Free Solution Capillary Electrophoresis Research Articles

Quantitative Analysis in Capillary Electrophoresis: Transformation of Raw Electropherograms into Continuous Distributions

Quantitative analysis in capillary electrophoresis based on time-scale electropherograms generally uses time-corrected peak areas to account for the differences in apparent velocities between solutes. However, it could be convenient and much more relevant to change the time-scale electropherograms into mass relative distribution of the effective mobility or any other characteristic parameter (molar mass, chemical composition, charge density, ...). In this study, the theoretical background required to perform the variable change on the electropherogram was developed with an emphasis on the fact that both x and y axes should be changed when the time scale electropherograms are modified to get the distributions. Applications to the characterization of polymers and copolymers by different modes of capillary electrophoresis (CE) are presented, including the molar mass distribution of poly-L-lysine oligomers by capillary gel electrophoresis (CGE), molar mass distribution of end-charged poly-l-alanine by free solution CE, molar mass distribution of evenly charged polyelectrolytes by CGE, and charge density distribution of variously charged polyelectrolytes by free solution CE.

Purity of double hydrophilic block copolymers revealed by capillary electrophoresis in the critical conditions

Block copolymers enable combining properties of different polymers; double hydrophilic block copolymers are innovative examples. Size-exclusion chromatography (SEC or GPC) has a quasi-monopoly in separation-based characterization methods for polymers, including block copolymers. However, in terms of purity determination (unintended homopolymers present in the copolymers), SEC resolution proves insufficient except for the extreme compositions for which the second block is much larger than the first one. The free solution capillary electrophoresis (capillary zone electrophoresis) technique does not separate charged homopolymers by their molar mass and we thus named the corresponding method capillary electrophoresis in the critical condition (CE–CC). CE–CC provides a means to assess the purity of poly(acrylic acid-b-acrylamide) – P(AA-b-AM) – copolymers, as well as of the more challenging cationic poly(acrylamido-N-propyltrimethylammonium chloride-b-N-isopropylacrylamide)—P(APTAC-b-NIPAM). In addition it can identify that a block copolymer has been produced. It is to be noted that P(APTAC-b-NIPAM) block copolymers cannot be eluted in SEC due to their exceptional ability to adsorb onto surfaces, while some information is obtained from CE–CC. Both possible parent homopolymers can be detected and their quantity estimated in a single injection by CE–CC. In both cases, one of the parent homopolymers is neutral and comes with the electro-osmotic flow. If the electro-osmotic flow is weak (conditions used for the cationic copolymer) then pressure assisted CE–CC is used to detect this homopolymer.

Thermodynamics-based Rational Design of DNA Block Copolymers for Quantitative Detection of Single-Nucleotide Polymorphisms by Affinity Capillary Electrophoresis

Diblock copolymers composed of allele-specific oligodeoxyribonucleotide (ODN) and poly(ethylene glycol) (PEG) are used as an affinity probe of free-solution capillary electrophoresis to quantitatively detect single-base substitutions in genetic samples. During electrophoresis, the probe binds strongly to a wild-type single-stranded DNA analyte (WT) through hybridization, while it binds weakly to its single-base-mutated DNA analyte (MT) due to a mismatch. Complex formation with the probe augments the hydrodynamic friction of either analyte, thereby retarding its migration. The difference in affinity strength leads to separation of the WT, MT, and contaminants, including the PCR primers used for sample preparation. The optimal sequence of the probe's ODN segment is rationally determined in such a way that the binding constant between the ODN segment and MT at the capillary temperature is on the order of 10(6) M(-1). The validity of this guideline is verified using various chemically synthesized DNA analytes, as well as those derived from a bacterial genome. The peak area ratio of MT agrees well with its feed ratio, suggesting the prospective use of the present method in SNP allele frequency estimation.

Physico-chemical characterization of polymeric micelles loaded with platinum derivatives by capillary electrophoresis and related methods

(1,2-diamino-cyclohexane)Platinum(II) ((DACH)Pt) loaded polymeric micelles of poly(ethylene glycol-b-sodium glutamate) (PEG-b-PGlu) are currently studied as a potential candidate to replace oxaliplatin in the treatment of cancers with the aim to reduce side effects like cumulative peripheral distal neurotoxicity and acute dysesthesias. As for all synthetic polymeric drug delivery systems, the characterization of the (co)polymer precursors and of the final drug delivery system (polymeric micelles) is crucial to control the repeatability of the different batches and to get correlation between physico-chemical structure and biological activity. In this work, the use of capillary electrophoresis (CE) and related methods for the characterization of (DACH)Pt-loaded polymeric micelles and their precursor (PEG-b-PGlu copolymer) has been investigated in detail. The separation and quantification of residual PGlu homopolymer in the PEG-b-PGlu sample were performed by free solution capillary zone electrophoresis mode. This mode brought also information on the PEG-b-PGlu copolymer composition and polydispersity. It also permitted to monitor the decomposition of polymeric micelles in the presence of NaCl at room temperature. Interactions between PEG-b-PGlu unimers, on one hand, and polymeric micelles or surfactants, on the other hand, were studied by using the Micellar Electrokinetic Chromatography and Frontal Analysis Capillary Electrophoresis modes. Finally, weight-average hydrodynamic radii of the loaded polymeric micelles and of the PEG-b-PGlu unimers were determined by Taylor Dispersion Analysis (an absolute size determination method that can be easily implemented on CE apparatus).

Separation and Characterization of Synthetic Polyelectrolytes and Polysaccharides with Capillary Electrophoresis

The development of macromolecular engineering and the need for renewable and sustainable polymer sources make polymeric materials progressively more sophisticated but also increasingly complex to characterize. Size-exclusion chromatography (SEC or GPC) has a monopoly in the separation and characterization of polymers, but it faces a number of proven, though regularly ignored, limitations for the characterization of a number of complex samples such as polyelectrolytes and polysaccharides. Free solution capillary electrophoresis (CE), or capillary zone electrophoresis, allows usually more robust separations than SEC due to the absence of a stationary phase. It is, for example, not necessary to filter the samples for analysis with CE. CE is mostly limited to polymers that are charged or can be charged, but in the case of polyelectrolytes it has similarities with liquid chromatography in the critical conditions: it does not separate a charged homopolymer by molar mass. It can thus characterize the topology of a branched polymer, such as poly(acrylic acid), or the purity or composition of copolymers, either natural ones such as pectin, chitosan, and gellan gum or synthetic ones.

Simple and robust monitoring of ethanol fermentations by capillary electrophoresis.

Free-solution capillary electrophoresis (CE), or capillary zone electrophoresis, with direct UV detection was used for the first time for the determination of mono- and disaccharides, sugar alcohols, and ethanol in fermentation broths. Sample preparation proved to be minimal: no derivatization or specific sample purification was needed. The CE conditions can be adapted to the type of fermentation by simply altering the background electrolyte (BGE). KOH (130 mM) or NaOH (130 mM) as the BGE led to the fastest analysis time when monitoring simple fermentations. A mixture of 65 mM NaOH and 65 mM LiOH led to a 19% improvement in resolution for a complex mixture of carbohydrates. Quantification of a simple carbohydrate fermentation by CE showed values in close agreement with that of high-performance anion exchange chromatography and high-performance liquid chromatography (HPLC) on a cation exchange resin. For complex fermentations, quantification of carbohydrates by HPLC and CE led to similar results, whereas CE requires an injection volume of only 10-20 nL. Analysis of an ethanol fermentation of hydrolyzed plant fiber demonstrated the robustness of the separation and detection of carbohydrates, as well as ethanol. Ethanol determination is achieved by coupling the CE method to pressure mobilization, using the same instrument and the same sample.

Ethanol determination using pressure mobilization and free solution capillary electrophoresis by photo-oxidation assisted ultraviolet detection

Free solution capillary electrophoresis (CE) can separate and quantify carbohydrates using a simple direct UV detection based on a photo-oxidation reaction taking place in the detection window without any labeling. Ethanol interferes with this photo-oxidation reaction. We thus present the first detection and quantification of ethanol using either a simple pressure mobilization set-up or CE. Ethanol can be detected down to 34.9mgL−1 and quantified in the range 117–1850mgL−1 through the interference with photo-oxidization of 2gL−1 sucrose. CE can thus separate and quantify both carbohydrates and ethanol, for example to monitor a lignocellulosic fermentation process. The method is not limited to ethanol and applies to alkyl amines and other alcohols and likely to most molecules possessing the ability to react with free radicals assuming they can be sufficiently separated from each other.

Understanding and improving direct UV detection of monosaccharides and disaccharides in free solution capillary electrophoresis

Direct UV detection of carbohydrates in free solution capillary electrophoresis at 270nm is made possible by a photo-oxidation reaction. Glucose, rhamnose and xylose were shown to have unique UV absorption spectra hypothesizing different UV absorbing intermediates for their respective photo-oxidation. NMR spectroscopy of the photo-oxidation end products proved they consisted of carboxylates and not malondialdehyde as previously theorized and that oxygen thus plays a key role in the photo-oxidation pathway. Adding the photo-initiator Irgacure® 2959 in the background electrolyte increased sensitivity by 40% at an optimum concentration of 1×10−4mM and 1×10−8mM for conventional 50μm i.d. capillaries and for the corresponding extended light path capillaries, respectively.

Separation of chitosan by degree of acetylation using simple free solution capillary electrophoresis

Chitosan is a biopolymer of increasing significance, as well as a renewable and sustainable material. Its main molecular characteristics are molar mass and degree of acetylation (composition). Precise average degrees of acetylation were measured by quantitative (1)H solution-state NMR spectroscopy. While number-average degrees of acetylation had already been determined by (1)H NMR spectroscopy, weight-average degrees of acetylation are also determined and may be more relevant for some properties, such as mechanical properties. We report the first separation of chitosan according to its degree of acetylation using free solution capillary electrophoresis. Capillary electrophoresis separates chitosan in the 'critical conditions': the molar mass plays little role and the separation is by the degree of acetylation. It characterises the heterogeneity of chitosan samples in terms of composition (dispersity of the distribution of degrees of acetylation). This heterogeneity (broad distribution of degrees of acetylation) cannot be neglected contrary to a common assumption found in the literature. This fast and easy separation will allow establishing a structure-property relationships.

Separation of poly(acrylic acid) salts according to topology using capillary electrophoresis in the critical conditions

Branching was detected in polyacrylates synthesised through radical polymerization via solution-state NMR, while inconsistencies have been reported for the determination of the molar mass of hydrophilic polyacrylates using aqueous-phase and organic-phase size-exclusion chromatography. In this work, poly(sodium acrylate)s, PNaAs, of various topologies were separated for the first time using free-solution capillary electrophoresis (CE). Free-solution CE does not separate the PNaAs by their molar mass, similarly to separations by liquid chromatography in the critical conditions, rather by different topologies (linear, star branched, and hyperbranched). The electrophoretic mobility of PNaAs increases as the degree of branching decreases. Separation is shown to be not only by the topology but also by the end groups as expected for a separation in the critical conditions: replacing a relatively bulky nitroxide end group with hydrogen atom yielded a higher electrophoretic mobility. This novel method, capillary electrophoresis in the critical conditions enabled, for the first time, the separation of hydrophilic polyacrylates according to their topology (branching) and their chain ends. This will allow meaningful and accurate characterization of their branched topologies as well as molar masses and progress in for advanced applications such as drug delivery or flocculation.

Simple and robust determination of monosaccharides in plant fibers in complex mixtures by capillary electrophoresis and high performance liquid chromatography

Carbohydrates partially liberated by acid hydrolysis of plant fiber can be separated by hydrophilic interaction liquid chromatography (HILIC), ligand-exchange liquid chromatography or other forms of LC with ion-exchange columns. However, the robust hydrogen-exchange columns show co-elution of galactose, xylose and mannose. Free solution capillary electrophoresis (CE) can be used without derivatization at pH 12.6 and was found to provide a higher resolution of galactose and xylose than common LC with no sample pre-treatment required, other than dilution, within 26min. CE was able to provide resolution higher than 0.79 for all separated carbohydrates, and the RSDs of determined concentrations lower than 10% for concentrations above 1.3gL−1. A quantitative comparison between CE and HPLC revealed that up to 22% more carbohydrates are quantified with CE. Direct UV detection in CE of mono- and disaccharides is unexpectedly possible at 270nm. NMR analysis shows that alkaline degradation is too slow to explain this detection. This CE detection sensitivity is increased by the electric field and our CE and NMR analyses are consistent with a photo-oxidation process.

Heterogeneous modification of chitosan via nitroxide-mediated polymerization

Chitosan (CS) was modified by SG1-based nitroxide-mediated polymerization under heterogeneous conditions. After introduction of acrylamide and/or acrylate functions onto the CS backbone followed by intermolecular 1,2 radical addition of the BlocBuilder alkoxyamine (CS–BB), methyl methacrylate (MMA) in the presence of a small amount of acrylonitrile (AN) or sodium 4-styrenesulfonate (SS) was polymerized by nitroxide-mediated polymerization (NMP). ESR and free-solution capillary electrophoresis confirmed the synthesis of CS–BB. The successful synthesis of CS-g-P(MMA-co-AN) and CS-g-PSS grafted copolymers was proved by TGA and solid-state NMR spectroscopy with ca. 20 to 30 wt% of grafted synthetic polymer in the final product.

Quantitative experimental determination of primer–dimer formation risk by free‐solution conjugate electrophoresis

DNA barcodes are short, unique ssDNA primers that "mark" individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 base-pairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive base-pairs formed, yet non-consecutive base-pairs did not create stable dimers even when 20 out of 30 possible base-pairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation.

Thermal Melting of Duplex DNA in the Slow Exchange Regime

The thermal melting of small DNA duplexes usually occurs as a two-state transition in which the duplex and its component single strands are co-populated throughout the transition. Since a duplex and its single-stranded forms can be resolved by free solution capillary electrophoresis, this technique can be used to monitor the kinetics of denaturation. When a duplex and its component single-strands are in fast exchange, a single peak is observed in the electropherograms, with a mobility that is proportional to the weighted average of the mobilities of the duplex and its component single strands at various temperatures. However, if melting occurs in the slow exchange regime, resolved peaks are observed for the duplex and its component single strands. The area under the peak corresponding to the duplex progressively diminishes with increasing temperature, while the area under the peak(s) characteristic of the single strands progressively increases. Although the melting of small DNA hairpins and duplexes often occurs in the fast exchange regime, the melting of DNA duplexes containing internal loops occurs in the slow exchange regime. The equilibrium and kinetic parameters accompanying the melting of DNA duplexes with internal loops will be reported.

Characterization of Gellan Gum by Capillary Electrophoresis

Gellan gums were characterised for the first time using free-solution capillary electrophoresis (CE) or CE under critical conditions (CE-CC). CE-CC is a fast method that separates the polysaccharide. Gellan gums are shown to be heterogeneous in terms of their electrophoretic mobility at 55°C revealing: oligomer peak(s), broad peaks of polymers with a random coil conformation with different degrees of acylation (composition), aggregates, and polymers with double-helix conformation. CE-CC is complementary with the rheological analysis also performed in this work. Sonication of gellan gums is shown to decrease the viscosity of gellan gum mainly by breaking up aggregates. The effect of sonication is stronger on the high-acyl gellan gum since the latter has a far higher tendency to aggregate.

Synthesis, free solution capillary electrophoresis separation and toxicity of seven potential impurities of dobutamine.

The seven O-methylated analogs of dobutamine [(+/-)-4-[2-[[3-(p-hydroxyphenyl)-1-methylpropyl]amino]ethyl]pyrocatechol, CAS 34368-04-2), a trihydroxy secondary amine, can be considered potential impurities of the latter, the ultimate step of the synthesis of dobutamine being the deprotection of the three phenolic groups. In order to enable the detection and the identification of such a contamination, the di- and the monomethylated derivatives of dobutamine were prepared and their mixture with dobutamine and its trimethoxy precursor completely resolved by Free Solution Capillary Electrophoresis (FSCE). Indeed, the above impurities proved to occur in dobutamine in consequence of improper demethylation conditions and to be significantly more toxic than the latter.

Characterization of nanoparticles by capillary electromigration separation techniques

Considering the large number of papers presented in the last years in the field of CE and plate gel electrophoresis (PGE) of nanoparticles and nanoparticle-biomolecule conjugates, this review article gives an overview on the application of capillary electromigration separation techniques for the separation of nanoparticles and nanoparticle-biomolecule conjugates according to differences in size and/or zeta potential and for the determination of several physicochemical parameters characterizing a population of nanoparticles. A brief introduction is given into the theoretical background of the size-selective electrophoretic separation of nanospheres in free-solution and sieving media including involvement of interparticle interactions. Some aspects of colloidal system stability are discussed. Applications of capillary electromigration techniques in the field of size-selective and/or surface charge density-selective separation of nanoparticles and nanoparticle-biomolecule conjugates are described, including free-solution CE, CGE, IEF, and ITP, complemented by an overview on the application of (semipreparative) PGE in the field of nanotechnology and nanobiotechnology. The review also includes actual developments regarding the coupling of capillary electromigration techniques to (wavelength-resolved) LIF and other selective detection methods and studies concerning individual-particle CE.

Capillary Electrophoretic Analysis of Synthetic Copolymers with Indirect UV Detection and Contactless Conductivity Detection

Methods for the determination of non-UV-absorbing charged copolymers and polyelectrolytes by capillary electrophoresis with indirect UV detection (IUV) and contactless conductivity detection (CCD) were developed. Synthetic poly(ethylene glycol)-poly(methacrylic acid) copolymers (PEG-MAA) with different charge densities were separated in an arginine/sorbate buffer by free solution capillary electrophoresis. Blends of PEG-MAA copolymers and a high molar mass poly(methacrylic acid) sodium salt standard PMAA75k (M w = 75 kg/mol) have been separated, and the effect of the charge density on the detector signals has been investigated. The new experimental protocol enables the simultaneous recording of the CCD and the IUV signal in one single run. The arginine/sorbate buffer provides sufficient signal-to-noise ratios for indirect UV detection and contactless conductivity detection.

Size-Based Characterization of an Ionic Polydiacetylene by Taylor Dispersion Analysis and Capillary Electrophoresis

This work focuses on the size-based characterization of a water-soluble ionic polydiacetylene with a polycationic structure, poly-[(1,6-bis(N-methylimidazolium)hexa-2,4-diyne)dibromide]. This polymer could not be characterized using classical analytical techniques such as size-exclusion chromatography and MALDI−TOF mass spectrometry due to problems of purification, low quantities available, and difficult laser desorption. The work presented here demonstrates the interest and the complementarity of two independent analytical methods, Taylor dispersion analysis (TDA) and capillary electrophoresis (CE), that require only very small amounts of sample (only a few nanoliters are injected) and that can be easily implemented on commercially available capillary electrophoresis apparatus. TDA is a nonseparative method that allows the absolute determination of the average hydrodynamic radius of the polymer. This method does not require the determination of the polymer concentration in the sample and is not perturbed by the presence of residual monomer. Since the average hydrodynamic radius determined by this method is a weight average value, it also gives information complementary to the average value derived from dynamic light scattering measurements. Simple hydrodynamic modeling allows estimation of a minimal value for the average degree of polymerization. Free solution CE can be used for monitoring the polymerization process and quantifying the degree of conversion. Furthermore, entangled polymer solution CE was used as a size-based separation technique for the characterization of the molar mass distribution using calibration with polyvinylpyridine standards. Number and weight molar mass distributions of the sample were obtained relative to this calibration.

Estratégias de pré-concentração em eletroforese capilar (CE): parte 1. Manipulação da velocidade eletroforética do analito

Capillary electrophoresis has become a well-established and routine-based separation technique. It is based on the differences between charged analyte mobility in aqueous or organic electrolytes. Its major limitation is the sensitivity due to small sample injection volumes and the narrow diameter of the capillaries, especially when UV detection is used. There are a number of ways to increase the concentration sensitivity. This report shows some on-line preconcentration strategies to perform it in free solution capillary electrophoresis that are based on manipulation of the analyte electrophoretic velocity during the sample introduction (stacking, field amplification and transient isotachophoresis).