Separation of polyphenolic compounds extracted from plant matrices using capillary electrophoresis

Phenolic compounds constitute a large group of secondary plant products whose chemical structure may range from quite simple compounds to highly polymerized substances. The polyphenols content have been investigated in the alcoholic extract of the fruits of three different plants: sweet gale, sea buckthorn, hiprose. The trans-resveratrol content we have studied in roots, stems, leaves and flowers of Japanese knotweed grown in Estonia. Plant material was pre-treated in two different ways: by infusing with methanol and by supercritical fluid extraction with carbon dioxide modified with different alcohols. The relationship between variables (pressure, temperature, modifier amount) and yields are examined. The capillary zone electrophoresis methods were developed for the separation of polyphenolic anti-oxidative compounds. Using both water based borate buffer and acetonitrile based non-aqueous media it was possible to get reliable separation of several polyphenolic compounds. Based on that there has been identified such as flavone, trans-resveratrol, catechin, chlorogenic acid, quercetin and myricetin in plant extracts. Changes in the relative concentrations of trans-resveratrol in different parts of the knotweed have been established.     

Use of dynamically coated capillaries with added cyclodextrins for the analysis of opium using capillary electrophoresis

A rapid, precise, accurate, and robust method using capillary electrophoresis (CE) with dynamically coated capillaries for the analysis of the major opium alkaloids in opium is presented. Dynamic coating of the capillary surface is accomplished using a commercially available reagent kit (polycation coating followed by polyanion coating). The addition of dual cyclodextrins (hydroxypropyl-beta-cyclodextrin and dimethyl-beta-cyclodextrin) to the run buffer imparts excellent selectivity for the opium alkaloids. For the determination of morphine, papaverine, codeine, noscapine and thebaine in opium gum and opium latex samples (using tetracaine as an internal standard) good agreement with values obtained by gradient high-performance liquid chromatography is obtained. Compared to the latter technique, CE affords better resolution with significantly faster analysis time (12 min versus 29 min). Dynamically coated capillaries, which give rise to a relatively high and robust electroosmotic flow (EOF) at the background electrolyte pH of 2.5, allow for rapid analysis and excellent migration time and peak area precision (RSDs < or = 0.12% and < or = 1.2%, respectively). Reproducible separations (relative migration times) for over 500 samples have been obtained on a single capillary. The nature of the injection solvent, the injection time and the contents of the waste vials have a profound effect on the pressure injection precision of the relatively hydrophobic solutes. The CE conditions reported in this study are also applicable to the analysis of lysergic acid diethylamide (LSD) exhibits.     

Preparative capillary zone electrophoresis using a dynamic coated wide-bore capillary

Preparative capillary zone electrophoresis separations of cytochrome c from bovine and horse heart are performed efficiently in a surfactant-coated capillary. The surfactant, dimethylditetradecylammonium bromide (2C(14)DAB), effectively eliminated protein adsorption from the capillary surface, such that symmetrical peaks with efficiencies of 0.7 million plates/m were observed in 50-microm id capillaries when low concentrations of protein were injected. At protein concentrations greater than 1 g/L, electromigration dispersion became the dominant source of band broadening and the peak shape distorted to triangular fronting. Matching of the mobility of the buffer co-ion to that of the cytochrome c resulted in dramatic improvements in the efficiency and peak shape. Using 100 mM bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane phosphate buffer at pH 7.0 with a 100-microm id capillary, the maximum sample loading capacity in a single run was 160 pmol (2.0 microg) of each protein.     

Separation of isomers of acyl glucuronides using micellar electrokinetic capillary chromatography in dynamically coated capillaries

The acyl glucuronide metabolites of endogenous as well as of xenobiotic compounds may undergo isomerization in vitro as well as in the human body. The parent acyl glucuronide and the isomerization products may react with endogenous protein to form products which in worst cases may act as antigens and thus create an allergic response. In the present paper new methods based on micellar electrokinetic or microemulsion electrokinetic chromatography for the separation of all isomerization products as well as the hydrolysis product of acyl glucuronides are described. In order to perform the separation at lower pH values in a reasonable time dynamically coated capillaries were used. This enables the electroosmotic flow to be high and constant even at low pH. The methods were developed using S-naproxen-beta-1-O-acyl glucuronide as the model substance. The assignment of the single peaks in the electropherogram was performed tentatively based on the sequential appearance of the isomerization products with time.     

Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary

A capillary zone electrophoresis (CZE) method was developed for the rapid analysis of charge heterogeneity of immunoglobulin G (IgG) monoclonal antibodies (mAbs). The separation was carried out in a short, dynamically coated fused-silica capillary. A number of separation parameters were investigated and optimized, including pH, concentration of the separation buffer (ε-amino caproic acid), concentration of the triethylenetetramine (TETA) dynamic coating, the capillary internal diameter and the field strength used for the separation. The effects of between-run flushing of the capillary and the data acquisition rate were also evaluated. Under the optimized conditions, a fast (<5 min), selective and reproducible separation of mAb charge variants was achieved under a very high electric field strength (1000 V/cm). This method also requires only a short conditioning of the capillary, with between-run conditioning completed within 2 min. The method was evaluated for specificity, sensitivity, linearity, accuracy and precision. The same separation conditions were applied to the rapid separation (2-5 min) of charge variants of multiple monoclonal antibodies with pI in the range of 7.0-9.5. Compared with other existing methods for charge variants analysis, this method has several advantages including a short run time, rapid capillary conditioning and simple sample preparation.     

Design and evaluation of capillary electrophoresis in dynamically coated capillaries coupled with chemiluminescence detection

A dynamic coating capillary electrophoresis coupled with a simplified on-line chemiluminescence detection system was designed and evaluated. In the proposed system, poly-vinylpyrrolidone was used as dynamic coating substance in the separation buffer to reduce the unwanted protein non-specific adsorption, which was first applied in capillary electrophoresis coupling with on-line chemiluminescence detection. In order to avoid complex processing, an ordinary plastic cuvette was modified as a three-way joint. The chemiluminescence reaction conditions and capillary electrophoresis separation conditions were investigated in detail. The results showed that the coated capillary can be injected protein samples at least 30 times continuously with good repeatability. Under optimal conditions, the chemiluminescence relative intensity was linear with the concentration of hemoglobin in the range of 4-1850 μg mL(-1) and the detection limit was 2.0 μg mL(-1) (S/N=3). The relative standard deviation of migration times and peak heights for 40 μg mL(-1) hemoglobin were 2.5% and 4.1% (n=11) respectively. Interference of matrix effects was overcome by the calibration according to standard addition methods. Afterwards, the method was validated successfully and was applied to detect the concentration of hemoglobin in the serum of haemolytic patients.     

Separation of enantiomers by capillary electrophoresis using pentosan polysulfate

Pentosan polysulfate, a semisynthetic polysaccharide, was employed as a chiral run buffer additive in capillary electrophoresis. Twenty-eight racemic analytes were resolved. The separations were successful only at low pH when the analytes were significantly protonated. This suggests that ionic interactions were the dominant associative interactions between the anionic pentosan polysulfate and the positively charged analytes. Compared to other linear, carbohydrate-based chiral selectors (i.e., chondroitin sulfates, heparin and dextran sulfate) pentosan polysulfate has some characteristics common of anionic polysaccharides; yet it has several differences in its structure and properties which account for its unusual enantioselectivity. The effects of pH, concentration of phosphate buffer, concentration of pentosan polysulfate and the type and concentration of organic modifier on the enantiomeric separations were investigated. The optimization of these separations were dependent on the nature of the analytes and could be achieved by the proper choice of experimental conditions.     

Separation of globins using free zone capillary electrophoresis.

This paper describes the use of high-performance capillary electrophoresis for the separation of globin chains. Adult and newborn haemolysates from normal individuals and children suspected of having thalassaemia were analysed using free zone electrophoresis. Separation of globins was accomplished using a 25 mM phosphate buffer at pH 11.8. Distinct peaks of alpha-, beta- and gamma-chains were resolved within 8 min. The coefficient of variation for within-day and between-day runs was 4.1% and 5.7%, respectively. This method is simple and rapid, and it can be used to screen for thalassaemia and for the clinical study of various haemoglobinopathies.     

Separation of fluorescein dyes by capillary electrophoresis using β-cyclodextrin

Summary A capillary electrophoresis method for the separation and determination of five synthetic dyes used in pharmaceutical preparations, cosmetics and as food additives is described. The dyes, fluorescein, dichlorofluorescein, Rose Bengal erythrosine and eosine are well separated in less than 12 min using an electrolyte of 50 mM phosphate buffer (pH 7.5), 10 mM β-cyclodextrin and 5% (v/v) methanol. A linear relationship between concentration and peak area for each dye was obtained in the concentration range 0.3–500 μg mL⁻¹, with a correlation coefficient greater than 0.999. Intra- and inter-day precision of about 0.2–2.6% RSD (n=11) and 4.9–9.7% RSD (n=30), respectively, were obtained. The method has been used for determining the purity of fluorescein and erythrosine in practical samples.     

Separation of cocaine stereoisomers by capillary electrophoresis using sulfated cyclodextrins

A capillary electrophoretic method for the separation of cocaine and its stereoisomers was developed. In this study, the effect of organic modifier was also investigated. The separation was achieved using 1% sulfated cyclodextrin, 10 mmol L(-1) phosphate buffer, 10% methanol at pH 3. The method provides good reproducibility and easy application.     

Separation of siderophores by capillary electrophoresis

Capillary electrophoresis (CE) was applied as a fast method of siderophore separation. Siderophores are iron binding and regulating cell products, which facilitate iron transport into cells. A fast and efficient method of siderophore analysis is important for better understanding of the iron pathways in a sea environment or marine organisms. The best results of CE analysis were obtained using free zone CE in 25 mM phosphate buffer at basic pH using a constant voltage of 20 kV. Under these conditions it was possible to detect the presence of siderophores in seawater.     

Separation of r-hirudin from similar substances by capillary electrophoresis

The thrombin inhibitor r-hirudin is a peptide of 65 amino acids and with a pI of 4.4. Capillary electrophoresis (CE) was used to separate r-hirudin from seven closely related substances which may be found as by-products or from degradation. Possibly two of these substances differ only in an isoaspartyl instead of an aspartyl binding. A baseline separation was possible with an acetate buffer (pH 4.4, 60 mM) containing 0.3% (m/m) PEG 20 000 and 0.1 mM Zn²⁺. The possibilities to prevent wall adsorption are discussed.     

Separation of oligonucleotides and DNA fragments by capillary electrophoresis in dynamically and permanently coated capillaries,using a copolymer of acrylamide and β-D-glucopyranoside as a new low viscosity matrix with high sieving capacity

New copolymers of acrylamide and β-D -glucopyranoside were synthesized and characterized. The different reactivity of the two monomers towards radical polymerization meant we could control the growth of the polymer chains whose length was inversely related to the number of glucose residues incorporated in the copolymers. The properties of these polymers were investigated in the separation of oligonucleotides and double-stranded DNA by capillary electrophoresis (CE) in coated and uncoated capillaries. The new copolymers were a suitable matrix for CE due to their high-resolving capacity and low viscosity. We also looked into the advantages of a new method of dynamic suppression of electroosmotic flow based on the addition of small amounts (0.03–0.05%) of dimethylacrylamide to the sieving and to the running buffer. A complete test was run on the reproducibility and efficiency of separations carried out in a permanently and dynamically coated capillary, and the advantages and disadvantages of the two methods were compared.     

Separation of bacteria by capillary electrophoresis

Differences in the surface charges of bacteria can be exploited for their separation by capillary electrophoresis. Because of their low electrophoretic mobility, the separation is not always easy to perform, especially in the presence of the electroosmotic flow. Elimination of electroosmotic flow by capillary wall modification with γ‐(trimethoxysilyl)propyl methacrylate followed by acrylamide bonding permits separation over a distance of 8.5 cm.     

Separation and determination of phospholipids in plant seeds by nonaqueous capillary electrophoresis

A method has been developed for the separation and determination of phospholipids by nonaqueous capillary electrophoresis in a separation medium of acetonitrile-2-proponol (3:2, v/v), 0.3% acetic acid and 60 mM ammonium acetate. To optimize the separation conditions, the composition of separation medium including alcohols, acetic acid, n-hexane and ammonium acetate was studied. The solvation interaction and ion-dipole interaction were also investigated. The contents of phospholipids in soybean, sunflower, peanut, apricot kernel, filbert and walnut were determined by the recommended method. The results obtained by the nonaqueous capillary electrophoreses were in good agreement with those determined by micellar electrokinetic chromatography.     

Separation of antidepressants by capillary electrophoresis with in-line solid-phase extraction using a novel monolithic adsorbent

The separation of three selective serotonin reuptake inhibitors (SSRIs) by capillary electrophoresis (CE) with fully integrated solid-phase extraction (SPE) is described. Polymeric monolithic SPE modules were prepared in situ within a fused silica capillary from either butyl methacrylate-co-ethylene dimethacrylate or 3-sulfopropyl methacrylate-co-butyl methacrylate-co-ethylene dimethacrylate. Using a 1 cm SPE module placed at the inlet of the capillary, a mixture of sertraline, fluoxetine and fluvoxamine was extracted from aqueous solution by applying a simple pressure rinse. Under pressure-driven conditions, efficient elution was possible from both SPE materials investigated using 50 mM phosphate buffer, pH 3.5 in acetonitrile (20/80, v/v). Two different strategies were investigated for the efficient elution and subsequent CE separation. Injection of an aqueous sample plug directly into the non-aqueous elution/separation buffer was found to be unsuitable with poor elution profiles observed in the electrodriven mode. Alternatively, a sample plug equivalent to several capillary volumes could be injected by pressure followed by filling the capillary with the non-aqueous elution/separation buffer from the outlet end using a combination of pressure and electrodriven flow. Using a neutral monolith, efficient elution/separation was not possible due to an unstable electroosmotic flow (EOF), however, by adding the ionisable monomer, 3-sulfopropyl methacrylate to the SPE module to increase and stabilise the EOF, it was possible to achieve efficient elution from the SPE module, followed by baseline separation by CE using a 200 mM acetate buffer, pH 3.5 in acetonitrile (10/90, v/v). With enrichment factors of over 500 achieved for each of the analytes this demonstrates the potential of in-line SPE-CE for the sensitive analysis of these drugs.     

Separation of perfluorocarboxylic acids using capillary electrophoresis with UV detection

A capillary electrophoretic method with UV detection for separation and quantitation of perfluorocarboxylic acids (PFCAs) from C6-PFCA to C12-PFCA has been developed. The optimization of measurement conditions included the choice of the most appropriate type and concentration of buffer in the background electrolyte (BGE), as well as the type and the content of an organic modifier. The optimal separation of investigated PFCAs was achieved with 50 mM phosphate buffer and 40% isopropanol in the BGE using direct UV detection. The optimum wavelength for direct UV detection was optimized at 190 nm. For indirect detection, several chromophores were studied. Five mM 3,5-Dinitrobenzoic acid (3,5-DNBA) in 20 mM phosphate buffer BGE and indirect UV detection at 280 nm gave the optimal detection and separation performance for the investigated PFCAs. The possibility of on-line preconcentration of solutes by stacking has been examined for indirect detection. The detection limits (LODs) determined for direct UV detection ranged from 2 microg/mL for C6-PFCA to 33 microg/mL for C12-PFCA. The LODs obtained for indirect UV detection were comparable to those obtained for direct UV detection.     

Separation of metal ions by capillary electrophoresis with a complexing electrolyte

Excellent separations of metal ions can be obtained very quickly by capillary electrophoresis provided a weak completing reagent is incorporated into the electrolyte to alter the effective mobilities of the sample ions. Indirect photometric detection is possible by also adding a UV-sensitive ion to the electrolyte. Separations are described using phthalate, tartrate, lactate or hydroxyisobutyrate as the complexing reagent. A separation of twenty-seven metal ions was achieved in only 6 min using a lactate system. A mechanism for the separation of lanthanides is proposed for the hydroxyisobutyrate system.     

Improvement of reproducibility and sensitivity of CE analysis by using the capillary coated dynamically with carboxymethyl chitosan

Analysis reproducibility and detection sensitivity of capillary electrophoresis (CE) are often questioned by applied scientists, which has hindered its application as a routine method. To address these issues, a simple, precise, and reproducible dynamic coating method was developed by applying carboxymethyl chitosan (CMC) dynamic coating on fused silica capillary. The proposed coating was accomplished by simply rinsing the capillary with CMC solution for 1 min in between runs, with no regeneration procedure or buffer additives needed. Electroosmotic flow could be well controlled by adjusting the pH of background electrolyte, and the adsorption of analytes onto the capillary inner wall was effectively eliminated. The main parameters of the coating condition were optimized, and extensive applications of these CMC-dynamically coated capillaries in CE separations were then firmly confirmed. By using proteins, aristolochic acids, and inorganic anions as model analytes, the coating showed a good stability, high reproducibility, as well as improved sensitivity. Baseline separations could be obtained with high efficiency. The reduced adsorption was impressively effective for basic proteins, with an average plate number of 90,000/m for each protein, apart from the good resolution on the chromatogram. A high sensitive detection of α-lactalbumin was achieved with a limit of detection (S/N = 3) of 3.5 nM, and the number of theoretical plates was as high as 1,200,000/m. In addition, the combination of the CMC coating with nonaqueous CE and CE-mass spectrometry proved to be practical. All results showed that the CMC-dynamically coated capillary has special properties and obvious superiority over the uncoated ones for CE analysis.     

Separation of basic proteins and peptides by capillary electrophoresis using a cationic surfactant

Incorporation of a low concentration of cetyltrimethylammonium bromide (CTAB) in the running electrolyte is shown to dynamically coat the silica capillary and to reverse the direction of electroosmotic flow. The CTAB coating prevented interaction of proteins with the capillary surface and enabled sharp peaks to be obtained in the electropherograms. A systematic study of experimental parameters demonstrated the importance of selecting a suitable buffer electrolyte and an appropriate pH. Excellent separations were obtained for five proteins, three enkephalins, and six dipeptides with an efficiency of approximately 500,000 theoretical plates per meter. The method developed is very simple to perform and was found to give excellent reproducibility.