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A novel method for applying cavity ring-down spectroscopy in the liquid phase, compatible with LC analyses, is presented. The core of the setup is a home-built cavity ring-down flow cell (cell volume 12 [micro]L) that is constructed using a silicon rubber spacer, which is clamped leak-tight between two high-reflectivity mirrors. The mirrors are in direct contact with the liquid flow, which provides for a small path length and short ring-down times. Inside the cavity there are no windows, reflection losses, or Brewster angles to be considered. Due to the small size of the presented cavity geometry, the setup can be implemented in conventional-size LC apparatuses. With a flow injection setup, a detection limit of 2.5 nM was obtained for Crystal Violet in ethanol, and the linear dynamic range of the system is at least 2 orders of magnitude. The method has the potential to become a powerful alternative for commercial LC UV/visible absorbance detectors.

University researchers investigated the transformation of pyrene fluorescence spectra in the presence of humic acid at low temperatures using groundwater samples collected from Germany.

Binding of the antihistamine drug brompheniramine (BPA) to human serum albumin (HSA) is studied by measuring quenching of the fluorescence and room temperature phosphorescence (RTP) of tryptophan. The modified Stern-Volmer equation was used to derive association constants and accessible fractions from the steady-state fluorescence data. Decay associated spectra (DAS) revealed three tryptophan fluorescence lifetimes, indicating the presence of three HSA conformations. BPA causes mainly static quenching of the long-living, solvent-exposed conformer. RTP spectra and lifetimes, recorded under deoxygenated conditions in the presence of 0.2 M KI, provided additional kinetic information about the HSA-BPA interactions. Fluorescence DAS that were also recorded in the presence of 0.2 M KI revealed that the solvent-exposed conformer is the major contributor to the RTP signal. The phosphorescence quenching is mostly dynamic at pH 7 and mostly static at pH 9, presumably related to the protonation state of the alkylamino chain of BPA. This provides direct insight into the binding mode of the antihistamine drug, as well as kinetic information at both the nanosecond and the millisecond time scales.

UV resonance Raman spectroscopy was used to characterize the binding of three first-generation histamine H(1) receptor antagonists-tripelennamine (TRP), mepyramine (MEP), and brompheniramine (BPA)-to human serum albumin (HSA) at pH 7.2 and pH 9.0. Binding constants differ at these pH values, which can be ascribed to the different extent of protonation of the ethylamino side chain of the ligands. We have recently shown [Tardioli et al. J. Raman Spectrosc. 2011, 42, 1016-1024] that for the solution conformation of TRP and MEP the side chain plays an important role by allowing an internal hydrogen bond with the aminopyridine nitrogen in TRP and MEP. Results presented in this paper suggest that the existence of such molecular structures has serious biological significance on the binding affinity of those ligands to HSA. At pH 7.2, only the stretched conformers of protonated TRP and MEP bind in HSA binding site I. Using UV absorption data, we derived binding constants for the neutral and protonated forms of TRP to HSA. The neutral species seems to be conjugated to a positive group of the protein, affecting both the tryptophan W214 and some of the tyrosine (Y) vibrations. BPA, for which the structure with an intramolecular hydrogen bonded side chain is not possible, is H bound to the indole ring nitrogen of W214, of which the side chain rotates over a certain angle to accommodate the drug in site I. We propose that the protonated BPA is also bound in site I, where the Y150 residue stabilizes the presence of this compound in the binding pocket. No spectroscopic evidence was found for conformational changes of the protein affecting the spectroscopic properties of W and Y in this pH range.

The detection of explosives concealed behind opaque, diffusely scattering materials is a challenge that requires noninvasive analytical techniques for identification without having to manipulate the package. In this context, this study focuses on the application of time-resolved Raman spectroscopy (TRRS) with a picosecond pulsed laser and an intensified charge-coupled device (ICCD) detector for the noninvasive identification of explosive materials through several millimeters of opaque polymers or plastic packaging materials. By means of a short (250 ps) gate which can be delayed several hundred picoseconds after the laser pulse, the ICCD detector allows for the temporal discrimination between photons from the surface of a sample and those from deeper layers. TRRS was applied for the detection of the two main isomers of dinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene as well as for various other components of explosive mixtures, including akardite II, diphenylamine, and ethyl centralite. Spectra were obtained through different diffuse scattering white polymer materials: polytetrafluoroethylene (PTFE), polyoxymethylene (POM), and polyethylene (PE). Common packaging materials of various thicknesses were also selected, including polystyrene (PS) and polyvinyl chloride (PVC). With the demonstration of the ability to detect concealed, explosives-related compounds through an opaque first layer, this study may have important applications in the security and forensic fields.

Isoflavones are known to show fluorescence with intensities that depend strongly on the solvent properties and exhibit Stokes' shifts as large as 1.4 eV. While some of this behavior can be explained by (excited state) deprotonation, this mechanism does not apply for all isoflavones. The aim of this study is to computationally and experimentally investigate the reasons for this anomalous behavior of neutral isoflavones, taking the daidzein molecule as a model compound. We find that the absence in fluorescence in aprotic solvents and the weak fluorescence in protic solvents can be explained by a change of order of the lowest singlet states in which a fluorescent charge-transfer state lies below the nonfluorescent locally excited state in water but not in acetonitrile. The large Stokes' shift is partly due to a significant rotation among the chromone-phenyl bond in the excited state.

In the present paper a detailed analysis of high-resolution luminescence spectra of Eu(III)-H(2)O species in frozen aqueous solution (T = 5 K) is presented. From the total luminescence spectra (TLS, excitation vs emission) and the luminescence decay matrixes (time vs emission), fundamental species-selective spectroscopic parameters are determined: excitation wavelength λ(exc), decay time τ, crystal field energy splitting ΔE (crystal field strength parameter N(ν)(B(2q))), crystal field parameters B(20) and B(22), asymmetry ratio r, and point symmetry group. The spectroscopic findings clearly show the presence of two distinct Eu(III) aquo species. Samples prepared with different counterions (Cl(-), ClO(4)(-)) and at different pH values (2 and 5) yielded comparable results. Furthermore, in D(2)O solutions the same two species were found, with similar spectral properties but much longer decay times. On the basis of the spectroscopic analysis, the two species were attributed to 8- and 9-fold coordinated Eu(III) aquo ions.

The sensitivity of enantioselective cyclodextrin-induced room-temperature phosphorescence detection of camphorquinone (CQ) is enhanced using sensitization via a donor with a high extinction coefficient. The enantiomeric distinction is based on the different phosphorescence lifetimes of (+)-CQ and (-)-CQ after their complexation with α-cyclodextrin (α-CD). The collisional Dexter energy transfer from the selected donor 2,6-naphthalenedisulfonic acid (2,6-NS) to the acceptor CQ is still very efficient despite the inclusion of the acceptor into CD. For coupling to the chiral separation of (±)-CQ in cyclodextrin-based electrokinetic chromatography, the donor was added to the deoxygenated background electrolyte that consisted of 20 mM α-CD, 10 mM carboxymethyl-β-CD, and 25 mM borate buffer at pH 9.0. Time-resolved batch studies on sensitized phosphorescence show a significant enantioselectivity for (+)- and (-)-CQ in the presence of both α-CD and CM-β-CD although the lifetime difference is somewhat reduced with respect to direct excitation. The enantiomers were distinguished after their separation using an online time-resolved detection system. Excitation was performed at 266 nm with a pulsed, small-sized, quadrupled Nd:YAG laser. With 1 × 10(-5) M 2,6-NS, limits of detection of 4.1 × 10(-8) M and 5.2 × 10(-8) M were found for (+)-CQ and (-)-CQ, respectively. The online measured lifetimes were 238 ± 8 μs for (+)-CQ and 126 ± 10 μs for (-)-CQ. The method was used to determine the concentration of (±)-CQ leaching from a cured dental resin into water. The extracts contained 4.7 ± 0.1 × 10(-7) M of both (+)-CQ and (-)-CQ.

Earlier steady-state fluorescence studies showed that 2-butylamino-6-methyl-4-nitropyridine N-oxide (2B6M) can undergo fast excited-state intramolecular proton transfer (ESIPT). In a nonpolar solvent such as n-octane, both normal and tautomeric fluorescence was observed. Strikingly, the relative ratio of those two emission bands and the fluorescence quantum yield of the normal emission were found to depend on the excitation wavelength in violation of the Kasha-Vavilov rule. In this work, the system was investigated further by means of transient absorption spectroscopy, followed by global and target analysis. Upon excitation at 420 nm, a normal excited singlet state S(1)(N) is reached, which decays in about 12 ps via fluorescence and ESIPT (minor pathways) and to a long-lived "dark" state (major pathway) that is most probably the triplet T(1)(N). Upon 330 nm excitation, however, a more complex pattern emerges and additional decay channels are opened. A set of four excited-state species is required to model the data, including a hot state S(1)(N)* that decays in about 3 ps to the tautomer, to the long-lived "dark" state and to the relaxed S(1)(N) state. A kinetic scheme is presented that can explain the observed transient absorption results as well as the earlier fluorescence data.

Protein adsorption to silica surfaces is a notorious problem in analytical separations. Evanescent-wave cavity ring-down spectroscopy (EW-CRDS) and capillary electrophoresis (CE) were employed to investigate the capability of positively charged polymer coatings to minimize the adsorption of basic proteins. Adsorption of cytochrome c (cyt c) to silica coated with a single layer of polybrene (PB), or a triple layer of PB, dextran sulfate (DS), and PB, was studied and compared to bare silica. Direct analysis of silica surfaces by EW-CRDS revealed that both coatings effectively reduce irreversible protein adsorption. Significant adsorption was observed only for protein concentrations above 400 microM, whereas the PB-DS-PB coating was shown to be most effective and stable. CE analyses of cyt c were performed with and without the respective coatings applied to the fused-silica capillary wall. Monitoring of the electroosmotic flow and protein peak areas indicated a strong reduction of irreversible protein adsorption by the positively charged coatings. Determination of the electrophoretic mobility and peak width of cyt c revealed reversible protein adsorption to the PB coating. It is concluded that the combination of results from EW-CRDS and CE provides highly useful information on the adsorptive characteristics of bare and coated silica surfaces toward basic proteins.

Enantioselective phosphorescence lifetime detection was combined with chiral cyclodextrin-based electrokinetic chromatography for the analysis of camphorquinone (CQ). A time-gated detection system based on a pulsed light-emitting diode for excitation at 465 nm was developed for the online lifetime determination. The background electrolyte for the chiral separation consisted of 20 mM alpha-cyclodextrin (alpha-CD), 10 mM carboxymethyl-beta-CD, and 25 mM borate buffer at pH 9.0. The separation of (+)-CQ and (-)-CQ is caused by a difference in association constants of these enantiomers with alpha-CD. Under the separation conditions, different phosphorescence lifetimes were obtained for (+)-CQ and (-)-CQ (tau = 384 +/- 8 and 143 +/- 5 micros, respectively), which could be used to distinguish the enantiomers. This selectivity in detection is based on a difference in protection of the enantiomers against phosphorescence quenching after their complexation with alpha-CD. Concentration detection limits were 2 x 10(-7) and 1 x 10(-6) M for (+)-CQ and (-)-CQ, respectively. After correction for the lifetime shortening by triplet-triplet annihilation at higher CQ concentrations, a linear dynamic range was obtained from the detection limit up to 2 mM. The system was used to determine the enantiomeric impurity levels of commercial samples of (+)-CQ and (-)-CQ; 0.2% and 0.1%, respectively.

Electronic absorption, emission, and excitation spectra, and fluorescence lifetimes of two H1 antihistamines--tripelennamine and mepyramine--are investigated in detail to ascertain their usefulness as fluorescent probes for ligand binding to G-protein coupled receptors. The photophysical behavior of these compounds in aqueous solution is complex due to the presence of three protonable nitrogens, intramolecular hydrogen bonding, quenching due to the formation of a charge transfer state, and intramolecular fluorescence resonance energy transfer. At physiological pH values, anomalous photophysical behavior is observed: the compounds are found to be in a ground-state equilibrium mixture of two species, one with the alkylamine tail involved in an intramolecular hydrogen bond and a second without such a bond. This internal hydrogen-bonded tail has a profound effect on the ground and excited-state properties of both tripelennamine and mepyramine, which is further elucidated by comparing them to the reference compounds 2-aminopyridine and 2-(N,N-dimethylamino)pyridine.

Pyrazoloquinolines are highly fluorescent, both in liquid solutions and in the solid state, which makes them good candidates for various optical devices. The aim of the current work is to understand the photochemical behavior of pyrazolo[3,4-b]quinoline (PQ), which is quite complicated since in n-alkane solvents PQ tends to form strong complexes with protic solvent constituents (often present as minor impurities), as well as dimers. Both types of H-bond complexes were studied systematically by temperature-dependent conventional absorption and fluorescence spectroscopy; the effect of protic solvent constituents was mimicked by varying the ethanol concentration in n-octane in the range from 0.0 to 0.8%. At room temperature the PQ:ethanol association constant was estimated at 80 M(-1) and the dimerization constant at 2 x 10(3) M(-1). Dimer formation is enhanced upon lowering the temperature in pure n-alkane down to 220 K, and the fluorescence is strongly reduced since the dimer is nonfluorescent. Surprisingly, when irradiating a frozen sample for several minutes at very low temperatures (<40 K), a narrow-banded Shpol'skii-type fluorescence spectrum gradually appears. To explain this unusual photochemical behavior, PQ and its deuterated analogue were studied using low-temperature absorption and fluorescence spectroscopy over the 300-5 K temperature range. In the case of normal (protonated) PQ, very fast excited-state intermolecular double proton transfer is responsible for the efficient quenching of PQ dimer fluorescence. Deuteration significantly slows down this proton transfer process, and in that case under cryogenic conditions a fluorescent dimer is observed. Photoirradiation under cryogenic conditions leads to molecular rearrangement of the dimers and the appearance of monomer spectra. For both H-PQ and D-PQ, these processes were found to be reversible. A simplified reaction scheme, in which the excited tautomeric dimer plays a crucial role, is presented to explain the observations.

The influence of pH on the redox properties of cytochrome c (cyt c) adsorbed on roughened silver electrodes chemically modified with a self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (MUA) was studied with voltammetric techniques in combination with surface-enhanced resonance Raman scattering (SERRS). The experiments were performed simultaneously on the same electrode sample in a homemade spectroelectrochemical cell suitable for such applications. At pH 7.0 cyt c was found in its native state; at higher pH values (ranging from 8.0 to 9.0) the redox properties of the adsorbed protein varied considerably, featuring a redox behavior which does not resemble the one reported for the alkaline transition. Our results instead indicate the presence of an electrochemically inactive 6cLS species immobilized on MUA at pH 9.0. The pH-induced conformational changes observed for cyt c immobilized on the SAM of MUA were found to be repeatable and chemically reversible, meaning that the recovery of the electrochemical signal due to the native protein occurred instantaneously (on the second time scale) when the electrode was switched back to pH 7.0. The pH-induced changes observed were attributed to a conformational change involving a heme reorientation with respect to the electrode surface.

This article describes the exceptional photophysics of 2-butylamino-6-methyl-4-nitropyridine N-oxide (2B6M). It is known from the literature that this compound may undergo excited-state intra- or intermolecular proton-transfer reactions. In nonpolar solvents, 2B6M exhibits an unusual fluorescence behavior: there is a substantial difference between the relative band intensities of the excitation and absorption spectra. Furthermore, in emission two bands are observed, and their relative intensities depend on the excitation wavelength, thus violating the Kasha-Vavilov rule. It is the objective of this research to interpret these results. For this purpose, steady-state fluorescence excitation and emission spectra in the liquid state were recorded and quantum yields were determined for the two types of emission. In addition, absorption spectra were measured at room temperature and under low-temperature conditions. Finally, fluorescence lifetimes of the emitting species were determined using the time-correlated single photon counting technique. The results suggest that in the liquid state only one (monomeric) ground state species dominates, which can emit via two different pathways (from the normal and the tautomeric excited state). The excitation spectra point at two different internal proton-transfer processes, one starting at the S1 state and one starting at the S2 state. On the basis of the measured lifetimes and fluorescence quantum yields, a kinetic scheme was completed that can quantitatively explain the observations.

To combine voltammetric techniques with surface-enhanced resonance Raman scattering (SERRS), cytochrome c (cyt c) was immobilized on a roughened silver electrode chemically modified with a self-assembled monolayer (SAM) of 4-mercaptopyridine (PySH). All measurements were performed on the same electrode in a homemade spectroelectrochemical cell suitable for such applications. Cyt c on a PySH-SAM shows a quasi-reversible, monoelectronic, adsorption-controlled CV response with a formal reduction potential of -0.061 V (vs SCE), which is comparable to the values found for native cyt c adsorbed on different SAMs. SERRS spectra proved that cyt c adsorbed on a PySH monolayer is present in the native conformer (the B1 state). Voltammetric and SERRS experiments at high ionic strength revealed that the interaction between the SAM and the protein is electrostatic in nature. In conclusion, PySH was found to be suitable for adsorption of cyt c at SERRS-active silver surfaces. In comparison with other SAMs, PySH requires less time (10 min vs 12-18 h) to form a long-time durable and reproducible coating on the roughened electrode surface.

Surface-enhanced resonance Raman scattering (SERRS) from dilute solutions (down to nanomolar concentrations) of human mono-oxygenase CYP2D6 is observed using aqueous dispersions of Ag nanoparticles (hydrosol) coated with self-assembled monolayers (SAMs) of mercaptoalkanoic acids of two different lengths. From a direct comparison with its resonance Raman spectrum in solution, CYP2D6 appears to fully retain its native structure upon adsorption on coated hydrosol through electrostatic interaction, while a structural change in the active site is observed when uncoated citrate-reduced hydrosol is used. Using SERRS on these biocompatible coated hydrosols, the effects of dextromethorphan on the enzyme's active site can be observed, demonstrating that CYP2D6 ability of binding substrates is preserved. Moreover, by tuning the wavelength of the exciting laser away from the main absorption band of the heme, the vibrational bands of the SAM coating are observed and analyzed to see how the presence of the protein affects the SAM structure.

The bacteriophage T4 capsid protein gp23 was studied using time-resolved and steady-state fluorescence of the intrinsic protein fluorophore tryptophan. In-vitro gp23 consists mostly of monomers at low temperature but forms hexamers at room temperature. To extend our knowledge of the structure and hexamerization characteristics of gp23, the temperature-dependent fluorescence properties of a tryptophan mutant (W13V) were compared to those of wild-type gp23. The W13V mutation is located in the N-terminal part of the protein, which is cleaved off after prohead formation in the live bacteriophage. Results show that W13 plays a role in the hexamerization process but is not needed to stabilize the hexamer once it is formed. Furthermore, besides the monomer-to-hexamer temperature transition (15-23 degrees C and 12-43 degrees C for wild-type and W13V gp23, respectively), we were able to observe denaturation of the N-terminus in hexameric wild-type gp23 around 40 degrees C. In addition, with the aid of a recently published homology model of gp23, the lifetimes obtained from time-resolved fluorescence measurements could tentatively be assigned to specific tryptophan residues.

To date, a successful combination of surface-enhanced resonance Raman spectroscopy (SERRS) and electrochemistry to study heme proteins is inhibited by the problems raised by the prerequisite to use silver as electrode metal. This paper indicates an approach to overcome these problems. It describes a quick and reproducible procedure to prepare silver electrodes chemically modified with 4-mercaptopyridine suitable to perform diffusion electrochemistry of cytochrome c (cyt c). The method involves the employment of a mechanical and a chemical treatment and avoids the use of alumina slurries and any electrochemical pretreatment. Cyclic voltammetry (CV) was used to test the electrochemical response of cyt c, and the CV signals were found identical with those obtained on gold electrodes under the same experimental conditions. Compared to previous literature, a significant improvement of the CV signal of cyt c at silver electrodes was achieved. Preliminary results show that this treatment can be also successfully employed for the preparation of SERRS-active electrodes.

Ultraviolet resonance Raman spectroscopy is carried out using a continuous wave frequency-doubled argon ion laser operated at 229, 244, and 257 nm in order to characterize the overtones and combination bands for several classes of organic compounds in liquid solutions. Contrary to what is generally anticipated, for molecules such as pyrene and anthracene, strong overtones and combination bands can show up; it is demonstrated that their intensity depends critically on the applied laser wavelength. If the excitation wavelength corresponds with a purely electronic transition--this applies to a good approximation for 244-nm excitation in the case of pyrene and for 257-nm excitation in the case of anthracene--mostly fundamental vibrations (up to 1700 cm(-1)) are observed. Overtones and combination bands are detected but are rather weak. However, if the laser overlaps with the vibronic region--as holds for 229- and 257-nm excitation for pyrene and 244-nm excitation for anthracene--very strong bands are found in the region 1700-3400 cm(-1). As illustrated for pyrene at 257 nm, all these bands can be assigned to first overtones or binary combinations of fundamental vibrations. Their intensity distribution can roughly be simulated by multiplying the relative intensities of the fundamental bands. Significant bands can also be found in the region 3400-5000 cm(-1), corresponding with second overtones and ternary combinations. It is shown that these findings are not restricted to planar and rigid molecules with high symmetry. Substituted pyrenes exhibit similar effects, and relatively strong overtones are also observed for adenosine monophosphate and for abietic acid. The reasons for these observations are discussed, as well as the potential applicability for analytical purposes.

A novel approach is presented for studying the chemical interaction between receptor binding sites and ligands. Monohydroxylated polyaromatic compounds were found to be environmentally sensitive ligands when applying a special mode of fluorescence: fluorescence line-narrowing spectroscopy (FLNS). With this technique, solvent dependencies and ligand-receptor interactions can be studied in great detail, due to the high spectral resolution and the fact that at cryogenic temperatures (4 K), no solvent reorientation effects complicate the interpretation. The FLN spectrum of a ligand bound to the receptor is compared to the spectra of the free ligand in solvent mixtures that mimic the functionalities present within the receptor's binding site. It is shown that for the well-known estrogen receptor (ER), the orientations of two xenoestrogenic ligands 3- and 9-hydroxybenzo[a]pyrene (3- and 9-OH-BaP) can be determined. The FLN results clearly indicate that an H-bond accepted by HIS524 plays a major role in the binding of these ligands to the ER. Furthermore, the spectra indicated a pi-pi stacking aromatic interaction for 9-OH-BaP with PHE404. These results are in line with molecular modeling studies published earlier.

The results of time-dependent density functional theory (TDDFT) calculations of resonance Raman intensities are combined with experimental deep-ultraviolet resonance Raman measurements at a single wavelength, i.e., 244 nm, in order to test the possibility to distinguish several very similar compounds. Pyrene and three of its substituted derivatives, in which a single hydrogen atom has been replaced by a halogen atom, are compared. The fixed 244 nm excitation wavelength overlapped with the same electronic transition of the four pyrenes. Ground-state calculations using the BP86 exchange-correlation functional were used to predict the Raman frequencies, whereas excited-state calculations have been carried out employing the "statistical averaging of (model) orbital potentials" (SAOP) potential within a linear-response TDDFT framework in combination with the short-time approximation of resonance Raman intensities. In view of the simplistic theoretical approach, we find a surprisingly good agreement between the simulated and measured resonance Raman spectra of pyrene and its substituted analogues in terms of frequencies and intensities, which shows that the calculations can be used reliably to interpret the experimental spectra. With this combined information, it is possible to find criteria to distinguish the compounds under investigation, although many features of their vibrational spectra are similar.

Excited-state intramolecular proton transfer (ESIPT) occurring in the salicylic acid (SA) derivative 5-methoxysalicylic acid (5-MeOSA) in an apolar solvent (cyclohexane) and in the presence of the hydrogen bond accepting agent diethyl ether (DEE) is investigated. Analysis of the directly measured subnanosecond time-resolved emission spectra (TRES) together with conventional steady-state fluorescence and time-correlated single-photon-counting (TCSPC) decays indicates that ESIPT in this system occurs much faster than fluorescence, and that the equilibrium between normal and tautomeric excited states is established before the emission from both states takes place. However, changes in time- and frequency-resolved fluorescence of the 5-MeOSA/DEE complex are observed due to structural relaxation within the complex, which is reflected in the dynamic Stokes shift of the tautomeric fluorescence band. The normal fluorescence band of 5-MeOSA/DEE does not exhibit marked changes within the investigated time range. A single-exponential relaxation time of 460 ps was determined for the dynamic Stokes shift of the tautomeric band, and it is attributed to a geometric change within the 5-MeOSA/DEE complex upon excitation. Since both tautomeric and normal emission bands are well resolved and exhibit different time-dependent behaviors, a double-well potential appears to be adequate to describe the excited state of the system studied.

A flow injection analysis (FIA) system for biochemical assays using time-resolved fluorescence resonance energy transfer (TR-FRET) in the millisecond time scale was developed. As a model system, we studied a kinase assay, measuring the phosphorylation of poly(GT)-biotin (substrate) by a receptor tyrosine kinase (epidermal growth factor receptor). A streptavidin labeled with XL665 (SA-XL665)-the acceptor-was coupled to the biotin moiety, and an antiphosphotyrosine antibody labeled with europium cryptate (Ab-EuK)-the donor-was coupled to the phosphorylated tyrosine group(s). Long-lived FRET can only occur if the substrate is successfully phosphorylated. For the time-resolved detection of such long-lived luminescence phenomena in a flow system, the repetition rate of the excitation source plays a crucial role. Good results were obtained for a small-sized commercially available quadrupled Nd:YAG laser emitting at 266 nm with a repetition rate of 7.8 kHz and a pulse width of 0.3 ns. The long-lived emissions of the donor at 625 nm and that of the acceptor at 665 nm were monitored simultaneously with two photomultipliers, using a delay time of 50 micros and a gate time of 75 micros to exclude background fluorescence interferences. In the FIA experiments, the Ab-EuK concentration was 6 nM and the substrate concentration and SA-XL665 concentrations were 7 nM. By monitoring the intensity changes at 625 and 665 nm, the inhibition of tyrosine kinase by tyrphostin AG1478 was studied and an IC(50) value of 5.1 +/- 0.4 nM obtained.

A new citrate-reduced silver hydrosol coated with omega-mercaptoalkanoic acids (mercaptopropionic and mercaptoundecanoic acids) self-assembled monolayers was prepared and characterized with surface-enhanced Raman spectroscopy. The structure and the quality of the coating monolayers are discussed and compared to similar coated and uncoated silver hydrosols previously developed. As an application, the new hydrosol was used as a biocompatible and efficient metal substrate for a surface-enhanced resonance Raman scattering (SERRS) study of cytochrome c. The high-quality SERRS spectra reported of cytochrome c (obtained using only 1 microL of a micromolar cytochrome solution) are discussed and compared with data available from literature studies.

Surface-enhanced resonance Raman spectra of cytochrome c on silver electrodes coated with self-assembled monolayers of mercaptopropionic acid were recorded at different potentials using 50 microL of a micromolar solution. For this purpose, a linearly moving, low-volume, small spectroelectrochemical cell was designed and used together with a Raman microprobe. The quality of the spectra obtained is good, and the spectra show essentially the same features reported by other authors using much larger volumes. The cell described in this paper is shown to be useful for studying the spectroelectrochemistry of photosensitive compounds such as heme proteins, which are available only in very small amounts (nanomoles to picomoles).

A new approach for studying antibody-antigen interactions of DNA adducts and metabolites of polycyclic aromatic hydrocarbons (PAHs) is demonstrated in which fluorescence line-narrowing spectroscopy (FLNS) is used. It is based on the fact that in an FLN spectrum the relative intensities of the line-narrowed bands (that correspond to the excited-state vibrations) are, in general, strongly dependent on the local environment of the fluorophore. Information on the nature of the interactions can be obtained by comparing the FLN spectra of the antigen-antibody complexes to the spectra of the antigen in different types of solvents (H-bonding, aprotic, and pi-electron-containing solvent molecules) recorded under the same conditions. The antigens used were the DNA adduct 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) and the metabolite (+)-trans-anti-7,8,9,10-benzo[a]pyrenetetrol (BP-tetrol) of benzo[a]pyrene; two monoclonal antibodies (MAbs) have been developed to selectively bind these compounds. It is shown that, for BP-tetrol, H-bonding solvents have a pronounced effect on the FLN spectra. The presence of pi electrons in the solvent molecules results in relatively small but still significant changes in the spectra. When BP-tetrol is bound to its MAb, however, neither of these effects is observed; its spectrum is very similar to the one obtained with an aprotic solvent, methylcyclohexane. Therefore, we can conclude that this MAb has an internal binding site in which the interaction with BP-tetrol is of a hydrophobic character. For BP-6-N7Gua, however, there is a strong effect of the presence of pi electrons in the solvent molecules. The FLN spectrum of this antigen bound to its MAb is very similar to its spectrum in acetone, indicating that pi-pi interactions play an important role in the binding.

Quenched phosphorescence detection of camphorquinone in cyclodextrin-based electrokinetic chromatography provides very favorable detection limits, i.e., 7 x 10(-)(7) M, 3 orders of magnitude lower than conventional UV absorption detection at 200 nm. The detection is based on the dynamic quenching by the analyte of the strong phosphorescence emission of brominated naphthalenesulfonate, under deoxygenated buffer solution conditions. This approach has been used to detect (1S)-(+)- and (1R)-(-)-camphorquinone after enantiomeric separation by CE. Although the use of the negatively charged carboxymethyl beta-cyclodextrin (CM-beta-CD) alone was not successful, the addition of a second, neutral cyclodextrin, alpha-CD, provided an adequate enantiomeric separation of camphorquinone. Using 25 mM borate buffer (pH 8.5) with 10 mM CM-beta-CD and 20 mM alpha-CD (applied voltage 20 kV, ambient temperature), the enantiomeric separation was performed in approximately 14 min. The chiral method was applied to monitor the stereoselectivity of the biotransformation of a racemic mixture of camphorquinone by yeast. It was found that the enantiomeric ratio calculated from the peak areas in the electropherogram (RSD = 5%) after 24 h of incubation decreased from 0.92 for the control solution (culture medium without yeast) to 0.24 for the culture medium; a similar ratio of 0.25 was observed for cell extract solutions. Therefore, racemic camphorquinone is enantioselectively degraded by yeast, the biodegradation of (1S)-(+)-camphorquinone being faster than that of the (1R)-(-)-enantiomer.

Capillary electrophoresis (CE) and resonance Raman spectroscopy (RRS) with excitation in the deep ultraviolet (UV) region (lambda(ex): 244 or 257 nm) were coupled on-line. The potential of this hyphenated technique, denoted as CE-UV-RRS, for analyte confirmation/identification purposes was explored with aromatic sulfonic acids and nucleotides as test compounds. Good-quality UV-RRS spectra could be recorded on-the-fly. Identification limits for the nucleotides were in the 10-125 microg/mL range. The RRS spectra showed sufficient characteristic features to enable analyte confirmation. In addition, the identification power of UV-RRS was studied with substituted pyrenes as model compounds. The compounds were distinguishable on the basis of their RRS spectra at 244 nm.

Quenched phosphorescence detection is a sensitive detection method recently introduced in capillary zone electrophoresis. It is based on the dynamic quenching interaction of the analytes (quenchers) with a phosphorophore, 1-bromo-4-naphthalenesulfonate (BrNS), present in the separation buffer. In this study, it is shown that this detection method can also be used in cyclodextrin-based electrokinetic chromatography (CD-EKC) despite the presence in the buffer solution of cyclodextrins, which are known to reduce the luminescence quenching rate constants. Experiments indicate that BrNS mainly resides in the aqueous phase, while the analytes are distributed between both phases. In principle, the observed quenching might arise from the interaction of BrNS with uncomplexed as well as complexed analytes. However, from the dependence of the fractional quenching on the capacity factor (the normalized fractional quenching was found to be equal to the fraction of analyte in the aqueous phase), it was concluded that only aqueous-phase quenching contributes significantly to the observed quenching. Nevertheless, separation and detection can be regarded as fully compatible, because the capacity factors encountered in CD-EKC are generally low (in this study they ranged from about 0.1 to 2.5). Indeed, with nitroaromatic compounds as the target analytes, limits of detection in the 10(-8) M range were achieved.

A capillary electrophoresis detection technique for (small) peptides is presented, i.e. quenched phosphorescence, a method that is generally applicable and does not require chemical derivatization. For this purpose, a novel phosphorophore, 1-bromo-4-naphthalenesulfonic acid (BrNS), was synthesized. BrNS has sufficient water solubility and provides strong phosphorescence at room temperature over a wide pH range. The detection is based on the dynamic quenching of the BrNS phosphorescence background signal by electron transfer from the amino group of the peptides at pH 9.5-10. For the di- and tripeptides Val-Tyr-Val, Val-Gly-Gly, Ala-Ser, Gly-Asn, Gly-Ala, and Gly-Tyr, detection limits in the range of 5-20 microg/L were obtained. The novel technique is even a good alternative for the (limited) group of peptides containing tyrosine and, thus, exhibiting native fluorescence as well as strong UV absorption: using Gly-Tyr, Val-Tyr-Val, methionine enkephalin, and human angiotensin II as test compounds, quenched phosphorescence detection was found to compare favorably with absorption detection at 190- and 266-nm laser-induced fluorescence detection, as performed with a recently developed, small-size, quadrupled Nd:YAG laser.

Ultraviolet resonance Raman spectroscopy (RRS) is presented as a novel identification tool for conventional-size column liquid chromatography (LC). The on-line coupling was made using a standard Z-shaped flow cell. A continuous-wave frequency-doubled argon ion laser operating at a wavelength of 244 nm was used for excitation. "On-the-fly" resonance Raman spectra of four model compounds, fluorene, phenanthrene, fluoranthene, and pyrene, were recorded after a standard acetonitrile/water reversed-phase LC separation. When applying a large-volume-injection procedure (32 mL), detection limits were at the nanogram per milliliter level. The results indicate that UV-RRS gives detailed spectral information at an appropriate sensitivity level so that coupling with LC becomes feasible.

The compatibility of ion-pair reversed-phase column liquid chromatography and surface-enhanced resonance Raman spectroscopy (SERRS) for separation and identification of anionic dyes has been investigated, with emphasis on the at-line coupling via a thin-layer chromatography (TLC) plate. SERR spectra using silver sols were recorded both for aqueous solutions and for samples deposited on aluminum oxide and silica TLC plates at 514.5- and 457.9-nm laser excitation. For some dyes, the shorter wavelength was needed to diminish the fluorescence background. For aqueous solutions and for samples deposited on aluminum oxide, clear SERR spectra were obtained upon addition of poly(L-lysine); for the silica plates, the addition of nitric acid was required. Upon drying the plates, the SERRS signals decreased in intensity; simply adding a drop of water could largely restore them. At-line coupling of LC and SERRS was successfully achieved when using silica, but not aluminum oxide, plates. The application of a gradient, a high water content, and the presence of ion-pair reagents needed for the separation did not adversely affect the deposition and the recording of SERR spectra. The identification limits were 10-20 ng of deposited material, depending on the dye selected, which corresponded to injected concentrations of 5-10 microg mL(-1).

The potential of sensitized and quenched phosphorescence of biacetyl as an optical detection mode in capillary electrophoresis (CE), complementary to absorption and fluorescence detection, was explored. From 24 naphthalenesulfonates (NS) that were studied in batch experiments, 5 NS were used as test compounds in CE. The technique is based on the intense phosphorescence emission of biacetyl (present as a constituent of the CE buffer) at room temperature in deoxygenated liquid solutions. A simple device, based on purging with nitrogen gas, was developed to meet this deoxygenation requirement in CE. A standard liquid chromatography luminescence detector, provided with a pulsed xenon light source, was used for detection. In view of the phosphoresence lifetime of biacetyl (70 μs under present solution conditions), the background caused by scattered excitation light could be readily suppressed by using a delay time for detection. Both phosphorescence modes can be applied at a 0.02 M biacetyl concentration, though in the quenched mode a biacetyl concentration of 0.05 M yields better results. From the five test analytes considered, three show sensitized phosphorescence and two dynamically quenched phosphorescence. Though various experimental parameters still have to be optimized further, the results are quite encouraging:  under stacking conditions (pt = 768 mbar·s), detection limits ranged from 5 × 10(-)(8) to 4 × 10(-)(7) M.