High performance separations of nucleic acids using capillary electrophoresis and high-performance liquid chromatography

High resolution separations of nucleic acids have been performed using high performance capillary electrophoresis (HPCE) and high performance liquid chromatography (HPLC). Electropherograms showing HPCE separations of single and double stranded DNA are presented and compared with HPLC separations. Single base resolution of poly(dA) oligonucleotides in the size range of 12 to 60-mers was achieved in 35 min using HPCE. Plate numbers for HPCE are in the hundreds of thousands and reproducibility is about 1–2 % (RSD). In comparison with HPLC separations, the resolution of nucleic acids obtained using HPCE is much better than that using HPLC, while reproducibility of HPCE is comparable with that of HPLC.     

Peptide nucleic acid and amino acid modified peptide nucleic acid analysis by capillary zone electrophoresis

A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax‐boric acid (pH 8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50 μmol/L. LOD and LOQ of PNA were 0.2 and 1.0 μmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP–HPLC, and also lower than 94.8% determined with RP–HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 μL PNA in RP–HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.     

Capillary gel electrophoresis of oligonucleotides using polymer solutions

Summary Capillary gel electrophoresis (CGE) has been recognized as an effective method for the analysis of oligonucleotides. CGE using polymer solutions is especially useful and effective compared with that using crosslinked gels, because of easy change of media. Replacement of media leads to the reproducible separation of analytes. We have investigated CGE analysis of oligonucleotides of less than 20 bases employing various kinds of polymers. Polyacrylamide, dextrin, dextran, pullakin, and poly(ethylene glycol) were used as sieving matrixes at concentrations of 0–30 %. Polydeoxythymidylic acids [p(dT)_11–20] were used as a test sample. These small oligonucleotides were successfully resolved on the basis of their base number by CGE using some of these polymer solutions. In particular, dextran was found to be effective and baseline separation was observed when a 30 % dextran solution was employed. Some validations such as linearity and reproducibility were also established and this method was found to be an adequate quality control method for small oligonucleotides. Finally, CGE using a 30 % dextran solution was successfully applied to impurity profiling of some synthetic oligonucleotides.     

Fast separation of single-stranded oligonucleotides by capillary electrophoresis using OliGreen as fluorescence inducing agent

The fast separation of oligonucleotide (oligos) sizing marker by CE using OliGreen and including effects due to the concentration of separation medium and urea denaturant is presented. OliGreen dye is found to be more sensitive than ethidium bromide (by a factor of about 6 based on S/N considerations) for the oligos' separations. Higher concentration of F127 in 1xTris-boricacid-EDTA (TBE) up to 30% w/v leads to better resolution of oligos separations. The addition of urea into the separation medium decreases the sensitivity. With an optimized running condition, the oligos sizing marker could be successfully separated with 1-base resolution within 1.3 min by using 30% w/v F127/1xTBE solution as the separation medium at an applied electric field of 800 V/cm in a 3 cm long capillary, the fastest capillary gel electrophoresis separation with high resolution reported to date for oligos in the similar size range.     

Sequence requirements of oligonucleotide chiral selectors for the capillary electrophoresis resolution of low‐affinity DNA binders

We recently reported that a great variety of DNA oligonucleotides (ONs) used as chiral selectors in partial‐filling capillary electrophoresis (CE) exhibited interesting enantioresolution properties toward low‐affinity DNA binders. Herein, the sequence prerequisites of ONs for the CE enantioseparation process were studied. First, the chiral resolution properties of a series of homopolymeric sequences (Poly‐dT) of different lengths (from 5 to 60‐mer) were investigated. It was shown that the size increase‐dependent random coil‐like conformation of Poly‐dT favorably acted on the apparent selectivity and resolution. The base‐unpairing state constituted also an important factor in the chiral resolution ability of ONs as the switch from the single‐stranded to double‐stranded structure was responsible for a significant decrease in the analyte selectivity range. Finally, the chemical diversity enhanced the enantioresolution ability of single‐stranded ONs. The present work could lay the foundation for the design of performant ON chiral selectors for the CE separation of weak DNA binder enantiomers.     

Capillary zone electrophoresis study of cyclodextrin--lipoic acid host-guest interaction

Lipoic acid is a naturally occurring compound which is being widely investigated for its therapeutic effects in the treatment or prevention of a variety of diseases associated with oxidative injury, particularly diabetes. The diversity of therapeutic applications of lipoic acid requires an appropriate formulation to control its bioavailability, site-targeting delivery and to overcome its inherent chemical instability. In this regard, cyclodextrins (CDs) are ideally suitable due to their well-documented ability to include in their cavity proper guest molecules and protect them from physical or chemical damages. Lipoic acid forms 1:1 inclusion complexes with betaCD as shown in a previous report of an extended investigation that also indicated the suitability of capillary zone electrophoresis (CZE) for the study of such host-guest interactions. In view of these possible applications, we extended the CZE analysis to determine the strength of binding, in a pH 9 phosphate buffer, of lipoic acid with other CD derivatives such as alphaCD, gammaCD and the alkylated derivatives of betaCD, namely (2-hydroxypropyl)-beta-CD (HPbetaCD), and heptakis(2,3,6-tri-O-methyl)-beta-CD (TMbetaCD). Once established that the easily available betaCD is the most suitable receptor for lipoic acid, we set up and here describe a simple and reliable procedure for the quantitative determination of lipoic acid in commercial dietary supplement tablets containing also other active substances and excipients.     

Characterization of single-stranded DNA separation by capillary gel electrophoresis

We have investigated the effect of polymer gel reconditioning, the shape of the capillary, the applied electric field, and the capillary length for single-stranded DNA. The polyethylene oxide gel had deformed under the high electric field causing the degradation of the separation power. By the reintroduction of the fresh polyethylene oxide gel for the next run, one-base resolution was recovered. It turned out that the tip of the capillary at the injection side needed to be clean and symmetric for much improved resolution. Changing DNA motion by the pulsed electric field resulted in the separation of DNA far more than 500 bases.     

Capillary electrophoresis with laser-induced fluorescence detection of main polyamines and precursor amino acids in saliva

A hollow-fiber liquid-phase microextraction （HF-LPME） method has been developed for the purification and preconcentration of biogenic polyamines and their precursor amino acids in human saliva. Putrescine （Put）, cadaverine （Cad）, spermidine （Spe）, ornithine （Orn）, lysine （Lys）, and arginine （Arg） were determined by the CE-LIF detection after microextraction. Several factors that affect extraction efficiency, separation, and detection were investigated. Under the optimum conditions, six analytes could achieve baseline separation within 30 min, exhibiting a linear calibration at three orders of magnitude （r2 〉 0.998）; the obtained enrichment factors of HF-LPME were between 19 （for Orn） and 2] 8 （for Cad）, and the LODs were in the range of 0.0072-0.26 nmol/L. The proposed HF-LPME/CE-LIF method has been successfully applied for the sensitive analyses of the real-world saliva samples collected from healthy volunteers and different patients with oral diseases, providing a potential method for primary non-invasive diagnosis of some oral diseases.     

Capillary electrophoresis for the monitoring of carboxylic acid production by Gluconobacter oxydans

Determination of carboxylic acids in Gluconobacter oxydans fermentations of wheat straw hydrolyzate was carried out. This matrix is of complex composition containing carbohydrates, organic compounds (e.g., amino acids, toxins), and inorganic salts making the analysis challenging even with separation techniques. A method based on capillary electrophoresis with indirect UV detection was developed for the simultaneous quantification of 18 carboxylic acids. The background electrolyte solution of ammonia, 2,3-pyridinedicarboxylic acid, and Ca²⁺ and Mg²⁺ salts, containing myristyltrimethylammonium hydroxide as a dynamic capillary coating reagent, was validated for the robust and repeatable separation of the carboxylic acids. Intraday relative standard deviations in the optimized method were less than 1.6% for migration times and between 1.0% and 5.9% for peak area. Interday relative standard deviations were less than 5.0% for migration times and between 5.7% and 9.3% for peak area. With 11 nl injected, detection limits for the analytes were between 10 and 43 μmol/l. Detection limits ranged from 0.1 to 0.5 pmol at signal-to-noise ratio of 3. The results demonstrated that wheat straw hydrolyzate was a suitable substrate for G. oxydans with a product yield of 45% for the formation of xylonic acid from xylose and 96% for the formation of gluconic acid from glucose.     

Capillary electrophoresis of DNA for molecular diagnostics

A number of applications of capillary zone electrophoresis (CZE) in sieving liquid polymers (notably linear polyacrylamides and cellulose) for the analysis of polymerase chain reaction (PCR) products of clinically relevant, diagnostic DNA, are reviewed. The fields covered are: human genetics, quantitative gene dosage, microbiology and virology, forensic medicine and therapeutic DNA (notably, antisense nucleotides). Some unique, novel developments are highlighted, such as: (i) nonisocratic CZE, i.e., temperature-programmed CZE for detection of DNA point mutations; (ii) the synthesis of novel N-substituted acrylamides, offering extreme resistance to alkaline hydrolysis coupled to high hydrophilicity. In the field of denaturing gradient gel electrophoresis (DGGE), as routinely performed in gel slabs, a novel methodology is described in CZE: double-gradient DGGE. In this technique, two gradients are simultaneously applied along the migration direction: a chemical (or thermal) denaturing gradient, for partially unwinding homo- and hetero-duplexes of DNA, and a porosity gradient, for recompacting diffuse bands melting over a broader range of denaturing conditions. It is thus demonstrated that chemical gradients, in addition to temperature gradients, can be easily implemented even in a capillary format.     

Isolation of cycloamylose by iodine affinity capillary electrophoresis

In contrast to α-, β- and γ-cyclodextrins, little information is available on the isolation and separation of cycloamylose (CA) with degree of polymerization (DP) larger than 22. The objective of the current study was to develop a new iodine affinity capillary electrophoresis (CE) for separation of CA with DP of 22-42, which was based on the formation of CA-iodine inclusion complexes, CA with twisted conformations made complicated mobility behaviors on CE instead of merely size dependent. The influences of iodide/iodine ratio, iodine concentration, pH, ion strength of running phosphate buffer, voltage, and temperature on the peak resolution and electrophoretic mobility were further investigated. Our results suggest that iodine affinity capillary electrophoresis provides a versatile and selective tool for the isolation and analysis of CA with DP from 22 to 42.     

Capillary electrophoresis separation of peptide diastereomers that contain methionine sulfoxide by dual cyclodextrin‐crown ether systems†

A dual‐selector system employing achiral crown ethers in combination with cyclodextrins has been developed for the separation of peptide diastereomers that contain methionine sulfoxide. The combinations of the crown ethers 15‐crown‐5, 18‐crown‐6, Kryptofix® 21 and Kryptofix® 22 and β‐cyclodextrin, carboxymethyl‐β‐cyclodextrin, and sulfated β‐cyclodextrin were screened at pH 2.5 and pH 8.0 using a 40/50.2 cm, 50 μm id fused‐silica capillary and a separation voltage of 25 kV. No diastereomer separation was observed in the sole presence of crown ethers, while only sulfated β‐cyclodextrin was able to resolve some peptide diastereomers at pH 8.0. Depending on the amino acid sequence of the peptide and the applied cyclodextrin, the addition of crown ethers, especially the Krpytofix® diaza‐crown ethers, resulted in significantly enhanced chiral recognition. Keeping one selector of the dual system constant, increasing concentrations of the second selector resulted in increased peak resolution and analyte migration time for peptide‐crown ether‐cyclodextrin combinations. The simultaneous diastereomer separation of three structurally related peptides was achieved using the dual selector system.     

Capillary electrophoresis of synthetic peptide standards varying in charge and hydrophobicity

A mixture of eight structurally closely related synthetic peptides as capillary electrophoretic (CE) standards is introduced. The almost identical mass-to-charge ratio of the standards, coupled with their random-coil (i.e., no secondary structure) nature, offer a potent analytical test for CE to separate peptides varying only subtly in hydrophobicity. Parameters varied to effect a separation included background electrolyte concentration, temperature, applied voltage in capillary zone electrophoresis (CZE in uncoated capillaries), as well as the introduction of hydrophobic mechanisms to the separation either through the use of micelles or C8-coated capillaries. Our step-by-step approach culminated in an optimized combination of a CZE mechanism for separation of differently charged peptide groups (based on common mass-to-charge ratio) and an ion-pairing mechanism (effecting a separation within each group of identically charged peptides), which we have termed ion-interaction CZE or II-CZE. The study clearly shows how the peptide standards allow an excellent assessment of the resolving power of CE.     

A covalent modified hydrophilic capillary for enhanced capillary electrophoresis of biopolymers

δ-Gluconolactone was covalently coupled to aminopropyl derivatized capillary,which created hydrophilic brushes on the inner wall of the capillary.The coated capillary was shown to generate a stable electroosmotic flow(EOF) in the investigated pH range of 2.0-9.0 and to suppress effectively the adsorption of proteins.And it enabled separation of some biopolymer mixtures including basic proteins,DNA and tryptic digested bovine serum albumin(BSA) within 15 min with efficiencies up to 450,000 plates/m.The in...     

Capillary electrophoresis with contactless conductivity detection for uric acid determination in biological fluids

The suitability of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) for the direct determination of uric acid in human plasma and urine was investigated. It was found that a careful optimization of the buffer composition and pH was necessary to achieve selective determination in the complex sample matrices. An electrolyte solution consisting of 10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM histidine and 0.1 mM hexadecyltrimethylammonium bromide (CTAB), pH 6.0, was finally found suitable for use as running buffer for both sample matrices. The limit of detection (3 S/N) was determined as 3.3 μM. The linearity of the response was tested for the range between 10 and 500 μM and a correlation coefficient of 0.9996 was obtained. Intra- and inter-day variabilities were <10%. Quantitative analysis of urine and plasma samples showed a good correlation with the routine enzymatic method currently used at the University Hospital of Basel.     

Capillary electrophoresis of liposomes functionalized for protein binding

CE enabled assessing the attachment of hexa-histidine-tagged proteins to functionalized phospholipid liposomes. The liposomes were made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, phosphatidyl-ethanolamine, cholesterol and distearoyl-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) in a molar ratio of 29:26:40:5. The unilamellar vesicles, which had an average diameter of 170 nm, were labelled by inclusion of FITC-dextran for fluorescence detection. CE was carried out in poly(vinyl alcohol) (PVA)-coated capillaries at 25 degrees C with a BGE consisting of Tris-HCl (50 mM, pH 8.0). For conjugation of the liposomes with the proteins (soluble synthetic receptor fragments with molecular mass of 60 and 70 kDa, respectively), Ni(2+) was implanted into the vesicle surface by an anchor lipid containing a nitrilotriacetate acid (NTA) group as complexation agent for the metal ions. The difference in surface charge enabled the separation of the different species of interest by CE: plain vesicles, vesicles functionalised with Ni-NTA, vesicle-protein complexes and the species formed upon removal of the Ni-ions by complexation with EDTA. Loss of the Ni-ions resulted in the release of the proteins and the reappearance of the plain Ni-free NTA-liposome species in the electropherograms.     

Determination of hydroxy acids in cosmetics by chemometric experimental design and cyclodextrin‐modified capillary electrophoresis

A CD‐modified CE method was established for quantitative determination of seven hydroxy acids in cosmetic products. This method involved chemometric experimental design aspects, including fractional factorial design and central composite design. Chemometric experimental design was used to enhance the method's separation capability and to explore the interactions between parameters. Compared to the traditional investigation that uses multiple parameters, the method that used chemometric experimental design was less time‐consuming and lower in cost. In this study, the influences of three experimental variables (phosphate concentration, surfactant concentration, and methanol percentage) on the experimental response were investigated by applying a chromatographic resolution statistic function. The optimized conditions were as follows: a running buffer of 150 mM phosphate solution (pH 7) containing 0.5 mM CTAB, 3 mM γ‐CD, and 25% methanol; 20 s sample injection at 0.5 psi; a separation voltage of ?15 kV; temperature was set at 25°C; and UV detection at 200 nm. The seven hydroxy acids were well separated in less than 10 min. The LOD (S/N = 3) was 625 nM for both salicylic acid and mandelic acid. The correlation coefficient of the regression curve was greater than 0.998. The RSD and relative error values were all less than 9.21%. After optimization and validation, this simple and rapid analysis method was considered to be established and was successfully applied to several commercial cosmetic products.     

Capillary electrophoresis coupled to chemometrics for analysis of carbon dots in nanoparticles mixtures

Since their discovery in 2004, carbon dots (CDs) have attracted attention due to their intrinsic physicochemical properties and the easy synthesis from simple precursors. However, quantification of CDs in mixtures of nanoparticles with similar sizes and surface functionality is still a challenging issue for research applications or regulatory purposes. In this work, CDs and silver nanoparticles were first synthesized under alkaline conditions by using glucose as precursor and capping agent, respectively. Mixtures of these nanoparticles were made at micromolar range, without purification, and then analyzed by CE–DAD, using an electrolyte solution composed of 20 mM sodium borate and 20 mM SDS at pH 8.5, in a total time of <15 min. The three-way electrophoretic data were then decomposed by advanced chemometric models, parallel factor analysis and multivariate curve resolution–alternating least-squares. The explained variances for both models were 95.8% (parallel factor analysis) and 85.3% (multivariate curve resolution–alternating least-squares). In both cases, the quality of the results was verified by the root mean square standard deviation coefficient variation, which resulted lower than 5%, and no significant bias was observed at 95% of statistical confidence. Satisfactory prediction for CDs concentration was obtained with recovery values between 80.0% and 115%.     

Capillary zone electrophoresis for simultaneous determination of seven nonsteroidal anti-inflammatory drugs in pharmaceuticals

A simple capillary zone electrophoresis (CZE) method has been developed for analyzing seven nonsteroidal anti-inflammatory drugs (NSAIDs)—sulindac (SU), ketoprofen (KE), indomethacin (IN), piroxicam (PI), nimesulide (NI), ibuprofen (IB), and naproxen (NA). The separation was run using borate buffer (60 mmol L^–1, pH 8.5) containing 13% (v/v) methanol at 20 kV, and detected at 200 nm. Several conditions were studied, including concentration and pH of borate buffer, methanol percentage, and separation voltage. In method validation, the calibration plots were linear over the range 40.0–500.0 mol L^–1. In intra-day and inter-day analysis, relative standard deviations (RSD) and relative errors (RE) were all less than 5%. The limits of detection were 10 mol L^–1 for SU, IN, PI, and 20 mol L^–1 for KE, NI, IB, NA (S/N = 3, sampling 6 s by pressure). All recoveries were greater than 95%. This method was applied to the quality control of six NSAIDs in pharmaceuticals using NI as internal standard (IS). The assay results were within the labeled amount required by USP 25.     

Capillary electrophoresis for the direct determination of cephalosporins in clinical samples

Summary The possibility of determination of four cephalosporin antibiotics in clinical samples by capillary electrophoresis has been investigated. The separation conditions for capillary zone electrophoresis (CZE) were studied in detail. The precision of migration times measured by use of the optimized method was satisfactory (RSD<1%) and response was linearly dependent on concentration over the approximate range 2–150 mg L⁻¹ for all the compounds studied (cefuroxime, cefotaxime, ceftriaxone, and ceftazidime). Complete separation could be achieved within 5 min. The CZE method was found to be highly suitable for direct determination of the antibiotics in clinical samples such as wound drainage, cerebrospinal fluid, and urine; for serum, however, the use of micellar electrokinetic capillary chromatography (MECC) was more advantageous. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001