Ken K.-C. Yeung

Suppression of Electroosmotic Flow and Prevention of Wall Adsorption in Capillary Zone Electrophoresis Using Zwitterionic Surfactants

Addition of zwitterionic surfactants such as dodecyldimethyl(3-sulfopropyl)ammonium hydroxide, hexadecyldimethyl(3-sulfopropyl)ammonium hydroxide, and coco (amidopropyl)hydroxyldimethylsulfobetaine (Rewoteric AM CAS U) to an electrophoretic buffer suppress the electroosmotic flow by 50-90%. Onset of suppression occurs at approximately the critical micelle concentration of the surfactant. CAS U effectively suppresses the electroosmotic flow over the pH range 3-12. Addition of 2 mM CAS U to the electrophoretic buffer prevents adsorption of cationic proteins lysozyme, α-chymotrypsinogen A, cytochrome c, and ribonuclease A. Migration time reproducibility for these proteins is ∼1% RSD within 1 day and 2-5% from day to day. Efficiencies in excess of 750 000 plates/m and recoveries of >80% were observed for protein injections from distilled water. Alternatively if 2 mM CAS U is added to samples, recoveries were quantitative, although efficiencies decreased to 325 000-600 000 plates/m. The natural electroosmotic flow of the capillaries is regenerated simply by rinsing with sodium hydroxide.

Recent applications of capillary electrophoresis-mass spectrometry (CE-MS): CE performing functions beyond separation.

Capillary electrophoresis is one of the separation tools commonly used in conjugation with mass spectrometry. Its primary purpose is to resolve the components in a sample mixture prior to mass spectral identification. Moreover, an increasing number of applications reported in the literature involve the use of CE for additional purposes, such as sample preparation and derivatization, and the study of biochemical properties. This review provides an overview on the various roles of CE beyond that of a simple separation tool. While the scope focuses on the area of interest rather than a predefined time period, the majority of the references highlighted were initially published within the past five years.

Improved resolution of inorganic anions in capillary electrophoresis by modification of the reversed electroosmotic flow and the anion mobility with mixed surfactants

Abstract The standard method for analysis of inorganic anions by capillary electrophoresis involves adding tetradecyltrimethylammonium bromide (TTAB) to the buffer to reverse the electroosmotic flow (EOF). The resolution achieved using this procedure is greatly improved by adding the zwitterionic surfactant, coco amidopropylhydroxydimethylsulfobetaine (CAS U) to lower the magnitude of the reversed EOF and alter the anion mobilities. In a mixed surfactant system, varying the ratio of TTAB to CAS U allows monotonic alteration of the EOF from fully reversed (TTAB alone) to near zero (CAS U alone). The total surfactant concentration (if greater than the critical micelle concentration) and buffer pH have minimal effect on the EOF. In addition, the anion mobilities can be altered to a minor degree by varying the ratio of TTAB to CAS U, which contributes to the improved anion resolution. The effect on the EOF of other surfactant systems involving CAS U and other cationic or anionic surfactants is also studied.

Capillary electrophoresis using a surfactant-treated capillary coupled with offline matrix-assisted laser desorption ionization mass spectrometry for high efficiency and sensitivity detection of proteins.

A method of combining capillary electrophoresis (CE) using a surfactant-modified capillary with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is described for protein analysis. The CE-MALDI-MS coupling is based on CE fraction collection of nanoliter volume samples in less than 5 microl of dilute acid. This offline coupling does not require any special instrumentation and can be readily performed with commercial instruments. Protein adsorption during CE separation is prevented by coating the capillary with the surfactant didodecyldimethylammonium bromide. This surfactant binds strongly with the capillary wall, hence it does not desorb significantly to interfere with subsequent MALDI-MS analysis. It is shown that the use of a dilute acid for CE fraction collection is advantageous in lowering the detection limit of MALDI-MS compared to using an electrophoretic buffer. The detection limit for proteins such as cytochrome c is 23 fmol injected for CE, or 1.2 fmol spotted for MALDI-MS. This sensitivity is comparable to alternative CE-MALDI-MS coupling techniques using direct CE sample deposition on the MALDI target. In addition, the fraction collection approach has the advantage of allowing multiple reactions to be carried out on the fractioned sample. These reactions are very important in protein identification and structure analysis.

Separation of positional and structural isomers by cyclodextrin-mediated capillary zone electrophoresis

Previously derived models for optimization of cyclodextrin (CD)-mediated capillary zone electrophoresis (CZE) referred only to the separations of enantiomers. These models assume that the mobility of the inclusion complexes of the two solutes are equal (i.e., μACD = μBCD). With other types of solutes, such as positional and structural isomers, this assumption is not valid (i.e., μACD ≠ μBCD). In this work, the effectiveness of the model of Wren and Rowe, which was developed for enantiometric separations, is evaluated for cyclodextrin-mediated CZE of other types of solutes. Experimental data is obtained for the α-cyclodextrin-mediated separation of positional and structural isomers, modelled by nitrophenols and phenylbutyric acids, respectively. It was found that the mobilities of the inclusion complexes of the isomers differed from one another (μACD ≠ μBCD) and that the complex mobility did not correlate with the solute mobility, the formation constant or the “quality of fit”. Despite the complex mobilities for the positional and structural isomers not being equal, the Wren and Rowe model is nonetheless effective for predicting the optimum α-cyclodextrin concentration. Only when the formation constants for two isomers are approximately equal (KACD ≈ KBCD) does the optimum α-cyclodextrin concentration differ from that predicted.

Isotopic Separation of [14N]- and [15N]Aniline by Capillary Electrophoresis Using Surfactant- Controlled Reversed Electroosmotic Flow

Separation of isotopically labeled [(14)N]- and [(15)N]aniline was achieved using capillary electrophoresis based on the isotopic effect on pK(a). The effects of the buffer co-ion, pH, and electroosmotic mobility on the resolution are investigated in this paper. Electroosmotic flow (EOF) was controlled using the zwitterionic surfactant Rewoteric AM CAS U as buffer additive. The resultant EOF was anodic (reversed) and low in magnitude (0.6 × 10(-)(4) cm(2)/(V·s)). The resolution of [(14)N]- and [(15)N]aniline was 1.22. Addition of a cationic surfactant, cetyltrimethylammonium bromide, to the zwitterionic surfactant increased the magnitude of the anodic EOF. This EOF improved the resolution to 1.33 based on mobility counterbalance.

In-capillary protein enrichment and removal of nonbuffering salts using capillary electrophoresis with discontinuous buffers.

Salt is abundant in biological samples and can cause problems in capillary electrophoresis (CE) due to excessive Joule heating and electrodispersion. Desalting with solid phase minibeds is currently most compatible with the small sample volumes of CE. They are however difficult to prepare and suffer from poor bed-to-bed reproducibility. Alternatively, enrichment of proteins and peptides was developed using CE, by trapping them at their isoelectric points with a discontinuous buffer of mismatched pH. Ionic salts, such as sodium chloride, do not possess isoelectric points and therefore are not retained by the discontinuous buffer. In this work, the removal of ionic salt during protein enrichment using CE with discontinuous buffers was investigated. Nonbuffering ions were found to electromigrate through the pH junction without disrupting the enrichment process and were eventually removed from the capillary. Mass spectral data obtained from the enriched and desalted sample confirmed a significant signal enhancement. Finally, a strong acid was introduced to remove the pH junction and thus facilitated a subsequent capillary zone electrophoresis separation. An integrated procedure of enrichment, desalting, and separation was demonstrated on a mixture of three protein standards.

Increased Expression of Simple Ganglioside Species GM2 and GM3 Detected by MALDI Imaging Mass Spectrometry in a Combined Rat Model of Aβ Toxicity and Stroke

The aging brain is often characterized by the presence of multiple comorbidities resulting in synergistic damaging effects in the brain as demonstrated through the interaction of Alzheimer’s disease (AD) and stroke. Gangliosides, a family of membrane lipids enriched in the central nervous system, may have a mechanistic role in mediating the brain’s response to injury as their expression is altered in a number of disease and injury states. Matrix-Assisted Laser Desorption Ionization (MALDI) Imaging Mass Spectrometry (IMS) was used to study the expression of A-series ganglioside species GD1a, GM1, GM2, and GM3 to determine alteration of their expression profiles in the presence of beta-amyloid (Aβ) toxicity in addition to ischemic injury. To model a stroke, rats received a unilateral striatal injection of endothelin-1 (ET-1) (stroke alone group). To model Aβ toxicity, rats received intracerebralventricular (icv) injections of the toxic 25-35 fragment of the Aβ peptide (Aβ alone group). To model the combination of Aβ toxicity with stroke, rats received both the unilateral ET-1 injection and the bilateral icv injections of Aβ₂₅₋₃₅ (combined Aβ/ET-1 group). By 3 d, a significant increase in the simple ganglioside species GM2 was observed in the ischemic brain region of rats who received a stroke (ET-1), with or without Aβ. By 21 d, GM2 levels only remained elevated in the combined Aβ/ET-1 group. GM3 levels however demonstrated a different pattern of expression. By 3 d GM3 was elevated in the ischemic brain region only in the combined Aβ/ET-1 group. By 21 d, GM3 was elevated in the ischemic brain region in both stroke alone and Aβ/ET-1 groups. Overall, results indicate that the accumulation of simple ganglioside species GM2 and GM3 may be indicative of a mechanism of interaction between AD and stroke.

Ultrahigh-resolution capillary electrophoretic separation with indirect ultraviolet detection: isotopic separation of [14N]- and [15N]ammonium.

Separation of isotopically labeled [14N]/[15N] ammonium was performed with capillary electrophoresis. This ultrahigh‐resolution separation was based on mobility counterbalance with precise control of the anodic electroosmotic flow. Mixtures of zwitterionic surfactant (Rewoteric AM CAS U) and cationic surfactant (cetyltrimethylammonium bromide) were used as buffer additives to modify the electroosmotic mobility. Indirect ultraviolet detection was used with benzyltributylammonium as the buffer coion. Baseline‐resolved peaks of [14N]‐ and [15N]ammonium were obtained within 11 min. The detection limit was 0.01 mM for both [14N]‐ and [15N]ammonium. Linear calibration in concentration was observed up to 1.0 mM for [15N]ammonium and 2.0 mM for [14N]ammonium. Calibration of the isotopic ratio, [15N]ammonium concentration to total ([14N]‐ and [15N])ammonium, was valid from 5 to 95%.

Bioenergy II: Characterization of the Pesticide Properties of Tobacco Bio-Oil

Pyrolysis converts biomass such as agricultural and forestry waste into bio-oil, preserving some chemicals while creating other, new ones. Nicotine, a chemical present in tobacco leaves and a known pesticide, was found to remain intact during pyrolysis. As expected, insecticidal properties were observed for tobacco bio-oil. Pesticide characteristics of tobacco bio-oil have been observed on the Colorado potato beetle (CPB), a pest currently resistant to all major insecticides, as well as a few bacteria and fungi that do not currently respond well to chemical treatment. Unexpectedly, nicotine-free fractions of the bio-oil were also found to be highly lethal to the beetles and successful at inhibiting the growth of select microorganisms. Through GC-MS, it was found that the active, nicotine-free fractions were rich in phenolics, chemicals likely created from lignin during pyrolysis. While bio-oils in general are known to contain phenolic chemicals, such as cresols, to our best knowledge, quantitative analysis has not been performed to determine if these chemicals are solely responsible for the observed pesticide activities. Based on GC-MS results, ten of the most abundant chemicals, eight of which were phenolic chemicals, were identified and examined through bio-assays. A mixture of these chemicals at the concentration levels found in the bio-oil did not account for the bio-oil activity towards the microorganisms. Tobacco bio-oil may have potential as a pesticide, however, further analyses using liquid chromatography is necessary to identify the remaining active chemicals.