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

Abstract

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.     

Solute-solvent interactions in micellar electrokinetic chromatography: V. Factors that produce peak splitting

The experimental conditions that produce analyte peak splitting in micellar electrokinetic capillary chromatography (MEKC) have been systematically investigated. The system studied was a neutral phosphate buffer and sodium dodecyl sulfate (SDS) micelles as pseudostationary phase. A number of analytes showing a wide variety of hydrophobicity values and several organic solvents as sample diluents have been tested. Peak splitting phenomena are mainly due to the presence of organic solvent in the sample solution. They increase with the hydrophobicity of the analyte and decrease with the increase of the surfactant concentration. When hydrophobic compounds are analyzed the suggested ways to avoid split peaks are: (i) the use of 1-propanol or 1-butanol as sample diluent instead of methanol or acetonitrile or (ii) the use of high concentration of surfactant in the separating solution when the analyte must be dissolved in pure methanol or acetonitrile.     

Optimization of the separation conditions of tetracyclines on a preselected reversed-phase column with embedded urea group

The use of a C12 stationary phase with embedded polar group has been investigated for the separation of seven tetracyclines. The influence of pH, organic modifier, buffer, and temperature on the peak shape and analyte separation was discussed. It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5/ACN (84:16 v/v) or 5 mM perchlorate buffer pH 2.5/ACN (75:25 v/v) at a temperature not exceeding 20 degrees C. This study reveals that the retention mechanism is ion-pairing.     

Peak distortion in the column liquid chromatographic determination of omeprazole dissolved in borax buffer.

Injection of a sample containing omeprazole dissolved in borax buffer (pH 9.2) into a reversed-phase liquid chromatographic system consisting of a mixture of acetonitrile and phosphate buffer (pH 7.6) as the mobile phase and a C18 surface-modified silica as the solid phase resulted under special conditions in split peaks of omeprazole. The degree of peak split and the retention time of omeprazole varied with the concentration of borax in the sample solution and the ionic strength of the mobile phase buffer as well as with the column used. Borax is eluted from the column in a broad zone starting from the void volume of the column. The retention is probably due to the presence of polyborate ions. The size of the zone varies with the concentration of borax in the sample injected. In the borax zone the pH is increased compared with the pH of the mobile phase, and when omeprazole (a weak acid) is co-eluting in the borax zone its retention is affected. In the front part and in the back part of the borax zone, pH gradients are formed, and these gradients can induce the peak splitting. When the dissolving medium is changed to a phosphate buffer or an ammonium buffer at pH 9 no peak distortion of omeprazole is observed.     

Quality control of benserazide-levodopa and carbidopa-levodopa tablets by capillary zone electrophoresis

In modern practice, the treatment of Parkinson's disease and syndrome is carried out using pharmaceutical formulations containing a combination of levodopa and a decarboxylation inhibitor (carbidopa or benserazide). Two pharmaceutical formulations were quantified by capillary zone electrophoresis using two procedures which differed only in the kind of background electrolyte used. One procedure used a 25 mM phosphate buffer, pH 2.5, while the second one used a 25 mM borate buffer, pH 8.5. The electrophoretic analysis was carried out using an uncoated fused- silica capillary, a separation voltage of 20 kV with currents typically less than 60 microA, and spectrophotometric detection at 205 nm. Calibration curves were performed for levodopa (concentration range 1-100 microg/mL), for carbidopa and benserazide (1-50 microg/mL), and the plots of the peak area versus concentration were found to be linear with a correlation coefficient better than 0.9990. Satisfactory results were obtained when commercial tablets were analyzed in terms of accuracy (98-102%), repeatability (0.6-2.0%), and intermediate precision (1.1-2.6%).     

Electrochemical analysis of d-penicillamine using a boron-doped diamond thin film electrode applied to flow injection system

The electroanalysis of d-penicillamine in 0.1 phosphate buffer (pH 7) was studied at a boron-doped diamond thin film (BDD) electrode using cyclic voltammetry as a function of concentration of analyte and pH of analyte solution. Comparison experiments were performing using a glassy carbon (GC) electrode. The BDD electrode exhibited a well-resolved and irreversible oxidation voltammogram, but the GC electrode provided only an ill-defined response. The BDD electrode provided a linear dynamic range from 0.5 to 10 mM and a detection limit of 25 muM (S/B>/=3) in voltammetric measurement. It was also found that the peak potentials were decreased when the pH of the analyte solution was increased. In addition, penicillamine has been studied by hydrodynamic voltammetry and flow injection analysis with amperometric detection using the BDD electrode. The flow injection analysis results at the diamond electrode indicated a linear dynamic range from 0.5 to 50 muM and a detection limit of 10 nM (S/N approximately 4). The proposed method was applied to determine d-penicillamine in dosage form (capsules), the results obtained in the recovery study (255+/-2.50 mg per tablet) were comparable to those labeled (250 mg per tablet).     

On-line preconcentration for capillary electrophoresis-atomic fluorescence spectrometric determination of arsenic compounds

An on-line preconcentration method was developed for capillary electrophoresis (CE) with hydride generation-atomic fluorescence spectrometric (HG-AFS) detection of arsenite, arsenate, dimethylarsenic acid, and monomethylarsenic acid. These arsenic species were negatively charged in the sample solution with high pH. When the potential was applied to the electrophoretic capillary, the negatively charged analyte ions moved faster and stacked at the boundary of sample and CE buffer with low pH. So, high sample pH in combination with low buffer pH allowed the injection of large sample volumes (approximately 1100 nL). Comparison of the preconcentration of analyte solution, prepared with doubly deionized water and that prepared with lake or river water, indicated that preconcentration was independent on the original matrix. With injection of approximately 1100 nL sample, an enrichment factor of 37-50-fold was achieved for the four species. Detection limits for the four arsenic species ranged from 5.0 to 9.3 microg.L(-1). Precisions (RSDs, n = 5) were in the range of 4.9-6.7% for migration time, 4.7-11% for peak area, and 4.3-7.1% for peak height, respectively. The recoveries of the four species in locally collected water solution spiked with 0.1 microg.mL(-1) (as As) ranged from 83 to 109%.     

Overloaded elution band profiles of ionizable compounds in reversed‐phase liquid chromatography: Influence of the competition between the neutral and the ionic species

The parameters that affect the shape of the band profiles of acido‐basic compounds under moderately overloaded conditions (sample size less than 500 nmol for a conventional column) in RPLC are discussed. Only analytes that have a single pK_a are considered. In the buffer mobile phase used for their elution, their dissociation may, under certain conditions, cause a significant pH perturbation during the passage of the band. Two consecutive injections (3.3 and 10 μL) of each one of three sample solutions (0.5, 5, and 50 mM) of ten compounds were injected on five C₁₈‐bonded packing materials, including the 5 μm Xterra‐C₁₈ (121 Å), 5 μm Gemini‐C₁₈ (110 Å), 5 μm Luna‐C₁₈(2) (93 Å), 3.5 μm Extend‐C₁₈ (80 Å), and 2.7 μm Halo‐C₁₈ (90 Å). The mobile phase was an aqueous solution of methanol buffered at a constant _W^WpH of 6, with a phosphate buffer. The total concentration of the phosphate groups was constant at 50 mM. The methanol concentration was adjusted to keep all the retention factors between 1 and 10. The compounds injected were phenol, caffeine, 3‐phenyl 1‐propanol, 2‐phenyl butyric acid, amphetamine, aniline, benzylamine, p‐toluidine, procainamidium chloride, and propranololium chloride. Depending on the relative values of the analyte pK_a and the buffer solution pH, these analytes elute as the neutral, the cationic, or the anionic species. The influence of structural parameters such as the charge, the size, and the hydrophobicity of the analytes on the shape of its overloaded band profile is discussed. Simple but general rules predict these shapes. An original adsorption model is proposed that accounts for the unusual peak shapes observed when the analyte is partially dissociated in the buffer solution during its elution.     

Electrochemical analysis of -penicillamine using a boron-doped diamond thin film electrode applied to flow injection system

The electroanalysis of -penicillamine in 0.1 phosphate buffer (pH 7) was studied at a boron-doped diamond thin film (BDD) electrode using cyclic voltammetry as a function of concentration of analyte and pH of analyte solution. Comparison experiments were performing using a glassy carbon (GC) electrode. The BDD electrode exhibited a well-resolved and irreversible oxidation voltammogram, but the GC electrode provided only an ill-defined response. The BDD electrode provided a linear dynamic range from 0.5 to 10 mM and a detection limit of 25 μM (S/B≥3) in voltammetric measurement. It was also found that the peak potentials were decreased when the pH of the analyte solution was increased. In addition, penicillamine has been studied by hydrodynamic voltammetry and flow injection analysis with amperometric detection using the BDD electrode. The flow injection analysis results at the diamond electrode indicated a linear dynamic range from 0.5 to 50 μM and a detection limit of 10 nM (S/N≈4). The proposed method was applied to determine -penicillamine in dosage form (capsules), the results obtained in the recovery study (255±2.50 mg per tablet) were comparable to those labeled (250 mg per tablet).     

The chromatographic interaction and separation of metal ions with 8-Quinolinol stationary phases in several aqueous eluents

Several 8-quinolinol silica gel (QSG) columns were used, with metal-uptake capacities of 10–156 μmol g^?1. Various transition and heavy metal ions were used as analytes in nitrate, sulfate, phosphate, citrate, tartrate, oxalate, phthalate, and maleate mobile phases. Metal-ion retention increased with column capacity and pH. Optimum capacity factors were obtained on columns of intermediate capacity (27 and 46 μmol g^?1). Retention times decreased with an increase in eluent buffer concentration, typically by half with a doubling of buffer. Evidence is presented for the occurence of mobile-phase complexation of analyte ions by eluent buffer species. Multiple or split peaks were often observed when the analyte solvent differed from the mobile phase. Chromatographic separation of up to six metals on the QSG columns is demonstrated in tartrate and maleate mobile phases.     

A study of thermal peak splitting in capillary GC

Fluctuating thermal gradients are recognized as a distinct source of peak splitting in capillary column gas chromatography. In this study, the influence of analyte signal level, temperature oscillation amplitude, and oscillation frequency on splitting are quantitatively examined. Stable gradients are also considered. A simple, effective method is proposed for virtually eliminating thermal peak splitting in isothermal capillary GC analysis.     

Phase-transfer catalytic determination of phenols as methylated derivatives by gas chromatography with flame ionization and mass-selective detection

A microemulsion electrokinetic chromatographic (MEEKC) method was developed for the separation of six catechins, specific marker phytochemicals of Cistus species. The MEEKC method involved the use of sodium dodecyl sulfate (SDS) as surfactant, heptane as organic solvent and butan-1-ol as co-solvent. In order to have a better stability of the studied catechins, the separation was performed under acidic conditions (pH 2.5 phosphate buffer). The effects of SDS concentration and of the amount of organic solvent and co-solvent on the analyte resolution were evaluated. The optimized conditions (heptane 1.36% (w/v), SDS 2.31% (w/v), butan-1-ol 9.72% (w/v) and 50 mM sodium phosphate buffer (pH 2.5) 86.61% (w/v)) allowed a useful and reproducible separation of the studied analytes to be achieved. These conditions provided a different separation profile compared to that obtained under conventional micellar electrokinetic chromatography (MECK) using SDS. The method was validated and applied to the determination of catechin and gallocatechin in lyophilized extracts of Cistus incanus and Cistus monspeliensis.     

A fresh look into background electrolyte selection for capillary electrophoresis‐laser induced fluorescence of peptides and proteins

This study reports a reinvestigation of background electrolyte selection strategy for performance improvement in CE‐LIF of peptides and proteins. This strategy is based on the employment of high concentrations of organic species in BGE possessing high buffer capacity and low specific conductivity in order to ensure excellent stacking preconcentration and separation resolution of fluorescently tagged peptides and proteins. Unlike universal UV detection, the use of such BGEs at high concentrations does not lead to degradation of LIF detection signals at the working excitation and emission wavelengths. At the same buffer ionic strength, pH and electric field, an “inorganic‐species‐free” BGE (or ISF BGE) for CE‐LIF of fluorescently labeled beta amyloid peptide Aβ 1–42 (a model analyte) offered a signal intensity and peak efficiency at least three‐times higher than those obtained with a conventional BGE normally used for CE‐LIF, while producing an electric current twice lower. Good peak performance (in terms of height and shape) was maintained when using ISF BGEs even with samples prepared in high‐conductivity phosphate buffer saline matrix. The advantageous features of such BGEs used at high concentrations over conventional ones in terms of high separation resolution, improved signal intensities, tuning of EOF magnitudes and minimization of protein adsorption on an uncoated fused silica capillary are demonstrated using Alexa‐488‐labelled trypsin inhibitor. Such BGE selection approach was applied for investigation of separation performance for CE‐LIF of ovalbumin labelled with different fluorophores.     

Simultaneous determination of aminothiols, ascorbic acid and uric acid in biological samples by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection has been employed for the separation and determination of homocysteine, cysteine, reduced glutathione, ascorbic acid and uric acid. Effects of several important factors such as the acidity and concentration of the running buffer, separation voltage, injection time and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 500 microm diameter platinum disk electrode at a working potential of +1.05 V (vs saturated calomel electrode). The five analytes were well separated within 10 min in a 50 cm long fused silica capillary at a separation voltage of 18 kV in a 100 mm phosphate buffer (pH 7.8). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with the detection limits (S/N = 3) ranging from 0.83 to 2.58 microm. The proposed method was successfully applied to determine cysteine, reduced glutathione, ascorbic acid and uric acid in human whole blood and rat brain tissues with satisfactory assay results and should find a wide range of bioanalytical applications.     

Separation and determination of the macrolide antibiotics (erythromycin, spiramycin and oleandomycin) by capillary electrophoresis coupled with fast reductive voltammetric detection

Separation and determination of erythromycin, spiramycin and oleandomycin by capillary zone electrophoresis coupled with fast reductive voltammetric detection using an Hg-film electrode was investigated in a simple aqueous phosphate buffer system. The influence of pH, concentration of phosphate, applied voltage, capillary length and dimension on the separation was examined and optimized. The entire separation of erythromycin, spiramycin, and oleandomycin was achieved in a 0.2 mol/L phosphate buffer system without organic modifiers. The electrochemical detection parameters, such as electrode material, applied waveform, scan rate, preconcentration potentials and preconcentration times, were investigated and discussed. This approach provides high separation efficiency and high sensitivity for all compounds, with detection limits (3 x peak-to-peak baseline noise) of 7.5 x 10(-8) mol/L for spiramycin, and 3 x 10(-7) mol/ L for erythromycin and oleandomycin. The calibration plot of peak areas for each separated peak vs. concentration of analyte was found to be linear over three orders of magnitude.     

Determination of active constituents in Lonicera confusa DC. by capillary electrophoresis with amperometric detection

A method based on capillary electrophoresis with amperometric detection has been developed for the determination of luteolin, chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeic acid in the dried flower buds, leaves and stems (three medicinal parts) of Lonicera confusa DC., respectively. The effects of several important factors such as detection potential, the concentration of the running buffer, separation voltage and injection time were investigated to acquire the optimum conditions. The detection electrode was a 300 microm diameter carbon disc electrode at a working potential of + 0.90 V (vs saturated calomel electrode). The four analytes can be well separated within 10 min in a 40 cm-long fused silica capillary at a separation voltage of 12 kV in a 50 mM borate-25 mM phosphate buffer (pH 8.0). The relationship between peak current and analyte concentration was linear over about 3 orders of magnitude with detection limits (S/N = 3) ranging from 0.35 to 0.52 microM for all analytes. The proposed method has been successfully applied to the monitoring of bioactive constituents in the real plant samples with satisfactory assay results.     

早期生物矿化过程中紫外吸收动力学曲线上的负峰现象

近年来, Jens等[1]利用紫外光度法测定生物矿化溶液的吸光度(即混浊度)的变化, 实时地记录生物矿物形成过程的信息, 从而研究其矿化规律. 实验发现, 胶原/羟基磷灰石矿化的紫外吸收动力学曲线并不是胶原和磷酸钙沉淀混浊度的简单迭加, 而是一条平滑的阶梯形曲线, 它反映了有机相和无机相相互作用的过程特征. 在采用紫外光度法研究生物矿化过程中首次发现, 阶梯形矿化曲线上还有更精细的变化: 在一定条件下, 矿化曲线起峰时出现一个负峰. 本文还研究了磷酸钙浓度、胶原浓度及入射光波长对该负峰峰位和峰值的影响. 研究该负峰与系统参数的相关性, 对于了解早期生物矿化的机理和优化新型骨及牙齿等组织工程框架材料的制备工艺[2]都具有重要的意义.     

Application of a novel micro-injector in the determination of indole derivatives in the rat pineal gland by capillary electrophoresis with electrochemical detection

A novel micro-injector has been fabricated for capillary electrophoresis (CE). It was successfully used for the determination of some indole derivatives for example melatonin (MT), serotonin (5-HT), tryptophan (Trp), and 5-hydroxy-tryptophane (5-HTrp) in the rat pineal gland by capillary electrophoresis with electrochemical detection (CE-EC). CE was performed in 0.20 mol L(-1) phosphate buffer (pH 8.0). The compounds investigated can be well separated and detected within 15 min. The working electrode used was a 300-microm diameter carbon electrode positioned opposite the outlet of the capillary. The relationship between peak current and analyte concentration was highly linear in the range from 0.10 to 500 micromol L(-1); detection limits (S/N = 3) were 0.03-0.13 micromol L(-1). The proposed method has been successfully used to analyze real biological samples.     

Optimization of background electrolytes for capillary electrophoresis I. Mathematical and computational model

A mathematical and computational model is introduced for optimization of background electrolyte systems for capillary zone electrophoresis of anions. The model takes into account mono- or di- or trivalent ions and allows also for modeling of highly acidic or alkaline electrolytes, where a presence of hydrogen and hydroxide ions is significant. At maximum, the electrolyte can contain two co-anions and two counter-cations. The mathematical relations of the model are formulated to enable an easy algorithmization and programming in a computer language. The model assesses the composition of the background electrolyte in the analyte zone, which enables prediction of the parameters of the system that are experimentally available, like the transfer ratio, which is a measure of the sensitivity in the indirect photometric detection or the molar conductivity detection response, which expresses the sensitivity of the conductivity detection. Furthermore, the model also enables the evaluation of a tendency of the analyte to undergo electromigration dispersion and allows the optimization of the composition of the background electrolyte to reach a good sensitivity of detection while still having the dispersion properties in the acceptable range. Although the model presented is aimed towards the separation of anions, it can be straightforwardly rearranged to serve for simulation of electromigration of cationic analytes. The suitability of the model is checked by inspecting the behavior of a phosphate buffer for analysis of anions. It is shown that parameters of the phosphate buffer when used at neutral and alkaline pH values possess singularities that indicate a possible occurrence of system peaks. Moreover, if the mobility of any analyte of the sample is close to the mobilities of the system peaks, the indirect detector signals following the background electrolyte properties will be heavily amplified and distorted. When a specific detector sensitive on presence of the analyte were used, the signal would be almost lost due to the excessive dispersion of the peak.     

Insights into electrolyte effects on photoactivities of dye-sensitized photoelectrochemical cells for water splitting

Dye-sensitized photoelectrochemical cell(DS-PEC) is an especially attractive method to generate hydrogen via visible light driven water splitting. Electrolyte, an essential component of DS-PEC, plays a great role in determining the photoactivities of devices for water splitting. When using phosphate buffer(pH = 6.4)as electrolyte, the DS-PEC displayed much higher photoactivity than using 0.1 M Na_2SO_4(pH = 6.4) as electrolyte. The insight is phosphate anion gathers together to form a negative electrostatic field on TiO_2 surface, which increases the resistance in the TiO_2/catalyst and electrolyte interface and validly reduces the charge recombination from TiO_2 to the oxidized catalyst.