Fe³⁺-immobilized nanoparticle-modified capillary for capillary electrophoretic separation of phosphoproteins and non-phosphoproteins

A fused-silica capillary modified with Fe3?-immobilized magnetic nanoparticles (Fe3?-IMAN) has been investigated for the capillary electrophoretic (CE) separation of phosphoproteins and non-phosphoproteins. The Fe3?-IMAN capillary was achieved by covalently immobilising epoxy-based magnetic silica nanoparticles (160?nm) on the prederivatized 3-aminopropyl-trimethoxysilane (APTMS) fused-silica capillary (75?μm id), followed by disodium iminodiacetate and Fe3?. The buildup process was examined by measuring the streaming potentials of the bare capillary, APTMS capillary, epoxy-based nanoparticle capillary and Fe3?-IMAN capillary by varying the buffer pH. An inverted fluorescence microscope was used to determine the surface features of the Fe3?-IMAN capillary derivatized with morin. Further experimental results confirmed that Fe3?-IMAN bonded on the inner wall of the APTMS capillary could provide sufficient solute-bonded phase interactions to allow for the CE separation of phosphoproteins and non-phosphoproteins at concentration levels down to 50?μg/mL. The highest number of theoretical plates obtained was about 233,000/m, and the relative standard deviation (RSD) for migration times was <2.57% for eight consecutive runs, respectively. Additionally, the Fe3?-IMAN modifing method was also applied to the analyses of bovine milk proteins. With simplicity, high resolving power, and high repeatability, the proposed method has shown great potential for phosphoproteomics applications.     

Analyzing small samples with high efficiency: capillary batch injection–capillary electrophoresis–mass spectrometry

We present an experimental approach to conducting fast capillary electrophoresis-mass spectrometry (CE-MS) measurements of very small samples in the nanoliter range. This is achieved by injecting sample very efficiently into a CE-MS system. Injection efficiency represents the ratio of injected sample to the amount of sample needed for carrying out the injection process (v/v). In order to increase this injection efficiency from typical values of 10(-3) to 10(-7), the concept of capillary batch injection is used to build an automated, small-footprint injection device for CE-MS. This device is capable of running true multi-sample measurement series, using minimal sample volumes and delivering an injection efficiency of up to 100?%. It is compatible with both aqueous and non-aqueous background electrolytes. As an additional benefit, CE-MS separations of a catecholamine model system in capillaries of 15?cm length under conditions of high electric field strength could be accomplished in 20?s with high separation efficiency. This report details design and specifications of the injection device and shows optimal parameter choices for injections with both high injection efficiency and high separation efficiency. Furthermore, a procedure is presented to coat the tip of a fused silica capillary with a silicone elastomer which acts as a seal between two capillaries.     

Sulfated and sulfonated polysaccharide as chiral stationary phases for capillary electrochromatography and capillary electrochromatography–mass spectrometry

The applications of polysaccharide phenyl carbamate derivatives as chiral stationary phases (CSPs) for capillary electrochromatography (CEC) are often hindered by longer retention times, especially using a normal-phase (NP) eluent due to very low electroosmotic flow (EOF). Therefore, in this study, we propose an approach for the aforementioned problems by introducing two new types of negatively charged sulfate and sulfonated groups for polysaccharide CSPs. These CSPs were utilized to pack CEC columns for enantioseparation with a NP eluent. Compared to conventional cellulose tris(3,5-dimethylphenyl carbamate) or CDMPC CSPs, the sulfated CDMPC CSP (sulfur content 4.25%, w/w) shortened the analysis time up to 50% but with a significant loss of enantiomeric resolution (∼60%). On the other hand, the sulfonated CDMPC CSP (sulfur content 1.76%, w/w) not only provided fast throughput but also maintained excellent resolving power. In addition, its synthesis is much more straightforward than the sulfated one. Furthermore, we studied several stationary phase parameters (CSP loading and silica gel pore size) and mobile phase parameters (including type of mobile phase and its composition) to evaluate the throughput and enantioselectivity. Using the optimized conditions, a chiral pool containing 66 analytes was screened to evaluate the enantioselectivity under three different mobile phase modes (i.e., NP, polar organic phase (POP) and reversed-phase (RP) eluents). Among these mobile phase modes, the RP mode showed the highest success rate, whereas some degree of complementary enantioselectivity was observed with NP and POP. Finally, the feasibility of applying this CSP for CEC–MS enantioseparation using internal tapered column was evaluated with NP, POP and RP eluents. In particular, the NP-CEC–MS provided significantly enhanced sensitivity when methanol was replaced with isopropanol in the sheath liquid. Using aminoglutethimide as model chiral analyte, all three modes of CEC–MS demonstrated excellent durability as well as excellent reproducibility of retention time and enantioselectivity.     

The new approach of standardization of capillary electrophoresis

In this paper, we develope the new standardization methods to eliminate the influence in capillary electrophoresis (CE). The markers were used to determine the basis position and then correct the data of sample by the migration time of standard sample, and make the migration time of samples consistent with the standard sample by the criterion of the marker. The problem of time transition was corrected in this way. Then according to the peak height or peak area of the marker in the sample (peak height was used here) compared with the standard sample, the sample data was zoomed appropriately. The absorbance error was made to be correct. The wavelet de-noise method was also used to make the data smooth and get a good baseline.     

Sol–gel-based molecularly imprinted xerogel for capillary microextraction

A novel molecularly imprinted xerogel (MIX) based on organically modified silica (ORMOSIL) was successfully prepared for on-line capillary microextraction (CME) coupled with high-performance liquid chromatography (HPLC). The sol-gel-based xerogel was prepared using only one precursor and exhibited extensive selectivity towards triazines along with significant thermal and chemical stability. Atrazine was selected as a model template molecule and 3-(trimethoxysilyl)propylmethacrylate (TMSPMA) as a precursor in which the propylmethacrylate moiety was responsible for van der Waals, dipole-dipole, and hydrogen-bond interactions with the template. This moiety plays a key role in creation of selective sites while methoxysilyl groups in TMSPMA acted as crosslinkers between the template and the propylmethacrylate moiety. Moreover, a non-imprinted xerogel (NIX) was also prepared in the absence of the template for evaluating the extraction efficiency of the prepared MIX. Then, the prepared imprinted and non-imprinted xerogels were used for extraction of three selected analytes of triazines class including atrazine, ametryn, and terbutryn, which have rather similar structures. The extraction efficiency of the prepared xerogel for atrazine, the template molecule, was found to be ten times greater than the efficiency achieved by the non-imprinted one. In the meantime, the extraction efficiency ratio of MIX to NIX for ametryn and terbutryn was also rather significant (eight times). Moreover, other compounds from different classes including dicamba, mecoprop, and estriol were also analyzed to evaluate the selectivity of the prepared MIX towards triazines. The ratio of enrichment factors (EF) of MIX to NIX for atrazine, ametryn, terbutryn, dicamba, mecoprop, and estriol were about 10, 8, 8, 2, 2, and 3, respectively. The linearity for the analytes was in the range of 5-700 μg L(-1). Limit of detection was in the range of 1-5 μg L(-1) and the RSD% values (n = 5) were all below 6.6% at the 20 μg L(-1) level. The developed method was also applied to real water samples and the relative recovery percentages obtained for the spiked water samples were from 92 to 104%. The CME loop, containing the prepared MIX, exhibited a rather long life time due to its remarkable solvent and mechanical stability. Even after 100 runs, no decrease in the peak areas was observed. The developed method could easily provide the possibility of preparing a selective sorbent in a unique way with the lowest possible cost and time.     

Parallel and serial dual electrode detectors for capillary electrophoresis

Application of parallel and serial dual electrode detectors for capillary electrophoresis was first described. In parallel dual electrode approach, two 100 μm-diameter Cu disks arranged side by side were used as the dual working electrode for the simultaneous determination of a mixture of carbohydrates and amino acids. In serial dual electrode approach, two working electrodes were arranged in a disk-ring manner for the simultaneous determination of both cysteine and cystine; the disk electrode was Hg/Au serving as the upstream electrode, the ring electrode was 5% CoPC carbon paste serving as the downstream electrode.     

Why not using capillary electrophoresis in drug analysis?

CE and related methods are well-established techniques in the analysis of biomolecules, such as DNA and proteins. Even though CE is a rather good alternative to HPLC for the evaluation of the impurity profile and the enantiomeric purity of a drug, it is rarely applied. This might be due to the reservation of national licensing authorities and the pharmacopoeia commissions for several reasons. In this review containing some experimental data we report on several drug examples which demonstrate the superiority of CE over HPLC in special cases, i.e., in the analysis of antibiotics, amino acids and peptides, and the determination of enantiomeric purity. However, in order to make the CE techniques more suitable for pharmacopoeial purposes the general methods describing separation methods have to be complemented with the adjustment of the electrophoretic conditions being necessary to satisfy the system suitability criteria without fundamentally modifying the methods. Taken together CE should be more often applied in drug quality control.     

Ionic liquid-mediated sol–gel coatings for capillary microextraction

Ionic liquid (IL)-mediated sol–gel hybrid organic–inorganic materials present enormous potential for effective use in analytical microextraction. This opportunity, however, has not yet been explored. One obstacle to materializing this prospect arises from high viscosity of ILs significantly slowing down sol–gel reactions. In this work, we developed a method that overcomes this hurdle and provides IL-mediated advanced sol–gel materials for capillary microextraction (CME). We examined two different ILs: (a) a phosphonium-based IL, trihexyltetradecylphosphonium tetrafluoroborate, and (b) a pyridinium-based ionic liquid, N-butyl-4-methylpyridinium tetrafluoroborate. These ILs were evaluated in conjunction with two types of hydroxy-terminated polymers: (a) two Si–OH terminated polymers (PDMS and BMPO), and (b) two C–OH terminated polymers (PEG and polyTHF) that differ in their sol–gel reactivity. Scanning electron microscopy results demonstrate that ILs can serve as porogenic agents in sol–gel reactions. The IL-mediated sol–gel coatings prepared with silanol-terminated polymers provided up to 28 times higher extractions in off-line CME-GC compared to analogous sol–gel coatings prepared without any IL in the sol solution. Contrary to this, the IL-mediated sol–gel coatings prepared with C–OH terminated polymers provided lower extraction efficiencies compared to their IL-free counterparts. These observations were explained by (a) lower sol–gel reactivity of C–OH groups in PEG and polyTHF compared to Si–OH groups in PDMS and in hydrolyzed alkoxysilane precursors and (b) extremely high viscosity of ionic liquids. This study shows that IL-generated porous morphology alone is not enough to provide effective extraction media: careful choice of the organic polymer and the precursor with close sol–gel reactivity must be made to ensure effective chemical bonding of the organic polymer to the created sol–gel material to be able to provide the desired sorbent characteristics. Additionally, IL-mediated sol–gel PDMS coatings provided run-to-run RSD values of 4.2–5.0% and detection limits ranging from 3.2 ng/L to 17.4 ng/L. PDMS sol–gels prepared without ILs provided RSD values of 2.8–14.1%, and detection limits ranging from 4.9 ng/L to 487.0 ng/L.     

Dead volume–free flow splitting in capillary electrophoresis

In recent years, several dual detection concepts (DDCs) for CE were developed, which consisted of at least one nondestructive detector. For these DDCs, a linear detector arrangement could be used, which is not possible when both detectors are destructive. To overcome this problem, we developed a concept for the splitting of the CE stream utilizing commercially available flow splitters (FSs) that allow the parallel positioning of two destructive detectors. In this proof-of-concept study, T- and Y-shaped FSs were characterized regarding their suitability for DDCs. To keep it simple, a UV detector (UV) and a C⁴D were used for the characterization. The model system consisted of an acetonitrile-based background electrolyte and the two model substances, (ferrocenylmethyl)trimethylammonium iodide and caffeine. CE hyphenated to a UV detector (CE-UV) measurements revealed that the split ratio was about 50% for both FSs. CE-C⁴D was used to evaluate the peak shape in front of and behind the FSs. These measurements showed that there was no significant peak broadening introduced by the FSs. Additionally, there were no changes in the LODs in front of and behind the FSs. Furthermore, the flexibility of the new FS approach allowed the usage of capillaries with different ids (25–75 µm) for injection and detection.     

Low-conductivity background electrolytes in capillary zone electrophoresis--myth or reality?

The asymmetric triangle (fronting or tailing) concentration profiles and their broadening are the typical results of the electromigrational zone dispersion characterizing a system of the analyte in the background electrolyte (BGE). The present contribution suggests the parameter named the relative velocity slope, SBGE,X, which was introduced here as a quantity characterizing the peak broadening and the asymmetry. SBGE,X VS. analyte ionic mobility diagrams are suitable for the comparison of BGEs of given pH and the conductivity composed of electrolytes of different pKaS and ionic mobilities. The concept of SBGE,X diagrams is verified by capillary zone electrophoresis of the model analytes, which involve (i) the series of sulfobenzoylated poly(ethylene glycols) as examples of the strong electrolytes with different ionic mobilities and (ii) the series of monobasic phenols as weak electrolytes with different pKaS and similar ionic mobilities. It follows from both theoretical predictions of peak symmetry and their experimental verification that the optimum composition of BGEs is determined mostly by the suitable ionic mobility of the coion in dependence on the ionic mobility of the analyte. The low-conductivity BGEs based on low-molecular carrier ampholytes are at best only comparable with the properly chosen monobasic electrolytes.     

Offline and online capillary electrophoresis enzyme assays of β-N-acetylhexosaminidase

Enzyme assays of β-N-acetylhexosaminidase from Aspergillus oryzae using capillary electrophoresis in the offline and online setup have been developed. The pH value and concentration of the borate-based background electrolyte were optimized in order to achieve baseline separation of N,N′,N″-triacetylchitotriose, N,N′-diacetylchitobiose, and N-acetyl-d-glucosamine. The optimized method using 25 mM tetraborate buffer, pH 10.0, was evaluated in terms of repeatability, limits of detection, quantification, and linearity. The method was successfully applied to the offline enzyme assay of β-N-acetylhexosaminidase, which was demonstrated by monitoring the hydrolysis of N,N′,N″-triacetylchitotriose. The presented method was also utilized to study the pH dependence of enzyme activity. An online assay with N,N′-diacetylchitobiose as a substrate was developed using the Transverse Diffusion of Laminar Flow Profiles model to optimize the injection sequence and in-capillary mixing of substrate and enzyme plugs. The experimental results were in good agreement with predictions of the model. The online assay was successfully used to observe the inhibition effect of N,N′-dimethylformamide on the activity of β-N-acetylhexosaminidase with nanoliter volumes of reagents used per run and a high degree of automation. After adjustment of background electrolyte pH, an online assay with N,N′,N″-triacetylchitotriose as a substrate was also performed.

Methoxypropylamino β-cyclodextrin clicked AC regioisomer for enantioseparations in capillary electrophoresis

In this work, a novel methoxypropylamino β-cyclodextrin (β-CD) clicked AC regioisomer, 6^A-4-hydroxyethyl-1,2,3-triazolyl-6^C-3-methoxypropylamino β-cyclodextrin (HETz-MPrAMCD), was synthesized via nucleophilic addition and click chemistry. The chiral separation ability of this AC regioisomer cationic CD was evaluated toward 7 ampholytic and 13 acidic racemates by capillary electrophoresis. Dependence of enantioselectivity and resolution on buffer pH (5.5–8.0) and chiral selector concentration (0.5–7.5 mM) was investigated. Enantioselectivities (α ≥ 1.05) could be achieved for most analytes under optimal conditions except dansyl-dl-noreleucine and dansyl-dl-serine. The highest resolutions for 2-chloromandelic acid p-hydroxymandelic acid were 15.6 and 9.7 respectively. The inclusion complexation between HETz-MPrAMCD and each 3-phenyllactic acid enantiomer was also revealed with nuclear magnetic resonance study.     

Determination of tetramethrin in water by liquid microextraction–capillary electrophoresis

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Fast counter-electroosmotic capillary electrophoresis–time-of-flight mass spectrometry of hyaluronan oligosaccharides

Fast capillary electrophoresis–mass spectrometry measurements under counter-electroosmotic analyte migration conditions are presented. Efficient separations of a homologous series of six hyaluronan oligosaccharides (comprising 1–6 hyalobiuronic acid moieties) could be completed in 65 s. Separations were achieved in short-length fused silica capillaries under high electric field strengths of up to 1.25 kV·cm⁻¹. Capillary inner diameters ranging from 5 to 50 μm were investigated, resulting in an optimal value of 15 μm. The influence of capillary dimensions and buffer composition on separation efficiency and sensitivity are discussed. Optimal separations were achieved using a 28 cm × 15 μm capillary, a separation high voltage of 35 kV, a background electrolyte of 25 mM ammonium acetate adjusted to pH 8.5, and negative ionization mode. The optimized method was successfully applied to a bovine testicular hyaluronidase digest of hyaluronan. Only minimal sample pretreatment for protein-containing samples is required. The simple manual injection procedure and fast separations allow for a sample throughput of 35 samples per hour.     

Probing affinity via capillary electrophoresis: advances in 2003–2004

This review addresses recent advances in capillary electrophoresis of biological-based molecular interaction from a broader perspective, based on applications reported during the period 2003–2004. These capillary electrophoresis-based studies of molecular interactions include affinity capillary electrophoresis, electrokinetic chromatography, and free zone electrophoresis. The review is written as a general synopsis of applications and does not cover the theory or protocol involved in the implementation of the analyses.The caption to Fig. 3 was incorrect. This revised version replaces the article published online on 10 February 2005.     

Separation of chiral drugs with β-CD phosphate by capillary electrophoresis

In capillary electrophoresis (CE) chiral separation is accomplished by adding suitableselector in the running electrolyte"'. The type of selector is of primary importance forachieving successful resolution. Selector concentration has considerable influence aswell3'. Charged cyclodextrins were first introduced for chiral separation of aminoacidss and used for enantioseparation of drugs by Terabe group'. Many kinds ofcharged CDs are now commercially available7-11. The charged CD commonly use…     

Chiral metal–organic framework used as stationary phases for capillary electrochromatography

Metal–organic frameworks (MOFs) have received great attention as novel media in separation sciences because of their fascinating structures and unusual properties. However, to the best of our knowledge, there has been no attempt to utilize chiral MOFs as stationary phases in capillary electrochromatography (CEC). In this study, a homochiral helical MOF [Zn₂(D-Cam)₂(4,4′-bpy)]_n (D-Cam = D-(+)-camphoric acid, 4,4′-bpy = 4,4′-bipyridine) was explored as the chiral stationary phase in open tubular capillary electrochromatography (OT-CEC) for separation of chiral compounds and isomers. The MOFs coated column has been developed using a simple procedure via MOFs post-coated on the sodium silicate layer. The baseline separations of flavanone and praziquantel were achieved on the MOFs coated column with high resolution of more than 2.10. The influences of pH, organic modifier content and buffer concentration on separation were investigated. Besides, the separations of isomers (nitrophenols and ionones) were evaluated. The relative standard deviations (RSDs) for the retention time of run-to-run, day-to-day and column-to-column were 1.04%, 2.16% and 3.07%, respectively. The results demonstrated that chiral MOFs are promising for enantioseparation in CEC.