Electrokinetic injection of DNA from gel micropads: basis for coupling polony technology with CE separation

We propose a novel method for electrokinetic injection of DNA samples into capillaries from nanoliter gel micropads, deposited on glass slides, which are coated with electroconducting film. Theoretical and experimental proof is presented for the proposed method. The method allows efficient and highly precise injection without physical contact between the gel pad and the capillary. Read length of more than 700 bp at Q20 has been reproducibly demonstrated in fused-silica capillaries using the proposed injection technique. Based on the obtained results we discuss a novel DNA sequencing system which combines DNA amplification and cycle sequencing in arrays of subnanoliter gel micropads and high-throughput electrophoretic separation in monolith multicapillary arrays.     

Electrokinetic supercharging in CE for the separation and preconcentration of barbiturate drugs in urine samples

Three barbiturate drugs, barbital, phenobarbital, and secobarbital were separated and analyzed by electrokinetic supercharging. The influence of different parameters on electrokinetic supercharging performance was evaluated using both univariated and multivariated optimization processes. The parameters studied were sample pH, concentration, and length of the leading and terminating electrolytes, electrokinetic injection of the sample and composition and hydrodynamic injection of the solvent plug. The leading electrolyte (50 mM NaCl) was hydrodynamically injected (50 mbar × 120 s) prior to the sample that was adjusted to pH 9.6 and electrokinetically injected at ?8.5 kV for 300 s. The terminating electrolyte (100 mM of 2‐(cyclohexylamino) ethanesulphonic acid) was then hydrodynamically injected (50 mbar × 140 s). The results showed that this strategy enhanced detection sensitivity around 1050‐fold compared with normal hydrodynamic injection, providing detection limits ranging between 1.5 and 2.1 ng/mL for standard samples with good repeatability in terms of peak area (values of relative standard deviation, %RSD < 3). The applicability of the optimized method was demonstrated by the analysis of human urine samples spiked with the studied compounds at different concentration levels and further liquid–liquid extraction step. The estimated detection limits obtained in the urine samples extract ranged between 8 and 15 ng/mL.     

Speciation of inorganic tellurium from seawater by ICP-MS following magnetic SPE separation and preconcentration

A new method was developed for the speciation of inorganic tellurium species in seawater by inductively coupled plasma-MS (ICP-MS) following selective magnetic SPE (MSPE) separation. Within the pH range of 2-9, tellurite (Te(IV)) could be quantitatively adsorbed on gamma-mercaptopropyltrimethoxysilane (gamma-MPTMS) modified silica-coated magnetic nanoparticles (MNPs), while the tellurate (Te(VI)) was not retained and remained in solution. Without filtration or centrifugation, these tellurite-loaded MNPs could be separated easily from the aqueous solution by simply applying external magnetic field. The Te(IV) adsorbed on the MNPs could be recovered quantitatively using a solution containing 2 mol/L HCl and 0.03 mol/L K2Cr2O7. Te(VI) was reduced to Te(IV) by L-cysteine prior to the determination of total tellurium, and its assay was based on subtracting Te(IV) from total tellurium. The parameters affecting the separation were investigated systematically and the optimal separation conditions were established. Under the optimal conditions, the LOD obtained for Te(IV) was 0.079 ng/L, while the precision was 7.0% (C = 10 ng/L, n = 7). The proposed method was successfully applied to the speciation of inorganic tellurium in seawater.     

CE coupling with end-column electrochemiluminescence detection for chiral separation of disopyramide

CE with electrochemiluminescence (ECL) detection technique was successfully applied for the chiral separation of a kind of class IA antiarrhythmic racemic drug. To the best of our knowledge, this is the first report of ECL detection used in chiral CE. To get better detection sensitivity and good enantioresolution at the same time, the conditions of capillary inlet and outlet buffer were systematically optimized. Unlike the traditional chiral separation method, the buffers we used in the capillary inlet and outlet differed from each other in terms of buffer pH, ionic strength, type of BGE as well as buffer composition. Under the optimum conditions, baseline enantioseparation and highly sensitive detection of the enantiomers were achieved. Wide linear relationship of each enantiomer was achieved in the range of 5 x 10(-7) to 2 x 10(-5) mol/L with relative coefficients of 0.996 and 0.997, respectively. The detection limits were estimated to be 8 x 10(-8) and 1.0 x 10(-7) mol/L (S/N = 3) for the enantiomers, respectively. In addition, a successful application of this new method to the chiral separation of the racemic drug in spiked plasma samples confirmed the validity and applicability of the chiral CE-ECL method.     

Ion exchange preconcentration and separation of mercury species by CE with indirect contactless conductometric detection

Capillary electrophoretic separation of four mercury species (inorganic Hg(2+), methyl-, ethyl-, and phenylmercury) was achieved in less than 8 min with an electrolyte consisting of 150 mM 2-amino-2-methyl-propanol (AMP) at pH 11.6. The analytes were complexed with 0.1% mercaptopropionic acid (MPA), separated in counter-EOF as anionic complexes, and detected by a contactless conductometric detector. Ion exchange preconcentration with on-column formation of MPA-mercury complexes was developed. Preconcentration factors of 25-150 were achieved and LODs in the range of 2.9-6.9 ng/mL were feasible. This method may prove to be applicable as a rapid screening method for mercury speciation in environmental samples.     

Novel hydroxyethylcellulose-graft-poly acrylamide copolymer for separation of double-stranded DNA fragments by CE

A novel separation medium, hydroxyethylcellulose-graft-polyacrylamide (HEC-g-PAM) synthesized by atom transfer radical polymerization (ATRP), used for dsDNA separation by CE is presented. The separation performance of HEC-g-PAM, which has the same graft density and different graft length, has been investigated in Tris-boric acid-EDTA (TBE) buffer solvent mixtures. The temperature-dependent rheological behavior of HEC-g-PAM was also studied by steady-shear rheometry. The results showed that dsDNA fragments between 72 and 1353 bp was achieved with a 30 cm effective capillary length at 150 V/cm using this type of graft copolymer as a separation medium in bare fused-silica capillaries, and separation improvement is obtained in HEC-g-PAM compared with HEC and poly(dimethylacrylamide (PDMA).     

On-line coupling of SPE and CE-MS for peptide analysis

An on-line SPE-CE-MS system has been developed for the analysis of peptides. Analytes are preconcentrated using a C(18) microcolumn (5 x 0.5 mm id), and then introduced into the CE system via a valve interface. The CE system with a Polybrene-poly(vinylsulfonate) bilayer coated capillary is combined with an ion-trap mass spectrometer via ESI using a coaxial sheath-liquid sprayer. The on-line coupling of the SPE and CE step by the valve interface is advantageous because it allows an independent functioning of the system parts. Optimization of the SPE-CE system was performed using UV detection. Subsequently, the SPE-CE system has been coupled to the ion-trap mass spectrometer. Test solutions with enkephalin peptides (50 ng/mL) were used for evaluation of system performance. Repeatability of effective mobility and peak area ratio of the two enkephalins were within 1.2% and 9% RSD, respectively. The analysis of 1:1 v/v diluted cerebrospinal fluid samples spiked with enkephalin peptides showed detection limits (S/N = 3) in the range of 1.5-3 ng/mL (around 5 nM), which were similar to those obtained for enkephalin test solutions. Moreover, the potential of the on-line SPE-CE-MS system was demonstrated by the analysis of a cytochrome C digest. Some hydrophilic peptides did not show sufficient retention on the SPE column, and were lost during preconcentration. Nonetheless, positive identification of the protein was achieved, indicating the feasibility of the system for proteomics.     

Transport extraction for trace element separation and preconcentration

Summary A new method of sorbent supported transport extraction based on the use of open-cell polyurethane foam sorbents in solvent sublation separation has been developed. The method has been shown to be effective for the separation and preconcentration of radioiodine and/or stable iodine from 41 of deionized, drinking (tap) and surface (river) waters using N-cetylpyridinium chloride as the cationic surfactant and N₂ carrier gas as the bubbling medium. A transport extraction efficiency of about 94% was obtained in 60 to 90 min; a 300-fold preconcentration was found for radioiodine as measured by direct radioactivity counting. Both radioiodine and stable iodine can be back-extracted from the polyurethane foam support (e.g. into acetone with an efficiency of almost 95%).Part II: This journal (1993) 346:905On leave from the Institute of Radioecology and Applied Nuclear Techniques, P.O. Box A-41, 04061 Koice, Slovakia     

Transport extraction for trace element separation and preconcentration

Summary Transport extraction based on solvent sublation has been proposed for separation and preconcentration of trace analytes from aqueous solutions. The experimental capabilities of this method were investigated using a complex of molecular iodine with ionic surfactants. A comparison of transport extraction with conventional solvent extraction was also made. The theoretical principles of transport extraction have been revealed and the basic difference between these two extractive separations has been explained. The results showed that the non-equilibrium nature of transport extraction gives it some distinctive advantages over equilibrium solvent extraction.On leave from the Institute of Radioecology and Applied Nuclear Techniques, P.O.Box A-41, CS-04061 Kosice, Czechoslovakia     

Recentchiral selectors for separation in HPLC and CE

Enantiomers (stereoisomers) can exhibit substantially different properties if present in chiral environments. Since chirality is a basic property of nature, the different behaviors of the individual enantiomers must be carefully studied and properly treated. Therefore, enantioselective separations are a very important part of separation science.     

Determination of gadolinium in river water by SPE preconcentration and ICP-MS

An analytical scheme was developed for the determination of Gd-diethylenetriaminepentaacetate (Gd-DTPA), Gd and the other rare earth elements (REE) in river water by inductively coupled plasma (quadrupole) mass spectrometry (ICP-Q-MS). The preconcentration step was essential, since the limits of detection of this multielemental analytical technique are higher than the trace concentrations of the interesting elements in river water.Solid phase extraction (SPE) with different commercially available complexing agents (Chelex 100, Toyopearl and ethylhexylphosphates) was employed for the preconcentration of REE. The investigations revealed that complex stability (varying in dependence of the pH value) has a strong influence on the degree of the enrichment of Gd-DTPA. Based on acidified water samples (pH<3) a procedure using ethylhexylphosphates was proposed for the preconcentration of Gd and REE from surface water samples. For this purpose C₁₈-cartridges loaded with ethylhexylphosphates were used, resulting in an enrichment factor of 40.     

In-line system containing porous polymer monoliths for protein digestion with immobilized pepsin, peptide preconcentration and nano-liquid chromatography separation coupled to electrospray ionization mass spectroscopy

The use of two different monoliths located in capillaries for on-line protein digestion, preconcentration of peptides and their separation has been demonstrated. The first monolith was used as support for covalent immobilization of pepsin. This monolith with well-defined porous properties was prepared by in situ copolymerization of 2-vinyl-4,4-dimethylazlactone and ethylene dimethacrylate. The second, poly(lauryl methacrylate-co-ethylene dimethacrylate) monolith with a different porous structure served for the preconcentration of peptides from the digest and their separation in reversed-phase liquid chromatography mode. The top of the separation capillary was used as a preconcentrator, thus enabling the digestion of very dilute solutions of proteins in the bioreactor and increasing the sensitivity of the mass spectrometric detection of the peptides using a time-of-flight mass spectrometer with electrospray ionization. Myoglobin, albumin, and hemoglobin were digested to demonstrate feasibility of the concept of using the two monoliths in-line. Successive protein injections confirmed both the repeatability of the results and the ability to reuse the bioreactor for at least 20 digestions.     

Etched bare fused-silica capillaries for online preconcentration of amino acids in CE

An online preconcentration method based on electrostatic interaction between the analytes and inner surface of the capillary column was developed for the determination of zwitterionic analytes such as amino acids in CE coupled with a DAD. The amino acids possessed positive charges when they were dissolved in an acidic solvent. When they were injected into the column, they were attracted by the negatively charged inner surface of the fused-silica capillary column. An etched column was used to increase the area of the capillary's inner surface and, consequently increase the electrostatic interaction between the amino acids and the inner surface of the capillary column. It was found that when the sample was injected at 10 psi for 1 min and the pH value of the sample was 4, the amount of amino acids attracted to the inner surface of the capillary was maximum. Under these optimized experimental conditions, the detection sensitivity of CE-DAD was enhanced by 5200, 2800, and 3100 times for asparagine, tryptophan and phenylalanine, respectively, compared with normal CE separation. The method provided good reproducibility in terms of both migration time and peak height. It can be successfully used for the preconcentration zwitterion.     

Bioanalytical separation and preconcentration using ionic liquids

Ionic liquids (ILs) are novel solvents that display a number of unique properties, such as negligible vapor pressure, thermal stability (even at high temperatures), favorable viscosity, and miscibility with water and organic solvents. These properties make them attractive alternatives to environmentally unfriendly solvents that produce volatile organic compounds. In this article, a critical review of state-of-the-art developments in the use of ILs for the separation and preconcentration of bioanalytes in biological samples is presented. Special attention is paid to the determination of various organic and inorganic analytes—including contaminants (e.g., pesticides, nicotine, opioids, gold, arsenic, lead, etc.) and functional biomolecules (e.g., testosterone, vitamin B₁₂, hemoglobin)—in urine, blood, saliva, hair, and nail samples. A brief introduction to modern microextraction techniques based on ILs, such as dispersive liquid–liquid microextraction (DLLME) and single-drop microextraction (SDME), is provided. A comparison of IL-based methods in terms of their limits of detection and environmental compatibilities is also made. Finally, critical issues and challenges that have arisen from the use of ILs in separation and preconcentration techniques are also discussed.

HPLC method with on-line SPE preconcentration for quantification of permethric acid sorption to goethite

Pesticide metabolites are often found to be more mobile in soil than their parent compounds. Pyrethroids are bound strongly to soil and therefore sorption of the pyrethroid metabolite permethric acid (PA) to a typical soil sorbent, goethite, was investigated. An on-line solid-phase extraction (SPE)-HPLC-UV procedure was developed for quantification of trans- and cis-permethric acid in aqueous samples. Limits of detection (LOD) were 500 times lower than those obtained with conventional HPLC-UV, resulting in LODs of 1.4 and 0.3?nM for the trans- and cis-isomers, respectively. Sorption of nanomolar concentrations of PA to goethite was found to be specific up to less than 1% surface coverage. In this range the data was described by a Langmuir equation with K _ads = 7.1 × 10^?9?L/mol and Γ_max = 7.1 × 10^?9?mol/m² for total PA (trans + cis) at pH = 3. K _ads,?cis (1.4 × 10⁶?L/mol) was approximately twice K _ads,?trans (7.9 × 10⁵?L/mol). At higher PA concentrations the slope of the sorption isotherm increased, which is ascribed to hydrophobic interactions between adsorbed and dissolved PA molecules. Based on comparison with reported K _om values, metal oxides are expected to have a relatively greater significance to the retention of PA than soil organic matter.     

In-line coupling headspace liquid-phase microextraction with capillary electrophoresis

An analytical technique of in-line coupling headspace liquid-phase microextraction (HS-LPME) with capillary electrophoresis (CE) was proposed to determine volatile analytes. A special cover unit of the sample vial was adopted in the coupling method. To evaluate the proposed method, phenols were used as model analytes. The parameters affecting the extraction efficiency were investigated, including the configuration of acceptor phase, kind and concentration of acceptor solution, extraction temperature and time, salt-out effect, sample volume, etc. The optimal enrichment factors of HS-LPME were obtained with the sample volume of about half of sample vials, which were confirmed by both the theoretical prediction and experimental results. The enrichment factors were obtained from 520 to 1270. The limits of detection (LODs, S/N = 3) were in the range from 0.5 to 1 ng/mL each phenol. The recoveries were from 87.2% to 92.7% and the relative standard deviations (RSDs) were lower than 5.7% (n = 6). The proposed method was successfully applied to the quantitative analysis of the phenols in tap water, and proved to be a simple, convenient and reliable sample preconcentration and determination method for volatile analytes in water samples.     

Iron protoporphyrin-modified monolithic support for on-line preconcentration of angiotensin prior to CE analysis

An on-line preconcentration method using a polymeric monolithic support is proposed for the retention of the decapeptide angiotensin I and its subsequent analysis by CZE. Monolithic capillary columns were prepared in fused-silica (FS) capillaries of 150 microm id by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iron protoporphyrin IX (Fe-ProP). Monolithic microcolumns (8 mm long) were coupled on-line to the inlet of the separation capillary (FS capillary, 75 microm id x10 cm from the inlet to the microcolumn and 27 cm from the microcolumn to the detector). Angiotensin I was released from the sorbent by a 50 mM sodium phosphate, pH 2.5/ACN, 75:25 v/v solution and then analyzed by CZE with UV absorption detection at 214 nm. The concentration LOQ (CLOQ) was 0.5 ng/mL. The Fe-ProP-derivatized monolithic microcolumn coupled to the separation capillary exhibited a high retention capacity for peptide angiotensin I, and showed as much as 10,000-fold improvement in concentration sensitivity.     

Mercury speciation by coupling cold vapour atomic absorption spectrometry with flow injection on-line preconcentration and liquid chromatographic separation

A fully automated system for the direct determination of methylmercury (MeHg), ethylmercury (EtHg), phenylmercury (PhHg), and inorganic mercury (Hg(II)) at the ng/L level is described. It is based on solid phase extraction preconcentration incorporated in a flow injection (FI) system, high performance liquid chromatography (HPLC) separation, reduction combined with thermolysis and determination by cold vapour atomic absorption spectrometry (CVAAS). For preconcentration a microcolumn of bonded silica with octadecyl functional groups (C18 reversed phase material) was used as a sorbent for the mercury complexes formed on-line with ammonium pyrrolidine dithiocarbamate. Retained mercury species are eluted with a methanol-acetonitrile-water mixture and subjected to separation on an octadecylsilane (ODS) column before determination by CVAAS. The sensitivity of organo-mercury determination could be improved by using NaBH₄ as a reductant combined with a thermolysis step. In order to perform on-line measurements the preconcentration microcolumn was mounted in a pressure-tight casing. Limits of detection for MeHg, EtHg, PhHg and Hg(II) employing a sample volume of 58.5 mL were 9, 6, 10 and 5 ng/L, respectively. The relative standard deviation (RSD) calculated from 9 repeated measurements was found to be 3.6%, 5.5%, 10.4% and 7.6% for MeHg, EtHg, PhHg and Hg(II), respectively. Finally, the application of this method for speciation of mercury in fish and human urine is described.