Polyamidoamine‐grafted silica nanoparticles as pseudostationary phases for capillary electrochromatographic separation of proteins

Polyamidoamine‐grafted silica nanoparticles were synthesized, characterized and investigated for the feasibility as pseudostationary phases in alkaline buffer for separation of cationic and anionic proteins, viz., lysozyme, cytochrome C, gamma globulin, and myoglobin. Neither bare silica nanoparticles nor polyamidoamines nor their mixtures as pseudostationary phases could lead to simultaneous separation of the four proteins. However, polyamidoamine‐grafted silica nanoparticles not only suppressed the irreversible wall adsorption of the cationic lysozyme and cytochrome C, but also provided selectivity toward all the proteins. We found that polyamidoamine generation two‐modified silica nanoparticles were the optimum pseudostationary phases with respect to detection sensitivity and separation efficiency; presence of the nanoparticles at 0.01% in the running buffer of 12.5 mM tetraborate/phosphate at pH 9.1 resulted in baseline resolution of the four proteins.     

Vesicles as pseudostationary phase for enantiomer separation by capillary electrophoresis

The suitability of noncovalently bilayer-coated capillaries for the analysis of proteins by capillary electrophoresis (CE) at medium pH was investigated. Fused-silica capillaries were coated simply by successively flushing with a polybrene (PB) and a poly(vinyl sulfonate) (PVS) solution. A protein test mixture was used to evaluate the performance of the coated capillaries. Comparisons with bare fused-silica capillaries were made. Several background electrolytes (BGEs) were tested in combination with the PB-PVS coating, showing that optimum performance was obtained for the proteins using high BGE concentrations. With a 300 mM Tris phosphate buffer (pH 7.0), good plate numbers (150,000-300,000), symmetrical peaks, and favorable migration-time repeatabilities (RSDs below 0.8%) were obtained for the proteins. Using bare fused-silica capillaries, the protein peaks were significantly broadened and the migration-time RSDs often exceeded 5%. It is concluded that the PB-PVS coating effectively minimizes adverse protein adsorption and provides a very stable electroosmotic flow (EOF). We also investigated the potential of a commercially available bilayer coating (CEofix) for protein analysis. It is demonstrated that with this coating, good plate numbers and peak symmetries for proteins can be achieved when the CEofix BGE ("accelerator") is replaced by a common BGE such as sodium or Tris phosphate. Apparently, the negatively charged polymer present in the "accelerator" interacts with the proteins causing band broadening. The utility of the bilayer coatings is further illustrated by the separation of proteins such as interferon-alpha 2b, myoglobin and carbonic anhydrase, by the analysis of a degraded insulin sample in time, and by the profiling of the glycoprotein ovalbumin. In addition, it is demonstrated that even in the presence of concentrations of human serum albumin in the sample of up to 60 mg/mL, the PB-PVS coating still provides reproducible protein separations of good performance.     

Amphiphilic silica nanoparticles as pseudostationary phase for capillary electrophoresis separation

Amphiphilic silica nanoparticles surface-functionalized by 3-aminopropyltriethoxysilane (APTES) and octyltriethoxylsilane (OTES) were successfully prepared and characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectrometry (FT-IR) and thermogravimetry (TG) techniques. The potential use of these bifunctionalized nanoparticles as pseudostationary phases (PSPs) in capillary electrophoresis (CE) for the separation of charged and neutral compounds was evaluated in terms of their suitability. As expected, fast separation of representative aromatic acids was fulfilled with high separation efficiency, because they migrate in the same direction with the electroosmotic flow (EOF) under optimum experimental conditions. Using a buffer solution of 30mmol/L phosphate (pH 3.0) in the presence of 0.5mg/mL of the synthesized bifunctionalized nanoparticles, the investigated basic compounds were baseline-resolved with symmetrical peaks. Due to the existence of amino groups on the surface of nanoparticles, "silanol effect" that occurs between positively charged basic analytes and the silanols on the inner surface of capillary was greatly suppressed. Furthermore, the separation systems also exhibited reversed-phase (RP) behavior when neutral analytes were tested.     

Silica nanoparticles as pseudostationary phase for protein separation

This paper investigated the potential use of silica nanoparticles (SNPs) as pseudostationary phase (PSP) for protein separation. The wall adsorption of SNPs as well as the influences of SNPs and poly(ethylene oxide) (PEO) were studied. The SNPs showed selectivity toward the proteins, and the concentration ratio of SNPs to PEO influenced the separation. The proteins that could not be baseline-resolved under conventional CZE mode were separated in a buffer consisting of 30 mM phosphoric acid, 0.05% PEO, and 0.05% SNPs at pH 2.37, with detection limits ranging from 2 to 45.5 ppm. The intraday and interday RSDs of the migration times were in the ranges of 2.1-2.8% (n = 5) and 2.5-3.4% (three days, n = 3 x 5 = 15), respectively.     

Copolymer solutions as separation media for DNA capillary electrophoresis

Capillary electrophoresis techniques offer high plate numbers and are highly suited for the efficient separations of a wide variety of chemical components in diverse matrices. Because of the small capillary and detection cell dimensions, together with the minute volumes of samples to be injected, sensitive detection schemes based on different physicochemical principles are being developed. One logical approach to increased sensitivity in capillary electrophoresis detection has been the development of chemiluminescence-based detectors. The development of on-line ultrasensitive chemiluminescence detection (referred to the concentration detection limit of nM order of magnitude or mass detection limit of amol order of magnitude) in capillary electrophoresis system is reviewed. The applications and limitations of the current detection methodology are briefly considered and future prospects for the development are discussed.     

Diamino moiety functionalized silica nanoparticles as pseudostationary phase in capillary electrochromatography separation of plant auxins

A novel and simple method for the preparation of silica nanoparticles having surface-functionalized diamino moiety (dASNPs) was reported in our paper and characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectrometry, and thermogravimetry techniques. To test this method practically, in this contribution we describe the enhanced separation of four plant auxins - indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (dCPAA), and 2-(1-naphthyl) acetic acid (NAA) - by capillary electrochromatography using diamino moiety functionalized silica nanoparticles as pseudostationary phase (PSP) in the running buffer. The effect of pH, buffer concentration, and diamino moiety functionalized silica nanoparticles concentration on the selectivity of separation was investigated. A combination of the nanoparticles and running buffer reversed the electroosmotic direction making possible the rapid and efficient separation of the auxins from the auxins migrated in the same direction with the EOF under optimum experimental conditions. A good resolution of four auxins was obtained within 5.5 min under optimum experimental conditions. The precision (RSD, n = 5) was in the range of 0.72-0.91% and 1.89-2.23% for migration time and peak area response, respectively. The detection limits were 0.48, 0.44, 0.46, and 0.42 μM for NAA, IBA, IAA, and dCPAA, respectively. Furthermore, the method was successfully tested for the determination of IAA in the grapes.     

New particle-loaded monoliths for chiral capillary electrochromatographic separation

Fritless particle-loaded monoliths for chiral capillary electrochromatographic (CEC) separation were prepared. Silica particles containing a chiral selector are suspended in a monomer solution, which is drawn into the capillary followed by in situ polymerization. Thereby the silica-based particles containing the chiral selector are embedded in a nonchiral continuous bed. This kind of chiral stationary phase is inexpensive, easy, and reproducible to prepare and circumvents the preparation of frits. As a model, teicoplanin aglycone as chiral selector bonded to 3 microm silica particles was used. The applicability of this approach is demonstrated by means of the chiral separation of aliphatic and aromatic amino acids and dipeptides. As a further application, the chiral selector ristocetin A bonded to 3 microm silica particles was used for the enantiomeric separation of chiral alpha-hydroxy acids. Since alpha-hydroxy acids migrate toward the anode, a cationic charge-providing agent was copolymerized with the matrix. This served to reverse the direction of the electroosmatic flow (EOF).     

Surfactant-coated single-walled carbon nanotubes as a novel pseudostationary phase in capillary EKC

The analytical potential of the use of surfactant-coated single-walled carbon nanotubes (SC-SWNTs) as pseudostationary phase in CE is described. The pseudostationary phase shows an efficient alternative in enhancing electrochromatographic resolution of compounds which are capable of interacting with a nanotube surface, such as aromatic compounds. In general, the resolution is enhanced by increasing nanotube concentration in the buffer but the maximum amount of SWNTs that can be added to background electrolyte was found limited by compatibility with the UV/visible detection. As an alternative, a low-extension partial filling was used, consisting of the introduction into the capillary of concentrated SC-SWNT, just before the sample, with a plug length similar to the sample one. This has been showed as a reliable procedure in increasing resolution and sensitivity by sweeping phenomena. Finally, the potential of SC-SWNTs to perform chiral separations is discussed.     

A novel monolithic column for capillary electrochromatographic separation of oligopeptides

A monolithic column was prepared using l-phenylalanine as template and a covalent approach through the formation of Schiff base with o-phthalaldehyde (OPA). OPA, allylmercaptan, l-phenylalanine, and triethylamine were stirred at first, then methacrylic acid, 2-vinylpyridine, ethyleneglycol dimethacrylate, α,α-azobisisobutyronitrile, and 1-propanol were added to the reaction mixture. The resulting material was introduced into a capillary column. Following thermal polymerization, the template was then extracted with a mixture of HCl and methanol. The column was employed for the capillary electrochromatographic separation of oligopeptides. A capillary column of 75 (50) cm × 75 μm ID with a mobile phase of phosphate buffer (pH 7.0, 40 mM)/methanol (5%, v/v), an applied voltage of +15 kV, and detection at 214 nm, could baseline separate angiotensin I, angiotensin II, [Sar¹, Thr⁸] angiotensin, oxytocin, vasopressin, tocinoic acid, β-casomorphin bovine, β-casomorphin human, and FMRF amide within 20 min. The separation behavior of the templated polymer was also compared with that of the non-templated polymer. As a result, it can be concluded that the electrochromatographic separation of this set of peptides was mediated by a combination of electrophoretic migration and chromatographic retention involving hydrophobic, hydrogen bonding, electrostatic as well as the Schiff base formation with OPA in the cavity of the templated polymer.     

Capillary electrochromatographic separation of bovine milk proteins using a G-quartet DNA stationary phase

DNA oligonucleotides that form G-quartet structures were used as stationary phase reagents for separation of bovine milk proteins, including alpha-casein, beta-casein, kappa-casein, alpha-lactalbumin and beta-lactoglobulin. Both artificial protein mixtures and a skim milk sample were analyzed. The separations were performed using open-tubular capillary electrochromatography, in which the oligonucleotides were covalently attached to the inner surface of a fused-silica capillary. Better resolution was achieved using the G-quartet-coated capillaries than was achieved using either a bare capillary or a capillary coated with an oligonucleotide that does not form a G-quartet structure. A 4-plane G-quartet-forming stationary phase was able to resolve three peaks for alpha-casein and to detect thermal denaturation of the proteins in the milk sample. The results suggest that G-quartet stationary phases could be used to separate very similar protein structures, such as those arising from genetic variations or post-translational modifications.     

Molecularly imprinted microparticles for capillary electrochromatographic enantiomer separation of propranolol

Molecularly imprinted microparticles imprinted against (S)-propranolol were synthesised and studied for use in capillary electrochromatographic separation of propranolol enantiomers. The imprinted microparticles were in the size range of 0.2-0.5 micron as determined by scanning electron microscopy. The microparticles were suspended, in high concentration, in the electrolyte and used to perform enantiomer separation by a partial filling technique.     

Titanium dioxide nanoparticles-coated column for capillary electrochromatographic separation of oligopeptides

A novel column made through the condensation reaction of TiO2 nanoparticles (TiO2 NPs) with silanol groups of the fused-silica capillary is described. EOF measurements under various buffer constitutions were used to monitor the completion of reactions. The results indicated that the EOF was dependent on the interactions between buffers and the bonded TiO2 NPs. With formate/Tris buffer, EOF reversal at pH below 5 and cathodic EOF at pH above 5 were indicated. The pI of the bonded TiO2 NPs was found at approximately ph 5. Only cathodic EOF was illustrated by substituting the mobile phase with either glutamate or phosphate buffer. It was elucidated that both glutamate and phosphate buffer yield a negative charge layer on the surface of TiO2 NPs attributable to the formation of a titanium complex. The CEC performance of the column was tested with angiotensin-type oligopeptides. Some parameters that would affect the retention behavior were investigated. The interactions between the bonded phases and the analytes were explicated by epitomized acid-base functional groups of the oligopepetides and the speciation of the surface oxide in different pH ranges. The average separation efficiencies of 3.1 x 10(4) plates/m is readily achieved with a column of 70 cm (50 cm) x 50 mum ID under an applied voltage of 15 kV, phosphate buffer (pH 6.0, 40 mM), and UV detection at 214 nm.     

Preparation of a positively charged cellulose derivative chiral stationary phase with copolymerization reaction for capillary electrochromatographic separation of enantiomers

A positively charged chiral stationary phase (CSP) was prepared by chemically immobilizing cellulose 3,5-dimethylphenylcarbamate onto methacryloyldiethylenetriaminopropylated silica (MCDEAPS) via a radical copolymerization reaction. The prepared CSP was evaluated for enantiomer separation in nonaqueous capillary electrochromatography (CEC). Electroosmotic flow (EOF) generated on the prepared CSP could be significantly improved with introduction of positive charges into the CSP, and separation of enantiomers in CEC has been achieved with mobile phases of ethanol and hexane-ethanol, respectively. In addition, we investigated the solvent versatility of the immobilized CSP on enantioseparations in CEC and capillary liquid chromatography (CLC) due to the elimination of dissolution of chiral selector in a number of solvents. Chiral resolution of some enantiomers was improved by adopting tetrahydrofuran (THF) and chloroform as mobile-phase modifiers, respectively.     

Chiral ion-exchange capillary electrochromatography of arylglycine amides with dextran sulfate as a pseudostationary phase

A low-cost tunable chiral ion-exchange capillary electrochromatographic method has been developed for the separation of arylglycine amide racemic mixtures with dextran sulfate (DS) as an anionic and chiral pseudostationary phase and Tris-tartrate as a buffer system. The concentrations of DS and Tris had opposite influences on retention and resolution and could serve as ideal factors to finely tune the running speed and chiral resolution. Tartrate and pH largely impact the separation but pH should be confined within 3.0-5.5, only suitable for coarse tuning, while tartrate was preserved as the key buffering reagent, normally maintained at 40 mmol/L. With a working system composed of 0.1-1.0% DS, 20-60 mmol/L Tris, and 40 mmol/L tartrate at pH 3.50-4.50, the enantioresolution of arylglycine amides was shown to be dependent on their chemical structure: The chiral resolution increased when the hydrogen at the alpha-amino group or at the p-position of phenyl ring was replaced by other larger group(s) but the resolution decreased when the group at the o- or m-site on the phenyl ring was enlarged. Further, the electronegative substitute of -Cl had larger resolution increment than methyl or methoxy at the position p- of phenyl ring but much lower increment at position m-. It is possible to well explain the resolution variation phenomenon by considering the group resistance and the variation of hydrogen-bonds formed inside the amino amides and between the solutes and DS. The amido group was shown irreplaceable to have chiral resolution with DS alone as an ionic and chiral pseudostationary phase.     

DNA mutation analysis based on capillary electrochromatography using colloidal poly(N-isopropylacrylamide) particles as pseudostationary phase

Poly(N-isopropylacrylamide) (PNIPAM) is an interesting class of temperature sensitive, water soluble polymer that has a lower critical solution temperature (LCST) of 32 °C. Above the LCST, PNIPAM gets phase-separated and precipitates out from water. The fascinating temperature-sensitive property of PNIPAM has led to a growing interest in diverse fields of applications. Recently, capillary electrochromatography (CEC) has gained attention due to the wide range of applications based on the use of open tubular capillaries. In this paper, the use of phase-separated PNIPAM as a pseudostationary phase for CEC is demonstrated for the detection of single nucleotide polymorphisms (SNPs). Owing to the dynamic coating, the phase-separated PNIPAM particles did not require any immobilization technique and could exist as a mobile stationary phase in the open tubular capillary. The heteroduplex analyses of mutation samples could be successfully performed based on the phase-separated PNIPAM particles in the constructed CEC system. The CEC system, based on PNIPAM particles capable of having a narrow size distribution, shows great potential as an alternative to conventional DNA mutation systems.     

Pressurized gradient capillary electrochromatographic separation of eighteen amino acid derivatives

A pressurized gradient capillary electrochromatography (pCEC) instrument was developed to separate 18 amino acid derivatives. A reversed-phase C18 column (3 microm, 130 mm x 75 microm I.D.) and an acetate buffer (50 mmol/l NaAc, pH 6.4) with an ion-pair reagent (1% N,N-dimethylformamide) were used to separate derivatized amino acids from a standard solution (2 microg/ml), and the wavelength of the UV-Vis detector was 360 nm. The pressure on the capillary column was kept at approx. 70 Pa and 3 kV positive voltage was added on the outlet end of column. The effect of voltage on the eluting order of amino acids and the resolution of separation were studied, and it was found that when the voltage was higher than 3 kV, the adsorption of amino acids in the porous C18 column occurred. The effect of salt concentration, injection volume, and column length on the separation of amino acids was determined. The amino acid sample was separated by pCEC, and RSDs of the migration times of each amino acid were all less than 2.5%.     

Validation of a chiral capillary electrochromatographic method for metoprolol on a teicoplanin stationary phase

Summary A chiral capillary electrochromatographic (CEC) method for determination of the enantiomeric purity of either enantiomer of metoprolol has been validated. High resolution and efficiency separations (R _s =2.5 and 80000 plates m⁻¹, respectively) were achieved by use of a teicoplanin chiral stationary phase in the polar organic mode. Method validation showed that detection linearity, robustness, accuracy, and repeatability were adequate. The method was also shown to be sufficiently sensitive for the determination of a minor enantiomer; the limit of quantitation (LOQ) was determined to be 0.09% of the peak area of the enantiomer under investigation. A similar commercial column was subsequently evaluated by use of the validated method and found to yield results for metoprolol comparable with those obtained on the homepacked columns. Acceptable separations on this commercial column were also obtained for other β-blocking drugs; those for alprenolol were particularly noteworthy (R _S =3.8 and 265000 plates m⁻¹).     

A macrocyclic polyamine as an anion receptor in the capillary electrochromatographic separation of carbohydrates

An analytical approach of the 32-membered macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N8) was described for the capillary electrochromatographic (CEC) separation of derivatized mono- and disaccharides. The column displayed reversal electroosmotic flow (EOF) at pH below 7.0, while a cathodic EOF was shown at pH above 7.0. The reductive amination of saccharides was carried out with p-aminobenzoic acid. Some parameters that affect the CEC separations were investigated. Several competitive ligands, such as Tris, EDTA and phosphate were also examined for the effect on the performance. We achieved a complete separation of all compounds as well as the excess derivatizing agent by using borate buffer (pH 9.0) in a mode of concentration gradient (60 mM inlet side and 70 mM outlet side). The relative standard deviation of the retention time measured for each sample was less than 4% in six continuous runs, suggesting that the bonded phase along with the gradient formed inside the column was quite stable. With the mixing modes of anion coordination, anion exchange, and shape discrimination, the interaction adequately accomplishes the separation of carbohydrates which are epimers or have different glycosidic linkage, although the electrophoretic migration is also involved in the separation mechanism.     

Cationic double-chained surfactant as pseudostationary phase in micellar electrokinetic capillary chromatography for drug separations

The cationic double-chained surfactant didodecyldimethylammonium bromide (DDAB) was used as pseudostationary phase (PSP) in micellar electrokinetic capillary chromatography (MEKC). Its performance on the three kinds of drugs, i.e., basic, acidic, and neutral drugs, was systematically investigated. Nicotine, cotinine, caffeine, lidocaine, and procaine were selected as the model basic drugs. Good baseline separation and high efficiency were obtained under the optimal separation condition that consisted of 50mM phosphate (pH 4.0) and 0.08 mM DDAB. Three basic phenylenediamine isomers can also be well separated with DDAB in buffer. In addition, DDAB can form cationic bilayer on the capillary wall, thus the wall adsorption of basic analytes was greatly suppressed. Compared with commonly used CTAB, the separation of basic drugs was significantly improved with a much lower amount of DDAB present in the buffer. The DDAB-involved MEKC also showed superiority to CTAB upon the separation of acidic drugs, amoxicillin and ampicillin. In the case of neutral compounds, a good separation of resorcinol, 1-naphthol and 2-naphthol was achieved with 0.1mM DDAB and 30% (v/v) acetonitrile (ACN) present in buffer. Hence, it was concluded that the double-chained cationic surfactant DDAB can be a good substitute for traditional single-chained surfactant CTAB in MEKC.