Ultraviolet absorbance detection of colchicine and related alkaloids on a capillary electrophoresis microchip

The separation and UV absorbance detection of four toxic alkaloids, colchicine, thiocolchicine, colchicoside, and thiocolchicoside, on a microchip-based capillary electrophoresis device are reported. To increase the sensitivity of UV absorbance detection, optical cells with extended path lengths were integrated into the separation channel during the microfabrication process. The absorbance values realized on the microchip using these optical cells were proportional to the increase in average depths according to the Beer-Lambert Law, resulting in sensitivity enhancements by as much as five times. Linearity of response was observed from 5.0 to 500 mg L⁻¹ of colchicine, with detection limits ranging from 2 to 6 mg L⁻¹ depending upon the specific alkaloid and the dimension of the optical cell. The extraction of colchicine from spiked milk samples was performed and an average recovery rate of 83% with a relative standard deviation of 3.8% was determined using the optimized conditions on the microchip.     

Investigation of ionene adsorption on quartz surfaces by thermal-lens spectrometry

Thermal-lens spectrometry was used for the investigation of the adsorption of ionene to quartz surfaces. The thermooptical analysis of the surface makes it possible to distinguish the modified surface from a clean quartz surface and to provide sensitive direct concentration measurements of the light absorbing co-adsorbed substance. The co-adsorption of chromate ions and 2,10-ionene from aqueous solutions to quartz surfaces was investigated and the desorption procedure proposed.     

Rapid capillary zone electrophoresis method for the determination of metal cations in beverages

A rapid and reliable capillary zone electrophoresis method for the determination of inorganic cations was developed. The complete separation of K⁺, Ba²⁺, Ca²⁺, Na⁺, Mg²⁺, Mn²⁺, Ni²⁺, Cd²⁺, Li⁺ and Cu²⁺ can be achieved in 4 min with a simple electrolyte composed by 10 mM imidazole as the carrier buffer and background absorbance provider and acetic acid as the complexing agent (pH 3.60). Injection was performed hydrostatically by elevating the sample at 10 cm for 30 s. The running voltage was +25 kV at room temperature. Indirect UV-absorption detection was achieved at 185 nm. The detection limit was in the range between 0.06 mg/l (Mg²⁺) and 0.57 mg/l (K⁺) and the quantification limits ranged from 0.10 mg/l (Ni²⁺) to 0.80 mg/l (Cu²⁺). The calibration graphs were linear in the concentration range from the quantification limit till at least 1 g/l in K⁺, 10 mg/l in Ba²⁺, Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺ and Cd²⁺, 40 mg/l in Na⁺ and 12 mg/l in Li⁺ and Cu²⁺. The repeatability, intraday and interday analysis were ≤1.55% and ≤3.64% for migration time and ≤3.38% and ≤3.63% for peak area. The method developed has been applied to several beverage samples with only a simple dilution and filtration treatment of the sample. The proposed method is simple, fast, cheap and it is achieved with common products in either laboratory. For these reasons, it is a very useful method for routine analysis.     

Capillary electrophoretic system incorporating an UV/CL dual detector

We developed a capillary electrophoretic system incorporating an ultra-violet absorption (UV)/chemiluminescence (CL) dual detector, taking advantage of the CL reaction of luminol-hydrogen peroxide and the batch-type CL detection cell. UV detection was carried out using the on-capillary method while CL detection was performed using the end-capillary method. Examination of isoluminol isothiocyanate (ILITC) as a model sample revealed two main peaks with UV detection and one main peak with CL detection. The first peak in the UV detection data corresponded to the main peak in the CL detection data. We then determined that the ILITC sample included natural ILITC as well as an impurity that had absorption behavior but did not have CL properties and labeling ability. Furthermore, the components of a mixture containing glycine, glycylglycine and glycylglycylglycine, all labeled with ILITC, were well separated and detected using the present system. The present system easily, rapidly, and simultaneously produces useful information due to the presence of both UV and CL detectors.     

Determination of flavonoids in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. by capillary electrophoresis with electrochemical detection

Four flavonoids (rutin, hyperoside, quercitrin and quercetin) in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. were determined by capillary electrophoresis with wall-jet amperometric detection. The working electrode was a 500 μm diameter carbon disc electrode and the detection potential was +0.95 V (versus Ag/AgCl). Effects of several important factors, such as the running buffer and its corresponding pH and concentration, separation voltage, injection time were investigated to acquire the optimum conditions for separation of these four flavonoids. Baseline separation for the four flavonoids was obtained within 21 min in a 60 cm length capillary at a separation voltage of 15 kV with a 60 mmoL/L Na₂B₄O₇-120 mmoL/L NaH₂PO₄ buffer (pH 8.8) as running buffer. The relationship between peak currents and analyte concentrations was linear over about two orders of magnitude with detection limits (defined as S/N = 3) ranging from 0.02 to 0.05 μg/mL for all analytes. This method was applied for the determination of the above four flavonoids in H. cordata Thunb. and S. chinensis (Lour.) Bail. with simple extraction procedures, and the assay results were satisfactory.     

A flow-based enzyme-linked immunosorbent assay on a polydimethylsiloxane microchip for the rapid determination of immunoglobulin A

A flow-based enzyme-linked immunosorbent assay (ELISA) on a polydimethylsiloxane (PDMS) microchip has been developed for the rapid determination of immunoglobulin A (IgA). The analytical principle of this integrated method is the same as the conventional sandwich-type ELISA. A primary antibody (anti-IgA) was adsorbed on the surface of a PDMS microchannel, and then an antigen (IgA) and a secondary antibody (anti-IgA HRP labeled) were reacted successively. The resulting antigen-antibody complex, fixed on the surface of the microchannel, was detected using Amplex^® Red and a fluorescent imaging system. The calibration curve of the IgA standard solution was linear in the range of 0-50 ng/mL at the flow rate of 10 μL/min. This flow rate corresponds to the reaction time of 4.8 s. Compared to the conventional assay on a 96-well microtiter plate, the present assay on the microchip dramatically shortened the reaction time necessary for the enzyme-substrate reaction from 30 min to 4.8 s, i.e., to 1/375. The amounts of the reagent and sample were also reduced to 1/100 compared to the 96-well microtiter plate.     

Identification and quantitation of iodotyrosines and iodothyronines in hydrolysate of iodinated casein by capillary electrophoresis

In this paper, a new method for separation, identification and quantitation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine (MIT), 3,5-diiodo-L-tyrosine (DIT), L-thyronine (T₀), 3,5-diiodo-L-thyronine (T₂), 3,5,3′-triiodo-L-thyronine (T₃) and 3,3′,5,5′-tetraiodo-L-thyronine (T₄)] was described by using capillary electrophoresis with photodiode-array ultraviolet-visible detection (CE-UV). The certain influence factors were systematically investigated, including the type, concentration and pH of buffer, and additive. We found that 10 mM sodium borate running buffer (pH 8.5) containing 0.10 mM β-CD as additive reagent allowed the best instrumental conditions for the optimum separation of the iodotyrosines and iodothyronines. Under optimized conditions, the analytical time was within 6 min, using an uncoated fused-silica capillary of 75 μm inner diameter with an effective length of 30 cm. The reproducibility of the migration time and peak area was less than 0.6% and 6.8%, respectively. A linear range from 10-1000 μg/mL and low limits of detection from 1.3-3.4 μg/mL were obtained at the detection wavelength of 280 nm. Our preliminary results show that the method is well suitable for determination of the hydrolysate of iodinated casein.     

Microchip capillary electrophoresis coupled with an end-column electrochemiluminescence detection

An easy and universal wall-jet configuration for microchip CE-ECL detection system was constructed and investigated in this work. Two detection modes of pre-column and post-column were applied to the above system. TPA, tramadol and lidocaine were chosen as model analytes to estimate the system in both modes. The important operational parameters such as the concentration of luminescent reagent and the distance between the separation outlet and the working electrode were optimally obtained and compared for the first time.     

Dynamics of Arabidopsis thaliana soluble proteome in response to different nutrient culture conditions

In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.     

Separation of phenolic acids by capillary electrophoresis with indirect contactless conductometric detection

A new method for the electrophoretic separation of nine phenolic acids (derivatives of benzoic and cinnamic acids) with contactless conductometric detection is presented. Based on theoretical calculations, in which the mobility of the electrolyte co- and counterions and mobility of analytes are taken into consideration, the electrolyte composition and detection mode was selected. This approach was found to be especially valuable for optimization of the electrolyte composition for the separation of analytes having medium mobility. Indirect conductometric detection mode was superior to the direct mode as predicted theoretically. The best performance was achieved with 150 mM 2-amino-2-methylpropanol electrolyte at pH 11.6. The separation was carried out in a counter-electroosmotic mode and completed in less than 6 min. The LODs achieved were about 2.3-3.3 microM and could be further improved to 0.12-0.17 microM by using a sample stacking procedure. The method compares well to the UV-Vis detection.