Sample Injection Time Research Articles

Determination of L-arginine in amniotic fluid by capillary zone electrophoresis

Conditions for the determination of L-arginine in a biological fluid using capillary zone electrophoresis were optimized. The effects of the pH of a running buffer, the time of sample injection into a capillary, the operating voltage, the temperature, and the wavelength on the results of the determination were studied. The procedure made it possible to evaluate the concentration of L-arginine over a range of 6–1000 μg/mL (c = 3 μg/mL; RSD = 2%). The duration of analysis including sample preparation was no longer than 30 min. The analysis of amniotic fluid samples in the cases of physiological pregnancy and pregnancy complicated by placental insufficiency demonstrated that the arginine content of amniotic fluid increased in the case of placental insufficiency, as compared to normal values.

End-to-End Differential Contactless Conductivity Sensor for Microchip Capillary Electrophoresis

In this contribution, a novel measurement approach for miniaturized capillary electrophoresis (CE) devices is presented: End-to-end differential capacitively coupled contactless conductivity measurement. This measurement technique is applied to a miniaturized CE device fabricated in low-temperature cofired ceramics (LTCC) multilayer technology. The working principle is based on the placement of two distinct detector areas near both ends of the fluid inlet and outlet of the separation channel. Both output signals are subtracted from each other, and the resulting differential signal is amplified and measured. This measurement approach has several advantages over established, single-end detectors: The high baseline level resulting from parasitic stray capacitance and buffer conductivity is reduced, leading to better signal-to-noise ratio and hence higher measurement sensitivity. Furthermore, temperature and, thus, baseline drift effects are diminished owing to the differentiating nature of the system. By comparing the peak widths measured with both detectors, valuable information about zone dispersion effects arising during the separation is obtained. Additionally, the novel measurement scheme allows the determination of dispersion effects that occur at the time of sample injection. Optical means of dispersion evaluation are ineffective because of the opaque LTCC substrate. Electrophoretic separation experiments of inorganic ions show sensitivity enhancements by about a factor of 30-60 compared to the single-end measurement scheme.

Simultaneous Determination of Phenolic Additives in Cosmetics by Micellar Electrokinetic Capillary Chromatography with Electrochemical Detection

AbstractA micellar electrokinetic capillary chromatography method with electrochemical detection (MECC‐ED) has been developed for the simultaneous determination of eight phenolic additives, including propyl gallate (PG), tert‐butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP) in cosmetic products. Method development involved optimization of the working electrode, the pH value of running buffer, the concentration of sodium dodecyl sulfate (SDS), the separation voltage, and the sample injection time. Under the optimum conditions, all analytes can be well separated within 26 min at the separation voltage of 18 kV in a 9 mmol·L−1 sodium dodecyl sulfate (SDS) −60 mmol·L−1 borate running buffer (pH 8.0). A 300 μm diameter carbon disk electrode generated good response at +0.90 V (vs. SCE) for all analytes. Linearity of the present method was over three orders of magnitude of analyte concentration with detection limits (S/N=3) ranging from 1.1×10−7 to 1.2×10−6 g·mL−1 for all analytes. This proposed method has been successfully applied to the simultaneous determination of the above additives in commercial cosmetics, and the assay results were satisfactory.

Residue analysis of melamine in milk products by micellar electrokinetic capillary chromatography with amperometric detection

A high-performance micellar electrokinetic capillary chromatography with amperometric detection (MECC–AD) method for the fast determination of melamine (MM), occasionally used to increase the apparent protein content of milk products, has been developed. Method development involved optimisation of the working electrode, the running buffer system and acidity, the concentration of sodium dodecyl sulphate (SDS), the separation voltage, the applied potential and the sample injection time. Under the optimum conditions, MM can be well separated from its co-existing interferences in real-world samples within 9 min at the separation voltage of 20 kV in a 8 mmol/L SDS/20 mmol/L H3BO3–Na2B4O7 running buffer (pH 7.4). Satisfactory recovery (83.3–105.5%), repeatability of the peak current (3.2%) and migration time (3.8%), and the limit of detection (2.1 × 10−6 g/mL) for the method were achieved. This proposed procedure has been successfully used for the determination of MM in milk products.

On‐chip coupling of free‐solution transient ITP and CGE for highly efficient separation of dsDNA with variable sample loading amounts

We developed an on-chip DNA analysis method, in which free-solution transient isotachophoresis (FstITP) were coupled with CGE. Using chloride ions in the sample matrix and HEPES in the background electrolyte, respectively, as the leading and terminating ions, dsDNAs were isotachophoretically preconcentrated in free-solution and then separated in sieving polymer. The coupling of FstITP and CGE enabled higher separation efficiency due to higher preconcentration rate in free-solution. The FstITP-CGE analysis offered adjustable signal intensities by varying sample injection time. With the maximum sample loading volume, the LOD of the FstITP-CGE analysis was determined to be 0.24 ng/mL by confocal laser-induced fluorescence detection. The FstITP-CGE method is simple, robust and flexible, thus well suited to the analysis of highly saline DNA samples at different concentration level.

Study on the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chloro-phenoxyacetic sodium (MCPA sodium) in natural agriculture-soils of Fuzhou, China using capillary electrophoresis

A new method of analyzing trace 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2-methy-4-chloro-lphenoxyacetic sodium (MCPA sodium) in soils by capillary electrophoresis (CE) has been developed in this study. The optimum analytical conditions including chemical component and concentration of buffer solution, pH, separation voltage and sample injection time were studied in detail. Under the optimum conditions, 2,4-D and MCPA sodium in soils can be speedy separated and determined within 20 min with detection limits of 0.15 µg/g (2,4-D) and 0.25 µg/g (MCPA sodium) , a RSD ( n = 6) < 5% and a recovery > 89%. With the help of analytical method developed in this study, the degradations of 2,4-D and MCPA sodium in natural agriculture-soils of Fuzhou were studied. The experimental results indicated that the degradations of 2,4-D and MCPA sodium follow first-order kinetics with degradation constants of 0.1509 day –1 (2,4-D) and 0.2722 day –1 (MCPA sodium) respectively. The degradation half-life were calculated to be 4.6 days (2,4-D) and 2.6 days (MCPA sodium) at 27 °C, implied that 2,4-D and MCPA sodium can be speedy degraded in natural agriculture-soils of Fuzhou, China.

Automated sampling system for the analysis of amino acids using microfluidic capillary electrophoresis

An improved automated continuous sample introduction system for microfluidic capillary electrophoresis (CE) is described. A sample plate was designed into gear-shaped and was fixed onto the shaft of a step motor. Twenty slotted reservoirs for containing samples and working electrolytes were fabricated on the “gear tooth” of the plate. A single 7.5-cm long Teflon AF-coated silica capillary serves as separation channel, sampling probe, as well as liquid-core waveguide (LCW) for light transmission. Platinum layer deposited on the capillary tip serves as the electrode. Automated continuous sample introduction was achieved by scanning the capillary tip through the slots of reservoirs. The sample was introduced into capillary and separated immediately in the capillary with only about 2-nL gross sample consumption. The laser-induced fluorescence (LIF) method with LCW technique was used for detecting fluorescein isothiocyanate (FITC)-labeled amino acids. With electric-field strength of 320 V/cm for injection and separation, and 1.0-s sample injection time, a mixture of FITC-labeled arginine and leucine was separated with a throughput of 60/h and a carryover of 2.7%.

On‐line preconcentration and quantitative analysis of peptide hormone of brain and intestine using on‐column transient isotachophoresis coupled with capillary electrophoresis/electrospray ionization mass spectrometry

A new approach was described to achieve very sensitive analysis of peptide hormone of brain and intestine by capillary electrophoresis coupled with a transient isotachophoresis (tITP) preconcentration method. The system used was electrospray ionization mass spectrometry (ESI-MS) as detector and equipped with a sheath flow configuration. The effects of sample matrix, pH and concentration of leading electrolyte (LE), sample injection time, and ESI-MS instrumental parameters on the efficiency of the sample stacking were investigated in detail. Under the optimized conditions, lower than micromole (0.01 microM) concentrations of the peptides were easily detected. Compared to traditional hydrodynamic injection methods, about 40-230-fold increase in detection sensitivity was obtained by this technique. A distinguishing feature of the described approach is that the background electrolyte can serve as terminating electrolyte (TE), which simplifies the process of the experiments. The method was further evaluated by the analysis of low concentration active peptide mixtures spiked in hypothalamus tissue of the rat.

Rapid separation and characterization of grain water-soluble proteins in bread wheat cultivars (Triticum aestivum L.) by capillary electrophoresis

Water-soluble (WS) proteins in wheat grain are considered to represent the suite of biologically active enzymes and enzyme inhibitors in the grain. In this study, a rapid capillary electrophoresis (CE) method for WS protein separations was developed using untreated fused-silica columns and an acidic phosphate-glycine buffer system. In order to optimize the resolution and reproducibility of CE separation, different protein extraction methods, organic modifiers in phosphate-glycine buffer and capillary electrophoresis conditions, including capillary length and inner diameter (ID), operating temperature, performance voltages, sample injection times, etc., were investigated. High resolution and reproducibility of WS proteins were achieved using 20% ethanol as the extracting buffer. The optimal condition to separate these proteins was 50 μm ID × 31.5 cm (26.5 cm to the detector) capillary at 11.0 kV and 35°C. The optimum buffer was 0.1 M phosphate-glycine (pH 2.5) containing 20% acetonitrile (ACN) and 0.05% hydroxylpropylmethylcellulose. Using this method, the WS proteins were well separated in less than 10 min. A total of 120 Chinese bread wheat cultivars were analyzed. The CE patterns of most bread wheat cultivars showed a higher level of polymorphisms compared with SDS-PAGE patterns. All cultivars analyzed could be readily differentiated based on their WS protein profiles. Results indicate that the WS proteins are useful biochemical markers for wheat genetics and breeding research and CE is expected to become a new and powerful tool for the separation and characterization of grain WS proteins in bread wheat. Key words: Triticum aestivum, bread wheat, water-soluble proteins, capillary electrophoresis, biochemical markers

Simultaneous determination of flavonoids in chrysanthemum by capillary zone electrophoresis with running buffer modifiers

Despite the separation efficiency of capillary electrophoresis (CE) is much higher than other chromatographic methods, it is sometimes difficult to perfectly separate the complex ingredients in biological samples. One possible and simple way to develop the separation effect in CE is to add some modifiers in the running buffer. In this paper, the suitable running buffer modifiers were explored to simultaneously separate and detect six typical flavonoids (apigenin, luteolin, kaempferol, quercetin, (+)-catechin and (−)-epicatechin) which are the main active ingredients in chrysanthemum by capillary zone electrophoresis with amperometric detection (CZE-AD). It was found that when β-cyclodextrin (β-CD) and the mixture of methanol and ethanol were used as running buffer modifiers, a baseline separation of the six analytes could be accomplished in less than 20 min and the detection limits were as low as 10 −7 or 10 −8 g ml −1. Other factors affecting the CZE separation, such as working potential, pH value and ionic strength of running buffer, separation voltage and sample injection time were extensively investigated. Under the optimum conditions, a successful practical application on the determination of chrysanthemum samples confirmed the validity and practicability of this method.

Preparation and Evaluation of Capillary Electrophoresis Column Bonded with Carbosilane Dendrimers

A new chiral capillary electrophoresis column coated with carbosilane dendrimers with peripheral Si–Cl groups and β-cyclodextrin was prepared. The separation characteristics of stability and longevity of capillaries modified by coating carbosilane dendrimers and β-cyclodextrin ( β-CD) were satisfactory, and the variation of separation efficiency was less than 5% after continuous running for a month. The optimum conditions include a running voltage of 16 kV, a UV detector wavelength of 214 nm, sample injection time of 7 s, and a phosphate buffer solution concentration of 40 × 10 −3 M. Chlortrimeton, Hydrochloric Promethazine, and Benzedrine were selected as the separation model targets. The G2P modified column was especially effective in separating Chlortrimeton enantiomers. Under the specified conditions, the column efficiency was 2.5 × 10 5 Plates m −1 with a resolution ( R S) of 1.43 and baseline separation was obtained. The results were satisfactory.

Measurement of glycogen synthase activity in crude extracts by CE

Glycogen synthase catalyzes the incorporation of UDP-glucose into glycogen. The activity of the enzyme is usually measured either by a spectrophotometric method or by a radioassay. The first one is not suitable because of the difficulties regarding the use of coupled enzymes in crude extracts, while the second is a time-consuming method involving glycogen isolation and manipulation of radioactivity. We have used a CZE technique as a novel approach to measure glycogen synthase activity. The separations were performed at 22 kV (36 microA) in uncoated capillaries (53 cmx50 microm). Sample injection time was 30 s and nucleotides were monitored at 254 nm. Best resolution was achieved in 20 mM tetraborate buffer, pH 9.2. Curves of absorbance as a function of UDP and UDP-glucose concentration were linear. Enzyme activity in oocyte extracts was linear with respect to time (up to15 min) and enzyme concentration. The K(m app.) for UDP-glucose was 0.87 mM, a value identical to the one reported using the radioassay. CZE enables easy quantitation of compounds, high sensitivity, and automation of the process. Small sample sizes are required, interferences by auxiliary enzymes and manipulation of radioactivity are avoided, and analysis time is significantly diminished.

Validation of Cystic Fibrosis Mutation Analysis Using ABI 3130XL Genetic Analyzer

Cystic fibrosis (CF) is one of the most common autosomal recessive diseases in the white population, with a prevalence estimate of 1 in 2500 to 3300 live births. CF is characterized by viscous mucus in the lungs with involvement of digestive and reproductive systems as well as sweat glands (excess salt loss). Treatment for CF patients is palliative. Over 1300 mutations have been identified in the CFTR gene. However, most of the mutations are at frequencies of <0.1% or represent private mutations. Although other methodologies are available for CF testing, the oligonucleotide ligation assay is a unique approach to mutation detection of point mutations, small deletions, and small insertions, and consists of 2 phases. Applied Biosystems 3130 Series Genetic Analyzers are the next-generation platform for low to medium throughput laboratories and deliver improved performance. One disadvantage of the Genetic Analyzers is that there is no template of instrument settings for POP-6 polymer using 36-cm array. The Abbott CF oligonucleotide ligation assay ASRs can be run only using POP-6 polymer. We are the first to have optimized the instrument settings for POP-6 polymer based on the template of Rapidseq36-POP6 for Abbott Diagnostics CF V3 ASRs. Several conditions were tried, and the conditions of sample injection voltage at 10,000 v and sample injection time at 5 seconds gave better results, which were with clearer peaks and lower background signals. Twenty cell line DNA samples from Coriell were analyzed, and the results were matched. In addition, Synthetic Controls from AcroMetrix were analyzed, and the results were same as expected. Also, about 1500 clinical samples were analyzed, and high-quality reportable results were obtained. In conclusion, our modified protocol is robust and reliable on this ABI 3130XL instrument.

Optimization of Field-Amplified Sample Injection for Analysis of Peptides by Capillary Electrophoresis−Mass Spectrometry

A versatile experimental approach is described to achieve very high sensitivity analysis of peptides by capillary electrophoresis-mass spectrometry with sheath flow configuration based on optimization of field-amplified sample injection. Compared to traditional hydrodynamic injection methods, signal enhancement in terms of detection sensitivity of the bioanalytes by more than 3000-fold can be achieved. The effects of injection conditions, composition of the acid and organic solvent in the sample solution, length of the water plug, sample injection time, and voltage on the efficiency of the sample stacking have been systematically investigated, with peptides in the low-nanomolar (10(-9) M) range readily detected under the optimized conditions. Linearity of the established stacking method was found to be excellent over 2 orders of magnitude of concentration. The method was further evaluated for the analysis of low concentration bioactive peptide mixtures and tryptic digests of proteins. A distinguishing feature of the described approach is that it can be employed directly for the analysis of low-abundance protein fragments generated by enzymatic digestion and a reversed-phase-based sample-desalting procedure. Thus, rapid identification of protein fragments as low-abundance analytes can be achieved with this new approach by comparison of the actual tandem mass spectra of selected peptides with the predicted fragmentation patterns using online database searching algorithms.

Electroinjection determination of aluminum in aqueous solutions

A procedure is proposed for determining aluminum(III) in water over a concentration range of 0.1–2.0 mg/L in the presence of commensurable amounts of iron(III) using electroinjection analysis. The following parameters were varied in the experiments: the composition and pH of a working buffer solution, the reagent-to-metal concentration ratio, the time of sample injection, the composition of a buffer solution in sample preparation for analysis, the concentration of the reagent (Xylenol Orange), and the reaction time. As a result of this, the sensitivity and selectivity of determining aluminum were improved as compared with the currently available procedures.

An Insight into Peak‐Splitting Phenomenon in On‐Column Concentration‐Micellar Electrokinetic Capillary Chromatography for Aqueous Sample Solution

A detailed study was carried out to investigate the origin of the peak‐splitting phenomena in on‐column concentration‐micellar electrokinetic capillary chromatography for aqueous sample solution. The system studied was a basic phosphate and borax mixed buffer with sodium dodecyl sulfate (SDS) as micellar phase. Phenol, benzyl alcohol, phenyl ethanol, salicylic acid, and p‐hydroxy benzyl acid were selected as the analytes. Several factors that affect peak splitting were investigated. The injection time, SDS micellar concentration, hydrophobicity of the analytes, and analytes concentration were the most important factors. A hypothesis was proposed to explain the peak‐splitting phenomena. Several means to avoid peak‐splitting phenomena were proposed, such as controlling sample injection time and hydrophobicity of the analyte, decreasing SDS concentration and increasing sample concentration. However, the most practical method for avoiding peak splitting was to control the sample injection time.

Combination of cationic surfactant-assisted solid-phase extraction with field-amplified sample stacking for highly sensitive analysis of chlorinated acid herbicides by capillary zone electrophoresis

This report describes a novel online field-amplified sample stacking (FASS) procedure to analyze 16 chlorinated acid herbicides. By using a poly(vinyl alcohol) (PVA)-coated capillary to reduce electroosmotic flow and introducing a methanol-water plug before sample loading, the sample injection time could be very long without loss of sample and separation efficiency. Under the optimized condition, the FASS procedure could provide great sensitivity enhancement (5000-10 000-fold) and satisfactory reproducibility (relative standard deviations of migration times less than 2.4%, relative standard deviations of peak areas less than 8.0%). Combined with cationic surfactant-assisted solid-phase extraction (CSA-SPE), the limit of detection of the herbicides ranged from 0.269 to 20.3 ppt, which are two orders lower than those of the US Environmental Protection Agency standard method 515.1. The CSA-SPE-FASS-CE method was successfully applied to the analysis of local pond water.

Development and validation of a capillary electrophoresis method for the determination of phenothiazines in human urine in the low nanogramper milliliter concentration range using field-amplified sample injection

A capillary zone electrophoresis (CZE) method with ultraviolet-visible detection has been established and validated for the determination of five phenothiazines: thiazinamium methylsulfate, promazine hydrochloride, chlorpromazine hydrochloride, thioridazine hydrochloride, and promethazine hydrochloride in human urine. Optimum separation was obtained on a 64.5 cm x 75 microm bubble cell capillary using a buffer containing 150 mM tris(hydroxymethyl)aminomethane and 25% acetonitrile at pH 8.2, with temperature and voltage of 25 degrees C and 20 kV, respectively. Naphazoline hydrochloride was used as an internal standard. Field-amplified sample injection (FASI) has been applied to improve the sensitivity of the detection. Considering the influence of parameters affecting the on-line preconcentration (nature of preinjection plug, sample solvent composition, injection times, and injection voltage) and due to the significant interactions among them, in this paper we propose for the first time the application of a multivariate approach to carry out the study. The optimized conditions were as follows: preinjection plug of water for 7 s at 50 mbar, electrokinetic injection for 40 s at 6.2 kV, and 32 microm of H3PO4 in the sample solvent. Also, a solid-phase extraction (SPE) procedure is developed to obtain low detection limits and an adequate selectivity for urine samples. The combination of SPE and FASI-CZE-UV allows adequate linearities and recoveries, low detection limits (from 2 to 5 ng/mL), and satisfactory precisions (3.0-7.2% for an intermediate RSD %).

PH-mediated acid stacking with reverse pressure for the analysis of cationic pharmaceuticals in capillary electrophoresis

When using capillary electrophoresis (CE) for the analysis of biological samples, it is often necessary to employ techniques to overcome peak-broadening that results from having a high-conductivity sample matrix. To improve the concentration detection limits and separation efficiency of cationic pharmaceuticals in CE, pH-mediated acid stacking was performed to electrofocus the sample, improving separation sensitivity for the analyzed cations by 60-fold. However, this method introduces a large titrated acid plug into the capillary. To overcome the limitations this low-conductivity plug poses to stacking, the plug was removed prior to the separation step by applying reverse pressure to force it out of the anode of the capillary. Employing this technique allows for roughly twice the volume of sample to be injected. A maximum sample injection time of 240 s was attainable with baseline peak resolution compared to a maximum sample injection time of 120 s without reverse pressure, leading to a twofold decrease in the limits of detection of the analytes used. Separation efficiency overall is also improved when utilizing the reverse pressure step. For example, a 60 s sample injection time results in 94,000 theoretical plates as compared to 60,500 theoretical plates without reverse pressure. This reverse-pressure method was used for detection and quantitation of several cationic pharmaceuticals that were prepared in Ringer's solution to simulate microdialysis sampling conditions.

Anticipated Diagnosis of Adverse Events after Stem Cell Transplantation Using Highly Sensitive STR-PCR for Chimerism Quantification.

Introduction: The current gold standard for chimerism quantification after allo-stem cell transplantation (SCT) is based on the polymerase chain reaction of DNA short tandem repeats (STR-PCR). This methodology is sex-independent, highly informative and quantitatively accurate. However, this method has a moderate sensitivity (1–3%).Objective: To test the usefulness of increasing the assay sensitivity by 3 different approaches in order to detect recipient residual cells.Material and Methods: Two sets of 20 samples, belonging to artificial mixtures of male and female peripheral blood (PB) cells in different proportions (% male: 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0) were prepared to test the assay sensitivity and reproducibility. Additionally 58 samples from 10 selected patients showing different clinical evolution post-SCT were retrospectively studied. Chimerism was analyzed on genomic DNA (purified QIAamp DNA blood kit, Qiagen) by multiplex STR-PCR (AmpFlSTR SGM Plus revealed on capillary electrophoresis in an ABI Prism 3100 automated DNA sequencer; Applied Biosystems). Three different approaches aimed to increase the sensitivity of STR-PCR were tested: (1) Increasing DNA concentration in the PCR (1x, 2.5x, 5x), (2) Increasing the concentration of the PCR product for the capillary electrophoresis (1x, 2.5x, 5x) and (3) Increasing the sample injection time into the capillary for the electrophoresis (10s, standard time, 30s and 50s).Results: The standard STR-PCR showed a sensitivity of 1% in both sets of male-female PB artificial mixtures. Both approaches increasing DNA concentration in the PCR and the PCR product in the capillary electrophoresis did not achieved better sensitivity. Moreover, appropriate analysis of the samples was frequently hampered due to fluorescence saturation. However, longer injection times (30s, 50s) showed that currently underrepresented DNA fragments were conspicuously overestimated, giving a one-log increase in the sensitivity of the STR-PCR assay, which reached a 0.1%. This approach was used to re-analyze the 23 SCT patient samples previously identified as in complete chimerism by standard STR-PCR. The 0.1% sensitivity methodological approach allowed to detect residual/reappearing recipient cells in three of the 23 analyzed samples. Therefore this new approach would have allowed to anticipate the detection of graft rejection and disease relapse in 2/10 patients, 7 and 77 days respectively.Conclusion: Long injection times (30s and 50s) in the capillary electrophoresis after STR-PCR result in a one log sensitivity increase (from 1% to 0.1%). This would allow earlier diagnosis of adverse events after SCT favoring a prompt introduction of therapeutic interventions.