Separation of acidic and basic compounds in capillary electrochromatography with polymethacrylate-based monolithic columns

Methacrylate-based monolithic columns with electroosmotic flow (EOF) or very weak EOF are prepared by in situ copolymerization in the presence of a porogen in fused-silica capillaries pretreated with a bifunctional reagent. Satisfactory separations of acidic and basic compounds on the column with EOF at either low or high pH are achieved, respectively. With sulfonic groups as dissociation functionalities, sufficient EOF mobility still remains as high as 1.74 x 10(-4) cm2 s(-1) V(-1) at low pH. Under this condition, seven acidic compounds are readily separated within 5.7 min. Moreover, at high pH, the peak shape of basic compounds is satisfactory without addition of any masking amines into running mobile phase since the secondary interaction between the basic compounds and the monolithic stationary phase are minimized at high pH. Reversed-phase mechanism for both acidic and basic compounds is observed under investigated separation conditions. In addition, possibilities of acidic and basic compound separations on a monolithic column with extremely low EOF are discussed.     

Separation of basic and acidic compounds by capillary electrochromatography using monolithic silica capillary columns with zwitterionic stationary phase

A novel monolithic silica column with zwitterionic stationary phase was prepared by in-situ covalent attachment of phenylalanine to a 3-glycidoxypropyltriethoxysilane-modified silica monolith. Due to the zwitterionic nature of the resulting stationary phase, the density and sign of the net surface charge, and accordingly the direction and magnitude of electroosmotic flow in this column during capillary electrochromatography could be manipulated by adjusting the pH values of the mobile phase. CEC separations of various acidic and basic compounds were performed on the prepared column in anodic and weakly cathodic EOF modes, respectively. The peak tailing of basic compounds in CEC on a silica column could be alleviated at optimized buffer compositions. Besides the electrophoretic mechanism and weak hydrophobic interaction, weak cation- and anion-exchange interactions are also involved in the separations of acids and bases, respectively, on the zwitterionic column.     

Simultaneous analysis of cationic, anionic, and neutral compounds using monolithic CEC columns

A new capillary electrochromatography (CEC) column for the simultaneous analysis of cationic, neutral, and anionic compounds using CEC-ESI-MS is described. Three different silica monolith columns were prepared by changing the poly(ethylene glycol) (PEG) contents for comparison of the separation property of these columns. Different separation programs were used for the simultaneous separation of different charged compounds under the same conditions. The column prepared with 80 mg of PEG separated typical compounds within 15 min using 1 M formic acid as the electrolyte. The analytes migrated in the order of cationic, neutral, and anionic compounds, which means that the migration order was mainly determined by the electrophoresis. The hydrodynamic flow by pressure from the inlet side was significant for a stable analysis to be achieved. The effect of the composition of the sheath liquid was also examined. All analytes (14 amino acids, thiourea, urea, citric acid, and ATP) were detectable when 1% acetic acid in 50% (v/v) methanol was used as the sheath liquid.     

Separation of acidic and neutral compounds by strong anion-exchange capillary electrochromatography dynamically modified with sodium dodecylsulfate

Summary Acidic and neutral compounds have been separated by strong anion-exchange capillary electrochromatography (SAXCEC) dynamically modified by addition of sodium dodecylsulfate (SDS). It was found that separation of neutral solutes by SAXCEC without addition of SDS is difficult because of the weak interaction of the solutes with the hydrophilic packing surface. The hydrophobicity of the packing surface increases on addition of SDS to the mobile phase, however, and the capacity of separating neutral solutes increases. Acidic solutes are retained, mainly because of the ion-exchange properties of this system. The influence of mobile phase composition, e.g. SDS concentration, ionic strength, and organic modifier fraction, on the retention of acidic and neutral solutes was investigated. Three acids and five neutral solutes were separated in only 5 min under optimized conditions, because the direction of the strong electroosmotic flow (EOF) was the same as that of electrophoretic migration of the acids under the conditions used. The repeatability of this system in terms of migration time relative standard deviation (RSD) is good—less than 0.48% for 10 consecutive runs of all the solutes tested. Column efficiencies for acids were > 125000 plates m⁻¹; those for neutral solutes varied from 25000 to 100000 plates m⁻¹.     

Separation of selected humic degradation compounds by capillary electrochromatography with monolithic and packed columns

The separation of selected lignin/humic substance (HS) degradation compounds by capillary electrochromatography (CEC) with a methacrylate-based monolithic column and a conventional column packed with 5 microm octadecyl silica (ODS) particles is presented. The effects of organic modifier concentration, pH of the mobile phase, ionic strength, applied voltage, and temperature on the separation were investigated to determine the optimal separation conditions. With the increase of pH in the mobile phase, some of analytes start to ionize and both chromatographic partition and electrophoresis can play roles in separation simultaneously. Accordingly, different selectivity from high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) could be achieved. The performances of both kinds of columns were compared. The results showed that the peaks of compounds obtained on the former column were much wider than those on the latter one, although good separation efficiency of alkylbenzenes could be readily achieved; the most probable reasons for this behavior and method to solve this problem were briefly discussed. The CEC of a soil fulvic acid with a monolithic column produced partly resolved broad bands; by means of nuclear magnetic resonance (NMR) analysis a wide range of oxygen derived aromatic substitution patterns was found with prominent contributions from phenolic and carboxylic groups.     

Separation of acidic compounds by strong anion-exchange capillary electrochromatography

Separation of the acidic compounds in the ion-exchange capillary electrochromatography (IE-CEC) with strong anion-exchange packing as the stationary phase was studied. It was observed that the electroosmotic flow (EOF) in strong anion-exchange CEC moderately changed with increase of the eluent ionic strength and decrease of the eluent pH, but the acetonitrile concentration in the eluent had almost no effect on the EOF. The EOF in strong anion-exchange CEC with eluent of low pH value was much larger than that in RP-CEC with Spherisorb-ODS as the stationary phase. The retention of acidic compounds on the strong anion-exchange packing was relatively weak due to only partial ionization of them, and both chromatographic and electrophoretic processes contributed to separation. It was observed that the retention values of acidic compounds decreased with the increase of phosphate buffer and acetonitrile concentration in the eluent as well as the decrease of the applied voltage, and even the acidic compounds could elute before the void time. These factors also made an important contribution to the separation selectivity for tested acidic compounds, which could be separated rapidly with high column efficiency of more than 220000 plates/m under the optimized separation conditions.     

Investigation of neutral monolithic capillary columns with varying n-alkyl chain lengths in capillary electrochromatography

Two different neutral nonpolar monolithic columns series (designated as A and B columns series) each consisting of three columns at varying n-alkyl chain length were prepared by the copolymerization of the functional monomers C8-methacrylate, C12-acrylate, or C16-methacrylate with the cross-linking monomer pentaerythritol triacrylate (PETA) to yield monoliths with surface bound C8, C12, and C16 chains. In the A columns series, the composition of the functional monomers and crosslinker was adjusted to yield comparable chromatographic retention regardless of the alkyl chain length. In the B columns series, the composition of the functional monomers and crosslinker was kept constant yielding chromatographic retention, which increased as expected in the order of increasing the n-alkyl chain length. Due to their direct influences on the monolith porosity and retention energetic, the nature and composition of the monomers at a given porogen composition have largely affected the solute's mass transfer characteristics and sorption kinetics, as assessed by the van Deemter plots and separation efficiencies. The C16-monolith of the A series yielded the highest separation efficiency toward small solutes, but the A columns series were inadequate for protein separation. The C8-monolith of the B series provided the best separation efficiency for proteins while for tryptic peptide mapping, the C16-monolith of the A series seems to provide the best separation. For large protein molecules, the energetically "softer" C8 surface allowed faster sorption kinetics and in turn improved efficiency, while an energetically "harder" C16 surface favored better separation of the smaller size peptide solutes.     

Analyses of benzophenones by capillary electrochromatography using methacrylate ester-based monolithic columns

In this study, eight benzophenones, which are commonly used as UV filters in various cosmetics and plastics, were analyzed by capillary electrochromatography with a methacrylate ester-based monolithic column. The effects of the composition and pH of mobile phase, porogenic solvent ratio, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) content on benzophenone separations were examined. For all benzophenones, separation performances were markedly improved in monolithic columns with larger 1-propanol ratio and higher AMPS content. Furthermore, a twofold increase in AMPS content almost reduced the separation time in half when a monolithic column had an adequately high surface area, i.e. monolithic column was produced in a higher ratio of 1-propanol. As well, the retention behaviors of these analytes in the monolithic column were strongly influenced by the level of acetonitrile in the mobile phase, and the pH of the mobile phase also had an apparent influence on separation resolution.     

Analyses of preservatives by capillary electrochromatography using methacrylate ester-based monolithic columns

Five common food preservatives were analyzed by capillary electrochromatography, utilizing a methacrylate ester-based monolithic capillary as separation column. In order to optimize the separation of these preservatives, the effects of the pore size of the polymeric stationary phase, the pH and composition of the mobile phase on separation were examined. For all analytes, it was found that an increase in pore size caused a reduction in retention time. However, separation performances were greatly improved in monolithic columns with smaller pore sizes. The pH of the mobile phase had little influence on separation resolution, but a dramatic effect on the amount of sample that was needed to be electrokinetically injected into the monolithic column. In addition, the retention behaviors of these analytes were strongly influenced by the level of acetonitrile in the mobile phase. An optimal separation of the five preservatives was obtained within 7.0 min with a pH 3.0 mobile phase composed of phosphate buffer and acetonitrile 35:65 v/v. Finally, preservatives in real commercial products, including cold syrup, lotion, wine, and soy sauces, were successfully determined by the methacrylate ester-based polymeric monolithic column under this optimized condition.     

Simultaneous analysis of basic, acidic and neutral compounds on an endcapped octadecylsilane silica-based monolith by pressure-assisted capillary electrochromatography

This simultaneous separation of basic, acidic and neutral analytes by pressure-assisted CEC (pCEC) using a hybrid (tetramethoxysilane and methyltrimethoxysilane) silica-based monolith, chemically modified with octadecyldimethylchlorosilane followed by endcapping with hexamethyldisilazane is described. The endcapping resulted in near Gaussian peaks for highly basic analytes such as nortriptyline without a significant loss in the EOF. The migration behaviour of analytes on this phase could be rationalised based on hydrophobicity, electrophoretic mobility and ion-exchange interactions. The high porosity of the monolith allowed manipulation of the linear velocity of mobile phases by the addition of varying amounts of pressure at the inlet to reduce analysis times and overcome the reversed migration of anionic species towards the detection window in cathodic EOF mode. The concomitant programmed application of pressure (2-4 bar) and voltage (27 kV) facilitated the simultaneous separation of four cationic, four neutral and two anionic compounds in 6 min with efficiencies ranging from 41 000 to 94 000, 57 000 to 77 000 and 180 000 to 210 000 theoretical plates/metre, respectively. The % RSD values of migration times and efficiencies in pCEC mode were less than 3.6 and 7.9%, respectively (n = 5).     

Practical aspects of using methacrylate-ester-based monolithic columns in capillary electrochromatography

Methacrylate-ester-based monoliths containing quaternary ammonium groups were prepared in situ in capillary columns and in simultaneous experiments in vials, employing thermal initiation. The chromatographic properties of the monoliths were determined with capillary electrochromatography (CEC), and their morphology was studied with mercury-intrusion porosimetry on the bulk materials. Materials with different, well repeatable pore-size distributions could be prepared. A satisfactory column-to-column and run-to-run repeatability was obtained for the electro-osmotic mobility, the retention characteristics (k-values) and the efficiency on the columns prepared and tested in the CEC mode. A relatively high electro-osmotic flow was observed in the direction of the positive electrode. The electro-osmotic mobility was found to be influenced only marginally by mobile-phase parameters such as the pH, ionic strength, and acetonitrile content. The retention behavior of the monolithic columns was similar to that of columns packed with C18-modified silica particles. Columns could be prepared with optimum plate heights ranging from 6 microm for unretained compounds to 20 microm for well retained (k=2.5) polyaromatic hydrocarbons. However, for specific analytes a - still unexplained - lower chromatographic column efficiency was observed.     

Enantiomeric separations of cationic and neutral compounds by capillary electrochromatography with monolithic chiral stationary phases of beta-cyclodextrin-bonded negatively charged polyacrylamide gels

Enantiomeric separation by capillary electrochromatography with beta-cyclodextrin-bonded negatively charged polyacrylamide gels was examined. The columns used are capillaries filled with a negatively charged polyacrylamide gel, a so-called monolithic stationary phase, to which allyl carbamoylated beta-CD (AC-beta-CD) derivatives covalently bind. The capillary wall is activated first with a bifunctional reagent to make the resulting gel bind covalently to the inner surface of the fused-silica tubing. Enantiomeric separations of 15 cationic compounds were achieved using the above-mentioned columns and mobile phases of 200 mmol l(-1) Tris-300 mmol I(-1) boric acid buffer (pH 7.0 or 9.0) or 200 mmol l(-1) Tris-300 mmol l(-1) boric acid buffer (pH 7.0) containing an achiral crown ether (18-crown-6). Enantiomeric separations of two neutral compounds were also achieved using 200 mmol l(-1) Tris-300 mmol l(-1) boric acid buffer (pH 9.0) as a mobile phase. High efficiencies of up to 150,000 plates m(-1) were obtained. Both the within- and between-run reproducibilities of retention time and separation factor were good. The reproducibilities of retention time and separation factor for three different columns prepared from a different batch of monomers were acceptable. The gel-filled capillaries were stable for at least 3 months with intermittent use, utilizing the mobile phase of 200 mmol I(-1) Tris-300 mmol I(-1) boric acid buffer (pH 9.0).     

Preparation of monolithic capillary columns for capillary electrochromatography by γ-ray irradiation

A novel approach is introduced and evaluated for the preparation of silica-based monoliths by a sol–gel technique where in situ polymerization was carried out by γ-ray irradiation within the capillary. The γ-radiation-initiated synthesis generated radicals directly on the monomer, thereby avoiding use of any initiator. The chromatographic behavior of the capillary monolithic columns was studied in the modes of CEC, p-CEC and low pressure-driven separation, all of which exhibited reversed-phase character. Various operational parameters, such as column temperature, separation voltage, acetonitrile content and buffer pH, were varied to assess their influence on column performance in the separation of a series of neutral compounds including thiourea, benzene, toluene, ethyl benzene, biphenyl and naphthalene. A scanning electron micrograph of a cross-section of the capillary column showed that the gel took the form of a spherical particle aggregate and adhered to the column inner wall. It provided a viable alternative to either thermally initiated or photo polymerization for the preparation of monolithic columns.     

Investigations on the behaviour of acidic, basic and neutral compounds in capillary electrochromatography on a mixed-mode stationary phase

This work describes the separation of acidic, basic and neutral organic compounds as well as inorganic anions in a single run by capillary electrochromatography employing a stationary phase which exhibits both strong anion-exchange and reversed-phase chromatographic characteristics. The positive surface charge of this stationary phase provided a substantial anodic electroosmotic flow. The analytes were separated by a mixed-mode mechanism which comprised chromatographic interactions (hydrophobic interactions, ion-exchange) as well as electrophoretic migration. The influence of ion-exchange and hydrophobic interactions on the retention/migration of the analytes could be manipulated by varying the concentration of a competing ion and/or the amount of organic modifier present in the background electrolyte. Additionally the effects of pH changes on both the chromatographic interactions as well as the electrophoretic migration of the analytes were investigated.     

Separation of related opiate compounds using capillary electrochromatography

Capillary electrophoretic separations have been investigated for six controlled narcotic analgesic compounds having related structures. Owing to the similar charge-to-mass ratios of these compounds, capillary zone electrophoresis failed to provide a satisfactory separation, whereas a baseline-resolved separation was achieved in 10 min using micellar electrokinetic chromatography. Column efficiencies of 40,000-150,000 plates/m were obtained with a 50 cm long, 50 microm inner diameter (ID) capillary using 50 mM sodium dodecyl sulfate (SDS) in a 50 mM borate solution containing 12% isopropanol. In contrast, separation of this mixture by capillary electrochromatography proved to be significantly superior. The capillary was 15 cm long, with an ID of 75 microm, and was packed with 1.5 microm nonporous octadecyl silica (ODS) particles. The mobile phase consisted of 80% 10 mM tris(hydroxymethyl)aminomethane (Tris) and 20% acetonitrile, and contained 5 mM SDS. A complete separation was obtained in 2.5 min with an efficiency of 250,000-500,000 plates/m.     

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 infinity.     

Preparation and characterization of monolithic polymer columns for capillary electrochromatography

A series of micro-monolithic columns with different porosities were prepared for capillary electrochromatography (CEC) by in-situ copolymerization of butyl methacrylate, ethylene glycol dimethacrylate, and 2-acrylamido-2-methyl-1-propane-sulfonic acid in the presence of a porogen in fused-silica capillaries of 100 microm I.D. Different column porosities were obtained by changing the ratios of monomers to porogenic solvents. Columns were investigated and evaluated under both pressure-driven (high-performance liquid chromatography, HPLC) and electro-driven (capillary electrochromatography, CEC) conditions. Each column exhibited different efficiency and dependency on flow velocity under electro-driven conditions. Abnormally broad peaks for some relatively bulky molecules were observed. Possible explanations are discussed. The differences in column efficiency and retention behavior between the two eluent-driven modes were studied in detail. In addition, other column properties, such as morphology, porosity, stability and reproducibility, were extensively tested.     

双子表面活性剂及其保护的纳米金作为缓冲添加剂用于毛细管电泳蛋白质分离

报道了使用阳离子双子表面活性剂作为毛细管电泳的缓冲添加剂用于同时分离酸性和碱性蛋白质.在酸性的缓冲条件下,只需要使用低浓度的阳离子双子表面活性剂（0.1mmol/L18-s-18）作为缓冲液的添加剂,就可以有效地抑制酸性和碱性蛋白质在毛细管壁的吸附,从而得到高效的蛋白质分离.实验表明,较小的胶束尺寸（如s=5～8）比大的胶束尺寸（如s〈4或〉10）能更有效地抑制酸性蛋白质的吸附.改变双子表面活性剂的中间基的长度能够对蛋白质的电泳淌度进行一定的调节,从而对分离的选择性进行一定的优化.在最优的实验条件下,蛋白质迁移时间的日内和日间标准偏差（RSD）分别小于0.8%和2.2%,回收率为79%到100.4%.另外,还考察了双子表面活性剂保护的金纳米颗粒用作毛细管电泳缓冲添加剂在蛋白质分离中的应用.实验表明,在缓冲液中加入纳米金能够缩短分析时间,并能小幅度地提高分离效率.最后,使用该方法分析了一系列复杂生物样品,包括血浆、红细胞和鸡蛋清样品,均得到了满意的结果.     

Separation of acidic and basic proteins by capillary electrophoresis using gemini surfactants and gemini-capped nanoparticles as buffer additives

This paper demonstrated simultaneous separation of acidic and basic proteins using cationic gemini surfactants as buffer additives in capillary electrophoresis. We showed that even at a low concentration (0.1 mmol·L-1) of alkanediyl-α,ω-bis(dimethyloctadecylammonium bromide) (18-s-18), the wall adsorption of both acidic and basic proteins could be effectively suppressed under acidic conditions. Smaller micelle size (e.g., s=5-8) is more effective for the separation of acidic proteins than larger micelle siz...     

Preparation and characterization of C16 monolithic columns for capillary electrochromatography

A series of methacrylamide-based C16 monolithic columns were prepared and characterized to determine how their porous structural properties and chromatographic behavior are affected by the percentages of functional monomer, base monomer, and cross-linker in the polymerization solution. Baseline separation of 6 neutral compounds can be readily obtained in an optimized column. Furthermore, the effects of organic additive in the mobile phase, operating voltages, and temperature on retention behaviors and separation efficiencies were also studied. The separation mechanism is also discussed. High column efficiency and good reproducibility indicate that the monolithic columns hold considerable promise.