Capillary Electrochromatography Column Research Articles

Acrylamide-based monoliths as robust stationary phases for capillary electrochromatography

A method is described for the synthesis of rigid, macroporous polymers (monoliths) to be used as stationary phases in capillary electrochromatography (CEC). The procedure reproducibly results in columns with good mechanical and chemical stability. Once the procedure was optimized, it yielded the desired CEC columns in nearly 100% of the cases. The batch-to-batch standard deviation of the migration of the electroosmotic flow (EOF) marker for nine randomly chosen columns was 5%. The polymerization is carried out inside the capillary, an aqueous phase is used as solvent. Monomers based on acrylamides with varying hydrophilicity were used to introduce the interactive moieties together with piperazine diacrylamide as cross-linker and vinylsulfonic acid as provider of the charged, EOF-producing moieties. The pore size of the monoliths was adjusted by adding varying amounts of ammonium sulfate to the reaction mixture. In this manner, the average pore size of a given monolith could be reproducibly adjusted to values ranging from 50 nm to 1.3 μm. The procedure was optimized for four particular types of monoliths, which differed in hydrophobicity. The latter was adjusted by introducing suitable co-monomers, such as alkyl chain-bearing molecules, into the monolithic structure. Attempts to systematically investigate the chromatographic behavior of the monolithic stationary phases were made, using a model mixture of aromatic compounds as sample. The standard deviations for the run-to-run reproducibility of the retention times for unretained and retained analytes were <1.5%. Flat Van Deemter curves were measured even at elevated flow-rates (2 mm/s). Plate heights between 10 and 15 μm were measured in this range. The retention order was taken as the principal indication for the chromatographic mode. The separation was found to be governed neither by pure reversed-phase nor by pure normal-phase chromatography, even on monoliths, where large amounts of C 6 ligands had been introduced.

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.

Column technology for capillary electrochromatography

Column technologies for capillary electrochromatography (CEC) are reviewed. To achieve high efficiency, the inner diameters of open-tubular and packed columns should be less than 25 and 200 μm, respectively. To obtain acceptable separation speed under typical CEC conditions (e.g. 30 kV, 1 mm s −1 electroosmotic flow velocity, and 2–4×10 −8 m 2 V −1 s −1 electroosmotic mobility) the column lengths for open-tubular and packed columns should be less than 120 and 60 cm, respectively. Capillary CEC columns are generally classified into three types: packed, open-tubular, and continuous-bed or monolithic. The various column preparation procedures and the advantages and disadvantages of each column type are discussed in detail.

Life-time studies with capillary electrochromatography columns operated under different conditions

A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k cec, peak symmetry coefficients, λ sym, and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 μm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 μm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 – 8.0, similar to those employed to study the retention behaviour of peptides that we have previously reported. The relative standard deviation (RSD) of the retention factors ( k cec values) of these n-alkylbenzenes, acquired with an eluent of (25 m M Tris–HCl, pH 8.0,)–acetonitrile (1:4, v/v), when the CEC capillary columns were used for the first time (virgin values), were 4% (based on data acquired with 4 CEC capillary columns) for the n-octyl bonded silica capillary columns, and 6% (based on 8 columns) for n-octadecyl bonded silica capillary columns. The RSD values of the k cec values of the n-alkylbenzenes for one set of replicates ( n=6) with one CEC capillary column was <0.5%. The theoretical plate numbers, N, for the virgin CEC capillary columns were ca. 60 000, whilst the observed N values for all new CEC capillary columns were ≥40 000 for n-octyl bonded silica capillary columns and ≥50 000 for n-octadecyl bonded silica capillary columns. The peak symmetry coefficients, λ sym, of the n-alkylbenzenes for virgin CEC capillary columns and for CEC capillary columns used for more than 1000 injections were always in the range 0.95–1.05. The experimental results clearly document that the life-time performance of the CEC capillary columns depends on the eluent composition, as well as the nature of the analytes to which the CEC capillary columns are exposed.

Chiral monolithic columns for enantioselective capillary electrochromatography prepared by copolymerization of a monomer with quinidine functionality. 1. Optimization of polymerization conditions, porous properties, and chemistry of the stationary phase.

Monolithic columns for chiral capillary electrochromatography have been prepared within the confines of untreated fused-silica capillaries in a single step by a simple copolymerization of mixtures of O-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine , ethylene dimethacrylate, and glycidyl methacrylate or 2-hydroxyethyl methacrylate in the presence of mixture of cyclohexanol and 1-dodecanol as a porogenic solvent. The porous properties of the monolithic columns can easily be controlled through changes in the composition of the binary porogenic solvent. Although both thermal- and UV light-initiated polymerizations afford useful capillary columns, monoliths prepared using the former approach exhibit better chromatographic properties. The ability to control pore size independently of the polymerization mixture composition enables the preparation of monoliths with varying percentages of the chiral monomer and cross-linker, as well as the optimization of their separation properties. Very good separations of model racemate (R,S)-N-3,5-dinitrobenzoylleucine were achieved using an optimized monolithic CEC column, with high efficiencies of up to 74000 plates/m for the retained peaks.

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.

Separation of rhubarb anthraquinones by capillary electrochromatography

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.

A simple procedure for the preparation of fritless columns by entrapping conventional high performance liquid chromatography sorbents.

A rapid and direct method for immobilizing conventional high performance liquid chromatography (HPLC) packing material inside fritless capillaries has been developed. Due to the simple composition of the entrapment matrix (tetraethoxysilane, alkyltriethoxysilane, ethanol and water), straightforward manufacturing procedure and modest equipment requirement, the method can readily be transferred to any laboratory and easily automated. The entrapment procedure has minimal influence on the structure and chromatographic properties of the original reverse-phase sorbent. Various immobilization solutions have been tested, and a comparison between columns entrapped with different immobilization mixtures and conventional packed capillaries is presented. High efficiency separations were obtained using tert-butyl-triethoxysilane entrapped columns in both capillary electrochromatography (reduced plate heights of 1.1-1.4 were measured) and microliquid chromatography (reduced plate heights of 2.2-2.6 were observed) formats. Elimination of frits, stabilization of the packed bed and on-the-fly customization of column length render mechanically robust columns that are remarkably stable over time, from which manufacturing imperfections can be removed easily.

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.

Comparison of separation behavior of benzodiazepines in packed capillary electrochromatography and open-tubular capillary electrochromatography

Packed column capillary electrochromatography (CEC), open-tubular CEC and microcolumn liquid chromatography (LC) using a cholesteryl silica bonded phase have been studied to compare the retention behavior for benzodiazepines. It has been found that packed column CEC gives better resolution, faster analysis time than microcolumn LC for benzodiazepines maintaining similar selectivity except for some solutes which are charged species under the separation conditions. However, open-tubular CEC gave different selectivities to a larger extent for charged benzodiazepines from that which should be produced by the chromatographic properties of the cholesteryl silica phase. Charged species migration times are mainly influenced by electrophoretic mobility rather than the chromatographic interactions.

Steroid profiles determined by capillary electrochromatography, laser-induced fluorescence detection and electrospray–mass spectrometry

Macroporous, monolithic capillary electrochromatography (CEC) columns, featuring a hydrophobic stationary phase, have been applied to the separations of steroids with good column efficiency. Using isocratic and gradient elution runs, mixtures of neutral or conjugated steroids could be resolved. While dansylated ketosteroids were detectable through laser-induced fluorescence at attomole levels, the CEC columns coupled to electrospray–ion-trap mass spectrometry featured femtomole detection limits.

Characteristics of particle-loaded monolithic sol–gel columns for capillary electrochromatography: I. Structural, electrical and band-broadening properties

Particle-loaded (3 μm, C 18) monolithic sol–gel columns have been prepared and selected characteristics measured. They have a surprisingly high permeability, allowing their operation in the microLC mode at pressures as low as 69 kPa where their efficiency is about 50 000 plates per meter and the CEC mode where efficiency is at least 106 000 plates per meter. These columns can withstand over 13.8 MPa pressure without compression or movement within the 75 μm capillary. Field strengths in the packed segments are approximately 50% greater than those in the open segments, due to the higher resistivity of the particle-laden regions. There is a relatively rapid loss of efficiency with increasing linear velocity in both the CEC and microLC modes, which may be due to a tortuosity effect in the inter- and intra-particulate voids. Chromatographic behavior is characteristic of conventional C 18 particles, indicating that analytes have significant access to the surface within the pores of the immobilized bonded phase.

Comparison of different packing methods for capillary electrochromatography columns

A study was carried out in which 50 μm I.D. fused-silica capillaries were packed with 3 μm octadecylsilane bonded silica, from the same batch, by four methods; liquid slurry and carbon dioxide supercritical carrier, each with and without the use of an ultrasonic probe. A neutral test mixture was analysed by capillary column in reversed-phase mode, and the reproducibility of the electroosmotic flow and of migration time, column efficiency and retention factors, was determined. Initially results suggested that there was no significant difference between properties of columns packed by different methods, and a more thorough statistical evaluation confirmed this; differences observed in the column performance were attributed to random variations between replicate columns, and not between packing methods. However, the variation was least when applying the ultrasonication during liquid slurry.

Control of dispersion in capillary electrochromatography coupled to UV and mass spectrometric detection

Capillary electrochromatography (CEC), along with other miniaturised chromatography techniques, such as capillary LC, offers the most benefit when efficiently coupled to mass spectrometric (MS) detectors. In conventional one-piece CEC columns, dispersion in the open connecting tube between the packed column and MS source reduces chromatographic performance to unacceptable levels. This paper examines the effect on dispersion of various column-tube arrangements and offers suggestions as to the most practical way of connecting CEC–UV–MS. Comparisons of theoretical and measured values for these different arrangements are shown.

Laser-induced dispersed fluorescence detection of polycyclic aromatic compounds in soil extracts separated by capillary electrochromatography

Polycyclic aromatic hydrocarbons (PAHs) and nitrogen containing aromatic compounds (NCACs) are characterized in soil extracts and laboratory standards by capillary electrochromatography (CEC) with laser-induced dispersed fluorescence (LIDF) detection using a liquid-nitrogen cooled charge-coupled device detector. The LIDF detection technique provides information on compound identity and, when coupled with the high separation efficiencies of the CEC technique, proves useful in the analysis of complex mixtures. Differences in fluorescence spectra also provide a means of identifying co-eluting compounds by using deconvolution algorithms. Detection limits range from 0.5 to 96×10 −10 M for selected PAHs and 0.9–3.7×10 −10 M for selected NCACs. Soil extracts are also injected onto the CEC column to evaluate chromatographic method performance with respect to complex samples and the ability to withstand exposure to environmental samples.

In-line coupling capillary electrochromatography with amperometric detection for analysis of explosive compounds.

Amperometric detection at a bare gold electrode has been in-line coupled with capillary electrochromatography (CEC) for analysis of nitroaromatic and nitroamine explosives in contaminated soils and ground water. The CEC column packed with 3 microm C18 particles performed best using a mobile phase containing 70-80% methanol, 30 or 20% water, 5 mM sodium dodecyl sulfate (SDS) and 10mM 2-(N-morpholino)ethanesulfonic acid (MES). In contrast, the separation column packed with 1.5 km C18 particles exhibited the best separation when only 30% methanol was added to a mobile phase containing 70% water, 7 mM SDS, and 10 mM MES. The detection, based on electrochemical reduction of the explosives (-0.7 or -1 V vs. Ag/AgCl, depending upon the level of methanol in the mobile phase), was compatible with such mobile phases. The detection limits for 13 explosives ranged from 100 to 200 ppb, i.e., about twofold better than those obtained with electrokinetic chromatography (EKC)/amperometric detection. From an operational viewpoint, exhaustive column conditioning was a prerequisite and care should be taken to prevent bubble formation and current breakdown during the course of separation. The CEC column equipped with amperometric detection successfully measured explosives in ground water and extracts prepared from contaminated soils and the results obtained agreed well with those of the U.S. Environmental protection Agency (EPA) method.

Electric Resistance Variation of Packing Materials in Capillary Electrochromatography by Using Fluorinated-Bonded Silica and Octadecylsilane Phases

The linear relationship between electric resistance of the packed column for capillary electrochromatography and applied voltage was observed in several different effluents and packing materials (fluorinated-bonded silica and octadecylsilane). These linear relationships had different slope values which depended on the charges on the surface of the stationary phase. If there are the negative charges on the stationary phase during the application of the electric field, the slope was negative value as observed in the fluorinated-bonded silica packed column. On the other hand, the slope was positive value in the fluorinated-bonded silica packed column using 0.1% tetrabutyammonium chloride as an effluent. The inclination of the linear relationship between the electric resistance and applied voltage also depended on the kind of the effluent, the concentration of effluent and the ratio of the organic modifier used. In the case of octadecylsilane packed column, the electric resistance of the column did not depend on the applied voltage. The relationship between the electric resistance and the electroosmotic mobility was also studied.

Pseudo-electrokinetic packing of high efficiency columns for capillary electrochromatography

An improved and easy electrokinetic packing procedure is presented for the production of stable capillary columns suitable for capillary electrochromatography (CEC). In pseudo-electrokinetic packing a high electric field is used in conjunction with a hydrodynamic flow. The packing of silica-based reversed-phase columns can be achieved with basic, commercially available capillary electrophoresis (CE) equipment in approximately 15 min. The procedure is robust and a high success rate is achieved. No steps which might damage the stationary phase are involved and only a minimum amount of packing material is required. Columns packed according to the developed procedure are operated at high electric field strengths during the CEC separation, without the application of a stabilising pressure. Columns are stable for at least hundred runs and were tested using mixtures of polycyclic aromatic hydrocarbons and positively charged drugs. Separations were performed in a relatively high conducting ammonium acetate buffer, with efficiencies of up to 283 000 plates/m.

Preparation of various silica-based columns for capillary electrochromatography by in-column derivatization

Chemically bonded silica gels were prepared in a capillary by pumping an ethanolic solution of a silylating reagent, such as octadecyltrimethoxysilane, 3-aminopropyltrimethoxysilane and dimethyloctadecyltrimethoxysilylpropylammonium chloride into a heated capillary packed with bare silica particles. The silylation reactions were completed in a short time and thus-prepared columns showed high column efficiency and high reproducibility. Examples are shown for the separation of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of aldopentoses on a 3-aminopropylated silica column and benzoate homologues as well as PMP derivatives of the component monosaccharides of glycoproteins on an octadecylammonium column. Since the presence of frit filters hampers high efficiency separation, an attempt was made to fix the bed of modified silica gel particles to the capillary inner wall by a cross-linking technique. The results indicated that this technique is promising.

Analysis of channel-geometry effects on separation efficiency in rectangular-capillary electrochromatography columns

A three-dimensional random walk model was developed to evaluate the impact of column geometry on separation efficiency in chromatography systems driven by electroosmotic flow. Contributions of injection plug length, cross-sectional area of channels, and aspect ratio of rectangular channels were examined in these simulation studies. Sample plug length had no impact on efficiency until it exceeded roughly 0.4% of the channel length. Plate height increased rapidly with increasing k′ as expected, almost doubling in going from k′=0.25 to 0.35. Channel geometry also had a major effect on efficiency. Plate height increased sharply in rectangular channel columns until the channel aspect ratio reached 4–8. But the effect of channel depth was even more dramatic. Minimum plate height ( H min) was roughly half that of the channel depth in ideal cases. H min in a 10×2 μm channel was at 1.6 mm s −1. Rectangular channels comparable to those obtained by microfabrication are equivalent to packed column capillary electrochromatography columns in all cases.