DNA separation by capillary electrophoresis with hydrophilic substituted celluloses as coating and sieving polymers. Application to the analysis of genetically modified meals

A coating procedure based on the physical adsorption of hydroxypropyl cellulose onto the wall of a capillary column has been successfully used for the separation of DNA fragments up to 500 bp. The method uses a running Tris-phosphate-EDTA buffer containing 2-hydroxyethyl cellulose as sieving polymer. The separation procedure shows good reproducibility (measured as RSD%) for consecutive runs (<0.64), for different days (< 1.15) and capillaries (<2.15), short analysis times, and a long coating lifetime. Good reproducibility and efficiency are even achieved by performing the separation in the presence of additives such as ethidium bromide and mannitol. The method is applied to the detection of GMOs in soybean and maize meals with an accurate evaluation of the length of DNA sequences, previously amplified by polymerase chain reaction.     

Capillary waveguide fluoroimmunosensor with improved repeatability and detection sensitivity

An optical capillary waveguide fluoroimmunosensor based on glass capillaries internally coated with an ultrathin poly(dimethylsiloxane) (PDMS) film is presented. The evaluation of the capillaries developed was done in comparison with aminosilanized [3-(aminopropyl)triethoxysilane, APTES] glass and poly(methylpentene) (PMP) capillaries by immobilizing rabbit γ-globulins on the internal capillary wall. Following reaction with (R)-phycoerythrin-labelled antibody, the capillary was scanned with a laser beam and the fluorescence waveguided through the capillary wall was detected by a photomultiplier placed at one of its ends. The capillaries developed provided considerably improved protein coating homogeneity (intracapillary coefficients of variation 2.9–6.6%) and repeatability (intercapillary coefficients of variation 2.1–5.0%) compared with APTES-treated ones (7.9–13.4 and 8.5–15.2%, respectively). With use of these capillaries in a sandwich-type immunosensor for the determination of rabbit γ-globulins, the assay detection limit was improved eightfold (4.4 ng/mL) compared with that obtained using PMP capillaries (35.3 ng/mL), whereas the assay repeatability was improved threefold (intra-assay coefficients of variation 5.9–13.1%) compared with APTES-treated capillaries (15.6–36%). Optoelectronic set-up used to scan the capillaries (left) and representative fluorescence scannings of dual-band poly(methylpentene) (PMP), PDMS-modified glass and APTES treated glass capillaries     

Electrophoretic separation of environmentally important phenolic compounds using montomorillonite-coated fused-silica capillaries

This paper reports a simple procedure for coating fused-silica capillaries with poly(diallyldimethyl ammonium chloride) and montmorillonite. The coated capillaries were characterized by performing EOF measurements as a function of buffer pH, number of layers of coating, and number of runs (stability). The coated capillaries showed a highly stable mu(EOF) (run-to-run RSD less than 1.5%, n = 20), allowing continuous use for several days without conditioning. The coated capillaries were then used for the effective separation of nine environmentally important phenolic compounds showing a significant improvement in the resolution, when compared to bare fused-silica capillaries. The EOF of the coated capillaries was constant in alkaline solutions (pH > or = 7), allowing the optimization of the separation conditions of phenolic compounds without significantly affecting the mu(EOF).     

Facile preparation of polysaccharide‐coated capillaries using a room temperature ionic liquid for chiral separations

In this study, the dissolution of polysaccharides into an ionic liquid was investigated and applied as a coating onto the capillary walls of a fused‐silica capillary in open‐tubular CEC. The coating was evaluated by examining the chiral separation of two analytes (thiopental, sotalol) with three cellulose derivatives (cellulose acetate, cellulose acetate phthalate, and cellulose acetate butyrate). Baseline separation of thiopental enantiomers was achieved by use of each polysaccharide coating (Rs: 7.0, 8.1, 7.1), while sotalol provided partial resolution (Rs: 0.7, 1.0, 0.9). In addition, reproducibility of the cellulose‐coated capillaries was evaluated by estimating the run‐to‐run and capillary‐to‐capillary RSD values of the EOF. Both stability and reproducibility were very good with RSD values of less than 7%.     

Enantioselective separation of indole derivatives by liquid chromatography using immobilized cellulose (3,5-dimethylphenylcarbamate) chiral stationary phase

A new chiral stationary phase of 3,5-dimethylphenylcarbamates of cellulose chemically bonded to 3-aminopropyl silica gel was prepared, which may be used with a wide range of solvents including standard and non-standard ones. Several racemic indole derivatives have been resolved using standard and non-standard solvents on the immobilized chiral column (15 cm × 0.46 cm) at a flow rate of 1.0 mL/min or 0.5 mL/min with a UV detection at 230 nm. Separation of indole derivatives on immobilized and coated chiral stationary phases (CSP) in HPLC using a mixture of hexane/2-propanol as mobile phase was compared. The resolution factors for immobilized and coated chiral column were 0.57–2.02 and 0.61–4.03, respectively. It was found that both coated and immbolized chiral stationary phases were suitable for the separation of indole derivatives; however, the coated CSP possesses a higher resolving power than the immobilized one. The article is published in the original.     

Determination of adrenolytic drugs by SPME–LC–MS

Five adrenolytic drugs have been analyzed by liquid chromatography–mass spectrometry (LC–MS). Samples were prepared by solid-phase microextraction (SPME) using polypyrrole fibers coated on stainless steel support as an adsorbent for the drugs. Adsorption efficiencies were 95% and were close for all the drugs investigated. Relative standard deviations (RSD), calculated for samples prepared in standard solutions, were in the range 2.5–13%, however RSD values for the drugs in human plasma were 2.5–4.5%. Using LC–MS the limit of detection (LOD) and the limit of quantification (LOQ) were in the ranges 0.11–0.18 and 0.39–0.54 ng mL⁻¹, respectively, for the five drugs.     

Cellulose derivative/barium titanate composites with high refractive index,conductivity and energy density

Cellulose - This work deals with hydroxypropyl cellulose (HPC)/barium titanate (BT) composites, having small levels of perovskite-like BT loading, i.e. 0.5–2% and being designed for electric...     

Stability of capillaries coated with highly charged polyelectrolyte monolayers and multilayers under various analytical conditions--application to protein analysis

The stability of capillaries coated with highly charged polyelectrolytes under various analytical conditions was studied, as well as their performance for the analysis of proteins by Capillary Electrophoreis (CE) over a wide range of pH (2.5-9.3). In this study, fused silica capillaries were modified either with a poly(diallyldimethylammonium) chloride (PDADMAC) monolayer or PDADMAC/poly(sodium 4-styrenesulfonate) (PSS) multilayer coatings, using optimal coating conditions previously determined. Results show that the coated capillaries are remarkably stable and efficient to limit protein adsorption under a variety of extreme electrophoretic conditions even in the absence of the coating agent in the background electrolyte which is exceptional for non-covalent coatings. Monolayer coated capillaries were demonstrated for the first time to be stable to acidic rinses and to organic solvents which proves that the stability of the capillaries is highly dependent on the coating procedure used. In addition, PDADMAC/PSS multilayer coatings were found to be stable to alkaline treatments. PDADMAC/PSS coated capillaries gave excellent performances for the analysis of proteins covering a large range of pI (4-11) and of molecular weight (14-65 kDa) over a wide pH range (i.e. 2.5-9.3). Even at high pH 9.3, protein analysis was possible with very good repeatabilities (RSD(tm)<1% and RSD(CPA)<2.6% (n ≥ 8)) and high peak efficiencies in the order of 700,000.     

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.     

Effect of Chiral Additives in the Preparation of Cellulose-Based Chiral Stationary Phases in HPLC, and Effect on Enantiomer Resolution

Chiral stationary phases (CSPs) for high-performance liquid chromatographic (HPLC) have been prepared by coating silica gel with cellulose tribenzoate or cellulose trisphenylcarbamate. The effect of chiral additives on preparation of the CSPs was studied with (+)-l-mandelic acid, (−)-2-phenyl-1-propanol, (+)-1-phenyl-1,2-ethanediol and (−)-1-(1-naphthyl)ethanol as chiral additives for cellulose tribenzoate and (−)-2-phenyl-1-propanol and (+)-phenylsuccinic acid as chiral additives for cellulose trisphenylcarbamate. The results showed that chiral recognition by these stationary phases was increased in comparison with the original CSPs, especially the resolution (R _S) obtained. The method can be used to improve the efficiency of enantiomer separation by silica gel stationary phases coated with polymers.     

Development of a new flow-through bulk optode for the determination of manganese(II)

A flow-through bulk optode based on the use of 1-(2-pyridylazo)-2-naphthol (PAN) immobilized in a plasticized poly(vinyl chloride) membrane entrapped in a cellulose support, in conjuntion with the flow injection analysis technique, is proposed for the determination of manganese(II). The calibration graph obtained at 570 nm was linear in the range 0.27–27.5 mg L^–1 (5 × 10^–6– 5 × 10^–4 M) Mn(II) with a detection limit of 0.18 mg L^–1. The coefficients of variation of the sensor response for 5.5 mg L^–1 of Mn(II) were ±0.22% for consecutive measurements (n = 10), ±0.48% between days (n = 5) and ±0.38% between different membranes (n = 6). The sensor was readily regenerated with the carrier acetic acid/acetate buffer of pH 4.5. The method was applied to the determination of manganese in steels, waters and lemon tree leaves. Received: 13 December 2000 / Revised: 25 January 2001 / Accepted: 26 January 2001     

Routine serum protein analysis by capillary zone electrophoresis: Clinical evaluation

Summary To measure the five classical protein fractions in human serum several electrophoretic techniques are available. Besides separation on cellulose acetate membrane or agarose gel, capillary zone electrophoresis (CZE) may be a useful analytical alternative in clinical routine. We have compared the Dionex CES I capillary electrophoresis system with that of the Olympus Fractoscan using specimens submitted for routine analysis. For clinical evaluation 102 samples from patients with various diseases have been analysed. Serum protein fractions were judged on separation performance, precision and the regression method ofBablok-Passing. Regression analysis revealed variable agreement between both methods with a slope ± intercept of 2.10–0.52 (α₁-fraction) and 1.0–0.20 (α₂-fraction) as worse and best, resectively; and the coefficient of variation of migration time: 5.9 %–6.8 % (between-run imprecision). Differences in the comparison of fractions are mainly caused by the improved resolution of CZE; e.g. one β-globulin peak on cellulose acetate is separated into two distinct protein fractions in CZE, including more detailed diagnostic information—as is also the case with γ-fraction. In some cases monoclonal gammopathy with low concentrations of immunglobulin clone can only be detected in CZE, whereas the cellulose acetate membrane (CAME) electropherogram is inconspicuous. The within-run precision (N=18) gave coefficients of variation of peak areas 1.3–5.9 % (CZE) and 1.0–3.8 % (cellulose acetate membrane). This is the first time that a complete clinical evaluation of CZE serum protein fraction analysis has been performed. CZE with its higher resolution and hence more detailed diagnostic information in some cases, showed good separation patterns, precision and correlation. Interchangeability of results showed that this CZE method is well suited for analysis of serum protein fractions in clinical routine. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Evaluation of an analytical method for determining phthalate esters in wine samples by solid-phase extraction and gas chromatography coupled with ion-trap mass spectrometer detector

A solid-phase extraction (SPE) method was developed for extraction and analysis of six phthalate esters in wine samples using Carbograph 1 sorbent. The SPE procedure allowed efficient recovery of the investigated phthalates ranging between 78% and 105% with a relative standard deviation (RSD) ≤6.5 for an ethanolic phthalic acid ester (PAE) standard solution and between 73–71% and 96–99% with a RSD ≤8.4 for red wine samples spiked with 20 and 50 ng mL⁻¹ of PAE, respectively. The adsorption isotherms and breakthrough curves for Carbograph 1/water solution were reported. Gas chromatography coupled with an ion-trap mass spectrometer detector (GC/IT-MS) was used for analysis. The instrumental analytical protocol was found to yield a linear calibration in the range 0.01-10.0 μg mL⁻¹ with R ² values ≥0.9992. The limits of detection in GC/IT-MS (SIM mode) vary between 0.2 and 14 ng mL⁻¹ (RSD ≤5.6) whereas the limits of quantification range between 0.5 and 25 ng mL⁻¹ (RSD ≤5.9); the intra- and inter-day repeatabilities calculated as RSD for wine samples, were between 0.9–7.8 and 1.0–10.5, respectively. The analytical method developed was applied to several commercial wine samples. Furthermore, the investigated methods are simple, reliable, reproducible, and not expensive.     

Response Surface Methodology-Based Optimisation of Agarose Gel Electrophoresis for Screening and Electropherotyping of Rotavirus

Management of rotavirus diarrhoea cases and prevention of nosocomial infection require rapid diagnostic method at the patient care level. Diagnostic tests currently available are not routinely used due to economic or sensitivity/specificity constraints. Agarose-based sieving media and running conditions were modulated by using central composite design and response surface methodology for screening and electropherotyping of rotaviruses. The electrophoretic resolution of rotavirus genome was calculated from input parameters characterising the gel matrix structure and running conditions. Resolution of rotavirus genome was calculated by densitometric analysis of the gel. The parameters at critical values were able to resolve 11 segmented rotavirus genome. Better resolution and electropherotypic variation in 11 segmented double-stranded RNA genome of rotavirus was detected at 1.96% (w/v) agarose concentration, 0.073 mol l⁻¹ ionic strength of Tris base–boric acid–ethylenediamine tetraacetic acid buffer (1.4×) and 4.31 h of electrophoresis at 4.6 V cm⁻¹ electric field strength. Modified agarose gel electrophoresis can replace other methods as a simplified alternative for routine detection of rotavirus where it is not in practice.     

Rapid and sensitive spectrophotometric determination of trace amounts of iron(III) using leuco Xylene cyanol FF

A new, simple, sensitive, and reliable method is presented for the rapid spectrophotometric determination of trace amounts of iron(III) using leuco Xylene cyanol FF. The method is based on the oxidation of leuco Xylene cyanol FF (LXCFF) to its blue form of xylene cyanol FF by iron(III) in sulfuric acid medium (pH 2.0–3.0), the absorbance of the formed dye is measured in an acetate buffer medium (pH 2.8–4.4) at 615 nm. The method obeys Beer's law over a concentration range of 0.15–0.9 g mL^-1 iron, having a molar absorptivity of 5.6×10⁴ L mol^-1 cm^-1 and a Sandell's sensitivity of 0.0001 g cm^-2. The optimum reaction conditions and other analytical parameters have been evaluated. The developed method has been successfully applied to the determination of iron in water, soil, industrial effluent, plant material, pharmaceutical preparations, synthetic mixtures, and aluminum alloys.     

Cellulose II nanoelements prepared from fully mercerized, partially mercerized and regenerated celluloses by 4-acetamido-TEMPO/NaClO/NaClO2 oxidation

A softwood bleached kraft pulp (SBKP) and cotton lint cellulose were fully or partially mercerized, and these along with celluloses and commercially available regenerated cellulose fiber and beads were oxidized by 4-acetamido-TEMPO/NaClO/NaClO₂ at 60 °C and pH 4.8. Weight recovery ratios and carboxylate contents of the oxidized celluloses were 65–80% and 1.8–2.2 mmol g⁻¹, respectively. Transparent and viscous dispersions were obtained by mechanical disintegration of the TEMPO-oxidized celluloses in water. These aqueous dispersions showed birefringence between cross-polarizers, indicating that mostly individualized cellulose nanoelements dispersed in water were obtained by these procedures. Transmission electron microscopy observation showed that the cellulose nanoelements prepared from mercerized SBKP, repeatedly mercerized SBKP, mercerized cotton lint cellulose, regenerated cellulose beads and 18% NaOH-treated SBKP, i.e. partially mercerized SBKP, had similar morphologies and sizes, 4–12 nm in width and 100–200 nm in length. The 18% NaOH-treated SBKP was converted to cellulose nanoelements consisting of both celluloses I and II.     

Separation of amino acids. Possibilities offered by optimized associations of low-voltage electrophoresis and chromatography on finely textured paper and on cellulose thin-layers

Summary Three optimized small-scale methods for amino acid separation are described and compared. Two dimensional separations are based on paper or cellulose thin-layer ionophoresis and chromatography with simple and inexpensive apparatus. The influence of experimental conditions on resolution and amount required for amino acids possessing almost identical mobilities, is studied. The quickest procedure based on the coupling of 200×200×0.25 mm thin-layer, low-voltage electrophoresis (20 V/cm) with chromatography, and the system: cellulose −1.04 mol dm⁻³ formic acid — tertbutanol/methanol/pyridine/formic acid/water (33∶43∶9.6∶0.4∶20), provides very reproducible results with high separating power for small samples (2–10 nmoles).     

A simple capillary gel electrophoresis approach for efficient and reproducible DNA separations. Analysis of genetically modified soy and maize

It is generally assumed that in order to achieve suitable separations of DNA fragments, capillary gel electrophoresis (CGE)-coated capillaries should be used. In this work, a new method is presented that allows to obtain reproducible CGE separations of DNA fragments using bare fused-silica capillaries without any previous coating step. The proposed method only requires: (i) a capillary washing with 0.1 M hydrochloric acid between injections and (ii) a running buffer composed of Tris-phosphate-ethylenediamine tetraacetic acid (EDTA) and 4.5% of 2-hydroxyethyl cellulose (HEC) as sieving polymer. The use of this new CGE procedure gives highly resolved and reproducible separations of DNA fragments ranging from 50 to 750 bp. The separation of these DNA fragments is accomplished in less than 30 min with efficiencies up to 1.7 x 10(6) plates/m. Reproducibility values of migration times (given as %RSD) for the analyzed DNA fragments are better than 1.0% (n = 4) for the same day, 2.2% (n = 16) for four different days, and 2.3% (n = 16) for four different capillaries. The usefulness of this separation method is demonstrated by detecting genetically modified maize and genetically modified soy after DNA amplification by PCR. This new CGE procedure together with LIF as detector provides sensitive analysis of 0.9% of Bt11 maize, Mon810 maize, and Roundup Ready soy in flours with S/ N up to 542. These results demonstrate the usefulness of this procedure to fulfill the European regulation on detection of genetically modified organisms in foods.     

Capillary electrophoresis methods for the determination of covalent polyphenol–protein complexes

The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein–polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (−)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein–polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine.