Separation of amino acids and amines by capillary electrophoresis using poly(ethylene oxide) solution containing cetyltrimethylammonium bromide

We describe simultaneous analysis of naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and amine derivatives by capillary electrophoresis in conjunction with light-emitting diode-induced fluorescence (LEDIF) detection using poly(ethylene oxide) (PEO) containing cetyltrimethylammonium bromide (CTAB). In the presence of CTAB and acetonitrile (ACN), adsorption of PEO on the capillary wall is suppressed, leading to generation of a fast and reproducible electroosmotic flow (EOF). In order to optimize separation resolution and speed, 100 mM Tris–borate solution (pH 7.0) containing 20 mM CTAB and 25% ACN was used to fill the capillary and to prepare 1.2% PEO that entered the capillary via EOF. The analysis of 14 NDA-amino acid and -amine derivatives by this approach is rapid (< 4 min), efficient ((0.9–6.4) × 10⁵ theoretical plates), and sensitive (the LODs (S/N = 3) range from 9.5 to 50.5 nM). The RSD values (n = 5) of the migration times and peak heights of the analytes for the intraday analysis are less than 1.5 and 1.2%, respectively. We have validated the practicality of this approach by quantitative determination of 10 amino acids and amines in a beer samples within 4 min.     

Swelling of poly(ethylene oxide) gel in aqueous solutions of sodium dodecyl sulfate with added sodium chloride

The swelling behavior of poly(ethylene oxide) (PEO) gels in aqueous solutions of sodium dodecyl sulfate (SDS) with and without NaCl was investigated. In the absence of NaCl, PEO gels with different degrees of cross-linking began to swell from a concentration lower than the critical micelle concentration (cmc) of SDS, then showed sigmoidal enhancements of swelling in a higher SDS concentration region until the degrees of swelling reached maximum values. The SDS concentration at which the swelling began to appear was in reasonable agreement with the critical aggregation concentration (cac) value reported for the aqueous PEO system. For the cases where NaCl was present, the swelling behavior of PEO gel was different from that when NaCl was absent in the following way. The concentrations where the swelling begins to appear, and hence those where the degree of swelling rises steeply, decreased with an increase in NaCl concentration. The ultimate degrees of swelling at higher concentration regions also decreased with an increase in the NaCl concentration. The lowering of the SDS concentrations at which the PEO gel began to swell is in line with the decreases in the cmc of SDS solutions containing NaCl and also with the decreases in the cac of PEO solution. Electronic Publication     

Separation and selectivity in micellar electrokinetic chromatography using sodium dodecyl sulfate micelles or Tween 20-modified mixed micelles

The separation and selectivity of eight aromatic compounds ranging from hydrophilic to hydrophobic properties in micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) micelles or Tween 20-modified mixed micelles were investigated. The effect of different operation conditions such as SDS and Tween 20 modifier surfactant concentration, buffer pH, and applied voltage was studied. The resolution and selectivity of analytes could be markedly affected by changing the SDS micelle concentration or Tween 20 content in the mixed micelles. Applied voltage and pH of running buffers were used mainly to shorten the separation time. Complete separation of eight analytes could be achieved with an appropriate choice of the concentration of SDS micelles or Tween 20-modified mixed micelles. Quicker elution and better precision could be obtained with SDS-Tween 20 mixed micelles than with SDS micelles. The mechanisms that migration order of those analytes was mainly based on their structures and solute-micelle interactions, including hydrophobic, electrostatic, and hydrogen bonding interactions, were discussed.     

A simple,rapid, and sensitive method for analysis of SYPRO Red labeled sodium dodecyl sulfate-protein complexes by capillary electrophoresis with laser-induced fluorescence

We describe a segmental filling method for the analysis of SYPRO Red labeled sodium dodecyl sulfate (SDS)-proteins (SRSPs) by capillary electrophoresis-laser induced fluorescence (CE-LIF) with electroosmotic counterflow of poly(ethylene oxide) (PEO). It is shown that SDS and salt play a crucial role in determining the fluorescence intensity of the SRSP. Although the fluorimetric measurements reveal that the SRSPs fluoresce strongly in Tris-borate (TB) buffer containing 0.1% SDS and high concentrations of NaCl (100 mM), these conditions are not appropriate to CE in view of Joule heating. To overcome that impediment, we applied a plug of 0.1% SDS (1/5 to 1/3 of the injection volume) prior to injection of samples (0.64 microL) prepared in TB buffer containing 50 mM NaCl and SYPRO Red. When using a background electrolyte of 0.6% PEO in TB buffer containing NaCl, electroosmotic counterflow of the analytes allows one to concentrate large sample volumes (up to 1/3 of effective capillary length) in 21 min, with detection of 0.35 and 0.10 nM for bovine serum albumin and casein, respectively. With a linear dynamic range from 10 nM to 5 microM, this method provides the capability of determining the concentration of casein in cow's milk as 0.45 +/- 0.03 mM (n = 5).     

Sensitive determination of biogenic amines by capillary electrophoresis with a new fluorogenic reagent 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde

An effective approach was proposed to the derivatization of seven biogenic amines using 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde (FBQCA) as a fluorogenic reagent. The sensitive determinations of these derivatives were achieved by micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection. The derivatization and electrophoretic conditions have been optimized. A running buffer was composed of mixtures of 25 mM pH 9.5 boric acid, 25 mM SDS, and 27% ACN. At 25 °C and 22.5 kV, the baseline separation of the derivatives was accomplished in 13 min. The detection limit (S/N = 3) was found as low as 0.4 nM. The proposed method was validated by the linearity of two orders magnitude and correlation coefficient in the range 0.9969–0.9998. Also, the procedure was successfully applied to the determination of biogenic amines in soy sauce, fish and wine samples.     

On‐line Concentration and Separation of Parabens by Micellar Electrokinetic Chromatography Using Polymer Solutions

Simultaneous separation and quantification of seven parabens commonly used as preservatives in cosmetic products, by micellar electrokinetic chromatography with a stacking technique has been demonstrated. An effective on‐line concentration strategy involving a combination of sweeping and the use of polymer solutions is a key feature of the proposed method, which successfully determined individual parabens. The analysis parameters such as injection time, pH and concentration of phosphate solution, and concentration of sodium dodecyl sulphate (SDS) and poly(ethylene oxide) (PEO) were examined. The optimum conditions were found to be as follows: a 15 mM phosphate solution (pH 9.5) containing 20 mM SDS for filling the capillary, and for the separation electrolytes, 0.100% PEO (8 MDa) added to the phosphate and SDS solution of the same composition as for the capillary. The entire analysis process was completed in 13 min and a 930‐2200‐fold enhancement factor was achieved. The LODs (S/N = 3) for this approach were in the range from 4.32 to 7.78 nM. The linear range for each paraben was between 50 nM and 5.0 μM (R² > 0.990). The optimized method was then successfully applied to the determination of parabens in commercial cosmetic products.     

Simultaneous monitoring of inorganic cations, amines and amino acids in human sweat by capillary electrophoresis

The determination of cationic constituents of sweat is widely recognized as a difficult analytical task due to its complex composition and minute sample volumes available for the individual analysis. Capillary electrophoresis (CE) has been evaluated as a simple routine method to measure sweat metal cations, biogenic amines, and amino acids using a sampling procedure previously developed in one of collaborative teams. The carrier electrolyte, which consisted of 10 mM 4-methylbenzylamine, 6.5 mM α-hydroxyisobutyric acid, and 2 mM 18-crown-6 at pH 4.25, allowed the separation of five cations (NH₄⁺, K⁺, Ca²⁺, Na⁺, Mg²⁺) and four amino acids (ornithine, histidine, lysine, arginine) to be completed in about 13 min with a positive polarity of the applied voltage (30 kV). By increasing the sample volume (due to employing hydrodynamic instead of hydrostatic injection mode), it was also possible to detect indirect UV signals of Zn²⁺, diethanolamine, and trithanolamine. Sweat samples were collected from the fingers and forearms of three healthy male volunteers and analyzed by CE. A good repeatability and reproducibility of peak area responses based on five intraday and three inter-day assays (average %R.S.D. less than 3.5 and 2.5, respectively) were obtained. The limits of detection were in the range of 3.2-5.8 μM for alkali and alkaline-earth cations (hydrostatic injection) and 0.27-0.79 μM for other target analytes (hydrodynamic injection). The analytical results for particular analytes were found to vary, depending on the sampling spot and individual, but in general correspond well to clinical concentration ranges.     

Determination of lead in the presence of morin-5′-sulfonic acid and sodium dodecyl sulfate by adsorptive stripping voltammetry

A simple and sensitive electroanalytical method is developed for the determination of lead by adsorptive stripping voltammetry (AdSV) in the presence of morin-5′-sulfonic acid (MSA) and sodium dodecyl sulfate (SDS). The Pb-MSA complex accumulates on the surface of a hanging mercury drop electrode (HMDE) and peak current is measured by square wave voltammetry (SWV). The complex is reduced at −0.48 V and peak current increases when low concentrations of SDS are added to the sample solution. The experimental variables pH, MSA concentration (C_MSA); accumulation time (t_acc); accumulation potential (E_acc), and SDS concentration (C_SDS), as well as potential interferences, are investigated. Under the optimized conditions (pH 3.2; C_MSA: 0.5 μmol L⁻¹; t_acc: 60 s; E_acc: −0.35 V, and C_SDS: 20 μmol L⁻¹), peak current is proportional to the concentration of Pb(II) over the 0.1-32.0 μg L⁻¹ range, with a detection limit of 0.04 μg L⁻¹. The relative standard deviation for a solution containing 5.0 μg L⁻¹ of Pb(II) solution was 1.5% for seven successive assays. The method was validated by determining Pb(II) in synthetic sea water (ASTM D665) spiked with ICP multi-element standard solution and in certified reference water (GBW08607). Finally, the method was successfully applied to the determination of Pb(II) in tap water and sea water after UV digestion.     

Shear and extensional rheology of solutions of mixtures of poly(ethylene oxide) and anionic surfactants in ionic environments

Interactions between a high molecular weight poly(ethylene oxide) (PEO) and the anionic surfactant sodium dodecyl benzene sulfonate (SDBS) in aqueous solutions were investigated by shear and extensional rheometry. Results for mixtures between PEO and sodium dodecyl sulfate (SDS) are also presented for comparison purposes. Addition of anionic surfactants to PEO solutions above the critical aggregation concentration (CAC), at which micellar aggregates attach to the polymer chain, results in an increase in shear viscosity due to PEO coil expansion, and a strengthening of interchain interactions. In extensional flows, these interactions result in a decrease of the critical shear rate for the onset of the characteristic extension thickening of the PEO solutions that is due to transient entanglements of polymer molecules. The relaxation times associated with these transient entanglements are not directly proportional to the shear viscosity of the solutions, but rather vary more rapidly with surfactant concentration. In the presence of an electrolyte, coil contraction results in lower shear viscosities and a decrease in the extension thickening effects at surfactant concentrations just beyond the CAC. The relaxation times associated with transient entanglement reach a minimum at the same surfactant concentration as the shear viscosity, which indicates that coil contraction is responsible for the observed effects in both types of flow. However, the increase in extensional-flow entanglement relaxation times is much more abrupt than the decrease in shear viscosity. All these results point to a greater sensitivity of extensional flows on the molecular conformation of PEO/surfactant complexes.     

Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser-induced fluorescence detection

A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 microm erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser-induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8-32 amol/single cell.     

Simultaneous determination of metal ions, amino acids, and other small biogenic molecules in human serum by capillary zone electrophoresis with transient isotachophoretic preconcentration

CE with indirect UV detection was used for the simultaneous determination of lithium, magnesium, calcium, creatinine, carnitine, and a number of amino acids in human serum. The target analytes, positively charged under acidic electrolyte conditions, were separated with positive separation voltage polarity using 10 mM 4-methylbenzylamine, 4.5 mM citric acid, 25% (v/v) methanol at pH 4.05 as background electrolyte providing optimal separation. When analyzing real samples, however, some peaks were broadened due to essentially destacking conditions. In order to maintain the separation efficiency and also enhance the detection sensitivity, transient isotachophoresis (tITP) sample stacking was applied and yielded theoretical plate numbers in the range from 160,000 (arginine) to 350,000 (creatinine). The limit of detection values with tITP preconcentration were 0.11-0.26 mg L(-1) for metal cations, 1.0 mg L(-1) for creatinine, and 1.3-3.9 mg L(-1) for histidine, lysine, arginine, and ornithine. The method precision for peak areas was from 0.4 to 5.0% relative standard deviation using the matrix sodium as internal standard. The accuracy of the developed tITP-CZE system was verified by consistent results for Li+, Mg2+, Ca2+, and creatinine obtained on analyzing two serum certified reference materials. The only sample preparation required was ultrafiltration and acidification (to release protein-bound alkaline earths), and working ranges for individual analytes corresponded well to clinical concentration ranges.     

Micellar electrokinetic chromatography-chemiluminescent detection of biogenic amines using N-(4-aminobutyl)-N-ethylisoluminol as derivatization reagent and trivalent copper chelate as chemiluminescence enhancer

A facile, sensitive and universal method was established for analysis of biogenic amines using micellar electrokinetic chromatography coupled with chemiluminescent (CL) detection. It was found that diperiodatocuprate (III) (K₅[Cu(HIO₆)₂], DPC), a transition metal chelate at unstable high oxidation state, could effectively enhance the reaction between luminol-type compound and hydrogen peroxide, to produce very strong CL signal. In addition, triethylamine was found to be able to effectively improve the yield of the derivatization reaction between biogenic amines and a luminol-type derivatization reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). Based on these facts, three biogenic amines were pre-column derivatized with ABEI, and post-column detected using high sensitive luminol-hydrogen peroxide-DPC CL system. Since the background was quite low, and the signal was quite strong, a considerable improved sensitivity was obtained. The presented method had been successfully applied to simultaneously analyze glycine, proline and phenylalanine with the detection limits (S/N = 3) of 0.030 μmol L⁻¹, 0.23 μmol L⁻¹ and 0.21 μmol L⁻¹, respectively. To evaluate its potential application value, glycine in saliva and urine samples was detected using this method, and satisfied results were obtained. This approach can be further extended to detection of many other compounds such as peptides and drugs by using luminol-type derivatization reagent.     

毛细管电泳-激光诱导荧光检测法分析胃癌组织及癌旁正常组织蛋白质的差异性

胃癌是临床常见的恶性肿瘤之一。近年来寻找肿瘤相关特异蛋白质是蛋白质组学研究的热点。本文通过考察毛细管动态涂层方法、筛分介质聚环氧乙烷(PEO)的浓度、缓冲液的pH值、分离电压、温度及荧光染料对分离效果的影响,建立了毛细管电泳-激光诱导荧光法分离胃癌组织及癌旁正常组织蛋白质的方法;通过分离检测,获得两者的蛋白质指纹图谱。经分析,两者的指纹图谱相似度达到0.8以上,差异蛋白质分子质量集中在50000~100000 Da之间,提示某些小分子蛋白质可能是和肿瘤发生相关的特异蛋白质,从而缩小了特异性分子标记物的筛选范围。病理组织学分型及蛋白质电泳峰数目的统计结果验证了该方法的可靠性。该方法具有临床应用的潜力。     

Solubilization of 1-butanol in a sodium dodecyl sulfate-poly(ethylene oxide) system by NMR and conductivity at 298.1 and 283.1 K

 The effects of adding 0.1 molal 1-butanol to the aqueous SDS system at 298.1 K and the aqueous PEO–SDS system at 298.1 and 283.1 K have been studied. NMR NOESY experiments on the PEO– SDS–1-butanol system in D₂O were obtained. NMR self-diffusion experiments and measurements of NMR chemical shifts and specific conductivity were carried out on the samples, i.e. on samples with PEO and without PEO. The addition of 1-butanol to an aqueous SDS–PEO system decreases the critical aggregation concentration (c.a.c). Determination of the second critical concentration (c ₂) depends on the method of measurements, i.e. the molecular species monitored. Conductivity measurements will give c ₂ as the SDS concentration where free micelles (micelles not bound to the polymer) are formed. PEO self-diffusion measurements, on the other hand, determine c ₂ as the SDS concentration where the polymer is saturated with SDS. Both the c.a.c and the c ₂ decrease upon 1-butanol addition. However, the c ₂ value exhibits a larger decrease than the c.a.c value. Thus, the amount of polymer bound surfactant molecules decreases upon addition of 1-butanol. Micellar solubilization of 1-butanol starts at c.a.c., but the solubilization capacity is low until the surfactant concentration reaches c ₂, where the increase in solubilization is significant. Thus, solubilization data can be used to detect c ₂, the concentration where free micelles form. Received: 21 July 1997 Accepted: 9 February 1998     

Simultaneous separation of five major ribonucleic acids by capillary electrophoresis with laser-induced fluorescence in the presence of electroosmotic flow: Application to the rapid screening of 5S rRNA from ovarian cancer cells

RNA integrity is important in RNA studies because poor RNA quality may impact downstream methodologies. This study proposes a rapid and cost-effective method for the determination of RNA integrity based on CE-LIF in the presence of electroosmotic flow. The proposed method uses poly(ethylene) oxide (M_avg = 4,000,000 Da) as a sieving matrix for total RNA separation. Ethidium bromide (μg mL⁻¹) was dissolved in a polymer solution as an interchelating dye for on-column fluorescent labeling. The 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNA from the total human RNA extracted from the cells were fully separated using the proposed method. The lowest detectable concentration of total RNA achieved was 100 pg μL⁻¹ with a 6 min sample injection followed by on-column concentration. In addition, the temperature-induced degradation of total RNA was observed by CE-LIF. The electropherograms revealed more fragmentation of 28S and 18S rRNAs by temperature-induced hydrolysis compared with the 5.8S rRNA, 5S rRNA and tRNA. Therefore, the results indicated that RNA degradation should be considered for long-term, high-temperature incubations in RNA-related experiments involving RNA hybridization. The proposed method is furthermore, applied to the determination of 5S rRNA overexpressed in ovarian cancer cells as compared to the cervical cancer cells. Overall, CE-LIF is highly promising for rapid screening of ovarian cancers without tedious pre-amplification steps.