Reversed-phase Capillary Electrochromatography Research Articles

Analysis of Polycyclic Aromatic Hydrocarbons by Reversed Phase Capillary Electrochromatography

Reversed phase capillary electrochromatography (RP-CEC) was used to achieve the rapid analysis of six polycyclic aromatic hydrocarbons (PAHs). Capillary column packed with 5 μm ODS stationary phase in the 10 cm packed length (31 cm total length) and UV detector was used. The effect of the separation voltage, organic modifier on the electroosmotic flow (EOF) and column performance was investigated. Baseline separation could be reached in the 90% acetonitrile content and 10kV-separation voltage, and theoretical plate numbers per meter for six PAHs were all above 70,000. The total analytical time was less than 4 min with RSD ≤0.60% (in retention factor) and 9.83% (in peak area) under the optimized experimental conditions. When the solute strength of the sample solution was less than the elution strength of the mobile phase, on-column concentration of the analytes could be occurred.

Effects of pore flow on the separation efficiency in capillary electrochromatography with porous particles.

The effect of pore flow on the separation efficiency of capillary electrochromatography (CEC) has been studied using columns packed with particles with different pore sizes. A previously developed model was used to predict the (relative) pore flow velocity in these columns under various experimental conditions. Equations are derived describing the effect of pore flow on peak broadening in CEC. The theory has been compared with practice in the reversed-phase CEC separation of various polyaromatic hydrocarbons. It is shown, by theory and experimentally, that the mass-transfer resistance contribution to peak dispersion can be effectively eliminated when using porous particles with a high (> or =50 nm) average pore diameter. Moreover, at high pore-to-interstitial flow ratios the flow inhomogeneity contribution (the A term in the plate height equation) is also shown to decrease. Under optimal conditions, a reduced plate height of 0.3 for the nonretained compound could be obtained. It is argued that fully perfusive porous particles can be a more efficient separation medium in CEC than nonporous particles.

Separation of 4-dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine derivatives of carbonyl compounds by reversed-phase capillary electrochromatography.

4-Dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine (DMNTH) is a novel derivatizing reagent specially designed for the determination of carbonyl compounds. In this work, we describe the separation of DMNTH-derivatized carbonyl compounds by reversed-phase capillary electrochromatography (CEC). After systematic investigations of the effects of experimental conditions viz. pH and concentration of buffer, type of stationary phase, injection volume of sample, organic modifier, and temperature, optimal conditions were found. The sample compounds, which were separated with gradient high performance liquid chromatography (HPLC), were separated by CEC under isocratic elution due to the high efficiency. Comparisons of separations by CEC and micellar electrokinetic chromatography (MEKC) were made.

Reversed-phase capillary electrochromatography of aloins and related constituents of aloe

The separation of some anthrone-C-glucosyl constituents of aloe was carried out by isocratic capillary electrochromatography using a 50 μm l.D. column packed with 3 μm ODS silica gel. In comparison to HPLC mode, CEC resolution of aloins was obtained in a very short time with a high selectivity factor. The influence of mobile phase parameters (pH and ionic strength of the buffer, acetonitrile amount in the eluent etc.) on the retention of aloins was examined in order to improve the separation conditions and to study the retention mechanism. Efficiency was found to increase with decreasing the EOF up to a reduced plate height of about 2 at a reduced linear velocity of 0.3 mm s−1. This effect was ascribed to the fact that, under the buffer conditions used (pH 8), aloins are partially anionically charged, with a consequent low mass transfer between the mobile and stationary phases. However, resolution remains almost the same in a wide pH range permitting baseline separations of aloins in short analysis times. Since aloins are known to undergo fast degradation reaction dependent on pH, a kinetic study was also carried out. The method was extended to the analysis of aloins in food and cosmetic products.

Enantioselective reversed-phase and non-aqueous capillary electrochromatography using a teicoplanin chiral stationary phase

Enantiomeric separation of chiral pharmaceuticals is carried out in aqueous and non-aqueous packed capillary electrochromatography (CEC) using a teicoplanin chiral stationary phase (CSP). Capillaries were slurry packed with 5 μm 100-Å porous silica particles modified with teicoplanin and initially evaluated using a non-aqueous polar organic mode system suitability test for the separation of metoprolol enantiomers ( R s=2.3 and 53 000 plates m −1). A number of pharmaceutical drugs were subsequently screened with enantioselectivity obtained for 25 racemic solutes including examples of neutral, acidic and basic molecules such as coumachlor ( R s=3.0 and 86 000 plates m −1) and alprenolol ( R s=3.3 and 135 000 plates m −1) in reversed-phase and polar organic mode, respectively. A statistical experimental design was used to investigate the effects of non-aqueous polar organic mobile phase parameters on the CEC electroosmotic flow, resolution and peak efficiency for two model solutes. Results primarily indicated that higher efficiency and resolution values could be attained at higher methanol contents which is similar to findings obtained on this phase in liquid chromatography.

Separation of basic solutes by reversed-phase capillary electrochromatography

The separation of basic solutes at low pH by capillary electrochromatography (CEC) has been investigated. The feasibility of separation of basic solutes by CEC was demonstrated. Influence of operational parameters, solvent composition, pH, temperature on retention and selectivity of the separation of a mixture of basic, neutral and acidic drug standards has been investigated. The observed elution behavior has been modeled to account for both chromatographic retention and differential electrophoretic mobility of the solutes. This model was verified experimentally. It is demonstrated in this work that the elution window of solutes in reversed-phase CEC is expanded to range from −1 to ∞.

High temperature and temperature programming in capillary electrochromatography

In electrochromatography, solvent electrophoretic mobility and solute partitioning are temperature dependent processes. If temperature variations are controlled, solute selectivity and analysis times can be tailored. In this study the feasibility of temperature programming in capillary electrochromatography (CEC) was demonstrated using a reversed-phase CEC mode. The outcome of programmed separations was compared with isothermal, isocratic and isorheic (constant flow) separations. The combined effects of column temperature and mobile phase flow-rate changes during the separation run, resulted in up to a 50% reduction in the separation run time, without adversely affecting the quality of separation. For capillary electrochromatography, temperature programming may be a valuable alternative to solvent programming modes because of the great technical difficulties associated with carrying out solvent gradient elution.

Immobilisation and evaluation of a vancomycin chiral stationary phase for capillary electrochromatography

The macrocyclic antibiotic, vancomycin, is covalently bonded to LiChrospher ® diol silica packed columns and evaluated in capillary electrochromatography (CEC) both in the reversed-phase and polar organic mode. Initially, capillaries were packed with 5 μm LiChrospher ® 100 Å diol silica and evaluated in CEC with a reversed-phase biphenyl-pyrene achiral test resulting in resolution and efficiency values of ca. 2.5 and 100 000 plates meter −1, respectively. Repeatability for this test (resolution and efficiency) was also examined and found to be acceptable for both run-to-run ( n=5, 0.74% and 1.5%) and column-to-column ( n=5, 3.4% and 9.0%), respectively. Similar results were obtained when the 10 μm LiChrospher ® 1000 Å diol silica was examined with the exception of efficiency, where a reduced plate height value of four times lower was obtained compared to the 100 Å material. A simple three step in-situ vancomycin immobilisation procedure was subsequently carried out on these packed diol columns. Selectivity was obtained for thalidomide enantiomers on this vancomycin chiral stationary phase in reversed-phase CEC with resolution and efficiency values of ca. 2.5 and 80 000 plates meter −1, with acceptable repeatability ( n=8) 0.9% and 3.0%, respectively. Selectivity was also obtained for thalidomide enantiomers on this phase in the polar organic mode with resolution and efficiency values of ca. 2.5 and 120 000 plates meter −1, with acceptable repeatability ( n=7) 0.9% and 2.0%, respectively. It was possible to deduce from a chemometric design carried out for evaluating the mobile phase component effects that organic modifier ratio, MeOH/MeCN, played a significant role in controlling both resolution and efficiency. It was also possible to separate a number of basic analytes including four β-adrenergic blocking agents in the polar organic mode albeit with lower resolution and efficiency values, ca. 1.5 and 45 000 plates meter −1, respectively.

Influence of mobile phase composition on electroosmotic flow velocity, solute retention and column efficiency in open-tubular reversed-phase capillary electrochromatography

The effects of some experimental parameters, such as the volume fraction and type of organic modifier in the mobile phase, and the concentration, type and pH of the buffer on the electroosmotic flow velocity, the retention behavior of test solutes, and the column efficiency have been investigated in capillary electrochromatography (CEC) using an open-tubular column of 9.60 μm I.D. with a porous silica layer chemically modified with C 18 as stationary phase. The retention of a group of polycyclic aromatic hydrocarbons (PAHs) used as a test mixture varied significantly by changing the organic modifier content in the hydroorganic mobile phase according to the reversed-phase-like selectivity of the stationary phase. In addition, an increase in the percentage of organic modifier resulted in a slight increase in the linear velocity of the EOF. On the other hand, when the phosphate buffer concentration was increased over the range 1–50 m M, the electroosmotic mobility fell dramatically, the retention of the solutes decreased steadily, and the plate height showed a significant increase. The results obtained with phosphate, trishydroxymethylaminomethane or 2-morpholinoethanesulfonic acid as buffers were similar when pH remained constant. Optimization in CEC was essential to achieve further enhancement of separation performance, because the analysis time and separation resolution are essentially affected when varying operating parameters. Separations of seven PAHs with more than 100 000 plates are presented within 4 min analysis time.

Capillary Electrochromatography with Novel Stationary Phases. 3. Retention Behavior of Small and Large Nucleic Acids on Octadecyl-Sulfonated-Silica

In this investigation, the potentials of porous and nonporous octadecyl-sulfonated-silica (ODSS) microparticles were demonstrated in the capillary electrochromatography (CEC) of small (e.g., nucleotides and dinucleotides) and large (e.g., transfer ribonucleic acids (t-RNAs)) nucleic acids. The ODSS stationary phase comprised two layers: a hydrophilic sulfonated (permanently charged) sublayer and an octadecyl top layer. While the sublayer is to provide a relatively strong electroosmotic flow, the octadecyl top layer is to ensure the retentivity and selectivity required for the separation of the analytes. Mono-, di-, and triphosphate nucleotides were best separated when a small amount of tetrabutylammonium bromide was added to the mobile phase. The tetrabutylammonium bromide functioned as an ion-pairing agent and consequently allowed the rapid separation of 12 different nucleotides. It is believed that the dynamic complex exchange model explains the basis of retention in ion pair reversed-phase CEC. Eight different dinucleotides, which have similar mass-to-charge ratios, separated very well by CEC. These solutes exhibited similar migration times (i.e., little or no separation) in capillary zone electrophoresis (CZE). Similarly, t-RNAs that did not separate by CZE were well resolved in CEC with nonporous ODSS. This demonstrates that CEC is very suitable for the separation of solutes that have similar mass-to-charge ratios but differ in their hydrophobicity.

Enantioseparations in normal- and reversed-phase nano-high-performance liquid chromatography and capillary electrochromatography using polyacrylamide and polysaccharide derivatives as chiral stationary phases

Enantioseparations of chiral compounds with different structures (β-blockers, benzodiazepines, diuretica, etc.) were performed in fused-silica capillaries packed with silica gel which was modified by covalent attachment of poly-N-acryloyl- l-phenylalanineethylester (Chiraspher ®) or by coating with cellulose tris(3,5-dimethylphenylcarbamate). Three different separation modes, normal- and reversed-phase nano-HPLC and capillary electrochromatography (CEC) with and without pressure-assistance were performed in these capillaries using essentially the same experimental set-up. This allows a more reliable comparison of the different techniques. Nano-HPLC separations in the normal-phase mode gave relatively high peak efficiency compared to HPLC in regular size columns. However, under the experimental conditions of this study no significant gain in separation efficiency could be obtained by changing from pressure-driven to electrically-driven migration of the solutes.

Retention Prediction Based on the Solvatochromic Comparison Method in Reversed-Phase Capillary Electrochromatography

Retention behavior of eleven neutral compounds in reversed phase capillary electrochromatography (RP-CEC) was investigated by using solvatochromic comparison method. The retention behavior in RP-CEC can be predicted quite well based on either multiple solvatochromic parameters of solutes or solvatochromic solvent polarity measurement ET (30) of mobile phase.

Fritless Packed Columns for Capillary Electrochromatography: Separation of Uncharged Compounds on Hydrophobic Hydrogels

A novel column is described that does not require frits to keep packing material within a capillary. A continuous bed is prepared in situ in aqueous solution by radical copolymerization of N-isopropylacrylamide and 2-acrylamido-2-methylpropanesulfonic acid (the resultant gel is denoted poly(AMPS-co-IPAAm). N,N'-Methylenebisacrylamide is used for cross-linking. On the application of an electrical field, electroosmotic flow (EOF) is developed in the bed along the capillary, where fluid propulsion would be otherwise difficult to achieve. The resultant EOF transports neutral compounds through the column without forcing the gel out of the capillary. Examination of the fluid motion in the continuous bed using a video microscope system and an image processor shows a relatively flat flow profile of EOF. The bed functions as the stationary phase for reversed-phase capillary electrochromatography (CEC). This new approach is an alternative to packed capillary columns which have been used previously in CEC. A high efficiency is obtained for a steroid which is separated on a 4.0% total monomer concentration (T), 10.0% degree of cross-linking (C), and 10.0% mole fraction of AMPS in the total monomer (S), poly(AMPS-co-IPAAm) column. A mixture of polyaromatic hydrocarbons is separated on a 6.9% T, 5.8% C, and 5.5% S poly(AMPS-co-IPAAm) column. The capacity factor of benzo[a]pyrene increases from 0.63 to 1.91 as the acetonitrile content in a Tris-boric acid buffer is decreased from 45 to 30% (v/v). The run-to-run RSD of analyte migration time is less than 0.73%, and the day-to-day RSD is acceptable. Potential benefits of this approach are also mentioned.