Characterization of open tubular capillary electrochromatography columns for the analysis of synthetic peptides using isocratic conditions.

In this paper, we report on investigations related to the performance characteristics of two different types of etched chemically (n-octadecyl- and cholesterol-) modified capillaries in the open tubular format of capillary electrochromatography (CEC) for the analysis of synthetic peptides. The results confirm that the nature of the surface chemistry used to modify the capillary wall and type of chemically bonded group employed can affect the selectivity as well as the resolution of peptide samples. The results are consistent with the participation of selective peptide interactions with the bonded phase, although other factors, such as the morphology of the capillary wall surfaces, appear to be also involved. Moreover, several surprising observations related to peptide-specific multi-zoning effects have been observed. Additional experimental variables that can also be utilized to affect the retention of peptides in this approach to OTCEC include the type and percentage of organic solvent modifier employed in the eluent and the pH of the buffer system. To evaluate the reproducibility of different batches of the n-octadecyl- and cholesterol-modified capillaries and the stability of the chemically modified surface, the OTCEC selectivity and peak shape behavior of two small basic molecules (serotonin and tryptamine) and two proteins (turkey and chicken lysozyme) were also investigated. Finally, the use of the "bubble" cell technology for creating the detector window has been shown to provide significantly higher detection sensitivity with peptides, as compared with the conventional capillary format.

Characterization and applications of etched chemically modified capillaries for open-tubular capillary electrochromatography.

In this article, the effects of the stationary phase, buffer pH, organic modifier type, organic modifier composition, applied voltage, and temperature on the migration of several synthetic peptides in etched chemically modified open-tubular capillaries are discussed. With these solutes, migration is due to two effects: electrophoretic mobility and solute/bonded phase interactions. In addition, relative migration rates are evaluated for the peptide samples as a function of these experimental variables in order to determine which parameters might be useful for optimizing separations in open-tubular capillary electrochromatography (OTCEC). Some examples of synthetic peptide separations are presented where the sample contains a major component and several minor species, demonstrating how the resolution of these mixtures can be affected by the appropriate choice of experimental variables.

Electrochromatographic characterization of etched chemically-modified capillaries with small synthetic peptides.

In this investigation, various capillary electrochromatographic (CEC) experiments have been employed to characterize the properties of etched, chemically-modified surfaces of open tubular capillary columns with peptides as solute probes and under conditions of variable voltage, temperature and solvent composition. The separation performance of etched capillaries with either n-octadecyl or liquid crystal moieties derived from a cholesterol phase bonded to the surface were compared. With the liquid crystal bonded species, interesting and significantly different variations in retention behavior of peptides are obtained compared to those observed with the corresponding n-octadecyl modified surfaces by changes in temperature, solvent composition and field strength. These peptide separations illustrate the usefulness of this CEC approach for practical applications, where both the retention characteristics of the charged analytes as well as the selectivity differences due to the surface properties of the etched chemically-modified surfaces of open tubular capillary columns can be rationally modulated. As in HPLC, appropriate choice of CEC experimental variables, including the chemical properties of the immobilized ligand(s), represents a powerful tool for optimizing resolution.

Open tubular capillary electrokinetic chromatography in etched fused-silica tubes.

This review describes an open tubular approach to capillary electrochromatography (OTCEC) that first etches the inner surface of the fused-silica tube using ammonium hydrogen diflouride. This process can increase the inner surface area significantly. The new surface is then chemically modified to attach a bonded stationary phase using a silanization/hydrosilation reaction process. The surfaces are characterized spectroscopically by diffuse reflectance infrared Fourier transform and by electroosmotic flow measurements. Applications of OTCEC columns with C18, diol and chiral stationary phases are described.

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

Packed column capillary electrochromatography (CEC), open-tubular CEC and microcolum 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.

Screening method for determining the presence of N-nitrosodiethanolamine in cosmetics by open-tubular capillary electrochromatography.

The presence of the carcinogenic N-nitrosodiethanolamine (NDELA; CAS No. 1116-54-7) in cosmetic samples was determined using an etched, C18 modified capillary in the open-tubular capillary electrochromatography technique. A very simple extraction procedure leads to a sample matrix free from interferences. The calibration curve was created using UV detection at 214 nm. The detector response was linear in the range of 5-120 ppm total amount injected. Minimum detection limits (1 ppm NDELA injected on capillary) are suitable for screening a large number of cosmetic samples. Diethanolamine and triethanolamine precursors of nitrosamines are not detected at the wavelength used. Cosmetic samples were analyzed unspiked and after addition of 60 ppm of NDELA. In spiked samples recoveries varied from 94% (hand and body lotion) to 55% (lipstick sample). NDELA was found in unspiked samples of old (5-15 years old) cosmetics at concentrations of 14.0 ppm and 35.0 ppm.

Fullerenes separation with monomeric type C30 stationary phase in high-performance liquid chromatography.

The temperature effect on the separation of fullerenes in LC was examined using monomeric type C30, C18 and C8 alkyl bonded stationary phases. It appears that the C30 phase exhibits superior separation ability for fullerenes. It is observed that the maximum retention temperature of fullerenes on the C30 phase is around 20 degrees C. A strong correlation between the changes in NMR spectra and the retention behavior of the solutes was found. The interpretation of the retention behavior of fullerenes on the alkyl bonded stationary phases, including the behavior in subambient temperature, is discussed using the information obtained by CP-MAS solid-state NMR spectroscopy and LC.

Chiral separations by open tubular capillary electrokinetic chromatography.

The inner walls of 50 microns fused-silica capillaries are etched by ammonium hydrogendifluoride and then modified by the silanization/hydrosilation method with a chiral selector. Three different types of selectors were evaluated: lactone, beta-cyclodextrin and naphthylethylamine. Each of the bonded chiral stationary phases provided at least partial separation for one type of racemic solute. These results confirm that bonded organic moieties on the etched inner wall of a capillary can provided sufficient solute-bonded phase interactions to influence the retention of molecules driven through a capillary by electroosmosis or a combination of electroosmosis and electrophoretic mobility.

Protein and peptide separations on high surface area capillaries.

A 50 microm capillary that has been etched with ammonium hydrogen difluoride is evaluated as a separation medium for capillary electrochromatography. For a tryptic digest of transferrin, the etched capillary gave better resolution (more peaks in the overall peptide map) and longer retention than separations done under identical experimental conditions on an unetched fused-silica capillary. Resolution on the etched capillary was improved by lowering the voltage from 300 to 267 V/cm. A four-component protein sample also resulted in longer retention on an echted capillary than on an unetched fused-silica capillary that were both coated with Polybrene. After correction for differences in electroosmotic flow between the two capillaries, the calculated electrophoretic mobilities for all four proteins were lower on the etched capillary than on the unetched fused-silica capillary. The results of both the tryptic digest and protein experiments strongly indicate the presence of chromatographic effects on the etched capillary that contribute to the increased retention and improved resolution with respect to the unetched fused-silica capillary.

Open tubular capillary electrochromatography in etched, chemically modified 20 microns I.D. capillaries.

Fused silica capillaries with an I.D. of 20 microns are etched and then chemically modified by the silanization/hydrosilation method to attach an octadecyl moiety for use in electrokinetic chromatography. The etched capillaries after chemical modification are shown to have an anodic electroosmotic flow below pH 4.5. In comparison to bare 20 microns capillaries and unetched but chemically modified 20 microns capillaries, the etched C18 fused silica tubes show better separation of mixtures of lysozymes and cytochrome c's under identical conditions of buffer, pH and applied voltage. It was also demonstrated that this open tubular approach to capillary electrochromatography was amenable to a number of different types of basic compounds ranging in size from typical small amines to biomolecules. As expected, pH is an important variable that must be controlled in order to obtain an optimized separation. Reproducibility studies verify the stability of the silicon-carbon linkage produced in this modification method so that column lifetimes of at least 300 injections can be expected.

Separation of benzodiazepines using cholesterol-modified fused-silica capillaries in capillary electrochromatography.

This paper describes the use of cholesteryl-10-undecenoate as a capillary column modifier for capillary electrochromatography. This bonded phase has shown an entirely different selectivity as compared to octadecyl silica (ODS) phases in reversed-phase liquid chromatography, specifically in the analysis of the benzodiazepines. This difference serves as the basis for the continuing study as well as the present focus on electrochromatography as a relatively new separation technique. To significantly increase the surface area, etching of the inner wall of a 75-micron capillary is performed using ammonium hydrogen difluoride and is then subsequently modified via a silation reaction with triethoxysilane and subjected to hydrosilation with cholesteryl-10-undecenoate to form the bonded phase. The performance of this capillary is compared with unetched cholesterol-modified and bare, fused-silica capillaries. Efficient resolution was noted for the etched capillary showing the effectiveness of the etching process as well as the selective property of the bonded phase.

Separation of proteins and peptides by capillary electrochromatography in diol- and octadecyl-modified etched capillaries.

This study involves the evaluation of a capillary electrochromatography method based on etching the inner walls of a fused-silica tube, which is subsequently modified by a silanization/hydrosilation reaction scheme. Two different organic moieties, octadecyl and diol, are attached to the etched capillary wall. The performance of these two columns is compared to a bare capillary using peptide (angiotensins) and protein samples. It is concluded that the etching process increases the surface area of the inner wall sufficiently to induce solute-bonded phase interactions for the capillaries modified with the octadecyl and diol moieties. The separation capabilities of the two modified capillaries are not the same, presumably due to differences in the chemical properties of the two ligands. When compared to a bare capillary where separation is due only to electrophoretic mobility effects, the bonded etched capillaries also exhibit significant differences in separation factors for the same solutes under identical experimental conditions.

A new open-tubular approach to capillary electrochromatography.

While most capillary electrochromatography is based on the use of packing materials similar to those for HPLC, this method creates a new surface on the inner wall of the fused-silica tubing that is then modified with various organic moieties. In this format there is no need for frits, and bubble formation is not a serious problem. With 50-microns-i.d. capillaries, molecules are separated by a combination of differences in electrophoretic mobility and solute/bonded phase interactions. A number of examples are presented, which include tetracyclines, peptides, and proteins. Peaks for all compounds, even basic ones, exhibit both good efficiency and symmetry. The reproducibility of migration times is excellent even after extended use of the column.

Column technology in capillary electrophoresis and capillary electrochromatography.

A review of column technology in capillary electrophoresis (CE) including wall modification processes and open tubular as well as packed column formats in capillary electrochromatography (CEC) are presented. There are many approaches which can be used to solve separation problems which provide higher efficiency and/or shorter analysis times in comparison to other chromatographic techniques. However, both CE and CEC are still relatively undeveloped in comparison to a more mature method such as high performance liquid chromatography (HPLC) and improvement in column technology is changing rapidly.

Electrochromatography in chemically modified etched fused-silica capillaries.

A new method of electrochromatography is described in which a 50-microns capillary is etched with ammonium hydrogen difluoride, followed by modification of the new surface via a silation reaction with triethoxysilane to produce a hydride intermediate, and then subsequently subjected to hydrosilation using 1-octadecene in the presence of a platinum complex catalyst. The C18 bonded phase is then compared with bare capillaries, etched bare capillaries and the hydride etched capillary to determine if any solute-bonded phase interactions are present. With bradykinin as a test solute, peak efficiencies are quite similar for all capillaries without C18 but become noticeably broader when the organic moiety is attached to the etched capillary wall. A test mixture of peptides and proteins displays shorter retention for some of the components when methanol is added to the mobile phase which is typical of reversed-phase behavior. The same result is also obtained when a mixture of tetracyclines is separated on the C18 capillary with and without methanol as part of the mobile phase. The reproducibility of retention times for two proteins is +/- 1.5%. A few results for several neutral compounds indicate small but measurable k' values.

Separation of tetracyclines by high-performance capillary electrophoresis and capillary electrochromatography.

Separations of various tetracycline mixtures by high-performance capillary electrophoresis (HPCE) and a new form of electrochromatography (CEC) are compared. The new CEC method involves etching the inner wall of the capillary surface with an appropriate reagent (ammonium dihydrogen fluoride) in order to produce a significant increase in surface area. The etched surface is then modified by a silation/hydrosilation reaction sequence to first produce a hydride intermediate which is then further reacted to attach a C18 moiety. The bare and hydride capillaries are tested under HPCE conditions while the C18 capillary functions in the CEC mode. The effects of pH and the presence of an organic modifier (methanol) are also studied. Detection limits ( < 10 micrograms/ml) are comparable to previous HPLC and HPCE results. Resolutions for mixtures which simulate real analytical problems are equal to or better than those reported for separations on polymeric and diol columns by HPLC and in earlier studies by HPCE and MECC.

Improvement of Gas Chromatographic Performance of Cyclic Siloxane Bonded Phases by Incorporation of n-Alkyl Groups.

Cyclic siloxane-based silica columns of high retention capacity for light hydrocarbons were prepared by incorporation of octyl and octadecyl groups on the siloxane skeleton. Spectroscopic studies revealed the presence of the alkyl groups on the silica surface, which results in high surface coverage of the packing materials. The retention characteristics of the columns are significantly higher than those of cyclic siloxane phases due to their high surface coverage and better solute-stationary phase interactions. In agreement with previous data for micropacked columns, the H vs u curves, on passing through the optimum region, remained substantially constant at high linear gas velocities, indicating their usefulness for fast analysis of light hydrocarbons at high flow rates without any appreciable loss in column performance. Presumably due to greater availability of the bonded alkyl groups for interactions, a 10-component mixture of C(1)-C(4) saturated and unsaturated hydrocarbons was sufficiently separated on the hexamethylcyclotrisiloxane-octadecylsilyl phase compared to the conventional octadecyldimethylsilyl phase, irrespective of the high carbon content of the latter.