Separation of derivatized carbohydrates by co-electroosmotic capillary electrophoresis

Summary The separation of derivatized carbohydrates has been performed by co-electroosmotic capillary electrophoresis. Derivatization was performed by reductive amination of the carbohydrates with ethylp-aminobenzoate or withp-aminobenzonitrile. Separation selectivity is optimized using buffer electrolytes containing high concentrations of borate, organic solvents, and mixtures thereof; this enabled separation of the carbohydrate derivatives then direct UV detection. Co-directional migration of the anionic analytes with the electroosmotic flow was achieved by adding a cationic polyer (hexadimethrine bromide, HDB) to the electrolyte. With this method it is possible to determine specific carbohydrates, such as arabinose, mannose, and glucose, which are difficult to separate by other CE methods. The applicability of the method is demonstrated for the analysis of plant hydrolyzates     

Study on liposomes by capillary electrophoresis

Liposomes made of mixtures of zwitterionic and anionic lipids were investigated by means of capillary electrophoresis and dynamic light scattering. The influence of the molar lipid ratio and of the buffers, used in the running electrolyte solution, on the physical characteristics of the liposomes were investigated. Data on effective electrophoretic mobilities, total charges as well as sizes of the liposomes are given. In addition, examples on the use of liposomes as carriers in electrokinetic capillary electrophoresis for the separation of benzene derivatives, steroids, and phenols are shown.     

柱前衍生化-非水毛细管电泳法高效分离饮料中的脂肪醛

以9,10-菲醌作为柱前衍生试剂,采用非水毛细管电泳模式考察并优化了脂肪醛的分离条件。实验采用毛细管(总长58.5 cm,有效长度50 cm,内径50 μm),应用80 mmol/L NH4Ac、1.4 mol/L HAc缓冲体系,于20 ℃、5 kPa(50 mbar)下压力进样 8 s,在不加入其他添加剂的情况下,实现了7种脂肪醛的高效基线分离,检出限为0.37～0.87 μmol/L,线性范围为0.78～25 μmol/L。应用所建立的方法对实际样品进行了测定,结果令人满意。     

On-line pre-concentration of atrazine by antibody immobilization capillary electrophoresis

Antibody coupled capillary electrophoresis was employed in the pre-concentration and detection of atrazine at the parts per billion (ppb) level. A 1000-fold increase in detection has been demonstrated in the use of IgG anti-atrazine monoclonal antibodies for the analysis of atrazine in well water by capillary zone electrophoresis (CZE) with UV-VIS detection. These results were confirmed by an enzyme linked immunosorbent assay (ELISA) specific for atrazine.     

Quaternary ammonium substituted agarose as surface coating for capillary electrophoresis

A novel positively charged polymer of quaternary ammonium substituted agarose (Q-agarose) has been synthesized and explored for use as a coating in capillary electrophoresis. The fast and simple coating procedure is based on a multi-site electrostatic interaction between the polycationic agarose polymer and the negatively charged fused-silica surface. By simply flushing fused-silica capillaries with hot polymer solution a positively charged, hydrophilic deactivation layer is achieved. The polymer surface provides an intermediate electroosmotic flow of reversed direction, over a range of pH 2-11, compared to unmodified fused-silica. The coating procedure was highly reproducible with an RSD of 4%, evaluated as the electroosmotic flow mobility for 30 capillaries prepared at 10 different occasions. The application of Q-agarose coated capillaries in separation science was investigated using a set of basic drugs and model proteins and peptides. Due to the intermediate electroosmotic flow generated, the resolution of basic drugs could be increased, compared to using bare fused-silica capillaries. Moreover, the coating enabled separation of proteins and peptides with efficiencies up to 300.000 plates m(-1).     

Evaluation of a novel hydrophilic derivatized capillary for protein analysis by capillary electrophoresis —electrospray mass spectrometry

A new type of hydrophilic derivatized capillary has been used to enable the on-line capillary electrophoresis separation and electrospray mass spectrometric detection of a mixture of proteins containing bovine cytochrome c, tuna cytochrome c and horse heart myoglobin. Less than 40 fmol of each compound were loaded into the capillary. Baseline resolution of components was achieved, as were accurate assignments of molecular masses. The hydrophilic derivatized capillaries were taken through extensive testing procedures to characterize their performance and capabilities for protein analysis. A mixture of six proteins (cytochrome c, ribonuclease A, -chymotrypsinogen, myoglobin, carbonic anhydrase II and -lactalbumin) in acetic acid—sodium acetate buffer was used to delineate the relationships between migration time and pH, along with migration time and buffer concentration for each protein. The variations in capillary efficiency as a function of pH and as a function of buffer concentration were also characterized for the same six proteins in the acetic acid–sodium acetate system. A pH of 4.8 was found to offer an excellent compromise between separation efficiency (up to 500 000 theoretical plates) and analysis time. Capillary efficiencies were also found to be very good when employing a Tris · HCl electrolyte adjusted to pH 4.8. Lastly, electropherogram reproducibility and capillary durability were examined with the finding that little deterioration of the capillary occurred over the course of 400 injections (200 h run time). This represents a notable improvement over previously documented derivatization procedures designed to reduce protein adsorption to fused-silica capillary walls     

Preparation approaches of the coated capillaries with liposomes in capillary electrophoresis

The use of liposomes as coating materials in capillary electrophoresis has recently emerged as an important and popular research area. There are three preparation methods that are commonly used for coating capillaries with liposomes, namely physical adsorption, avidin–biotin binding and covalent coupling. Herein, the three different coating methods were compared, and the liposome-coated capillaries prepared by these methods were evaluated by studying systematically their EOF characterization and performance (repeatability, reproducibility and lifetime). The amount of immobilized phospholipids and the interactions between liposome or phospholipid membrane and neutral compounds for the liposome-coated capillaries prepared by these methods were also investigated in detail. Finally, the merits and disadvantages for each coating method were reviewed.     

A novel covalent coupling method for coating of capillaries with liposomes in capillary electrophoresis

A novel covalent coupling method for coating of capillaries with liposomes has been developed, which includes three steps: (i) epoxy-diol coating, (ii) activation with 2,2,2-trifluoroethanesulfonyl chloride, and (iii) liposome coupling. The coating conditions, such as the reaction time and temperature of liposome coupling, the content of dimyristoylphosphatidylethanolamine in liposomes, were optimized. Vesicles were visualized on the inner silica wall as confirmed by atomic force microscopy. The effectiveness of the coating was demonstrated by investigating the effect of pH of BGE on EOF and separating neutral compounds. The intra- and inter-capillary variations in EOF are 4.02% RSD (n=30) and 6.72% RSD (n=4) respectively, and the coated capillaries can be used to perform analysis at least for one month without any performance deterioration when stored at 4 degrees C. A set of drugs with diverse structures was applied into the developed liposome-coated CE. The normalized capacity factor (K) was introduced to quantitatively evaluate drug-membrane interactions. The relationship between log K and the fraction dose absorbed in humans (Fa%) shows that the liposome-coated CE can be utilized for in vitro prediction of Fa% of drugs that follow the transcellular passive transport route.     

Study of DNA-ellipticine interaction by capillary electrophoresis with laser-induced fluorescence detection

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), an alkaloid isolated from Apocynaceae plants, exhibits an antitumor activity, which is exceptionally high against several specific types of tumors. Ellipticine is also interesting as an anticancer drug as it has limited side effects and lacks of hematological toxicity. Various methods to study intercalating activity of this drug have been developed. However, to our best knowledge, capillary electrophoresis (CE) as a technique combining high separation resolution with various detection options has never been used for these purposes. In this study, a novel separation method based on CE with laser-induced fluorescence (CE-LIF) detection has been developed for the determination of ellipticine and for the monitoring of ellipticine-DNA interaction. Sodium acetate (50 mM, pH 4.5) was used as a background electrolyte and LIF detection at λ(ex) = 488 nm. The limit of detection for ellipticine was determined to be 5 × 10?? M. A total of 20% dimethyl sulfoxide was found optimal as sample solvent. Additionally, intercalation of ellipticine into the double-stranded DNA was investigated. Signal corresponding to ellipticine was decreasing and a new peak appeared and was growing. It can be concluded that CE-LIF is a method applicable to in vitro studies of ellipticine-DNA complexes.     

Partial-filling affinity capillary electrophoresis of glycoprotein oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid

Partial-filling affinity capillary electrophoresis has been applied to the simultaneous analysis of interactions between glycoprotein oligosaccharides and certain plant lectins. A lectin solution and a mixture of glycoprotein-derived oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid were introduced to a neutrally coated capillary in this order, and separated by application of a negative voltage. Interaction of a lectin with each oligosaccharide in the mixture was observed as the specific retardation or dissipation of peaks, in addition to the size/charge separation of oligosaccharides by zone electrophoresis in the remainder (≈90%) of the capillary. The strength of the interaction with lectin was controlled by introducing an appropriate volume of lectin solution. Application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Moreover, sequential injection of four lectins (Maachia amurensis mitogen, Sambucus sieboldiana agglutinin, Erythrina cristagalli agglutinin, Aleuria aurantia lectin) induced complete dissipation of complex-type oligosaccharides and enabled specific determination of the presence of high-mannose oligosaccharides without the interference or alteration of the electropherogram in porcine thyroglobulin. This method was also applied to determine the binding constants of ovalbumin-derived oligosaccharides to wheat germ agglutinin.     

Effect of sample composition on electrophoretic migration application to hemoglobin analysis by capillary electrophoresis and agarose electrophoresis

The electrophoretic migration, in routine analysis, is crucial for compound identification especially when multiple components are present in the sample. In complex or crude samples, such as those obtained from biological fluids, electrophoretic migration often does not correspond well to that of a pure standard compound. Several factors, related to the sample itself, have been identified as modulating the electrophoretic migration in zone electrophoresis both in gel and capillary electrophoresis (CE): solute mobility and concentrations, salt content, and protein interaction in the sample. Peak shape asymmetry often signals changes in migration especially when comparing samples with wide differences in concentration or those containing high ionic strength. Also, the migration of a protein can be influenced by the presence of a high concentration of another slowly migrating protein in the sample. A weak interaction during the separation between the two proteins which lead to a decreased velocity has been postulated. This was confirmed by finding a curve-linear relationship between the ratio of the two hemoglobin (Hb) variants, hemoglobin F (Hb F) and hemoglobin S (Hb S), and the distance between the two in gel electrophoresis (GE); and also by the observation of formation of a new small peak based on the analysis of hemoglobin F by capillary electrophoresis upon the addition of Hb S to the separation buffer. These factors when present together have an additive effect on the migration. As an example, Hb F, present in low but variable concentration in patients with sickle cell disease (Hb S), migrates in gel electrophoresis slightly slower than it is expected; enough to be confused with other unknown variants. However, the small peaks with different migration distances between Hb S and the adult Hb (Hb A) correlated well (r = 0.98) with Hb F performed by an alkali-denaturing assay indicating that these peaks are indeed Hb F in spite of the difference in their migration.     

Bioanalytical interaction studies executed by preincubation affinity capillary electrophoresis

The versatility of CE is beneficial for the study of many types of molecular interactions, because different experimental designs can be made to suit the characteristics of a particular interaction. A very versatile starting point is the preequilibration type of affinity CE that has been used extensively for characterizing biomolecular interactions in the last 15 years. We review this field here and include a comprehensive overview of the existing preincubation ACE modes including their advantages and limitations as well as the methodological developments and applications within the bioanalytical field.     

Laser-induced fluorescence and UV detection of derivatized aldehydes in air samples using capillary electrophoresis

In this work, two capillary zone electrophoresis methodologies using UV absorption detection (214 nm) and laser-induced fluorescence detection (He/Cd laser, 325 nm excitation, 520 nm emission) of selected aldehydes (formaldehyde, acetaldehyde, propionaldehyde and acrolein) derivatized with dansylhydrazine (DNSH, 5-dimethylaminonaphthalene-1-sulfohydrazide) were proposed and validated. The aldehydes react with DNSH to form negatively charged molecules in methanolic medium. In both methodologies, nine DNSH-derivatives, including isomers of acetaldehyde, propionaldehyde and acrolein and two impurities were baseline separated in 20 mmol l(-1) phosphate buffer at pH 7.02, in less than 9 min. The limits of detection for the UV and LIF methodologies ranged from 1.1-9.5 microg l(-1) and 0.29-5.3 microg l(-1), respectively. The applicability of both methodologies to contemplate real samples was confirmed in the analysis of aldehyde-DNSH derivatives in indoor and outdoor air samples.     

Method development approaches for capillary ion electrophoresis

Capillary ion electrophoresis (CIE) is a capillary electrophoretic technique optimized for rapid determination of low-molecular-mass inorganic and organic ions. CIE predominantly employs indirect UV detection since the majority of the analytes lack specific chromophores. Described are three methods for detection and electrolyte optimization. The first method discussed approaches for optimizing sensitivity, selectivity and peak confirmation using a chromate electrolyte and selected detection wavelengths. Peak confirmation is aided by using both direct detection of analytes. The second and third methods involve an unattended electrolyte development approach for instruments that only provide fresh electrolyte on the injection side of the capillary. The electrolyte composition is changed in both the injection side vial and in capillary before each sample injection while leaving the receiving side electrolyte vial constant at the initial electrolyte composition. In one mode, the concentration of the electroosmotic flow (EOF) modifier used to induce anodic flow is varied while keeping the background electrolyte composition constant. In a second experiment, the background electrolyte co-ion is sequentially changed from high mobility to low mobility while keeping the EOF modifier concentration constant. The end effect is to achieve a broad range of controlled peak symmetry for analytes in a simple matrix. The results are compared to separations obtained when the injection side and receiving side electrolytes are manually matched.     

毛细管电泳研究抗癌药物紫杉醇与人血清蛋白结合作用

采用毛细管区带电泳(CZE)技术, 研究了天然抗癌药物紫杉醇(Paclita-xel)与人血清白蛋白(HSA)的结合机制. 在以硼砂-碳酸钠(pH 10, 50 mmoL)为运行缓冲溶液, 运行电压21 kV, 进样时间5.0 s, 紫外检测器(214 nm)的条件下检测, 结合常数和结合位点数在298和310 K分别为K298 K=1.7×104 L/mol, n298 K=4.1, K310 K=3.4×104 L/mol, n310 K=3.0.     

Membrane-mediated capillary electrophoresis: interaction of cationic peptides with bicelles

Electrokinetic capillary chromatography is applied to determine the membrane affinity of peptides using both 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles and DHPC/1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bicelles under controlled conditions. The effect of temperature and the bicelle q value in surface association with cationic peptides is studied. The cationic peptides selected have a well-defined membrane structure (indolicidin), induced secondary structure (melittin, magainin 2), or do not possess classical secondary structure (atrial natriuretic peptide (ANP) 1-28, 4-28, 5-27). Electrokinetic capillary chromatography facilitated by DMPC and DHPC additives provides a rapid means of estimating lipophilicity and screening for peptides that have membrane affinity.     

Altering the selectivity of inorganic anion separations using electrostatic capillary electrophoresis

A new method for altering the selectivity of inorganic anion separations in capillary electrophoresis is described. Addition of the zwitterionic surfactant 3-(N,N-dimethyldodecylammonio)propane sulfonate (DDAPS) to the background electrolyte modifies the migration order via electrostatic ion chromatography type interactions. Variation of the DDAPS surfactant concentration from 4 to 120 mM monotonically alters the selectivity from electrophoretic mobility based to that of electrostatic ion chromatography, without increasing Joule heating. This technique was applied to the determination of nitrate, nitrite, bromide and iodide in artificial seawater. Detection limits for the anions in 1:5 diluted seawater were 11, 5, 7 and 11 microM, respectively.     

Method for enantiomeric purity of a quinuclidine candidate drug by capillary electrophoresis

A chiral procedure based on EKC was developed and validated for determination of the enantiomeric purity of PHA-543613, a drug candidate that was under development for treatment of the cognitive deficits of Alzheimer's disease and schizophrenia. Separation of enantiomers is accomplished via differential, enantiospecific complexation with a single-isomer, precisely sulfated beta-CD and heptakis-6-sulfato-beta-CD (HpS-beta-CD). Both neutral and sulfated CDs were screened before selecting HpS-beta-CD as the chiral selector. The separation is conducted in a 61 cm x 50 microm uncoated fused silica capillary with 25 mM HpS-beta-CD in pH 2.50, 25 mM lithium phosphate as the separation buffer with detection at 220 nm. Application of reverse polarity at -30 kV results in an elution time of about 12 min for PHA-543613 and 13 min for the undesired S-enantiomer. Quantification is versus an authentic reference S-enantiomer as an external standard in combination with an internal standard. The procedure was validated over the range 0.1-2.0% w/w. The detection limit is 0.01-0.02%. The amount of distomer intrinsic to the drug substance is about 0.1% or less. The developed method was used to generate stability data on multiple lots: in one case for up to 3 years.     

Studying protein-drug interaction by microfluidic chip affinity capillary electrophoresis with indirect laser-induced fluorescence detection

We developed a microfluidic chip-affinity CE method based on indirect LIF detection to study protein-drug interactions. The interaction between heparin and BSA was quantitatively studied, as a model system. In our method, sodium fluorescein was chosen as background, and redistilled water as marker to monitor EOF. The electrophoretic mobility changes of BSA were measured, with various concentrations of heparin added to the running buffer. Each run was completed within 80 s. The binding constant was determined to be (1.24 +/- 0.05) x 10(3) M(-1), which was in good agreement with that reported in the literature.