This report presents an analytical strategy for the identification of beta-lactoglobulin (beta-Lg) variants or isoforms in bovine milk by using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) and nano-liquid chromatography-electrospray-mass spectrometry-mass spectrometry (nano-LC-ESI-MS-MS). beta-Lg isoforms were separated and collected by CE, and their molecular masses were determined by CE-ESI-MS. Protein isoforms collected by CE were digested with trypsin and analyzed by nano-LC-ESI-MS-MS. Isoforms analyzed by CE-ESI-MS presented multiply charged ions that after deconvolution resulted in molecular masses of 18,364 +/- 1.0 Da and 18,279 +/- 2.0 Da. Fragmentation of parent ions in MS-MS experiments of specific tryptic peptides were carried out to identify beta-Lg variants in milk samples. The milk samples analyzed were indeed phenotypes AA, BB, or AB.
Multicapillary electrophoresis continues to see improvements in speed, robustness, and reliability. This paper reports on our work on two components belonging to a multicapillary sequencer developed in our group. Injection of the DNA samples into the capillaries was optimized to make it reproducible and to reduce the amount of sample volume required. An alternative laser illumination of the capillaries was also developed. Light intensity in the capillaries was increased as a result of a step-by-step scanning of the laser and the use of microlenses in front of the capillaries.
Researchers came together for the 24th Annual Meeting of the American Electrophoresis Society (AES), which was held November 4-9, 2007, at the Salt Palace Convention Center in Salt Lake City, UT, U.S.A. The Annual AES meeting is held in conjunction with the annual meeting of the American Institute of Chemical Engineers (AIChE). This year's meeting had a significant emphasis on theoretical and experimental advances in Biological Micro Electro Mechanical Systems (BioMEMS), electrokinetics, and proteomics technologies. A total of 15 sessions were held, within which 71 presentations and 18 posters were discussed. This review provides a brief sampling of the exciting research presented at the conference.
Accurate authentication of related species is necessary to prevent the illegal replacement of species of higher quality and/or price with lower-quality, less expensive species. Atlantic salmon (Salmo salar), an expensive and valuable species, can be intentionally substituted with rainbow trout (Oncorhynchus mykiss). Using the technique of capillary zone electrophoresis (CZE), the identification of raw species was based on the analysis of muscle sarcoplasmic (water-soluble) proteins, while for canned species identification was based on muscle proteins solubilized with urea and sodium dodecyl sulfate (SDS). Either water or urea-SDS extracted proteins gave reproducible and distinct electrophoretic patterns of proteins for both species independent of storage conditions. Although data analysis indicated changes in protein profiles for both species during storage, species identification was still possible. In addition, quality determination during refrigerated and frozen storage involved the use of two freshness indexes: K value (the relationship between inosine monophosphate, inosine, and hypoxantine) and pH. A linear correlation was found between changes in electrophoretic patterns and K values for refrigerated salmon species. Trout kept in storage for six days reached a constant K value higher than the maximum limit for a product of good quality, preventing determination of any correlation with electrophoretic pattern. The protein profiles obtained by CZE during long storage revealed their potential for monitoring both differences between fish species and changes in quality during refrigerated, frozen, and canned storage of the species under study.
A simple and rapid method based on capillary electrophoresis with amperometric detection (CE-AD) to calculate the dissociation constant of salicyl alcohol and phenol is described. The effects of several factors, such as the potential applied to the working electrode, injection time, and separation voltage, were investigated to find the optimum conditions for pKa determination. Operating in a wall-jet configuration, a 300-pm carbon disk electrode used as the working electrode exhibited good response at 0.86 V (vs a saturated calomel electrode ISCE]) for salicyl alcohol and phenol. The effective mobilities of salicyl alcohol at different temperatures were determined by monitoring the migration time changes of salicyl alcohol and phenol. The dissociation constants of salicyl alcohol and phenol were calculated to be 9.94 and 9.96, respectively, which are in good agreement with the literature.
The development and experimental optimization of a novel flow injection-capillary electrophoresis (FI-CE) analyzer employing UV-visible fiber optic detection is described. The analyzer incorporates a miniature charge-coupled device (CCD) spectrometer and operates in a graphical programming environment. Data from experimental optimization studies and small molecule separations involving affinity capillary electrophoresis (ACE) and indirect detection of anions are presented. Future directions in terms of instrument automation and incorporation into a microfluidic format are also discussed.
Capillary electrophoresis is still widely used for DNA sequencing. The quality of the replaceable sieving matrix is a key area for massive sequencing with regard to speed and efficiency. The T25 polymer has been tested extensively and compared to poly(N,N-dimethylacrylamide) (PDMA). In terms of peak resolution, both polymers perform similarly. On the other hand, the run time is much shorter with the T25 polymer.
Voltage gradient partial-filling affinity capillary electrophoresis (VGPFACE) is used to determine binding constants between carbonic anhydrase B (CAB, E.C.4.2.1.1) and arylsulfonamides, and vancomycin (Van) from Streptomyces orientalis and teicoplanin (Teic) from Actinoplanes teicomyceticus and D-Ala-D-Ala terminus peptides. Two variations of VGPFACE are described herein. In the first technique, the capillary is partially filled with ligand at increasing concentrations followed by a sample containing receptor and two noninteracting standards and electrophoresed in buffer using a voltage gradient that increases from 0 to 25 kV over the duration of the experiment. Upon continued electrophoresis, zones of solution overlap, and equilibrium is established between the ligand and receptor, causing a shift in the migration time of the receptor with respect to the noninteracting standards. This change in migration time is utilized for estimating a binding constant (K(b)). In the second technique, voltage gradient partial-filling multiple-injection ACE (VGPFMIACE), a multiple-injection sequence is used whereby the capillary is partially filled with ligand at increasing concentrations, a noninteracting standard, three or four separate plugs of receptor each separated by small plugs of buffer, and a plug containing a second noninteracting standard; this is then electrophoresed in buffer with a similar voltage gradient. Upon continued electrophoresis, a similar equilibrium is established and a value for K(b) is obtained for the interaction. The VGPFACE technique expands the functionality and potential of ACE as an analytical tool to examine various receptor-ligand interactions.
C18-sphingosine 1-phosphate (C18-SPP) is a sphingolipid with important functional and structural roles in cells. In this paper we report a new capillary electrophoresis technique that is coupled to a laser-induced fluorescence detection (CE-LIF) method to quantify the level of C18-SPP in biological samples. The method utilizes a commercial standard C17-sphingosine 1-phosphate (C17-SPP) derivatized with naphthalene-2,3-dicarboxaldehyde (NDA), a fluorogenic dye used to label primary amines. The detection limit for the C17-SPP NDA was 0.54 fmol. We quantified the C18-SPP in leukemic human cells before and after irradiation by gamma rays. We demonstrated that the amounts of this sphingolipid decreased after the irradiation. In a second part of this work, we used the technique to evaluate the ability of a novel transparent fused-silica capillary, which allows the use of fused-silica capillaries without burning a window. Solarization, homogeneity, and sensitivity were studied using this capillary. The results demonstrate that this new durable capillary can provide a sensitive and reproducible quantitation procedure for CE-LIF studies.
A simple and efficient method based on micellar electrokinetic capillary electrophoresis (MECC) with electrochemical detection for the determination of catechin, rutin, hyperin, and kaempferol in Plumula Nelumbini is described. Under optimum conditions, all analytes were well separated within 20 min at a separation voltage of 16 kV in a 60-mmol/L borate running buffer (pH 8.0) containing 20 mmol/L sodium dodecyl sulfate (SDS). The current response was linear over two orders of magnitude with detection limits (S/N = 3) ranging from 6.90 x 10(-8) g/mL to 1.30 x 10(-7) g/mL for all analytes. This work provides a useful method for the analysis of Plumula Nelumbini.
Capillary-zone electrophoresis with electrochemical detection has been used for the separation and determination of (-)-epicatechin, rutin, hyperin, chlorogenic acid, and quercetin in hawthorn and hawthorn piece. The effects of several important factors, including the running buffer acidity, the separation voltage, and the working electrode potential, were evaluated to acquire the optimum analytical conditions. The working electrode was a 300-μm carbon-disk electrode at a working potential of +0.95 V (vs. SCE). Under the optimum conditions, the analytes can be well separated within 16 min in a 75-cm-long fused-silica capillary. The current response was linear over two orders of magnitude with detection limits (S/N = 3) ranging from 6.00 × 10−8 to 3.75 × 10−7 g/mL for all analytes. The method was successfully used in the analysis of hawthorn and hawthorn piece and the assay results were satisfactory.
Corn has been known for its accumulation of flavones and phenolic acids. However, many parts of corn, except kernel, have not drawn much attention. In this work, a method based on capillary zone electrophoresis with electrochemical detection has been used for the separation and determination of epicatechin, rutin, ascorbic acid (Vc), kaempferol, chlorogenic acid, and quercetin in corn silk, leaf, and kernel. The distribution comparison of the ingredients among silk, leaf, and kernel is discussed. Several important factors--including running buffer acidity, separation voltage, and working electrode potential--were evaluated to acquire the optimum analysis conditions. Under the optimum conditions, the analytes could be well separated within 19 min in a 40-mmol/L borate buffer (pH 9.2). The response was linear over three orders of magnitude with detection limits (S/N = 3) ranging from 4.97 x 10(-8) to 9.75 x 10(-8) g/mL. The method has been successfully applied for the analysis of corn silk, leaf, and kernel with satisfactory results.
Heteroduplex analysis is the most popular method for double-stranded DNA mutation detection thus far. Since different DNA fragments have various melting temperatures due to different base pair compositions and sizes, the PCR-amplified DNA fragments need to be denatured, generally, over 90 degrees C and reannealed to give a mixture of four duplexes, two homoduplexes, and two heteroduplexes for electrophoresis or chromatographic analysis. To separate homoduplex and heteroduplex DNA fragments, the column temperature must be controlled at the DNA melting temperature. This is tedious for DNA mutation study, since the melting point has to be measured before heteroduplex analysis. A novel heteroduplex analysis method using a capillary electrophoresis-laser-induced fluorescence (CE-LIF) detection system is described in this paper for the separation of all homo- and heteroduplex DNA fragments, which have different melting temperatures, at a single temdegrees C, 64 degrees C, and 70 degrees C--were separated and detected. The assay is simple, accurate, and sensitive, giving it potential for multiplex analysis for DNA mutation study.
In this report, the most recent results regarding the use of microchip-capillary electrophoresis and pulsed electrochemical detection are reviewed. This article is particularly focused on the analysis of three groups of compounds: phenolic contaminants, phenolic acids, and phenolic antioxidants. Background information and a brief discussion covering other related analytical strategies are also included.
A chiral separation model of gel electrochromatography in a polydimethylsiloxane (PDMS) microfluidic device for amino acids (AAs) is presented. Six pairs of fluorescein isothiocyanate (FITC)-labeled dansyl amino acids (Dns-AAs) were separated in a 36-mm effectual separation channel in less than 120 sec, with resolutions all above 0.96. This highly efficient PDMS chiral microfluidic chip was prepared by inserting the mixture solution of monomers, crosslinkers, and radical initiation into the microchannel via syringe. Specifically, allyl-gamma-cyclodextrin (CD) as a chiral selector and crosslinker was bonded in gamma-CD-bonded polyacrylamide (PAA) gel, which was the separation media, and was immobilized in a PDMS microchannel through the stable linkage of 3-(trimethoxysilyl)-propyl methacrylate (Bind-Silane, Sigma, St. Louis, MO, U.S.A.). The preparation not only permitted the prompt chiral separation of AAs, but also extended application of the PDMS microfluidic device by restraining its hydrophobicity through the PAA gel monolithic column. Furthermore, the longevity of the PDMS microfluidic device was prolonged significantly. This can also be a powerful way to develop a rapid and efficient bioanalysis method and portable analytical apparatus.
In this study, a novel electrochemical detection system for capillary electrophoresis was proposed. In the proposed system, sheath flow would transport analytes to the working electrode surface to allow electrochemical detection. The sheath-flow electrochemical detector would require no modification of capillaries and could accommodate capillaries larger than 25 microm i.d.
A simple and reliable analytical procedure using capillary electrophoresis with UV absorbance detection for the direct enantiomeric resolution and quantitation of chiral fungicide metalaxyl is described. Several native cyclodextrins and modified beta-cyclodextrins were investigated as chiral additives to 50 mM sodium tetraborate buffer pH 9.3. The effect of their concentration on the resolution of metalaxyl enantiomeric forms was studied. The best results were achieved when 55 mM succynyl-beta-cyclodextrin in 50 mM sodium tetraborate buffer pH 9.3 was used. The optimized method showed satisfactory intraday repeatability as far as relative migration times and relative peak areas are concerned, with a detection limit of 0.1 mM for both enantiomers, and has been successfully applied to the analysis of a real plant protection product containing metalaxyl-M (metalaxyl R-form) as active ingredient.
A review of microchip-based dielectrophoretic separations is presented. Benefits of miniaturization and applications of these devices are discussed. Device geometries employed to date are described.
