urine. For the coupling of CEC with ESI-MS a laboratory-made electrophoretic device was used in order to overcome some technical disadvantages of commercial instrumentation.

Applications of capillary electrochromatography

Applications performed by capillary electrochromatography (CEC) in all its modes, namely packed column CEC (packed-CEC), open tubular CEC (OT-CEC) and pressure-assisted CEC (pseudo-CEC), and published by June 1999 are reviewed. The review is divided into (i) separation of neutral, acidic and basic analytes with the main goal of evaluating column and system performance, (ii) separation according to field of application and/or chemical class, and (iii) separation of chiral analytes.

Chiral separations in capillary high-performance liquid chromatography and nonaqueous capillary electrochromatography using helically chiral poly(diphenyl-2-pyridylmethyl methacrylate) as chiral stationary phase.

Enantioseparations in nonaqueous capillary electrochromatography (CEC) are reported in this study for the first time, using wide-pore aminopropyl silica gel coated with helically chiral poly(diphenyl-2-pyridylmethyl methacrylate) (PDPM) as chiral stationary phase (CSP). The anodic electroosmotic flow (EOF) in a methanolic solution of ammonium acetate was used for the migration of neutral analytes through the packed bed in the capillaries. Four different techniques, high-performance liquid chromatography (HPLC) in common-size columns, capillary HPLC, pressure-assisted CEC and CEC were compared from the viewpoint of separation parameters. The latter three were performed with the same experimental setup, varying the relative contribution of the pressure-driven and the electrically driven flow to the overall mobility of the analyte. Capillary HPLC offers clear advantages compared to enantioseparations in common-size columns. However, for a given particle size of the packing material, CEC was not obviously advantageous compared to pressure-driven separations.

Applications of high-speed data acquisition for semiconductor device yield analysis, Part I : John M. Charles and Mikkel W. Lantz. Solid St. Technol., 119 (March 1982)

A novel fritting technology was introduced for the fused-silica capillary. The technique involved sintering of stainless steel (SS) particles at the tip of capillary through flame heating. A simple butane gas based welding torch was used for sintering the SS particles. The new fritting technique, flame induced sintering of SS particles (FIS/SSP), was applied for making frits with different inlet diameters (75 μm, 100 μm, 250 μm and 530 μm). The changes in morphologies of SS particles during sintering process were identified by scanning electron microscopy (SEM). Frits with the length of 0.5–1 mm and capillaries with inner diameter about 50–100 μm were fabricated through suitable selection of experimental conditions (size of SS particles and heating mode). The frits prepared by FIS/SSP technique exhibited adequate separation properties and mechanical strength. Columns packed with C18 particles were stable with these frits in a few important chromatographic operations. Frits prepared by FIS/SSP technique was used in three typical separation modes namely, capillary electrochromatography (CEC), p-assisted CEC (p-CEC) and low pressure liquid chromatography (LPLC). Importantly, no bubble formation was noticed with the frit over a period of one week. A good peak symmetry and high efficiency for separation were obtained using pressure-assisted CEC, p-CEC and low pressure-driven separation modes.

Manufacture of chilled iron car-wheels: F.C. Weber. ( Metal. and Chem. Eng., xi, 92.)

A novel fritting technology was introduced for the fused-silica capillary. The technique involved sintering of stainless steel (SS) particles at the tip of capillary through flame heating. A simple butane gas based welding torch was used for sintering the SS particles. The new fritting technique, flame induced sintering of SS particles (FIS/SSP), was applied for making frits with different inlet diameters (75 μm, 100 μm, 250 μm and 530 μm). The changes in morphologies of SS particles during sintering process were identified by scanning electron microscopy (SEM). Frits with the length of 0.5–1 mm and capillaries with inner diameter about 50–100 μm were fabricated through suitable selection of experimental conditions (size of SS particles and heating mode). The frits prepared by FIS/SSP technique exhibited adequate separation properties and mechanical strength. Columns packed with C18 particles were stable with these frits in a few important chromatographic operations. Frits prepared by FIS/SSP technique was used in three typical separation modes namely, capillary electrochromatography (CEC), p-assisted CEC (p-CEC) and low pressure liquid chromatography (LPLC). Importantly, no bubble formation was noticed with the frit over a period of one week. A good peak symmetry and high efficiency for separation were obtained using pressure-assisted CEC, p-CEC and low pressure-driven separation modes.

“Duriron”: a non-corrosive iron alloy: J.R. Pitman. ( Eng. News, lxix, 563.)

A novel fritting technology was introduced for the fused-silica capillary. The technique involved sintering of stainless steel (SS) particles at the tip of capillary through flame heating. A simple butane gas based welding torch was used for sintering the SS particles. The new fritting technique, flame induced sintering of SS particles (FIS/SSP), was applied for making frits with different inlet diameters (75 μm, 100 μm, 250 μm and 530 μm). The changes in morphologies of SS particles during sintering process were identified by scanning electron microscopy (SEM). Frits with the length of 0.5–1 mm and capillaries with inner diameter about 50–100 μm were fabricated through suitable selection of experimental conditions (size of SS particles and heating mode). The frits prepared by FIS/SSP technique exhibited adequate separation properties and mechanical strength. Columns packed with C18 particles were stable with these frits in a few important chromatographic operations. Frits prepared by FIS/SSP technique was used in three typical separation modes namely, capillary electrochromatography (CEC), p-assisted CEC (p-CEC) and low pressure liquid chromatography (LPLC). Importantly, no bubble formation was noticed with the frit over a period of one week. A good peak symmetry and high efficiency for separation were obtained using pressure-assisted CEC, p-CEC and low pressure-driven separation modes.