Fluorescently Labeling Amino Acids in a Deep Eutectic Solvent.

The increased use of deep eutectic solvents (DESs) in recent years has been significant and provides new approaches to sample collection and preparation. At the same time, the use of these new solvents to prepare samples can present challenges for subsequent analyses. Common analytical approaches, such as fluorescent labeling, may not be compatible with the solvents. In this work, we explore how effective three traditional fluorescent labels can be at derivatizing amino acids in the most common DESs, formed from choline chloride and ethylene glycol. We demonstrate that the unique solvent characteristics of the DESs still allow for two of the fluorophores, fluorescein isothiocyanate and 5-carboxyfluorescein succinimidyl ester, to effectively label amino acids. Initial optimizations of the reaction conditions demonstrate that we can effectively label both d- and l-amino acids, in solution with concentrations of amino acids down to 4 µM. Capillary electrophoretic separations following this preparation can detect as little as 50 nM. This is possible without removal of any DES from the sample matrix. These results represent the first complete fluorescent labeling reaction in a DES and subsequent capillary electrophoretic separation of the analytes.

Metal cation control of electroosmotic flow magnitude in phospholipid-coated capillaries.

CZE has become widespread for the separation and analysis of biomolecules such as proteins and peptides, due to factors such as, the speed of the separations, low sample volume, and high resolution associated with the technique. However, the separation of biomolecules by CZE does present a significant challenge due to the electrostatic attraction and adsorption of cationic, or cation containing, biomolecules to the capillary surface. To that end numerous methods have been developed to passivate, or protect the surface, in order to prevent the adsorption of analytes. Yet, in the process of protecting the capillary surface, the potential for further modification of the EOF, a factor crucial to effective analyte resolution, is greatly diminished. In seeking to overcome this limitation we have explored the potential of incorporating a range of metal cations into a phospholipid bilayer capillary coating. It has previously been established that the inclusion of calcium into the separation buffer with a phospholipid coating will reverse the EOF in the capillary. Here, we present our investigation of a broader range of metal cations included in the separation buffer (Ca(2+) , Mg(2+) , Co(2+) , Ni(2+) , Sr(2+) , Ba(2+) , and Ce(3+) ) revealing that the choice of metal cation can drastically influence the EOF, with observed values between -3.80 × 10(-4) and -5.74 × 10(-5) cm(2) /V·s.

Towards the identification of autologous blood transfusions through capillary electrophoresis.

The use of autologous blood transfusions by endurance athletes has remained one of the most difficult doping practices to detect. The implementation of the Athlete's Biological Passport by some sporting bodies has proved to be effective; however, the analysis relies on the long-term monitoring of numerous biological markers, looking for abnormal variations in a number of biological markers to indicate doping. This work introduces an approach to identify autologous blood transfusions by examining the red blood cells (RBCs) directly. By using high-speed capillary electrophoretic separations, the relative distribution of the sizes of the RBCs in a sample can be established in under 3 min, following the preparation of the cells. As RBCs that have been stored for transfusions undergo vesiculation, the relative size of the transfused cells differs from the native cells. The capillary electrophoretic separation allows for a rapid examination of this distribution and the changes that are seen when transfused RBCs are mixed with native cells. In this work, the effectiveness of this approach is demonstrated in the identification of simulated (in vitro) autologous blood transfusions performed with blood samples from three highly trained cyclists; it was possible to rapidly identify when as little as 5 % of the RBCs in the sample were from a simulated autologous transfusion.

Role of capillary electrophoresis in the fight against doping in sports.

At present the role of capillary electrophoresis in the detection of doping agents in athletes is, for the most part, nonexistent. More traditional techniques, namely gas and liquid chromatography with mass spectrometric detection, remain the gold standard of antidoping tests. This Feature will investigate the in-roads that capillary electrophoresis has made, the limitations that the technique suffers from, and where the technique may grow into being a key tool for antidoping analysis.

Adsorption of buffer ion pairs can alter long-term electroosmotic flow stability.

Dynamic capillary coatings have become widespread due to their efficacy in modifying the EOF in capillary electrophoretic separations and ability to limit unwanted analyte-surface interactions. However, our understanding of exactly what types of interactions are taking place at the surface of a capillary when these dynamic additives are present is limited. In this work, we have chosen a simple, small molecule additive, tetramethylammonium to examine its influence on the EOF under typical separation conditions. What we have revealed is that this simple compound does not interact with the capillary surface in a very simple manner. Our initial hypothesis of a direct ionic interaction with the silanol surface has evolved with evidence of complex ion pairing between the silanols, the tetramethylammonium, and the buffer ions. This ion pairing can result in drastic changes in the EOF over time, and that the EOF can only be restored to initial levels with harsh rinses containing sodium hydroxide.

Capillary electrophoretic analysis of whole blood samples for hemoglobin-based oxygen carriers without the use of immunoprecipitation.

Hemoglobin-based oxygen carriers (HBOCs) are blood substitutes, synthesized by polymerizing hemoglobin, which are being developed and investigated as alternatives to blood for medical purposes. However, due to their ability to increase the oxygen carrying capacity when taken by healthy individuals, HBOCs have been used as a doping agent among endurance athletes and are included in the World Anti-Doping Agency's Prohibited List. To maintain the fairness of competitions and continue the battle against doping, it is essential to be able to detect HBOCs if present in an athlete's blood. To achieve this goal, it is necessary to differentiate HBOCs from the native hemoglobin and to do so in a cost and time effective manner. We have developed a rapid capillary zone electrophoresis (CZE), UV absorbance, method capable of detecting HBOCs, in whole blood samples, at levels below those considered necessary to provide a performance enhancement. Our approach to the analysis for HBOCs utilizes the whole blood sample, not just the plasma, and does not require the use of immunoprecipitants to ensure accurate analysis. By lysing the red blood cells and using centrifugal filtration, followed by our CZE separation, we are able to effectively distinguish between native hemoglobin and HBOCs. Through this method, we have been able to reliably detect concentrations of HBOCs at the equivalent of 5.5 g/L, the equivalent to a 3.5% increase in blood hemoglobin concentration for an athlete.

Sulfonium and phosphonium, new ion-pairing agents with unique selectivity towards polarizable anions.

Ion-pair chromatography (IPC) almost universally relies upon ammonium-based ion-pairing agents (IPAs) for anion separations. This work compares tetrabutylammonium (TBA) with tetrabutylphosphonium (TBP) and tributylsulfonium (TBS). To best understand the retention behavior analytes used for characterization of the IPAs spanned the Hofmeister series; from kosmotropic monoanions (iodate, chloride, nitrite) and intermediate anions (nitrate, bromide) to chaotropic ions (perchlorate, thiocyanate, iodide). The studies demonstrate that tetrabutylphosphonium is the most chaotropic IPA, followed by tetrabutylammonium and finally tributylsulfonium is the least chaotropic. In the case of the chaotropic anions, the retention of perchlorate was least with tributylsulfonium, and greatest for tetrabutylphosphonium, with tetrabutylammonium being intermediate. The multivalent kosmotropic anions (sulfate, chromate, thiosulfate) demonstrated unique selectivity changes depending on the kosmotropic/chaotropic nature of the IPA. Demonstrating increases in retention with increasing IPA concentration only with tributylsulfonium, whereas the more chaotropic IPAs universally decreased the retention of the multivalent anions.

Determination of zwitterionic and cationic surfactants by high-performance liquid chromatography with chemiluminescenscent nitrogen detection.

The use of chemiluminescent nitrogen specific detection (CLND) combined with an HPLC separation allows for the identification and quantification of cationic and zwitterionic surfactants. The CLND provides equimolar responses, based on the amount of nitrogen within any compound. This allows for the detection of any nitrogen containing surfactant. Reversed-phase separation methods using cyano columns are developed for cationic and zwitterionic (sulfobetaine) surfactant mixtures. The limits of detection for these surfactants are in the single micromolar range (1 ng N). A linear response was obtained (R2=0.9981) between 50 microM and 5 mM. The methodology was then applied to the determination of an industrial zwitterionic surfactant, Rewoteric AM CAS U [coco(amidopropyl)hydroxyldimethylsulfobetaine].