Tris(2,2'-bipyridyl)ruthenium can be excited to fluorescence by visible light (lambda abs 454 nm, lambda em 607 nm) when in the M(II) oxidation state, but not in the M(III) state. A novel chromatographic detection method using the non-fluorescent M(III) form of the complex as a postcolumn fluorogenic reagent is demonstrated. The M(III) form is a powerful oxidizing agent (E degree = 1.27 V vs NHE, 1.05 V vs Ag/AgCl). The M(III) reagent is generated on-line from the M(II) form of the complex by a highly efficient porous carbon electrode and then reacted briefly with chromatographic effluent; the M(II) created by electron transfer from oxidation-susceptible analytes is then detected by fluorescence. The fluorescence detector can be calibrated for number of electrons transferred by injection of either M(II) or an oxidative standard such as ferrocyanide. It is hoped that this redox-based detection scheme will provide an alternative to electrochemical detection. Among the advantages are freedom from surface fouling and the potential for extremely low detection limits. The scheme was applied to detection of the peptide dynorphin A and several of its fragments. Dynorphin A contains tyrosine at the N-terminus (position 1) and tryptophan in position 15; these amino acid residues are susceptible to oxidation and peptides containing them can be detected on that basis. Flow injection testing of the model compounds Tyr-Gly-Gly-Phe-Leu and Gly-Gly-Trp-Gly indicated that tyrosine transferred approximately 1 electron to the M(III) reagent and that tryptophan transferred approximately 4 electrons. Similar results were obtained from the chromatographic runs. Dynorphin A and six dynorphin A fragments containing the N-terminal tyrosine were detected easily at 100 nM concentration (14 pmol) using laser-induced fluorescence. As expected, one fragment that did not contain tryptophan or tyrosine was not detected. A mass detection limit of 80 fmol was estimated for the tyrosine-containing fragments.

A new technique for fabrication of channel structures with diameters down to 13 μm in fluorinated ethylene propylene (also known as poly(tetrafluoroethylene-co-hexafluoropropylene), FEP) is described. The technique is based on the unique property of a dual-layer fluoropolymer tubing consisting of an outer layer of poly(tetrafluoroethylene) (PTFE) and an inner layer of FEP. When heated (>350 °C), the outer PTFE layer shrinks while the inner FEP layer melts, resulting in filling of all empty space inside the tubing with FEP. The channel structures are formed using tungsten wires as templates that are pulled out after completion of the shrinking and melting process. While several analytical devices have been reproducibly prepared and shown to function, this report describes a single example. A microreactor coupled to an electrochemical flow cell detects the biuret complex of the natively electroinactive peptide des-Tyr-Leu-enkephalin.Microfluidic systems with channel dimensions below 100 μm have received enormous attention in the last twenty years. Different types of separations can be performed in these channels including capillary liquid chromatography, 1 capillary electrophoresis, 2 and capillary electrochromatography. 3 Several types of tubing with minimum diameters between 5 and 100 μm are commercially available in many materials. To combine operations (including, for example, injection, separation, detection, dilution, chemical reactions, cell lysis, and PCR amplification), techniques for fabrication of more complex channel structures on so-called chips have been developed. Initial applications were simple one-dimensional separations with optical detection in glass. More recently, one-dimensional separations have been carried out on polymeric chips 4,5 and glass chips with a polymeric separation medium, 3 multiple separation channels with mass spectrometric detection, 6 two-dimensional separations, 7,8 mixing, 9 adsorption, 10 and electrochemical detection 11,12 have also been carried out on chips. Fabrication techniques for channels with dimensions in the 10-100-μm range include photolithography and wet etching, radiation-induced etching, molding, imprinting, and casting and are applied to materials such as glass, plastics, and polymeric materials. [13][14][15][16][17][18] Decreasing the dimensions of channels and reservoirs results in an increase in the wall areato-solution volume ratio. Hence, interaction between solvents and solutes with the wall surface can be a major problem in miniaturized flow systems. Several types of interactions are possible including, for example, adsorption, absorption, and leakage of compounds from the wall material. In addition, several typical materials used in microfluidic devices are only resistant to a limited number of chemicals.Fluoropolymers have some unique properties originating from the C-F bond that make them particularly suitable as material for chemical containers. 19 These materials are thermally stable and chemically inert to acids, bases, oxidizing and r...

The reversible electrochemistry of the Cu(II)/Cu(III) couple was investigated for the copper(II) complexes of triglycine (G3), tetraglycine (G4), and pentaglycine (G5) in alkaline solution using a rotating ring-disk electrode (RRDE). The study was motivated by the need to elucidate electrochemical processes occurring in dual electrode postcolumn detection of peptides. The disk electrode served as the anode and the ring electrode as the cathode. The electrode was used in both linear sweep voltammetry and constant potential, varied rotation speed modes. Redox waves for two generic forms of the complexes, Cu(II)-NNNN and Cu(II)-NNNO, were identified, with respective E1/2 values of approximately 0.45 and 0.7 V. It was found that the G3 complex underwent an ECE-like process at the anode that magnified the anodic signal and suppressed the cathodic signal. The G4 and G5 complexes were subject to two CE processes, the C reactions being (i) deprotonation of Cu(II)-NNNO to Cu(II)-NNNN and (ii) loss of hydroxide ion by Cu(II)-NNN(OH-), a variant of Cu(II)-NNNO. An additional C reaction, dissociation of a carbonate variant of Cu(II)-NNNO, occurred in 0.2 M carbonate buffer. Visible absorbance measurements assisted in assignment of these forms. Measurements of diffusion coefficients of the complexes were performed by Taylor-Aris laminar flow axial dispersion measurements. The analytical implications for these findings are discussed.